Note: Descriptions are shown in the official language in which they were submitted.
CA 02692819 2010-01-07
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HUMANIZED ANTI-CD79B ANTIBODIES AND IMMUNOCONJUGATES AND METHODS OF
USE
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This non-provisional application filed under 37 CFR 1.53(b), claims
the benefit under 35
USC 119(e) of U.S. Provisional Application Serial No. 60/950,088 filed on
July 16, 2007, which is
incorporated by reference in entirety.
FIELD OF THE INVENTION
[0002] The present invention is directed to compositions of matter useful for
the treatment of
hematopoietic tumor in mammals and to methods of using those compositions of
matter for the same.
BACKGROUND OF THE INVENTION
[0003] Malignant tumors (cancers) are the second leading cause of death in the
United States, after
heart disease (Boring et al., CA Cancel J. Clin. 43:7 (1993)). Cancer is
characterized by the increase in the
number of abnormal, or neoplastic, cells derived from a normal tissue which
proliferate to form a tumor mass,
the invasion of adjacent tissues by these neoplastic tumor cells, and the
generation of malignant cells which
eventually spread via the blood or lymphatic system to regional lymph nodes
and to distant sites via a process
called metastasis. In a cancerous state, a cell proliferates under conditions
in which normal cells would not
grow. Cancer manifests itself in a wide variety of forms, characterized by
different degrees of invasiveness
and aggressiveness.
[0004] Cancers which involve cells generated during hematopoiesis, a process
by which cellular
elements of blood, such as lymphocytes, leukocytes, platelets, erythrocytes
and natural killer cells are
generated are referred to as hematopoietic cancers. Lymphocytes which can be
found in blood and lymphatic
tissue and are critical for immune response are categorized into two main
classes of lymphocytes: B
lymphocytes (B cells) and T lymphocytes ( T cells), which mediate humoral and
cell mediated immunity,
respectively.
[0005] B cells mature within the bone marrow and leave the marrow expressing
an antigen-binding
antibody on their cell surface. When a naive B cell first encounters the
antigen for which its membrane-bound
antibody is specific, the cell begins to divide rapidly and its progeny
differentiate into memory B cells and
effector cells called "plasma cells". Memory B cells have a longer life span
and continue to express
membrane-bound antibody with the same specificity as the original parent cell.
Plasma cells do not produce
membrane-bound antibody but instead produce the antibody in a form that can be
secreted. Secreted
antibodies are the major effector molecule of humoral immunity.
[0006] B cell-related disorders include, but are not limited to, malignant
lymphoma (Non-
Hodgkin's Lymphoma, NHL), multiple myeloma (MM) and chronic lymphocytic
leukemia (CLL, B cell
leukemia (CD5+ B lymphocytes). Non-Hodgkin's lymphomas (NHLs), a heterogeneous
group of cancers
principally arising from B lymphocytes, represent approximately 4% of all
newly diagnosed cancers (Jemal, A.
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et al., CA-Cancer J Clin., 52: 23-47 (2002)). Aggressive NHL comprises
approximately 30-40% of adult NHL
(Harris, N.L. et al., Hematol. J., 1:53-66 (2001)) and includes diffuse large
B-cell lymphoma (DLBCL),
mantle cell lymphoma (MCL), peripheral T-cell lymphoma, and anaplastic large
cell lymphoma. Frontline
combination chemotherapy cures less than half of the patients with aggressive
NHL, and most patients
eventually succumb to their disease (Fischer, R.I., Semin. Oncol., 27 (suppl
12): 2-8 (2000)).
[0007] T cells mature within the thymus which provides an environment for the
proliferation and
differentiation of immature T cells. During T cell maturation, the T cells
undergo the gene rearrangements
that produce the T-cell receptor and the positive and negative selection which
helps determine the cell-surface
phenotype of the mature T cell. Characteristic cell surface markers of mature
T cells are the CD3:T-cell
receptor complex and one of the coreceptors, CD4 or CD8.
[0008] In attempts to discover effective cellular targets for cancer therapy,
researchers have sought
to identify transmembrane or otherwise membrane-associated polypeptides that
are specifically expressed on
the surface of one or more particular type(s) of cancer cell as compared to on
one or more normal non-
cancerous cell(s). Often, such membrane-associated polypeptides are more
abundantly expressed on the
surface of the cancer cells as compared to on the surface of the non-cancerous
cells. The identification of such
tumor-associated cell surface antigen polypeptides has given rise to the
ability to specifically target cancer
cells for destruction via antibody-based therapies. In this regard, it is
noted that antibody-based therapy has
proved very effective in the treatment of certain cancers. For example,
HERCEPTIN and RITUXAN
(both from Genentech Inc., South San Francisco, California) are antibodies
that have been used successfully to
treat breast cancer and non-Hodgkin's lymphoma, respectively. More
specifically, HERCEPTIN is a
recombinant DNA-derived humanized monoclonal antibody that selectively binds
to the extracellular domain
of the human epidermal growth factor receptor 2 (HER2) proto-oncogene. HER2
protein overexpression is
observed in 25-30% of primary breast cancers. RITUXAN is a genetically
engineered chimeric
murine/human monoclonal antibody directed against the CD20 antigen found on
the surface of normal and
malignant B lymphocytes. Both these antibodies are recombinantly produced in
CHO cells.
[0009] In other attempts to discover effective cellular targets for cancer
therapy, researchers have
sought to identify (1) non-membrane-associated polypeptides that are
specifically produced by one or more
particular type(s) of cancer cell(s) as compared to by one or more particular
type(s) of non-cancerous normal
cell(s), (2) polypeptides that are produced by cancer cells at an expression
level that is significantly higher
than that of one or more normal non-cancerous cell(s), or (3) polypeptides
whose expression is specifically
limited to only a single (or very limited number of different) tissue type(s)
in both the cancerous and non-
cancerous state (e.g., normal prostate and prostate tumor tissue). Such
polypeptides may remain
intracellularly located or may be secreted by the cancer cell. Moreover, such
polypeptides may be expressed
not by the cancer cell itself, but rather by cells which produce and/or
secrete polypeptides having a
potentiating or growth-enhancing effect on cancer cells. Such secreted
polypeptides are often proteins that
provide cancer cells with a growth advantage over normal cells and include
such things as, for example,
angiogenic factors, cellular adhesion factors, growth factors, and the like.
Identification of antagonists of such
non-membrane associated polypeptides would be expected to serve as effective
therapeutic agents for the
treatment of such cancers. Furthermore, identification of the expression
pattern of such polypeptides would be
useful for the diagnosis of particular cancers in mammals.
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[0010] Despite the above identified advances in mammalian cancer therapy,
there is a great need for
additional therapeutic agents capable of detecting the presence of tumor in a
mammal and for effectively
inhibiting neoplastic cell growth, respectively. Accordingly, it is an
objective of the present invention to
identify polypeptides, cell membrane-associated, secreted or intracellular
polypeptides whose expression is
specifically limited to only a single (or very limited number of different)
tissue type(s), hematopoietic tissues,
in both a cancerous and non-cancerous state, and to use those polypeptides,
and their encoding nucleic acids,
to produce compositions of matter useful in the therapeutic treatment and/or
detection of hematopoietic cancer
in mammals.
[0011] CD79 is the signaling component of the B-cell receptor consisting of a
covalent heterodimer
containing CD79a (Iga, mb-1) and CD79b (Ig(3, B29). CD79a and CD79b each
contain an extracellular
immunoglobulin (Ig) domain, a transmembrane domain, and an intracellular
signaling domain, an
immunoreceptor tyrosine-based activation motif (ITAM) domain . CD79 is
expressed on B cells and in Non-
Hodgkin's Lymphoma cells (NHLs) (Cabezudo et al., Haematologica, 84:413-418
(1999); D'Arena et al., Am.
J. Hematol., 64: 275-281 (2000); Olejniczak et al., Immunol. Invest., 35: 93-
114 (2006)). CD79a and CD79b
and slg are all required for surface expression of the CD79 (Matsuuchi et al.,
Curr. Opin. Inmunol., 13(3):
270-7)). The average surface expression of CD79b on NHLs is similar to that on
normal B-cells, but with a
greater range (Matsuuchi et al., Curr. Opin. Immunol., 13(3): 270-7 (2001)).
[0012] Given the expression of CD79b, it is beneficial to produce therapeutic
antibodies to the
CD79b antigen that create minimal or no antigenicity when administered to
patients, especially for chronic
treatment. The present invention satisfies this and other needs. The present
invention provides anti-CD79b
antibodies that overcome the limitations of current therapeutic compositions
as well as offer additional
advantages that will be apparent from the detailed description below.
[0013] The use of antibody-drug conjugates (ADC), i.e. immunoconjugates, for
the local delivery of
cytotoxic or cytostatic agents, i.e. drugs to kill or inhibit tumor cells in
the treatment of cancer (Lambert, J.
(2005) Curr. Opinion in Pharmacology 5:543-549; Wu et al (2005) Nature
Biotechnology 23(9):1137-1146;
Payne, G. (2003) Cancer Ce113:207-212; Syrigos and Epenetos (1999) Anticancer
Research 19:605-614;
Niculescu-Duvaz and Springer (1997) Adv. Drug Del. Rev. 26:151-172; US
4975278) allows targeted
delivery of the drug moiety to tumors, and intracellular accumulation therein,
where systemic administration
of these unconjugated drug agents may result in unacceptable levels of
toxicity to normal cells as well as the
tumor cells sought to be eliminated (Baldwin et al (1986) Lancet pp. (Mar. 15,
1986):603-05; Thorpe, (1985)
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in
Monoclonal Antibodies '84:
Biological And Clinical Applications, A. Pinchera et al (ed.s), pp. 475-506).
Efforts to improve the
therapeutic index, i.e. maximal efficacy and minimal toxicity of ADC have
focused on the selectivity of
polyclonal (Rowland et al (1986) Cancer Immunol. Immunother., 21:183-87) and
monoclonal antibodies
(mAbs) as well as drug-linking and drug-releasing properties (Lambert, J.
(2005) Curr. Opinion in
Pharmacology 5:543-549). Drug moieties used in antibody drug conjugates
include bacterial protein toxins
such as diphtheria toxin, plant protein toxins such as ricin, small molecules
such as auristatins, geldanamycin
(Mandler et al (2000) J. of the Nat. Cancer Inst. 92(19):1573-1581; Mandler et
al (2000) Bioorganic & Med.
Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem. 13:786-
791), maytansinoids (EP
1391213; Liu et al (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623),
calicheamicin (Lode et al (1998) Cancer
Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342), daunomycin,
doxorubicin, methotrexate, and
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vindesine (Rowland et al (1986) supra). The drug moieties may affect cytotoxic
and cytostatic mechanisms
including tubulin binding, DNA binding, or topoisomerase inhibition. Some
cytotoxic drugs tend to be
inactive or less active when conjugated to large antibodies or protein
receptor ligands.
[0014] The auristatin peptides, auristatin E (AE) and monomethylauristatin
(MMAE), synthetic
analogs of dolastatin (WO 02/088172), have been conjugated as drug moieties
to: (i) chimeric monoclonal
antibodies cBR96 (specific to Lewis Y on carcinomas); (ii) cAC10 which is
specific to CD30 on
hematological malignancies (Klussman, et al (2004), Bioconjugate Chemistry
15(4):765-773; Doronina et al
(2003) Nature Biotechnology 21(7):778-784; Francisco et al (2003) Blood
102(4):1458-1465; US
2004/00 1 8 1 94; (iii) anti-CD20 antibodies such as rituxan (WO 04/032828)
for the treatment of CD20-
expressing cancers and immune disorders; (iv) anti-EphB2R antibody 2H9 for
treatment of colorectal cancer
(Mao et al (2004) Cancer Research 64(3):781-788); (v) E-selectin antibody
(Bhaskar et al (2003) Cancer Res.
63:6387-6394); (vi) trastuzumab (HERCEPTIN , US 2005/0238649), and (vi) anti-
CD30 antibodies (WO
03/043583). Variants of auristatin E are disclosed in US 5767237 and US
6124431. Monomethyl auristatin E
conjugated to monoclonal antibodies are disclosed in Senter et al, Proceedings
of the American Association
for Cancer Research, Volume 45, Abstract Number 623, presented March 28, 2004.
Auristatin analogs
MMAE and MMAF have been conjugated to various antibodies (US 2005/0238649).
[0015] Conventional means of attaching, i.e. linking through covalent bonds, a
drug moiety to an
antibody generally leads to a heterogeneous mixture of molecules where the
drug moieties are attached at a
number of sites on the antibody. For example, cytotoxic drugs have typically
been conjugated to antibodies
through the often-numerous lysine residues of an antibody, generating a
heterogeneous antibody-drug
conjugate mixture. Depending on reaction conditions, the heterogeneous mixture
typically contains a
distribution of antibodies with from 0 to about 8, or more, attached drug
moieties. In addition, within each
subgroup of conjugates with a particular integer ratio of drug moieties to
antibody, is a potentially
heterogeneous mixture where the drug moiety is attached at various sites on
the antibody. Analytical and
preparative methods may be inadequate to separate and characterize the
antibody-drug conjugate species
molecules within the heterogeneous mixture resulting from a conjugation
reaction. Antibodies are large,
complex and structurally diverse biomolecules, often with many reactive
functional groups. Their reactivities
with linker reagents and drug-linker intermediates are dependent on factors
such as pH, concentration, salt
concentration, and co-solvents. Furthermore, the multistep conjugation process
may be nonreproducible due
to difficulties in controlling the reaction conditions and characterizing
reactants and intermediates.
[0016] Cysteine thiols are reactive at neutral pH, unlike most amines which
are protonated and less
nucleophilic near pH 7. Since free thiol (RSH, sulfhydryl) groups are
relatively reactive, proteins with
cysteine residues often exist in their oxidized form as disulfide-linked
oligomers or have internally bridged
disulfide groups. Extracellular proteins generally do not have free thiols
(Garman, 1997, Non-Radioactive
Labelling: A Practical Approach, Academic Press, London, at page 55). Antibody
cysteine thiol groups are
generally more reactive, i.e. more nucleophilic, towards electrophilic
conjugation reagents than antibody
amine or hydroxyl groups. Cysteine residues have been introduced into proteins
by genetic engineering
techniques to form covalent attachments to ligands or to form new
intramolecular disulfide bonds (Better et al
(1994) J. Biol. Chem. 13:9644-9650; Bernhard et al (1994) Bioconjugate Chem.
5:126-132; Greenwood et al
(1994) Therapeutic Immunology 1:247-255; Tu et al (1999) Proc. Natl. Acad. Sci
USA 96:4862-4867; Kanno
et al (2000) J. of Biotechnology, 76:207-214; Chmura et al (2001) Proc. Nat.
Acad. Sci. USA 98(15):8480-
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8484; US 6248564). However, engineering in cysteine thiol groups by the
mutation of various amino acid
residues of a protein to cysteine amino acids is potentially problematic,
particularly in the case of unpaired
(free Cys) residues or those which are relatively accessible for reaction or
oxidation. In concentrated solutions
of the protein, whether in the periplasm of E. coli, culture supernatants, or
partially or completely purified
protein, unpaired Cys residues on the surface of the protein can pair and
oxidize to form intermolecular
disulfides, and hence protein dimers or multimers. Disulfide dimer formation
renders the new Cys unreactive
for conjugation to a drug, ligand, or other label. Furthermore, if the protein
oxidatively forms an
intramolecular disulfide bond between the newly engineered Cys and an existing
Cys residue, both Cys thiol
groups are unavailable for active site participation and interactions.
Furthermore, the protein may be rendered
inactive or non-specific, by misfolding or loss of tertiary structure (Zhang
et al (2002) Anal. Biochem. 311:1-
9).
[0017] Cysteine-engineered antibodies have been designed as FAB antibody
fragments (thioFab)
and expressed as full-length, IgG monoclonal (thioMab) antibodies (Junutula,
J.R. et al. (2008) J Immunol
Methods 332:41-52; US 2007/0092940, the contents of which are incorporated by
reference). ThioFab and
ThioMab antibodies have been conjugated through linkers at the newly
introduced cysteine thiols with thiol-
reactive linker reagents and drug-linker reagents to prepare antibody drug
conjugates (Thio ADC).
[0018] All references cited herein, including patent applications and
publications, are incorporated
by reference in their entirety.
SUMMARY OF THE INVENTION
[0019] The invention provides anti-CD79b antibodies or functional fragments
thereof, and their
method of use in the treatment of hematopoietic tumors.
[0020] In one aspect, the invention provides an antibody which binds,
preferably specifically, to any
of the above or below described polypeptides. Optionally, the antibody is a
monoclonal antibody, antibody
fragment, including Fab, Fab', F(ab')2, and Fv fragment, diabody, single
domain antibody, chimeric antibody,
humanized antibody, single-chain antibody or antibody that competitively
inhibits the binding of an anti-
CD79b polypeptide antibody to its respective antigenic epitope. Antibodies of
the present invention may
optionally be conjugated to a growth inhibitory agent or cytotoxic agent such
as a toxin, including, for
example, an auristatin, a maytansinoid, a dolostatin derivative or a
calicheamicin, an antibiotic, a radioactive
isotope, a nucleolytic enzyme, or the like. The antibodies of the present
invention may optionally be produced
in CHO cells or bacterial cells and preferably induce death of a cell to which
they bind. For detection
purposes, the antibodies of the present invention may be detectably labeled,
attached to a solid support, or the
like.
[0021] In one aspect, the invention provides a humanized anti-CD79b antibody
wherein the
monovalent affinity (e.g. affinity of the antibody as a Fab fragment to CD79b)
or affinity in its bivalent form
of the antibody to CD79b (e.g. affinity of the antibody as an IgG fragment to
CD79b) is substantially the same
as, lower than, or greater than, the monovalent affinity or affinity in its
bivalent form, respectively, of a murine
antibody (e.g. affinity of the murine antibody as a Fab fragment or as an IgG
fragment to CD79b) or a
chimeric antibody (e.g. affinity of the chimeric antibody as a Fab fragment or
as an IgG fragment to CD79b),
comprising, consisting or consisting essentially of a light chain and heavy
chain variable domain sequence as
depicted in Figure 7 (SEQ ID NO: 10) and Figures 8A-B (SEQ ID NO: 14).
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[0022] In another aspect, the invention provides a humanized anti-CD79b
antibody wherein the
affinity of the antibody in its bivalent form to CD79b (e.g., affinity of the
antibody as an IgG to CD79b) is 2.0
nM.
[0023] In one aspect, an antibody that binds to CD79b is provided, wherein the
antibody comprises:
(a) at least one, two, three, four, five or six HVRs selected from the group
consisting of:
(i) HVR-L1 comprising sequence A1-A15, wherein A1-A16 is KSSQSLLDSDGKTYLN
(SEQ ID NO: 59)
(ii) HVR-L2 comprising sequence B1-B7, wherein B1-B7 is LVSKLDS (SEQ ID NO:
60)
(iii) HVR-L3 comprising sequence C1-C9, wherein C1-C9 is WQGTHFPYT (SEQ ID NO:
61)
(iv) HVR-H1 comprising sequence D1-D10, wherein D1-D10 is GYTFTSYWMN (SEQ ID
NO: 62)
(v) HVR-H2 comprising sequence E1-E18, wherein E1-E18 is GMIDPSDSETHYNHIFKD
(SEQ ID NO: 63) and
(vi) HVR-H3 comprising sequence F1-F6, wherein F1-F10 IS ARNLYL (SEQ ID NO:
64).
[0024] In one aspect, an antibody that binds to CD79b is provided, wherein the
antibody comprises
at least one variant HVR wherein the variant HVR sequence comprises
modification of at least one residue of
the sequence depicted in SEQ ID NOs: 59, 60, 61, 62, 63 or 64.
[0025] In one aspect, the invention provides an antibody comprising a heavy
chain variable domain
comprising the HVR1-HC, HVR2-HC and/or HVR3-HC sequence depicted in Figure 13
(SEQ ID NO: 31-33).
[0026] In one aspect, the invention provides an antibody comprising a light
chain variable domain
comprising HVR1-LC, HVR2-LC and/or HVR3-LC sequence depicted in Figure 13 (SEQ
ID NO: 23-25).
[0027] In one aspect, the invention includes an anti-CD79b antibody comprising
a heavy chain
variable domain of SEQ ID NO: 16. In another aspect, the invention includes an
anti-CD79b antibody
comprising a light chain variable domain of SEQ ID NO: 12.
[0028] In one aspect, the invention includes a cysteine engineered anti-CD79b
antibody comprising
one or more free cysteine amino acids and a sequence selected from SEQ ID NOs:
91-122. The cysteine
engineered anti-CD79b antibody may bind to a CD79b polypeptide. The cysteine
engineered anti-CD79b
antibody may be prepared by a process comprising replacing one or more amino
acid residues of a parent anti-
CD79b antibody by cysteine.
[0029] In one aspect, the invention includes a cysteine engineered anti-CD79b
antibody comprises
one or more free cysteine amino acids wherein the cysteine engineered anti-
CD79b antibody binds to a CD79b
polypeptide and is prepared by a process comprising replacing one or more
amino acid residues of a parent
anti-CD79b antibody by cysteine wherein the parent antibody comprises at least
one HVR sequence selected
from:
(a) HVR-L1 comprising sequence A1-A15, wherein A1-A16 is KSSQSLLDSDGKTYLN (SEQ
ID
NO: 59);
(b) HVR-L2 comprising sequence B1-B7, wherein B1-B7 is LVSKLDS (SEQ ID NO:
60);
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(c) HVR-L3 comprising sequence C1-C9, wherein C1-C9 is WQGTHFPYT (SEQ ID NO:
61) or
FQGTHFPFT (SEQ ID NO: 79);
(d) HVR-H1 comprising sequence D1-D10, wherein D1-D10 is GYTFTSYWMN (SEQ ID
NO: 62);
(e) HVR-H2 comprising sequence E1-E18, wherein E1-E18 is
GMIDPSDSETHYNHIFKD(SEQ ID
NO: 63); and
(f) HVR-H3 comprising sequence F1-F6, wherein F1-F10 IS ARNLYL (SEQ ID NO:
64).
[0030] The cysteine engineered anti-CD79b antibody may be a monoclonal
antibody, antibody
fragment, chimeric antibody, humanized antibody, single-chain antibody or
antibody that competitively
inhibits the binding of an anti-CD79b polypeptide antibody to its respective
antigenic epitope. Antibodies of
the present invention may optionally be conjugated to a growth inhibitory
agent or cytotoxic agent such as a
toxin, including, for example, an auristatin or maytansinoid. The antibodies
of the present invention may
optionally be produced in CHO cells or bacterial cells and preferably inhibit
the growth or proliferation of or
induce the death of a cell to which they bind. For diagnostic purposes, the
antibodies of the present invention
may be detectably labeled, attached to a solid support, or the like.
[0031 ] In one aspect, the invention provides methods for making an antibody
of the invention. For
example, the invention provides a method of making a CD79b antibody (which, as
defined herein includes full
length and fragments thereof), said method comprising expressing in a suitable
host cell a recombinant vector
of the invention encoding said antibody (or fragment thereof), and recovering
said antibody.
[0032] In one aspect, the invention is a pharmaceutical formulation comprising
an antibody of the
invention or an antibody-drug conjugate of the invention, and a
pharmaceutically acceptable diluent, carrier or
excipient.
[0033] In one aspect, the invention provides an article of manufacture
comprising a container; and a
composition contained within the container, wherein the composition comprises
one or more CD79b
antibodies of the invention.
[0034] In one aspect, the invention provides a kit comprising a first
container comprising a
composition comprising one or more CD79b antibodies of the invention; and a
second container comprising a
buffer.
[0035] In one aspect, the invention provides use of a CD79b antibody of the
invention in the
preparation of a medicament for the therapeutic and/or prophylactic treatment
of a disease, such as a cancer, a
tumor and/or a cell proliferative disorder.
[0036] In one aspect, the invention provides use of an article of manufacture
of the invention in the
preparation of a medicament for the therapeutic and/or prophylactic treatment
of a disease, such as a cancer, a
tumor and/or a cell proliferative disorder.
[0037] In one aspect, the invention provides use of a kit of the invention in
the preparation of a
medicament for the therapeutic and/or prophylactic treatment of a disease,
such as a cancer, a tumor and/or a
cell proliferative disorder.
[0038] In one aspect, the invention provides a method of inhibiting the growth
of a cell that
expresses CD79b, said method comprising contacting said cell with an antibody
of the invention thereby
causing an inhibition of growth of said cell. In one embodiment, the antibody
is conjugated to a cytotoxic
agent. In one embodiment, the antibody is conjugated to a growth inhibitory
agent.
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[0039] In one aspect, the invention provides a method of therapeutically
treating a mammal having
a cancerous tumor comprising a cell that expresses CD79b, said method
comprising administering to said
mammal a therapeutically effective amount of an antibody of the invention,
thereby effectively treating said
mammal. In one embodiment, the antibody is conjugated to a cytotoxic agent. In
one embodiment, the
antibody is conjugated to a growth inhibitory agent.
[0040] In one aspect, the invention provides a method for treating or
preventing a cell proliferative
disorder associated with increased expression of CD79b, said method comprising
administering to a subject in
need of such treatment an effective amount of an antibody of the invention,
thereby effectively treating or
preventing said cell proliferative disorder. In one embodiment, said
proliferative disorder is cancer. In one
embodiment, the antibody is conjugated to a cytotoxic agent. In one
embodiment, the antibody is conjugated
to a growth inhibitory agent.
[0041] In one aspect, the invention provides a method for inhibiting the
growth of a cell, wherein
growth of said cell is at least in part dependent upon a growth potentiating
effect of CD79b, said method
comprising contacting said cell with an effective amount of an antibody of the
invention, thereby inhibiting the
growth of said cell. In one embodiment, the antibody is conjugated to a
cytotoxic agent. In one embodiment,
the antibody is conjugated to a growth inhibitory agent.
[0042] In one aspect, the invention provides a method of therapeutically
treating a tumor in a
mammal, wherein the growth of said tumor is at least in part dependent upon a
growth potentiating effect of
CD79b, said method comprising contacting said cell with an effective amount of
an antibody of the invention,
thereby effectively treating said tumor. In one embodiment, the antibody is
conjugated to a cytotoxic agent.
In one embodiment, the antibody is conjugated to a growth inhibitory agent.
[0043] In one aspect, the invention provides a method of treating cancer
comprising administering
to a patient the pharmaceutical formulation comprising an immunoconjugate
described herein, acceptable
diluent, carrier or excipient.
[0044] In one aspect, the invention provides a method of inhibiting B cell
proliferation comprising
exposing a cell to an immunoconjugate comprising an antibody of the invention
under conditions permissive
for binding of the immunoconjugate to CD79b.
[0045] In one aspect, the invention provides a method of determining the
presence of CD79b in a
sample suspected of containing CD79b, said method comprising exposing said
sample to an antibody of the
invention, and determining binding of said antibody to CD79b in said sample
wherein binding of said antibody
to CD79b in said sample is indicative of the presence of said protein in said
sample.
[0046] In one aspect, the invention provides a method of diagnosing a cell
proliferative disorder
associated with an increase in cells, such as B cells, expressing CD79b is
provided, the method comprising
contacting a test cells in a biological sample with any of the above
antibodies; determining the level of
antibody bound to test cells in the sample by detecting binding of the
antibody to CD79b; and comparing the
level of antibody bound to cells in a control sample, wherein the level of
antibody bound is normalized to the
number of CD79b-expressing cells in the test and control samples, and wherein
a higher level of antibody
bound in the test sample as compared to the control sample indicates the
presence of a cell proliferative
disorder associated with cells expressing CD79b.
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[0047] In one aspect, the invention provides a method of detecting soluble
CD79b in blood or
serum, the method comprising contacting a test sample of blood or serum from a
mammal suspected of
experiencing a B cell proliferative disorder with an anti-CD79b antibody of
the invention and detecting a
increase in soluble CD79b in the test sample relative to a control sample of
blood or serum from a normal
mammal.
[0048] In one aspect, the invention provides a method of binding an antibody
of the invention to a
cell that expresses CD79b, said method comprising contacting said cell with an
antibody of the invention. In
one embodiment, the antibody is conjugated to a cytotoxic agent. In one
embodiment, the antibody is
conjugated to a growth inhibitory agent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0049] Figure 1 shows a nucleotide sequence (SEQ ID NO: 1) of a PR036249 cDNA,
wherein SEQ
ID NO: 1 is a clone designated herein as "DNA225786" (also referred here in as
"CD79b"). The nucleotide
sequence encodes for CD79b with the start and stop codons shown in bold and
underlined.
[0050] Figure 2 shows the amino acid sequence (SEQ ID NO: 2) derived from the
coding sequence
of SEQ ID NO: 1 shown in Figure 1.
[0051] Figure 3 shows the nucleotide sequence (SEQ ID NO: 3) of the light
chain of chimeric 2F2
(ch2F2) IgGl (2F2 is a murine monoclonal anti-CD79b antibody). The nucleotide
sequence encodes for the
light chain of ch2F2 shown in Figure 4 with the first codon (encoding for the
first amino acid of SEQ ID NO:
4) shown in bold and underlined.
[0052] Figure 4 shows the amino acid sequence (SEQ ID NO: 4) derived from the
coding sequence
of SEQ ID NO: 3 shown in Figure 3. Variable regions are regions not
underlined.
[0053] Figure 5 shows the nucleotide sequence (SEQ ID NO: 5) of the heavy
chain of chimeric 2F2
(ch2F2) IgGl (2F2 is a murine monoclonal anti-CD79b antibody). The nucleotide
sequence encodes for the
heavy chain of ch2F2 shown in Figure 6 with the first codon (encoding for the
first amino acid of SEQ ID NO:
6) shown in bold and underlined.
[0054] Figure 6 shows the amino acid sequence (SEQ ID NO: 6) derived from the
coding sequence
of SEQ ID NO: 5 shown in Figure 5. Variable regions are regions not
underlined.
[0055] Figure 7 shows the alignment of sequences of the variable light chains
for the following:
light chain human kappa I consensus sequence (labeled as "huKI"; SEQ ID NO: 9)
with VL-FR1, VL-FR2,
VL-FR3, VL-FR4 (SEQ ID NOs: 65-68, respectively), murine 2F2 anti-CD79b
antibody (labeled as "mu2F2"
and herein also referred to as "2F2"; SEQ ID NO: 10), 2F2-grafted "humanized"
antibody (labeled as "hu2F2
graft"; SEQ ID NO: 11) and 2F2-grafted "humanized" antibody variant 7 (labeled
as "hu2F2.D7"; SEQ ID
NO: 12) (containing 71A,73T and 78A). Positions are numbered according to
Kabat and hypervariable
regions (HVRs) grafted from murine 2F2 to the variable light Kappa I consensus
framework are boxed.
[0056] Figures 8A-B show the alignment of sequences of the variable heavy
chains for the
following: heavy chain human subgroup III consensus sequence (labeled as
"humlIP"; SEQ ID NO: 13) with
VH-FR1, VH-FR2, VH-FR3, and VH-FR4 (SEQ ID NOs: 69-72, respectively), murine
2F2 anti-CD79b
antibody (labeled as "mu2F2" and herein also referred to as "2F2"; SEQ ID NO:
14), 2F2-grafted
"humanized" antibody (labeled as "hu2F2 graft"; SEQ ID NO: 15) (containing
71A, 73T and 78A) and 2F2-
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WO 2009/012256 PCT/US2008/070061
grafted "humanized" antibody variant 7 (labeled as "hu2F2.D7"; SEQ ID NO: 16)
(containing 71A, 73T and
78A). Positions are numbered according to Kabat and hypervariable regions
(HVRs) grafted from mu2F2 to
the variable heavy subgroup III consensus framework are boxed.
[0057] Figure 9 shows the HVR sequence of a selected 2F2-grafted "humanized"
antibody variant
(SEQ ID NO: 18), wherein the variant has multiple amino acid changes in a
single HVR region of the 2F2-
grafted "humanized" antibody (portion of HVR-L3 (SEQ ID NO: 61) is shown in
Figure 9 as SEQ ID NO: 17).
The sequences of the variable light and variable heavy chains outside of the
shown amino acid changes were
identical to the 2F2 graft and are not shown. No changes were observed in HVR-
L1 (SEQ ID NO: 59), HVR-
L2 (SEQ ID NO: 60), HVR-H1 (SEQ ID NO: 62); HVR-H2 (SEQ ID NO: 63) or HVR-H3
(SEQ ID NO: 64)
of the 2F2-grafted "humanized" antibody.
[0058] Figure 10 shows Biacore analysis of selected anti-CD79b antibodies,
including ch2F2
antibody (labeled as "ch2F2"), 2F2-grafted "humanized" antibody (labeled as
"hu2F2 graft" and also referred
to herein as "2F2 graft"), and 2F2-grafted "humanized" antibody variant 7
(hu2F2.D7) (89F, 96F; SEQ ID
NO: 18) to designated antigens, including the extracellular domain of human
CD79b (huCD79be,d), the
extracellular domain of human CD79b fused to Fc (huCD79be,d-Fc) and a 16 amino
acid peptide containing
the epitope for 2F2 (16mer peptide; SEQ ID NO: 78; epitope at amino acids 1-11
of SEQ ID NO: 78). No
detected binding is designated in the figure as "NB".
[0059] Figure 11A-B (variable heavy (VH) consensus frameworks) and Figure 12
(variable light
(VL) consensus frameworks) depict exemplary acceptor human consensus framework
sequences for use in
practicing the instant invention with sequence identifiers as follows: (Figure
11A-B) human VH subgroup I
consensus framework minus Kabat CDRs (SEQ ID NO: 36), human VH subgroup I
consensus framework
minus extended hypervariable regions (SEQ ID NOs: 37-39), human VH subgroup II
consensus framework
minus Kabat CDRs (SEQ ID NO: 40), human VH subgroup II consensus framework
minus extended
hypervariable regions (SEQ ID NOs: 41-43), human VH subgroup III consensus
framework minus Kabat
CDRs (SEQ ID NO: 44), human VH subgroup III consensus framework minus extended
hypervariable regions
(SEQ ID NOs: 45-47), human VH acceptor framework minus Kabat CDRs (SEQ ID NO:
48), human VH
acceptor framework minus extended hypervariable regions (SEQ ID NOs: 49-50),
human VH acceptor 2
framework minus Kabat CDRs (SEQ ID NO: 51) and human VH acceptor 2 framework
minus extended
hypervariable regions (SEQ ID NOs: 52-54) and (Figure 12) human VL kappa
subgroup I consensus
framework (SEQ ID NO: 55), human VL kappa subgroup II consensus framework (SEQ
ID NO: 56), human
kappa subgroup III consensus framework (SEQ ID NO: 57) and human kappa
subgroup IV consensus
framework (SEQ ID NO: 58).
[0060] Figure 13A (light chain) and 13B (heavy chain) shows amino acid
sequence of an antibody
of the invention (2F2.D7). Figures 13A (light chain) and 13B (heavy chain)
shows amino acid sequences of
the framework (FR), hypervariable region (HVR), first constant domain (CL or
CH1) and Fc region (Fc) of
one embodiment of an antibody of the invention (hu2F2.D7) (SEQ ID NOs: 19-26
(Figure 13A) and SEQ ID
NOs: 27-35 (Figure 13B)). Full-length amino acid sequences (variable and
constant regions) of the light and
heavy chains of 2F2.D7 are shown (SEQ ID NO: 89 (Figure 13A) and 90 (Figure
13B), respectively, with the
constant domains underlined. Amino acid sequences of the variable domains are
shown (SEQ ID NO: 12
(Figure 13A for light chain) and SEQ ID NO: 16 (Figure 13B for heavy chain)).
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[0061] Figure 14 shows the alignment of the amino acid sequences of CD79b from
human (SEQ ID
NO: 2), cynomolgus monkey (cyno) (SEQ ID NO: 7) and mouse (SEQ ID NO: 8).
Human and cyno-CD79b
have 85% amino acid identity. The signal sequence, test peptide (the 11 amino
acid epitope for 2F2 antibody
described in Example 1; amino acids 1-11 (ARSEDRYRNPK) of SEQ ID NO: 78),
transmembrane (TM)
domain and immunoreceptor tyrosine-based activation motif (ITAM) domain are
indicated. The region boxed
is the region of CD79b that is absent in the splice variant of CD79b
(described in Example 1).
[0062] Figure 15 shows depictions of cysteine engineered anti-CD79b antibody
drug conjugates
(ADC) where a drug moiety is attached to an engineered cysteine group in: the
light chain (LC-ADC); the
heavy chain (HC-ADC); and the Fc region (Fc-ADC).
[0063] Figure 16 shows the steps of: (i) reducing cysteine disulfide adducts
and interchain and
intrachain disulfides in a cysteine engineered anti-CD79b antibody (ThioMab)
with reducing agent TCEP
(tris(2-carboxyethyl)phosphine hydrochloride); (ii) partially oxidizing, i.e.
reoxidation to reform interchain
and intrachain disulfides, with dhAA (dehydroascorbic acid); and (iii)
conjugation of the reoxidized antibody
with a drug-linker intermediate to form a cysteine anti-CD79b drug conjugate
(ADC).
[0064] Figure 17 shows (A) the light chain sequence (SEQ ID NO: 86) and (B)
heavy chain
sequence (SEQ ID NO: 85) of humanized cysteine engineered anti-CD79b antibody
(thio-hu2F2.D7-HC-
A118C), in which an alanine at EU position 118 (sequential position alanine
118; Kabat position 114) of the
heavy chain was altered to a cysteine. A drug moiety may be attached to the
engineered cysteine group in the
heavy chain. In each figure, the altered amino acid is shown in bold text with
double underlining. Single
underlining indicates constant regions. Variable regions are regions not
underlined. Fc region is marked by
italic. "Thio" refers to cysteine-engineered antibody while "hu" refers to
humanized antibody.
[0065] Figure 18 shows (A) the light chain sequence (SEQ ID NO: 88) and (B)
heavy chain
sequence (SEQ ID NO: 87) of cysteine engineered anti-CD79b antibody (Thio-
hu2F2.D7-LC-V205C), in
which a valine at Kabat position 205 (sequential position Valine 210) of the
light chain was altered to a
cysteine. Amino acid D at EU position 6 (shaded in Figure) of the heavy chain
may alternatively be E. A
drug moiety may be attached to the engineered cysteine group in the heavy
chain. In each figure, the altered
amino acid is shown in bold text with double underlining. Single underlining
indicates constant regions.
Variable regions are regions not underlined. Fc region is marked by italic.
"Thio" refers to cysteine-
engineered antibody.
[0066] Figure 19 is a graph of inhibition of in vivo tumor growth in a BJAB-
luciferase xenograft
model which shows that administration of anti-CD79b antibodies ((a) ch2F2-SMCC-
DM1, drug load was
approximately 3 (Table 7) and (b) hu2F2.D7-SMCC-DM1, drug load was
approximately 2.3 (Table 7)), to
SCID mice having human B cell tumors significantly inhibited tumor growth.
Controls included
HERCEPTIN (trastuzumab)-SMCC-DM1 (anti-HER2-SMCC-DM1). "hu" refers to
humanized antibody
and "ch" refers to chimeric antibody.
[0067] Figure 20A is a graph of inhibition of in vivo tumor growth in Granta-
519 (Human Mantle
Cell Lymphoma) xenograft model which shows that administration of anti-CD79b
antibody, hu2F2.D7-
SMCC-DM1, drug load was approximately 2.8 (Table 8), to SCID mice having human
B cell tumors
significantly inhibited tumor growth. Controls included HERCEPTIN
(trastuzumab)-SMCC-DM1 (anti-
HER2-SMCC-DM1). Figure 20B is a plot of percent weight change in the mice from
the Granta-519
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xenograft study (Figure 20A and Table 8) showing that there was no significant
change in weight during the
first 7 days of the study. "hu" refers to humanized antibody.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0068] The invention provides methods, compositions, kits and articles of
manufacture for
identifying compositions useful for the treatment of hematopoietic tumor in
mammals and to methods of using
those compositions of matter for the same.
[0069] Details of these methods, compositions, kits and articles of
manufacture are provided herein.
1. General Techniques
[0070] The practice of the present invention will employ, unless otherwise
indicated, conventional
techniques of molecular biology (including recombinant techniques),
microbiology, cell biology, biochemistry, and
immunology, which are within the skill of the art. Such techniques are
explained fully in the literature, such as,
"Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al.,
1989); "Oligonucleotide Synthesis"
(M. J. Gait, ed., 1984); "Animal Cell Culture" (R. I. Freshney, ed., 1987);
"Methods in Enzymology" (Academic
Press, Inc.); "Current Protocols in Molecular Biology" (F. M. Ausubel et al.,
eds., 1987, and periodic updates);
"PCR: The Polymerase Chain Reaction", (Mullis et al., ed., 1994); "A Practical
Guide to Molecular Cloning"
(Perbal Bernard V., 1988); "Phage Display: A Laboratory Manual" (Barbas et
al., 2001).
II. Definitions
[0071] For purposes of interpreting this specification, the following
definitions will apply and
whenever appropriate, terms used in the singular will also include the plural
and vice versa. In the event that
any definition set forth conflicts with any document incorporated herein by
reference, the definition set forth
below shall control.
[0072] A "B-cell surface marker" or "B-cell surface antigen" herein is an
antigen expressed on the
surface of a B cell that can be targeted with an antagonist that binds
thereto, including but not limited to,
antibodies to a B-cell surface antigen or a soluble form a B-cell surface
antigen capable of antagonizing
binding of a ligand to the naturally occurring B-cell antigen. Exemplary B-
cell surface markers include the
CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD40, CD53, CD72, CD73, CD74,
CDw75, CDw76,
CD77, CDw78, CD79a, CD79b, CD80, CD81, CD82, CD83, CDw84, CD85 and
CD861eukocyte surface
markers (for descriptions, see The Leukocyte Antigen Facts Book, 2"d Edition.
1997, ed. Barclay et al.
Academic Press, Harcourt Brace & Co., New York). Other B-cell surface markers
include RP105, FcRH2, B-
cell CR2, CCR6, P2X5, HLA-DOB, CXCR5, FCER2, BR3, BAFF, BLyS, Btig, NAG14,
SLGC16270,
FcRH1, IRTA2, ATWD578, FcRH3, IRTA1, FcRH6, BCMA, and 239287. The B-cell
surface marker of
particular interest is preferentially expressed on B cells compared to other
non-B-cell tissues of a mammal and
may be expressed on both precursor B cells and mature B cells.
[0073] The term "CD79b", as used herein, refers to any native CD79b from any
vertebrate source,
including mammals such as primates (e.g. humans, cynomolgus monkey (cyno)) and
rodents (.e.g., mice and
rats), unless otherwise indicated. Human CD79b is also referred herein to as
"PRO36249" (SEQ ID NO: 2)
and encoded by the nucleotide sequence (SEQ ID NO: 1) also referred herein to
as "DNA225786". The term
"CD79b" encompasses "full-length," unprocessed CD79b as well as any form of
CD79b that results from
12
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processing in the cell. The term also encompasses naturally occurring variants
of CD79b, e.g., splice variants,
allelic variants and isoforms. The CD79b polypeptides described herein may be
isolated from a variety of
sources, such as from human tissue types or from another source, or prepared
by recombinant or synthetic
methods. A "native sequence CD79b polypeptide" comprises a polypeptide having
the same amino acid
sequence as the corresponding CD79b polypeptide derived from nature. Such
native sequence CD79b
polypeptides can be isolated from nature or can be produced by recombinant or
synthetic means. The term
"native sequence CD79b polypeptide" specifically encompasses naturally-
occurring truncated or secreted
forms of the specific CD79b polypeptide (e.g., an extracellular domain
sequence), naturally-occurring variant
forms (e.g., alternatively spliced forms) and naturally-occurring allelic
variants of the polypeptide. In certain
embodiments of the invention, the native sequence CD79b polypeptides disclosed
herein are mature or full-
length native sequence polypeptides comprising the full-length amino acids
sequences shown in the
accompanying figures. Start and stop codons (if indicated) are shown in bold
font and underlined in the
figures. Nucleic acid residues indicated as "N" in the accompanying figures
are any nucleic acid residue.
However, while the CD79b polypeptides disclosed in the accompanying figures
are shown to begin with
methionine residues designated herein as amino acid position 1 in the figures,
it is conceivable and possible
that other methionine residues located either upstream or downstream from the
amino acid position 1 in the
figures may be employed as the starting amino acid residue for the CD79b
polypeptides.
[0074] The term "2F2" is used herein to refer to murine anti-CD79b monoclonal
antibody (also
herein referred to as "mu2F2" or "murine 2F2") or chimeric antibody (also
herein referred to as "ch2F2").
[0075] "mu2F2" or "murine 2F2" is used herein to specifically refer to murine
anti-CD79b
monoclonal antibody wherein the murine antibody comprises the light chain
variable domain of SEQ ID NO:
(Figure 7) and the heavy chain variable domain of SEQ ID NO: 14 (Figures 8A-
B). mu2F2 may be
generated from hybridomas deposited with the ATCC as PTA-7712 on July 11,
2006.
[0076] "ch2F2" or "chimeric 2F2 antibody" is used herein to specifically refer
to chimeric antibody
(as previously described in US Application No. 11/462,336, filed August 3,
2006) wherein the chimeric
antibody comprises the light chain of SEQ ID NO: 4 (Figure 4) wherein the
light chain comprises the variable
domain of SEQ ID NO: 10 (Figure 7) and the light chain constant domain of
human IgGl. The chimeric
antibody further comprises the heavy chain of SEQ ID NO: 6 (Figure 6) wherein
the light chain comprises the
variable domain of SEQ ID NO: 14 (Figures 8A-B) and the heavy chain constant
domain of human IgGl.
[0077] "21`2-graft " or "2F2-grafted `humanized' antibody" or "hu2F2 graft" is
used herein to
specifically refer to the graft generated by grafting the hypervariable
regions from murine 2F2 antibody
(mu2F2) into the acceptor human consensus VL kappa I (huKI) and human subgroup
III consensus VH (huIII)
with R71A, N73T and L78A (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285
(1992)) (See Example 1A and
Figures 7 (SEQ ID NO: 11) and 8 (SEQ ID NO: 15)).
[0078] A "modification" of an amino acid residue/position, as used herein,
refers to a change of a
primary amino acid sequence as compared to a starting amino acid sequence,
wherein the change results from
a sequence alteration involving said amino acid residue/positions. For
example, typical modifications include
substitution of the residue (or at said position) with another amino acid
(e.g., a conservative or non-
conservative substitution), insertion of one or more (generally fewer than 5
or 3) amino acids adjacent to said
residue/position, and deletion of said residue/position. An "amino acid
substitution", or variation thereof,
refers to the replacement of an existing amino acid residue in a predetermined
(starting) amino acid sequence
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WO 2009/012256 PCT/US2008/070061
with a different amino acid residue. Generally and preferably, the
modification results in alteration in at least
one physicobiochemical activity of the variant polypeptide compared to a
polypeptide comprising the starting
(or "wild type") amino acid sequence. For example, in the case of an antibody,
a physicobiochemical activity
that is altered can be binding affinity, binding capability and/or binding
effect upon a target molecule.
[0079] The term "antibody" is used in the broadest sense and specifically
covers, for example,
single anti-CD79b monoclonal antibodies (including agonist, antagonist,
neutralizing antibodies, full length or
intact monoclonal antibodies), anti-CD79b antibody compositions with
polyepitopic specificity, polyclonal
antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific
antibodies so long as they exhibit
the desired biological activity), formed from at least two intact antibodies,
single chain anti-CD79b antibodies,
and fragments of anti-CD79b antibodies (see below). including Fab, Fab',
F(ab')2 and Fv fragments, diabodies,
single domain antibodies (sdAbs), as long as they exhibit the desired
biological or immunological activity.
The term "immunoglobulin" (Ig) is used interchangeable with antibody herein.
An antibody can be human,
humanized and/or affinity matured.
:0080J The term "anti-CD79b antibody" or "an antibody that binds to CD79b"
refers to an antibody
that is capable of binding CD79b with sufficient affinity such that the
antibody is useful as a diagnostic and/or
therapeutic agent in targeting CD79b. Preferably, the extent of binding of an
anti-CD79b antibody to an
unrelated, non-CD79b protein is less than about 10% of the binding of the
antibody to CD79b as measured,
e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibody that
binds to CD79b has a
dissociation constant (Kd) of < 1 M, < 100 nM, < 10 nM, < 1 nM, or < 0.1 nM.
In certain embodiments,
anti-CD79b antibody binds to an epitope of CD79b that is conserved among CD79b
from different species.
[0081] An "isolated antibody" is one which has been identified and separated
and/or recovered from
a component of its natural environment. Contaminant components of its natural
environment are materials
which would interfere with therapeutic uses for the antibody, and may include
enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred embodiments, the
antibody will be purified (1) to
greater than 95% by weight of antibody as determined by the Lowry method, and
most preferably more than
99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-
terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-
PAGE under reducing or
nonreducing conditions using Coomassie blue or, preferably, silver stain.
Isolated antibody includes the
antibody in situ within recombinant cells since at least one component of the
antibody's natural environment
will not be present. Ordinarily, however, isolated antibody will be prepared
by at least one purification step.
[0082] The basic 4-chain antibody unit is a heterotetrameric glycoprotein
composed of two identical
light (L) chains and two identical heavy (H) chains (an IgM antibody consists
of 5 of the basic heterotetramer
unit along with an additional polypeptide called J chain, and therefore
contain 10 antigen binding sites, while
secreted IgA antibodies can polymerize to form polyvalent assemblages
comprising 2-5 of the basic 4-chain
units along with J chain). In the case of IgGs, the 4-chain unit is generally
about 150,000 daltons. Each L
chain is linked to a H chain by one covalent disulfide bond, while the two H
chains are linked to each other by
one or more disulfide bonds depending on the H chain isotype. Each H and L
chain also has regularly spaced
intrachain disulfide bridges. Each H chain has at the N-terminus, a variable
domain (VH) followed by three
constant domains (CH) for each of the a and y chains and four CH domains for
and s isotypes. Each L chain
has at the N-terminus, a variable domain (VL) followed by a constant domain
(CL) at its other end. The VL is
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WO 2009/012256 PCT/US2008/070061
aligned with the VH and the CL is aligned with the first constant domain of
the heavy chain (CH1). Particular
amino acid residues are believed to form an interface between the light chain
and heavy chain variable
domains. The pairing of a VH and VL together forms a single antigen-binding
site. For the structure and
properties of the different classes of antibodies, see, e.g., Basic and
Clinical Immunology, 8th edition, Daniel
P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange,
Norwalk, CT, 1994, page 71 and
Chapter 6.
[0083] The L chain from any vertebrate species can be assigned to one of two
clearly distinct types,
called kappa and lambda, based on the amino acid sequences of their constant
domains. Depending on the
amino acid sequence of the constant domain of their heavy chains (CH),
immunoglobulins can be assigned to
different classes or isotypes. There are five classes of immunoglobulins: IgA,
IgD, IgE, IgG, and IgM, having
heavy chains designated a, b, s, y, and , respectively. The y and a classes
are further divided into subclasses
on the basis of relatively minor differences in CH sequence and function,
e.g., humans express the following
subclasses: IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.
[0084] The "variable region" or "variable domain" of an antibody refers to the
amino-terminal
domains of the heavy or light chain of the antibody. The variable domain of
the heavy chain may be referred
to as "VH." The variable domain of the light chain may be referred to as "VL."
These domains are generally
the most variable parts of an antibody and contain the antigen-binding sites.
[0085] The term "variable" refers to the fact that certain segments of the
variable domains differ
extensively in sequence among antibodies. The V domain mediates antigen
binding and defines specificity of
a particular antibody for its particular antigen. However, the variability is
not evenly distributed across the
110-amino acid span of the variable domains. Instead, the V regions consist of
relatively invariant stretches
called framework regions (FRs) of 15-30 amino acids separated by shorter
regions of extreme variability
called "hypervariable regions" that are each 9-12 amino acids long. The
variable domains of native heavy and
light chains each comprise four FRs, largely adopting a(3-sheet configuration,
connected by three
hypervariable regions, which form loops connecting, and in some cases forming
part of, the (3-sheet structure.
The hypervariable regions in each chain are held together in close proximity
by the FRs and, with the
hypervariable regions from the other chain, contribute to the formation of the
antigen-binding site of
antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest,
5th Ed. Public Health Service,
National Institutes of Health, Bethesda, MD. (1991)). The constant domains are
not involved directly in
binding an antibody to an antigen, but exhibit various effector functions,
such as participation of the antibody
in antibody dependent cellular cytotoxicity (ADCC).
[0086] An "intact" antibody is one which comprises an antigen-binding site as
well as a CL and at
least heavy chain constant domains, CH1, CH2 and CH3. The constant domains may
be native sequence
constant domains (e.g. human native sequence constant domains) or amino acid
sequence variant thereof.
Preferably, the intact antibody has one or more effector functions.
[0087] A "naked antibody" for the purposes herein is an antibody that is not
conjugated to a
cytotoxic moiety or radiolabel.
[0088] "Antibody fragments" comprise a portion of an intact antibody,
preferably the antigen
binding or variable region of the intact antibody. Examples of antibody
fragments include Fab, Fab', F(ab')2,
and Fv fragments; diabodies; linear antibodies (see U.S. Patent No. 5,641,870,
Example 2; Zapata et al.,
Protein Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules; and
multispecific antibodies formed
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from antibody fragments. In one embodiment, an antibody fragment comprises an
antigen binding site of the
intact antibody and thus retains the ability to bind antigen.
[0089] Papain digestion of antibodies produces two identical antigen-binding
fragments, called
"Fab" fragments, and a residual "Fc" fragment, a designation reflecting the
ability to crystallize readily. The
Fab fragment consists of an entire L chain along with the variable region
domain of the H chain (VH), and the
first constant domain of one heavy chain (CH1). Each Fab fragment is
monovalent with respect to antigen
binding, i.e., it has a single antigen-binding site. Pepsin treatment of an
antibody yields a single large F(ab')2
fragment which roughly corresponds to two disulfide linked Fab fragments
having divalent antigen-binding
activity and is still capable of cross-linking antigen. Fab' fragments differ
from Fab fragments by having
additional few residues at the carboxy terminus of the CH1 domain including
one or more cysteines from the
antibody hinge region. Fab'-SH is the designation herein for Fab' in which the
cysteine residue(s) of the
constant domains bear a free thiol group. F(ab')2 antibody fragments
originally were produced as pairs of Fab'
fragments which have hinge cysteines between them. Other chemical couplings of
antibody fragments are
also known.
[0090] The Fc fragment comprises the carboxy-terminal portions of both H
chains held together by
disulfides. The effector functions of antibodies are determined by sequences
in the Fc region, which region is
also the part recognized by Fc receptors (FcR) found on certain types of
cells.
[0091 ]"Fv" is the minimum antibody fragment which contains a complete antigen-
recognition and
-binding site. This fragment consists of a dimer of one heavy- and one light-
chain variable region domain in
tight, non-covalent association. In a single-chain Fv (scFv) species, one
heavy- and one light-chain variable
domain can be covalently linked by a flexible peptide linker such that the
light and heavy chains can associate
in a "dimeric" structure analogous to that in a two-chain Fv species. From the
folding of these two domains
emanate six hypervariable loops (3 loops each from the H and L chain) that
contribute the amino acid residues
for antigen binding and confer antigen binding specificity to the antibody.
However, even a single variable
domain (or half of an Fv comprising only three CDRs specific for an antigen)
has the ability to recognize and
bind antigen, although at a lower affinity than the entire binding site.
[0092] "Single-chain Fv" also abbreviated as "sFv" or "scFv" are antibody
fragments that comprise
the VH and VL antibody domains connected into a single polypeptide chain.
Preferably, the sFv polypeptide
further comprises a polypeptide linker between the VH and VL domains which
enables the sFv to form the
desired structure for antigen binding. For a review of sFv, see Pluckthun in
The Pharmacology of Monoclonal
Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.
269-315 (1994);
Borrebaeck 1995, infra.
[0093] The term "diabodies" refers to antibody fragments with two antigen-
binding sites, which
fragments comprise a heavy-chain variable domain (VH) connected to a light-
chain variable domain (VL) in
the same polypeptide chain (VH-VL). The small antibody fragments are prepared
by constructing sFv
fragments (see preceding paragraph) with short linkers (about 5-10 residues)
between the VH and VL domains
such that inter-chain but not intra-chain pairing of the V domains is
achieved, resulting in a bivalent fragment,
i.e., fragment having two antigen-binding sites. Diabodies may be bivalent or
bispecific. Bispecific diabodies
are heterodimers of two "crossover" sFv fragments in which the VH and VL
domains of the two antibodies are
present on different polypeptide chains. Diabodies are described more fully
in, for example, EP 404,097; WO
93/11161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al.,
Proc. Natl. Acad. Sci. USA,
16
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WO 2009/012256 PCT/US2008/070061
90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson
et al., Nat. Med. 9:129-134
(2003).
[0094] The term "monoclonal antibody" as used herein refers to an antibody
obtained from a
population of substantially homogeneous antibodies, i.e., the individual
antibodies comprising the population
are identical except for possible naturally occurring mutations that may be
present in minor amounts.
Monoclonal antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in
contrast to polyclonal antibody preparations which include different
antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed against a single
determinant on the antigen. In
addition to their specificity, the monoclonal antibodies are advantageous in
that they may be synthesized
uncontaminated by other antibodies. The modifier "monoclonal" is not to be
construed as requiring
production of the antibody by any particular method. For example, the
monoclonal antibodies useful in the
present invention may be prepared by the hybridoma methodology first described
by Kohler et al., Nature,
256:495 (1975), or may be made using recombinant DNA methods in bacterial,
eukaryotic animal or plant
cells (see, e.g., U.S. Patent No. 4,816,567). The "monoclonal antibodies" may
also be isolated from phage
antibody libraries using the techniques described in Clackson et al., Nature,
352:624-628 (1991) and Marks et
al., J. Mol. Biol., 222:581-597 (1991), for example.
[0095] The monoclonal antibodies herein include "chimeric" antibodies in which
a portion of the
heavy and/or light chain is identical with or homologous to corresponding
sequences in antibodies derived
from a particular species or belonging to a particular antibody class or
subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences in
antibodies derived from another
species or belonging to another antibody class or subclass, as well as
fragments of such antibodies, so long as
they exhibit the desired biological activity (see U.S. Patent No. 4,816,567;
and Morrison et al., Proc. Natl.
Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein
include "primatized" antibodies
comprising variable domain antigen-binding sequences derived from a non-human
primate (e.g. Old World
Monkey, Ape etc), and human constant region sequences.
[0096] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric
antibodies that
contain minimal sequence derived from the non-human antibody. For the most
part, humanized antibodies are
human immunoglobulins (recipient antibody) in which residues from a
hypervariable region of the recipient
are replaced by residues from a hypervariable region of a non-human species
(donor antibody) such as mouse,
rat, rabbit or non-human primate having the desired antibody specificity,
affinity, and capability. In some
instances, framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-
human residues. Furthermore, humanized antibodies may comprise residues that
are not found in the recipient
antibody or in the donor antibody. These modifications are made to further
refine antibody performance. In
general, the humanized antibody will comprise substantially all of at least
one, and typically two, variable
domains, in which all or substantially all of the hypervariable loops
correspond to those of a non-human
immunoglobulin and all or substantially all of the FRs are those of a human
immunoglobulin sequence. The
humanized antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc),
typically that of a human immunoglobulin. For further details, see Jones et
al., Nature 321:522-525 (1986);
Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct.
Biol. 2:593-596 (1992). See also
the following review articles and references cited therein: Vaswani and
Hamilton, Ann. Allergy, Asthma and
17
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WO 2009/012256 PCT/US2008/070061
Immunol., 1:105-115 (1998); Harris, Biochem. Soc. Transactions, 23:1035-1038
(1995); Hurle and Gross,
Curr. Op. Biotech., 5:428-433 (1994).
[0097] "Thio" when used herein to refer to an antibody refers to a cysteine-
engineered antibody
while "hu" when used herein to refer to an antibody refers to a humanized
antibody.
[0098] A "human antibody" is one which possesses an amino acid sequence which
corresponds to
that of an antibody produced by a human and/or has been made using any of the
techniques for making human
antibodies as disclosed herein. This definition of a human antibody
specifically excludes a humanized
antibody comprising non-human antigen-binding residues. Human antibodies can
be produced using various
techniques known in the art, including phage-display libraries. Hoogenboom and
Winter, J. Mol. Biol.,
227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also available
for the preparation of human
monoclonal antibodies are methods described in Cole et al., Monoclonal
Antibodies and Cancer Therapy,
Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991).
See also van Dijk and van de
Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be
prepared by administering the
antigen to a transgenic animal that has been modified to produce such
antibodies in response to antigenic
challenge, but whose endogenous loci have been disabled, e.g., immunized
xenomice (see, e.g., U.S. Pat. Nos.
6,075,181 and 6,150,584 regarding XENOMOUSETM technology). See also, for
example, Li et al., Proc. Natl.
Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodies generated via
a human B-cell hybridoma
technology.
[0099] The term "hypervariable region", "HVR", or "HV", when used herein
refers to the regions
of an antibody variable domain which are hypervariable in sequence and/or form
structurally defined loops.
Generally, antibodies comprise six hypervariable regions; three in the VH (H1,
H2, H3), and three in the VL
(L1, L2, L3). A number of hypervariable region delineations are in use and are
encompassed herein. The
Kabat Complementarity Determining Regions (CDRs) are based on sequence
variability and are the most
commonly used (Kabat et al., Sequences ofProteins ofbnmunologicalInterest, 5th
Ed. Public Health Service,
National Institutes of Health, Bethesda, MD. (1991)). Chothia refers instead
to the location of the structural
loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The end of the
Chothia CDR-H1 loop when
numbered using the Kabat numbering convention varies between H32 and H34
depending on the length of the
loop (this is because the Kabat numbering scheme places the insertions at H35A
and H35B; if neither 35A nor
35B is present, the loop ends at 32; if only 35A is present, the loop ends at
33; if both 35A and 35B are present,
the loop ends at 34). The AbM hypervariable regions represent a compromise
between the Kabat CDRs and
Chothia structural loops, and are used by Oxford Molecular's AbM antibody
modeling software. The "contact"
hypervariable regions are based on an analysis of the available complex
crystal structures. The residues from
each of these hypervariable regions are noted below.
Loop Kabat AbM Chothia Contact
------- -------- ------- ---------- ----------
L1 L24-L34 L24-L34 L24-L34 L30-L36
L2 L50-L56 L50-L56 L50-L56 L46-L55
L3 L89-L97 L89-L97 L89-L97 L89-L96
H1 H31-H35B H26-H35B H26-H32..34 H30-H35B
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WO 2009/012256 PCT/US2008/070061
(Kabat Numbering)
H1 H31-H35 H26-H35 H26-H32 H30-H35
(Chothia Numbering)
H2 H50-H65 H50-H58 H52-H56 H47-H58
H3 H95-H102 H95-H102 H95-H102 H93-H101
[0100] Hypervariable regions may comprise "extended hypervariable regions" as
follows: 24-36 or
24-34 (L1), 46-56 or 50-56 (L2) and 89-97 (L3) in the VL and 26-35B (H1), 50-
65, 47-65 or 49-65 (H2) and
93-102, 94-102 or 95-102 (H3) in the VH. The variable domain residues are
numbered according to Kabat et
al., supra for each of these definitions.
[0101 ]"Framework" or "FR" residues are those variable domain residues other
than the
hypervariable region residues herein defined.
[0102] The term "variable domain residue numbering as in Kabat" or "amino acid
position
numbering as in Kabat", and variations thereof, refers to the numbering system
used for heavy chain variable
domains or light chain variable domains of the compilation of antibodies in
Kabat et al., Sequences of Proteins
of Immunological Interest, 5th Ed. Public Health Service, National Institutes
of Health, Bethesda, MD. (1991).
Using this numbering system, the actual linear amino acid sequence may contain
fewer or additional amino
acids corresponding to a shortening of, or insertion into, a FR or CDR of the
variable domain. For example, a
heavy chain variable domain may include a single amino acid insert (residue
52a according to Kabat) after
residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc
according to Kabat) after heavy
chain FR residue 82. The Kabat numbering of residues may be determined for a
given antibody by alignment
at regions of homology of the sequence of the antibody with a "standard" Kabat
numbered sequence.
[0103] The Kabat numbering system is generally used when referring to a
residue in the variable
domain (approximately residues 1-107 of the light chain and residues 1-113 of
the heavy chain) (e.g, Kabat et
al., Sequences ofbnmunologicalInterest. 5th Ed. Public Health Service,
National Institutes of Health,
Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally
used when referring to a
residue in an immunoglobulin heavy chain constant region (e.g., the EU index
reported in Kabat et al., supra).
The "EU index as in Kabat" refers to the residue numbering of the human IgGl
EU antibody. Unless stated
otherwise herein, references to residue numbers in the variable domain of
antibodies means residue numbering
by the Kabat numbering system. Unless stated otherwise herein, references to
residue numbers in the constant
domain of antibodies means residue numbering by the EU numbering system (e.g.,
see United States
Provisional Application No. 60/640,323, Figures for EU numbering).
[0104] An "affinity matured" antibody is one with one or more alterations in
one or more HVRs
thereof which result in an improvement in the affinity of the antibody for
antigen, compared to a parent
antibody which does not possess those alteration(s). Preferred affinity
matured antibodies will have
nanomolar or even picomolar affinities for the target antigen. Affinity
matured antibodies are produced by
procedures known in the art. Marks et al. Bio/Technology 10:779-783 (1992)
describes affinity maturation by
VH and VL domain shuffling. Random mutagenesis of HVR and/or framework
residues is described by:
Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier et al. Gene
169:147-155 (1995); Yelton
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CA 02692819 2010-01-07
WO 2009/012256 PCT/US2008/070061
et al. J. bnmunol. 155:1994-2004 (1995); Jackson et al., J. bnmunol.
154(7):3310-9 (1995); and Hawkins et al,
J. Mol. Biol. 226:889-896 (1992).
[0105] A "blocking" antibody or an "antagonist" antibody is one which inhibits
or reduces
biological activity of the antigen it binds. Preferred blocking antibodies or
antagonist antibodies substantially
or completely inhibit the biological activity of the antigen.
[0106] An "agonist antibody", as used herein, is an antibody which mimics at
least one of the
functional activities of a polypeptide of interest.
[0107] A "species-dependent antibody," e.g., a mammalian anti-human IgE
antibody, is an antibody
which has a stronger binding affinity for an antigen from a first mammalian
species than it has for a
homologue of that antigen from a second mammalian species. Normally, the
species-dependent antibody
"bind specifically" to a human antigen (i.e., has a binding afflnity (Kd)
value of no more than about 1 x 10-' M,
preferably no more than about 1 x 10 and most preferably no more than about 1
x 10-9 M) but has a binding
affinity for a homologue of the antigen from a second non-human mammalian
species which is at least about
50 fold, or at least about 500 fold, or at least about 1000 fold, weaker than
its binding affinity for the human
antigen. The species-dependent antibody can be of any of the various types of
antibodies as defined above,
but preferably is a humanized or human antibody.
[0108] "Binding affinity" generally refers to the strength of the sum total of
noncovalent
interactions between a single binding site of a molecule (e.g., an antibody)
and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding affinity"
refers to intrinsic binding affinity
which reflects a 1:1 interaction between members of a binding pair (e.g.,
antibody and antigen). The affinity
of a molecule X for its partner Y can generally be represented by the
dissociation constant (Kd). Affinity can
be measured by common methods known in the art, including those described
herein. Low-affinity antibodies
generally bind antigen slowly and tend to dissociate readily, whereas high-
affinity antibodies generally bind
antigen faster and tend to remain bound longer. A variety of methods of
measuring binding affinity are known
in the art, any of which can be used for purposes of the present invention.
Specific illustrative embodiments
are described in the following.
[0109] "Or better" when used herein to refer to binding affinity refers to a
stronger binding between
a molecule and its binding partner. "Or better" when used herein refers to a
stronger binding, represented by a
smaller numerical Kd value. For example, an antibody which has an affinity for
an antigen of ".6 nM or
better", the antibody's affinity for the antigen is <.6 nM, i.e. .59 nM, .58
nM, .57 nM etc. or any value less
than.6 nM.
[0110] In one embodiment, the "Kd" or "Kd value" according to this invention
is measured by a
radiolabeled antigen binding assay (RIA) performed with the Fab version of an
antibody of interest and its
antigen as described by the following assay that measures solution binding
affinity of Fabs for antigen by
equilibrating Fab with a minimal concentration of (1251)-labeled antigen in
the presence of a titration series of
unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-
coated plate (Chen, et al., (1999) J.
Mol Biol 293:865-881). To establish conditions for the assay, microtiter
plates (Dynex) are coated overnight
with 5 g/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium
carbonate (pH 9.6), and
subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five
hours at room temperature
(approximately 23 C). In a non-adsorbant plate (Nunc #269620), 100 pM or 26 pM
[I25I]-antigen are mixed
CA 02692819 2010-01-07
WO 2009/012256 PCT/US2008/070061
with serial dilutions of a Fab of interest (e.g., consistent with assessment
of an anti-VEGF antibody, Fab-12, in
Presta et al., (1997) Cancer Res. 57:4593-4599). The Fab of interest is then
incubated overnight; however, the
incubation may continue for a longer period (e.g., 65 hours) to insure that
equilibrium is reached. Thereafter,
the mixtures are transferred to the capture plate for incubation at room
temperature (e.g., for one hour). The
solution is then removed and the plate washed eight times with 0.1 % Tween-20
in PBS. When the plates have
dried, 150 Uwell of scintillant (MicroScint-20; Packard) is added, and the
plates are counted on a Topcount
gamma counter (Packard) for ten minutes. Concentrations of each Fab that give
less than or equal to 20% of
maximal binding are chosen for use in competitive binding assays. According to
another embodiment the Kd
or Kd value is measured by using surface plasmon resonance assays using a
BlAcoreTM-2000 or a
BlAcoreTM-3000 (BlAcore, Inc., Piscataway, NJ) at 25C with immobilized antigen
CM5 chips at -10
response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5,
BlAcore Inc.) are activated
with N-ethyl-N'- (3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and
N-hydroxysuccinimide
(NHS) according to the supplier's instructions. Antigen is diluted with 10mM
sodium acetate, pH 4.8, into
5ug/ml (-0.2uM) before injection at a flow rate of 5u1/minute to achieve
approximately 10 response units
(RU) of coupled protein. Following the injection of antigen, 1M ethanolamine
is injected to block unreacted
groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM
to 500 nM) are injected in PBS
with 0.05% Tween 20 (PBST) at 25 C at a flow rate of approximately 25u1/min.
Association rates (kon) and
dissociation rates (koff) are calculated using a simple one-to-one Langmuir
binding model (BlAcore
Evaluation Software version 3.2) by simultaneous fitting the association and
dissociation sensorgram. The
equilibrium dissociation constant (Kd) is calculated as the ratio kofflkon.
See, e.g., Chen, Y., et al., (1999) J.
Mol Biol 293:865-881. If the on-rate exceeds 106 M-I S-I by the surface
plasmon resonance assay above,
then the on-rate can be determined by using a fluorescent quenching technique
that measures the increase or
decrease in fluorescence emission intensity (excitation = 295 nm; emission =
340 nm, 16 nm band-pass) at
25 C of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the
presence of increasing
concentrations of antigen as measured in a spectrometer, such as a stop-flow
equipped spectrophometer (Aviv
Instruments) or a 8000-series SLM-Aminco spectrophotometer (ThermoSpectronic)
with a stir red cuvette.
[0111] An "on-rate" or "rate of association" or "association rate" or
"konaccording to this
invention can also be determined with the same surface plasmon resonance
technique described above using a
BlAcoreTM-2000 or a BlAcoreTM-3000 (BlAcore, Inc., Piscataway, NJ) as
described above.
[0112] The phrase "substantially similar," or "substantially the same", as
used herein, denotes a
sufficiently high degree of similarity between two numeric values (generally
one associated with an antibody
of the invention and the other associated with a reference/comparator
antibody) such that one of skill in the art
would consider the difference between the two values to be of little or no
biological and/or statistical
significance within the context of the biological characteristic measured by
said values (e.g., Kd values). The
difference between said two values is preferably less than about 50%,
preferably less than about 40%,
preferably less than about 30%, preferably less than about 20%, preferably
less than about 10% as a function
of the value for the reference/comparator antibody.
[0113] The phrase "substantially reduced," or "substantially different", as
used herein, denotes a
sufficiently high degree of difference between two numeric values (generally
one associated with an antibody
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WO 2009/012256 PCT/US2008/070061
of the invention and the other associated with a reference/comparator
antibody) such that one of skill in the art
would consider the difference between the two values to be of statistical
significance within the context of the
biological characteristic measured by said values (e.g., Kd values, HAMA
response). The difference between
said two values is preferably greater than about 10%, preferably greater than
about 20%, preferably greater
than about 30%, preferably greater than about 40%, preferably greater than
about 50% as a function of the
value for the reference/comparator antibody.
[0114] An "antigen" is a predetermined antigen to which an antibody can
selectively bind. The
target antigen may be polypeptide, carbohydrate, nucleic acid, lipid, hapten
or other naturally occurring or
synthetic compound. Preferably, the target antigen is a polypeptide.
[0115] An "acceptor human framework" for the purposes herein is a framework
comprising the
amino acid sequence of a VL or VH framework derived from a human
immunoglobulin framework, or from a
human consensus framework. An acceptor human framework "derived from" a human
immunoglobulin
framework or human consensus framework may comprise the same amino acid
sequence thereof, or may
contain pre-existing amino acid sequence changes. Where pre-existing amino
acid changes are present,
preferably no more than 5 and preferably 4 or less, or 3 or less, pre-existing
amino acid changes are present.
Where pre-existing amino acid changes are present in a VH, preferably those
changes are only at three, two or
one of positions 71H, 73H and 78H; for instance, the amino acid residues at
those positions may be 71A, 73T
and/or 78A. In one embodiment, the VL acceptor human framework is identical in
sequence to the VL human
immunoglobulin framework sequence or human consensus framework sequence.
[0116] A "human consensus framework" is a framework which represents the most
commonly
occurring amino acid residue in a selection of human immunoglobulin VL or VH
framework sequences.
Generally, the selection of human immunoglobulin VL or VH sequences is from a
subgroup of variable
domain sequences. Generally, the subgroup of sequences is a subgroup as in
Kabat et al. In one embodiment,
for the VL, the subgroup is subgroup kappa I as in Kabat et al. In one
embodiment, for the VH, the subgroup
is subgroup III as in Kabat et al.
[0117] A "VH subgroup III consensus framework" comprises the consensus
sequence obtained
from the amino acid sequences in variable heavy subgroup III of Kabat et al.
In one embodiment, the VH
subgroup III consensus framework amino acid sequence comprises at least a
portion or all of each of the
following sequences: EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 69)-H1-
WVRQAPGKGLEWV
(SEQ ID NO: 70)-H2-RFTISRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO: 71)-H3-
WGQGTLVTVSS (SEQ ID NO: 72).
[0118] A "VL subgroup I consensus framework" comprises the consensus sequence
obtained from
the amino acid sequences in variable light kappa subgroup I of Kabat et al. In
one embodiment, the VL
subgroup I consensus framework amino acid sequence comprises at least a
portion or all of each of the
following sequences:
DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 65)-L1-WYQQKPGKAPKLLIY (SEQ ID NO: 66)-L2-
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 67)-L3-FGQGTKVEIKR (SEQ ID NO:
68).
[0119] An "unmodified human framework" is a human framework which has the same
amino acid
sequence as the acceptor human frameworlc, e.g. lacking human to non-human
amino acid substitution(s) in
the acceptor human framework.
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[0120] An "altered hypervariable region" for the purposes herein is a
hypervariable region
comprising one or more (e.g. one to about 16) amino acid substitution(s)
therein.
[0121] An "un-modified hypervariable region" for the purposes herein is a
hypervariable region
having the same amino acid sequence as a non-human antibody from which it was
derived, i.e. one which
lacks one or more amino acid substitutions therein.
[0122] An antibody "which binds" an antigen of interest, e.g. a tumor-
associated polypeptide
antigen target, is one that binds the antigen with sufficient affinity such
that the antibody is useful as a
therapeutic agent in targeting a cell or tissue expressing the antigen, and
does not significantly cross-react with
other proteins. In such embodiments, the extent of binding of the antibody to
a "non-target" protein will be
less than about 10% of the binding of the antibody to its particular target
protein as determined by
fluorescence activated cell sorting (FACS) analysis or
radioimmunoprecipitation (RIA). With regard to the
binding of an antibody to a target molecule, the term "specific binding" or
"specifically binds to" or is
"specific for" a particular polypeptide or an epitope on a particular
polypeptide target means binding that is
measurably different from a non-specific interaction. Specific binding can be
measured, for example, by
determining binding of a molecule compared to binding of a control molecule,
which generally is a molecule
of similar structure that does not have binding activity. For example,
specific binding can be determined by
competition with a control molecule that is similar to the target, for
example, an excess of non-labeled target.
In this case, specific binding is indicated if the binding of the labeled
target to a probe is competitively
inhibited by excess unlabeled target. The term "specific binding" or
"specifically binds to" or is "specific for"
a particular polypeptide or an epitope on a particular polypeptide target as
used herein can be exhibited, for
example, by a molecule having a Kd for the target of at least about 10-4 M,
alternatively at least about 10-5 M,
alternatively at least about 10-6 M, alternatively at least about 10-' M,
alternatively at least about 10-8 M,
alternatively at least about 10-9 M, alternatively at least about 10-10 M,
alternatively at least about 10-" M,
alternatively at least about 10-12 M, or greater. In one embodiment, the term
"specific binding" refers to
binding where a molecule binds to a particular polypeptide or epitope on a
particular polypeptide without
substantially binding to any other polypeptide or polypeptide epitope.
[0123] An antibody that "inhibits the growth of tumor cells expressing a CD79b
polypeptide" or a
"growth inhibitory" antibody is one which results in measurable growth
inhibition of cancer cells expressing
or overexpressing the appropriate CD79b polypeptide. The CD79b polypeptide may
be a transmembrane
polypeptide expressed on the surface of a cancer cell or may be a polypeptide
that is produced and secreted by
a cancer cell. Preferred growth inhibitory anti-CD79b antibodies inhibit
growth of CD79b-expressing tumor
cells by greater than 20%, preferably from about 20% to about 50%, and even
more preferably, by greater than
50% (e.g., from about 50% to about 100%) as compared to the appropriate
control, the control typically being
tumor cells not treated with the antibody being tested. In one embodiment,
growth inhibition can be measured
at an antibody concentration of about 0.1 to 30 g/ml or about 0.5 nM to 200
nM in cell culture, where the
growth inhibition is determined 1-10 days after exposure of the tumor cells to
the antibody. Growth inhibition
of tumor cells in vivo can be determined in various ways such as is described
in the Experimental Examples
section below. The antibody is growth inhibitory in vivo if administration of
the anti-CD79b antibody at
about 1 g/kg to about 100 mg/kg body weight results in reduction in tumor
size or tumor cell proliferation
within about 5 days to 3 months from the first administration of the antibody,
preferably within about 5 to 30
days.
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[0124] An antibody which "induces apoptosis" is one which induces programmed
cell death as
determined by binding of annexin V, fragmentation of DNA, cell shrinkage,
dilation of endoplasmic reticulum,
cell fragmentation, and/or formation of membrane vesicles (called apoptotic
bodies). The cell is usually one
which overexpresses a CD79b polypeptide. Preferably the cell is a tumor cell,
e.g., a hematopoietic cell, such
as a B cell, T cell, basophil, eosinophil, neutrophil, monocyte, platelet or
erythrocyte. Various methods are
available for evaluating the cellular events associated with apoptosis. For
example, phosphatidyl serine (PS)
translocation can be measured by annexin binding; DNA fragmentation can be
evaluated through DNA
laddering; and nuclear/chromatin condensation along with DNA fragmentation can
be evaluated by any
increase in hypodiploid cells. Preferably, the antibody which induces
apoptosis is one which results in about 2
to 50 fold, preferably about 5 to 50 fold, and most preferably about 10 to 50
fold, induction of annexin binding
relative to untreated cell in an annexin binding assay.
[0125] An antibody which "induces cell death" is one which causes a viable
cell to become
nonviable. The cell is one which expresses a CD79b polypeptide and is of a
cell type which specifically
expresses or overexpresses a CD79b polypeptide. The cell may be cancerous or
normal cells of the particular
cell type. The CD79b polypeptide may be a transmembrane polypeptide expressed
on the surface of a cancer
cell or may be a polypeptide that is produced and secreted by a cancer cell.
The cell may be a cancer cell, e.g.,
a B cell or T cell. Cell death in vitro may be determined in the absence of
complement and immune effector
cells to distinguish cell death induced by antibody-dependent cell-mediated
cytotoxicity (ADCC) or
complement dependent cytotoxicity (CDC). Thus, the assay for cell death may be
performed using heat
inactivated serum (i.e., in the absence of complement) and in the absence of
immune effector cells. To
determine whether the antibody is able to induce cell death, loss of membrane
integrity as evaluated by uptake
of propidium iodide (PI), trypan blue (see Moore et al. Cytotechnology 17:1-11
(1995)) or 7AAD can be
assessed relative to untreated cells. Preferred cell death-inducing antibodies
are those which induce PI uptake
in the PI uptake assay in BT474 cells.
[0126] Antibody "effector functions" refer to those biological activities
attributable to the Fc region
(a native sequence Fc region or amino acid sequence variant Fc region) of an
antibody, and vary with the
antibody isotype. Examples of antibody effector functions include: C1 q
binding and complement dependent
cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated
cytotoxicity (ADCC); phagocytosis;
down regulation of cell surface receptors (e.g., B cell receptor); and B cell
activation.
[0127] The term "Fc region" herein is used to define a C-terminal region of an
immunoglobulin
heavy chain, including native sequence Fc regions and variant Fc regions.
Although the boundaries of the Fc
region of an immunoglobulin heavy chain might vary, the human IgG heavy chain
Fc region is usually defined
to stretch from an amino acid residue at position Cys226, or from Pro230, to
the carboxyl-terminus
thereof. The C-terminal lysine (residue 447 according to the EU numbering
system) of the Fc region may be
removed, for example, during production or purification of the antibody, or by
recombinantly engineering the
nucleic acid encoding a heavy chain of the antibody. Accordingly, a
composition of intact antibodies may
comprise antibody populations with all K447 residues removed, antibody
populations with no K447 residues
removed, and antibody populations having a mixture of antibodies with and
without the K447 residue.
[0128] A "functional Fc region" possesses an "effector function" of a native
sequence Fc region.
Exemplary "effector functions" include C 1 q binding; CDC; Fc receptor
binding; ADCC; phagocytosis; down
regulation of cell surface receptors (e.g. B cell receptor; BCR), etc. Such
effector functions generally require
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the Fc region to be combined with a binding domain (e.g., an antibody variable
domain) and can be assessed
using various assays as disclosed, for example, in definitions herein.
[0129] A "native sequence Fc region" comprises an amino acid sequence
identical to the amino acid
sequence of an Fc region found in nature. Native sequence human Fc regions
include a native sequence human
IgGl Fc region (non-A and A allotypes); native sequence human IgG2 Fc region;
native sequence human
IgG3 Fc region; and native sequence human IgG4 Fc region as well as naturally
occurring variants thereof.
[0130] A "variant Fc region" comprises an amino acid sequence which differs
from that of a native
sequence Fc region by virtue of at least one amino acid modification,
preferably one or more amino acid
substitution(s). Preferably, the variant Fc region has at least one amino acid
substitution compared to a native
sequence Fc region or to the Fc region of a parent polypeptide, e.g. from
about one to about ten amino acid
substitutions, and preferably from about one to about five amino acid
substitutions in a native sequence Fc
region or in the Fc region of the parent polypeptide. The variant Fc region
herein will preferably possess at
least about 80% homology with a native sequence Fc region and/or with an Fc
region of a parent polypeptide,
and most preferably at least about 90% homology therewith, more preferably at
least about 95% homology
therewith.
[0131 ]"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a
form of
cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on
certain cytotoxic cells (e.g.,
Natural Killer (NK) cells, neutrophils, and macrophages) enable these
cytotoxic effector cells to bind
specifically to an antigen-bearing target cell and subsequently kill the
target cell with cytotoxins. The
antibodies "arm" the cytotoxic cells and are absolutely required for such
killing. The primary cells for
mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express
FcyRI, FcyRII and FcyRIII.
FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of
Ravetch and Kinet, Annu.
Rev. Immunol. 9:457-92 (1991). To assess ADCC activity of a molecule of
interest, an in vitro ADCC assay,
such as that described in US Patent No. 5,500,362 or 5,821,337 may be
performed. Useful effector cells for
such assays include peripheral blood mononuclear cells (PBMC) and Natural
Killer (NK) cells. Alternatively,
or additionally, ADCC activity of the molecule of interest may be assessed in
vivo, e.g., in a animal model
such as that disclosed in Clynes et al. (USA) 95:652-656 (1998).
[0132] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region
of an antibody. The
preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is one
which binds an IgG
antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII and
FcyRIII subclasses, including
allelic variants and alternatively spliced forms of these receptors. FcyRII
receptors include FcyRIIA (an
"activating receptor") and FcyRIIB (an "inhibiting receptor"), which have
similar amino acid sequences that
differ primarily in the cytoplasmic domains thereof. Activating receptor
FcyRIIA contains an immunoreceptor
tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting
receptor FcyRIIB contains an
immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic
domain. (see review M. in Daeron,
Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and
Kinet, Annu. Rev. Immunol.
9:457-492 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et
al., J. Lab. Clin. Med.
126:330-41 (1995). Other FcRs, including those to be identified in the future,
are encompassed by the term
"FcR" herein. The term also includes the neonatal receptor, FcRn, which is
responsible for the transfer of
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WO 2009/012256 PCT/US2008/070061
maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim
et al., J. Immunol. 24:249
(1994)).
[0133] Binding to human FcRn in vivo and serum half life of human FcRn high
affinity binding
polypeptides can be assayed, e.g., in transgenic mice or transfected human
cell lines expressing human FcRn,
or in primates to which the polypeptides with a variant Fc region are
administered. WO 2000/42072 (Presta)
describes antibody variants with improved or diminished binding to FcRs. See
also, e.g., Shields et al. J. Biol.
Chem. 9(2):6591-6604 (2001).
[0134] "Human effector cells" are leukocytes which express one or more FcRs
and perform effector
functions. Preferably, the cells express at least FcyRIII and perform ADCC
effector function. Examples of
human leukocytes which mediate ADCC include peripheral blood mononuclear cells
(PBMC), natural killer
(NK) cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK
cells being preferred. The
effector cells may be isolated from a native source, e.g., from blood.
[0135] "Complement dependent cytotoxicity" or "CDC" refers to the lysis of a
target cell in the
presence of complement. Activation of the classical complement pathway is
initiated by the binding of the
first component of the complement system (C1q) to antibodies (of the
appropriate subclass) which are bound
to their cognate antigen. To assess complement activation, a CDC assay, e.g.,
as described in Gazzano-
Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed.
Polypeptide variants with altered Fc
region amino acid sequences (polypeptides with a variant Fc region) and
increased or decreased Cl q binding
capability are described, e.g., in US Patent No. 6,194,551 B 1 and WO
1999/51642. See also, e.g., Idusogie et
al. J. Immunol. 164: 4178-4184 (2000).
[0136] The term "Fc region-comprising antibody" refers to an antibody that
comprises an Fc region.
The C-terminal lysine (residue 447 according to the EU numbering system) of
the Fc region may be removed,
for example, during purification of the antibody or by recombinant engineering
of the nucleic acid encoding
the antibody. Accordingly, a composition comprising an antibody having an Fc
region according to this
invention can comprise an antibody with K447, with all K447 removed, or a
mixture of antibodies with and
without the K447 residue.
[0137] The CD79b polypeptide "extracellular domain" or "ECD" refers to a form
of the CD79b
polypeptide which is essentially free of the transmembrane and cytoplasmic
domains. Ordinarily, a CD79b
polypeptide ECD will have less than 1% of such transmembrane and/or
cytoplasmic domains and preferably,
will have less than 0.5% of such domains. It will be understood that any
transmembrane domains identified
for the CD79b polypeptides of the present invention are identified pursuant to
criteria routinely employed in
the art for identifying that type of hydrophobic domain. The exact boundaries
of a transmembrane domain
may vary but most likely by no more than about 5 amino acids at either end of
the domain as initially
identified herein. Optionally, therefore, an extracellular domain of a CD79b
polypeptide may contain from
about 5 or fewer amino acids on either side of the transmembrane
domain/extracellular domain boundary as
identified in the Examples or specification and such polypeptides, with or
without the associated signal
peptide, and nucleic acid encoding them, are contemplated by the present
invention.
[0138] The approximate location of the "signal peptides" of the CD79b
polypeptide disclosed
herein may be shown in the present specification and/or the accompanying
figures. It is noted, however, that
the C-terminal boundary of a signal peptide may vary, but most likely by no
more than about 5 amino acids on
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either side of the signal peptide C-terminal boundary as initially identified
herein, wherein the C-terminal
boundary of the signal peptide may be identified pursuant to criteria
routinely employed in the art for
identifying that type of amino acid sequence element (e.g., Nielsen et al.,
Prot. Eng. 10:1-6 (1997) and von
Heinje et al., Nucl. Acids. Res. 14:4683-4690 (1986)). Moreover, it is also
recognized that, in some cases,
cleavage of a signal sequence from a secreted polypeptide is not entirely
uniform, resulting in more than one
secreted species. These mature polypeptides, where the signal peptide is
cleaved within no more than about 5
amino acids on either side of the C-terminal boundary of the signal peptide as
identified herein, and the
polynucleotides encoding them, are contemplated by the present invention.
[0139] "CD79b polypeptide variant" means a CD79b polypeptide, preferably an
active CD79b
polypeptide, as defined herein having at least about 80% amino acid sequence
identity with a full-length native
sequence CD79b polypeptide sequence as disclosed herein, a CD79b polypeptide
sequence lacking the signal
peptide as disclosed herein, an extracellular domain of a CD79b polypeptide,
with or without the signal
peptide, as disclosed herein or any other fragment of a full-length CD79b
polypeptide sequence as disclosed
herein (such as those encoded by a nucleic acid that represents only a portion
of the complete coding sequence
for a full-length CD79b polypeptide). Such CD79b polypeptide variants include,
for instance, CD79b
polypeptides wherein one or more amino acid residues are added, or deleted, at
the N- or C-terminus of the
full-length native amino acid sequence. Ordinarily, a CD79b polypeptide
variant will have at least about 80%
amino acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence
identity, to a full-length
native sequence CD79b polypeptide sequence as disclosed herein, a CD79b
polypeptide sequence lacking the
signal peptide as disclosed herein, an extracellular domain of a CD79b
polypeptide, with or without the signal
peptide, as disclosed herein or any other specifically defined fragment of a
full-length CD79b polypeptide
sequence as disclosed herein. Ordinarily, CD79b variant polypeptides are at
least about 10 amino acids in
length, alternatively at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,
120, 130, 140, 150, 160, 170, 180,
190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330,
340, 350, 360, 370, 380, 390, 400,
410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,
560, 570, 580, 590, 600 amino
acids in length, or more. Optionally, CD79b variant polypeptides will have no
more than one conservative
amino acid substitution as compared to the native CD79b polypeptide sequence,
alternatively no more than 2,
3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitution as compared to
the native CD79b polypeptide
sequence.
[0140] "Percent (%) amino acid sequence identity" with respect to a peptide or
polypeptide
sequence, i.e. CD79b polypeptide sequences identified herein, is defined as
the percentage of amino acid
residues in a candidate sequence that are identical with the amino acid
residues in the specific peptide or
polypeptide sequence, i.e. CD79b polypeptide sequence, after aligning the
sequences and introducing gaps, if
necessary, to achieve the maximum percent sequence identity, and not
considering any conservative
substitutions as part of the sequence identity. Alignment for purposes of
determining percent amino acid
sequence identity can be achieved in various ways that are within the skill in
the art, for instance, using
publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign
(DNASTAR) software.
Those skilled in the art can determine appropriate parameters for measuring
alignment, including any
algorithms needed to achieve maximal alignment over the full length of the
sequences being compared. For
purposes herein, however, % amino acid sequence identity values are generated
using the sequence
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comparison computer program ALIGN-2, wherein the complete source code for the
ALIGN-2 program is
provided in Table 1 below. The ALIGN-2 sequence comparison computer program
was authored by
Genentech, Inc. and the source code shown in Table 1 below has been filed with
user documentation in the
U.S. Copyright Office, Washington D.C., 20559, where it is registered under
U.S. Copyright Registration No.
TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc.,
South San Francisco,
California or may be compiled from the source code provided in Table 1 below.
The ALIGN-2 program
should be compiled for use on a UNIX operating system, preferably digital UNIX
V4.0D. All sequence
comparison parameters are set by the ALIGN-2 program and do not vary.
[0141] In situations where ALIGN-2 is employed for amino acid sequence
comparisons, the %
amino acid sequence identity of a given amino acid sequence A to, with, or
against a given amino acid
sequence B (which can alternatively be phrased as a given amino acid sequence
A that has or comprises a
certain % amino acid sequence identity to, with, or against a given amino acid
sequence B) is calculated as
follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by
the sequence alignment program
ALIGN-2 in that program's alignment of A and B, and where Y is the total
number of amino acid residues in
B. It will be appreciated that where the length of amino acid sequence A is
not equal to the length of amino
acid sequence B, the % amino acid sequence identity of A to B will not equal
the % amino acid sequence
identity of B to A.
[0142] "CD79b variant polynucleotide" or "CD79b variant nucleic acid sequence"
means a nucleic
acid molecule which encodes a CD79b polypeptide, preferably an active CD79b
polypeptide, as defined
herein and which has at least about 80% nucleic acid sequence identity with a
nucleotide acid sequence
encoding a full-length native sequence CD79b polypeptide sequence as disclosed
herein, a full-length native
sequence CD79b polypeptide sequence lacking the signal peptide as disclosed
herein, an extracellular domain
of a CD79b polypeptide, with or without the signal peptide, as disclosed
herein or any other fragment of a full-
length CD79b polypeptide sequence as disclosed herein (such as those encoded
by a nucleic acid that
represents only a portion of the complete coding sequence for a full-length
CD79b polypeptide). Ordinarily, a
CD79b variant polynucleotide will have at least about 80% nucleic acid
sequence identity, alternatively at
least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%,
98%, or 99% nucleic acid sequence identity with a nucleic acid sequence
encoding a full-length native
sequence CD79b polypeptide sequence as disclosed herein, a full-length native
sequence CD79b polypeptide
sequence lacking the signal peptide as disclosed herein, an extracellular
domain of a CD79b polypeptide, with
or without the signal sequence, as disclosed herein or any other fragment of a
full-length CD79b polypeptide
sequence as disclosed herein. Variants do not encompass the native nucleotide
sequence.
[0143] Ordinarily, CD79b variant polynucleotides are at least about 5
nucleotides in length,
alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115,
120, 125, 130, 135, 140, 145, 150,
155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250,
260, 270, 280, 290, 300, 310, 320,
330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470,
480, 490, 500, 510, 520, 530, 540,
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550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690,
700, 710, 720, 730, 740, 750, 760,
770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910,
920, 930, 940, 950, 960, 970, 980,
990, or 1000 nucleotides in length, wherein in this context the term "about"
means the referenced nucleotide
sequence length plus or minus 10% of that referenced length.
[0144] "Percent (%) nucleic acid sequence identity" with respect to CD79b-
encoding nucleic acid
sequences identified herein is defined as the percentage of nucleotides in a
candidate sequence that are
identical with the nucleotides in the CD79b nucleic acid sequence of interest,
after aligning the sequences and
introducing gaps, if necessary, to achieve the maximum percent sequence
identity. Alignment for purposes of
determining percent nucleic acid sequence identity can be achieved in various
ways that are within the skill in
the art, for instance, using publicly available computer software such as
BLAST, BLAST-2, ALIGN or
Megalign (DNASTAR) software. For purposes herein, however, % nucleic acid
sequence identity values are
generated using the sequence comparison computer program ALIGN-2, wherein the
complete source code for
the ALIGN-2 program is provided in Table 1 below. The ALIGN-2 sequence
comparison computer program
was authored by Genentech, Inc. and the source code shown in Table 1 below has
been filed with user
documentation in the U.S. Copyright Office, Washington D.C., 20559, where it
is registered under U.S.
Copyright Registration No. TXU510087. The ALIGN-2 program is publicly
available through Genentech,
Inc., South San Francisco, California or may be compiled from the source code
provided in Table 1 below.
The ALIGN-2 program should be compiled for use on a UNIX operating system,
preferably digital UNIX
V4.0D. All sequence comparison parameters are set by the
ALIGN-2 program and do not vary.
[0145] In situations where ALIGN-2 is employed for nucleic acid sequence
comparisons, the %
nucleic acid sequence identity of a given nucleic acid sequence C to, with, or
against a given nucleic acid
sequence D (which can alternatively be phrased as a given nucleic acid
sequence C that has or comprises a
certain % nucleic acid sequence identity to, with, or against a given nucleic
acid sequence D) is calculated as
follows:
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the
sequence alignment program
ALIGN-2 in that program's alignment of C and D, and where Z is the total
number of nucleotides in D. It will
be appreciated that where the length of nucleic acid sequence C is not equal
to the length of nucleic acid
sequence D, the % nucleic acid sequence identity of C to D will not equal the
% nucleic acid sequence identity
of D to C. Unless specifically stated otherwise, all % nucleic acid sequence
identity values used herein are
obtained as described in the immediately preceding paragraph using the ALIGN-2
computer program.
[0146] In other embodiments, CD79b variant polynucleotides are nucleic acid
molecules that
encode a CD79b polypeptide and which are capable of hybridizing, preferably
under stringent hybridization
and wash conditions, to nucleotide sequences encoding a full-length CD79b
polypeptide as disclosed herein.
CD79b variant polypeptides may be those that are encoded by a CD79b variant
polynucleotide.
[0147] The term "full-length coding region" when used in reference to a
nucleic acid encoding a
CD79b polypeptide refers to the sequence of nucleotides which encode the full-
length CD79b
polypeptide of the invention (which is often shown between start and stop
codons, inclusive thereof, in the
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accompanying figures). The term "full-length coding region" when used in
reference to an ATCC deposited
nucleic acid refers to the CD79b polypeptide-encoding portion of the cDNA that
is inserted into the vector
deposited with the ATCC (which is often shown between start and stop codons,
inclusive thereof, in the
accompanying figures (start and stop codons are bolded and underlined in the
figures)).
[0148] "Isolated," when used to describe the various CD79b polypeptides
disclosed herein, means
polypeptide that has been identified and separated and/or recovered from a
component of its natural
environment. Contaminant components of its natural environment are materials
that would typically interfere
with therapeutic uses for the polypeptide, and may include enzymes, hormones,
and other proteinaceous or
non-proteinaceous solutes. In preferred embodiments, the polypeptide will be
purified (1) to a degree
sufficient to obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup
sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing
conditions using Coomassie
blue or, preferably, silver stain. Isolated polypeptide includes polypeptide
in situ within recombinant cells,
since at least one component of the CD79b polypeptide natural environment will
not be present. Ordinarily,
however, isolated polypeptide will be prepared by at least one purification
step.
[0149] An "isolated" CD79b polypeptide-encoding nucleic acid or other
polypeptide-encoding
nucleic acid is a nucleic acid molecule that is identified and separated from
at least one contaminant nucleic
acid molecule with which it is ordinarily associated in the natural source of
the polypeptide-encoding nucleic
acid. An isolated polypeptide-encoding nucleic acid molecule is other than in
the form or setting in which it is
found in nature. Isolated polypeptide-encoding nucleic acid molecules
therefore are distinguished from the
specific polypeptide-encoding nucleic acid molecule as it exists in natural
cells. However, an isolated
polypeptide-encoding nucleic acid molecule includes polypeptide-encoding
nucleic acid molecules contained
in cells that ordinarily express the polypeptide where, for example, the
nucleic acid molecule is in a
chromosomal location different from that of natural cells.
[0150] The term "control sequences" refers to DNA sequences necessary for the
expression of an
operably linked coding sequence in a particular host organism. The control
sequences that are suitable for
prokaryotes, for example, include a promoter, optionally an operator sequence,
and a ribosome binding site.
Eukaryotic cells are known to utilize promoters, polyadenylation signals, and
enhancers.
[0151] Nucleic acid is "operably linked" when it is placed into a functional
relationship with
another nucleic acid sequence. For example, DNA for a presequence or secretory
leader is operably linked to
DNA for a polypeptide if it is expressed as a preprotein that participates in
the secretion of the polypeptide; a
promoter or enhancer is operably linked to a coding sequence if it affects the
transcription of the sequence; or
a ribosome binding site is operably linked to a coding sequence if it is
positioned so as to facilitate translation.
Generally, "operably linked" means that the DNA sequences being linked are
contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However, enhancers do not
have to be contiguous. Linking
is accomplished by ligation at convenient restriction sites. If such sites do
not exist, the synthetic
oligonucleotide adaptors or linkers are used in accordance with conventional
practice.
[0152] "Stringency" of hybridization reactions is readily determinable by one
of ordinary skill in
the art, and generally is an empirical calculation dependent upon probe
length, washing temperature, and salt
concentration. In general, longer probes require higher temperatures for
proper annealing, while shorter
probes need lower temperatures. Hybridization generally depends on the ability
of denatured DNA to
reanneal when complementary strands are present in an environment below their
melting temperature. The
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higher the degree of desired homology between the probe and hybridizable
sequence, the higher the relative
temperature which can be used. As a result, it follows that higher relative
temperatures would tend to make
the reaction conditions more stringent, while lower temperatures less so. For
additional details and
explanation of stringency of hybridization reactions, see Ausubel et al.,
Current Protocols in Molecular
Biology, Wiley Interscience Publishers, (1995).
[0153 ]"Stringent conditions" or "high stringency conditions", as defined
herein, may be identified
by those that: (1) employ low ionic strength and high temperature for washing,
for example 0.015 M sodium
chloride/0.0015 M sodium citrate/0.1 % sodium dodecyl sulfate at 50 C; (2)
employ during hybridization a
denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1
% bovine serum
albumin/0. 1 % FicolU0.1 % polyvinylpyrrolidone/50mM sodium phosphate buffer
at pH 6.5 with 750 mM
sodium chloride, 75 mM sodium citrate at 42 C; or (3) overnight hybridization
in a solution that employs 50%
formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 m1V1 sodium
phosphate (pH 6.8), 0.1 %
sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50
g/ml), 0.1 % SDS, and
10% dextran sulfate at 42 C, with a 10 minute wash at 42 C in 0.2 x SSC
(sodium chloride/sodium citrate)
followed by a 10 minute high-stringency wash consisting of 0.1 x SSC
containing EDTA at 55 C.
[0154] "Moderately stringent conditions" may be identified as described by
Sambrook et al.,
Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press,
1989, and include the use of
washing solution and hybridization conditions (e.g., temperature, ionic
strength and %SDS) less stringent that
those described above. An example of moderately stringent conditions is
overnight incubation at 37 C in a
solution comprising: 20% formamide, 5 x SSC (150 m1V1 NaCl, 15 mM trisodium
citrate), 50 mM sodium
phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/ml
denatured sheared salmon
sperm DNA, followed by washing the filters in 1 x SSC at about 37-50 C. The
skilled artisan will recognize
how to adjust the temperature, ionic strength, etc. as necessary to
accommodate factors such as probe length
and the like.
[0155] The term "epitope tagged" when used herein refers to a chimeric
polypeptide comprising a
CD79b polypeptide or anti-CD79b antibody fused to a "tag polypeptide". The tag
polypeptide has enough
residues to provide an epitope against which an antibody can be made, yet is
short enough such that it does not
interfere with activity of the polypeptide to which it is fused. The tag
polypeptide preferably also is fairly
unique so that the antibody does not substantially cross-react with other
epitopes. Suitable tag polypeptides
generally have at least six amino acid residues and usually between about 8
and 50 amino acid residues
(preferably, between about 10 and 20 amino acid residues).
[0156] "Active" or "activity" for the purposes herein refers to form(s) of a
CD79b polypeptide
which retain a biological and/or an immunological activity of native or
naturally-occurring CD79b, wherein
"biological" activity refers to a biological function (either inhibitory or
stimulatory) caused by a native or
naturally-occurring CD79b other than the ability to induce the production of
an antibody against an antigenic
epitope possessed by a native or naturally-occurring CD79b and an
"immunological" activity refers to the
ability to induce the production of an antibody against an antigenic epitope
possessed by a native or naturally-
occurring CD79b.
[0157] The term "antagonist" is used in the broadest sense, and includes any
molecule that partially
or fully blocks, inhibits, or neutralizes a biological activity of a native
CD79b polypeptide. In a similar
manner, the term "agonist" is used in the broadest sense and includes any
molecule that mimics a biological
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activity of a native CD79b polypeptide. Suitable agonist or antagonist
molecules specifically include agonist
or antagonist antibodies or antibody fragments, fragments or amino acid
sequence variants of native CD79b
polypeptides, peptides, antisense oligonucleotides, small organic molecules,
etc. Methods for identifying
agonists or antagonists of a CD79b polypeptide, may comprise contacting a
CD79b polypeptide, with a
candidate agonist or antagonist molecule and measuring a detectable change in
one or more biological
activities normally associated with the CD79b polypeptide.
[0158] "Purified" means that a molecule is present in a sample at a
concentration of at least 95% by
weight, or at least 98% by weight of the sample in which it is contained.
[0159] An "isolated" nucleic acid molecule is a nucleic acid molecule that is
separated from at least
one other nucleic acid molecule with which it is ordinarily associated, for
example, in its natural environment.
An isolated nucleic acid molecule further includes a nucleic acid molecule
contained in cells that ordinarily
express the nucleic acid molecule, but the nucleic acid molecule is present
extrachromasomally or at a
chromosomal location that is different from its natural chromosomal location.
[0160] The term "vector," as used herein, is intended to refer to a nucleic
acid molecule capable of
transporting another nucleic acid to which it has been linked. One type of
vector is a "plasmid", which refers
to a circular double stranded DNA loop into which additional DNA segments may
be ligated. Another type
of vector is a phage vector. Another type of vector is a viral vector, wherein
additional DNA segments may
be ligated into the viral genome. Certain vectors are capable of autonomous
replication in a host cell into
which they are introduced (e.g., bacterial vectors having a bacterial origin
of replication and episomal
mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can
be integrated into the
genome of a host cell upon introduction into the host cell, and thereby are
replicated along with the host
genome. Moreover, certain vectors are capable of directing the expression of
genes to which they are
operatively linked. Such vectors are referred to herein as "recombinant
expression vectors" (or simply,
"recombinant vectors"). In general, expression vectors of utility in
recombinant DNA techniques are often in
the form of plasmids. In the present specification, "plasmid" and "vector" may
be used interchangeably as
the plasmid is the most commonly used form of vector.
[0161] "Polynucleotide," or "nucleic acid," as used interchangeably herein,
refer to polymers of
nucleotides of any length, and include DNA and RNA. The nucleotides can be
deoxyribonucleotides,
ribonucleotides, modified nucleotides or bases, and/or their analogs, or any
substrate that can be incorporated
into a polymer by DNA or RNA polymerase, or by a synthetic reaction. A
polynucleotide may comprise
modified nucleotides, such as methylated nucleotides and their analogs. If
present, modification to the
nucleotide structure may be imparted before or after assembly of the polymer.
The sequence of nucleotides
may be interrupted by non-nucleotide components. A polynucleotide may be
further modified after synthesis,
such as by conjugation with a label. Other types of modifications include, for
example, "caps", substitution of
one or more of the naturally occurring nucleotides with an analog,
internucleotide modifications such as, for
example, those with uncharged linkages (e.g., methyl phosphonates,
phosphotriesters, phosphoamidates,
carbamates, etc.) and with charged linkages (e.g., phosphorothioates,
phosphorodithioates, etc.), those
containing pendant moieties, such as, for example, proteins (e.g., nucleases,
toxins, antibodies, signal peptides,
ply-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen,
etc.), those containing chelators (e.g.,
metals, radioactive metals, boron, oxidative metals, etc.), those containing
alkylators, those with modified
linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified
forms of the polynucleotide(s).
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Further, any of the hydroxyl groups ordinarily present in the sugars may be
replaced, for example, by
phosphonate groups, phosphate groups, protected by standard protecting groups,
or activated to prepare
additional linkages to additional nucleotides, or may be conjugated to solid
or semi-solid supports. The 5' and
3' terminal OH can be phosphorylated or substituted with amines or organic
capping group moieties of from 1
to 20 carbon atoms. Other hydroxyls may also be derivatized to standard
protecting groups. Polynucleotides
can also contain analogous forms of ribose or deoxyribose sugars that are
generally known in the art,
including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azido-
ribose, carbocyclic sugar
analogs, .alpha.-anomeric sugars, epimeric sugars such as arabinose, xyloses
or lyxoses, pyranose sugars,
furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs
such as methyl riboside. One
or more phosphodiester linkages may be replaced by alternative linking groups.
These alternative linking
groups include, but are not limited to, embodiments wherein phosphate is
replaced by P(O)S("thioate"), P(S)S
("dithioate"), "(O)NR2 ("amidate"), P(O)R, P(O)OR', CO or CH2
("formacetal"), in which each R or
R' is independently H or substituted or unsubstituted alkyl (1-20 C.)
optionally containing an ether (-0-)
linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages
in a polynucleotide need be
identical. The preceding description applies to all polynucleotides referred
to herein, including RNA and
DNA.
[0162] "Oligonucleotide," as used herein, generally refers to short, generally
single stranded,
generally synthetic polynucleotides that are generally, but not necessarily,
less than about 200 nucleotides in
length. The terms "oligonucleotide" and "polynucleotide" are not mutually
exclusive. The description above
for polynucleotides is equally and fully applicable to oligonucleotides.
[0163] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in
mammals that is typically characterized by unregulated cell growth. Examples
of cancer include, but are not
limited to, hematopoietic cancers or blood-related cancers, such as lymphoma,
leukemia, myeloma or
lymphoid malignancies, but also cancers of the spleen and cancers of the lymph
nodes and also carcinoma,
blastoma and sarcoma. More particular examples of cancer include B-cell
associated cancers, including for
example, high, intermediate and low grade lymphomas (including B cell
lymphomas such as, for example,
mucosa-associated-lymphoid tissue B cell lymphoma and non-Hodgkin's lymphoma
(NHL), mantle cell
lymphoma, Burkitt's lymphoma, small lymphocytic lymphoma, marginal zone
lymphoma, diffuse large cell
lymphoma, follicular lymphoma, and Hodgkin's lymphoma and T cell lymphomas)
and leukemias (including
secondary leukemia, chronic lymphocytic leukemia (CLL), such as B cell
leukemia (CD5+ B lymphocytes),
myeloid leukemia, such as acute myeloid leukemia, chronic myeloid leukemia,
lymphoid leukemia, such as
acute lymphoblastic leukemia (ALL) and myelodysplasia), and other
hematological and/or B cell- or T-cell-
associated cancers. Also included are cancers of additional hematopoietic
cells, including polymorphonuclear
leukocytes, such as basophils, eosinophils, neutrophils and monocytes,
dendritic cells, platelets, erythrocytes
and natural killer cells. Also included are cancerous B cell proliferative
disorders selected from the following:
lymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL, relapsed aggressive
NHL, relapsed indolent
NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia
(CLL), small lymphocytic
lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia
(ALL), and mantle cell
lymphoma. The origins of B-cell cancers include as follows: marginal zone B-
cell lymphoma origins in
memory B-cells in marginal zone, follicular lymphoma and diffuse large B-cell
lymphoma originates in
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centrocytes in the light zone of germinal centers, chronic lymphocytic
leukemia and small lymphocytic
leukemia originates in B 1 cells (CD5+), mantle cell lymphoma originates in
naive B-cells in the mantle zone
and Burkitt's lymphoma originates in centroblasts in the dark zone of germinal
centers. Tissues which include
hematopoietic cells referred herein to as "hematopoietic cell tissues" include
thymus and bone marrow and
peripheral lymphoid tissues, such as spleen, lymph nodes, lymphoid tissues
associated with mucosa, such as
the gut-associated lymphoid tissues, tonsils, Peyer's patches and appendix and
lymphoid tissues associated
with other mucosa, for example, the bronchial linings. Further particular
examples of such cancers include
squamous cell cancer, small-cell lung cancer, non-small cell lung cancer,
adenocarcinoma of the lung,
squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular
cancer, gastrointestinal cancer,
pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer,
bladder cancer, hepatoma, breast
cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma,
salivary gland carcinoma, kidney
cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic
carcinoma, leukemia and other
lymphoproliferative disorders, and various types of head and neck cancer.
[0164] A "B-cell malignancy" herein includes non-Hodgkin's lymphoma (NHL),
including low
grade/follicular NHL, small lymphocytic (SL) NHL, intermediate
grade/follicular NHL, intermediate grade
diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high
grade small non-cleaved
cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and
Waldenstrom's
Macroglobulinemia, non-Hodgkin's lymphoma (NHL), lymphocyte predominant
Hodgkin's disease (LPHD),
small lymphocytic lymphoma (SLL), chronic lymphocytic leukemia (CLL), indolent
NHL including relapsed
indolent NHL and rituximab-refractory indolent NHL; leukemia, including acute
lymphoblastic leukemia
(ALL), chronic lymphocytic leukemia (CLL), Hairy cell leukemia, chronic
myeloblastic leukemia; mantle cell
lymphoma; and other hematologic malignancies. Such malignancies may be treated
with antibodies directed
against B-cell surface markers, such as CD79b. Such diseases are contemplated
herein to be treated by the
administration of an antibody directed against a B cell surface marker, such
as CD79b, and includes the
administration of an unconjugated ("naked") antibody or an antibody conjugated
to a cytotoxic agent as
disclosed herein. Such diseases are also contemplated herein to be treated by
combination therapy including
an anti-CD79b antibody or anti-CD79b antibody drug conjugate of the invention
in combination with another
antibody or antibody drug conjugate, another cytoxic agent, radiation or other
treatment administered
simultaneously or in series. In exemplary treatment method of the invention,
an anti-CD79b antibody of the
invention is administered in combination with an anti-CD20 antibody,
immunoglobulin, or CD20 binding
fragment thereof, either together or sequentially. The anti-CD20 antibody may
be a naked antibody or an
antibody drug conjugate. In an embodiment of the combination therapy, the anti-
CD79b antibody is an
antibody of the present invention and the anti-CD20 antibody is Rituxan
(rituximab).
[0165] The term "non-Hodgkin's lymphoma" or "NHL", as used herein, refers to a
cancer of the
lymphatic system other than Hodgkin's lymphomas. Hodgkin's lymphomas can
generally be distinguished
from non-Hodgkin's lymphomas by the presence of Reed-Sternberg cells in
Hodgkin's lymphomas and the
absence of said cells in non-Hodgkin's lymphomas. Examples of non-Hodgkin's
lymphomas encompassed by
the term as used herein include any that would be identified as such by one
skilled in the art (e.g., an
oncologist or pathologist) in accordance with classification schemes known in
the art, such as the Revised
European-American Lymphoma (REAL) scheme as described in Color Atlas of
Clinical Hematology (3rd
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edition), A. Victor Hoffbrand and John E. Pettit (eds.) (Harcourt Publishers
Ltd., 2000). See, in particular, the
lists in Fig. 11.57, 11.58 and 11.59. More specific examples include, but are
not limited to, relapsed or
refractory NHL, front line low grade NHL, Stage III/IV NHL, chemotherapy
resistant NHL, precursor B
lymphoblastic leukemia and/or lymphoma, small lymphocytic lymphoma, B cell
chronic lymphocytic
leukemia and/or prolymphocytic leukemia and/or small lymphocytic lymphoma, B-
cell prolymphocytic
lymphoma, immunocytoma and/or lymphoplasmacytic lymphoma, lymphoplasmacytic
lymphoma, marginal
zone B cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone
- MALT lymphoma, nodal
marginal zone lymphoma, hairy cell leukemia, plasmacytoma and/or plasma cell
myeloma, low
grade/follicular lymphoma, intermediate grade/follicular NHL, mantle cell
lymphoma, follicle center
lymphoma (follicular), intermediate grade diffuse NHL, diffuse large B-cell
lymphoma, aggressive NHL
(including aggressive front-line NHL and aggressive relapsed NHL), NHL
relapsing after or refractory to
autologous stem cell transplantation, primary mediastinal large B-cell
lymphoma, primary effusion lymphoma,
high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small
non-cleaved cell NHL,
bulky disease NHL, Burkitt's lymphoma, precursor (peripheral) large granular
lymphocytic leukemia, mycosis
fungoides and/or Sezary syndrome, skin (cutaneous) lymphomas, anaplastic large
cell lymphoma, angiocentric
lymphoma.
[0166] A "disorder" is any condition that would benefit from treatment with a
substance/molecule
or method of the invention. This includes chronic and acute disorders or
diseases including those pathological
conditions which predispose the mammal to the disorder in question. Non-
limiting examples of disorders to
be treated herein include cancerous conditions such as malignant and benign
tumors; non-leukemias and
lymphoid malignancies; neuronal, glial, astrocytal, hypothalamic and other
glandular, macrophagal, epithelial,
stromal and blastocoelic disorders; and inflammatory, immunologic and other
angiogenesis-related disorders.
Disorders further include cancerous conditions such as B cell proliferative
disorders and/or B cell tumors, e.g.,
lymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL, relapsed aggressive
NHL, relapsed indolent
NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia
(CLL), small lymphocytic
lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia
(ALL), and mantle cell
lymphoma.
[0167] The terms "cell proliferative disorder" and "proliferative disorder"
refer to disorders that are
associated with some degree of abnormal cell proliferation. In one embodiment,
the cell proliferative disorder
is cancer.
[0168] "Tumor", as used herein, refers to all neoplastic cell growth and
proliferation, whether
malignant or benign, and all pre-cancerous and cancerous cells and tissues.
[0169] An "autoimmune disease" herein is a disease or disorder arising from
and directed against an
individual's own tissues or organs or a co-segregate or manifestation thereof
or resulting condition therefrom.
In many of these autoimmune and inflammatory disorders, a number of clinical
and laboratory markers may
exist, including, but not limited to, hypergammaglobulinemia, high levels of
autoantibodies, antigen-antibody
complex deposits in tissues, benefit from corticosteroid or immunosuppressive
treatments, and lymphoid cell
aggregates in affected tissues. Without being limited to any one theory
regarding B-cell mediated
autoimmune disease, it is believed that B cells demonstrate a pathogenic
effect in human autoimmune diseases
through a multitude of mechanistic pathways, including autoantibody
production, immune complex formation,
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dendritic and T-cell activation, cytokine synthesis, direct chemokine release,
and providing a nidus for ectopic
neo-lymphogenesis. Each of these pathways may participate to different degrees
in the pathology of
autoimmune diseases.
[0170] "Autoimmune disease" can be an organ-specific disease (i.e., the immune
response is
specifically directed against an organ system such as the endocrine system,
the hematopoietic system, the skin,
the cardiopulmonary system, the gastrointestinal and liver systems, the renal
system, the thyroid, the ears, the
neuromuscular system, the central nervous system, etc.) or a systemic disease
which can affect multiple organ
systems (for example, systemic lupus erythematosus (SLE), rheumatoid
arthritis, polymyositis, etc.).
Preferred such diseases include autoimmune rheumatologic disorders (such as,
for example, rheumatoid
arthritis, Sj6gren's syndrome, scleroderma, lupus such as SLE and lupus
nephritis,
polymyositis/dermatomyositis, cryoglobulinemia, anti-phospholipid antibody
syndrome, and psoriatic
arthritis), autoimmune gastrointestinal and liver disorders (such as, for
example, inflammatory bowel diseases
(e.g., ulcerative colitis and Crohn's disease), autoimmune gastritis and
pernicious anemia, autoimmune
hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, and
celiac disease), vasculitis (such as, for
example, ANCA-negative vasculitis and ANCA-associated vasculitis, including
Churg-Strauss vasculitis,
Wegener's granulomatosis, and microscopic polyangiitis), autoimmune
neurological disorders (such as, for
example, multiple sclerosis, opsoclonus myoclonus syndrome, myasthenia gravis,
neuromyelitis optica,
Parkinson's disease, Alzheimer's disease, and autoimmune polyneuropathies),
renal disorders (such as, for
example, glomerulonephritis, Goodpasture's syndrome, and Berger's disease),
autoimmune dermatologic
disorders (such as, for example, psoriasis, urticaria, hives, pemphigus
vulgaris, bullous pemphigoid, and
cutaneous lupus erythematosus), hematologic disorders (such as, for example,
thrombocytopenic purpura,
thrombotic thrombocytopenic purpura, post-transfusion purpura, and autoimmune
hemolytic anemia),
atherosclerosis, uveitis, autoimmune hearing diseases (such as, for example,
inner ear disease and hearing
loss), Behcet's disease, Raynaud's syndrome, organ transplant, and autoimmune
endocrine disorders (such as,
for example, diabetic-related autoimmune diseases such as insulin-dependent
diabetes mellitus (IDDM),
Addison's disease, and autoimmune thyroid disease (e.g., Graves' disease and
thyroiditis)). More preferred
such diseases include, for example, rheumatoid arthritis, ulcerative colitis,
ANCA-associated vasculitis, lupus,
multiple sclerosis, Sj6gren's syndrome, Graves' disease, IDDM, pernicious
anemia, thyroiditis, and
glomerulonephritis.
[0171] Specific examples of other autoimmune diseases as defined herein, which
in some cases
encompass those listed above, include, but are not limited to, arthritis
(acute and chronic, rheumatoid arthritis
including juvenile-onset rheumatoid arthritis and stages such as rheumatoid
synovitis, gout or gouty arthritis,
acute immunological arthritis, chronic inflammatory arthritis, degenerative
arthritis, type II collagen-induced
arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis,
psoriatic arthritis, Still's disease, vertebral
arthritis, osteoarthritis, arthritis chronica progrediente, arthritis
deformans, polyarthritis chronica primaria,
reactive arthritis, menopausal arthritis, estrogen-depletion arthritis, and
ankylosing spondylitis/rheumatoid
spondylitis), autoimmune lymphoproliferative disease, inflammatory
hyperproliferative skin diseases,
psoriasis such as plaque psoriasis, gutatte psoriasis, pustular psoriasis, and
psoriasis of the nails, atopy
including atopic diseases such as hay fever and Job's syndrome, dermatitis
including contact dermatitis,
chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis,
allergic contact dermatitis, hives,
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dermatitis herpetiformis, nummular dermatitis, seborrheic dermatitis, non-
specific dermatitis, primary irritant
contact dermatitis, and atopic dermatitis, x-linked hyper IgM syndrome,
allergic intraocular inflammatory
diseases, urticaria such as chronic allergic urticaria and chronic idiopathic
urticaria, including chronic
autoimmune urticaria, myositis, polymyositis/dermatomyositis, juvenile
dermatomyositis, toxic epidermal
necrolysis, scleroderma (including systemic scleroderma), sclerosis such as
systemic sclerosis, multiple
sclerosis (MS) such as spino-optical MS, primary progressive MS (PPMS), and
relapsing remitting MS
(RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis,
sclerosis disseminata, ataxic
sclerosis, neuromyelitis optica (NMO), inflammatory bowel disease (IBD) (for
example, Crohn's disease,
autoimmune-mediated gastrointestinal diseases, gastrointestinal inflammation,
colitis such as ulcerative colitis,
colitis ulcerosa, microscopic colitis, collagenous colitis, colitis polyposa,
necrotizing enterocolitis, and
transmural colitis, and autoimmune inflammatory bowel disease), bowel
inflammation, pyoderma
gangrenosum, erythema nodosum, primary sclerosing cholangitis, respiratory
distress syndrome, including
adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation
of all or part of the uvea, iritis,
choroiditis, an autoimmune hematological disorder, graft-versus-host disease,
angioedema such as hereditary
angioedema, cranial nerve damage as in meningitis, herpes gestationis,
pemphigoid gestationis, pruritis scroti,
autoimmune premature ovarian failure, sudden hearing loss due to an autoimmune
condition, IgE-mediated
diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis
such as Rasmussen's encephalitis and
limbic and/or brainstem encephalitis, uveitis, such as anterior uveitis, acute
anterior uveitis, granulomatous
uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis,
or autoimmune uveitis,
glomerulonephritis (GN) with and without nephrotic syndrome such as chronic or
acute glomerulonephritis
such as primary GN, immune-mediated GN, membranous GN (membranous
nephropathy), idiopathic
membranous GN or idiopathic membranous nephropathy, membrano- or membranous
proliferative GN
(MPGN), including Type I and Type II, and rapidly progressive GN (RPGN),
proliferative nephritis,
autoimmune polyglandular endocrine failure, balanitis including balanitis
circumscripta plasmacellularis,
balanoposthitis, erythema annulare centrifugum, erythema dyschromicum
perstans, eythema multiform,
granuloma annulare, lichen nitidus, lichen sclerosus et atrophicus, lichen
simplex chronicus, lichen spinulosus,
lichen planus, lamellar ichthyosis, epidermolytic hyperkeratosis, premalignant
keratosis, pyoderma
gangrenosum, allergic conditions and responses, food allergies, drug
allergies, insect allergies, rare allergic
disorders such as mastocytosis, allergic reaction, eczema including allergic
or atopic eczema, asteatotic
eczema, dyshidrotic eczema, and vesicular palmoplantar eczema, asthma such as
asthma bronchiale, bronchial
asthma, and auto-immune asthma, conditions involving infiltration of T cells
and chronic inflammatory
responses, immune reactions against foreign antigens such as fetal A-B-O blood
groups during pregnancy,
chronic pulmonary inflammatory disease, autoimmune myocarditis, leukocyte
adhesion deficiency, lupus,
including lupus nephritis, lupus cerebritis, pediatric lupus, non-renal lupus,
extra-renal lupus, discoid lupus
and discoid lupus erythematosus, alopecia lupus, SLE, such as cutaneous SLE or
subacute cutaneous SLE,
neonatal lupus syndrome (NLE), and lupus erythematosus disseminatus, juvenile
onset (Type I) diabetes
mellitus, including pediatric IDDM, adult onset diabetes mellitus (Type II
diabetes), autoimmune diabetes,
idiopathic diabetes insipidus, diabetic retinopathy, diabetic nephropathy,
diabetic colitis, diabetic large-artery
disorder, immune responses associated with acute and delayed hypersensitivity
mediated by cytokines and T-
lymphocytes, tuberculosis, sarcoidosis, granulomatosis including lymphomatoid
granulomatosis,
agranulocytosis, vasculitides (including large-vessel vasculitis such as
polymyalgia rheumatica and giant-cell
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(Takayasu's) arteritis, medium-vessel vasculitis such as Kawasaki's disease
and polyarteritis
nodosa/periarteritis nodosa, immunovasculitis, CNS vasculitis, cutaneous
vasculitis, hypersensitivity
vasculitis, necrotizing vasculitis such as fibrinoid necrotizing vasculitis
and systemic necrotizing vasculitis,
ANCA-negative vasculitis, and ANCA-associated vasculitis such as Churg-Strauss
syndrome (CSS),
Wegener's granulomatosis, and microscopic polyangiitis), temporal arteritis,
aplastic anemia, autoimmune
aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic
anemia or immune hemolytic
anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia (anemia
pemiciosa), Addison's
disease, pure red cell anemia or aplasia (PRCA), Factor VIII deficiency,
hemophilia A, autoimmune
neutropenia(s), cytopenias such as pancytopenia, leukopenia, diseases
involving leukocyte diapedesis, CNS
inflammatory disorders, Alzheimer's disease, Parkinson's disease, multiple
organ injury syndrome such as
those secondary to septicemia, trauma or hemorrhage, antigen-antibody complex-
mediated diseases, anti-
glomerular basement membrane disease, anti-phospholipid antibody syndrome,
motoneuritis, allergic neuritis,
Behget's disease/syndrome, Castleman's syndrome, Goodpasture's syndrome,
Reynaud's syndrome, Sjogren's
syndrome, Stevens-Johnson syndrome, pemphigoid or pemphigus such as pemphigoid
bullous, cicatricial
(mucous membrane) pemphigoid, skin pemphigoid, pemphigus vulgaris,
paraneoplastic pemphigus,
pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, and pemphigus
erythematosus, epidermolysis
bullosa acquisita, ocular inflammation, preferably allergic ocular
inflammation such as allergic conjunctivis,
linear IgA bullous disease, autoimmune-induced conjunctival inflammation,
autoimmune
polyendocrinopathies, Reiter's disease or syndrome, thermal injury due to an
autoimmune condition,
preeclampsia, an immune complex disorder such as immune complex nephritis,
antibody-mediated nephritis,
neuroinflammatory disorders, polyneuropathies, chronic neuropathy such as IgM
polyneuropathies or IgM-
mediated neuropathy, thrombocytopenia (as developed by myocardial infarction
patients, for example),
including thrombotic thrombocytopenic purpura (TTP), post-transfusion purpura
(PTP), heparin-induced
thrombocytopenia, and autoimmune or immune-mediated thrombocytopenia
including, for example,
idiopathic thrombocytopenic purpura (ITP) including chronic or acute ITP,
scleritis such as idiopathic cerato-
scleritis, episcleritis, autoimmune disease of the testis and ovary including
autoimmune orchitis and oophoritis,
primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases
including thyroiditis such as
autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's
thyroiditis), or subacute
thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Grave's
disease, Grave's eye disease
(ophthalmopathy or thyroid-associated ophthalmopathy), polyglandular syndromes
such as autoimmune
polyglandular syndromes, for example, type I (or polyglandular endocrinopathy
syndromes), paraneoplastic
syndromes, including neurologic paraneoplastic syndromes such as Lambert-Eaton
myasthenic syndrome or
Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, encephalomyelitis
such as allergic
encephalomyelitis or encephalomyelitis allergica and experimental allergic
encephalomyelitis (EAE),
myasthenia gravis such as thymoma-associated myasthenia gravis, cerebellar
degeneration, neuromyotonia,
opsoclonus or opsoclonus myoclonus syndrome (OMS), and sensory neuropathy,
multifocal motor neuropathy,
Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis,
giant-cell hepatitis, chronic
active hepatitis or autoimmune chronic active hepatitis, pneumonitis such as
lymphoid interstitial pneumonitis
(LIP), bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barre
syndrome, Berger's disease (IgA
nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile
neutrophilic dermatosis,
subcorneal pustular dermatosis, transient acantholytic dermatosis, cirrhosis
such as primary biliary cirrhosis
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and pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac or Coeliac
disease, celiac sprue (gluten
enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia such as
mixed cryoglobulinemia,
amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery
disease, autoimmune ear disease
such as autoimmune inner ear disease (AIED), autoimmune hearing loss,
polychondritis such as refractory or
relapsed or relapsing polychondritis, pulmonary alveolar proteinosis,
keratitis such as Cogan's
syndrome/nonsyphilitic interstitial keratitis, Bell's palsy, Sweet's
disease/syndrome, rosacea autoimmune,
zoster-associated pain, amyloidosis, a non-cancerous lymphocytosis, a primary
lymphocytosis, which includes
monoclonal B cell lymphocytosis (e.g., benign monoclonal gammopathy and
monoclonal gammopathy of
undetermined significance, MGUS), peripheral neuropathy, paraneoplastic
syndrome, channelopathies such as
epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness,
periodic paralysis, and
channelopathies of the CNS, autism, inflammatory myopathy, focal or segmental
or focal segmental
glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveoretinitis,
chorioretinitis, autoimmune
hepatological disorder, fibromyalgia, multiple endocrine failure, Schmidt's
syndrome, adrenalitis, gastric
atrophy, presenile dementia, demyelinating diseases such as autoimmune
demyelinating diseases and chronic
inflammatory demyelinating polyneuropathy, Dressler's syndrome, alopecia
areata, alopecia totalis, CREST
syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility,
sclerodactyly, and telangiectasia),
male and female autoimmune infertility, e.g., due to anti-spermatozoan
antibodies, mixed connective tissue
disease, Chagas' disease, rheumatic fever, recurrent abortion, farmer's lung,
erythema multiforme, post-
cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung, allergic
granulomatous angiitis, benign
lymphocytic angiitis, Alport's syndrome, alveolitis such as allergic
alveolitis and fibrosing alveolitis,
interstitial lung disease, transfusion reaction, leprosy, malaria, parasitic
diseases such as leishmaniasis,
kypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter's
syndrome, Caplan's syndrome, dengue,
endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary
fibrosis, interstitial lung fibrosis,
fibrosing mediastinitis, pulmonary fibrosis, idiopathic pulmonary fibrosis,
cystic fibrosis, endophthalmitis,
erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic
faciitis, Shulman's syndrome, Felty's
syndrome, flariasis, cyclitis such as chronic cyclitis, heterochronic
cyclitis, iridocyclitis (acute or chronic), or
Fuch's cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV)
infection, SCID, acquired
immune deficiency syndrome (AIDS), echovirus infection, sepsis (systemic
inflammatory response syndrome
(SIRS)), endotoxemia, pancreatitis, thyroxicosis, parvovirus infection,
rubella virus infection, post-vaccination
syndromes, congenital rubella infection, Epstein-Barr virus infection, mumps,
Evan's syndrome, autoimmune
gonadal failure, Sydenham's chorea, post-streptococcal nephritis,
thromboangitis ubiterans, thyrotoxicosis,
tabes dorsalis, chorioiditis, giant-cell polymyalgia, chronic hypersensitivity
pneumonitis, conjunctivitis, such
as vernal catarrh, keratoconjunctivitis sicca, and epidemic
keratoconjunctivitis, idiopathic nephritic syndrome,
minimal change nephropathy, benign familial and ischemia-reperfusion injury,
transplant organ reperfusion,
retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive
airway/pulmonary disease, silicosis,
aphthae, aphthous stomatitis, arteriosclerotic disorders (cerebral vascular
insufficiency) such as arteriosclerotic
encephalopathy and arteriosclerotic retinopathy, aspermiogenese, autoimmune
hemolysis, Boeck's disease,
cryoglobulinemia, Dupuytren's contracture, endophthalmia phacoanaphylactica,
enteritis allergica, erythema
nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome,
febris rheumatica, Hamman-Rich's
disease, sensoneural hearing loss, haemoglobinuria paroxysmatica,
hypogonadism, ileitis regionalis,
leucopenia, mononucleosis infectiosa, traverse myelitis, primary idiopathic
myxedema, nephrosis, ophthalmia
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symphatica (sympathetic ophthalmitis), neonatal ophthalmitis, optic neuritis,
orchitis granulomatosa,
pancreatitis, polyradiculitis acuta, pyoderma gangrenosum, Quervain's
thyreoiditis, acquired spenic atrophy,
non-malignant thymoma, lymphofollicular thymitis, vitiligo, toxic-shock
syndrome, food poisoning,
conditions involving infiltration of T cells, leukocyte-adhesion deficiency,
immune responses associated with
acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes,
diseases involving leukocyte
diapedesis, multiple organ injury syndrome, antigen-antibody complex-mediated
diseases, antiglomerular
basement membrane disease, autoimmune polyendocrinopathies, oophoritis,
primary myxedema, autoimmune
atrophic gastritis, rheumatic diseases, mixed connective tissue disease,
nephrotic syndrome, insulitis,
polyendocrine failure, autoimmune polyglandular syndromes, including
polyglandular syndrome type I, adult-
onset idiopathic hypoparathyroidism (AOIH), cardiomyopathy such as dilated
cardiomyopathy, epidermolisis
bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome,
primary sclerosing cholangitis,
purulent or nonpurulent sinusitis, acute or chronic sinusitis, ethmoid,
frontal, maxillary, or sphenoid sinusitis,
allergic sinusitis, an eosinophil-related disorder such as eosinophilia,
pulmonary infiltration eosinophilia,
eosinophilia-myalgia syndrome, Loffler's syndrome, chronic eosinophilic
pneumonia, tropical pulmonary
eosinophilia, bronchopneumonic aspergillosis, aspergilloma, or granulomas
containing eosinophils,
anaphylaxis, spondyloarthropathies, seronegative spondyloarthritides,
polyendocrine autoimmune disease,
sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis,
Bruton's syndrome, transient
hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia
telangiectasia syndrome, angiectasis,
autoimmune disorders associated with collagen disease, rheumatism such as
chronic arthrorheumatism,
lymphadenitis, reduction in blood pressure response, vascular dysfunction,
tissue injury, cardiovascular
ischemia, hyperalgesia, renal ischemia, cerebral ischemia, and disease
accompanying vascularization, allergic
hypersensitivity disorders, glomerulonephritides, reperfusion injury, ischemic
re-perfusion disorder,
reperfusion injury of myocardial or other tissues, lymphomatous
tracheobronchitis, inflammatory dermatoses,
dermatoses with acute inflammatory components, multiple organ failure, bullous
diseases, renal cortical
necrosis, acute purulent meningitis or other central nervous system
inflammatory disorders, ocular and orbital
inflammatory disorders, granulocyte transfusion-associated syndromes, cytokine-
induced toxicity, narcolepsy,
acute serious inflammation, chronic intractable inflammation, pyelitis,
endarterial hyperplasia, peptic ulcer,
valvulitis, and endometriosis. Such diseases are contemplated herein to be
treated by the administration of an
antibody which binds to a B cell surface marker, such as CD79b, and includes
the administration of an
unconjugated ("naked") antibody or an antibody conjugated to a cytotoxic agent
as disclosed herein. Such
diseases are also contemplated herein to be treated by combination therapy
including an anti-CD79b antibody
or anti-CD79b antibody drug conjugate of the invention in combination with
another antibody or antibody
drug conjugate, another cytoxic agent, radiation or other treatment
administered simultaneously or in series.
[0172] "Treating" or "treatment" or "alleviation" refers to both therapeutic
treatment and
prophylactic or preventative measures, wherein the object is to prevent or
slow down (lessen) the targeted
pathologic condition or disorder. Those in need of treatment include those
already with the disorder as well as
those prone to have
the disorder or those in whom the disorder is to be prevented. A subject or
mammal is successfully "treated"
for a CD79b polypeptide-expressing cancer if, after receiving a therapeutic
amount of an anti-CD79b antibody
according to the methods of the present invention, the patient shows
observable and/or measurable reduction
CA 02692819 2010-01-07
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in or absence of one or more of the following: reduction in the number of
cancer cells or absence of the cancer
cells; reduction in the tumor size; inhibition (i.e., slow to some extent and
preferably stop) of cancer cell
infiltration into peripheral organs including the spread of cancer into soft
tissue and bone; inhibition (i.e., slow
to some extent and preferably stop) of tumor metastasis; inhibition, to some
extent, of tumor growth; and/or
relief to some extent, one or more of the symptoms associated with the
specific cancer; reduced morbidity and
mortality, and improvement in quality of life issues. To the extent the anti-
CD79b antibody may prevent
growth and/or kill existing cancer cells, it may be cytostatic and/or
cytotoxic. Reduction of these signs or
symptoms may also be felt by the patient.
[0173] The above parameters for assessing successful treatment and improvement
in the disease are
readily measurable by routine procedures familiar to a physician. For cancer
therapy, efficacy can be
measured, for example, by assessing the time to disease progression (TTP)
and/or determining the response
rate (RR). Metastasis can be determined by staging tests and by bone scan and
tests for calcium level and
other enzymes to determine spread to the bone. CT scans can also be done to
look for spread to the pelvis and
lymph nodes in the area. Chest X-rays and measurement of liver enzyme levels
by known methods are used to
look for metastasis to the lungs and liver, respectively. Other routine
methods for monitoring the disease
include transrectal ultrasonography (TRUS) and transrectal needle biopsy
(TRNB).
[0174] For bladder cancer, which is a more localized cancer, methods to
determine progress of
disease include urinary cytologic evaluation by cystoscopy, monitoring for
presence of blood in the urine,
visualization of the urothelial tract by sonography or an intravenous
pyelogram, computed tomography (CT)
and magnetic resonance imaging (MRI). The presence of distant metastases can
be assessed by CT of the
abdomen, chest x-rays, or radionuclide imaging of the skeleton.
[0175] "Chronic" administration refers to administration of the agent(s) in a
continuous mode as
opposed to an acute mode, so as to maintain the initial therapeutic effect
(activity) for an extended period of
time. "Intermittent" administration is treatment that is not consecutively
done without interruption, but rather
is cyclic in nature.
[0176] An "individual" is a vertebrate. In certain embodiments, the vertebrate
is a mammal.
Mammals include, but are not limited to, farm animals (such as cows), sport
animals, pets (such as cats, dogs,
and horses), primates, mice and rats. In certain embodiments, a mammal is a
human.
[0177] "Mammal" for purposes of the treatment of, alleviating the symptoms of
a cancer refers to
any animal classified as a mammal, including humans, domestic and farm
animals, and zoo, sports, or pet
animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc.
Preferably, the mammal is human.
[0178] Administration "in combination with" one or more further therapeutic
agents includes
simultaneous (concurrent) and consecutive administration in any order.
[0179] "Carriers" as used herein include pharmaceutically acceptable carriers,
excipients, or
stabilizers which are nontoxic to the cell or mammal being exposed thereto at
the dosages and concentrations
employed. Often the physiologically acceptable carrier is an aqueous pH
buffered solution. Examples of
physiologically acceptable carriers include buffers such as phosphate,
citrate, and other organic acids;
antioxidants including ascorbic acid; low molecular weight (less than about 10
residues) polypeptide; proteins,
such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such
as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, arginine or lysine;
monosaccharides, disaccharides, and
other carbohydrates including glucose, mannose, or dextrins; chelating agents
such as EDTA; sugar alcohols
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such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or
nonionic surfactants such as
TWEEN , polyethylene glycol (PEG), and PLURONICS .
[0180] By "solid phase" or "solid support" is meant a non-aqueous matrix to
which an antibody of
the present invention can adhere or attach. Examples of solid phases
encompassed herein include those
formed partially or entirely of glass (e.g., controlled pore glass),
polysaccharides (e.g., agarose),
polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain
embodiments, depending on the
context, the solid phase can comprise the well of an assay plate; in others it
is a purification column (e.g., an
affinity chromatography column). This term also includes a discontinuous solid
phase of discrete particles,
such as those described in U.S. Patent No. 4,275,149.
[0181] A "liposome" is a small vesicle composed of various types of lipids,
phospholipids and/or
surfactant which is useful for delivery of a drug (such as an CD79b antibody)
to a mammal. The components
of the liposome are commonly arranged in a bilayer formation, similar to the
lipid arrangement of biological
membranes.
[0182] A "small" molecule or "small" organic molecule is defined herein to
have a molecular
weight below about 500 Daltons.
[0183] An "individual," "subject," or "patient" is a vertebrate. In certain
embodiments, the
vertebrate is a mammal. mammals include, but are not limited to, farm animals
(such as cows), sport animals,
pets (such as cats, dogs, and horses), primates, mice and rats. In certain
embodiments, a mammal is human.
[0184] The term "pharmaceutical formulation" refers to a preparation which is
in such form as to
permit the biological activity of the active ingredient to be effective, and
which contains no additional
components which are unacceptably toxic to a subject to which the formulation
would be administered. Such
formulation may be sterile.
[0185] A "sterile" formulation is aseptic of free from all living
microorganisms and their spores.
[0186] An "effective amount" of an antibody as disclosed herein is an amount
sufficient to carry out
a specifically stated purpose. An "effective amount" may be determined
empirically and in a routine manner,
in relation to the stated purpose.
[0187] The term "therapeutically effective amount" refers to an amount of an
antibody or other drug
effective to "treat" a disease or disorder in a subject or mammal. In the case
of cancer, the therapeutically
effective amount of the drug may reduce the number of cancer cells; reduce the
tumor size; inhibit (i.e., slow
to some extent and preferably stop) cancer cell infiltration into peripheral
organs; inhibit (i.e., slow to some
extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor
growth; and/or relieve to some
extent one or more of the symptoms associated with the cancer. See the
definition herein of "treating". To the
extent the drug may prevent growth and/or kill existing cancer cells, it may
be cytostatic and/or cytotoxic. A
"prophylactically effective amount" refers to an amount effective, at dosages
and for periods of time necessary,
to achieve the desired prophylactic result. Typically but not necessarily,
since a prophylactic dose is used in
subjects prior to or at an earlier stage of disease, the prophylactically
effective amount will be less than the
therapeutically effective amount.
[0188] A "growth inhibitory amount" of an anti-CD79b antibody is an amount
capable of inhibiting
the growth of a cell, especially tumor, e.g., cancer cell, either in vitro or
in vivo. A "growth inhibitory
amount" of an anti-CD79b antibody for purposes of inhibiting neoplastic cell
growth may be determined
empirically and in a routine manner.
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[0189] A "cytotoxic amount" of an anti-CD79b antibody is an amount capable of
causing the
destruction of a cell, especially tumor, e.g., cancer cell, either in vitro or
in vivo. A "cytotoxic amount" of an
anti-CD79b antibody for purposes of inhibiting neoplastic cell growth may be
determined empirically and in a
routine manner.
[0190] A "CD79b-expressing cell" is a cell which expresses an endogenous or
transfected CD79b
polypeptide either on the cell surface or in a secreted form. A "CD79b-
expressing cancer" is a cancer
comprising cells that have a CD79b polypeptide present on the cell surface or
that produce and secrete a
CD79b polypeptide. A "CD79b-expressing cancer" optionally produces sufficient
levels of CD79b
polypeptide on the surface of cells thereof, such that an anti-CD79b antibody
can bind thereto and have a
therapeutic effect with respect to the cancer. In another embodiment, a "CD79b-
expressing cancer" optionally
produces and secretes sufficient levels of CD79b polypeptide, such that an
anti-CD79b antibody antagonist
can bind thereto and have a therapeutic effect with respect to the cancer.
With regard to the latter, the
antagonist may be an antisense oligonucleotide which reduces, inhibits or
prevents production and secretion of
the secreted CD79b polypeptide by tumor cells. A cancer which "overexpresses"
a CD79b polypeptide is one
which has significantly higher levels of CD79b polypeptide at the cell surface
thereof, or produces and
secretes, compared to a noncancerous cell of the same tissue type. Such
overexpression may be caused by
gene amplification or by increased transcription or translation. CD79b
polypeptide overexpression may be
determined in a detection or prognostic assay by evaluating increased levels
of the CD79b protein present on
the surface of a cell, or secreted by the cell (e.g., via an
immunohistochemistry assay using anti-CD79b
antibodies prepared against an isolated CD79b polypeptide which may be
prepared using recombinant DNA
technology from an isolated nucleic acid encoding the CD79b polypeptide; FACS
analysis, etc.).
Alternatively, or additionally, one may measure levels of CD79b polypeptide-
encoding nucleic acid or mRNA
in the cell, e.g., via fluorescent in situ hybridization using a nucleic acid
based probe corresponding to a
CD79b-encoding nucleic acid or the complement thereof; (FISH; see W098/45479
published October, 1998),
Southern blotting, Northern blotting, or polymerase chain reaction (PCR)
techniques, such as real time
quantitative PCR (RT-PCR). One may also study CD79b polypeptide overexpression
by measuring shed
antigen in a biological fluid such as serum, e.g., using antibody-based assays
(see also, e.g., U.S. Patent No.
4,933,294 issued June 12, 1990; W091/05264 published April 18, 1991; U.S.
Patent 5,401,638 issued March
28, 1995; and Sias et al., J. Immunol. Methods 132:73-80 (1990)). Aside from
the above assays, various in
vivo assays are available to the skilled practitioner. For example, one may
expose cells within the body of the
patient to an antibody which is optionally labeled with a detectable label,
e.g., a radioactive isotope, and
binding of the antibody to cells in the patient can be evaluated, e.g., by
external scanning for radioactivity or
by analyzing a biopsy taken from a patient previously exposed to the antibody.
[0191] As used herein, the term "immunoadhesin" designates antibody-like
molecules which
combine the binding specificity of a heterologous protein (an "adhesin") with
the effector functions of
immunoglobulin constant domains. Structurally, the immunoadhesins comprise a
fusion of an amino acid
sequence with the desired binding specificity which is other than the antigen
recognition and binding site of an
antibody (i.e., is "heterologous"), and an immunoglobulin constant domain
sequence. The adhesin part of an
immunoadhesin molecule typically is a contiguous amino acid sequence
comprising at least the binding site of
a receptor or a ligand. The immunoglobulin constant domain sequence in the
immunoadhesin may be
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obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4
subtypes, IgA (including IgA-1
and IgA-2), IgE, IgD or IgM.
[0192] The word "label" when used herein refers to a detectable compound or
composition which is
conjugated directly or indirectly to the antibody so as to generate a
"labeled" antibody. The label may be
detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in
the case of an enzymatic label, may
catalyze chemical alteration of a substrate compound or composition which is
detectable.
[0193] The term "cytotoxic agent" as used herein refers to a substance that
inhibits or prevents the
function of cells and/or causes destruction of cells. The term is intended to
include radioactive isotopes (e.g.,
At211, 1131, I125 Y90, Re186, Re'88, Smi53 Bi 212, P32 and radioactive
isotopes of Lu), chemotherapeutic agents e.g.
methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine,
etoposide), doxorubicin, melphalan,
mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes
and fragments thereof such
as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins
or enzymatically active toxins of
bacterial, fungal, plant or animal origin, including fragments and/or variants
thereof, and the various antitumor
or anticancer agents disclosed below. Other cytotoxic agents are described
below. A tumoricidal agent causes
destruction of tumor cells.
[0194] A "toxin" is any substance capable of having a detrimental effect on
the growth or
proliferation of a cell.
[0195] A "chemotherapeutic agent" is a chemical compound useful in the
treatment of cancer,
regardless of mechanism of action. Classes of chemotherapeutic agents include,
but are not limited to:
alkyating agents, antimetabolites, spindle poison plant alkaloids,
cytoxic/antitumor antibiotics, topoisomerase
inhibitors, antibodies, photosensitizers, and kinase inhibitors.
Chemotherapeutic agents include compounds
used in "targeted therapy" and conventional chemotherapy. Examples of
chemotherapeutic agents include:
erlotinib (TARCEVA , Genentech/OSI Pharm.), docetaxel (TAXOTERE , Sanofi-
Aventis), 5-FU
(fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR , Lilly),
PD-0325901 (CAS No.
391210-10-9, Pfizer), cisplatin (cis-diamine,dichloroplatinum(II), CAS No.
15663-27-1), carboplatin (CAS
No. 41575-94-4), paclitaxel (TAXOL , Bristol-Myers Squibb Oncology, Princeton,
N.J.), trastuzumab
(HERCEPTIN , Genentech), temozolomide (4-methyl-5-oxo- 2,3,4,6,8-
pentazabicyclo [4.3.0] nona-2,7,9-
triene- 9-carboxamide, CAS No. 85622-93-1, TEMODAR , TEMODAL , Schering
Plough), tamoxifen
((Z)-2-[4-(1,2-diphenylbut-l-enyl)phenoxy]-N,N-dimethyl-ethanamine, NOLVADEX ,
ISTUBAL ,
VALODEX ), and doxorubicin (ADRIAMYCIN ), Akti- 1/2, HPPD, and rapamycin.
[0196] More examples of chemotherapeutic agents include: oxaliplatin (ELOXATIN
, Sanofi),
bortezomib (VELCADE , Millennium Pharm.), sutent (SUNITINIB , SU11248,
Pfizer), letrozole
(FEMARA , Novartis), imatinib mesylate (GLEEVEC , Novartis), XL-518 (Mek
inhibitor, Exelixis, WO
2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, Astra
Zeneca), SF-1126 (P13K
inhibitor, Semafore Pharmaceuticals), BEZ-235 (P13K inhibitor, Novartis), XL-
147 (P13K inhibitor, Exelixis),
PTK787/ZK 222584 (Novartis), fulvestrant (FASLODEX , AstraZeneca), leucovorin
(folinic acid),
rapamycin (sirolimus, RAPAMUNE , Wyeth), lapatinib (TYKERB , GSK572016, Glaxo
Smith Kline),
lonafarnib (SARASARTM, SCH 66336, Schering Plough), sorafenib (NEXAVAR , BAY43-
9006, Bayer
Labs), gefitinib (IRESSA , AstraZeneca), irinotecan (CAMPTOSAR , CPT-11,
Pfizer), tipifarnib
(ZARNESTRATM, Johnson & Johnson), ABRAXANETM (Cremophor-free), albumin-
engineered nanoparticle
formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, I1),
vandetanib (rINN, ZD6474,
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ZACTIMA , AstraZeneca), chloranmbucil, AG1478, AG1571 (SU 5271; Sugen),
temsirolimus (TORISEL ,
Wyeth), pazopanib (G1axoSmithKline), canfosfamide (TELCYTA , Telik), thiotepa
and cyclosphosphamide
(CYTOXAN , NEOSAR ); alkyl sulfonates such as busulfan, improsulfan and
piposulfan; aziridines such as
benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines including
altretamine, triethylenemelamine, triethylenephosphoramide,
triethylenethiophosphoramide and
trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a
camptothecin (including the
synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its
adozelesin, carzelesin and bizelesin
synthetic analogs); cryptophycins (particularly cryptophycin 1 and
cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogs, KW-2189 and CB1-TM1); eleutherobin;
pancratistatin; a sarcodictyin;
spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,
chlorophosphamide, estramustine,
ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine,
prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine,
chlorozotocin, fotemustine,
lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne
antibiotics (e.g., calicheamicin,
calicheamicin gammall, calicheamicin omegall (Angew Chem. Intl. Ed. Engl.
(1994) 33:183-186);
dynemicin, dynemicin A; bisphosphonates, such as clodronate; an esperamicin;
as well as neocarzinostatin
chromophore and related chromoprotein enediyne antibiotic chromophores),
aclacinomysins, actinomycin,
authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin,
carzinophilin, chromomycinis,
dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,
morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin),
epirubicin, esorubicin,
idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins,
peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,
streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-
fluorouracil (5-FU); folic acid
analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-
mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as
ancitabine, azacitidine, 6-azauridine,
carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;
androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-
adrenals such as aminoglutethimide,
mitotane, trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside;
aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene;
edatraxate; defofamine; demecolcine;
diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid;
gallium nitrate; hydroxyurea; lentinan;
lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone;
mitoxantrone; mopidanmol;
nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic
acid; 2-ethylhydrazide;
procarbazine; PSK polysaccharide complex (JHS Natural Products, Eugene, OR);
razoxane; rhizoxin;
sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-
trichlorotriethylamine; trichothecenes
(especially T-2 toxin, verracurin A, roridin A and anguidine); urethan;
vindesine; dacarbazine; mannomustine;
mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");
cyclophosphamide; thiotepa; 6-
thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin
and carboplatin; vinblastine;
etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine
(NAVELBINE ); novantrone;
teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA ,
Roche); ibandronate; CPT- 11;
topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids
such as retinoic acid; and
pharmaceutically acceptable salts, acids and derivatives of any of the above.
[0197] Also included in the definition of "chemotherapeutic agent" are: (i)
anti-hormonal agents
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that act to regulate or inhibit hormone action on tumors such as anti-
estrogens and selective estrogen receptor
modulators (SERMs), including, for example, tamoxifen (including NOLVADEX ;
tamoxifen citrate),
raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,
onapristone, and
FARESTON (toremifine citrate); (ii) aromatase inhibitors that inhibit the
enzyme aromatase, which
regulates estrogen production in the adrenal glands, such as, for example,
4(5)-imidazoles, aminoglutethimide,
MEGASE (megestrol acetate), AROMASIN (exemestane; Pfizer), formestanie,
fadrozole, RIVISOR
(vorozole), FEMARA (letrozole; Novartis), and ARIMIDEX (anastrozole;
AstraZeneca); (iii) anti-
androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and
goserelin; as well as troxacitabine (a
1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors such
as MEK inhibitors (WO
2007/044515); (v) lipid kinase inhibitors; (vi) antisense oligonucleotides,
particularly those which inhibit
expression of genes in signaling pathways implicated in aberrant cell
proliferation, for example, PKC-alpha,
Raf and H-Ras, such as oblimersen (GENASENSE , Genta Inc.); (vii) ribozymes
such as VEGF expression
inhibitors (e.g., ANGIOZYME ) and HER2 expression inhibitors; (viii) vaccines
such as gene therapy
vaccines, for example, ALLOVECTIN , LEUVECTIN , and VAXID ; PROLEUKIN rIL-2;
topoisomerase 1 inhibitors such as LURTOTECAN ; ABARELIX rmRH; (ix) anti-
angiogenic agents such
as bevacizumab (AVASTIN , Genentech); and pharmaceutically acceptable salts,
acids and derivatives of
any of the above.
[0198] Also included in the definition of "chemotherapeutic agent" are
therapeutic antibodies such
as alemtuzumab (Campath), bevacizumab (AVASTIN , Genentech); cetuximab
(ERBITUX , Imclone);
panitumumab (VECTIBIX , Amgen), rituximab (RITUXAN , Genentech/Biogen Idec),
pertuzumab
(OMNITARGTM, 2C4, Genentech), trastuzumab (HERCEPTIN , Genentech), tositumomab
(Bexxar,
Corixia), and the antibody drug conjugate, gemtuzumab ozogamicin (MYLOTARG ,
Wyeth).
[0199] A "growth inhibitory agent" when used herein refers to a compound or
composition which
inhibits growth of a cell, especially a CD79b-expressing cancer cell, either
in vitro or in vivo. Thus, the
growth inhibitory agent may be one which significantly reduces the percentage
of CD79b-expressing cells in S
phase. Examples of growth inhibitory agents include agents that block cell
cycle progression (at a place other
than S phase), such as agents that induce G1 arrest and M-phase arrest.
Classical M-phase blockers include
the vincas (vincristine and vinblastine), taxanes, and topoisomerase II
inhibitors such as doxorubicin,
epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest
G1 also spill over into S-phase
arrest, for example, DNA alkylating agents such as tamoxifen, prednisone,
dacarbazine, mechlorethamine,
cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be
found in The Molecular Basis of
Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle
regulation, oncogenes, and antineoplastic
drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995), especially p. 13.
The taxanes (paclitaxel and
docetaxel) are anticancer drugs both derived from the yew tree. Docetaxel
(TAXOTERE , Rhone-Poulenc
Rorer), derived from the European yew, is a semisynthetic analogue of
paclitaxel (TAXOL , Bristol-Myers
Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from
tubulin dimers and stabilize
microtubules by preventing depolymerization, which results in the inhibition
of mitosis in cells.
[0200] "Doxorubicin" is an anthracycline antibiotic. The full chemical name of
doxorubicin is (8S-
cis)-10-[(3 -amino-2,3,6-trideoxy-a-L-lyxo-hexapyranosyl)oxy] -7, 8,9,10-
tetrahydro-6, 8,11-trihydroxy-8-
(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione.
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[0201] The term "cytokine" is a generic term for proteins released by one cell
population which act
on another cell as intercellular mediators. Examples of such cytokines are
lymphokines, monokines, and
traditional polypeptide hormones. Included among the cytokines are growth
hormone such as human growth
hormone, N-methionyl human growth hormone, and bovine growth hormone;
parathyroid hormone; thyroxine;
insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as
follicle stimulating hormone (FSH),
thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic
growth factor; fibroblast growth
factor; prolactin; placental lactogen; tumor necrosis factor-a and -(3;
mullerian-inhibiting substance; mouse
gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth
factor; integrin; thrombopoietin
(TPO); nerve growth factors such as NGF-(3; platelet-growth factor;
transforming growth factors (TGFs) such
as TGF-a and TGF-(3; insulin-like growth factor-I and -II; erythropoietin
(EPO); osteoinductive factors;
interferons such as interferon -a, -(3, and -y; colony stimulating factors
(CSFs) such as macrophage-CSF (M-
CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);
interleukins (ILs) such as IL-
1, IL- 1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; a
tumor necrosis factor such as TNF-a or
TNF-13; and other polypeptide factors including LIF and kit ligand (KL). As
used herein, the term cytokine
includes proteins from natural sources or from recombinant cell culture and
biologically active equivalents of
the native sequence cytokines.
[0202] The term "package insert" is used to refer to instructions customarily
included in
commercial packages of therapeutic products, that contain information about
the indications, usage, dosage,
administration, contraindications and/or warnings concerning the use of such
therapeutic products.
[0203] The term "intracellular metabolite" refers to a compound resulting from
a metabolic process
or reaction inside a cell on an antibody-drug conjugate (ADC). The metabolic
process or reaction may be an
enzymatic process, such as proteolytic cleavage of a peptide linker of the
ADC, or hydrolysis of a functional
group such as a hydrazone, ester, or amide. Intracellular metabolites include,
but are not limited to, antibodies
and free drug which have undergone intracellular cleavage after entry,
diffusion, uptake or transport into a cell.
[0204] The terms "intracellularly cleaved" and "intracellular cleavage" refer
to a metabolic process
or reaction inside a cell on an antibody-drug conjugate (ADC) whereby the
covalent attachment, i.e. linker,
between the drug moiety (D) and the antibody (Ab) is broken, resulting in the
free drug dissociated from the
antibody inside the cell. The cleaved moieties of the ADC are thus
intracellular metabolites.
[0205] The term "bioavailability" refers to the systemic availability (i.e.,
blood/plasma levels) of a
given amount of drug administered to a patient. Bioavailability is an absolute
term that indicates measurement
of both the time (rate) and total amount (extent) of drug that reaches the
general circulation from an
administered dosage form.
[0206] The term "cytotoxic activity" refers to a cell-killing, cytostatic or
growth inhibitory effect of
an ADC or an intracellular metabolite of an ADC. Cytotoxic activity may be
expressed as the IC50 value,
which is the concentration (molar or mass) per unit volume at which half the
cells survive.
[0207] The term "alkyl" as used herein refers to a saturated linear or
branched-chain monovalent
hydrocarbon radical of one to twelve carbon atoms (Ci-C12), wherein the alkyl
radical may be optionally
substituted independently with one or more substituents described below. In
another embodiment, an alkyl
radical is one to eight carbon atoms (Ci-C8), or one to six carbon atoms (Ci-
C6). Examples of alkyl groups
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include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1 -
propyl (n-Pr, n-propyl, -
CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -
CH2CH2CH2CH3), 2-methyl-l-
propyl (i-Bu, i-butyl, -CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl, -CH(CH3)CH2CH3),
2-methyl-2-propyl (t-Bu, t-
butyl, -C(CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-
CH(CH3)CH2CH2CH3), 3-pentyl (-
CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-
CH(CH3)CH(CH3)2), 3-methyl-l-
butyl (-CH2CH2CH(CH3)2), 2-methyl-1 -butyl (-CH2CH(CH3)CH2CH3), 1 -hexyl (-
CH2CH2CH2CH2CH2CH3),
2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-
pentyl (-
C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-
pentyl (-
CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-
CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-
butyl (-CH(CH3)C(CH3)3,
1 -heptyl, 1-octyl, and the like.
[0208] The term "alkenyl" refers to linear or branched-chain monovalent
hydrocarbon radical of
two to eight carbon atoms (C2-C8) with at least one site of unsaturation,
i.e., a carbon-carbon, sp2 double bond,
wherein the alkenyl radical may be optionally substituted independently with
one or more substituents
described herein, and includes radicals having "cis" and "trans" orientations,
or alternatively, "E" and "Z"
orientations. Examples include, but are not limited to, ethylenyl or vinyl (-
CH=CH2), allyl (-CHzCH=CHz),
and the like.
[0209] The term "alkynyl" refers to a linear or branched monovalent
hydrocarbon radical of two to
eight carbon atoms (C2-C8) with at least one site of unsaturation, i.e., a
carbon-carbon, sp triple bond, wherein
the alkynyl radical may be optionally substituted independently with one or
more substituents described herein.
Examples include, but are not limited to, ethynyl (-C=CH), propynyl
(propargyl, -CHzC=CH), and the like.
[0210] The terms "carbocycle", "carbocyclyl", "carbocyclic ring" and
"cycloalkyl" refer to a
monovalent non-aromatic, saturated or partially unsaturated ring having 3 to
12 carbon atoms (C3-C12) as a
monocyclic ring or 7 to 12 carbon atoms as a bicyclic ring. Bicyclic
carbocycles having 7 to 12 atoms can be
arranged, for example, as a bicyclo [4,5], [5,5], [5,6] or [6,6] system, and
bicyclic carbocycles having 9 or 10
ring atoms can be arranged as a bicyclo [5,6] or [6,6] system, or as bridged
systems such as
bicyclo [2.2. 1 ]heptane, bicyclo[2.2.2]octane and bicyclo [3.2.2]nonane.
Examples ofmonocyclic carbocycles
include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1 -
cyclopent- 1 -enyl, 1 -cyclopent-2-enyl,
1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-l-enyl, 1-cyclohex-2-enyl, 1-
cyclohex-3-enyl, cyclohexadienyl,
cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl,
and the like.
[0211 ]"Aryl" means a monovalent aromatic hydrocarbon radical of 6-20 carbon
atoms (C6-C20)
derived by the removal of one hydrogen atom from a single carbon atom of a
parent aromatic ring system.
Some aryl groups are represented in the exemplary structures as "Ar". Aryl
includes bicyclic radicals
comprising an aromatic ring fused to a saturated, partially unsaturated ring,
or aromatic carbocyclic ring.
Typical aryl groups include, but are not limited to, radicals derived from
benzene (phenyl), substituted
benzenes, naphthalene, anthracene, biphenyl, indenyl, indanyl, 1,2-
dihydronaphthalene, 1,2,3,4-
tetrahydronaphthyl, and the like. Aryl groups are optionally substituted
independently with one or more
substituents described herein.
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[0212] The terms "heterocycle," "hetercyclyl" and "heterocyclic ring" are used
interchangeably
herein and refer to a saturated or a partially unsaturated (i.e., having one
or more double and/or triple bonds
within the ring) carbocyclic radical of 3 to 20 ring atoms in which at least
one ring atom is a heteroatom
selected from nitrogen, oxygen, phosphorus and sulfur, the remaining ring
atoms being C, where one or more
ring atoms is optionally substituted independently with one or more
substituents described below. A
heterocycle may be a monocycle having 3 to 7 ring members (2 to 6 carbon atoms
and 1 to 4 heteroatoms
selected from N, 0, P, and S) or a bicycle having 7 to 10 ring members (4 to 9
carbon atoms and 1 to 6
heteroatoms selected from N, 0, P, and S), for example: a bicyclo [4,5],
[5,5], [5,6], or [6,6] system.
Heterocycles are described in Paquette, Leo A.; "Principles of Modern
Heterocyclic Chemistry" (W.A.
Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; "The
Chemistry of Heterocyclic
Compounds, A series of Monographs" (John Wiley & Sons, New York, 1950 to
present), in particular
Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566.
"Heterocyclyl" also includes radicals
where heterocycle radicals are fused with a saturated, partially unsaturated
ring, or aromatic carbocyclic or
heterocyclic ring. Examples of heterocyclic rings include, but are not limited
to, pyrrolidinyl,
tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl,
dihydropyranyl,
tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxanyl,
piperazinyl, homopiperazinyl,
azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl,
oxazepinyl, diazepinyl, thiazepinyl, 2-
pyrrolinyl, 3-pyrroLinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-
dioxolanyl, pyrazolinyl, dithianyl,
dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl,
pyrazolidinylimidazolinyl, imidazolidinyl, 3-
azabicyco[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl,
azabicyclo[2.2.2]hexanyl, 3H-indolyl quinolizinyl and
N-pyridyl ureas. Spiro moieties are also included within the scope of this
definition. Examples of a
heterocyclic group wherein 2 ring carbon atoms are substituted with oxo (=0)
moieties are pyrimidinonyl and
1,1-dioxo-thiomorpholinyl. The heterocycle groups herein are optionally
substituted independently with one
or more substituents described herein.
[0213] The term "heteroaryl" refers to a monovalent aromatic radical of 5-, 6-
, or 7-membered rings,
and includes fused ring systems (at least one of which is aromatic) of 5-20
atoms, containing one or more
heteroatoms independently selected from nitrogen, oxygen, and sulfur. Examples
of heteroaryl groups are
pyridinyl (including, for example, 2-hydroxypyridinyl), imidazolyl,
imidazopyridinyl, pyrimidinyl (including,
for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl, pyrazinyl,
tetrazolyl, furyl, thienyl, isoxazolyl,
thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl,
indolyl, benzimidazolyl, benzofuranyl,
cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl,
isoindolyl, pteridinyl, purinyl,
oxadiazolyl, triazolyl, thiadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl,
benzothiophenyl, benzothiazolyl,
benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl.
Heteroaryl groups are optionally
substituted independently with one or more substituents described herein.
[0214] The heterocycle or heteroaryl groups may be carbon (carbon-linked), or
nitrogen (nitrogen-
linked) bonded where such is possible. By way of example and not limitation,
carbon bonded heterocycles or
heteroaryls are bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3,
4, 5, or 6 of a pyridazine, position 2,
4, 5, or 6 of a pyrimidine, position 2, 3, 5, or 6 of a pyrazine, position 2,
3, 4, or 5 of a furan, tetrahydrofuran,
thiofuran, thiophene, pyrrole or tetrahydropyrrole, position 2, 4, or 5 of an
oxazole, imidazole or thiazole,
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position 3, 4, or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3
of an aziridine, position 2, 3, or 4 of
an azetidine, position 2, 3, 4, 5, 6, 7, or 8 of a quinoline or position 1, 3,
4, 5, 6, 7, or 8 of an isoquinoline.
[0215] By way of example and not limitation, nitrogen bonded heterocycles or
heteroaryls are
bonded at position 1 of an aziridine, azetidine, pyrrole, pyrrolidine, 2-
pyrroline, 3-pyrroline, imidazole,
imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-
pyrazoline, 3-pyrazoline, piperidine,
piperazine, indole, indoline, 1H-indazole, position 2 of a isoindole, or
isoindoline, position 4 of a morpholine,
and position 9 of a carbazole, or (3-carboLine.
[0216] "Alkylene" refers to a saturated, branched or straight chain or cyclic
hydrocarbon radical of 1-
18 carbon atoms, and having two monovalent radical centers derived by the
removal of two hydrogen atoms from
the same or two different carbon atoms of a parent alkane. Typical alkylene
radicals include, but are not limited
to: methylene (-CH2-) 1,2-ethyl (-CHzCHz-), 1,3-propyl (-CH2CH2CH2-), 1,4-
butyl (-CH2CH2CH2CH2-), and the
like.
[0217] A"Ci-Cio alkylene" is a straight chain, saturated hydrocarbon group of
the formula -(CHz)i_
io-. Examples of a Ci-Cio alkylene include methylene, ethylene, propylene,
butylene, pentylene, hexylene,
heptylene, ocytylene, nonylene and decalene.
[0218] "Alkenylene" refers to an unsaturated, branched or straight chain or
cyclic hydrocarbon radical
of 2-18 carbon atoms, and having two monovalent radical centers derived by the
removal of two hydrogen atoms
from the same or two different carbon atoms of a parent alkene. Typical
alkenylene radicals include, but are not
limited to: 1,2-ethylene (-CH=CH-).
[0219] "Alkynylene" refers to an unsaturated, branched or straight chain or
cyclic hydrocarbon radical
of 2-18 carbon atoms, and having two monovalent radical centers derived by the
removal of two hydrogen atoms
from the same or two different carbon atoms of a parent alkyne. Typical
alkynylene radicals include, but are not
limited to: acetylene (-C=C-), propargyl (-CHzC=C-), and 4-pentynyl (-
CHzCHzCHzC=C-).
[0220] An "arylene" is an aryl group which has two covalent bonds and can be
in the ortho, meta,
or para configurations as shown in the following structures:
~ > >
in which the phenyl group can be unsubstituted or substituted with up to four
groups including, but not limited
to, -Ci-CB alkyl, -O-(Ci-C8 alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -
C(O)NH2, -C(O)NHR', -
C(O)N(R')2 -NHC(O)R', -S(O)zR', -S(O)R', -OH, -halogen, -N3, -NH2, -NH(R'), -
N(R')2 and -CN; wherein
each R' is independently selected from H, -Ci-CB alkyl and aryl.
[0221 ]"Arylalkyl" refers to an acyclic alkyl radical in which one of the
hydrogen atoms bonded to
a carbon atom, typically a terminal or sp3 carbon atom, is replaced with an
aryl radical. Typical arylalkyl
groups include, but are not limited to, benzyl, 2-phenylethan- 1 -yl, 2-
phenylethen- 1 -yl, naphthylmethyl, 2-
naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-
1-yl and the like. The
CA 02692819 2010-01-07
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arylalkyl group comprises 6 to 20 carbon atoms, e.g. the alkyl moiety,
including alkanyl, alkenyl or alkynyl
groups, of the arylalkyl group is 1 to 6 carbon atoms and the aryl moiety is 5
to 14 carbon atoms.
[0222] "Heteroarylalkyl" refers to an acyclic alkyl radical in which one of
the hydrogen atoms
bonded to a carbon atom, typically a terminal or sp3 carbon atom, is replaced
with a heteroaryl radical. Typical
heteroarylalkyl groups include, but are not limited to, 2-
benzimidazolylmethyl, 2-furylethyl, and the like. The
heteroarylalkyl group comprises 6 to 20 carbon atoms, e.g. the alkyl moiety,
including alkanyl, alkenyl or
alkynyl groups, of the heteroarylalkyl group is 1 to 6 carbon atoms and the
heteroaryl moiety is 5 to 14 carbon
atoms and 1 to 3 heteroatoms selected from N, 0, P, and S. The heteroaryl
moiety of the heteroarylalkyl
group may be a monocycle having 3 to 7 ring members (2 to 6 carbon atoms or a
bicycle having 7 to 10 ring
members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, 0, P, and
S), for example: a bicyclo
[4,5], [5,5], [5,6], or [6,6] system.
[0223] The term "prodrug" as used in this application refers to a precursor or
derivative form of a
compound of the invention that may be less cytotoxic to cells compared to the
parent compound or drug and is
capable of being enzymatically or hydrolytically activated or converted into
the more active parent form. See,
e.g., Wilman, "Prodrugs in Cancer Chemotherapy" Biochemical Society
Transactions, 14, pp. 375-382, 615th
Meeting Belfast (1986) and Stella et al., "Prodrugs: A Chemical Approach to
Targeted Drug Delivery,"
Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press
(1985). The prodrugs of this
invention include, but are not limited to, phosphate-containing prodrugs,
thiophosphate-containing prodrugs,
sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-
modified prodrugs, glycosylated
prodrugs, (3-lactam-containing prodrugs, optionally substituted
phenoxyacetamide-containing prodrugs,
optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine
and other 5-fluorouridine
prodrugs which can be converted into the more active cytotoxic free drug.
Examples of cytotoxic drugs that
can be derivatized into a prodrug form for use in this invention include, but
are not limited to, compounds of
the invention and chemotherapeutic agents such as described above.
[0224] A "metabolite" is a product produced through metabolism in the body of
a specified
compound or salt thereof. Metabolites of a compound may be identified using
routine techniques known in
the art and their activities determined using tests such as those described
herein. Such products may result for
example from the oxidation, reduction, hydrolysis, amidation, deamidation,
esterification, deesterification,
enzymatic cleavage, and the like, of the administered compound. Accordingly,
the invention includes
metabolites of compounds of the invention, including compounds produced by a
process comprising
contacting a compound of this invention with a mammal for a period of time
sufficient to yield a metabolic
product thereof.
[0225] A "liposome" is a small vesicle composed of various types of lipids,
phospholipids and/or
surfactant which is useful for delivery of a drug to a mammal. The components
of the liposome are commonly
arranged in a bilayer formation, similar to the lipid arrangement of
biological membranes.
[0226] "Linker" refers to a chemical moiety comprising a covalent bond or a
chain of atoms that
covalently attaches an antibody to a drug moiety. In various embodiments,
linkers include a divalent radical
such as an alkyldiyl, an aryldiyl, a heteroaryldiyl, moieties such as: -
(CRz)õO(CRz)ri , repeating units of
alkyloxy (e.g. polyethylenoxy, PEG, polymethyleneoxy) and alkylamino (e.g.
polyethyleneamino,
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JeffamineTM); and diacid ester and amides including succinate, succinamide,
diglycolate, malonate, and
caproamide.
[0227] The term "chiral" refers to molecules which have the property of non-
superimposability of
the mirror image partner, while the term "achiral" refers to molecules which
are superimposable on their
mirror image partner.
[0228] The term "stereoisomers" refers to compounds which have identical
chemical constitution,
but differ with regard to the arrangement of the atoms or groups in space.
[0229] "Diastereomer" refers to a stereoisomer with two or more centers of
chirality and whose
molecules are not mirror images of one another. Diastereomers have different
physical properties, e.g. melting
points, boiling points, spectral properties, and reactivities. Mixtures of
diastereomers may separate under high
resolution analytical procedures such as electrophoresis and chromatography.
[0230] "Enantiomers" refer to two stereoisomers of a compound which are non-
superimposable
mirror images of one another.
[0231] Stereochemical definitions and conventions used herein generally follow
S. P. Parker, Ed.,
McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New
York; and Eliel, E.
and Wilen, S., Stereochemistry of Organic Compounds (1994) John Wiley & Sons,
Inc., New York. Many
organic compounds exist in optically active forms, i.e., they have the ability
to rotate the plane of plane-
polarized light. In describing an optically active compound, the prefixes D
and L, or R and S, are used to
denote the absolute configuration of the molecule about its chiral center(s).
The prefixes d and I or (+) and (-)
are employed to designate the sign of rotation of plane-polarized light by the
compound, with (-) or 1 meaning
that the compound is levorotatory. A compound prefixed with (+) or d is
dextrorotatory. For a given chemical
structure, these stereoisomers are identical except that they are mirror
images of one another. A specific
stereoisomer may also be referred to as an enantiomer, and a mixture of such
isomers is often called an
enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a
racemic mixture or a racemate, which
may occur where there has been no stereoselection or stereospecificity in a
chemical reaction or process. The
terms "racemic mixture" and "racemate" refer to an equimolar mixture of two
enantiomeric species, devoid of
optical activity.
[0232] The term "tautomer" or "tautomeric form" refers to structural isomers
of different energies
which are interconvertible via a low energy barrier. For example, proton
tautomers (also known as prototropic
tautomers) include interconversions via migration of a proton, such as keto-
enol and imine-enamine
isomerizations. Valence tautomers include interconversions by reorganization
of some of the bonding
electrons.
[0233] The phrase "pharmaceutically acceptable salt" as used herein, refers to
pharmaceutically
acceptable organic or inorganic salts of a compound of the invention.
Exemplary salts include, but are not
limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide,
nitrate, bisulfate, phosphate, acid
phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate,
tannate, pantothenate, bitartrate,
ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate,
saccharate, formate, benzoate,
glutamate, methanesulfonate "mesylate", ethanesulfonate, benzenesulfonate, p-
toluenesulfonate, and pamoate
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(i.e., 1,1'-methylene-bis(2-hydroxy-3-naphthoate)) salts. A pharmaceutically
acceptable salt may involve the
inclusion of another molecule such as an acetate ion, a succinate ion or other
counter ion. The counter ion
may be any organic or inorganic moiety that stabilizes the charge on the
parent compound. Furthermore, a
pharmaceutically acceptable salt may have more than one charged atom in its
structure. Instances where
multiple charged atoms are part of the pharmaceutically acceptable salt can
have multiple counter ions. Hence,
a pharmaceutically acceptable salt can have one or more charged atoms and/or
one or more counter ion.
[0234] If the compound of the invention is a base, the desired
pharmaceutically acceptable salt may
be prepared by any suitable method available in the art, for example,
treatment of the free base with an
inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid,
nitric acid, methanesulfonic acid,
phosphoric acid and the like, or with an organic acid, such as acetic acid,
trifluoroacetic acid, maleic acid,
succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic
acid, glycolic acid, salicylic acid,
a pyranosidyl acid, such as glucuronic acid or galacturonic acid, an alpha
hydroxy acid, such as citric acid or
tartaric acid, an amino acid, such as aspartic acid or glutamic acid, an
aromatic acid, such as benzoic acid or
cinnamic acid, a sulfonic acid, such as p-toluenesulfonic acid or
ethanesulfonic acid, or the like.
[0235] If the compound of the invention is an acid, the desired
pharmaceutically acceptable salt
may be prepared by any suitable method, for example, treatment of the free
acid with an inorganic or organic
base, such as an amine (primary, secondary or tertiary), an alkali metal
hydroxide or alkaline earth metal
hydroxide, or the like. Illustrative examples of suitable salts include, but
are not limited to, organic salts
derived from amino acids, such as glycine and arginine, ammonia, primary,
secondary, and tertiary amines,
and cyclic amines, such as piperidine, morpholine and piperazine, and
inorganic salts derived from sodium,
calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and
lithium.
[0236] The phrase "pharmaceutically acceptable" indicates that the substance
or composition must
be compatible chemically and/or toxicologically, with the other ingredients
comprising a formulation, and/or
the mammal being treated therewith.
[0237] A "solvate" refers to an association or complex of one or more solvent
molecules and a
compound of the invention. Examples of solvents that form solvates include,
but are not limited to, water,
isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and
ethanolamine. The term "hydrate"
refers to the complex where the solvent molecule is water.
[0238] The term "protecting group" refers to a substituent that is commonly
employed to block or
protect a particular functionality while reacting other functional groups on
the compound. For example, an
"amino-protecting group" is a substituent attached to an amino group that
blocks or protects the amino
functionality in the compound. Suitable amino-protecting groups include
acetyl, trifluoroacetyl, t-
butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ) and 9-
fluorenylmethylenoxycarbonyl (Fmoc). Similarly, a
"hydroxy-protecting group" refers to a substituent of a hydroxy group that
blocks or protects the hydroxy
functionality. Suitable protecting groups include acetyl and silyl. A "carboxy-
protecting group" refers to a
substituent of the carboxy group that blocks or protects the carboxy
functionality. Common carboxy-
protecting groups include phenylsulfonylethyl, cyanoethyl, 2-
(trimethylsilyl)ethyl, 2-
(trimethylsilyl)ethoxymethyl, 2-(p-toluenesulfonyl)ethyl, 2-(p-
nitrophenylsulfenyl)ethyl, 2-
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(diphenylphosphino)-ethyl, nitroethyl and the like. For a general description
of protecting groups and their use,
see T. W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons,
New York, 1991.
[0239] "Leaving group" refers to a functional group that can be substituted by
another functional
group. Certain leaving groups are well known in the art, and examples include,
but are not limited to, a halide
(e.g., chloride, bromide, iodide), methanesulfonyl (mesyl), p-toluenesulfonyl
(tosyl), trifluoromethylsulfonyl
(triflate), and trifluoromethylsulfonate.
Abbreviations
[0240] LINKER COMPONENTS:
MC = 6-maleimidocaproyl
Val-Cit or "vc" = valine-citrulline (an exemplary dipeptide in a protease
cleavable linker)
Citrulline = 2-amino-5-ureido pentanoic acid
PAB = p-aminobenzyloxycarbonyl (an example of a "self immolative" linker
component)
Me-Val-Cit = N-methyl-valine-citrulline (wherein the linker peptide bond has
been modified to
prevent its cleavage by cathepsin B)
MC(PEG)6-OH = maleimidocaproyl- polyethylene glycol (can be attached to
antibody cysteines).
CYTOTOXIC DRUGS:
MMAE = mono-methyl auristatin E (MW 718)
MMAF = variant of auristatin E (MMAE) with a phenylalanine at the C-terminus
of the drug (MW
731.5)
MMAF-DMAEA = MMAF with DMAEA (dimethylaminoethylamine) in an amide linkage to
the C-
terminal phenylalanine (MW 801.5)
MMAF-TEG = MMAF with tetraethylene glycol esterified to the phenylalanine
MMAF-NtBu = N-t-butyl, attached as an amide to C-terminus of MMAF
DM1 = N(2')-deacetyl-N(2')-(3 -mercapto- 1 -oxopropyl)-maytansine
DM3 = N(2')-deacetyl-N2-(4-mercapto- 1 -oxopentyl)-maytansine
DM4 = N(2')-deacetyl-N2-(4-mercapto-4-methyl- 1 -oxopentyl)-maytansine
[0241] Further abbreviations are as follows: AE is auristatin E, Boc is N-(t-
butoxycarbonyl), cit is
citrulline, dap is dolaproine, DCC is 1,3-dicyclohexylcarbodrimide, DCM is
dichloromethane, DEA is
diethylamine, DEAD is diethylazodicarboxylate, DEPC is
diethylphosphorylcyanidate, DIAD is
diisopropylazodicarboxylate, DIEA is N,N-diisopropylethylamine, dil is
dolaisoleucine, DMA is
dimethylacetamide, DMAP is 4-dimethylaminopyridine, DME is ethyleneglycol
dimethyl ether (or 1,2-
dimethoxyethane), DMF is N,N-dimethylformamide, DMSO is dimethylsulfoxide, doe
is dolaphenine, dov is
N,N-dimethylvaline, DTNB is 5,5'-dithiobis(2-nitrobenzoic acid), DTPA is
diethylenetriaminepentaacetic
acid, DTT is dithiothreitol, EDCI is 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride, EEDQ is
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2-ethoxy-l-ethoxycarbonyl-1,2-dihydroquinoline, ES-MS is electrospray mass
spectrometry, EtOAc is ethyl
acetate, Fmoc is N-(9-fluorenylmethoxycarbonyl), gly is glycine, HATU is O-(7-
azabenzotriazol-l-yl)-
N,N,N',N'-tetramethyluronium hexafluorophosphate, HOBt is 1-
hydroxybenzotriazole, HPLC is high pressure
liquid chromatography, ile is isoleucine, lys is lysine, MeCN (CH3CN) is
acetonitrile, MeOH is methanol, Mtr
is 4-anisyldiphenylmethyl (or 4-methoxytrityl),nor is (IS, 2R)-(+)-
norephedrine, PBS is phosphate-buffered
saline (pH 7.4), PEG is polyethylene glycol, Ph is phenyl, Pnp is p-
nitrophenyl, MC is 6-maleimidocaproyl,
phe is L-phenylalanine, PyBrop is bromo tris-pyrrolidino phosphonium
hexafluorophosphate, SEC is size-
exclusion chromatography, Su is succinimide, TFA is trifluoroacetic acid, TLC
is thin layer chromatography,
UV is ultraviolet, and val is valine.
[0242] A "free cysteine amino acid" refers to a cysteine amino acid residue
which has been
engineered into a parent antibody, has a thiol functional group (-SH), and is
not paired as an intramolecular or
intermolecular disulfide bridge.
[0243] The term "thiol reactivity value" is a quantitative characterization of
the reactivity of free
cysteine amino acids. The thiol reactivity value is the percentage of a free
cysteine amino acid in a cysteine
engineered antibody which reacts with a thiol-reactive reagent, and converted
to a maximum value of 1. For
example, a free cysteine amino acid on a cysteine engineered antibody which
reacts in 100% yield with a
thiol-reactive reagent, such as a biotin-maleimide reagent, to form a biotin-
labelled antibody has a thiol
reactivity value of 1Ø Another cysteine amino acid engineered into the same
or different parent antibody
which reacts in 80% yield with a thiol-reactive reagent has a thiol reactivity
value of 0.8. Another cysteine
amino acid engineered into the same or different parent antibody which fails
totally to react with a thiol-
reactive reagent has a thiol reactivity value of 0. Determination of the thiol
reactivity value of a particular
cysteine may be conducted by ELISA assay, mass spectroscopy, liquid
chromatography, autoradiography, or
other quantitative analytical tests.
[0244] A "parent antibody" is an antibody comprising an amino acid sequence
from which one or
more amino acid residues are replaced by one or more cysteine residues. The
parent antibody may comprise a
native or wild type sequence. The parent antibody may have pre-existing amino
acid sequence modifications
(such as additions, deletions and/or substitutions) relative to other native,
wild type, or modified forms of an
antibody. A parent antibody may be directed against a target antigen of
interest, e.g. a biologically important
polypeptide. Antibodies directed against nonpolypeptide antigens (such as
tumor-associated glycolipid
antigens; see US 5091178) are also contemplated.
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Table 1
*
* C-C increased from 12 to 15
* Z is average of EQ
* B is average of ND
* match with stop is _M; stop-stop = 0; J(joker) match = 0
#define _M -8 /* value of a match with a stop
int _day[26][26] = {
ABCDEFGHIJKLMNOPQRSTUVWXYZ*/
/* A { 2, 0,-2, 0, 0,-4, 0,-1,-2,-1, 0, M, 1, 0,-2, 1, 1, 0, 0,-6, 0,-3, 0},
/* B { 0, 3,-4, 3, 2,-5, 0, 1,-2, 0, 0,-3,-2, 2, M,-1, 1, 0, 0, 0, 0,-2,-5, 0,-
3, 1},
/* C {-2,-4,15,-5,-5,-4,-3,-3,-2, 0,-5,-6,-5,-4, M,-3,-5,-4, 0,-2, 0,-2,-8, 0,
0,-5},
/* D { 0, 3,-5, 4, 3,-6, 1, 1,-2, 0, 0,-4,-3, 2, M,-1, 2,-1, 0, 0, 0,-2,-7, 0,-
4, 2},
/* E { 0, 2,-5, 3, 4,-5, 0, 1,-2, 0, 0,-3,-2, 1, M,-1, 2,-1, 0, 0, 0,-2,-7, 0,-
4, 3},
/* F {-4,-5,-4,-6,-5, 9,-5,-2, 1, 0,-5, 2, 0,-4, M,-5,-5,-4,-3,-3, 0,-1, 0, 0,
7,-5},
/* G { 1, 0,-3, 1, 0,-5, 5,-2,-3, 0,-2,-4,-3, 0, M,-1,-1,-3, 1, 0, 0,-1,-7, 0,-
5, 0},
/* H {-1, 1,-3, 1, 1,-2,-2, 6,-2, 0, 0,-2,-2, 2, M, 0, 3, 2,-1,-1, 0,-2,-3, 0,
0, 2},
/* I {-1,-2,-2,-2,-2, 1,-3,-2, 5, 0,-2, 2, 2,-2, M,-2,-2,-2,-1, 0, 0, 4,-5, 0,-
1,-2},
/*J {0,0,0,0,0,0,0,0,0,0,0,0,0,0, M,0,0,0,0,0,0,0,0,0,0,0},
/* K {-1, 0,-5, 0, 0,-5,-2, 0,-2, 0, 5,-3, 0, 1, M,-1, 1, 3, 0, 0, 0,-2,-3, 0,-
4, 0},
/* L {-2,-3,-6,-4,-3, 2,-4,-2, 2, 0,-3, 6, 4,-3, M,-3,-2,-3,-3,-1, 0, 2,-2, 0,-
1,-2},
/* M {-1,-2,-5,-3,-2, 0,-3,-2, 2, 0, 0, 4, 6,-2, M,-2,-1, 0,-2,-1, 0, 2,-4, 0,-
2,-1},
/* N { 0, 2,-4, 2, 1,-4, 0, 2,-2, 0, 1,-3,-2, 2, M,-1, 1, 0, 1, 0, 0,-2,-4, 0,-
2, 1},
/* O{_M,_M,_M,_M, M, M, M, M, M, M, M, M, M, M, 0, M, M,
M,_M,_M,_M,_M,_M,_M,_M,_M},
/* P{ 1,-0,-2, 0,-1,-3,-2,-1, M, 6, 0, 0, 1, 0, 0,-1,-6, 0,-5, 0},
/* Q{ 0, 1,-5, 2, 2,-5,-1, 3,-2, 0, 1,-2,-1, 1, M, 0, 4, 1,-1,-1, 0,-2,-5, 0,-
4, 3},
/* R {-2, 0,-4,-1,-1,-4,-3, 2,-2, 0, 3,-3, 0, 0, M, 0, 1, 6, 0,-1, 0,-2, 2, 0,-
4, 0},
/* S { 1, 0, 0, 0, 0,-3, 0, 0,-3,-2, 1, M, 1,-1, 0, 2, 1, 0,-1,-2, 0,-3, 0},
/* T { 1, 0,-2, 0, 0,-3, 0,-1, 0, 0, 0,-1,-1, 0, M, 0,-1,-1, 1, 3, 0, 0,-5, 0,-
3, 0},
/* U { 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, M, 0, 0, 0, 0, 0, 0, 0, 0, 0,
0, 0},
/* V{ 0,-2,-2,-2,-2,-1,-1,-2, 4, 0,-2, 2, 2,-2, M,-1,-2,-2,-1, 0, 0, 4,-6, 0,-
2,-2},
/* W{-6,-5,-8,-7,-7, 0,-7,-3,-5, 0,-3,-2,-4,-4, M,-6,-5, 2,-2,-5, 0,-6,17, 0,
0,-6},
/* X{ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, M, 0, 0, 0, 0, 0, 0, 0, 0, 0,
0, 0},
/* Y{-3,-3, 0,-4,-4, 7,-5, 0,-1, 0,-4,-1,-2,-2, M,-5,-4,-4,-3,-3, 0,-2, 0,
0,10,-4},
/* Z{ 0, 1,-5, 2, 3,-5, 0, 2,-2, 0, 0,-2,-1, 1, M, 0, 3, 0, 0, 0, 0,-2,-6, 0,-
4, 4}
}
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Table 1 (cont')
#include <stdio.h>
#include <ctype.h>
#define MAXJMP 16 /* max jumps in a diag
#define MAXGAP 24 /* don't continue to penalize gaps larger than this */
#define JMPS 1024 /* max jmps in an path */
#define MX 4 /* save if there's at least MX-1 bases since last jmp
#define DMAT 3 /* value of matching bases */
#define DMIS 0 /* penalty for mismatched bases */
#define DINSO 8 /* penalty for a gap
#define DINS1 1 /* penalty per base */
#define PINSO 8 /* penalty for a gap
#define PINS1 4 /* penalty per residue */
structjmp {
short n[MAXJMP]; /* size of jmp (neg for dely)
unsigned short x[MAXJMP]; /* base no. of jmp in seq x
/* timits seq to 2^16 -1
struct diag {
int score; /* score at last jmp
long offset; /* offset of prev block */
short ijmp; /* current jmp index */
struct jmp jp; /* list ofjmps struct path {
int spc; /* number of leading spaces
short n[JMPS]; /* size ofjmp (gap) */
int x[JMPS]; /* loc of jmp (last elem before gap) */
char *ofile; /* output file name
char *namex[2]; /* seq names: getseqsQ
char *prog; /* prog name for err msgs
char *seqx[2]; /* seqs: getseqsQ
int dmax; /* best diag: nwQ
int dmax0; /* final diag */
int dna; /* set if dna: mainQ
int endgaps; /* set if penalizing end gaps
int gapx, gapy; /* total gaps in seqs
int len0, len1; /* seq lens */
int ngapx, ngapy; /* total size of gaps
int smax; /* max score: nwQ
int *xbm; /* bitmap for matching */
long offset; /* current offset injmp file */
struct diag *dx; /* holds diagonals */
struct path pp[2]; /* holds path for seqs
char *callocQ, *mallocQ, *indexU, *strcpyQ;
char *getseqQ, *g_callocQ;
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Table 1 (cont')
/* Needleman-Wunsch alignment program
*
* usage: progs filel file2
* where filel and file2 are two dna or two protein sequences.
* The sequences can be in upper- or lower-case an may contain ambiguity
* Any lines beginning with ;', '>' or'<' are ignored
* Max file length is 65535 (limited by unsigned short x in the jmp struct)
* A sequence with 1/3 or more of its elements ACGTU is assumed to be DNA
* Output is in the file "align.out"
*
* The program may create a tmp file in /tmp to hold info about traceback.
* Original version developed under BSD 4.3 on a vax 8650
#include "nw.h"
#include "day.h"
static dbval[26]
1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0
};
static _pbval[26]
1, 21(1 ('D'-'A'))I(1 ('N'-'A')), 4, 8, 16, 32, 64,
128, 256, OxFFFFFFF, 1 10, 1 11, 1 12, 1 13, 1 14,
1 15, 1 16, 1 17, 1 18, 1 19, 1 20, 1 21, 1 22,
1 23, 1 24, 1 251(1 ('E'-'A'))I(1 ('Q'-'A'))
main(ac, av) main
int ac;
char *av[];
{
prog = av[0];
if(ac!=3){
fprintf(stderr,"usage: %s filel file2\n", prog);
fprintf(stderr,"where filel and file2 are two dna or two protein
sequences.An");
fprintf(stderr,"The sequences can be in upper- or lower-case\n");
fprintf(stderr,"Any lines beginning with ';' or'<' are ignored\n");
fprintf(stderr,"Output is in the file V"align.outV"\n");
exit(l);
}
namex[0] = av[1];
namex[l] = av[2];
seqx[0] = getseq(namex[0], &len0);
seqx[1] = getseq(namex[1], &lenl);
xbm = (dna)? dbval : pbval;
endgaps = 0; /* 1 to penalize endgaps */
ofile = "align.out"; /* output file */
nwQ; /* fill in the matrix, get the possible jmps */
readjmpsQ; /* get the actual jmps */
printU; /* print stats, alignment */
cleanup(0); /* unlink any tmp files */}
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Table 1 (cont')
/* do the alignment, return best score: mainQ
* dna: values in Fitch and Smith, PNAS, 80, 1382-1386, 1983
* pro: PAM 250 values
* When scores are equal, we prefer mismatches to any gap, prefer
* a new gap to extending an ongoing gap, and prefer a gap in seqx
*toagapinseqy.
nwQ nw
{
char *px, *py; /* seqs and ptrs int *ndely, *dely; /* keep track of dely
int ndelx, delx; /* keep track of delx */
int *tmp; /* for swapping rowO, rowl
int mis; /* score for each type
int insO, insl; /* insertion penalties */
register id; /* diagonal index */
register ij; /* jmp index */
register *co10, *coll; /* score for curr, last row
register xx, yy; /* index into seqs */
dx = (struct diag *)g_calloc("to get diags", len0+len1+1, sizeof(struct
diag));
ndely =(int *)g_calloc("to get ndely", len1+1, sizeof(int));
dely =(int *)g_calloc("to get dely", len1+1, sizeof(int));
colO =(int *)g_calloc("to get co10", lenl+l, sizeof(int));
coll =(int *)g_calloc("to get coll", lenl+l, sizeof(int));
insO = (dna)? DINSO : PINSO;
insl = (dna)? DINS1 : PINS1;
smax = -10000;
if (endgaps) {
for (co10[0] = dely[0] =-ins0, yy = 1; yy <=1en1; yy++) {
colO[yy] = dely[yy] = co10[yy-1] - insl;
ndely[yy] = yy;
}
co10[0] = 0; /* Waterman Bull Math Bio184
}
else
for (yy = 1; yy <=1en1; yy++)
dely[yy] = -insO;
/* fill in match matrix
for (px = seqx[0], xx = 1; xx <=1en0; px++, xx++) {
/* initialize first entry in col
if (endgaps) {
if (xx == 1)
coll[0] = delx = -(ins0+ins1);
else
coll[0] = delx = co10[0] - insl;
ndelx = xx;
}
else {
coll[0] = 0;
delx = -insO;
ndelx = 0;
}
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Table 1 (cont')
...nw
for (py = seqx[1], yy = 1; yy <=1en1; py++, yy++) {
mis = colO[yy-1];
if (dna)
mis +_ (xbm[*px-'A']&xbm[*py-'A'])? DMAT : DMIS;
else
mis +_ _day[ *px-'A'] [ *py-'A'];
/* update penalty for del in x seq;
* favor new del over ongong del
* ignore MAXGAP if weighting endgaps
if (endgaps 11 ndely[yy] < MAXGAP) {
if (colO[yy] - insO >= dely[yy]) {
dely[yy] = colO[yy] - (insO+insl);
ndely[yy] = 1;
} else {
dely[yy] -= insl;
ndely[yy]++;
}
} else {
if (colO[yy] - (ins0+ins1) >= dely[yy]) {
dely[yy] = colO[yy] - (insO+insl);
ndely[yy] = 1;
} else
ndely[yy]++;
}
/* update penalty for del in y seq;
* favor new del over ongong del
if (endgaps 11 ndelx < MAXGAP) {
if (coll [yy-1] - insO >= delx) {
delx = coll[yy-1] - (ins0+ins1);
ndelx = 1;
} else {
delx = insl;
ndelx++;
}
} else {
if (coll[yy-1] - (ins0+ins1) >= delx) {
delx = coll[yy-1] - (ins0+ins1);
ndelx = 1;
} else
ndelx++;
}
/* pick the maximum score; we're favoring
* mis over any del and delx over dely
...nw
id=xx - yy+lenl - 1;
if (mis >= delx && mis >= dely[yy])
coll[yy] = mis;
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Table 1 (cont')
else if (delx >= dely[yy]) {
coll[yy] = delx;
ii = dx[id].ijmp;
if (dx[id].jp.n[0] && (!dna 11 (ndelx >= MAXJMP
&& xx > dx[id].jp.x[ij]+MX) 11 mis > dx[id].score+DINSO)) {
dx[id].ijmp++=
if (++ij >= MAXJMP) {
writejmps(id);
ii = dx[id].ijmp = 0;
dx[id].offset = offset;
offset += sizeof(struct jmp) + sizeof(offset);
}
}
dx[id].jp.n[ij] = ndelx;
dx[id].jp.x[ij] = xx;
dx[id].score = delx;
}
else {
coll[yy] = dely[yy];
ij = dx[id].ijmp;
if (dx[id].jp.n[0] && (!dna 11 (ndely[yy] >= MAXJMP
&& xx > dx[id].jp.x[ij]+MX) 11 mis > dx[id].score+DINSO)) {
dx[id].ijmp++=
if (++ij >= MAXJMP) {
writejmps(id);
ij = dx[id].ijmp = 0;
dx[id].offset = offset;
offset += sizeof(struct jmp) + sizeof(offset);
}
}
dx[id].jp.n[ij] = -ndely[yy];
dx[id].jp.x[ij] = xx;
dx[id].score = dely[yy];
}
if (xx ==1en0 && yy < lenl) {
/* last col
if (endgaps)
coll[yy] -=ins0+ins1*(lenl-yy);
if (coll [yy] > smax) {
smax = coll [yy];
dmax = id;
}
}
}
if (endgaps && xx < lenO)
coll[yy-1] = ins0+ins1*(len0-xx);
if (coll[yy-1] > smax) {
smax = coll [yy-1];
dmax = id;
}
tmp = co10; co10 = coll; coll = tmp;
}
(void) free((char *)ndely);
(void) free((char *)dely);
(void) free((char *)colO);
(void) free((char *)coll); }
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Table 1 (cont')
*
* printQ -- only routine visible outside this module
*
* static:
* getmatQ -- trace back best path, count matches: printQ
* pr_alignO -- print alignment of described in array p[]: printO
* dumpblockQ -- dump a block of lines with numbers, stars: pr_alignQ
* numsQ -- put out a number line: dumpblockQ
* putlineO -- put out a line (name, [num], seq, [num]): dumpblockO
* starsQ - -put a line of stars: dumpblockO
* stripnameQ -- strip any path and prefix from a seqname
#include "nw.h"
#define SPC 3
#define P LINE 256 /* maximum output line */
#define P_SPC 3 /* space between name or num and seq
extern _day[26][26];
int olen; /* set output line length */
FILE *fx; /* output file */
printo print
{
int lx, ly, firstgap, lastgap; /* overlap */
if ((fx = fopen(ofile, "w")) == 0) {
fprintf(stderr,"%s: can't write %s\n", prog, ofile);
cleanup(l);
}
fprintf(fx, "<first sequence: %s (length = %d)\n", namex[0], lenO);
fprintf(fx, "<second sequence: %s (length = %d)\n", namex[1], lenl);
olen = 60;
lx =1en0;
ly =1en1;
firstgap = lastgap = 0;
if (dmax < lenl - 1) { /* leading gap in x
pp[0].spc = firstgap =1en1 - dmax - 1;
ly = pp[0].spc;
}
else if (dmax > lenl - 1) { /* leading gap in y
pp[l].spc = firstgap = dmax - (lenl - 1);
Ix -= pp[1]=spc;
}
if (dmax0 < len0 - 1) { /* trailing gap in x
lastgap =1en0 - dmax0 -1;
lx -=lastgap;
}
else if (dmax0 > lenO - 1) { /* trailing gap in y
lastgap = dmax0 - (lenO - 1);
ly = lastgap;
}
getmat(lx, ly, firstgap, lastgap);
pr_alignU; }
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Table 1 (cont')
* trace back the best path, count matches
static
getmat(lx, ly, firstgap, lastgap) getmat
int lx, ly; /* "core" (minus endgaps)
int firstgap, lastgap; /* leading trailing overlap */
{
int nm, iO, il, sizO, sizl;
char outx[32];
double pct;
register n0, nl;
register char *p0, *pl;
/* get total matches, score
i0 = il = siz0 = sizl = 0;
p0 = seqx[O] +pp[1].spc;
p1 = seqx[1] +pp[0].spc;
n0 = pp[1].spc + 1;
nl = pp[0].spc + 1;
nm=0;
while ( *p0 && *p1 ) {
if (siz0) {
pl++;
nl++;
siz0--;
}
else if (sizl) {
p0++;
nO++;
sizl--;
}
else {
if (xbm[*p0-'A']&xbm[*pl-'A'])
nm++;
if (n0++ == pp[0].x[i0])
sizO = pp[0].n[i0++];
if (nl++ == pp[l].x[il])
sizl =pp[1].n[il++];
p0++;
pl++;
}
}
/* pct homology:
* if penalizing endgaps, base is the shorter seq
* else, knock off overhangs and take shorter core
if (endgaps)
lx = (lenO < lenl)? len0 : lenl;
else
lx=(lx<ly)?lx:ly;
pct = 100.*(double)nm/(double)lx;
fprintf(fx, "\n");
fprintf(fx, "<%d match%s in an overlap of %d: %.2f percent similarity\n",
nm, (nm == 1)? "' . "es", lx, pct);
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Table 1 (cont')
fprintf(fx, "<gaps in first sequence: %d", gapx); ...getlriat
if (gapx) {
(void) sprintf(outx, " (%d %s%s)",
ngapx, (dna)? "base":"residue", (ngapx == 1)? " :"s");
fprintf(fx,"%s", outx);
fprintf(fx, ", gaps in second sequence: %d", gapy);
if (gapy) {
(void) sprintf(outx, " (%d %s%s)",
ngapy, (dna)? "base":"residue", (ngapy == 1)? " :"s");
fprintf(fx,"%s", outx);
}
if (dna)
fprintf(fx,
"\n<score: %d (match = %d, mismatch = %d, gap penalty = %d + %d per base)\n",
smax, DMAT, DMIS, DINSO, DINS1);
else
fprintf(fx,
"\n<score: %d (Dayhoff PAM 250 matrix, gap penalty = %d + %d per residue)\n",
smax, PINSO, PINS1);
if (endgaps)
fprintf(fx,
"<endgaps penalized. left endgap: %d %s%s, right endgap: %d %s%s\n",
firstgap, (dna)? "base" : "residue", (firstgap == 1)? "" : "s",
lastgap, (dna)? "base" : "residue", (lastgap == 1)? "s");
else
fprintf(fx, "<endgaps not penalized\n");
}
static nm; /* matches in core -- for checking */
static hnax; /* lengths of stripped file names
static ij [2]; /* jmp index for a path */
static nc[2]; /* number at start of current line */
static ni[2]; /* current elem number -- for gapping
static siz[2];
static char *ps[2]; /* ptr to current element */
static char *po[2]; /* ptr to next output char slot */
static char out[2][P_LINE]; /* output line */
static char star[P_LINE]; /* set by starsQ
* print alignment of described in struct path pp[]
static
pr_alignQ pr_align
{
int nn; /* char count
int more;
register i;
for (i = 0,1max=0;i<2;i++){
nn = stripname(namex[i]);
if (nn > lmax)
hnax = nn;
nc[i] = 1;
ni[i] = 1;
siz[i] = ij[i] = 0;
ps[i] = seqx[i];
po[i] = out[i]; }
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Table 1 (cont')
for (nn = nm = 0, more = 1; more; ) { ...pr_allgn
for(i=more=0;i<2;i++){
* do we have more of this sequence?
if (! *ps[i])
continue;
more++;
if (pp[i].spc) { /* leading space
*po [i]++ = ' ';
pp[i].spc--;
}
else if (siz[i]) { /* in a gap
*po [i]++
siz[i]--;
}
else { /* we're putting a seq element
*po[i] = *ps[i];
if (islower(*ps[i]))
*ps[i] = toupper(*ps[i]);
po[i]++;
ps[i]++;
* are we at next gap for this seq?
if (ni[i] == pp[i].x[ij[i]]) {
* we need to merge all gaps
* at this location
siz[i] = pp[i].n[ij[i]++];
while (ni[i] == pp[i].x[ij[i]])
siz[i] +=pp[i].n[ij[i]++];
}
ni[i]++;
}
}
if (++nn == olen I !more && nn) {
dumpblockQ;
for (i = 0; i < 2; i++)
po[i] = out[i];
nn = 0;
}
}
}
* dump a block of lines, including numbers, stars: pralignQ
static
dumpblockQ dumpblock
{
register i;
for (i = 0; i < 2; i++)
*po[i]-- ='\0 ;
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Table 1 (cont')
...dumpblock
(void) putc('\n', fx);
for(i=0;i<2;i++){
if (*out[i] && (*out[i] != " II *(po[i]) != ")) {
if (i == 0)
nums(i);
if (i == 0 && *out[l])
starsQ;
putline(i);
if (i == 0 && *out[l])
fprintf(fx, star);
if(i==1)
nums(i);
}
}
}
* put out a number line: dumpblockQ
static
nums(ix) nums
int ix; /* index in out[] holding seq line */
{
char nline[P_LINE];
register i, j;
register char *pn, *px, *py;
for (pn = nline, i 0; i < 1max+P_SPC; i++, pn++)
*pn =
for (i = nc[ix], py = out[ix]; *py; py++, pn++) {
if (*py 1 1 11 *py =_ )
*pn
else {
if (i%10 == 0 II (i ==1 && nc[ix] 1))
{
j = (i < 0)? -i: i;
for (px = pn; j; j10, px--)
*px = j%10 +'0';
if (i < 0)
*px
}
else
*pn =
i++;
}
}
*pn = '\0';
nc[ix] = i;
for (pn = nline; *pn; pn++)
(void) putc(*pn, fx);
(void) putc('\n', fx);
}
* put out a line (name, [num], seq, [num]): dumpblockQ
static
putline(ix) putline
int ix; {
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Table 1 (cont')
...putline
int i;
register char *px;
for (px = namex[ix], i = 0; *px && *px !=':'; px++, i++)
(void) putc(*px, fx);
for (; i < 1max+P_SPC; i++)
(void) putc(' ', fx);
/* these count from 1:
* ni[] is current element (from 1)
* nc[] is number at start of current line
for (px = out[ix]; *px; px++)
(void) putc(*px&Ox7F, fx);
(void) putc('\n', fx);
}
* put a line of stars (seqs always in out[0], out[1]): dumpblockQ
static
starso stars
{
int i;
register char *p0, *pl, cx, *px;
if (! *out[0] (*out[0] && *(po[0])
!*out[l] II (*out[l] && *(po[l]) return;
px = star;
for (i = hnax+P_SPC; i; i--)
*px++ _ ,
for (p0 = out[0], p1 = out[1]; *pO && *pl; pO++, pl++) {
if (isalpha(*pO) && isalpha(*p 1)) {
if (xbm[*p0-'A']&xbm[*pl-'A']) {
cx ='*';
nm++;
}
else if (!dna && _day[*p0-'A'][*pl-'A'] > 0)
cx=
else
cx= ,
}
else
,
cx
*px++ = cx;
}
*px++ _ '\n';
*px = '\0';
}
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Table 1 (cont')
* strip path or prefix from pn, return len: pr_alignO
static
stripname(pn) stripname
char *pn; /* file name (may be path) */
{
register char *px, *py;
py=0;
for (px = pn; *px; px++)
if (*px == '/')
py = px + 1;
if (py)
(void) strcpy(pn, py);
return(strlen(pn));
}
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Table 1 (cont')
* cleanupQ -- cleanup any tmp file
* getseqQ -- read in seq, set dna, len, maxlen
* g_callocQ -- callocQ with error checkin
* readjmpsQ -- get the good jmps, from tmp file if necessary
* writejmpsQ -- write a filled array of jmps to a tmp file: nwQ
#include "nw.h"
#include <sys/file.h>
char *jname = "/tmp/homgXXXXXX"; /* tmp file for jmps
FILE *fj;
int cleanupQ; /* cleanup tmp file */
long lseekQ;
* remove any tmp file if we blow
cleanup(i) cleanup
int i;
{
if (fj)
(void) unlink(jname);
exit(i);
}
* read, return ptr to seq, set dna, len, maxlen
* skip lines starting with '; ,'<', or '>'
* seq in upper or lower case
char *
getseq(file, len) getseq
char *file; /* file name
int *len; /* seq len */
{
char line[1024], *pseq;
register char *px, *py;
int natgc, tlen;
FILE *fp;
if ((fp = fopen(file,"r")) == 0) {
fprintf(stderr,"%s: can't read %s\n", prog, file);
exit(l);
}
tlen = natgc = 0;
while (fgets(line, 1024, fp)) {
if (*line == ;' 11 *line *line
continue;
for (px = line; *px !='\n'; px++)
if (isupper(*px) 11 islower(*px))
tlen++;
}
if ((pseq = malloc((unsigned)(tlen+6))) == 0) {
fprintf(stderr,"%s: mallocQ failed to get %d bytes for %s\n", prog, tlen+6,
file);
exit(l);
}
pseq[0] = pseq[1] = pseq[2] = pseq[3] ='\0';
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Table 1 (cont')
...getseq
py = pSeq + 4;
*len = tlen;
rewind(fp);
while (fgets(line, 1024, fp)) {
if (*line =_ ;' Il *line =='<' Il *line
continue;
for (px = line; *px !='\n'; px++) {
if (isupper(*px))
*py++ = *px;
else if (islower(*px))
*py++ = toupper(*px);
if (index("ATGCU", *(py- 1)))
natgc++;
}
}
*py++ _ '\0';
*py = 1\01 ;
(void) fclose(fp);
dna = natgc > (tlen/3);
return(pseq+4);
}
char *
g_calloc(msg, nx, sz) g_calloc
char *msg; /* program, calling routine
int nx, sz; /* number and size of elements */
{
char *px, *callocQ;
if ((px = calloc((unsigned)nx, (unsigned)sz)) == 0) {
if (*msg) {
fprintf(stderr, "%s: g_callocQ failed %s (n=%d, sz=%d)\n", prog, msg, nx, sz);
exit(l);
}
}
return(px);
}
* get final jmps from dx[] or tmp file, set pp[], reset dmax: mainO
readjmpso readj mps
{
int fd = -1;
int siz, i0, il;
register i, j, xx;
if (fj) {
(void) fclose(fj);
if ((fd = open(jname, ORDONLY, 0)) < 0) {
fprintf(stderr, "%s: can't openo %s\n", prog, jname);
cleanup(l);
}
}
for(i=i0=i1=0,dmax0=dmax,xx=len0;;i++){
while (1) {
for (j = dx[dmax].ijmp; j>= 0 && dx[dmax].jp.x[j] >= xx; j--)
CA 02692819 2010-01-07
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Table 1 (cont')
...readjmps
if (j < 0 && dx[dmax].offset && fj) {
(void) lseek(fd, dx[dmax].offset, 0);
(void) read(fd, (char *)&dx[dmax].jp, sizeof(struct jmp));
(void) read(fd, (char *)&dx[dmax].offset, sizeof(dx [dmax]. offset));
dx[dmax].ijmp = MAXJMP-1; }
else
break; }
if (i >= JMPS) {
fprintf(stderr, "%s: too many gaps in alignment\n", prog);
cleanup(1);
}
if(j>=0){
siz = dx[dmax].jp.n[j];
xx = dx[dmax] Jp.xU];
dmax += siz;
if (siz < 0) { /* gap in second seq
pp[1].n[il] = -siz;
xx += siz;
/*id=xx-yy+lenl - 1
pp[1].x[il] = xx - dmax + lenl - 1;
gapy++;
ngapy -= siz;
/* ignore MAXGAP when doing endgaps */
siz = (-siz < MAXGAP 11 endgaps)? -siz : MAXGAP;
il++;
}
else if (siz > 0) { /* gap in first seq
pp[0].n[i0] = siz;
pp[0].x[i0] = xx;
gapx++;
ngapx += siz;
/* ignore MAXGAP when doing endgaps */
siz = (siz < MAXGAP 11 endgaps)? siz : MAXGAP;
i0++;
}
}
else
break;
}
/* reverse the order of jmps
for (j = 0, iO--; j < i0; j++, iO--) {
= pp[0].n[j]; pp[0].n[j] = pp[0].n[i0]; pp[0].n[i0] = i;
= pp[0].x[j]; pp[0].x[j] = pp[0].x[i0]; pp[0].x[i0] = i;
}
for(j=0,i1--;j<il;j++,il--){
= pp[1].n[j]; pp[1].n[j] = pp[1].n[il]; pp[1].n[il] = i;
= pp[1].x[j]; pp[1].x[j] = pp[1].x[il]; pp[1].x[il] = i;
}
if (fd >= 0)
(void) close(fd);
if (fj) {
(void) unlink(jname);
fj = 0;
offset = 0;
} }
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Table 1 (cont')
* write a filled jmp struct offset of the prev one (if any): nwQ
writejmps(ix) writej mps
int ix;
{
char *mktempQ;
if (!fj) {
if (mktemp(jname) < 0) {
fprintf(stderr, "%s: can't mktempO %s\n", prog, jname);
cleanup(l);
}
if ((fj = fopen(jname, "w")) == 0) {
fprintf(stderr, "%s: can't write %s\n", prog, jname);
exit(l);
}
}
(void) fwrite((char *)&dx[ix].jp, sizeof(struct jmp), 1, fj);
(void) fwrite((char *)&dx[ix].offset, sizeof(dx[ix].offset), 1, fj);
}
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III. Compositions and Methods of the Invention
[0245] The invention provides anti-CD79b antibodies or functional fragments
thereof, and their
method of use in the treatment of hematopoietic tumors.
[0246] In one aspect, the invention provides an antibody which binds,
preferably specifically, to any
of the above or below described polypeptides. Optionally, the antibody is a
monoclonal antibody, antibody
fragment, including Fab, Fab', F(ab')2, and Fv fragment, diabody, single
domain antibody, chimeric antibody,
humanized antibody, single-chain antibody or antibody that competitively
inhibits the binding of an anti-
CD79b polypeptide antibody to its respective antigenic epitope. Antibodies of
the present invention may
optionally be conjugated to a growth inhibitory agent or cytotoxic agent such
as a toxin, including, for
example, an auristatin, a maytansinoid, a dolostatin derivative or a
calicheamicin, an antibiotic, a radioactive
isotope, a nucleolytic enzyme, or the like. The antibodies of the present
invention may optionally be produced
in CHO cells or bacterial cells and preferably induce death of a cell to which
they bind. For detection
purposes, the antibodies of the present invention may be detectably labeled,
attached to a solid support, or the
like.
[0247] In one aspect, the invention provides a humanized anti-CD79b antibody
wherein the
monovalent affinity of the antibody to CD79b (e.g. affinity of the antibody as
a Fab fragment to CD79b) is
substantially the same as the monovalent affinity of a murine antibody (e.g.
affinity of the murine antibody as
a Fab fragment to CD79b) or a chimeric antibody (e.g. affinity of the chimeric
antibody as a Fab fragment to
CD79b), comprising, consisting or consisting essentially of a light chain and
heavy chain variable domain
sequence as depicted in Figure 7 (SEQ ID NO: 10) and Figures 8A-B (SEQ ID NO:
14).
[0248] In another aspect, the invention provides a humanized anti-CD79b
antibody wherein the
monovalent affinity of the antibody to CD79b (e.g., affinity of the antibody
as a Fab fragment to CD79b) is
lower, for example at least 1, 2 or 3-fold lower, than the monovalent affinity
of a murine antibody (e.g.,
affinity of the murine antibody as a Fab fragment to CD79b) or a chimeric
antibody (e.g. affinity of the
chimeric antibody as a Fab fragment to CD79b), comprising, consisting or
consisting essentially of a light
chain and heavy chain variable domain sequence as depicted in Figure 7 (SEQ ID
NO: 10) and Figures 8A-B
(SEQ ID NO: 14).
[0249] In another aspect, the invention provides a humanized anti-CD79b
antibody wherein the
monovalent affinity of the antibody to CD79b (e.g., affinity of the antibody
as a Fab fragment to CD79b) is
greater, for example at least 1, 2 or 3-fold greater, than the monovalent
affinity of a murine antibody (e.g.,
affinity of the murine antibody as a Fab fragment to CD79b) or a chimeric
antibody (e.g. affinity of the
chimeric antibody as a Fab fragment to CD79b), comprising, consisting or
consisting essentially of a light
chain and heavy chain variable domain sequence as depicted in Figure 7 (SEQ ID
NO: 10) and Figures 8A-B
(SEQ ID NO: 14).
[0250] In one aspect, the invention provides a humanized anti-CD79b antibody
wherein the affinity
of the antibody in its bivalent form to CD79b (e.g. affinity of the antibody
as an IgG fragment to CD79b) is
substantially the same as the affinity of a murine antibody (e.g. affinity of
the murine antibody as an IgG
fragment to CD79b) or a chimeric antibody (e.g. affinity of the chimeric
antibody as an IgG fragment to
CD79b) in its bivalent form, comprising, consisting or consisting essentially
of a light chain and heavy chain
variable domain sequence as depicted in Figure 7 (SEQ ID NO: 10) and Figures
8A-B (SEQ ID NO: 14).
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[0251] In another aspect, the invention provides a humanized anti-CD79b
antibody wherein the
affinity of the antibody in its bivalent form to CD79b (e.g., affinity of the
antibody as an IgG fragment to
CD79b) is lower, for example at least 1, 2 or 3-fold lower, than the affinity
of a murine antibody (e.g., affinity
of the murine antibody as an IgG fragment to CD79b) or a chimeric antibody
(e.g. affinity of the chimeric
antibody as a Fab fragment to CD79b) in its bivalent form, comprising,
consisting or consisting essentially of
a light chain and heavy chain variable domain sequence as depicted in Figure 7
(SEQ ID NO: 10) and
Figures 8A-B (SEQ ID NO: 14).
[0252] In another aspect, the invention provides a humanized anti-CD79b
antibody wherein the
affinity of the antibody in its bivalent form to CD79b (e.g., affinity of the
antibody as an IgG fragment to
CD79b) is greater, for example at least 1, 2 or 3-fold greater, than the
affinity of a murine antibody (e.g.,
affinity of the murine antibody as an IgG fragment to CD79b) or a chimeric
antibody (e.g. affinity of the
chimeric antibody as a Fab fragment to CD79b) in its bivalent form,
comprising, consisting or consisting
essentially of a light chain and heavy chain variable domain sequence as
depicted in Figure 7 (SEQ ID NO:
10) and Figures 8A-B (SEQ ID NO: 14).
[0253] In another aspect, the invention provides a humanized anti-CD79b
antibody wherein the
affinity of the antibody in its bivalent form to CD79b (e.g., affinity of the
antibody as an IgG to CD79b) is 2.0
nM. In a further aspect, the invention provides a humanized anti-CD79b
antibody wherein the affinity of the
antibody in its bivalent form to CD79b (e.g., affinity of the antibody as an
IgG to CD79b) is 2.0 nM +/- 0.5.
[0254] In another aspect, the invention provides a humanized anti-CD79b
antibody wherein the
affinity of the antibody in its bivalent form to CD79b (e.g., affinity of the
antibody as an IgG to CD79b) is 1
nM or better. In another aspect, the invention provides a humanized anti-CD79b
antibody wherein the affinity
of the antibody in its bivalent form to CD79b (e.g., affinity of the antibody
as an IgG to CD79b) is 1.5 nM or
better. In another aspect, the invention provides a humanized anti-CD79b
antibody wherein the affinity of the
antibody in its bivalent form to CD79b (e.g., affinity of the antibody as an
IgG to CD79b) is 2 nM or better. In
another aspect, the invention provides a humanized anti-CD79b antibody wherein
the affinity of the antibody
in its bivalent form to CD79b (e.g., affinity of the antibody as an IgG to
CD79b) is 2.5 nM or better. In
another aspect, the invention provides a humanized anti-CD79b antibody wherein
the affinity of the antibody
in its bivalent form to CD79b (e.g., affinity of the antibody as an IgG to
CD79b) is 3 nM or better. In another
aspect, the invention provides a humanized anti-CD79b antibody wherein the
affinity of the antibody in its
bivalent form to CD79b (e.g., affinity of the antibody as an IgG to CD79b) is
between 1 nM and 3 nM. In
another aspect, the invention provides a humanized anti-CD79b antibody wherein
the affinity of the antibody
in its bivalent form to CD79b (e.g., affinity of the antibody as an IgG to
CD79b) is between 1.5 nM and 2.5
nM. In another aspect, the invention provides a humanized anti-CD79b antibody
wherein the affinity of the
antibody in its bivalent form to CD79b (e.g., affinity of the antibody as an
IgG to CD79b) is between 1.75 nM
and 2.25 nM.
[0255] In one aspect, the monovalent affinity of the murine antibody to CD79b
is substantially the
same as the binding affinity of a Fab fragment comprising variable domain
sequences of SEQ ID NO: 10
(Figure 7) and SEQ ID NO: 14 (Figures 8A-B). In another aspect, the monovalent
affinity of the murine
antibody to CD79b is substantially the same as the binding affinity of a Fab
fragment comprising variable
domain sequences of an antibody generated from hybridomas deposited with the
ATCC as PTA-7712 on July
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WO 2009/012256 PCT/US2008/070061
11, 2006 or chimeric antibodies comprising the variable domains from antibody
generated from hybridomas
deposited with the ATCC as PTA-7712 on July 11, 2006.
[0256] As is well-established in the art, binding affinity of a ligand to its
receptor can be determined
using any of a variety of assays, and expressed in terms of a variety of
quantitative values. Accordingly, in
one embodiment, the binding affinity is expressed as Kd values and reflects
intrinsic binding affinity (e.g.,
with minimized avidity effects). Generally and preferably, binding affinity is
measured in vitro, whether in a
cell-free or cell-associated setting. As described in greater detail herein,
fold difference in binding affinity can
be quantified in terms of the ratio of the monovalent binding affinity value
of a humanized antibody (e.g., in
Fab form) and the monovalent binding affinity value of a reference/comparator
antibody (e.g., in Fab form)
(e.g., a murine antibody having donor hypervariable region sequences), wherein
the binding affinity values are
determined under similar assay conditions. Thus, in one embodiment, the fold
difference in binding affinity is
determined as the ratio of the Kd values of the humanized antibody in Fab form
and said reference/comparator
Fab antibody. For example, in one embodiment, if an antibody of the invention
(A) has an affinity that is "3-
fold lower" than the affinity of a reference antibody (M), then if the Kd
value for A is 3x, the Kd value of M
would be lx, and the ratio of Kd of A to Kd of M would be 3:1. Conversely, in
one embodiment, if an
antibody of the invention (C) has an affinity that is "3-fold greater" than
the affinity of a reference antibody
(R), then if the Kd value for C is lx, the Kd value of R would be 3x, and the
ratio of Kd of C to Kd of R would
be 1:3. Any of a number of assays known in the art, including those described
herein, can be used to obtain
binding affinity measurements, including, for example, Biacore,
radioimmunoassay (RIA) and ELISA.
[0257] In one aspect, an antibody that binds to CD79b is provided, wherein the
antibody comprises:
(a) at least one, two, three, four, five or six HVRs selected from the group
consisting of:
(i) HVR-L1 comprising sequence A1-A15, wherein A1-A16 is KSSQSLLDSDGKTYLN
(SEQ ID NO: 59)
(ii) HVR-L2 comprising sequence B1-B7, wherein B1-B7 is LVSKLDS (SEQ ID NO:
60)
(iii) HVR-L3 comprising sequence Cl-C9, wherein C1-C9 is WQGTHFPYT (SEQ ID NO:
61)
(iv) HVR-H1 comprising sequence D1-D10, wherein D1-D10 is GYTFTSYWMN (SEQ ID
NO: 62)
(v) HVR-H2 comprising sequence E1-E18, wherein E1-E18 is GMIDPSDSETHYNHIFKD
(SEQ ID NO: 63) and
(vi) HVR-H3 comprising sequence F1-F6, wherein F1-F10 IS ARNLYL (SEQ ID NO:
64).
In one embodiment, HVR-L1 of an antibody of the invention comprises the
sequence of SEQ ID NO: 59. In
one embodiment, HVR-L2 of an antibody of the invention comprises the sequence
of SEQ ID NO: 60. In one
embodiment, HVR-L3 of an antibody of the invention comprises the sequence of
SEQ ID NO: 61. In one
embodiment, HVR-H1 of an antibody of the invention comprises the sequence of
SEQ ID NO: 62. In one
embodiment, HVR-H2 of an antibody of the invention comprises the sequence of
SEQ ID NO: 63. In one
embodiment, HVR-H3 of an antibody of the invention comprises the sequence of
SEQ ID NO: 64. In one
embodiment, an antibody of the invention comprising these sequences (in
combination as described herein) is
humanized or human.
[0258] In one aspect, an antibody that binds to CD79b is provided, wherein the
antibody comprises:
(a) at least one, two, three, four, five or six HVRs selected from the group
consisting of:
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(i) HVR-L1 comprising sequence A1-A15, wherein A1-A16 is KSSQSLLDSDGKTYLN
(SEQ ID NO: 59)
(ii) HVR-L2 comprising sequence B1-B7, wherein B1-B7 is LVSKLDS (SEQ ID NO:
60)
(iii) HVR-L3 comprising sequence C1-C9, wherein C1-C9 is WQGTHFPYT (SEQ ID NO:
61)
(iv) HVR-H1 comprising sequence D1-D10, wherein D1-D10 is GYTFTSYWMN (SEQ ID
NO: 62)
(v) HVR-H2 comprising sequence E1-E18, wherein E1-E18 is GMIDPSDSETHYNHIFKD
(SEQ ID NO: 63) and
(vi) HVR-H3 comprising sequence F1-F6, wherein F1-F10 IS ARNLYL (SEQ ID NO:
64);
and
(b) at least one variant HVR wherein the variant HVR sequence comprises
modification of at least
one residue of the sequence depicted in SEQ ID NOs: 59, 60, 61, 62, 63 or 64.
In one embodiment, HVR-L1
of an antibody of the invention comprises the sequence of SEQ ID NO: 59. In
one embodiment, HVR-L2 of
an antibody of the invention comprises the sequence of SEQ ID NO: 60. In one
embodiment, HVR-L3 of an
antibody of the invention comprises the sequence of SEQ ID NO: 61. In one
embodiment, HVR-H1 of an
antibody of the invention comprises the sequence of SEQ ID NO: 62. In one
embodiment, HVR-H2 of an
antibody of the invention comprises the sequence of SEQ ID NO: 63. In one
embodiment, HVR-H3 of an
antibody of the invention comprises the sequence of SEQ ID NO: 64. In one
embodiment, an antibody of the
invention comprising these sequences (in combination as described herein) is
humanized or human.
[0259] In one aspect, the invention provides an antibody comprising one, two,
three, four, five or
six HVRs, wherein each HVR comprises, consists or consists essentially of a
sequence selected from the
group consisting of SEQ ID NOs: 59, 60, 61, 62, 63, and 64, and wherein SEQ ID
NO: 59 corresponds to an
HVR-L1, SEQ ID NO: 60 corresponds to HVR-L2, SEQ ID NO: 61 corresponds to an
HVR-L3, SEQ ID NO:
62 corresponds to an HVR-H1, SEQ ID NO: 63 corresponds to an HVR-H2, and SEQ
ID NO: 64 corresponds
to an HVR-H3. In one embodiment, an antibody of the invention comprises HVR-
L1, HVR-L2, HVR-L3,
HVR-H1, HVR-H2, and HVR-H3, wherein each, in order, comprises SEQ ID NO: 59,
60, 61, 62, 63 and 64.
[0260] Variant HVRs in an antibody of an invention can have modifications of
one or more
residues within the HVR. In one embodiment, a HVR-L3 variant comprises 1-2 (1
or 2) substitutions in any
combination of the following positions: Cl (F) and C8 (F). Letter(s) in
parenthesis following each position
indicates an illustrative substitution (i.e., replacement) amino acid; as
would be evident to one skilled in the art,
suitability of other amino acids as substitution amino acids in the context
described herein can be routinely
assessed using techniques known in the art and/or described herein. In one
embodiment, C1 in a variant HVR-
L3 is F. In one embodiment, C8 in a variant HVR-L3 is F. In one embodiment an
antibody of the invention
comprises a variant HVR-L3 wherein C1 is F and C8 is F.
[0261] In one embodiment, an antibody of the invention comprises a variant HVR-
L3 wherein Cl is
F. In one embodiment, an antibody of the invention comprises a variant HVR-L3
wherein C8 is F. In some
embodiments, said variant HVR-L3 antibody further comprises HVR-L1, HVR-L2,
HVR-H1, HVR-H2 and
HVR-H3 wherein each comprises, in order, the sequence depicted in SEQ ID NOs:
59, 60, 62, 63 and 64. In
some embodiments, these antibodies further comprise a human subgroup III heavy
chain framework
consensus sequence. In one embodiment of these antibodies, the framework
consensus sequence comprises
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substitution at position 71, 73 and/or 78. In some embodiments of these
antibodies, position 71 is A, 73 is T
and/or 78 is A. In one embodiments of these antibodies, these antibodies
further comprise a human KI light
chain framework consensus sequence.
[0262] In one aspect, the invention provides an antibody comprising the HVR
sequence depicted in
Figure 9 (SEQ ID NO: 18).
[0263] A therapeutic agent for use in a host subject preferably elicits little
to no immunogenic
response against the agent in said subject. In one embodiment, the invention
provides such an agent. For
example, in one embodiment, the invention provides a humanized antibody that
elicits and/or is expected to
elicit a human anti-mouse antibody response (HAMA) at a substantially reduced
level compared to an
antibody comprising the sequence of SEQ ID NO: 10 and 14 in a host subject. In
another example, the
invention provides a humanized antibody that elicits and/or is expected to
elicit minimal or no human anti-
mouse antibody response (HAMA). In one example, an antibody of the invention
elicits anti-mouse antibody
response that is at or less than a clinically-acceptable level.
[0264] A humanized antibody of the invention may comprise one or more human
and/or human
consensus non-hypervariable region (e.g., framework) sequences in its heavy
and/or light chain variable
domain. In some embodiments, one or more additional modifications are present
within the human and/or
human consensus non-hypervariable region sequences. In one embodiment, the
heavy chain variable domain
of an antibody of the invention comprises a human consensus framework
sequence, which in one embodiment
is the subgroup III consensus framework sequence. In one embodiment, an
antibody of the invention
comprises a variant subgroup III consensus framework sequence modified at
least one amino acid position.
For example, in one embodiment, a variant subgroup III consensus framework
sequence may comprise a
substitution at one or more of positions 71, 73 and/or 78. In one embodiment,
said substitution is R71A, N73T
and/or L78A, in any combination thereof.
[0265] As is known in the art, and as described in greater detail herein
below, the amino acid
position/boundary delineating a hypervariable region of an antibody can vary,
depending on the context and
the various definitions known in the art (as described below). Some positions
within a variable domain may
be viewed as hybrid hypervariable positions in that these positions can be
deemed to be within a hypervariable
region under one set of criteria while being deemed to be outside a
hypervariable region under a different set
of criteria. One or more of these positions can also be found in extended
hypervariable regions (as further
defined below). The invention provides antibodies comprising modifications in
these hybrid hypervariable
positions. In one embodiment, these hypervariable positions include one or
more positions 26-30, 33-35B, 47-
49, 57-65, 93, 94 and 101-102 in a heavy chain variable domain. In one
embodiment, these hybrid
hypervariable positions include one or more of positions 24-29, 35-36, 46-49,
56 and 97 in a light chain
variable domain. In one embodiment, an antibody of the invention comprises a
human variant human
subgroup consensus framework sequence modified at one or more hybrid
hypervariable positions.
[0266] In one aspect, an antibody of the invention comprises a heavy chain
variable domain
comprising a variant human subgroup III consensus framework sequence modified
at one or more of positions
26-30, 33-35, 48-49, 58, 60-63, 93 and 101.
[0267] In one aspect, an antibody of the invention comprises a light chain
variable domain
comprising a variant human kappa subgroup I consensus framework sequenced
modified at one or more of
positions 24, 27-29, 56 and 97.
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[0268] In one aspect, an antibody of the invention comprises a heavy chain
variable domain
comprising a variant human subgroup III consensus framework sequence modified
at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16 or all of positions 26-30, 33-35, 48-49, 58, 60-63, 93
and 101. In some embodiments of
the invention, an antibody of the invention comprises a variant subgroup III
consensus framework sequence
modified at position 71, 73 and/or 78. In one embodiment, said substitution is
R71A, N73T and/or L78A.
[0269] In one aspect, an antibody of the invention comprises a light chain
variable domain
comprising a variant human kappa subgroup I consensus framework sequence
modified at 1, 2, 3, 4, 5 or all of
positions 24, 27-29, 56 and 97.
[0270] An antibody of the invention can comprise any suitable human or human
consensus light
chain framework sequences, provided the antibody exhibits the desired
biological characteristics (e.g., a
desired binding affinity). In one embodiment, an antibody of the invention
comprises at least a portion (or all)
of the framework sequence of human x light chain. In one embodiment, an
antibody of the invention
comprises at least a portion (or all) of human x subgroup I framework
consensus sequence.
[0271] In one aspect, an antibody of the invention comprises a heavy and/or
light chain variable
domain comprising framework sequence depicted in SEQ ID NO: 9 (Figure 7)
and/or 13 (Figures 8A-B).
[0272] In one aspect, an antibody of the invention is a humanized anti-CD79b
antibody conjugated
to a cytotoxic agent. In one aspect, the humanized anti-CD79b antibody
conjugated to a cytotoxic agent
inhibits tumor progression in xenografts.
[0273] In one aspect, both the humanized antibody and chimeric antibody are
monovalent. In one
embodiment, both the humanized and chimeric antibody comprise a single Fab
region linked to an Fc region.
In one embodiment, the reference chimeric antibody comprises variable domain
sequences depicted in Figure
7 (SEQ ID NO: 10) and Figures 8A-B (SEQ ID NO: 14) linked to a human Fc
region. In one embodiment, the
human Fc region is that of an IgG (e.g., IgGl, 2, 3 or 4).
[0274] In one aspect, the invention provides an antibody comprising a heavy
chain variable domain
comprising the HVR1-HC, HVR2-HC and/or HVR3-HC sequence depicted in Figure 13
(SEQ ID NO: 31-33).
In one embodiment, the variable domain comprises FR1-HC, FR2-HC, FR3-HC and/or
FR4-HC sequence
depicted in Figure 13 (SEQ ID NO: 27-30). In one embodiment, the antibody
comprises CH1 and/or Fc
sequence depicted in Figure 13 (SEQ ID NO: 34-35). In one embodiment, an
antibody of the invention
comprises a heavy chain variable domain comprising the HVR1-HC, HVR2-HC and/or
HVR3 -HC sequence
(Figure 13, SEQ ID NO: 31-33), and the FR1-HC, FR2-HC, FR3-HC and/or FR4-HC
sequence (Figure 13,
SEQ ID NO: 27-30). In one embodiment, an antibody of the invention comprises a
heavy chain variable
domain comprising the HVR1-HC, HVR2-HC and/or HVR3-HC sequence (Figure 13, SEQ
ID NO: 31-33),
and the CH1 and/or Fc sequence depicted in Figure 13 (SEQ ID NO: 34-35) In one
embodiment, an antibody
of the invention comprises a heavy chain variable domain comprising the HVR1-
HC, HVR2-HC and/or
HVR3-HC sequence (Figure 13, SEQ ID NO: 31-33), and the FR1-HC, FR2-HC, FR3-HC
and/or FR4-HC
sequence (Figure 13, SEQ ID NO:27-30), and the CH1 and/or Fc (Figure 13, SEQ
ID NO: 34-35).
[0275] In one aspect, the invention provides an antibody comprising a light
chain variable domain
comprising HVR1-LC, HVR2-LC and/or HVR3-LC sequence depicted in Figure 13 (SEQ
ID NO: 23-25). In
one embodiment, the variable domain comprises FR1-LC, FR2-LC, FR3-LC and/or
FR4-LC sequence
depicted in Figure 13 (SEQ ID NO: 19-22). In one embodiment, the antibody
comprises CL1 sequence
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depicted in Figure 13 (SEQ ID NO: 26). In one embodiment, the antibody of the
invention comprises a light
chain variable domain comprising the HVR1-LC, HVR2-LC and/or HVR3-LC sequence
(SEQ ID NO: 23-25),
and the FR1-LC, FR2-LC, FR3-LC and/or FR4-LC sequence (SEQ ID NO: 19-22)
depicted in Figure 13. In
one embodiment, an antibody of the invention comprises a light chain variable
domain comprising the HVR1-
LC, HVR2-LC and/or HVR3-LC sequence (SEQ ID NO: 23-25), and the CL1 sequence
(SEQ ID NO: 26)
depicted in Figure 13. In one embodiment, an antibody of the invention
comprises a light chain variable
domain comprising the HVR1-LC, HVR2-LC and/or HVR3-LC sequence (SEQ ID NO: 23-
26), and the FR1-
LC, FR2-LC, FR3-LC and/or FR4-LC (SEQ ID NO: 19-22) sequence depicted in
Figure 13, and the CL1
sequence depicted in Figure 13 (SEQ ID NO: 26).
[0276] In one aspect, the antibodies of the invention include cysteine
engineered antibodies where
one or more amino acids of a parent antibody are replaced with a free cysteine
amino acid as disclosed in
W02006/034488; US 2007/0092940 (herein incorporated by reference in its
entirety). Any form of anti-
CD79b antibody may be so engineered, i.e. mutated. For example, a parent Fab
antibody fragment may be
engineered to form a cysteine engineered Fab, referred to herein as "ThioFab."
Similarly, a parent monoclonal
antibody may be engineered to form a "ThioMab." It should be noted that a
single site mutation yields a
single engineered cysteine residue in a ThioFab, while a single site mutation
yields two engineered cysteine
residues in a ThioMab, due to the dimeric nature of the IgG antibody. The
cysteine engineered anti-CD79b
antibodies of the invention include monoclonal antibodies, humanized or
chimeric monoclonal antibodies, and
antigen-binding fragments of antibodies, fusion polypeptides and analogs that
preferentially bind cell-
associated CD79b polypeptides. A cysteine engineered antibody may
alternatively comprise an antibody
comprising a cysteine at a position disclosed herein in the antibody or Fab,
resulting from the sequence design
and/or selection of the antibody, without necessarily altering a parent
antibody, such as by phage display
antibody design and selection or through de novo design of light chain and/or
heavy chain framework
sequences and constant regions. A cysteine engineered antibody comprises one
or more free cysteine amino
acids having a thiol reactivity value in the ranges of 0.6 to 1.0; 0.7 to 1.0
or 0.8 to 1Ø A free cysteine amino
acid is a cysteine residue which has been engineered into the parent antibody
and is not part of a disulfide
bridge. Cysteine engineered antibodies are useful for attachment of cytotoxic
and/or imaging compounds at
the site of the engineered cysteine through, for example, a maleimide or
haloacetyl. The nucleophilic
reactivity of the thiol functionality of a Cys residue to a maleimide group is
about 1000 times higher compared
to any other amino acid functionality in a protein, such as amino group of
lysine residues or the N-terminal
amino group. Thiol specific functionality in iodoacetyl and maleimide reagents
may react with amine groups,
but higher pH (>9.0) and longer reaction times are required (Garman, 1997, Non-
Radioactive Labelling: A
Practical Approach, Academic Press, London).
[0277] In an aspect, a cysteine engineered anti-CD79b antibody of the
invention comprises an
engineered cysteine at any one of the following positions, where the position
is numbered according to Kabat
et al. in the light chain (see Kabat et al (1991) Sequences of Proteins of
Immunological Interest, 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, MD) and according to
EU numbering in the heavy
chain (including the Fc region) (see Kabat et al. (1991), supra) , wherein the
light chain constant region
depicted by underlining in Figure 17A and 18A begins at position 109 (Kabat
numbering) and the heavy chain
constant region depicted by underling in Figures 17B and 18B begins at
position 118 (EU numbering). The
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position may also be referred to by its position in sequential numbering of
the amino acids of the full length
light chain or heavy chain shown in Figures 17-18. According to one embodiment
of the invention, an anti-
CD79b antibody comprises an engineered cysteine at LC-V205C (Kabat number:
Va1205; sequential number
210 in Figure 18A engineered to be Cys at that position). The engineered
cysteine in the light chain is shown
in bold, double underlined text in Figure 18A. According to one embodiment, an
anti-CD79b antibody
comprises an engineered cysteine at HC-A118C (EU number: Ala 118; Kabat number
114; sequential number
118 in Figure 17B engineered to be Cys at that position). The engineered
cysteine in the heavy chain is shown
in bold, double underlined text in Figure 17B. According to one embodiment, an
anti-CD79b antibody
comprises an engineered cysteine at Fc-S400C (EU number: Ser 400; Kabat number
396; sequential number
400 in Figure 17-18B engineered to be Cys at that position). In other
embodiments, the engineered cysteine of
the heavy chain (including the Fc region) is at any one of the following
positions (according to Kabat
numbering with EU numbering in parenthesis): 5, 23, 84, 112, 114 (118 EU
numbering), 116 (120 EU
numbering), 278 (282 EU numbering), 371 (375 EU numbering) or 396 (400 EU
numbering). Thus, changes
in the amino acid at these positions for a parent humanized anti-CD79b
antibody of the invention are: V5C,
A23C, A84C, S112C, A114C (A118C EU Numbering), T116C (T120C EU numbering),
V278C (V282C EU
numbering), S371C (S375C EU numbering) or S396C (S400C EU numbering). Thus,
changes in the amino
acid at these positions for a parent chimeric anti-CD79b antibody of the
invention are: Q5C, K23C, S84C,
S112C, A114C (A118C EU Numbering), T116C (T120C EU numbering), V278C (V282C EU
numbering),
S371C (S375C EU numbering) or S396C (S400C EU numbering). In other
embodiments, the engineered
cysteine of the light chain is at any one of the following positions
(according to Kabat numbering): 15, 110,
114, 121, 127, 168, 205. Thus, changes in the amino acid at these positions
for a parent humanized anti-
CD79b antibody of the invention are: V15C, V110C, S114C, S121C, S127C, S168C,
or V205C. Thus,
changes in the amino acid at these positions for a parent chimeric anti-CD79b
antibody of the invention are:
I15C, V110C, S114C, S121C, S127C, S168C, or V205C.
[0278] In one aspect, the invention includes a cysteine engineered anti-CD79b
antibody comprises
one or more free cysteine amino acids wherein the cysteine engineered anti-
CD79b antibody binds to a
CD79b polypeptide and is prepared by a process comprising replacing one or
more amino acid residues of a
parent anti-CD79b antibody by cysteine wherein the parent antibody comprises
at least one HVR sequence
selected from:
(a) HVR-L1 comprising sequence A1-A15, wherein A1-A16 is KSSQSLLDSDGKTYLN (SEQ
ID
NO: 59)
(b) HVR-L2 comprising sequence B1-B7, wherein B1-B7 is LVSKLDS (SEQ ID NO: 60)
(c) HVR-L3 comprising sequence C1-C9, wherein C1-C9 is WQGTHFPYT (SEQ ID NO:
61) or
FQGTHFPFT (SEQ ID NO: 79)
(d) HVR-H1 comprising sequence D1-D10, wherein D1-D10 is GYTFTSYWMN (SEQ ID
NO: 62)
(e) HVR-H2 comprising sequence E1-E18, wherein E1-E18 is
GMIDPSDSETHYNHIFKD(SEQ ID
NO: 63) and
(f) HVR-H3 comprising sequence F1-F6, wherein F1-F10 is ARNLYL (SEQ ID NO:
64).
[0279] In a certain aspect, the invention concerns a cysteine engineered anti-
CD79b antibody,
comprising an amino acid sequence having at least about 80% amino acid
sequence identity, alternatively at
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least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%,
98%, 99% or 100% amino acid sequence identity, to a cysteine engineered
antibody having a full-length
amino acid sequence as disclosed herein, or a cysteine engineered antibody
amino acid sequence lacking the
signal peptide as disclosed herein.
[0280] In a yet further aspect, the invention concerns an isolated cysteine
engineered anti-CD79b
antibody comprising an amino acid sequence that is encoded by a nucleotide
sequence that hybridizes to the
complement of a DNA molecule encoding (a) a cysteine engineered antibody
having a full-length amino acid
sequence as disclosed herein, (b) a cysteine engineered antibody amino acid
sequence lacking the signal
peptide as disclosed herein, (c) an extracellular domain of a transmembrane
cysteine engineered antibody
protein, with or without the signal peptide, as disclosed herein, (d) an amino
acid sequence encoded by any of
the nucleic acid sequences disclosed herein or (e) any other specifically
defined fragment of a full-length
cysteine engineered antibody amino acid sequence as disclosed herein.
[0281] In a specific aspect, the invention provides an isolated cysteine
engineered anti-CD79b
antibody without the N-terminal signal sequence and/or without the initiating
methionine and is encoded by a
nucleotide sequence that encodes such an amino acid sequence as described in.
Processes for producing the
same are also herein described, wherein those processes comprise culturing a
host cell comprising a vector
which comprises the appropriate encoding nucleic acid molecule under
conditions suitable for expression of
the cysteine engineered antibody and recovering the cysteine engineered
antibody from the cell culture.
[0282] Another aspect of the invention provides an isolated cysteine
engineered anti-CD79b
antibody which is either transmembrane domain-deleted or transmembrane domain-
inactivated. Processes for
producing the same are also herein described, wherein those processes comprise
culturing a host cell
comprising a vector which comprises the appropriate encoding nucleic acid
molecule under conditions suitable
for expression of the cysteine engineered antibody and recovering the cysteine
engineered antibody from the
cell culture.
[0283] In other aspects, the invention provides isolated anti-CD79b chimeric
cysteine engineered
antibodies comprising any of the herein described cysteine engineered antibody
fused to a heterologous (non-
CD79b) polypeptide. Examples of such chimeric molecules comprise any of the
herein described cysteine
engineered antibodies fused to a heterologous polypeptide such as, for
example, an epitope tag sequence or a
Fc region of an immunoglobulin.
[0284] The cysteine engineered anti-CD79b antibody may be a monoclonal
antibody, antibody
fragment, chimeric antibody, humanized antibody, single-chain antibody or
antibody that competitively
inhibits the binding of an anti-CD79b polypeptide antibody to its respective
antigenic epitope. Antibodies of
the present invention may optionally be conjugated to a growth inhibitory
agent or cytotoxic agent such as a
toxin, including, for example, an auristatin, maytansinoid, a dolostatin
derivative or a calicheamicin, an
antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. The
antibodies of the present invention
may optionally be produced in CHO cells or bacterial cells and preferably
inhibit the growth or proliferation of
or induce the death of a cell to which they bind. For diagnostic purposes, the
antibodies of the present
invention may be detectably labeled, attached to a solid support, or the like.
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[0285] In other aspects of the present invention, the invention provides
vectors comprising DNA
encoding any of the herein described anti-CD79b antibodies and anti-CD79b
cysteine engineered antibodies.
Host cells comprising any such vector are also provided. By way of example,
the host cells may be CHO cells,
E. coli cells, or yeast cells. A process for producing any of the herein
described polypeptides is further
provided and comprises culturing host cells under conditions suitable for
expression of the desired polypeptide
and recovering the desired polypeptide from the cell culture.
[0286] Cysteine engineered antibodies may be useful in the treatment of cancer
and include
antibodies specific for cell surface and transmembrane receptors, and tumor-
associated antigens (TAA). Such
antibodies may be used as naked antibodies (unconjugated to a drug or label
moiety) or as antibody-drug
conjugates (ADC). Cysteine engineered antibodies of the invention may be site-
specifically and efficiently
coupled with a thiol-reactive reagent. The thiol-reactive reagent may be a
multifunctional linker reagent, a
capture label reagent, a fluorophore reagent, or a drug-linker intermediate.
The cysteine engineered antibody
may be labeled with a detectable label, immobilized on a solid phase support
and/or conjugated with a drug
moiety. Thiol reactivity may be generalized to any antibody where substitution
of amino acids with reactive
cysteine amino acids may be made within the ranges in the light chain selected
from amino acid ranges: L10-
L20, L105-L115, L109-L119, L116-L126, L122-L132, L163-L173, L200-L210; and
within the ranges in the
heavy chain selected from amino acid ranges: H1-H10, H18-H28, H79-H89, H107-
H117, H109-H119, H111-
H121, and in the Fc region within the ranges selected from H270-H280, H366-
H376, H391-401, where the
numbering of amino acid positions begins at position 1 of the Kabat numbering
system (Kabat et al. (1991)
Sequences of Proteins of Immunological Interest, 5th Ed. Public Health
Service, National Institutes of Health,
Bethesda, MD) and continues sequentially thereafter as disclosed in
W02006034488; US 2007/0092940.
Thiol reactivity may also be generalized to certain domains of an antibody,
such as the light chain constant
domain (CL) and heavy chain constant domains, CH1, CH2 and CH3. Cysteine
replacements resulting in thiol
reactivity values of 0.6 and higher may be made in the heavy chain constant
domains a, b, s, y, and of intact
antibodies: IgA, IgD, IgE, IgG, and IgM, respectively, including the IgG
subclasses: IgG1, IgG2, IgG3, IgG4,
IgA, and IgA2. Such antibodies and their uses are disclosed in W02006/034488;
US 2007/0092940.
[0287] Cysteine engineered antibodies of the invention preferably retain the
antigen binding
capability of their wild type, parent antibody counterparts. Thus, cysteine
engineered antibodies are capable
of binding, preferably specifically, to antigens. Such antigens include, for
example, tumor-associated antigens
(TAA), cell surface receptor proteins and other cell surface molecules,
transmembrane proteins, signalling
proteins, cell survival regulatory factors, cell proliferation regulatory
factors, molecules associated with (for
e.g., known or suspected to contribute functionally to) tissue development or
differentiation, lymphokines,
cytokines, molecules involved in cell cycle regulation, molecules involved in
vasculogenesis and molecules
associated with (for e.g., known or suspected to contribute functionally to)
angiogenesis. The tumor-
associated antigen may be a cluster differentiation factor (i.e., a CD
protein, including but not limited to
CD79b). Cysteine engineered anti-CD79b antibodies of the invention retain the
antigen binding ability of
their parent anti-CD79b antibody counterparts. Thus, cysteine engineered anti-
CD79b antibodies of the
invention are capable of binding, preferably specifically, to CD79b antigens
including human anti-CD79b
isoforms beta and/or alpha, including when such antigens are expressed on the
surface of cells, including,
without limitation, B cells.
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[0288] In one aspect, antibodies of the invention may be conjugated with any
label moiety which
can be covalently attached to the antibody through a reactive moiety, an
activated moiety, or a reactive
cysteine thiol group (Singh et al (2002) Anal. Biochem. 304:147-15; Harlow E.
and Lane, D. (1999) Using
Antibodies: A Laboratory Manual, Cold Springs Harbor Laboratory Press, Cold
Spring Harbor, NY; Lundblad
R.L. (1991) Chemical Reagents for Protein Modification, 2nd ed. CRC Press,
Boca Raton, FL). The attached
label may function to: (i) provide a detectable signal; (ii) interact with a
second label to modify the detectable
signal provided by the first or second label, e.g. to give FRET (fluorescence
resonance energy transfer); (iii)
stabilize interactions or increase affinity of binding, with antigen or
ligand; (iv) affect mobility, e.g.
electrophoretic mobility or cell-permeability, by charge, hydrophobicity,
shape, or other physical parameters,
or (v) provide a capture moiety, to modulate ligand affinity, antibody/antigen
binding, or ionic complexation.
[0289] Labelled cysteine engineered antibodies may be useful in diagnostic
assays, e.g., for
detecting expression of an antigen of interest in specific cells, tissues, or
serum. For diagnostic applications,
the antibody will typically be labeled with a detectable moiety. Numerous
labels are available which can be
generally grouped into the following categories:
[0290] Radioisotopes (radionuclides), such as 3H, "C, 14C, 18 F, 32 P, 35S,
64cU, 68Ga, 86Y, 99'Tc, " 'In
,
123I 124I 125I 131I 133Xe, 177 Lu, 2llAt, or 213Bi. Radioisotope labelled
antibodies are useful in receptor targeted
imaging experiments. The antibody can be labeled with ligand reagents that
bind, chelate or otherwise
complex a radioisotope metal where the reagent is reactive with the engineered
cysteine thiol of the antibody,
using the techniques described in Current Protocols in Immunology, Volumes 1
and 2, Coligen et al, Ed.
Wiley-Interscience, New York, NY, Pubs. (1991). Chelating ligands which may
complex a metal ion include
DOTA, DOTP, DOTMA, DTPA and TETA (Macrocyclics, Dallas, TX). Radionuclides can
be targeted via
complexation with the antibody-drug conjugates of the invention (Wu et al
(2005) Nature Biotechnology
23 (9):1137-1146).
[0291] Linker reagents such as DOTA-maleimide (4-maleimidobutyramidobenzyl-
DOTA) can be
prepared by the reaction of aminobenzyl-DOTA with 4-maleimidobutyric acid
(Fluka) activated with
isopropylchloroformate (Aldrich), following the procedure of Axworthy et al
(2000) Proc. Natl. Acad. Sci.
USA 97(4):1802-1807). DOTA-maleimide reagents react with the free cysteine
amino acids of the cysteine
engineered antibodies and provide a metal complexing ligand on the antibody
(Lewis et al (1998) Bioconj.
Chem. 9:72-86). Chelating linker labelling reagents such as DOTA-NHS (1,4,7,10-
tetraazacyclododecane-
1,4,7,10-tetraacetic acid mono (N-hydroxysuccinimide ester) are commercially
available (Macrocyclics,
Dallas, TX). Receptor target imaging with radionuclide labelled antibodies can
provide a marker of pathway
activation by detection and quantitation of progressive accumulation of
antibodies in tumor tissue (Albert et al
(1998) Bioorg. Med. Chem. Lett. 8:1207-1210). The conjugated radio-metals may
remain intracellular
following lysosomal degradation.
[0292] Metal-chelate complexes suitable as antibody labels for imaging
experiments are disclosed:
US 5342606; US 5428155; US 5316757; US 5480990; US 5462725; US 5428139; US
5385893; US 5739294;
US 5750660; US 5834456; Hnatowich et al (1983) J. Immunol. Methods 65:147-157;
Meares et al (1984)
Anal. Biochem. 142:68-78; Mirzadeh et al (1990) Bioconjugate Chem. 1:59-65;
Meares et al (1990) J.
Cancer1990, Suppl. 10:21-26; Izard et al (1992) Bioconjugate Chem. 3:346-350;
Nikula et al (1995) Nucl.
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Med. Biol. 22:387-90; Camera et al (1993) Nucl. Med. Biol. 20:955-62; Kukis et
al (1998) J. Nucl. Med.
39:2105-2110; Verel et al (2003) J. Nucl. Med. 44:1663-1670; Camera et al
(1994) J. Nucl. Med. 21:640-646;
Ruegg et al (1990) Cancer Res. 50:4221-4226; Verel et al (2003) J. Nucl. Med.
44:1663-1670; Lee et al
(2001) Cancer Res. 61:4474-4482; Mitchell, et al (2003) J. Nucl. Med. 44:1105-
1112; Kobayashi et al (1999)
Bioconjugate Chem. 10:103-111; Miederer et al (2004) J. Nucl. Med. 45:129-137;
DeNardo et al (1998)
Clinical Cancer Research 4:2483-90; Blend et al (2003) Cancer Biotherapy &
Radiopharmaceuticals 18:355-
363; Nikula et al (1999) J. Nucl. Med. 40:166-76; Kobayashi et al (1998) J.
Nucl. Med. 39:829-36;
Mardirossian et al (1993) Nucl. Med. Biol. 20:65-74; Roselli et al (1999)
Cancer Biotherapy &
Radiopharmaceuticals, 14:209-20.
[0293] Fluorescent labels such as rare earth chelates (europium chelates),
fluorescein types
including FITC, 5-carboxyfluorescein, 6-carboxy fluorescein; rhodamine types
including TAMRA; dansyl;
Lissamine; cyanines; phycoerythrins; Texas Red; and analogs thereof. The
fluorescent labels can be
conjugated to antibodies using the techniques disclosed in Current Protocols
in Immunology, supra, for
example. Fluorescent dyes and fluorescent label reagents include those which
are commercially available
from Invitrogen/Molecular Probes (Eugene, OR) and Pierce Biotechnology, Inc.
(Rockford, IL).
[0294] Various enzyme-substrate labels are available or disclosed (US
4275149). The enzyme
generally catalyzes a chemical alteration of a chromogenic substrate that can
be measured using various
techniques. For example, the enzyme may catalyze a color change in a
substrate, which can be measured
spectrophotometrically. Alternatively, the enzyme may alter the fluorescence
or chemiluminescence of the
substrate. Techniques for quantifying a change in fluorescence are described
above. The chemiluminescent
substrate becomes electronically excited by a chemical reaction and may then
emit light which can be
measured (using a chemiluminometer, for example) or donates energy to a
fluorescent acceptor. Examples of
enzymatic labels include luciferases (e.g., firefly luciferase and bacterial
luciferase; US 4737456), luciferin,
2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as
horseradish peroxidase
(HRP), alkaline phosphatase (AP), 0-galactosidase, glucoamylase, lysozyme,
saccharide oxidases (e.g.,
glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase),
heterocyclic oxidases (such as
uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.
Techniques for conjugating
enzymes to antibodies are described in O'Sullivan et al (1981) "Methods for
the Preparation of Enzyme-
Antibody Conjugates for use in Enzyme Immunoassay", in Methods in Enzym. (ed
J. Langone & H. Van
Vunakis), Academic Press, New York, 73:147-166.
[0295] Examples of enzyme-substrate combinations include, for example:
(i) Horseradish peroxidase (HRP) with hydrogen peroxidase as a substrate,
wherein the
hydrogen peroxidase oxidizes a dye precursor (e.g., orthophenylene diamine
(OPD) or 3,3',5,5'-
tetramethylbenzidine hydrochloride (TMB));
(ii) alkaline phosphatase (AP) with para-nitrophenyl phosphate as chromogenic
substrate; and
(iii) (3-D-galactosidase (0-D-Gal) with a chromogenic substrate (e.g., p-
nitrophenyl-(3-D-
galactosidase) or fluorogenic substrate 4-methylumbelliferyl-(3-D-
galactosidase.
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[0296] Numerous other enzyme-substrate combinations are available to those
skilled in the art. For
a general review, see US 4275149 and US 4318980.
[0297] A label may be indirectly conjugated with an amino acid side chain, an
activated amino acid
side chain, a cysteine engineered antibody, and the like. For example, the
antibody can be conjugated with
biotin and any of the three broad categories of labels mentioned above can be
conjugated with avidin or
streptavidin, or vice versa. Biotin binds selectively to streptavidin and
thus, the label can be conjugated with
the antibody in this indirect manner. Alternatively, to achieve indirect
conjugation of the label with the
polypeptide variant, the polypeptide variant is conjugated with a small hapten
(e.g., digoxin) and one of the
different types of labels mentioned above is conjugated with an anti-hapten
polypeptide variant (e.g., anti-
digoxin antibody). Thus, indirect conjugation of the label with the
polypeptide variant can be achieved
(Hermanson, G. (1996) in Bioconjugate Techniques Academic Press, San Diego).
[0298] The antibody of the present invention may be employed in any known
assay method, such as
ELISA, competitive binding assays, direct and indirect sandwich assays, and
immunoprecipitation assays
(Zola, (1987) Monoclonal Antibodies: A Manual of Techniques, pp.147-158, CRC
Press, Inc.).
[0299] A detection label may be useful for localizing, visualizing, and
quantitating a binding or
recognition event. The labelled antibodies of the invention can detect cell-
surface receptors. Another use for
detectably labelled antibodies is a method of bead-based immunocapture
comprising conjugating a bead with a
fluorescent labelled antibody and detecting a fluorescence signal upon binding
of a ligand. Similar binding
detection methodologies utilize the surface plasmon resonance (SPR) effect to
measure and detect antibody-
antigen interactions.
[0300] Detection labels such as fluorescent dyes and chemiluminescent dyes
(Briggs et al (1997)
"Synthesis of Functionalised Fluorescent Dyes and Their Coupling to Amines and
Amino Acids," J. Chem.
Soc., Perkin-Trans. 1:1051-1058) provide a detectable signal and are generally
applicable for labelling
antibodies, preferably with the following properties: (i) the labelled
antibody should produce a very high
signal with low background so that small quantities of antibodies can be
sensitively detected in both cell-free
and cell-based assays; and (ii) the labelled antibody should be photostable so
that the fluorescent signal may
be observed, monitored and recorded without significant photo bleaching. For
applications involving cell
surface binding of labelled antibody to membranes or cell surfaces, especially
live cells, the labels preferably
(iii) have good water-solubility to achieve effective conjugate concentration
and detection sensitivity and (iv)
are non-toxic to living cells so as not to disrupt the normal metabolic
processes of the cells or cause premature
cell death.
[0301] Direct quantification of cellular fluorescence intensity and
enumeration of fluorescently
labelled events, e.g. cell surface binding of peptide-dye conjugates may be
conducted on an system (FMAT
8100 HTS System, Applied Biosystems, Foster City, Calif.) that automates mix-
and-read, non-radioactive
assays with live cells or beads (Miraglia, "Homogeneous cell- and bead-based
assays for high throughput
screening using fluorometric microvolume assay technology", (1999) J. of
Biomolecular Screening 4:193-204).
Uses of labelled antibodies also include cell surface receptor binding assays,
inmmunocapture assays,
fluorescence linked immunosorbent assays (FLISA), caspase-cleavage (Zheng,
"Caspase-3 controls both
cytoplasmic and nuclear events associated with Fas-mediated apoptosis in
vivo", (1998) Proc. Natl. Acad. Sci.
CA 02692819 2010-01-07
WO 2009/012256 PCT/US2008/070061
USA 95:618-23; US 6372907), apoptosis (Vermes, "A novel assay for apoptosis.
Flow cytometric detection
of phosphatidylserine expression on early apoptotic cells using fluorescein
labelled Annexin V" (1995) J.
Immunol. Methods 184:39-51) and cytotoxicity assays. Fluorometric microvolume
assay technology can be
used to identify the up or down regulation by a molecule that is targeted to
the cell surface (Swartzman, "A
homogeneous and multiplexed immunoassay for high-throughput screening using
fluorometric microvolume
assay technology", (1999) Anal. Biochem. 271:143-51).
[0302] Labelled antibodies of the invention are useful as imaging biomarkers
and probes by the
various methods and techniques of biomedical and molecular imaging such as:
(i) MRI (magnetic resonance
imaging); (ii) MicroCT (computerized tomography); (iii) SPECT (single photon
emission computed
tomography); (iv) PET (positron emission tomography) Chen et al (2004)
Bioconjugate Chem. 15:41-49; (v)
bioluminescence; (vi) fluorescence; and (vii) ultrasound. Immunoscintigraphy
is an imaging procedure in
which antibodies labeled with radioactive substances are administered to an
animal or human patient and a
picture is taken of sites in the body where the antibody localizes (US
6528624). Imaging biomarkers may be
objectively measured and evaluated as an indicator of normal biological
processes, pathogenic processes, or
pharmacological responses to a therapeutic intervention. Biomarkers may be of
several types: Type 0 are
natural history markers of a disease and correlate longitudinally with known
clinical indices, e.g. MRI
assessment of synovial inflammation in rheumatoid arthritis; Type I markers
capture the effect of an
intervention in accordance with a mechanism-of-action, even though the
mechanism may not be associated
with clinical outcome; Type II markers function as surrogate endpoints where
the change in, or signal from,
the biomarker predicts a clinical benefit to "validate" the targeted response,
such as measured bone erosion in
rheumatoid arthritis by CT. Imaging biomarkers thus can provide
pharmacodynamic (PD) therapeutic
information about: (i) expression of a target protein, (ii) binding of a
therapeutic to the target protein, i.e.
selectivity, and (iii) clearance and half-life pharmacokinetic data.
Advantages of in vivo imaging biomarkers
relative to lab-based biomarkers include: non-invasive treatment,
quantifiable, whole body assessment,
repetitive dosing and assessment, i.e. multiple time points, and potentially
transferable effects from preclinical
(small animal) to clinical (human) results. For some applications, bioimaging
supplants or minimizes the
number of animal experiments in preclinical studies.
[0303] Peptide labelling methods are well known. See Haugland, 2003, Molecular
Probes
Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.;
Brinkley, 1992,
Bioconjugate Chem. 3:2; Garman, (1997) Non-Radioactive Labelling: A Practical
Approach, Academic Press,
London; Means (1990) Bioconjugate Chem. 1:2; Glazer et al (1975) Chemical
Modification of Proteins.
Laboratory Techniques in Biochemistry and Molecular Biology (T. S. Work and E.
Work, Eds.) American
Elsevier Publishing Co., New York; Lundblad, R. L. and Noyes, C. M. (1984)
Chemical Reagents for Protein
Modification, Vols. I and II, CRC Press, New York; Pfleiderer, G. (1985)
"Chemical Modification of
Proteins", Modern Methods in Protein Chemistry, H. Tschesche, Ed., Walter
DeGryter, Berlin and New York;
and Wong (1991) Chemistry of Protein Conjugation and Cross-linking, CRC Press,
Boca Raton, Fla.); De
Leon-Rodriguez et al (2004) Chem.Eur. J. 10:1149-1155; Lewis et al (2001)
Bioconjugate Chem. 12:320-324;
Li et al (2002) Bioconjugate Chem. 13:110-115; Mier et al (2005) Bioconjugate
Chem. 16:240-237.
[0304] Peptides and proteins labelled with two moieties, a fluorescent
reporter and quencher in
sufficient proximity undergo fluorescence resonance energy transfer (FRET).
Reporter groups are typically
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fluorescent dyes that are excited by light at a certain wavelength and
transfer energy to an acceptor, or
quencher, group, with the appropriate Stokes shift for emission at maximal
brightness. Fluorescent dyes
include molecules with extended aromaticity, such as fluorescein and
rhodamine, and their derivatives. The
fluorescent reporter may be partially or significantly quenched by the
quencher moiety in an intact peptide.
Upon cleavage of the peptide by a peptidase or protease, a detectable increase
in fluorescence may be
measured (Knight, C. (1995) "Fluorimetric Assays of Proteolytic Enzymes",
Methods in Enzymology,
Academic Press, 248:18-34).
[0305] The labelled antibodies of the invention may also be used as an
affinity purification agent.
In this process, the labelled antibody is immobilized on a solid phase such a
Sephadex resin or filter paper,
using methods well known in the art. The immobilized antibody is contacted
with a sample containing the
antigen to be purified, and thereafter the support is washed with a suitable
solvent that will remove
substantially all the material in the sample except the antigen to be
purified, which is bound to the
immobilized polypeptide variant. Finally, the support is washed with another
suitable solvent, such as glycine
buffer, pH 5.0, that will release the antigen from the polypeptide variant.
[0306] Labelling reagents typically bear reactive functionality which may
react (i) directly with a
cysteine thiol of a cysteine engineered antibody to form the labelled
antibody, (ii) with a linker reagent to form
a linker-label intermediate, or (iii) with a linker antibody to form the
labelled antibody. Reactive functionality
of labelling reagents include: maleimide, haloacetyl, iodoacetamide
succinimidyl ester (e.g. NHS, N-
hydroxysuccinimide), isothiocyanate, sulfonyl chloride, 2,6-dichlorotriazinyl,
pentafluorophenyl ester, and
phosphoramidite, although other functional groups can also be used.
[0307] An exemplary reactive functional group is N-hydroxysuccinimidyl ester
(NHS) of a
carboxyl group substituent of a detectable label, e.g. biotin or a fluorescent
dye. The NHS ester of the label
may be preformed, isolated, purified, and/or characterized, or it may be
formed in situ and reacted with a
nucleophilic group of an antibody. Typically, the carboxyl form of the label
is activated by reacting with
some combination of a carbodiimide reagent, e.g. dicyclohexylcarbodiimide,
diisopropylcarbodiimide, or a
uronium reagent, e.g. TSTU (O-(N-Succinimidyl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate, HBTU
(O-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate), or
HATU (O-(7-azabenzotriazol-
1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate), an activator, such as
1-hydroxybenzotriazole
(HOBt), and N-hydroxysuccinimide to give the NHS ester of the label. In some
cases, the label and the
antibody may be coupled by in situ activation of the label and reaction with
the antibody to form the label-
antibody conjugate in one step. Other activating and coupling reagents include
TBTU (2-(1H-benzotriazo-l-
yl)-1-1,3,3-tetramethyluronium hexafluorophosphate), TFFH (N,N',N",N"'-
tetramethyluronium 2-fluoro-
hexafluorophosphate), PyBOP (benzotriazole-1-yl-oxy-tris-pyrrolidino-
phosphonium hexafluorophosphate,
EEDQ (2-ethoxy-l-ethoxycarbonyl-1,2-dihydro-quinoline), DCC
(dicyclohexylcarbodiimide); DIPCDI
(diisopropylcarbodiimide), MSNT (1 -(mesitylene-2-sulfonyl)-3 -nitro- 1 H-
1,2,4-triazole, and aryl sulfonyl
halides, e.g. triisopropylbenzenesulfonyl chloride.
Albumin binding peptide-Fab compounds of the invention:
[0308] In one aspect, the antibody of the invention is fused to an albumin
binding protein. Plasma-
protein binding can be an effective means of improving the pharmacokinetic
properties of short lived
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molecules. Albumin is the most abundant protein in plasma. Serum albumin
binding peptides (ABP) can alter
the pharmacodynamics of fused active domain proteins, including alteration of
tissue uptake, penetration, and
diffusion. These pharmacodynamic parameters can be modulated by specific
selection of the appropriate
serum albumin binding peptide sequence (US 20040001827). A series of albumin
binding peptides were
identified by phage display screening (Dennis et al. (2002) "Albumin Binding
As A General Strategy For
Improving The Pharmacokinetics Of Proteins" J Biol Chem. 277:35035-35043; WO
01/45746). Compounds
of the invention include ABP sequences taught by: (i) Dennis et al (2002) J
Biol Chem. 277:35035-35043 at
Tables III and IV, page 35038; (ii) US 20040001827 at [0076] SEQ ID NOS: 9-22;
and (iii) WO 01/45746 at
pages 12-13, all of which are incorporated herein by reference. Albumin
Binding (ABP)-Fabs are engineered
by fusing an albumin binding peptide to the C-terminus of Fab heavy chain in
1:1 stoichiometric ratio (1 ABP
/ 1 Fab). It was shown that association of these ABP-Fabs with albumin
increased antibody half life by more
than 25 fold in rabbits and mice. The above described reactive Cys residues
can therefore be introduced in
these ABP-Fabs and used for site-specific conjugation with cytotoxic drugs
followed by in vivo animal studies.
[0309] Exemplary albumin binding peptide sequences include, but are not
limited to the amino acid
sequences listed in SEQ ID NOS: 80-84:
CDKTHTGGGSQRLMEDICLPRWGCLWEDDF SEQ ID NO: 80
QRLMEDICLPRWGCLWEDDF SEQ ID NO: 81
QRLIEDICLPRWGCLWEDDF SEQ ID NO: 82
RLIEDICLPRWGCLWEDD SEQ ID NO: 83
DICLPRWGCLW SEQ ID NO: 84
Antibody-Drug Conjugates
[0310] In another aspect, the invention provides immunoconjugates, or antibody-
drug conjugates
(ADC), comprising an antibody conjugated to a cytotoxic agent such as a
chemotherapeutic agent, a drug, a
growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of
bacterial, fungal, plant, or animal origin,
or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). In
another aspect, the invention further
provides methods of using the immunoconjugates. In one aspect, an
immunoconjugate comprises any of the
above anti-CD79b antibodies covalently attached to a cytotoxic agent or a
detectable agent.
[0311] In one aspect, a CD79b antibody of the invention binds to the same
epitope on CD79b
bound by another CD79b antibody. In another embodiment, a CD79b antibody of
the invention binds to the
same epitope on CD79b bound by the Fab fragment of, a monoclonal antibody
generated from hybridomas
deposited with the ATCC as PTA-7712 on July 11, 2006, a monoclonal antibody
comprising the variable
domains of SEQ ID NO: 10 (Figure 7) and SEQ ID NO: 14 (Figures 8A-B) or a
chimeric antibody comprising
the variable domains of either antibody gernated from PTA-7712 hybridomas
deposited with the ATCC on
July 11, 2006, and constant domains from IgG1, or the variable domains of
monoclonal antibody comprising
the sequences of SEQ ID NO: 10 (Figure 7) and SEQ ID NO: 14 (Figures 8A-B). In
another embodiment, a
CD79b antibody of the invention binds to the same epitope on CD79b bound by
another CD79b antibody (i.e.,
CB3.1 (BD Biosciences Catalog #555678; San Jose, CA), AT105-1 (AbD Serotec
Catalog #MCA2208;
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Raleigh, NC), AT 107-2 (AbD Serotec Catalog #MCA2209), anti-human CD79b
antibody (BD Biosciences
Catalog #557592; San Jose, CA)).
[0312] In another aspect, a CD79b antibody of the invention binds to an
epitope on CD79b distinct
from an epitope bound by another CD79b antibody. In another embodiment, a
CD79b antibody of the
invention binds to an epitope on CD79b distinct from an epitope bound by the
Fab fragment of, monoclonal
antibody generated from hybridomas deposited with the ATCC as PTA-7712 on July
11, 2006, monoclonal
antibody comprising the variable domains of SEQ ID NO: 10 (Figure 7) and SEQ
ID NO: 14 (Figures 8A-B),
or chimeric antibody comprising the variable domain of either antibody
generated from hybridomas deposited
with the ATCC as PTA-7712 on July 11, 2006, and constant domains from IgG1, or
the variable domains of
monoclonal antibody comprising the sequences of SEQ ID NO: 10 (Figure 7) and
SEQ ID NO: 14
(Figures 8A-B). In another embodiment, a CD79b antibody of the invention binds
an epitope on CD79b
distinct from an epitope on CD79b bound by another CD79b antibody (i.e., CB3.1
(BD Biosciences Catalog
#555678; San Jose, CA), AT105-1 (AbD Serotec Catalog #MCA2208; Raleigh, NC),
AT107-2 (AbD Serotec
Catalog #MCA2209), anti-human CD79b antibody (BD Biosciences Catalog #557592;
San Jose, CA)).
[0313] In another aspect, a CD79b antibody of the invention is distinct from
(i.e., it is not) a Fab
fragment of, the monoclonal antibody generated from hybridomas deposited with
the ATCC as PTA-7712 on
July 11, 2006, the monoclonal antibody comprising the variable domains of SEQ
ID NO: 10 (Figure 7) and
SEQ ID NO: 14 (Figures 8A-B), or chimeric antibody comprising the variable
domain of antibody generated
from hybridomas deposited with the ATCC as PTA-7712 on July 11, 2006, and
constant domains from IgG1,
or the variable domains of monoclonal antibody comprising the sequences of SEQ
ID NO: 10 (Figure 7) and
SEQ ID NO: 14 (Figures 8A-B). In another embodiment, a CD79b antibody of the
invention is distinct from
(i.e., it is not) a Fab fragment of another CD79b antibody ((i.e., CB3.1 (BD
Biosciences Catalog #555678; San
Jose, CA), AT105-1 (AbD Serotec Catalog #MCA2208; Raleigh, NC), AT107-2 (AbD
Serotec Catalog
#MCA2209), anti-human CD79b antibody (BD Biosciences Catalog #557592; San
Jose, CA)).
[0314] In one aspect, an antibody of the invention specifically binds to CD79b
of a first animal
species, and does not specifically bind to CD79b of a second animal species.
In one embodiment, the first
animal species is human and/or primate (e.g., cynomolgus monkey), and the
second animal species is murine
(e.g., mouse) and/or canine. In one embodiment, the first animal species is
human. In one embodiment, the
first animal species is primate, for example cynomolgus monkey. In one
embodiment, the second animal
species is murine, for example mouse. In one embodiment, the second animal
species is canine.
[0315] In one aspect, the invention provides compositions comprising one or
more antibodies of the
invention and a carrier. In one embodiment, the carrier is pharmaceutically
acceptable.
[0316] In one aspect, the invention provides nucleic acids encoding a CD79b
antibody of the
invention.
[0317] In one aspect, the invention provides vectors comprising a nucleic acid
of the invention.
[0318] In one aspect, the invention provides host cells comprising a nucleic
acid or a vector of the
invention. A vector can be of any type, for example a recombinant vector such
as an expression vector. Any
of a variety of host cells can be used. In one embodiment, a host cell is a
prokaryotic cell, for example, E. coli.
In one embodiment, a host cell is a eukaryotic cell, for example a mammalian
cell such as Chinese Hamster
Ovary (CHO) cell.
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[0319] In one aspect, the invention provides methods for making an antibody of
the invention. For
example, the invention provides a method of making a CD79b antibody (which, as
defined herein includes full
length and fragments thereof), said method comprising expressing in a suitable
host cell a recombinant vector
of the invention encoding said antibody (or fragment thereof), and recovering
said antibody.
[0320] In one aspect, the invention provides an article of manufacture
comprising a container; and a
composition contained within the container, wherein the composition comprises
one or more CD79b
antibodies of the invention. In one embodiment, the composition comprises a
nucleic acid of the invention. In
one embodiment, a composition comprising an antibody further comprises a
carrier, which in some
embodiments is pharmaceutically acceptable. In one embodiment, an article of
manufacture of the invention
further comprises instructions for administering the composition (e.g., the
antibody) to a subject.
[0321] In one aspect, the invention provides a kit comprising a first
container comprising a
composition comprising one or more CD79b antibodies of the invention; and a
second container comprising a
buffer. In one embodiment, the buffer is pharmaceutically acceptable. In one
embodiment, a composition
comprising an antagonist antibody further comprises a carrier, which in some
embodiments is
pharmaceutically acceptable. In one embodiment, a kit further comprises
instructions for administering the
composition (e.g., the antibody) to a subject.
[0322] In one aspect, the invention provides use of a CD79b antibody of the
invention in the
preparation of a medicament for the therapeutic and/or prophylactic treatment
of a disease, such as a cancer, a
tumor and/or a cell proliferative disorder. In one embodiment, cancer, tumor
and/or cell proliferative
disorder is selected from lymphoma, non-Hodgkins lymphoma (NHL), aggressive
NHL, relapsed aggressive
NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic
lymphocytic leukemia (CLL),
small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute
lymphocytic leukemia (ALL), and
mantle cell lymphoma.
[0323] In one aspect, the invention provides use of a nucleic acid of the
invention in the preparation
of a medicament for the therapeutic and/or prophylactic treatment of a
disease, such as a cancer, a tumor
and/or a cell proliferative disorder. In one embodiment, cancer, tumor and/or
cell proliferative disorder is
selected from lymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL, relapsed
aggressive NHL,
relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic
lymphocytic leukemia (CLL), small
lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic
leukemia (ALL), and mantle
cell lymphoma.
[0324] In one aspect, the invention provides use of an expression vector of
the invention in the
preparation of a medicament for the therapeutic and/or prophylactic treatment
of a disease, such as a cancer, a
tumor and/or a cell proliferative disorder. In one embodiment, cancer, tumor
and/or cell proliferative
disorder is selected from lymphoma, non-Hodgkins lymphoma (NHL), aggressive
NHL, relapsed aggressive
NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic
lymphocytic leukemia (CLL),
small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute
lymphocytic leukemia (ALL), and
mantle cell lymphoma.
[0325] In one aspect, the invention provides use of a host cell of the
invention in the preparation of
a medicament for the therapeutic and/or prophylactic treatment of a disease,
such as a cancer, a tumor and/or a
cell proliferative disorder. In one embodiment, cancer, tumor and/or cell
proliferative disorder is selected
from lymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL, relapsed
aggressive NHL, relapsed
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indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic
leukemia (CLL), small
lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic
leukemia (ALL), and mantle
cell lymphoma.
[0326] In one aspect, the invention provides use of an article of manufacture
of the invention in the
preparation of a medicament for the therapeutic and/or prophylactic treatment
of a disease, such as a cancer, a
tumor and/or a cell proliferative disorder. In one embodiment, cancer, tumor
and/or cell proliferative
disorder is selected from lymphoma, non-Hodgkins lymphoma (NHL), aggressive
NHL, relapsed aggressive
NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic
lymphocytic leukemia (CLL),
small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute
lymphocytic leukemia (ALL), and
mantle cell lymphoma.
[0327] In one aspect, the invention provides use of a kit of the invention in
the preparation of a
medicament for the therapeutic and/or prophylactic treatment of a disease,
such as a cancer, a tumor and/or a
cell proliferative disorder. In one embodiment, cancer, tumor and/or cell
proliferative disorder is selected
from lymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL, relapsed
aggressive NHL, relapsed
indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic
leukemia (CLL), small
lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic
leukemia (ALL), and mantle
cell lymphoma.
[0328] In one aspect, the invention provides a method of inhibiting the growth
of a cell that
expresses CD79b, said method comprising contacting said cell with an antibody
of the invention thereby
causing an inhibition of growth of said cell. In one embodiment, the antibody
is conjugated to a cytotoxic
agent. In one embodiment, the antibody is conjugated to a growth inhibitory
agent.
[0329] In one aspect, the invention provides a method of therapeutically
treating a mammal having
a cancerous tumor comprising a cell that expresses CD79b, said method
comprising administering to said
mammal a therapeutically effective amount of an antibody of the invention,
thereby effectively treating said
mammal. In one embodiment, the antibody is conjugated to a cytotoxic agent. In
one embodiment, the
antibody is conjugated to a growth inhibitory agent.
[0330] In one aspect, the invention provides a method for treating or
preventing a cell proliferative
disorder associated with increased expression of CD79b, said method comprising
administering to a subject in
need of such treatment an effective amount of an antibody of the invention,
thereby effectively treating or
preventing said cell proliferative disorder. In one embodiment, said
proliferative disorder is cancer. In one
embodiment, the antibody is conjugated to a cytotoxic agent. In one
embodiment, the antibody is conjugated
to a growth inhibitory agent.
[0331] In one aspect, the invention provides a method for inhibiting the
growth of a cell, wherein
growth of said cell is at least in part dependent upon a growth potentiating
effect of CD79b, said method
comprising contacting said cell with an effective amount of an antibody of the
invention, thereby inhibiting the
growth of said cell. In one embodiment, the antibody is conjugated to a
cytotoxic agent. In one embodiment,
the antibody is conjugated to a growth inhibitory agent.
[0332] In one aspect, the invention provides a method of therapeutically
treating a tumor in a
mammal, wherein the growth of said tumor is at least in part dependent upon a
growth potentiating effect of
CD79b, said method comprising contacting said cell with an effective amount of
an antibody of the invention,
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thereby effectively treating said tumor. In one embodiment, the antibody is
conjugated to a cytotoxic agent.
In one embodiment, the antibody is conjugated to a growth inhibitory agent.
[0333] In one aspect, the invention provides a method of treating cancer
comprising administering
to a patient the pharmaceutical formulation comprising an immunoconjugate
described herein, acceptable
diluent, carrier or excipient. In one embodiment, the cancer is selected from
the lymphoma, non-Hodgkins
lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent
NHL, refractory NHL,
refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic
lymphoma, leukemia, hairy
cell leukemia (HCL), acute lymphocytic leukemia (ALL) and mantle cell
lymphoma. In one embodiment, the
patient is administered a cytotoxic agent in combination with the antibody-
drug conjugate compound.
[0334] In one aspect, the invention provides a method of inhibiting B cell
proliferation comprising
exposing a cell to an immunoconjugate comprising an antibody of the invention
under conditions permissive
for binding of the immunoconjugate to CD79b. In one embodiment, the B cell
proliferation is selected from
lymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL, relapsed aggressive
NHL, relapsed indolent
NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia
(CLL), small lymphocytic
lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia
(ALL) and mantle cell
lymphoma. In one embodiment, the B cell is a xenograft. In one embodiment, the
exposing takes place in
vitro. In one embodiment, the exposing taxes place in vivo.
[0335] In one aspect, the invention provides a method of determining the
presence of CD79b in a
sample suspected of containing CD79b, said method comprising exposing said
sample to an antibody of the
invention, and determining binding of said antibody to CD79b in said sample
wherein binding of said antibody
to CD79b in said sample is indicative of the presence of said protein in said
sample. In one embodiment, the
sample is a biological sample. In a further embodiment, the biological sample
comprises B cells. In one
embodiment, the biological sample is from a mammal experiencing or suspected
of experiencing a B cell
disorder and/or a B cell proliferative disorder including, but not limited to,
lymphoma, non-Hodgkin's
lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent
NHL, refractory NHL,
refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic
lymphoma, leukemia, hairy
cell leukemia (HCL), acute lymphocytic leukemia (ALL) and mantle cell
lymphoma.
[0336] In one aspect, the invention provides a method of diagnosing a cell
proliferative disorder
associated with an increase in cells, such as B cells, expressing CD79b is
provided, the method comprising
contacting a test cells in a biological sample with any of the above
antibodies; determining the level of
antibody bound to test cells in the sample by detecting binding of the
antibody to CD79b; and comparing the
level of antibody bound to cells in a control sample, wherein the level of
antibody bound is normalized to the
number of CD79b-expressing cells in the test and control samples, and wherein
a higher level of antibody
bound in the test sample as compared to the control sample indicates the
presence of a cell proliferative
disorder associated with cells expressing CD79b.
[0337] In one aspect, the invention provides a method of detecting soluble
CD79b in blood or
serum, the method comprising contacting a test sample of blood or serum from a
mammal suspected of
experiencing a B cell proliferative disorder with an anti-CD79b antibody of
the invention and detecting a
increase in soluble CD79b in the test sample relative to a control sample of
blood or serum from a normal
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mammal. In an embodiment, the method of detecting is useful as a method of
diagnosing a B cell proliferative
disorder associated with an increase in soluble CD79b in blood or serum of a
mammal.
[0338] In one aspect, the invention provides a method of binding an antibody
of the invention to a
cell that expresses CD79b, said method comprising contacting said cell with an
antibody of the invention. In
one embodiment, the antibody is conjugated to a cytotoxic agent. In one
embodiment, the antibody is
conjugated to a growth inhibitory agent.
[0339] Methods of the invention can be used to affect any suitable
pathological state, for example,
cells and/or tissues associated with expression of CD79b. In one embodiment, a
cell that is targeted in a
method of the invention is a hematopoietic cell. For example, a hematopoietic
cell can be one selected from
the group consisting of a lymphocyte, leukocyte, platelet, erythrocyte and
natural killer cell. In one
embodiment, a cell that is targeted in a method of the invention is a B cell
or T cell. In one embodiment, a cell
that is targeted in a method of the invention is a cancer cell. For example, a
cancer cell can be one selected
from the group consisting of a lymphoma cell, leukemia cell, or myeloma cell.
[0340] Methods of the invention can further comprise additional treatment
steps. For example, in
one embodiment, a method further comprises a step wherein a targeted cell
and/or tissue (e.g., a cancer cell) is
exposed to radiation treatment or a chemotherapeutic agent.
[0341] As described herein, CD79b is a signaling component of the B cell
receptor. Accordingly,
in one embodiment of methods of the invention, a cell that is targeted (e.g.,
a cancer cell) is one in which
CD79b is expressed as compared to a cell that does not express CD79b. In a
further embodiment, the targeted
cell is a cancer cell in which CD79b expression is enhanced as compared to a
normal non-cancer cell of the
same tissue type. In one embodiment, a method of the invention causes the
death of a targeted cell.
[0342] In other aspects of the present invention, the invention provides
vectors comprising DNA
encoding any of the herein described antibodies. Host cell comprising any such
vector are also provided. By
way of example, the host cells may be CHO cells, E. coli cells, or yeast
cells. A process for producing any of
the herein described antibodies is further provided and comprises culturing
host cells under conditions suitable
for expression of the desired antibody and recovering the desired antibody
from the cell culture.
[0343] In a still further aspect, the invention concerns a composition of
matter comprising an anti-
CD79b antibody as described herein, in combination with a carrier. Optionally,
the carrier is a
pharmaceutically acceptable carrier.
[0344] Another aspect of the present invention is directed to the use of an
anti-CD79b polypeptide
antibody as described herein, for the preparation of a medicament useful in
the treatment of a condition which
is responsive to the anti-CD79b polypeptide antibody.
[0345] Another aspect of the invention is a composition comprising a mixture
of antibody-drug
compounds of Formula I where the average drug loading per antibody is about 2
to about 5, or about 3 to
about 4.
[0346] Another aspect of the invention is a pharmaceutical composition
including a Formula I ADC
compound, a mixture of Formula I ADC compounds, or a pharmaceutically
acceptable salt or solvate thereof,
and apharmaceutically acceptable diluent, carrier, or excipient.
[0347] Another aspect provides a pharmaceutical combination comprising a
Formula I ADC
compound and a second compound having anticancer properties or other
therapeutic effects.
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[0348] Another aspect is a method for killing or inhibiting the proliferation
of tumor cells or cancer
cells comprising treating the cells with an amount of an antibody-drug
conjugate of Formula I, or a
pharmaceutically acceptable salt or solvate thereof, being effective to kill
or inhibit the proliferation of the
tumor cells or cancer cells.
[0349] Another aspect is a method of treating cancer comprising administering
to a patient a
therapeutically effective amount of a pharmaceutical composition including a
Formula I ADC.
[0350] Another aspect includes articles of manufacture, i.e. kits, comprising
an antibody-drug
conjugate, a container, and a package insert or label indicating a treatment.
[0351] An aspect of the invention is a method for making a Formula I antibody
drug conjugate
compound comprising the steps of: (a) reacting an engineered cysteine group of
the cysteine engineered
antibody with a linker reagent to form antibody-linker intermediate Ab-L; and
(b) reacting Ab-L with an
activated drug moiety D; whereby the antibody-drug conjugate is formed; or
comprising the steps of: (c)
reacting a nucleophilic group of a drug moiety with a linker reagent to form
drug-linker intermediate D-L; and
(d) reacting D-L with an engineered cysteine group of the cysteine engineered
antibody; whereby the
antibody-drug conjugate is formed.
[0352] An aspect of the invention is an assay for detecting cancer cells
comprising: (a) exposing
cells to a cysteine engineered anti-CD79b antibody-drug conjugate; and (b)
determining the extent of binding
of the cysteine engineered anti-CD79b antibody-drug conjugate compound to the
cells.
A. Anti-CD79b Antibodies
[0353] In one embodiment, the present invention provides anti-CD79b antibodies
which may find
use herein as therapeutic agents. Exemplary antibodies include polyclonal,
monoclonal, humanized, bispecific,
and heteroconjugate antibodies.
1. Polyclonal Antibodies
[0354] Polyclonal antibodies are preferably raised in animals by multiple
subcutaneous (sc) or
intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It
may be useful to conjugate the
relevant antigen (especially when synthetic peptides are used) to a protein
that is immunogenic in the species
to be immunized. For example, the antigen can be conjugated to keyhole limpet
hemocyanin (KLH), serum
albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using a
bifunctional or derivatizing agent, e.g.,
maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine
residues), N-hydroxysuccinimide
(through lysine residues), glutaraldehyde, succinic anhydride, SOC12, or RI
N=C=NR, where R and RI are
different alkyl groups.
[0355] Animals are immunized against the antigen, immunogenic conjugates, or
derivatives by
combining, e.g., 100 g or 5 g of the protein or conjugate (for rabbits or
mice, respectively) with 3 volumes
of Freund's complete adjuvant and injecting the solution intradermally at
multiple sites. One month later, the
animals are boosted with 1/5 to 1/10 the original amount of peptide or
conjugate in Freund's complete
adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later,
the animals are bled and the
serum is assayed for antibody titer. Animals are boosted until the titer
plateaus. Conjugates also can be made
in recombinant cell culture as protein fusions. Also, aggregating agents such
as alum are suitably used to
enhance the immune response.
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2. Monoclonal Antibodies
[0356] Monoclonal antibodies may be made using the hybridoma method first
described by Kohler
et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods
(U.S. Patent No. 4,816,567).
[0357] In the hybridoma method, a mouse or other appropriate host animal, such
as a hamster, is
immunized as described above to elicit lymphocytes that produce or are capable
of producing antibodies that
will specifically bind to the protein used for immunization. Alternatively,
lymphocytes may be immunized in
vitro. After immunization, lymphocytes are isolated and then fused with a
myeloma cell line using a suitable
fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding,
Monoclonal Antibodies:
Principles and Practice, pp.59-103 (Academic Press, 1986)).
[0358] The hybridoma cells thus prepared are seeded and grown in a suitable
culture medium which
medium preferably contains one or more substances that inhibit the growth or
survival of the unfused, parental
myeloma cells (also referred to as fusion partner). For example, if the
parental myeloma cells lack the enzyme
hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the selective
culture medium for the
hybridomas typically will include hypoxanthine, aminopterin, and thymidine
(HAT medium), which
substances prevent the growth of HGPRT-deficient cells.
[0359] Preferred fusion partner myeloma cells are those that fuse efficiently,
support stable high-
level production of antibody by the selected antibody-producing cells, and are
sensitive to a selective medium
that selects against the unfused parental cells. Preferred myeloma cell lines
are murine myeloma lines, such as
those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk
Institute Cell Distribution
Center, San Diego, California USA, and SP-2 and derivatives e.g., X63-Ag8-653
cells available from the
American Type Culture Collection, Manassas, Virginia, USA. Human myeloma and
mouse-human
heteromyeloma cell lines also have been described for the production of human
monoclonal antibodies
(Kozbor, J. Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal Antibody
Production Techniques and
Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
[0360] Culture medium in which hybridoma cells are growing is assayed for
production of
monoclonal antibodies directed against the antigen. Preferably, the binding
specificity of monoclonal
antibodies produced by hybridoma cells is determined by immunoprecipitation or
by an in vitro binding assay,
such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
[0361] The binding affinity of the monoclonal antibody can, for example, be
determined by the
Scatchard analysis described in Munson et al., Anal. Biochem., 107:220 (1980).
[0362] Once hybridoma cells that produce antibodies of the desired
specificity, affinity, and/or
activity are identified, the clones may be subcloned by limiting dilution
procedures and grown by standard
methods (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103
(Academic Press, 1986)).
Suitable culture media for this purpose include, for example, D-MEM or RPMI-
1640 medium. In addition,
the hybridoma cells may be grown in vivo as ascites tumors in an animal e.g,,
by i.p. injection of the cells into
mice.
[0363] The monoclonal antibodies secreted by the subclones are suitably
separated from the culture
medium, ascites fluid, or serum by conventional antibody purification
procedures such as, for example,
affinity chromatography (e.g., using protein A or protein G-Sepharose) or ion-
exchange chromatography,
hydroxylapatite chromatography, gel electrophoresis, dialysis, etc.
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[0364] DNA encoding the monoclonal antibodies is readily isolated and
sequenced using
conventional procedures (e.g., by using oligonucleotide probes that are
capable of binding specifically to
genes encoding the heavy and light chains of murine antibodies). The hybridoma
cells serve as a preferred
source of such DNA. Once isolated, the DNA may be placed into expression
vectors, which are then
transfected into host cells such as E. coli cells, simian COS cells, Chinese
Hamster Ovary (CHO) cells, or
myeloma cells that do not otherwise produce antibody protein, to obtain the
synthesis of monoclonal
antibodies in the recombinant host cells. Review articles on recombinant
expression in bacteria of DNA
encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-
262 (1993) and Pluckthun,
Immunol. Revs. 130:151-188 (1992).
[0365] In a further embodiment, monoclonal antibodies or antibody fragments
can be isolated from
antibody phage libraries generated using the techniques described in
McCafferty et al., Nature, 348:552-554
(1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol.
Biol., 222:581-597 (1991)
describe the isolation of murine and human antibodies, respectively, using
phage libraries. Subsequent
publications describe the production of high affinity (nM range) human
antibodies by chain shuffling (Marks
et al., Bio/Technology, 10:779-783 (1992)), as well as combinatorial infection
and in vivo recombination as a
strategy for constructing very large phage libraries (Waterhouse et al., Nuc.
Acids. Res. 21:2265-2266 (1993)).
Thus, these techniques are viable alternatives to traditional monoclonal
antibody hybridoma techniques for
isolation of monoclonal antibodies.
[0366] The DNA that encodes the antibody may be modified to produce chimeric
or fusion
antibody polypeptides, for example, by substituting human heavy chain and
light chain constant domain (CH
and CL) sequences for the homologous murine sequences (U.S. Patent No.
4,816,567; and Morrison, et al.,
Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by fusing the immunoglobulin
coding sequence with all or
part of the coding sequence for a non-immunoglobulin polypeptide (heterologous
polypeptide). The non-
immunoglobulin polypeptide sequences can substitute for the constant domains
of an antibody, or they are
substituted for the variable domains of one antigen-combining site of an
antibody to create a chimeric bivalent
antibody comprising one antigen-combining site having specificity for an
antigen and another antigen-
combining site having specificity for a different antigen.
3. Human and Humanized Antibodies
[0367] The anti-CD79b antibodies of the invention may further comprise
humanized antibodies or
human antibodies. Humanized forms of non-human (e.g., murine) antibodies are
chimeric immunoglobulins,
immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or
other antigen-binding
subsequences of antibodies) which contain minimal sequence derived from non-
human immunoglobulin.
Humanized antibodies include human immunoglobulins (recipient antibody) in
which residues from a
complementary determining region (CDR) of the recipient are replaced by
residues from a CDR of a non-
human species (donor antibody) such as mouse, rat or rabbit having the desired
specificity, affinity and
capacity. In some instances, Fv framework residues of the human immunoglobulin
are replaced by
corresponding non-human residues. Humanized antibodies may also comprise
residues which are found
neither in the recipient antibody nor in the imported CDR or framework
sequences. In general, the humanized
antibody will comprise substantially all of at least one, and typically two,
variable domains, in which all or
substantially all of the CDR regions correspond to those of a non-human
immunoglobulin and all or
substantially all of the FR regions are those of a human immunoglobulin
consensus sequence. The humanized
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antibody optimally also will comprise at least a portion of an immunoglobulin
constant region (Fc), typically
that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986);
Riechmann et al., Nature,
332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
[0368] Methods for humanizing non-human antibodies are well known in the art.
Generally, a
humanized antibody has one or more amino acid residues introduced into it from
a source which is non-human.
These non-human amino acid residues are often referred to as "import"
residues, which are typically taken
from an "import" variable domain. Humanization can be essentially performed
following the method of
Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et
al., Nature, 332:323-327
(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting
rodent CDRs or CDR sequences for
the corresponding sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric
antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an
intact human variable domain has
been substituted by the corresponding sequence from a non-human species. In
practice, humanized antibodies
are typically human antibodies in which some CDR residues and possibly some FR
residues are substituted by
residues from analogous sites in rodent antibodies.
[0369] The choice of human variable domains, both light and heavy, to be used
in making the
humanized antibodies is very important to reduce antigenicity and HAMA
response (human anti-mouse
antibody) when the antibody is intended for human therapeutic use. Reduction
or elimination of a HAMA
response is a significant aspect of clinical development of suitable
therapeutic agents. See, e.g., Khaxzaeli et
al., J. Natl. Cancer Inst. (1988), 80:937; Jaffers et al., Transplantation
(1986), 41:572; Shawler et al., J.
Immunol. (1985), 135:1530; Sears et al., J. Biol. Response Mod. (1984), 3:138;
Miller et al., Blood (1983),
62:988; Hakimi et al., J. Immunol. (1991), 147:1352; Reichmann et al., Nature
(1988), 332:323; Junghans et
al., Cancer Res. (1990), 50:1495. As described herein, the invention provides
antibodies that are humanized
such that HAMA response is reduced or eliminated. Variants of these antibodies
can further be obtained
using routine methods known in the art, some of which are further described
below. According to the so-
called "best-fit" method, the sequence of the variable domain of a rodent
antibody is screened against the
entire library of known human variable domain sequences. The human V domain
sequence which is closest to
that of the rodent is identified and the human framework region (FR) within it
accepted for the humanized
antibody (Sims et al., J. Immunol. 151:2296 (1993); Chothia et al., J. Mol.
Biol., 196:901 (1987)). Another
method uses a particular framework region derived from the consensus sequence
of all human antibodies of a
particular subgroup of light or heavy chains. The same framework may be used
for several different
humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285
(1992); Presta et al., J. Immunol.
151:2623 (1993)).
[0370] For example, an amino acid sequence from an antibody as described
herein can serve as a
starting (parent) sequence for diversification of the framework and/or
hypervariable sequence(s). A selected
framework sequence to which a starting hypervariable sequence is linked is
referred to herein as an acceptor
human framework. While the acceptor human frameworks may be from, or derived
from, a human
immunoglobulin (the VL and/or VH regions thereof), preferably the acceptor
human frameworks are from, or
derived from, a human consensus framework sequence as such frameworks have
been demonstrated to have
minimal, or no, immunogenicity in human patients.
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[0371] Where the acceptor is derived from a human immunoglobulin, one may
optionally select a
human framework sequence that is selected based on its homology to the donor
framework sequence by
aligning the donor framework sequence with various human framework sequences
in a collection of human
framework sequences, and select the most homologous framework sequence as the
acceptor.
[0372] In one embodiment, human consensus frameworks herein are from, or
derived from, VH
subgroup III and/or VL kappa subgroup I consensus framework sequences.
[0373] Thus, the VH acceptor human framework may comprise one, two, three or
all of the
following framework sequences:
FRl comprising EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 69),
FR2 comprising WVRQAPGKGLEWV (SEQ ID NO: 70),
FR3 comprising FR3 comprises RFTISXiDX2SKNTX3YLQMNSLRAEDTAVYYC (SEQ ID NO:
73),
wherein Xi is A or R, X2 is T or N, and X3 is A or L,
FR4 comprising WGQGTLVTVSS (SEQ ID NO: 72).
[0374] Examples of VH consensus frameworks include:
human VH subgroup I consensus framework minus Kabat CDRs (SEQ ID NO: 36);
human VH subgroup I consensus framework minus extended hypervariable regions
(SEQ ID NOs: 37-39);
human VH subgroup II consensus framework minus Kabat CDRs (SEQ ID NO: 40);
human VH subgroup II consensus framework minus extended hypervariable regions
(SEQ ID NOs: 41-43);
human VH subgroup III consensus framework minus Kabat CDRs (SEQ ID NO: 44);
human VH subgroup III consensus framework minus extended hypervariable regions
(SEQ ID NO: 45-47);
human VH acceptor framework minus Kabat CDRs (SEQ ID NO: 48);
human VH acceptor framework minus extended hypervariable regions (SEQ ID NOs:
49-50);
human VH acceptor 2 framework minus Kabat CDRs (SEQ ID NO: 51); or
human VH acceptor 2 framework minus extended hypervariable regions (SEQ ID
NOs: 52-54).
[0375] In one embodiment, the VH acceptor human framework comprises one, two,
three or all of
the following framework sequences:
FRl comprising EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 69),
FR2 comprising WVRQAPGKGLEWV (SEQ ID NO: 70),
FR3 comprising RFTISADTSKNTAYLQMNSLRAEDTAVYYC (SEQ ID NO: 71),
RFTISADTSKNTAYLQMNSLRAEDTAVYYCA (SEQ ID NO: 74),
RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 75),
RFTISADTSKNTAYLQMNSLRAEDTAVYYCS (SEQ ID NO: 76), or
RFTISADTSKNTAYLQMNSLRAEDTAVYYCSR (SEQ ID NO: 77)
FR4 comprising WGQGTLVTVSS (SEQ ID NO: 72).
[0376] The VL acceptor human framework may comprise one, two, three or all of
the following
framework sequences:
FRl comprising DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 65),
FR2 comprising WYQQKPGKAPKLLIY (SEQ ID NO: 66),
FR3 comprising GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 67),
FR4 comprising FGQGTKVEIKR (SEQ ID NO: 68).
[0377] Examples of VL consensus frameworks include:
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human VL kappa subgroup I consensus framework (SEQ ID NO: 55);
human VL kappa subgroup II consensus framework (SEQ ID NO: 56);
human VL kappa subgroup III consensus framework (SEQ ID NO: 57); or
human VL kappa subgroup IV consensus framework (SEQ ID NO: 58)
[0378] While the acceptor may be identical in sequence to the human framework
sequence selected,
whether that be from a human immunoglobulin or a human consensus framework,
the present invention
contemplates that the acceptor sequence may comprise pre-existing amino acid
substitutions relative to the
human immunoglobulin sequence or human consensus framework sequence. These pre-
existing substitutions
are preferably minimal; usually four, three, two or one amino acid differences
only relative to the human
immunoglobulin sequence or consensus framework sequence.
[0379] Hypervariable region residues of the non-human antibody are
incorporated into the VL
and/or VH acceptor human frameworks. For example, one may incorporate residues
corresponding to the
Kabat CDR residues, the Chothia hypervariable loop residues, the Abm residues,
and/or contact residues.
Optionally, the extended hypervariable region residues as follows are
incorporated: 24-34 (L1), 50-56 (L2)
and 89-97 (L3), 26-35B (111), 50-65, 47-65 or 49-65 (H2) and 93-102, 94-102,
or 95-102 (H3).
[0380] While "incorporation" of hypervariable region residues is discussed
herein, it will be
appreciated that this can be achieved in various ways, for example, nucleic
acid encoding the desired amino
acid sequence can be generated by mutating nucleic acid encoding the mouse
variable domain sequence so
that the framework residues thereof are changed to acceptor human framework
residues, or by mutating
nucleic acid encoding the human variable domain sequence so that the
hypervariable domain residues are
changed to non-human residues, or by synthesizing nucleic acid encoding the
desired sequence, etc.
[0381] In the examples herein, hypervariable region-grafted variants were
generated by Kunkel
mutagenesis of nucleic acid encoding the human acceptor sequences, using a
separate oligonucleotide for each
hypervariable region. Kunkel et al., Methods Enzymol. 154:367-382 (1987).
Appropriate changes can be
introduced within the framework and/or hypervariable region, using routine
techniques, to correct and re-
establish proper hypervariable region-antigen interactions.
[03 82] Phage(mid) display (also referred to herein as phage display in some
contexts) can be used
as a convenient and fast method for generating and screening many different
potential variant antibodies in a
library generated by sequence randomization. However, other methods for making
and screening altered
antibodies are available to the skilled person.
[0383] Phage(mid) display technology has provided a powerful tool for
generating and selecting
novel proteins which bind to a ligand, such as an antigen. Using the
techniques of phage(mid) display allows
the generation of large libraries of protein variants which can be rapidly
sorted for those sequences that bind
to a target molecule with high affinity. Nucleic acids encoding variant
polypeptides are generally fused to a
nucleic acid sequence encoding a viral coat protein, such as the gene III
protein or the gene VIII protein.
Monovalent phagemid display systems where the nucleic acid sequence encoding
the protein or polypeptide is
fused to a nucleic acid sequence encoding a portion of the gene III protein
have been developed. (Bass, S.,
Proteins, 8:309 (1990); Lowman and Wells, Methods: A Companion to Methods in
Enzymology, 3:205
(1991)). In a monovalent phagemid display system, the gene fusion is expressed
at low levels and wild type
gene III proteins are also expressed so that infectivity of the particles is
retained. Methods of generating
peptide libraries and screening those libraries have been disclosed in many
patents (e.g. U.S. Patent No.
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5,723,286, U.S. PatentNo. 5,432, 018, U.S. PatentNo. 5,580,717, U.S. PatentNo.
5,427,908 andU.S. Patent
No. 5,498,530).
[0384] Libraries of antibodies or antigen binding polypeptides have been
prepared in a number of
ways including by altering a single gene by inserting random DNA sequences or
by cloning a family of related
genes. Methods for displaying antibodies or antigen binding fragments using
phage(mid) display have been
described in U.S. Patent Nos. 5,750,373, 5,733,743, 5,837,242, 5,969,108,
6,172,197, 5,580,717, and
5,658,727. The library is then screened for expression of antibodies or
antigen binding proteins with the
desired characteristics.
[0385] Methods of substituting an amino acid of choice into a template nucleic
acid are well
established in the art, some of which are described herein. For example,
hypervariable region residues can be
substituted using the Kunkel method. See, e.g., Kunkel et al., Methods
Enzymol. 154:367-382 (1987).
[0386] The sequence of oligonucleotides includes one or more of the designed
codon sets for the
hypervariable region residues to be altered. A codon set is a set of different
nucleotide triplet sequences used
to encode desired variant amino acids. Codon sets can be represented using
symbols to designate particular
nucleotides or equimolar mixtures of nucleotides as shown in below according
to the IUB code.
IUB CODES
[0387] G Guanine
A Adenine
T Thymine
C Cytosine
R (A or G)
Y (C or T)
M (A or C)
K (G or T)
S (C or G)
W (A or T)
H (A or C or T)
B (C or G or T)
V (A or C or G)
D (AorGorT)H
N (AorCorGorT)
[0388] For example, in the codon set DVK, D can be nucleotides A or G or T; V
can be A or G or
C; and K can be G or T. This codon set can present 18 different codons and can
encode amino acids Ala, Trp,
Tyr, Lys, Thr, Asn, Lys, Ser, Arg, Asp, Glu, Gly, and Cys.
[0389] Oligonucleotide or primer sets can be synthesized using standard
methods. A set of
oligonucleotides can be synthesized, for example, by solid phase synthesis,
containing sequences that
represent all possible combinations of nucleotide triplets provided by the
codon set and that will encode the
desired group of amino acids. Synthesis of oligonucleotides with selected
nucleotide "degeneracy" at certain
positions is well known in that art. Such sets of nucleotides having certain
codon sets can be synthesized
using commercial nucleic acid synthesizers (available from, for example,
Applied Biosystems, Foster City,
CA), or can be obtained commercially (for example, from Life Technologies,
Rockville, MD). Therefore, a
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set of oligonucleotides synthesized having a particular codon set will
typically include a plurality of
oligonucleotides with different sequences, the differences established by the
codon set within the overall
sequence. Oligonucleotides, as used according to the invention, have sequences
that allow for hybridization to
a variable domain nucleic acid template and also can include restriction
enzyme sites for cloning purposes.
[0390] In one method, nucleic acid sequences encoding variant amino acids can
be created by
oligonucleotide-mediated mutagenesis. This technique is well known in the art
as described by Zoller et al.
Nucleic Acids Res. 10:6487-6504(1987). Briefly, nucleic acid sequences
encoding variant amino acids are
created by hybridizing an oligonucleotide set encoding the desired codon sets
to a DNA template, where the
template is the single-stranded form of the plasmid containing a variable
region nucleic acid template
sequence. After hybridization, DNA polymerase is used to synthesize an entire
second complementary strand
of the template that will thus incorporate the oligonucleotide primer, and
will contain the codon sets as
provided by the oligonucleotide set.
[0391] Generally, oligonucleotides of at least 25 nucleotides in length are
used. An optimal
oligonucleotide will have 12 to 15 nucleotides that are completely
complementary to the template on either
side of the nucleotide(s) coding for the mutation(s). This ensures that the
oligonucleotide will hybridize
properly to the single-stranded DNA template molecule. The oligonucleotides
are readily synthesized using
techniques known in the art such as that described by Crea et al., Proc.
Nat'l. Acad. Sci. USA, 75:5765 (1978).
[0392] The DNA template is generated by those vectors that are either derived
from bacteriophage
M13 vectors (the commercially available M13mp18 and M13mp19 vectors are
suitable), or those vectors that
contain a single-stranded phage origin of replication as described by Viera et
al., Meth. Enzymol., 153:3
(1987). Thus, the DNA that is to be mutated can be inserted into one of these
vectors in order to generate
single-stranded template. Production of the single-stranded template is
described in sections 4.21-4.41 of
Sambrook et al., above.
[0393] To alter the native DNA sequence, the oligonucleotide is hybridized to
the single stranded
template under suitable hybridization conditions. A DNA polymerizing enzyme,
usually T7 DNA polymerase
or the Klenow fragment of DNA polymerase I, is then added to synthesize the
complementary strand of the
template using the oligonucleotide as a primer for synthesis. A heteroduplex
molecule is thus formed such
that one strand of DNA encodes the mutated form of gene 1, and the other
strand (the original template)
encodes the native, unaltered sequence of gene 1. This heteroduplex molecule
is then transformed into a
suitable host cell, usually a prokaryote such as E. coli JM101. After growing
the cells, they are plated onto
agarose plates and screened using the oligonucleotide primer radiolabelled
with a 32-Phosphate to identify the
bacterial colonies that contain the mutated DNA.
[0394] The method described immediately above may be modified such that a
homoduplex
molecule is created wherein both strands of the plasmid contain the
mutation(s). The modifications are as
follows: The single stranded oligonucleotide is annealed to the single-
stranded template as described above.
A mixture of three deoxyribonucleotides, deoxyriboadenosine (dATP),
deoxyriboguanosine (dGTP), and
deoxyribothymidine (dTT), is combined with a modified thiodeoxyribocytosine
called dCTP-(aS) (which can
be obtained from Amersham). This mixture is added to the template-
oligonucleotide complex. Upon addition
of DNA polymerase to this mixture, a strand of DNA identical to the template
except for the mutated bases is
generated. In addition, this new strand of DNA will contain dCTP-(aS) instead
of dCTP, which serves to
protect it from restriction endonuclease digestion. After the template strand
of the double-stranded
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heteroduplex is nicked with an appropriate restriction enzyme, the template
strand can be digested with ExoIII
nuclease or another appropriate nuclease past the region that contains the
site(s) to be mutagenized. The
reaction is then stopped to leave a molecule that is only partially single-
stranded. A complete double-stranded
DNA homoduplex is then formed using DNA polymerase in the presence of all four
deoxyribonucleotide
triphosphates, ATP, and DNA ligase. This homoduplex molecule can then be
transformed into a suitable host
cell.
[0395] As indicated previously the sequence of the oligonucleotide set is of
sufficient length to
hybridize to the template nucleic acid and may also, but does not necessarily,
contain restriction sites. The
DNA template can be generated by those vectors that are either derived from
bacteriophage M13 vectors or
vectors that contain a single-stranded phage origin of replication as
described by Viera et al. Meth. Enzymol.,
153:3 (1987). Thus, the DNA that is to be mutated must be inserted into one of
these vectors in order to
generate single-stranded template. Production of the single-stranded template
is described in sections 4.21-
4.41 of Sambrook et al., supra.
[0396] According to another method, antigen binding may be restored during
humanization of
antibodies through the selection of repaired hypervariable regions (See
Application No. 11/061,841, filed
February 18, 2005). The method includes incorporating non-human hypervariable
regions onto an acceptor
framework and further introducing one or more amino acid substitutions in one
or more hypervariable regions
without modifying the acceptor framework sequence. Alternatively, the
introduction of one or more amino
acid substitutions may be accompanied by modifications in the acceptor
framework sequence.
[0397] According to another method, a library can be generated by providing
upstream and
downstream oligonucleotide sets, each set having a plurality of
oligonucleotides with different sequences, the
different sequences established by the codon sets provided within the sequence
of the oligonucleotides. The
upstream and downstream oligonucleotide sets, along with a variable domain
template nucleic acid sequence,
can be used in a polymerase chain reaction to generate a "library" of PCR
products. The PCR products can be
referred to as "nucleic acid cassettes", as they can be fused with other
related or unrelated nucleic acid
sequences, for example, viral coat proteins and dimerization domains, using
established molecular biology
techniques.
[0398] The sequence of the PCR primers includes one or more of the designed
codon sets for the
solvent accessible and highly diverse positions in a hypervariable region. As
described above, a codon set is a
set of different nucleotide triplet sequences used to encode desired variant
amino acids.
[0399] Antibody selectants that meet the desired criteria, as selected through
appropriate
screening/selection steps can be isolated and cloned using standard
recombinant techniques.
[0400] It is further important that antibodies be humanized with retention of
high binding affinity
for the antigen and other favorable biological properties. To achieve this
goal, according to a preferred
method, humanized antibodies are prepared by a process of analysis of the
parental sequences and various
conceptual humanized products using three-dimensional models of the parental
and humanized sequences.
Three-dimensional immunoglobulin models are commonly available and are
familiar to those skilled in the art.
Computer programs are available which illustrate and display probable three-
dimensional conformational
structures of selected candidate immunoglobulin sequences. Inspection of these
displays permits analysis of
the likely role of the residues in the functioning of the candidate
immunoglobulin sequence, i.e., the analysis
of residues that influence the ability of the candidate immunoglobulin to bind
its antigen. In this way, FR
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residues can be selected and combined from the recipient and import sequences
so that the desired antibody
characteristic, such as increased affinity for the target antigen(s), is
achieved. In general, the hypervariable
region residues are directly and most substantially involved in influencing
antigen binding.
[0401] Various forms of a humanized anti-CD79b antibody are contemplated. For
example, the
humanized antibody may be an antibody fragment, such as a Fab, which is
optionally conjugated with one or
more cytotoxic agent(s) in order to generate an immunoconjugate.
Alternatively, the humanized antibody may
be an intact antibody, such as an intact IgG1 antibody.
[0402] As an alternative to humanization, human antibodies can be generated.
For example, it is
now possible to produce transgenic animals (e.g., mice) that are capable, upon
immunization, of producing a
full repertoire of human antibodies in the absence of endogenous
immunoglobulin production. For example, it
has been described that the homozygous deletion of the antibody heavy-chain
joining region (JH) gene in
chimeric and germ-line mutant mice results in complete inhibition of
endogenous antibody production.
Transfer of the human germ-line immunoglobulin gene array into such germ-line
mutant mice will result in
the production of human antibodies upon antigen challenge. See, e.g.,
Jakobovits et al., Proc. Natl. Acad. Sci.
USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann
et al., Year in Immuno.
7:33 (1993); U.S. Patent Nos. 5,545,806, 5,569,825, 5,591,669 (all of
GenPharm); 5,545,807; and WO
97/17852.
[0403] Alternatively, phage display technology (McCafferty et al., Nature
348:552-553 [1990]) can
be used to produce human antibodies and antibody fragments in vitro, from
immunoglobulin variable (V)
domain gene repertoires from unimmunized donors. According to this technique,
antibody V domain genes
are cloned in-frame into either a major or minor coat protein gene of a
filamentous bacteriophage, such as
M13 or fd, and displayed as functional antibody fragments on the surface of
the phage particle. Because the
filamentous particle contains a single-stranded DNA copy of the phage genome,
selections based on the
functional properties of the antibody also result in selection of the gene
encoding the antibody exhibiting those
properties. Thus, the phage mimics some of the properties of the B-cell. Phage
display can be performed in a
variety of formats, reviewed in, e.g., Johnson, Kevin S. and Chiswell, David
J., Current Opinion in Structural
Biology 3:564-571 (1993). Several sources of V-gene segments can be used for
phage display. Clackson et
al., Nature, 352:624-628 (1991) isolated a diverse array of anti-oxazolone
antibodies from a small random
combinatorial library of V genes derived from the spleens of immunized mice. A
repertoire of V genes from
unimmunized human donors can be constructed and antibodies to a diverse array
of antigens (including self-
antigens) can be isolated essentially following the techniques described by
Marks et al., J. Mol. Biol. 222:581-
597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993). See, also, U.S.
Patent Nos. 5,565,332 and
5,573,905.
[0404] As discussed above, human antibodies may also be generated by in vitro
activated B cells
(see U.S. Patents 5,567,610 and 5,229,275).
4. Antibody fragments
In certain circumstances there are advantages of using antibody fragments,
rather than whole
antibodies. The smaller size of the fragments allows for rapid clearance, and
may lead to improved access to
solid tumors.
[0405] Various techniques have been developed for the production of antibody
fragments.
Traditionally, these fragments were derived via proteolytic digestion of
intact antibodies (see, e.g., Morimoto
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et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and
Brennan et al., Science,
229:81 (1985)). However, these fragments can now be produced directly by
recombinant host cells. Fab, Fv
and ScFv antibody fragments can all be expressed in and secreted from E. coli,
thus allowing the facile
production of large amounts of these fragments. Antibody fragments can be
isolated from the antibody phage
libraries discussed above. Alternatively, Fab'-SH fragments can be directly
recovered from E. coli and
chemically coupled to form F(ab')2 fragments (Carter et al., Bio/Technology
10:163-167 (1992)). According
to another approach, F(ab')2 fragments can be isolated directly from
recombinant host cell culture. Fab and
F(ab')2 fragment with increased in vivo half-life comprising a salvage
receptor binding epitope residues are
described in U.S. Patent No. 5,869,046. Other techniques for the production of
antibody fragments will be
apparent to the skilled practitioner. In other embodiments, the antibody of
choice is a single chain Fv
fragment (scFv). See WO 93/16185; U.S. Patent No. 5,571,894; and U.S. Patent
No. 5,587,458. Fv and sFv
are the only species with intact combining sites that are devoid of constant
regions; thus, they are suitable for
reduced nonspecific binding during in vivo use. sFv fusion proteins may be
constructed to yield fusion of an
effector protein at either the amino or the carboxy terminus of an sFv. See
Antibody Engineering, ed.
Borrebaeck, supra. The antibody fragment may also be a "linear antibody",
e.g., as described in U.S. Patent
5,641,870 for example. Such linear antibody fragments may be monospecific or
bispecific.
5. Bispecific Antibodies
[0406] Bispecific antibodies are antibodies that have binding specificities
for at least two different
epitopes. Exemplary bispecific antibodies may bind to two different epitopes
of a CD79b protein as described
herein. Other such antibodies may combine a CD79b binding site with a binding
site for another protein.
Alternatively, an anti-CD79b arm may be combined with an arm which binds to a
triggering molecule on a
leukocyte such as a T-cell receptor molecule (e.g. CD3), or Fc receptors for
IgG (FcyR), such as FcyRI
(CD64), FcyRII (CD32) and FcyRIII (CD16), so as to focus and localize cellular
defense mechanisms to the
CD79b-expressing cell. Bispecific antibodies may also be used to localize
cytotoxic agents to cells which
express CD79b. These antibodies possess a CD79b-binding arm and an arm which
binds the cytotoxic agent
(e.g., saporin, anti-interferon-a, vinca alkaloid, ricin A chain, methotrexate
or radioactive isotope hapten).
Bispecific antibodies can be prepared as full length antibodies or antibody
fragments (e.g., F(ab')2 bispecific
antibodies).
[0407] WO 96/16673 describes a bispecific anti-ErbB2/anti-FcyRIII antibody and
U.S. Patent No.
5,837,234 discloses a bispecific anti-ErbB2/anti-FcyRI antibody. A bispecific
anti-ErbB2/Fca antibody is
shown in W098/02463. U.S. Patent No. 5,821,337 teaches a bispecific anti-
ErbB2/anti-CD3 antibody.
[0408] Methods for making bispecific antibodies are known in the art.
Traditional production of
full length bispecific antibodies is based on the co-expression of two
immunoglobulin heavy chain-light chain
pairs, where the two chains have different specificities (Millstein et al.,
Nature 305:537-539 (1983)). Because
of the random assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a
potential mixture of 10 different antibody molecules, of which only one has
the correct bispecific structure.
Purification of the correct molecule, which is usually done by affinity
chromatography steps, is rather
cumbersome, and the product yields are low. Similar procedures are disclosed
in WO 93/08829, and in
Traunecker et al., EMBO J. 10:3655-3659 (1991).
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[0409] According to a different approach, antibody variable domains with the
desired binding
specificities (antibody-antigen combining sites) are fused to immunoglobulin
constant domain sequences.
Preferably, the fusion is with an Ig heavy chain constant domain, comprising
at least part of the hinge, CH2,
and CH3 regions. It is preferred to have the first heavy-chain constant region
(CH1) containing the site
necessary for light chain bonding, present in at least one of the fusions.
DNAs encoding the immunoglobulin
heavy chain fusions and, if desired, the immunoglobulin light chain, are
inserted into separate expression
vectors, and are co-transfected into a suitable host cell. This provides for
greater flexibility in adjusting the
mutual proportions of the three polypeptide fragments in embodiments when
unequal ratios of the three
polypeptide chains used in the construction provide the optimum yield of the
desired bispecific antibody. It is,
however, possible to insert the coding sequences for two or all three
polypeptide chains into a single
expression vector when the expression of at least two polypeptide chains in
equal ratios results in high yields
or when the ratios have no significant affect on the yield of the desired
chain combination.
[0410] In a preferred embodiment of this approach, the bispecific antibodies
are composed of a
hybrid immunoglobulin heavy chain with a first binding specificity in one arm,
and a hybrid immunoglobulin
heavy chain-light chain pair (providing a second binding specificity) in the
other arm. It was found that this
asymmetric structure facilitates the separation of the desired bispecific
compound from unwanted
immunoglobulin chain combinations, as the presence of an immunoglobulin light
chain in only one half of the
bispecific molecule provides for a facile way of separation. This approach is
disclosed in WO 94/04690. For
further details of generating bispecific antibodies see, for example, Suresh
et al., Methods in Enzymology
121:210 (1986).
[0411] According to another approach described in U.S. Patent No. 5,731,168,
the interface
between a pair of antibody molecules can be engineered to maximize the
percentage of heterodimers which
are recovered from recombinant cell culture. The preferred interface comprises
at least a part of the CH3
domain. In this method, one or more small amino acid side chains from the
interface of the first antibody
molecule are replaced with larger side chains (e.g., tyrosine or tryptophan).
Compensatory "cavities" of
identical or similar size to the large side chain(s) are created on the
interface of the second antibody molecule
by replacing large amino acid side chains with smaller ones (e.g., alanine or
threonine). This provides a
mechanism for increasing the yield of the heterodimer over other unwanted end-
products such as homodimers.
[0412] Bispecific antibodies include cross-linked or "heteroconjugate"
antibodies. For example,
one of the antibodies in the heteroconjugate can be coupled to avidin, the
other to biotin. Such antibodies have,
for example, been proposed to target immune system cells to unwanted cells
(U.S. Patent No. 4,676,980), and
for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089).
Heteroconjugate antibodies
may be made using any convenient cross-linking methods. Suitable cross-linking
agents are well known in the
art, and are disclosed in U.S. Patent No. 4,676,980, along with a number of
cross-linking techniques.
[0413] Techniques for generating bispecific antibodies from antibody fragments
have also been
described in the literature. For example, bispecific antibodies can be
prepared using chemical linkage.
Brennan et al., Science 229:81 (1985) describe a procedure wherein intact
antibodies are proteolytically
cleaved to generate F(ab')2 fragments. These fragments are reduced in the
presence of the dithiol complexing
agent, sodium arsenite, to stabilize vicinal dithiols and prevent
intermolecular disulfide formation. The Fab'
fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
One of the Fab'-TNB
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derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an
equimolar amount of the other Fab'-TNB derivative to form the bispecific
antibody. The bispecific antibodies
produced can be used as agents for the selective immobilization of enzymes.
[0414] Recent progress has facilitated the direct recovery of Fab'-SH
fragments from E. coli, which
can be chemically coupled to form bispecific antibodies. Shalaby et al., J.
Exp. Med. 175: 217-225 (1992)
describe the production of a fully humanized bispecific antibody F(ab')2
molecule. Each Fab' fragment was
separately secreted from E. coli and subjected to directed chemical coupling
in vitro to form the bispecific
antibody. The bispecific antibody thus formed was able to bind to cells
overexpressing the ErbB2 receptor
and normal human T cells, as well as trigger the lytic activity of human
cytotoxic lymphocytes against human
breast tumor targets.
[0415]Various techniques for making and isolating bispecific antibody
fragments directly from
recombinant cell culture have also been described. For example, bispecific
antibodies have been produced
using leucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992).
The leucine zipper peptides
from the Fos and Jun proteins were linked to the Fab' portions of two
different antibodies by gene fusion. The
antibody homodimers were reduced at the hinge region to form monomers and then
re-oxidized to form the
antibody heterodimers. This method can also be utilized for the production of
antibody homodimers. The
"diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA
90:6444-6448 (1993) has
provided an alternative mechanism for making bispecific antibody fragments.
The fragments comprise a VH
connected to a VL by a linker which is too short to allow pairing between the
two domains on the same chain.
Accordingly, the VH and VL domains of one fragment are forced to pair with the
complementary VL and VH
domains of another fragment, thereby forming two antigen-binding sites.
Another strategy for making
bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has
also been reported. See Gruber
et al., J. Immunol., 152:5368 (1994).
[0416] Antibodies with more than two valencies are contemplated. For example,
trispecific
antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).
6. HeteroconLgate Antibodies
[0417] Heteroconjugate antibodies are also within the scope of the present
invention.
Heteroconjugate antibodies are composed of two covalently joined antibodies.
Such antibodies have, for
example, been proposed to target immune system cells to unwanted cells [U.S.
Patent No. 4,676,980], and for
treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089]. It is
contemplated that the antibodies
may be prepared in vitro using known methods in synthetic protein chemistry,
including those involving
crosslinking agents. For example, immunotoxins may be constructed using a
disulfide exchange reaction or
by forming a thioether bond. Examples of suitable reagents for this purpose
include iminothiolate and methyl-
4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No.
4,676,980.
7. Multivalent Antibodies
[0418] A multivalent antibody may be internalized (and/or catabolized) faster
than a bivalent
antibody by a cell expressing an antigen to which the antibodies bind. The
antibodies of the present invention
can be multivalent antibodies (which are other than of the IgM class) with
three or more antigen binding sites
(e.g. tetravalent antibodies), which can be readily produced by recombinant
expression of nucleic acid
encoding the polypeptide chains of the antibody. The multivalent antibody can
comprise a dimerization
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domain and three or more antigen binding sites. The preferred dimerization
domain comprises (or consists of)
an Fc region or a hinge region. In this scenario, the antibody will comprise
an Fc region and three or more
antigen binding sites amino-terminal to the Fc region. The preferred
multivalent antibody herein comprises
(or consists of) three to about eight, but preferably four, antigen binding
sites. The multivalent antibody
comprises at least one polypeptide chain (and preferably two polypeptide
chains), wherein the polypeptide
chain(s) comprise two or more variable domains. For instance, the polypeptide
chain(s) may comprise VD1-
(X1)ri VD2-(X2)ri Fc, wherein VD1 is a first variable domain, VD2 is a second
variable domain, Fc is one
polypeptide chain of an Fc region, Xl and X2 represent an amino acid or
polypeptide, and n is 0 or 1. For
instance, the polypeptide chain(s) may comprise: VH-CH1-flexible linker-VH-CH1-
Fc region chain; or VH-
CH 1 -VH-CH 1 -Fc region chain. The multivalent antibody herein preferably
further comprises at least two (and
preferably four) light chain variable domain polypeptides. The multivalent
antibody herein may, for instance,
comprise from about two to about eight light chain variable domain
polypeptides. The light chain variable
domain polypeptides contemplated here comprise a light chain variable domain
and, optionally, further
comprise a CL domain.
8. Effector Function Engineering
[0419] It may be desirable to modify the antibody of the invention with
respect to effector function,
e.g., so as to enhance antigen-dependent cell-mediated cyotoxicity (ADCC)
and/or complement dependent
cytotoxicity (CDC) of the antibody. This may be achieved by introducing one or
more amino acid
substitutions in an Fc region of the antibody. Alternatively or additionally,
cysteine residue(s) may be
introduced in the Fc region, thereby allowing interchain disulfide bond
formation in this region. The
homodimeric antibody thus generated may have improved internalization
capability and/or increased
complement-mediated cell killing and antibody-dependent cellular cytotoxicity
(ADCC). See Caron et al., J.
Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922 (1992).
Homodimeric
antibodies with enhanced anti-tumor activity may also be prepared using
heterobifunctional cross-linkers as
described in Wolff et al., Cancer Research 53:2560-2565 (1993). Alternatively,
an antibody can be engineered
which has dual Fc regions and may thereby have enhanced complement lysis and
ADCC capabilities. See
Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989). To increase the
serum half life of the antibody,
one may incorporate a salvage receptor binding epitope into the antibody
(especially an antibody fragment) as
described in U.S. Patent 5,739,277, for example. As used herein, the term
"salvage receptor binding epitope"
refers to an epitope of the Fc region of an IgG molecule (e.g., IgGI, IgG2,
IgG3, or IgG4) that is responsible
for increasing the in vivo serum half-life of the IgG molecule.
9. ImmunoconLgates
[0420] The invention also pertains to immunoconjugates (interchangeably
referred to as "antibody-
drug conjugates," or "ADCs") comprising an antibody conjugated to a cytotoxic
agent such as a
chemotherapeutic agent, a growth inhibitory agent, a toxin (e.g., an
enzymatically active toxin of bacterial,
fungal, plant, or animal origin, or fragments thereof), or a radioactive
isotope (i.e., a radioconjugate).
[0421] In certain embodiments, an immunoconjugate comprises an antibody and a
chemotherapeutic agent or other toxin. Chemotherapeutic agents useful in the
generation of such
immunoconjugates have been described above. Enzymatically active toxins and
fragments thereof that can be
used include diphtheria A chain, nonbinding active fragments of diphtheria
toxin, exotoxin A chain (from
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Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-
sarcin, Aleurites fordii
proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and
PAP-S), momordica charantia
inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin,
enomycin, and the tricothecenes. A variety of radionuclides are available for
the production of
radioconjugated antibodies. Examples include 212 Bi,13'I 131In, 90Y, and'86Re.
Conjugates of the antibody and
cytotoxic agent are made using a variety of bifunctional protein-coupling
agents such as N-succinimidyl-3-(2-
pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional
derivatives of imidoesters (such as
dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate),
aldehydes (such as
glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl)
hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates
(such as tolyene 2,6-
diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-
dinitrobenzene). For example, a
ricin immunotoxin can be prepared as described in Vitetta et al., Science,
238: 1098 (1987). Carbon-14-
labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-
DTPA) is an exemplary
chelating agent for conjugation of radionucleotide to the antibody. See
W094/11026.
[0422] Conjugates of an antibody and one or more small molecule toxins, such
as a calicheamicin,
auristatin peptides, such as monomethylauristatin (MMAE) (synthetic analog of
dolastatin), maytansinoids,
such as DM1, a trichothene, and CC1065, and the derivatives of these toxins
that have toxin activity, are also
contemplated herein.
Exemplary Immunoconjugates - Antibody-Drug Conjugates
[0423] An immunoconjugate (or "antibody-drug conjugate" ("ADC")) of the
invention may be of
Formula I, below, wherein an antibody is conjugated (i.e., covalently
attached) to one or more drug moieties
(D) through an optional linker (L). ADCs may include thioMAb drug conjugates
("TDC").
Ab-(L-D)p 1
[0424] Accordingly, the antibody may be conjugated to the drug either directly
or via a linker. In
Formula I, p is the average number of drug moieties per antibody, which can
range, e.g., from about 1 to about
20 drug moieties per antibody, and in certain embodiments, from 1 to about 8
drug moieties per antibody. The
invention includes a composition comprising a mixture of antibody-drug
compounds of Formula I where the
average drug loading per antibody is about 2 to about 5, or about 3 to about
4.
a. Exemplary Linkers
[0425] A linker may comprise one or more linker components. Exemplary linker
components
include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-
citrulline ("val-cit" or "vc"),
alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl (a "PAB"), and
those resulting from
conjugation with linker reagents: N-Succinimidyl 4-(2-pyridylthio) pentanoate
("SPP"), N-succinimidyl 4-(N-
maleimidomethyl) cyclohexane-1 carboxylate ("SMCC", also referred to herein as
"MCC"), and N-
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Succinimidyl (4-iodo-acetyl) aminobenzoate ("SIAB"). Various linker components
are known in the art, some
of which are described below.
[0426] A linker may be a "cleavable linker," facilitating release of a drug in
the cell. For example,
an acid-labile linker (e.g., hydrazone), protease-sensitive (e.g., peptidase-
sensitive) linker, photolabile linker,
dimethyl linker or disulfide-containing linker (Chari et al., Cancer Research
52:127-13 1 (1992); U.S. Patent
No. 5,208,020) may be used.
[0427] In certain embodiments, a linker is as shown in the following Formula
II:
-Aa WW-Yy 11
wherein A is a stretcher unit, and a is an integer from 0 to 1; W is an amino
acid unit, and w is an integer from
0 to 12; Y is a spacer unit, and y is 0, 1, or 2; and Ab, D, and p are defined
as above for Formula I. Exemplary
embodiments of such linkers are described in US 2005-0238649 Al, which is
expressly incorporated herein by
reference.
[0428] In some embodiments, a linker component may comprise a "stretcher unit"
that links an
antibody to another linker component or to a drug moiety. Exemplary stretcher
units are shown below
(wherein the wavy line indicates sites of covalent attachment to an antibody):
O
O
0 MC
O 0
N
0 MP
O 0
N' N~/O~~O
i
H
0 MPEG
O
Av K NH
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[0429] In some embodiments, a linker component may comprise an amino acid
unit. In one such
embodiment, the amino acid unit allows for cleavage of the linker by a
protease, thereby facilitating release of
the drug from the immunoconjugate upon exposure to intracellular proteases,
such as lysosomal enzymes. See,
e.g., Doronina et al. (2003) Nat. Biotechnol. 21:778-784. Exemplary amino acid
units include, but are not
limited to, a dipeptide, a tripeptide, a tetrapeptide, and a pentapeptide.
Exemplary dipeptides include: valine-
citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe);
phenylalanine-lysine (flc or phe-lys); or N-
methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include: glycine-
valine-citrulline (gly-val-cit) and
glycine-glycine-glycine (gly-gly-gly). An amino acid unit may comprise amino
acid residues that occur
naturally, as well as minor amino acids and non-naturally occurring amino acid
analogs, such as citrulline.
Amino acid units can be designed and optimized in their selectivity for
enzymatic cleavage by a particular
enzyme, for example, a tumor-associated protease, cathepsin B, C and D, or a
plasmin protease.
[0430] In some embodiments, a linker component may comprise a "spacer" unit
that links the
antibody to a drug moiety, either directly or by way of a stretcher unit
and/or an amino acid unit. A spacer
unit may be "self-immolative" or a "non-self-immolative." A "non-self-
immolative" spacer unit is one in
which part or all of the spacer unit remains bound to the drug moiety upon
enzymatic (e.g., proteolytic)
cleavage of the ADC. Examples of non-self-immolative spacer units include, but
are not limited to, a glycine
spacer unit and a glycine-glycine spacer unit. Other combinations of peptidic
spacers susceptible to sequence-
specific enzymatic cleavage are also contemplated. For example, enzymatic
cleavage of an ADC containing a
glycine-glycine spacer unit by a tumor-cell associated protease would result
in release of a glycine-glycine-
drug moiety from the remainder of the ADC. In one such embodiment, the glycine-
glycine-drug moiety is
then subjected to a separate hydrolysis step in the tumor cell, thus cleaving
the glycine-glycine spacer unit
from the drug moiety.
[0431] A "self-immolative" spacer unit allows for release of the drug moiety
without a separate
hydrolysis step. In certain embodiments, a spacer unit of a linker comprises a
p-aminobenzyl unit. In one
such embodiment, a p-aminobenzyl alcohol is attached to an amino acid unit via
an amide bond, and a
carbamate, methylcarbamate, or carbonate is made between the benzyl alcohol
and a cytotoxic agent. See, e.g.,
Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15:1087-1103. In one
embodiment, the spacer unit is
p-aminobenzyloxycarbonyl (PAB). In certain embodiments, the phenylene portion
of a p-amino benzyl unit is
substituted with Qm, wherein Q is -Ci-CB alkyl, -O-(C1-C8 alkyl), -halogen,-
nitro or -cyano; and m is an
integer ranging from 0-4. Examples of self-immolative spacer units further
include, but are not limited to,
aromatic compounds that are electronically similar to p-aminobenzyl alcohol
(see, e.g., US 2005/0256030 Al),
such as 2-aminoimidazol-5-methanol derivatives (Hay et al. (1999) Bioorg. Med.
Chem. Lett. 9:2237) and
ortho- or para-aminobenzylacetals. Spacers can be used that undergo
cyclization upon amide bond hydrolysis,
such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et
al., Chemistry Biology, 1995,
2, 223); appropriately substituted bicyclo[2.2. 1] and bicyclo[2.2.2] ring
systems (Storm, et al., J. Amer. Chem.
Soc., 1972, 94, 5815); and 2-aminophenylpropionic acid amides (Amsberry, et
al., J. Org. Chem., 1990, 55,
5867). Elimination of amine-containing drugs that are substituted at the a-
position of glycine (Kingsbury, et al.,
J. Med. Chem., 1984, 27, 1447) are also examples of self-immolative spacers
useful in ADCs.
[0432] In one embodiment, a spacer unit is a branched
bis(hydroxymethyl)styrene (BHMS) unit as
depicted below, which can be used to incorporate and release multiple drugs.
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0
11
Qm CH2(OC)n-D
Ab A -W -NH ~ I ~ / O
a `" CH2(OC)n-D
enzymatic
cleavage
2 drugs
wherein Q is -Ci-CB alkyl, -O-(Ci-C8 alkyl), -halogen, -nitro or -cyano; m is
an integer ranging from 0-4; n is 0
or 1; and p ranges raging from 1 to about 20.
[0433] In another embodiment, linker L may be a dendritic type linker for
covalent attachment of
more than one drug moiety through a branching, multifunctional linker moiety
to an antibody (Sun et al (2002)
Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al (2003)
Bioorganic & Medicinal
Chemistry 11:1761-1768). Dendritic linkers can increase the molar ratio of
drug to antibody, i.e. loading,
which is related to the potency of the ADC. Thus, where a cysteine engineered
antibody bears only one
reactive cysteine thiol group, a multitude of drug moieties may be attached
through a dendritic linker.
[0434] Exemplary linker components and combinations thereof are shown below in
the context of
ADCs of Formula II:
N N Yy-D
H Ab (Aa_ )
H O - P
HN
O~NH2
Val-Cit or VC
O
0 H 0
Ab N N N~Yy-D
O H O -
p
HN
O~NH2
MC-val-cit
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0
~
0 0 H 0 D
Ab N N N
p p
~ H p
HN
N H 2 MC-val-cit-PAB
[0435] Linkers components, including stretcher, spacer, and amino acid units,
may be synthesized
by methods known in the art, such as those described in US 2005-0238649 Al.
b. Exemplary Drug Moieties
(1) Maytansine and maytansinoids
[0436] In some embodiments, an immunoconjugate comprises an antibody
conjugated to one or
more maytansinoid molecules. Maytansinoids are mitototic inhibitors which act
by inhibiting tubulin
polymerization. Maytansine was first isolated from the east African shrub
Maytenus serrata (U. S. Patent No.
3896111). Subsequently, it was discovered that certain microbes also produce
maytansinoids, such as
maytansinol and C-3 maytansinol esters (U.S. Patent No. 4,151,042). Synthetic
maytansinol and derivatives
and analogues thereof are disclosed, for example, in U.S. Patent Nos.
4,137,230; 4,248,870; 4,256,746;
4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428;
4,313,946; 4,315,929;
4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254;
4,362,663; and 4,371,533.
[0437] Maytansinoid drug moieties are attractive drug moieties in antibody-
drug conjugates
because they are: (i) relatively accessible to prepare by fermentation or
chemical modification or
derivatization of fermentation products, (ii) amenable to derivatization with
functional groups suitable for
conjugation through disulfide and non-disulfide linkers to antibodies, (iii)
stable in plasma, and (iv) effective
against a variety of tumor cell lines.
[0438] Maytansine compounds suitable for use as maytansinoid drug moieties are
well known in
the art and can be isolated from natural sources according to known methods or
produced using genetic
engineering and fermentation techniques (US 6790952; US 2005/0170475; Yu et al
(2002) PNAS 99:7968-
7973). Maytansinol and maytansinol analogues may also be prepared
synthetically according to known
methods.
[0439] Exemplary maytansinoid drug moieties include those having a modified
aromatic ring, such
as: C-19-dechloro (US Pat. No. 4256746) (prepared by lithium aluminum hydride
reduction of ansamytocin
P2); C-20-hydroxy (or C-20-demethyl) +/-C-19-dechloro (US Pat. Nos. 4361650
and 4307016) (prepared by
demethylation using Streptomyces or Actinomyces or dechlorination using LAH);
and C-20-demethoxy, C-20-
acyloxy (-OCOR), +/-dechloro (U.S. Pat. No. 4,294,757) (prepared by acylation
using acyl chlorides). and
those having modifications at other positions.
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[0440] Exemplary maytansinoid drug moieties also include those having
modifications such as: C-
9-SH (US Pat. No. 4424219) (prepared by the reaction of maytansinol with HzS
or PzSs); C-14-
alkoxymethyl(demethoxy/CH2 OR)(US 4331598); C-14-hydroxymethyl or
acyloxymethyl (CHzOH or
CHzOAc) (US Pat. No. 4450254) (prepared from Nocardia); C-15-hydroxy/acyloxy
(US 4364866) (prepared
by the conversion of maytansinol by Streptomyces); C-15-methoxy (US Pat. Nos.
4313946 and 4315929)
(isolated from Trewia nudlflora); C-18-N-demethyl (US Pat. Nos. 4362663 and
4322348) (prepared by the
demethylation of maytansinol by Streptomyces); and 4,5-deoxy (US 4371533)
(prepared by the titanium
trichloride/LAH reduction of maytansinol).
[0441] Many positions on maytansine compounds are known to be useful as the
linkage position,
depending upon the type of link. For example, for forming an ester linkage,
the C-3 position having a
hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15
position modified with a hydroxyl
group and the C-20 position having a hydroxyl group are all suitable (US
5208020; US RE39151; US
6913748; US 7368565; US 2006/0167245; US 2007/0037972).
[0442] Maytansinoid drug moieties include those having the structure:
H3C (CR2)m-S-
0
~ O
H3C 0 0
CI N O
CH3O
0
N 11~
O
CH3OO H
where the wavy line indicates the covalent attachment of the sulfur atom of
the maytansinoid drug moiety to a
linker of an ADC. R may independently be H or a Ci-C6 alkyl. The alkylene
chain attaching the amide group
to the sulfur atom may be methanyl, ethanyl, or propyl, i.e., m is 1, 2, or 3
(US 633410; US 5208020; US
7276497; Chari et al (1992) CancerRes. 52:127-131; Liu et al (1996) Proc.
Natl. Acad. Sci USA 93:8618-
8623).
[0443] All stereoisomers of the maytansinoid drug moiety are contemplated for
the compounds of
the invention, i.e. any combination of R and S configurations at the chiral
carbons of D. In one embodiment,
the maytansinoid drug moiety will have the following stereochemistry:
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H3C (CR2)m-S-
O N--(
H3C 0 CI ~N
CH3O
N
CH3OHO H
[0444] Exemplary embodiments of maytansinoid drug moieities include: DM1; DM3;
and DM4,
having the structures:
H3C CH2CH2S
O N--~O
H3C 0 CI N = 0
\ =``~~~ DM1
CH30
O
N -'-~O
CH3OHO
H
ICH3
H3C CH2CH2C-S
O N-~
H3C 0 O
CI ~N = O
\ =~~~~~
CH30 DM3
O
~ ~ - N
CH3OHO
H
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ICH3
H3C CH2CH2C-S
O N4 I
>__~ O CH3
H3C O O_
CI ~N = 0
DM4
CH3O
O
~ ~ - N
CH3OHO
H
wherein the wavy line indicates the covalent attachment of the sulfur atom of
the drug to a linker (L) of an
antibody-drug conjugate. (WO 2005/037992; US 2005/0276812 Al).
[0445] Other exemplary maytansinoid antibody-drug conjugates have the
following structures and
abbreviations, (wherein Ab is antibody and p is 1 to about 8):
O
N Ab
S-S H p
OH3CN~
H3 O O
C O N O
~C
CH30
O
/~
HON O
CH3O H Ab -SPP-DM1
N Ab
I
S-S H p
H3q
O N
O
CIH3C N O O 0
CH3O ~ \
O
/ / HO NO
CH3O H
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Ab-SPDB-DM4
O
O N Ab
H p
N
S
H3C 0
O N
O
CIH3C O O O
N
CH3O
O
_
HON O
CH3O H
Ab-SMCC-DM1
[0446] Exemplary antibody-drug conjugates where DM1 is linked through a BMPEO
linker to a
thiol group of the antibody have the structure and abbreviation:
O
O Ab
NO 'O'~O~'
n O p
OH3C CH2CH2S
N
O
C 0 O O
CIH3 N
CH30
O
HO N~0
=
CH3O H
where Ab is antibody; n is 0, 1, or 2; and p is 1, 2, 3, or 4.
[0447] Immunoconjugates containing maytansinoids, methods of making the same,
and their
therapeutic use are disclosed, for example, in Erickson, et al (2006) Cancer
Res. 66(8):4426-4433; U.S. Patent
Nos. 5,208,020, 5,416,064, US 2005/0276812 Al, and European Patent EP 0 425
235 Bl, the disclosures of
which are hereby expressly incorporated by reference.
[0448] Antibody-maytansinoid conjugates are prepared by chemically linking an
antibody to a
maytansinoid molecule without significantly diminishing the biological
activity of either the antibody or the
maytansinoid molecule. See, e.g., U.S. Patent No. 5,208,020 (the disclosure of
which is hereby expressly
incorporated by reference). Maytansinoids can be synthesized by known
techniques or isolated from natural
sources. Suitable maytansinoids are disclosed, for example, in U.S. Patent No.
5,208,020 and in the other
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patents and nonpatent publications referred to hereinabove, such as
maytansinol and maytansinol analogues
modified in the aromatic ring or at other positions of the maytansinol
molecule, such as various maytansinol
esters.
[0449] There are many linking groups known in the art for making antibody-
maytansinoid
conjugates, including, for example, those disclosed in U.S. Patent No. 5208020
or EP Patent 0 425 235 B 1;
Chari et al. Cancer Research 52:127-131 (1992); and US 2005/016993 Al, the
disclosures of which are
hereby expressly incorporated by reference. Antibody-maytansinoid conjugates
comprising the linker
component SMCC may be prepared as disclosed in US 2005/0276812 Al, "Antibody-
drug conjugates and
Methods." The linkers comprise disulfide groups, thioether groups, acid labile
groups, photolabile groups,
peptidase labile groups, or esterase labile groups, as disclosed in the above-
identifled patents. Additional
linkers are described and exemplified herein.
[0450] Conjugates of the antibody and maytansinoid may be made using a variety
of bifunctional
protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate
(SPDP), succinimidyl-4-(N-
maleimidomethyl) cyclohexane- 1 -carboxylate (SMCC), iminothiolane (IT),
bifunctional derivatives of
imidoesters (such as dimethyl adipimidate HCI), active esters (such as
disuccinimidyl suberate), aldehydes
(such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl)
hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates
(such as toluene 2,6-
diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-
dinitrobenzene). In certain
embodiments, the coupling agent is N-succinimidyl-3-(2-pyridyldithio)
propionate (SPDP) (Carlsson et al.,
Biochem. J. 173:723-737 (1978)) or N-succinimidyl-4-(2-pyridylthio)pentanoate
(SPP) to provide for a
disulfide linkage.
[0451] The linker may be attached to the maytansinoid molecule at various
positions, depending on
the type of the link. For example, an ester linkage may be formed by reaction
with a hydroxyl group using
conventional coupling techniques. The reaction may occur at the C-3 position
having a hydroxyl group, the C-
14 position modified with hydroxymethyl, the C- 15 position modified with a
hydroxyl group, and the C-20
position having a hydroxyl group. In one embodiment, the linkage is formed at
the C-3 position of
maytansinol or a maytansinol analogue.
(2) Auristatins and dolastatins
[0452] In some embodiments, an immunoconjugate comprises an antibody
conjugated to dolastatin
or a dolastatin peptidic analog or derivative, e.g., an auristatin (US Pat.
Nos. 5635483; 5780588). Dolastatins
and auristatins have been shown to interfere with microtubule dynamics, GTP
hydrolysis, and nuclear and
cellular division (Woyke et al (2001) Antimicrob. Agents and Chemother.
45(12):3580-3584) and have
anticancer (US Pat. No.5663149) and antifungal activity (Pettit et al (1998)
Antimicrob. Agents Chemother.
42:2961-2965). The dolastatin or auristatin drug moiety may be attached to the
antibody through the N
(amino) terminus or the C (carboxyl) terminus of the peptidic drug moiety (WO
02/088172).
[0453] Exemplary auristatin embodiments include the N-terminus linked
monomethylauristatin
drug moieties DE and DF (US2005/0238649, disclosed in Senter et al,
Proceedings of the American
Association for Cancer Research, Volume 45, Abstract Number 623, presented
March 28, 2004, the disclosure
of which is expressly incorporated by reference in its entirety).
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[0454] A peptidic drug moiety may be selected from Formulas DE and DF below:
R3 0 R7 CH3 R9
H r I
~N- N ~
N
N N R1$
R2 O R4 R5 R6 R8 0 R8 O DE
R3 0 R7 CH3 R9 0
N N N Z R' 1
N N
R2 0 R4 R5 R6 R8 0 R8 0
R10 DF
wherein the wavy line of DE and DF indicates the covalent attachment site to
an antibody or antibody-linker
component, and independently at each location:
R2 is selected from H and Ci-Cs alkyl;
R3 is selected from H, Ci-C8 alkyl, C3-C8 carbocycle, aryl, Ci-C8 alkyl-aryl,
Ci-C8 a1ky1-(C3-C8
carbocycle), C3-C8 heterocycle and Ci-C8 a1ky1-(C3-C8 heterocycle);
R4 is selected from H, Ci-C8 alkyl, C3-C8 carbocycle, aryl, Ci-C8 alkyl-aryl,
Ci-C8 a1ky1-(C3-C8
carbocycle), C3-Cs heterocycle and Ci-Cs a1ky1-(C3-Cs heterocycle);
Rs is selected from H and methyl;
or R4 and Rs jointly form a carbocyclic ring and have the formula -(CRaRb)õ-
wherein Ra and Rb are
independently selected from H, Ci-C8 alkyl and C3-C8 carbocycle and n is
selected from 2, 3, 4, 5 and 6;
R6 is selected from H and Ci-C8 alkyl;
R' is selected from H, Ci-Cs alkyl, C3-Cs carbocycle, aryl, Ci-Cs alkyl-aryl,
Ci-Cs a1ky1-(C3-Cs
carbocycle), C3-Cs heterocycle and Ci-Cs a1ky1-(C3-Cs heterocycle);
each R8 is independently selected from H, OH, Ci-Cs alkyl, C3-Cs carbocycle
and O-(Ci-Cs alkyl);
R9is selected from H and Ci-C8 alkyl;
R10 is selected from aryl or C3-C8 heterocycle;
Z is 0, S, NH, or NR'2, wherein R'2 is Ci-C8 alkyl;
R" is selected from H, Ci-Czo alkyl, aryl, Cs-Cs heterocycle, -(R13O)mR14, or -
(R130)mCH(R's)
ti
m is an integer ranging from 1-1000;
R13 is C2-C8 alkyl;
R14 is H or Ci-C8 alkyl;
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each occurrence of R's is independently H, COOH, -(CH2)õ-N(R16)2, -(CH2)õ-
SO3H, or -(CHz)ri
S03-Ci-CB alkyl;
each occurrence of R16 is independently H, Ci-C8 alkyl, or -(CHz)ri COOH;
R18 is selected from -C(R8)z-C(R8)z-aryl, -C(R8)2-C(R8)2-(C3-C8 heterocycle),
and
-C(R8)2_C(R8)z (C3-C8 carbocycle); and
n is an integer ranging from 0 to 6.
[0455] In one embodiment, R3, R4 and R' are independently isopropyl or sec-
butyl and Rs is -H or
methyl. In an exemplary embodiment, R3 and R4 are each isopropyl, Rs is -H,
and R7 is sec-butyl.
[0456] In yet another embodiment, R2 and R6 are each methyl, and R9 is -H.
[0457] In still another embodiment, each occurrence of R8 is -OCH3.
[0458] In an exemplary embodiment, R3 and R4 are each isopropyl, R2 and R6 are
each methyl, Rs is
-H, R' is sec-butyl, each occurrence of R8 is -OCH3, and R9 is -H.
[0459] In one embodiment, Z is -0- or -NH-.
[0460] In one embodiment, R10 is aryl.
[0461] In an exemplary embodiment, R10 is -phenyl.
[0462] In an exemplary embodiment, when Z is -0-, Ri i is -H, methyl or t-
butyl.
[0463] In one embodiment, when Z is -NH, R" is -CH(R's)z, wherein R's is -
(CHz)ri N(R16)z, and
R16 is -Ci-CB alkyl or -(CHz)õ-COOH.
[0464] In another embodiment, when Z is -NH, R" is -CH(R's)z, wherein R's is -
(CH2)õ-S03H.
[0465] An exemplary auristatin embodiment of formula DE is MMAE, wherein the
wavy line
indicates the covalent attachment to a linker (L) of an antibody-drug
conjugate:
O H OH
N N N N N ~
I O O\ yy"
O O~ O /
MMAE
[0466] An exemplary auristatin embodiment of formula DF is MMAF, wherein the
wavy line
indicates the covalent attachment to a linker (L) of an antibody-drug
conjugate (see US 2005/0238649 and
Doronina et al. (2006) Bioconjugate Chem. 17:114-124):
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WO 2009/012256 PCT/US2008/070061
H 0
N,,, YN~( N H
N ' N
~ MMAF
I O I O\ O O~ O O OH ~
[0467] Other exemplary embodiments include monomethylvaline compounds having
phenylalanine
carboxy modifications at the C-terminus of the pentapeptide auristatin drug
moiety (WO 2007/008848) and
monomethylvaline compounds having phenylalanine sidechain modifications at the
C-terminus of the
pentapeptide auristatin drug moiety (WO 2007/008603).
[0468] Other drug moieties include the following MMAF derivatives, wherein the
wavy line
indicates the covalent attachment to a linker (L) of an antibody-drug
conjugate:
O O
N N N g
~ O H OCH3O OCH3O
O
H
N N N
y N
rr r
N
O O_ 0
O~ O I/
O O
O
N N N N N
I \
~
O O\ O O~ O O%'NH /
H ~
O \
10_,r O\
y r y- N N
N
O I OCH3 O H
OCH3 O 0
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O
N N N N N
O O_ O O1-1 O I/
O NH
~
N
O
/N N N N N
O O_ 0
O~ 0 0 O
~
HOOCI-/ N,---,COOH
O
N NN N H
O O'~, O
O1-1 O O NHI \
/
~
SO3H
~ O
H
N N N N N
O O_ 0
Ol-~ O I/
O NH
HOOC --ll COOH and
O
N NN N N
0 O_ O
O~ 0 O NHI \
/
NH2
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[0469] In one aspect, hydrophilic groups including but not limited to,
triethylene glycol esters
(TEG), as shown above, can be attached to the drug moiety at R". Without being
bound by any particular
theory, the hydrophilic groups assist in the internalization and non-
agglomeration of the drug moiety.
[0470] Exemplary embodiments of ADCs of Formula I comprising an
auristatin/dolastatin or
derivative thereof are described in US 2005-0238649 and Doronina et al. (2006)
Bioconjugate Chem. 17:114-
124, which is expressly incorporated herein by reference. Exemplary
embodiments of ADCs of Formula I
comprising MMAE or MMAF and various linker components have the following
structures and abbreviations
(wherein "Ab" is an antibody; p is 1 to about 8, "Val-Cit" or "vc" is a valine-
citrulline dipeptide; and "S" is a
sulfur atom. It will be noted that in certain of the structural descriptions
of sulfur linked ADC herein the
antibody is represented as "Ab-S" merely to indicate the sulfur link feature
and not to indicate that a particular
sulfur atom bears multiple linker-drug moieties. The left parentheses of the
following structures may also be
placed to the left of the sulfur atom, between Ab and S, which would be an
equivalent description of the ADC
of the invention described throughout herein.
Ab- O H O
S
N
O ONN
~I O O O
NV al-Cit-N" ' 00
I
H , O HO p
Ab-MC-vc-PAB-MMAF
AbS O H O ~~'N),X OH
O O ~ O~N~N N
I I O O O
I
N`~~Val-Cit-N" v ~ O, O
O H p
Ab-MC-vc-PAB-MMAE
Ab-S
O H OH
N N N,".. N N
O O O'O O\ O
Ab-MC-MMAE
Ab-S
O
O H H
-1-1-1)-' N NN N
V N
N
:~_X
O I I O"O O"O O O o
H
Ab-MC-MMAF
[0471] Exemplary embodiments of ADCs of Formula I comprising MMAF and various
linker
components further include Ab-MC-PAB-MMAF and Ab-PAB-MMAF. Interestingly,
immunoconjugates
comprising MMAF attached to an antibody by a linker that is not
proteolytically cleavable have been shown
to possess activity comparable to immunoconjugates comprising MMAF attached to
an antibody by a
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WO 2009/012256 PCT/US2008/070061
proteolytically cleavable linker. See, Doronina et al. (2006) Bioconjugate
Chem. 17:114-124. In such
instances, drug release is believed to be effected by antibody degradation in
the cell. Id.
[0472] Typically, peptide-based drug moieties can be prepared by forming a
peptide bond between
two or more amino acids and/or peptide fragments. Such peptide bonds can be
prepared, for example,
according to the liquid phase synthesis method (see E. Schr6der and K. Lubke,
"The Peptides", volume 1, pp
76-136, 1965, Academic Press) that is well known in the field of peptide
chemistry. Auristatin/dolastatin
drug moieties may be prepared according to the methods of: US 2005-0238649 Al;
US Pat. No.5635483; US
Pat. No.5780588; Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465; Pettit
et al (1998) Anti-Cancer Drug
Design 13:243-277; Pettit, G.R., et al. Synthesis, 1996, 719-725; Pettit et al
(1996) J. Chem. Soc. Perkin
Trans. 1 5:859-863; and Doronina (2003) Nat. Biotechnol. 21(7):778-784.
[0473] In particular, auristatin/dolastatin drug moieties of formula DF, such
as MMAF and
derivatives thereof, may be prepared using methods described in US 2005-
0238649 Al and Doronina et al.
(2006) Bioconjugate Chem. 17:114-124. Auristatin/dolastatin drug moieties of
formula DE, such as MMAE
and derivatives thereof, may be prepared using methods described in Doronina
et al. (2003) Nat. Biotech.
21:778-784. Drug-linker moieties MC-MMAF, MC-MMAE, MC-vc-PAB-MMAF, and MC-vc-
PAB-
MMAE may be conveniently synthesized by routine methods, e.g., as described in
Doronina et al. (2003) Nat.
Biotech. 21:778-784, and Patent Application Publication No. US 2005/0238649
Al, and then conjugated to
an antibody of interest.
(3) Calicheamicin
[0474] In other embodiments, the immunoconjugate comprises an antibody
conjugated to one or
more calicheamicin molecules. The calicheamicin family of antibiotics are
capable of producing double-
stranded DNA breaks at sub-picomolar concentrations. For the preparation of
conjugates of the
calicheamicin family, see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116,
5,767,285, 5,770,701, 5,770,710,
5,773,001, 5,877,296 (all to American Cyanamid Company). Structural analogues
of calicheamicin which
may be used include, but are not limited to, yi, azi, a3, N-acetyl-yii, PSAG
and 01i (Hinman et al., Cancer
Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928
(1998), and the aforementioned
U.S. patents to American Cyanamid). Another anti-tumor drug to which the
antibody can be conjugated is
QFA, which is an antifolate. Both calicheamicin and QFA have intracellular
sites of action and do not
readily cross the plasma membrane. Therefore, cellular uptake of these agents
through antibody-mediated
internalization greatly enhances their cytotoxic effects.
c. Other cytotoxic agents
[0475] Other antitumor agents that can be conjugated to an antibody include
BCNU, streptozocin,
vincristine and 5-fluorouracil, the family of agents known collectively as the
LL-E33288 complex, described
in US Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (US Pat. No.
5,877,296).
[0476] Enzymatically active toxins and fragments thereof which can be used
include diphtheria A
chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from
Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii
proteins, dianthin proteins,
Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia
inhibitor, curcin, crotin,
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sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin and the tricothecenes.
See, for example, WO 93/21232 published October 28, 1993.
[0477] The present invention further contemplates an immunoconjugate formed
between an
antibody and a compound with nucleolytic activity (e.g., a ribonuclease or a
DNA endonuclease such as a
deoxyribonuclease; DNase).
[0478] In certain embodiments, an immunoconjugate may comprise a highly
radioactive atom. A
variety of radioactive isotopes are available for the production of
radioconjugated antibodies. Examples
include At211, 1131, 1125 Y90 Re186, Re'88, Smi53 Bi212, P32, Pb212 and
radioactive isotopes of Lu. When the
immunoconjugate is used for detection, it may comprise a radioactive atom for
scintigraphic studies, for
example tc99m or I123, or a spin label for nuclear magnetic resonance (NMR)
imaging (also known as magnetic
resonance imaging, mri), such as iodine-123, iodine-13 1, indium-111, fluorine-
19, carbon-13, nitrogen-15,
oxygen-17, gadolinium, manganese or iron.
[0479] The radio- or other labels may be incorporated in the immunoconjugate
in known ways. For
example, the peptide may be biosynthesized or may be synthesized by chemical
amino acid synthesis using
suitable amino acid precursors involving, for example, fluorine-19 in place of
hydrogen. Labels such as tc99m
or I123, Re186, Re'88 and In"' can be attached via a cysteine residue in the
peptide. Yttrium-90 can be attached
via a lysine residue. The IODOGEN method (Fraker et al (1978) Biochem.
Biophys. Res. Commun. 80: 49-57
can be used to incorporate iodine- 123. "Monoclonal Antibodies in
Immunoscintigraphy" (Chatal, CRC Press
1989) describes other methods in detail.
[0480] In certain embodiments, an immunoconjugate may comprise an antibody
conjugated to a
prodrug-activating enzyme that converts a prodrug (e.g., a peptidyl
chemotherapeutic agent, see WO
81/01145) to an active drug, such as an anti-cancer drug. Such
immunoconjugates are useful in antibody-
dependent enzyme-mediated prodrug therapy ("ADEPT"). Enzymes that may be
conjugated to an antibody
include, but are not limited to, alkaline phosphatases, which are useful for
converting phosphate-containing
prodrugs into free drugs; arylsulfatases, which are useful for converting
sulfate-containing prodrugs into free
drugs; cytosine deaminase, which is useful for converting non-toxic 5-
fluorocytosine into the anti-cancer
drug, 5-fluorouracil; proteases, such as serratia protease, thermolysin,
subtilisin, carboxypeptidases and
cathepsins (such as cathepsins B and L), which are useful for converting
peptide-containing prodrugs into
free drugs; D-alanylcarboxypeptidases, which are useful for converting
prodrugs that contain D-amino acid
substituents; carbohydrate-cleaving enzymes such as (3-galactosidase and
neuraminidase, which are useful for
converting glycosylated prodrugs into free drugs; (3-lactamase, which is
useful for converting drugs
derivatized with (3-lactams into free drugs; and penicillin amidases, such as
penicillin V amidase and
penicillin G amidase, which are useful for converting drugs derivatized at
their amine nitrogens with
phenoxyacetyl or phenylacetyl groups, respectively, into free drugs. Enzymes
may be covalently bound to
antibodies by recombinant DNA techniques well known in the art. See, e.g.,
Neuberger et al., Nature
312:604-608 (1984).
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d. Drug Loading
[0481] Drug loading is represented by p, the average number of drug moieties
per antibody in a
molecule of Formula I. Drug loading may range from 1 to 20 drug moieties (D)
per antibody. ADCs of
Formula I include collections of antibodies conjugated with a range of drug
moieties, from 1 to 20. The
average number of drug moieties per antibody in preparations of ADC from
conjugation reactions may be
characterized by conventional means such as mass spectroscopy, ELISA assay,
and HPLC. The quantitative
distribution of ADC in terms of p may also be determined. In some instances,
separation, purification, and
characterization of homogeneous ADC where p is a certain value from ADC with
other drug loadings may be
achieved by means such as reverse phase HPLC or electrophoresis.
Pharmaceutical formulations of Formula
I antibody-drug conjugates may thus be a heterogeneous mixture of such
conjugates with antibodies linked to
1, 2, 3, 4 or more drug moieties.
[0482] For some antibody-drug conjugates, p may be limited by the number of
attachment sites on
the antibody. For example, where the attachment is a cysteine thiol, as in the
exemplary embodiments above,
an antibody may have only one or several cysteine thiol groups, or may have
only one or several sufficiently
reactive thiol groups through which a linker may be attached. In certain
embodiments, higher drug loading,
e.g. p >5, may cause aggregation, insolubility, toxicity, or loss of cellular
permeability of certain antibody-
drug conjugates. In certain embodiments, the drug loading for an ADC of the
invention ranges from 1 to
about 8; from about 2 to about 6; or from about 3 to about 5. Indeed, it has
been shown that for certain ADCs,
the optimal ratio of drug moieties per antibody may be less than 8, and may be
about 2 to about 5. See US
2005-0238649 Al.
[0483] In certain embodiments, fewer than the theoretical maximum of drug
moieties are
conjugated to an antibody during a conjugation reaction. An antibody may
contain, for example, lysine
residues that do not react with the drug-linker intermediate or linker
reagent, as discussed below. Generally,
antibodies do not contain many free and reactive cysteine thiol groups which
may be linked to a drug moiety;
indeed most cysteine thiol residues in antibodies exist as disulfide bridges.
In certain embodiments, an
antibody may be reduced with a reducing agent such as dithiothreitol (DTT) or
tricarbonylethylphosphine
(TCEP), under partial or total reducing conditions, to generate reactive
cysteine thiol groups. In certain
embodiments, an antibody is subjected to denaturing conditions to reveal
reactive nucleophilic groups such as
lysine or cysteine.
[0484] The loading (drug/antibody ratio) of an ADC may be controlled in
different ways, e.g., by:
(i) limiting the molar excess of drug-linker intermediate or linker reagent
relative to antibody, (ii) limiting the
conjugation reaction time or temperature, and (iii) partial or limiting
reductive conditions for cysteine thiol
modification.
[0485] It is to be understood that where more than one nucleophilic group
reacts with a drug-linker
intermediate or linker reagent followed by drug moiety reagent, then the
resulting product is a mixture of
ADC compounds with a distribution of one or more drug moieties attached to an
antibody. The average
number of drugs per antibody may be calculated from the mixture by a dual
ELISA antibody assay, which is
specific for antibody and specific for the drug. Individual ADC molecules may
be identified in the mixture
by mass spectroscopy and separated by HPLC, e.g. hydrophobic interaction
chromatography (see, e.g.,
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McDonagh et al (2006) Prot. Engr. Design & Selection 19(7):299-307; Hamblett
et al (2004) Clin. Cancer
Res. 10:7063-7070; Hamblett, K.J., et al. "Effect of drug loading on the
pharmacology, pharmacokinetics,
and toxicity of an anti-CD30 antibody-drug conjugate," Abstract No. 624,
American Association for Cancer
Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR,
Volume 45, March 2004;
Alley, S.C., et al. "Controlling the location of drug attachment in antibody-
drug conjugates," Abstract No.
627, American Association for Cancer Research, 2004 Annual Meeting, March 27-
31, 2004, Proceedings of
the AACR, Volume 45, March 2004). In certain embodiments, a homogeneous ADC
with a single loading
value may be isolated from the conjugation mixture by electrophoresis or
chromatography.
e. Certain Methods of Preparing Immunconjugates
[0486] An ADC of Formula I may be prepared by several routes employing organic
chemistry
reactions, conditions, and reagents known to those skilled in the art,
including: (1) reaction of a nucleophilic
group of an antibody with a bivalent linker reagent to form Ab-L via a
covalent bond, followed by reaction
with a drug moiety D; and (2) reaction of a nucleophilic group of a drug
moiety with a bivalent linker reagent,
to form D-L, via a covalent bond, followed by reaction with a nucleophilic
group of an antibody. Exemplary
methods for preparing an ADC of Formula I via the latter route are described
in US 2005-023 8649 Al, which
is expressly incorporated herein by reference.
[0487] Nucleophilic groups on antibodies include, but are not limited to: (i)
N-terminal amine
groups, (ii) side chain amine groups, e.g. lysine, (iii) side chain thiol
groups, e.g. cysteine, and (iv) sugar
hydroxyl or amino groups where the antibody is glycosylated. Amine, thiol, and
hydroxyl groups are
nucleophilic and capable of reacting to form covalent bonds with electrophilic
groups on linker moieties and
linker reagents including: (i) active esters such as NHS esters, HOBt esters,
haloformates, and acid halides;
(ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes,
ketones, carboxyl, and maleimide groups.
Certain antibodies have reducible interchain disulfides, i.e. cysteine
bridges. Antibodies may be made
reactive for conjugation with linker reagents by treatment with a reducing
agent such as DTT (dithiothreitol)
or tricarbonylethylphosphine (TCEP), such that the antibody is fully or
partially reduced. Each cysteine
bridge will thus form, theoretically, two reactive thiol nucleophiles.
Additional nucleophilic groups can be
introduced into antibodies through modification of lysine residues, e.g., by
reacting lysine residues with 2-
iminothiolane (Traut's reagent), resulting in conversion of an amine into a
thiol. Reactive thiol groups may
be introduced into an antibody by introducing one, two, three, four, or more
cysteine residues (e.g., by
preparing variant antibodies comprising one or more non-native cysteine amino
acid residues).
[0488] Antibody-drug conjugates of the invention may also be produced by
reaction between an
electrophilic group on an antibody, such as an aldehyde or ketone carbonyl
group, with a nucleophilic group
on a linker reagent or drug. Useful nucleophilic groups on a linker reagent
include, but are not limited to,
hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate,
and arylhydrazide. In one
embodiment, an antibody is modified to introduce electrophilic moieties that
are capable of reacting with
nucleophilic substituents on the linker reagent or drug. In another
embodiment, the sugars of glycosylated
antibodies may be oxidized, e.g. with periodate oxidizing reagents, to form
aldehyde or ketone groups which
may react with the amine group of linker reagents or drug moieties. The
resulting imine Schiff base groups
may form a stable linkage, or may be reduced, e.g. by borohydride reagents to
form stable amine linkages. In
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one embodiment, reaction of the carbohydrate portion of a glycosylated
antibody with either galactose
oxidase or sodium meta-periodate may yield carbonyl (aldehyde and ketone)
groups in the antibody that can
react with appropriate groups on the drug (Hermanson, Bioconjugate
Techniques). In another embodiment,
antibodies containing N-terminal serine or threonine residues can react with
sodium meta-periodate, resulting
in production of an aldehyde in place of the first amino acid (Geoghegan &
Stroh, (1992) Bioconjugate Chem.
3:138-146; US 5362852). Such an aldehyde can be reacted with a drug moiety or
linker nucleophile.
[0489] Nucleophilic groups on a drug moiety include, but are not limited to:
amine, thiol, hydroxyl,
hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and
arylhydrazide groups capable of
reacting to form covalent bonds with electrophilic groups on linker moieties
and linker reagents including: (i)
active esters such as NHS esters, HOBt esters, haloformates, and acid halides;
(ii) alkyl and benzyl halides
such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide
groups.
[0490] The compounds of the invention expressly contemplate, but are not
limited to, ADC
prepared with the following cross-linker reagents: BMPS, EMCS, GMBS, HBVS, LC-
SMCC, MBS, MPBH,
SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-
MBS, sulfo-
SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-
vinylsulfone)benzoate) which are
commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL.,
USA; see pages 467-498,
2003-2004 Applications Handbook and Catalog.
[0491] Immunoconjugates comprising an antibody and a cytotoxic agent may also
be made using a
variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-
pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),
iminothiolane (IT), bifunctional
derivatives of imidoesters (such as dimethyl adipimidate HCI), active esters
(such as disuccinimidyl suberate),
aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-
azidobenzoyl) hexanediamine), bis-
diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene
2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-
dinitrobenzene). For example,
a ricin immunotoxin can be prepared as described in Vitetta et al., Science
238:1098 (1987). Carbon-14-
labeled 1 -isothiocyanatobenzyl-3 -methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary
chelating agent for conjugation of radionucleotide to the antibody. See
W094/11026.
[0492] Alternatively, a fusion protein comprising an antibody and a cytotoxic
agent may be made,
e.g., by recombinant techniques or peptide synthesis. A recombinant DNA
molecule may comprise regions
encoding the antibody and cytotoxic portions of the conjugate either adjacent
to one another or separated by a
region encoding a linker peptide which does not destroy the desired properties
of the conjugate.
[0493] In yet another embodiment, an antibody may be conjugated to a
"receptor" (such as
streptavidin) for utilization in tumor pre-targeting wherein the antibody-
receptor conjugate is administered to
the patient, followed by removal of unbound conjugate from the circulation
using a clearing agent and then
administration of a "ligand" (e.g., avidin) which is conjugated to a cytotoxic
agent (e.g., a radionucleotide).
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Exemplary Immunoconjugates - Thio-Antibody Drug Conjugates
a. Preparation of Cysteine Engineered Anti-CD79b Antibodies
[0494] DNA encoding an amino acid sequence variant of the cysteine engineered
anti-CD79b
antibodies and parent anti-CD79b antibodies of the invention is prepared by a
variety of methods which
include, but are not limited to, isolation from a natural source (in the case
of naturally occurring amino acid
sequence variants), preparation by site-directed (or oligonucleotide-mediated)
mutagenesis (Carter (1985) et
al Nucleic Acids Res. 13:4431-4443; Ho et al (1989) Gene (Amst.) 77:51-59;
Kunkel et al (1987) Proc. Natl.
Acad. Sci. USA 82:488; Liu et al (1998) J. Biol. Chem. 273:20252-20260), PCR
mutagenesis (Higuchi,
(1990) in PCR Protocols, pp.177-183, Academic Press; Ito et al (1991) Gene
102:67-70; Bernhard et al
(1994) Bioconjugate Chem. 5:126-132; and Vallette et al (1989) Nuc. Acids Res.
17:723-733), and cassette
mutagenesis (Wells et al (1985) Gene 34:315-323) of an earlier prepared DNA
encoding the polypeptide.
Mutagenesis protocols, kits, and reagents are commercially available, e.g.
QuikChange Multi Site-Direct
Mutagenesis Kit (Stratagene, La Jolla, CA). Single mutations are also
generated by oligonucleotide directed
mutagenesis using double stranded plasmid DNA as template by PCR based
mutagenesis (Sambrook and
Russel, (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; Zoller et
al (1983) Methods Enzymol.
100:468-500; Zoller, M.J. and Smith, M. (1982) Nucl. Acids Res. 10:6487-6500).
Variants of recombinant
antibodies may be constructed also by restriction fragment manipulation or by
overlap extension PCR with
synthetic oligonucleotides. Mutagenic primers encode the cysteine codon
replacement(s). Standard
mutagenesis techniques can be employed to generate DNA encoding such mutant
cysteine engineered
antibodies (Sambrook et al Molecular Cloning, A Laboratory Manual, Cold Spring
Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989; and Ausubel et al Current Protocols in
Molecular Biology, Greene
Publishing and Wiley-Interscience, New York, N.Y., 1993).
[0495] Phage display technology (McCafferty et al (1990) Nature 348:552-553)
can be used to
produce anti-CD79b human antibodies and antibody fragments in vitro, from
immunoglobulin variable (V)
domain gene repertoires from unimmunized donors. According to this technique,
antibody V domain genes
are cloned in-frame into either a major or minor coat protein gene of a
filamentous bacteriophage, such as
M13 or fd, and displayed as functional antibody fragments on the surface of
the phage particle. Because the
filamentous particle contains a single-stranded DNA copy of the phage genome,
selections based on the
functional properties of the antibody also result in selection of the gene
encoding the antibody exhibiting those
properties. Thus, the phage mimics some of the properties of the B-cell
(Johnson et al (1993) Current Opinion
in Structural Biology 3:564-571; Clackson et al (1991) Nature, 352:624-628;
Marks et al (1991) J. Mol. Biol.
222:581-597; Griffith et al (1993) EMBO J. 12:725-734; US 5565332; US 5573905;
US 5567610; US
5229275).
[0496] Anti-CD79b antibodies may be chemically synthesized using known
oligopeptide synthesis
methodology or may be prepared and purified using recombinant technology. The
appropriate amino acid
sequence, or portions thereof, may be produced by direct peptide synthesis
using solid-phase techniques
(Stewart et al., Solid-Phase Peptide Synthesis, (1969)W.H. Freeman Co., San
Francisco, CA; Merrifield,
(1963) J. Am. Chem. Soc., 85:2149-2154). In vitro protein synthesis may be
performed using manual
techniques or by automation. Automated solid phase synthesis may be
accomplished, for instance, employing
t-BOC or Fmoc protected amino acids and using an Applied Biosystems Peptide
Synthesizer (Foster City, CA)
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using manufacturer's instructions. Various portions of the anti-CD79b antibody
or CD79b polypeptide may
be chemically synthesized separately and combined using chemical or enzymatic
methods to produce the
desired anti-CD79b antibody or CD79b polypeptide.
[0497] Various techniques have been developed for the production of antibody
fragments.
Traditionally, these fragments were derived via proteolytic digestion of
intact antibodies (Morimoto et al
(1992) Journal of Biochemical and Biophysical Methods 24:107-117; and Brennan
et al (1985) Science,
229:81), or produced directly by recombinant host cells. Fab, Fv and ScFv anti-
CD79b antibody fragments
can all be expressed in and secreted from E. coli, thus allowing the facile
production of large amounts of these
fragments. Antibody fragments can be isolated from the antibody phage
libraries discussed herein.
Alternatively, Fab'-SH fragments can be directly recovered from E. coli and
chemically coupled to form
F(ab')2 fragments (Carter et al (1992) Bio/Technology 10:163-167), or isolated
directly from recombinant host
cell culture. The anti-CD79b antibody may be a (scFv) single chain Fv fragment
(WO 93/16185; US
5571894; US. 5587458). The anti-CD79b antibody fragment may also be a "linear
antibody" (US 5641870).
Such linear antibody fragments may be monospecific or bispecific.
[0498] The description below relates primarily to production of anti-CD79b
antibodies by culturing
cells transformed or transfected with a vector containing anti-CD79b antibody-
encoding nucleic acid. DNA
encoding anti-CD79b antibodies may be obtained from a cDNA library prepared
from tissue believed to
possess the anti-CD79b antibody mRNA and to express it at a detectable level.
Accordingly, human anti-
CD79b antibody or CD79b polypeptide DNA can be conveniently obtained from a
cDNA library prepared
from human tissue. The anti-CD79b antibody-encoding gene may also be obtained
from a genomic library or
by known synthetic procedures (e.g., automated nucleic acid synthesis).
[0499] The design, selection, and preparation methods of the invention enable
cysteine engineered
anti-CD79b antibodies which are reactive with electrophilic functionality.
These methods further enable
antibody conjugate compounds such as antibody-drug conjugate (ADC) compounds
with drug molecules at
designated, designed, selective sites. Reactive cysteine residues on an
antibody surface allow specifically
conjugating a drug moiety through a thiol reactive group such as maleimide or
haloacetyl. The nucleophilic
reactivity of the thiol functionality of a Cys residue to a maleimide group is
about 1000 times higher compared
to any other amino acid functionality in a protein, such as amino group of
lysine residues or the N-terminal
amino group. Thiol specific functionality in iodoacetyl and maleimide reagents
may react with amine groups,
but higher pH (>9.0) and longer reaction times are required (Garman, 1997, Non-
Radioactive Labelling: A
Practical Approach, Academic Press, London). The amount of free thiol in a
protein may be estimated by the
standard Ellman's assay. Immunoglobulin M is an example of a disulfide-linked
pentamer, while
immunoglobulin G is an example of a protein with internal disulfide bridges
bonding the subunits together. In
proteins such as this, reduction of the disulfide bonds with a reagent such as
dithiothreitol (DTT) or selenol
(Singh et al (2002) Anal. Biochem. 304:147-156) is required to generate the
reactive free thiol. This approach
may result in loss of antibody tertiary structure and antigen binding
specificity.
[0500] The PHESELECTOR (Phage ELISA for Selection of Reactive Thiols) Assay
allows for
detection of reactive cysteine groups in antibodies in an ELISA phage format
thereby assisting in the design of
cysteine engineered antibodies (Junutula, J.R. et al. (2008) J Immunol Methods
332:41-52; WO 2006/034488;
US 2007/0092940). The cysteine engineered antibody is coated on well surfaces,
followed by incubation with
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phage particles, addition of HRP labeled secondary antibody, and absorbance
detection. Mutant proteins
displayed on phage may be screened in a rapid, robust, and high-throughput
manner. Libraries of cysteine
engineered antibodies can be produced and subjected to binding selection using
the same approach to identify
appropriately reactive sites of free Cys incorporation from random protein-
phage libraries of antibodies or
other proteins. This technique includes reacting cysteine mutant proteins
displayed on phage with an affinity
reagent or reporter group which is also thiol-reactive.
[0501] The PHESELECTOR assay allows screening of reactive thiol groups in
antibodies.
Identification of the A121C variant by this method is exemplary. The entire
Fab molecule may be effectively
searched to identify more ThioFab variants with reactive thiol groups. A
parameter, fractional surface
accessibility, was employed to identify and quantitate the accessibility of
solvent to the amino acid residues in
a polypeptide. The surface accessibility can be expressed as the surface area
(A2) that can be contacted by a
solvent molecule, e.g. water. The occupied space of water is approximated as a
1.4 A radius sphere. Software
is freely available or licensable (Secretary to CCP4, Daresbury Laboratory,
Warrington, WA4 4AD, United
Kingdom, Fax: (+44) 1925 603825, or by internet:
www.ccp4.ac.uk/dist/htmUINDEX.html) as the CCP4 Suite
of crystallography programs which employ algorithms to calculate the surface
accessibility of each amino acid
of a protein with known x-ray crystallography derived coordinates ("The CCP4
Suite: Programs for Protein
Crystallography" (1994) Acta. Cryst. D50:760-763). Two exemplary software
modules that perform surface
accessibility calculations are "AREAIMOL" and "SURFACE", based on the
algorithms of B.Lee and
F.M.Richards (1971) J.Mol.Biol. 55:379-400. AREAIMOL defines the solvent
accessible surface of a protein
as the locus of the centre of a probe sphere (representing a solvent molecule)
as it rolls over the Van der Waals
surface of the protein. AREAIMOL calculates the solvent accessible surface
area by generating surface points
on an extended sphere about each atom (at a distance from the atom centre
equal to the sum of the atom and
probe radii), and eliminating those that lie within equivalent spheres
associated with neighboring atoms.
AREAIMOL finds the solvent accessible area of atoms in a PDB coordinate file,
and summarizes the
accessible area by residue, by chain and for the whole molecule. Accessible
areas (or area differences) for
individual atoms can be written to a pseudo-PDB output file. AREAIMOL assumes
a single radius for each
element, and only recognizes a limited number of different elements.
[0502] AREAIMOL and SURFACE report absolute accessibilities, i.e. the number
of square
Angstroms (A). Fractional surface accessibility is calculated by reference to
a standard state relevant for an
amino acid within a polypeptide. The reference state is tripeptide Gly-X-Gly,
where X is the amino acid of
interest, and the reference state should be an `extended' conformation, i.e.
like those in beta-strands. The
extended conformation maximizes the accessibility of X. A calculated
accessible area is divided by the
accessible area in a Gly-X-Gly tripeptide reference state and reports the
quotient, which is the fractional
accessibility. Percent accessibility is fractional accessibility multiplied by
100. Another exemplary algorithm
for calculating surface accessibility is based on the SOLV module of the
program xsae (Broger, C., F.
Hoffman-LaRoche, Basel) which calculates fractional accessibility of an amino
acid residue to a water sphere
based on the X-ray coordinates of the polypeptide. The fractional surface
accessibility for every amino acid in
an antibody may be calculated using available crystal structure information
(Eigenbrot et al. (1993) J Mol Biol.
229:969-995).
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[0503] DNA encoding the cysteine engineered antibodies is readily isolated and
sequenced using
conventional procedures (e.g., by using oligonucleotide probes that are
capable of binding specifically to
genes encoding the heavy and light chains of murine antibodies). The hybridoma
cells serve as a source of
such DNA. Once isolated, the DNA may be placed into expression vectors, which
are then transfected into
host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary
(CHO) cells, or other mammalian
host cells, such as myeloma cells (US 5807715; US 2005/0048572; US
2004/0229310) that do not otherwise
produce the antibody protein, to obtain the synthesis of monoclonal antibodies
in the recombinant host cells.
[0504] After design and selection, cysteine engineered antibodies, e.g.
ThioFabs, with the
engineered, highly reactive unpaired Cys residues, "free cysteine amino
acids", may be produced by: (i)
expression in a bacterial, e.g. E. coli, system (Skerra et al (1993) Curr.
Opinion in Immunol. 5:256-262;
Pluckthun (1992) Immunol. Revs. 130:151-188) or a mammalian cell culture
system (WO 01/00245), e.g.
Chinese Hamster Ovary cells (CHO); and (ii) purification using common protein
purification techniques
(Lowman et al (1991) J. Biol. Chem. 266(17):10982-10988).
[0505] The engineered Cys thiol groups react with electrophilic linker
reagents and drug-linker
intermediates to form cysteine engineered antibody drug conjugates and other
labelled cysteine engineered
antibodies. Cys residues of cysteine engineered antibodies, and present in the
parent antibodies, which are
paired and form interchain and intrachain disulfide bonds do not have any
reactive thiol groups (unless treated
with a reducing agent) and do not react with electrophilic linker reagents or
drug-linker intermediates. The
newly engineered Cys residue, can remain unpaired, and able to react with,
i.e. conjugate to, an electrophilic
linker reagent or drug-linker intermediate, such as a drug-maleimide.
Exemplary drug-linker intermediates
include: MC-MMAE, MC-MMAF, MC-vc-PAB-MMAE, and MC-vc-PAB-MMAF. The structure
positions
of the engineered Cys residues of the heavy and light chains are numbered
according to a sequential
numbering system. This sequential numbering system is correlated to the Kabat
numbering system (Kabat et
al., (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public
Health Service, National Institutes
of Health, Bethesda, MD) starting at the N-terminus, differs from the Kabat
numbering scheme (bottom row)
by insertions noted by a,b,c. Using the Kabat numbering system, the actual
linear amino acid sequence may
contain fewer or additional amino acids corresponding to a shortening of, or
insertion into, a FR or CDR of the
variable domain. The cysteine engineered heavy chain variant sites are
identified by the sequential numbering
and Kabat numbering schemes.
[0506] In one embodiment, the cysteine engineered anti-CD79b antibody is
prepared by a process
comprising:
(a) replacing one or more amino acid residues of a parent anti-CD79b antibody
by cysteine; and
(b) determining the thiol reactivity of the cysteine engineered anti-CD79b
antibody by reacting the
cysteine engineered antibody with a thiol-reactive reagent.
[0507] The cysteine engineered antibody may be more reactive than the parent
antibody with the
thiol-reactive reagent.
[0508] The free cysteine amino acid residues may be located in the heavy or
light chains, or in the
constant or variable domains. Antibody fragments, e.g. Fab, may also be
engineered with one or more
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cysteine amino acids replacing amino acids of the antibody fragment, to form
cysteine engineered antibody
fragments.
[0509] Another embodiment of the invention provides a method of preparing
(making) a cysteine
engineered anti-CD79b antibody, comprising:
(a) introducing one or more cysteine amino acids into a parent anti-CD79b
antibody in order to
generate the cysteine engineered anti-CD79b antibody; and
(b) determining the thiol reactivity of the cysteine engineered antibody with
a thiol-reactive
reagent;
wherein the cysteine engineered antibody is more reactive than the parent
antibody with the thiol-reactive
reagent.
Step (a) of the method of preparing a cysteine engineered antibody may
comprise:
(i) mutagenizing a nucleic acid sequence encoding the cysteine engineered
antibody;
(ii) expressing the cysteine engineered antibody; and
(iii) isolating and purifying the cysteine engineered antibody.
[0510] Step (b) of the method of preparing a cysteine engineered antibody may
comprise expressing
the cysteine engineered antibody on a viral particle selected from a phage or
a phagemid particle.
[0511] Step (b) of the method of preparing a cysteine engineered antibody may
also comprise:
(i) reacting the cysteine engineered antibody with a thiol-reactive affinity
reagent to generate an
affinity labelled, cysteine engineered antibody; and
(ii) measuring the binding of the affinity labelled, cysteine engineered
antibody to a capture
media.
[0512] Another embodiment of the invention is a method of screening cysteine
engineered
antibodies with highly reactive, unpaired cysteine amino acids for thiol
reactivity comprising:
(a) introducing one or more cysteine amino acids into a parent antibody in
order to generate a
cysteine engineered antibody;
(b) reacting the cysteine engineered antibody with a thiol-reactive affinity
reagent to generate an
affinity labelled, cysteine engineered antibody; and
(c) measuring the binding of the affinity labelled, cysteine engineered
antibody to a capture
media; and
(d) determining the thiol reactivity of the cysteine engineered antibody with
the thiol-reactive
reagent.
[0513] Step (a) of the method of screening cysteine engineered antibodies may
comprise:
(i) mutagenizing a nucleic acid sequence encoding the cysteine engineered
antibody;
(ii) expressing the cysteine engineered antibody; and
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(iii) isolating and purifying the cysteine engineered antibody.
[0514] Step (b) of the method of screening cysteine engineered antibodies may
comprise expressing
the cysteine engineered antibody on a viral particle selected from a phage or
a phagemid particle.
[0515] Step (b) of the method of screening cysteine engineered antibodies may
also comprise:
(i) reacting the cysteine engineered antibody with a thiol-reactive affinity
reagent to generate an
affinity labelled, cysteine engineered antibody; and
(ii) measuring the binding of the affinity labelled, cysteine engineered
antibody to a capture
media.
b. Cysteine Engineering of Anti-CD79b IgG Variants
[0516] Cysteine was introduced at the heavy chain 118 (EU numbering)
(equivalent to heavy chain
position 118, sequential numbering) site into the full-length, chimeric parent
monoclonal anti-CD79b
antibodies or at the light chain 205 (Kabat numbering) (equivalent to light
chain position 210, sequential
numbering) site into the full-length, chimeric parental monoclonal anti-CD79b
antibodies by the cysteine
engineering methods described herein.
[0517] Cysteine engineered antibodies with cysteine at heavy chain 118 (EU
numbering) generated
were: (a) thio-hu2F2.D7-HC(A118C) with heavy chain sequence (SEQ ID NO: 85)
and light chain sequence
(SEQ ID NO: 86), Figure 17.
[0518] Cysteine engineered antibodies with cysteine at light chain 205 (Kabat
numbering)
generated were: (a) thio-hu2F2.D7-LC(V205C) with heavy chain sequence (SEQ ID
NO: 87) and light chain
sequence (SEQ ID NO: 88), Figure 18.
[0519] These cysteine engineered monoclonal antibodies were expressed in CHO
(Chinese Hamster
Ovary) cells by transient fermentation in media containing 1 mM cysteine.
[0520] According to one embodiment, humanized 2F2 cysteine engineered anti-
CD79b antibodies
comprise one or more of the following heavy chain sequences with a free
cysteine amino acid (SEQ ID NOs:
91-99, Table 2).
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Table 2: Comparison of heavy chain Sequential, Kabat and EU numbering for
humanized 2F2
cysteine engineered anti-CD79b antibody variants
SEQUENCE SEQUENTIAL KABAT EU NUMBERING SEQ ID NO:
NUMBERING NUMBERING
EVQLCESGGG V5C V5C 91
LRLSCCASGYT A23C A23C 92
MNSLRCEDTAV A88C A84C 93
TLVTVCSASTK S112C S112C 94
VTVSSCSTKGP A114C A114C A118C 95
VSSASCKGPSV T116C T116C T120C 96
WYVDGCEVHNA V278C V278C V282C 97
KGFYPCDIAVE S371C S371C S375C 98
PPVLDCDGSFF S396C S396C S400C 99
[0521] According to one embodiment, chimeric 2F2 cysteine engineered anti-
CD79b antibodies
comprise one or more of the following heavy chain sequences with a free
cysteine amino acid (SEQ ID NOs:
100-108, Table 3).
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Table 3: Comparison of heavy chain Sequential, Kabat and EU numbering for
ch2F2 cysteine
engineered anti-CD79b antibody variants:
SEQUENCE SEQUENTIAL KABAT EU NUMBERING SEQ ID NO:
NUMBERING NUMBERING
QVQLCQPGAE Q5C Q5C 100
VKLSCCASGYT K23C K23C 101
LSSLTCEDSAV S88C S84C 102
TSVTVCLASTK S112C S112C 103
VTVSSCSTKGP A114C A114C A118C 104
VSSASCKGPSV T116C T116C T120C 105
WYVDGCEVHNA V278C V278C V282C 106
KGFYPCDIAVE S371C S371C S375C 107
PPVLDCDGSFF S396C S396C S400C 108
[0522] According to one embodiment, humanized 2F2 cysteine-engineered anti-
CD79b antibodies
comprise one or more of the following light chain sequences with a free
cysteine amino acid (SEQ ID NOs:
109-115, Table 4).
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Table 4: Comparison of light chain Sequential and Kabat numbering for
humanized 2F2 cysteine
engineered anti-CD79b antibody variants
SEQUENCE SEQUENTIAL KABAT SEQ ID NO:
NUMBERING NUMBERING
SLSASCGDRVT V15C V15C 109
EIKRTCAAPSV V115C V110C 110
TVAAPCVFIFP S119C S114C 111
FIFPPCDEQLK S126C S121C 112
DEQLKCGTASV S132C S127C 113
VTEQDCKDSTY S173C S168C 114
GLSSPCTKSFN V210C V205C 115
[0523] According to one embodiment, chimeric 2F2 cysteine-engineered anti-
CD79b antibodies
comprise one or more of the following light chain sequences with a free
cysteine amino acid (SEQ ID NOs:
116-122, Table 5).
Table 5: Comparison of light chain Sequential and Kabat numbering for chimeric
2F2 cysteine
engineered anti-CD79b antibody variants
SEQUENCE SEQUENTIAL KABAT SEQ ID NO:
NUMBERING NUMBERING
TLSVTCGQPAS I15C I15C 116
EIKRTCAAPSV V115C V110C 117
TVAAPCVFIFP S119C S114C 118
FIFPPCDEQLK S126C S121C 119
DEQLKCGTASV S132C S127C 120
VTEQDCKDSTY S173C S168C 121
GLSSPCTKSFN V210C V205C 122
c. Labelled Cysteine Engineered Anti-CD79b Antibodies
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[0524] Cysteine engineered anti-CD79b antibodies may be site-specifically and
efficiently coupled
with a thiol-reactive reagent. The thiol-reactive reagent may be a
multifunctional linker reagent, a capture, i.e.
affinity, label reagent (e.g. a biotin-linker reagent), a detection label
(e.g. a fluorophore reagent), a solid phase
immobilization reagent (e.g. SEPHAROSETM, polystyrene, or glass), or a drug-
linker intermediate. One
example of a thiol-reactive reagent is N-ethyl maleimide (NEM). In an
exemplary embodiment, reaction of a
ThioFab with a biotin-linker reagent provides a biotinylated ThioFab by which
the presence and reactivity of
the engineered cysteine residue may be detected and measured. Reaction of a
ThioFab with a multifunctional
linker reagent provides a ThioFab with a functionalized linker which may be
further reacted with a drug
moiety reagent or other label. Reaction of a ThioFab with a drug-linker
intermediate provides a ThioFab drug
conjugate.
[0525] The exemplary methods described here may be applied generally to the
identification and
production of antibodies, and more generally, to other proteins through
application of the design and screening
steps described herein.
[0526] Such an approach may be applied to the conjugation of other thiol-
reactive reagents in which
the reactive group is, for example, a maleimide, an iodoacetamide, a pyridyl
disulfide, or other thiol-reactive
conjugation partner (Haugland, 2003, Molecular Probes Handbook of Fluorescent
Probes and Research
Chemicals, Molecular Probes, Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2;
Garman, 1997, Non-Radioactive
Labelling: A Practical Approach, Academic Press, London; Means (1990)
Bioconjugate Chem. 1:2;
Hermanson, G. in Bioconjugate Techniques (1996) Academic Press, San Diego, pp.
40-55, 643-671). The
thiol-reactive reagent may be a drug moiety, a fluorophore such as a
fluorescent dye like fluorescein or
rhodamine, a chelating agent for an imaging or radiotherapeutic metal, a
peptidyl or non-peptidyl label or
detection tag, or a clearance-modifying agent such as various isomers of
polyethylene glycol, a peptide that
binds to a third component, or another carbohydrate or lipophilic agent.
d. Uses of Cysteine Engineered Anti-CD79b Antibodies
[0527] Cysteine engineered anti-CD79b antibodies, and conjugates thereof may
find use as
therapeutic and/or diagnostic agents. The present invention further provides
methods of preventing, managing,
treating or ameliorating one or more symptoms associated with a B-cell related
disorder. In particular, the
present invention provides methods of preventing, managing, treating, or
ameliorating one or more symptoms
associated with a cell proliferative disorder, such as cancer, e.g., lymphoma,
non-Hodgkins lymphoma (NHL),
aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory
NHL, refractory indolent NHL,
chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia,
hairy cell leukemia (HCL),
acute lymphocytic leukemia (ALL), and mantle cell lymphoma. The present
invention still further provides
methods for diagnosing a CD79b related disorder or predisposition to
developing such a disorder, as well as
methods for identifying antibodies, and antigen-binding fragments of
antibodies, that preferentially bind B
cell-associated CD79b polypeptides.
Another embodiment of the present invention is directed to the use of a
cysteine engineered anti-CD79b
antibody for the preparation of a medicament useful in the treatment of a
condition which is responsive to a B
cell related disorder.
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e. Cysteine Engineered Antibody Drug Conjugates (Thio-antibody Drug
Conjugates (TDCs))
[0528] Another aspect of the invention is an antibody-drug conjugate compound
comprising a
cysteine engineered anti-CD79b antibody (Ab), and an auristatin drug moiety
(D) wherein the cysteine
engineered antibody is attached through one or more free cysteine amino acids
by a linker moiety (L) to D; the
compound having Formula I:
Ab-(L-D)p i
where p is 1, 2, 3, or 4; and wherein the cysteine engineered antibody is
prepared by a process comprising
replacing one or more amino acid residues of a parent anti-CD79b antibody by
one or more free cysteine
amino acids.
[0529] Another aspect of the invention is a composition comprising a mixture
of antibody-drug
compounds of Formula I where the average drug loading per antibody is about 2
to about 5, or about 3 to
about 4.
[0530] Figures 17-18 show embodiments of cysteine engineered anti-CD79b
antibody drug
conjugates (ADC) where an auristatin drug moiety is attached to an engineered
cysteine group in: the light
chain (LC-ADC) or the heavy chain (HC-ADC).
[0531] Potential advantages of cysteine engineered anti-CD79b antibody drug
conjugates include
improved safety (larger therapeutic index), improved PK parameters, the
antibody inter-chain disulfide bonds
are retained which may stabilize the conjugate and retain its active binding
conformation, the sites of drug
conjugation are defined, and the preparation of cysteine engineered antibody
drug conjugates from
conjugation of cysteine engineered antibodies to drug-linker reagents results
in a more homogeneous product.
Linkers
[0532] "Linker", "Linker Unit", or "linlC' means a chemical moiety comprising
a covalent bond or a
chain of atoms that covalently attaches an antibody to a drug moiety. In
various embodiments, a linker is
specified as L. A "Linker" (L) is a bifunctional or multifunctional moiety
which can be used to link one or
more Drug moieties (D) and an antibody unit (Ab) to form antibody-drug
conjugates (ADC) of Formula I.
Antibody-drug conjugates (ADC) can be conveniently prepared using a Linker
having reactive functionality
for binding to the Drug and to the Antibody. A cysteine thiol of a cysteine
engineered antibody (Ab) can form
a bond with an electrophilic functional group of a linker reagent, a drug
moiety or drug-linker intermediate.
[0533] In one aspect, a Linker has a reactive site which has an electrophilic
group that is reactive to
a nucleophilic cysteine present on an antibody. The cysteine thiol of the
antibody is reactive with an
electrophilic group on a Linker and forms a covalent bond to a Linker. Useful
electrophilic groups include,
but are not limited to, maleimide and haloacetamide groups.
[0534] Linkers include a divalent radical such as an alkyldiyl, an arylene, a
heteroarylene, moieties
such as: -(CRz)õO(CRz)ri , repeating units of alkyloxy (e.g. polyethylenoxy,
PEG, polymethyleneoxy) and
alkylamino (e.g. polyethyleneamino, JeffamineTM); and diacid ester and amides
including succinate,
succinamide, diglycolate, malonate, and caproamide.
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[0535] Cysteine engineered antibodies react with linker reagents or drug-
linker intermediates, with
electrophilic functional groups such as maleimide or a-halo carbonyl,
according to the conjugation method at
page 766 of Klussman, et al (2004), Bioconjugate Chemistry 15(4):765-773, and
according to the protocol of
Example 6.
[0536] The linker may be composed of one or more linker components. Exemplary
linker
components include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"),
valine-citrulline ("val-cit" or
"vc"), alanine-phenylalanine ("ala-phe" or "af'), p-aminobenzyloxycarbonyl
("PAB"), N-succinimidyl 4-(2-
pyridylthio) pentanoate ("SPP"), N-succinimidyl 4-(N-maleimidomethyl)
cyclohexane-1 carboxylate
("SMCC'), N-Succinimidyl (4-iodo-acetyl) aminobenzoate ("SIAB"), ethyleneoxy -
CHzCHzO- as one or more
repeating units ("EO" or "PEO"). Additional linker components are known in the
art and some are described
herein.
[0537] In one embodiment, linker L of an ADC has the formula:
-Aa WW Yy_
wherein:
-A- is a Stretcher unit covalently attached to a cysteine thiol of the
antibody (Ab);
a is 0 or 1;
each -W- is independently an Amino Acid unit;
w is independently an integer ranging from 0 to 12;
-Y- is a Spacer unit covalently attached to the drug moiety; and
y is 0, 1 or 2.
Stretcher unit
[0538] The Stretcher unit (-A-), when present, is capable of linking an
antibody unit to an amino
acid unit (-W-). In this regard an antibody (Ab) has a functional group that
can form a bond with a functional
group of a Stretcher. Useful functional groups that can be present on an
antibody, either naturally or via
chemical manipulation include, but are not limited to, sulfhydryl (-SH),
amino, hydroxyl, carboxy, the
anomeric hydroxyl group of a carbohydrate, and carboxyl. In one aspect, the
antibody functional groups are
sulfhydryl or amino. Sulfhydryl groups can be generated by reduction of an
intramolecular disulfide bond of
an antibody. Alternatively, sulfhydryl groups can be generated by reaction of
an amino group of a lysine
moiety of an antibody using 2-iminothiolane (Traut's reagent) or another
sulfhydryl generating reagent. In
one embodiment, an antibody (Ab) has a free cysteine thiol group that can form
a bond with an electrophilic
functional group of a Stretcher Unit. Exemplary stretcher units in Formula I
conjugates are depicted by
Formulas II and III, wherein Ab-, -W-, -Y-, -D, w and y are as defined above,
and R" is a divalent radical
selected from (CH2)r, C3-C8 carbocyclyl, O-(CHz)r, arylene, (CHz)r arylene, -
arylene-(CHz)r ,(CHz)r (C3-C8
carbocyclyl), (C3-C8 carbocyclyl)-(CH2)r, C3-C8 heterocyclyl, (CHz)r (C3-C8
heterocyclyl), -(C3-C8
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heterocyclyl)-(CHz)r , (CHz)rC(O)NRb(CHz)r , (CHzCHzO)r , (CHzCHzO)r CHz-,
-(CHz)rC(O)NRb(CHzCHzO)r , -(CHz)rC(O)NRb(CHzCHzO)r CHz , -
(CHzCHzO)rC(O)NRb(CHzCHzO)r ,
-(CHzCHzO)rC(O)NRb(CHzCHzO)r CHz-, and -(CHzCHzO)rC(O)NRb(CHz)r ; where Rb is
H, Ci-C6 alkyl,
phenyl, or benzyl; and r is independently an integer ranging from 1-10.
[0539] Arylene includes divalent aromatic hydrocarbon radicals of 6-20 carbon
atoms derived by
the removal of two hydrogen atoms from the aromatic ring system. Typical
arylene groups include, but are
not limited to, radicals derived from benzene, substituted benzene,
naphthalene, anthracene, biphenyl, and the
like.
[0540] Heterocyclyl groups include a ring system in which one or more ring
atoms is a heteroatom,
e.g. nitrogen, oxygen, and sulfur. The heterocycle radical comprises 1 to 20
carbon atoms and 1 to 3
heteroatoms selected from N, 0, P, and S. A heterocycle may be a monocycle
having 3 to 7 ring members (2
to 6 carbon atoms and 1 to 3 heteroatoms selected from N, 0, P, and S) or a
bicycle having 7 to 10 ring
members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, 0, P, and
S), for example: a bicyclo
[4,5], [5,5], [5,6], or [6,6] system. Heterocycles are described in Paquette,
Leo A.; "Principles of Modern
Heterocyclic Chemistry" (W.A. Benjamin, New York, 1968), particularly Chapters
1, 3, 4, 6, 7, and 9; "The
Chemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley &
Sons, New York, 1950 to
present), in particular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc.
(1960) 82:5566.
[0541] Examples of heterocycles include by way of example and not limitation
pyridyl,
dihydroypyridyl, tetrahydropyridyl (piperidyl), thiazolyl,
tetrahydrothiophenyl, sulfur oxidized
tetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl,
imidazolyl, tetrazolyl, benzofuranyl,
thianaphthalenyl, indolyl, indolenyl, quinolinyl, isoquinolinyl,
benzimidazolyl, piperidinyl, 4-piperidonyl,
pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, bis-
tetrahydrofuranyl, tetrahydropyranyl, bis-
tetrahydropyranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,
decahydroquinolinyl, octahydroisoquinolinyl,
azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H- 1,5,2-dithiazinyl, thienyl,
thianthrenyl, pyranyl,
isobenzofuranyl, chromenyl, xanthenyl, phenoxathinyl, 2H-pyrrolyl,
isothiazolyl, isoxazolyl, pyrazinyl,
pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl, 4H-
quinolizinyl, phthalazinyl,
naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, 4Ah-
carbazolyl, carbazolyl, (3-carbolinyl,
phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl,
phenothiazinyl, furazanyl, phenoxazinyl,
isochromanyl, chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl,
pyrazolinyl, piperazinyl, indolinyl,
isoindolinyl, quinuclidinyl, morpholinyl, oxazolidinyl, benzotriazolyl,
benzisoxazolyl, oxindolyl,
benzoxazolinyl, and isatinoyl.
[0542] Carbocyclyl groups include a saturated or unsaturated ring having 3 to
7 carbon atoms as a
monocycle or 7 to 12 carbon atoms as a bicycle. Monocyclic carbocycles have 3
to 6 ring atoms, still more
typically 5 or 6 ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms,
e.g. arranged as a bicyclo [4,5],
[5,5], [5,6] or [6,6] system, or 9 or 10 ring atoms arranged as a bicyclo
[5,6] or [6,6] system. Examples of
monocyclic carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, 1-
cyclopent-l-enyl, 1-cyclopent-2-enyl,
1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-l-enyl, 1-cyclohex-2-enyl, 1-
cyclohex-3-enyl, cycloheptyl, and
cyclooctyl.
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[0543] It is to be understood from all the exemplary embodiments of Formula I
ADC such as II-VI,
that even where not denoted expressly, from 1 to 4 drug moieties are linked to
an antibody ( p = 1-4),
depending on the number of engineered cysteine residues.
O
Ab-S
N-R17-C(O)-WW-Yy-D
O p
II
Ab-S CH2 CONH-R17-C(O)-WW-Yy-D
p III
[0544] An illustrative Formula II Stretcher unit is derived from maleimido-
caproyl (MC) wherein
R 17 is -(CH2)1-:
O
N
O
0 MC
[0545] An illustrative Stretcher unit of Formula II, and is derived from
maleimido-propanoyl (MP)
wherein R" is -(CH2)2-:
O O
N ~S
O MP
[0546] Another illustrative Stretcher unit of Formula II wherein R" is -
(CHzCHzO)r CHz - and r is
2:
O
O
O
[0547] Another illustrative Stretcher unit of Formula II wherein R" is
-(CHz)rC(O)NRb(CHzCHzO)r CHz- where Rb is H and each r is 2:
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O O
H O
0 MPEG
[0548] An illustrative Stretcher unit of Formula III wherein R" is -(CHz)s-:
O
H O
[0549] In another embodiment, the Stretcher unit is linked to the cysteine
engineered anti-CD79b
antibody via a disulfide bond between the engineered cysteine sulfur atom of
the antibody and a sulfur atom of
the Stretcher unit. A representative Stretcher unit of this embodiment is
depicted by Formula IV, wherein R",
Ab-, -W-, -Y-, -D, w and y are as defined above.
Ab S S-R17-C(O) WW-Yy-D
p Iv
[0550] In yet another embodiment, the reactive group of the Stretcher contains
a thiol-reactive
functional group that can form a bond with a free cysteine thiol of an
antibody. Examples of thiol-reaction
functional groups include, but are not limited to, maleimide, a-haloacetyl,
activated esters such as succinimide
esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl
esters, anhydrides, acid chlorides,
sulfonyl chlorides, isocyanates and isothiocyanates. Representative Stretcher
units of this embodiment are
depicted by Formulas Va and Vb, wherein -R"-, Ab-, -W-, -Y-, -D, w and y are
as defined above;
Ab S C(O)NH-R17-C(O)-WW-Yy- D
p Va
Ab S C(S)NH-R17-C(O)-WW-Yy- D
p Vb
[0551] In another embodiment, the linker may be a dendritic type linker for
covalent attachment of
more than one drug moiety through a branching, multifunctional linker moiety
to an antibody (Sun et al (2002)
Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al (2003)
Bioorganic & Medicinal
Chemistry 11:1761-1768; King (2002) Tetrahedron Letters 43:1987-1990).
Dendritic linkers can increase the
molar ratio of drug to antibody, i.e. loading, which is related to the potency
of the ADC. Thus, where a
cysteine engineered antibody bears only one reactive cysteine thiol group, a
multitude of drug moieties may be
attached through a dendritic linker.
Amino acid unit
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[0552] The linker may comprise amino acid residues. The Amino Acid unit (-Ww
), when present,
links the antibody (Ab) to the drug moiety (D) of the cysteine engineered
antibody-drug conjugate (ADC) of
the invention.
[0553] -Ww- is a dipeptide, tripeptide, tetrapeptide, pentapeptide,
hexapeptide, heptapeptide,
octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit.
Amino acid residues which
comprise the Amino Acid unit include those occurring naturally, as well as
minor amino acids and non-
naturally occurring amino acid analogs, such as citrulline. Each -W- unit
independently has the formula
denoted below in the square brackets, and w is an integer ranging from 0 to
12:
O
H
R19
w
wherein R19 is hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-
hydroxybenzyl, -CHzOH,
-CH(OH)CH3, -CH2CH2SCH3, -CH2CONH2, -CH2COOH, -CH2CH2CONH2, -CH2CH2COOH, -
(CH2)3NHC(=NH)NH2, -(CH2)3NH2, -(CH2)3NHCOCH3, -(CH2)3NHCHO, -
(CH2)4NHC(=NH)NH2, -
(CH2)4NH2, -(CH2)4NHCOCH3, -(CH2)4NHCHO, -(CH2)3NHCONH2, -(CH2)4NHCONH2, -
CHzCHzCH(OH)CHzNHz, 2-pyridylmethyl-, 3-pyridylmethyl-, 4-pyridylmethyl-,
phenyl, cyclohexyl,
oH
CH2 or $ CH2
H CN
H
[0554] When R19 is other than hydrogen, the carbon atom to which R19 is
attached is chiral. Each
carbon atom to which R19 is attached is independently in the (S) or (R)
configuration, or a racemic mixture.
Amino acid units may thus be enantiomerically pure, racemic, or
diastereomeric.
[0555] Exemplary -Ww Amino Acid units include a dipeptide, a tripeptide, a
tetrapeptide or a
pentapeptide. Exemplary dipeptides include: valine-citrulline (vc or val-cit),
alanine-phenylalanine (af or ala-
phe). Exemplary tripeptides include: glycine-valine-citrulline (gly-val-cit)
and glycine-glycine-glycine (gly-
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gly-gly). Amino acid residues which comprise an amino acid linker component
include those occurring
naturally, as well as minor amino acids and non-naturally occurring amino acid
analogs, such as citrulline.
[0556] The Amino Acid unit can be enzymatically cleaved by one or more
enzymes, including a
tumor-associated protease, to liberate the Drug moiety (-D), which in one
embodiment is protonated in vivo
upon release to provide a Drug (D). Amino acid linker components can be
designed and optimized in their
selectivity for enzymatic cleavage by a particular enzymes, for example, a
tumor-associated protease,
cathepsin B, C and D, or a plasmin protease.
Spacer unit
[0557] The Spacer unit (-Yy ), when present (y = 1 or 2), links an Amino Acid
unit (-Ww ) to the
drug moiety (D) when an Amino Acid unit is present (w = 1-12). Alternately,
the Spacer unit links the
Stretcher unit to the Drug moiety when the Amino Acid unit is absent. The
Spacer unit also links the drug
moiety to the antibody unit when both the Amino Acid unit and Stretcher unit
are absent (w, y = 0). Spacer
units are of two general types: self-immolative and non self-immolative. A non
self-immolative Spacer unit is
one in which part or all of the Spacer unit remains bound to the Drug moiety
after cleavage, particularly
enzymatic, of an Amino Acid unit from the antibody-drug conjugate or the Drug
moiety-linker. When an
ADC containing a glycine-glycine Spacer unit or a glycine Spacer unit
undergoes enzymatic cleavage via a
tumor-cell associated-protease, a cancer-cell-associated protease or a
lymphocyte-associated protease, a
glycine-glycine-Drug moiety or a glycine-Drug moiety is cleaved from Ab-Aa-Ww-
. In one embodiment, an
independent hydrolysis reaction takes place within the target cell, cleaving
the glycine-Drug moiety bond and
liberating the Drug.
[0558] In another embodiment, -Yy is a p-aminobenzylcarbamoyl (PAB) unit whose
phenylene
portion is substituted with Qm wherein Q is -Ci-CB alkyl, -O-(Ci-C8 alkyl), -
halogen,- nitro or -cyano; and m is
an integer ranging from 0-4.
[0559] Exemplary embodiments of a non self-immolative Spacer unit (-Y-) are: -
Gly-Gly- ; -Gly-
;
-Ala-Phe- ; -Val-Cit-.
[0560] In one embodiment, a Drug moiety-linker or an ADC is provided in which
the Spacer unit is
absent (y=0), or a pharmaceutically acceptable salt or solvate thereof.
[0561] Alternatively, an ADC containing a self-immolative Spacer unit can
release -D. In one
embodiment, -Y- is a PAB group that is linked to -Ww- via the amino nitrogen
atom of the PAB group, and
connected directly to -D via a carbonate, carbamate or ether group, where the
ADC has the exemplary
structure:
Qm
I-
Ab Aa WN,-NH
O-C-D
11
O p
wherein Q is -Ci-CB alkyl, -O-(C1-C8 alkyl), -halogen, -nitro or -cyano; m is
an integer ranging from
0-4; and p ranges from 1 to 4.
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[0562] Other examples of self-immolative spacers include, but are not limited
to, aromatic
compounds that are electronically similar to the PAB group such as 2-
aminoimidazol-5-methanol derivatives
(Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237), heterocyclic PAB analogs
(US 2005/0256030), beta-
glucuronide (WO 2007/011968), and ortho or para-aminobenzylacetals. Spacers
can be used that undergo
cyclization upon amide bond hydrolysis, such as substituted and unsubstituted
4-aminobutyric acid amides
(Rodrigues et al (1995) Chemistry Biology 2:223), appropriately substituted
bicyclo[2.2.1] and bicyclo[2.2.2]
ring systems (Storm et al (1972) J. Amer. Chem. Soc. 94:5815) and 2-
aminophenylpropionic acid amides
(Amsberry, et al (1990) J. Org. Chem. 55:5867). Elimination of amine-
containing drugs that are substituted at
glycine (Kingsbury et al (1984) J. Med. Chem. 27:1447) are also examples of
self-immolative spacer useful in
ADCs.
[0563] Exemplary Spacer units (-Yy-) are represented by Formulas X-XII:
H
-N
O x
-HN-CH2-CO-
xI
I-NHCH2C(O)-NHCH2C(O)-
xII
Dendritic linkers
[0564] In another embodiment, linker L may be a dendritic type linker for
covalent attachment of
more than one drug moiety through a branching, multifunctional linker moiety
to an antibody (Sun et al (2002)
Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al (2003)
Bioorganic & Medicinal
Chemistry 11:1761-1768). Dendritic linkers can increase the molar ratio of
drug to antibody, i.e. loading,
which is related to the potency of the ADC. Thus, where a cysteine engineered
antibody bears only one
reactive cysteine thiol group, a multitude of drug moieties may be attached
through a dendritic linker.
Exemplary embodiments of branched, dendritic linkers include 2,6-
bis(hydroxymethyl)-p-cresol and 2,4,6-
tris(hydroxymethyl)-phenol dendrimer units (WO 2004/01993; Szalai et al (2003)
J. Amer. Chem. Soc.
125:15688-15689; Shamis et al (2004) J. Amer. Chem. Soc. 126:1726-173 1; Amir
et al (2003) Angew. Chem.
Int. Ed. 42:4494-4499).
[0565] In one embodiment, the Spacer unit is a branched
bis(hydroxymethyl)styrene (BHMS),
which can be used to incorporate and release multiple drugs, having the
structure:
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0
11
QF CH2(OC)n-D
Ab A
4 a-Wv~,-NH c I O
CH2(OC)n-D
comprising a 2-(4-aminobenzylidene)propane- 1,3 -diol dendrimer unit (WO
2004/043493; de Groot et
al (2003) Angew. Chem. Int. Ed. 42:4490-4494), wherein Q is -Ci-CB alkyl, -O-
(Ci-C8 alkyl), -halogen, -nitro
or -cyano; m is an integer ranging from 0-4; n is 0 or 1; and p ranges ranging
from 1 to 4.
[0566] Exemplary embodiments of the Formula I antibody-drug conjugate
compounds include
XIIIa (MC), XIIIb (val-cit), XIIIc (MC-val-cit), and XIIId (MC-val-cit-PAB):
H O
Ab-S 4 Aa N N,,,,~LYy D
O H O p
O
Ab-S D HN
O O____ N H 2
XIIIa XIIIb
O
O H 0
Ab-S N N N~Yy D
O H O -
~ p
HN
O~NH2 xIIIc
O
O O Fi O 2- D
N,,,~ NA
Ab-S N N
O H O~ H
HN
0:-"-NH2 XIIId
[0567] Other exemplary embodiments of the Formula Ia antibody-drug conjugate
compounds
include XIVa-e:
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O
O
11
N-X-C-D
Ab-S
~ p XIVa
O 0
II II
Ab-S CH2C-Y-C-D
p XIVb
Ab-S CH2C ~~ D
0
p XIVc
O
/~ O
N-CH2~ rC-
Ab-S ~/
O Dp xlVd
4 H - O
Ab-S CH2C O -N C D
p XIVe
where X is:
-CH2-C>- (CH2)n- , - (CH2CH20),,-
11 O /
-CH2~C-N (CH2)n-
R
O
(CH2)n 11
or -(CH2)n-C-N-(CH2)n
Y is:
R R
N or -N-(CH2)n-
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and R is independently H or C1-C6 alkyl; and n is 1 to 12.
[0568] In another embodiment, a Linker has a reactive functional group which
has a nucleophilic
group that is reactive to an electrophilic group present on an antibody.
Useful electrophilic groups on an
antibody include, but are not limited to, aldehyde and ketone carbonyl groups.
The heteroatom of a
nucleophilic group of a Linker can react with an electrophilic group on an
antibody and form a covalent bond
to an antibody unit. Useful nucleophilic groups on a Linker include, but are
not limited to, hydrazide, oxime,
amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.
The electrophilic group on
an antibody provides a convenient site for attachment to a Linker.
[0569] Typically, peptide-type Linkers can be prepared by forming a peptide
bond between two or
more amino acids and/or peptide fragments. Such peptide bonds can be prepared,
for example, according to
the liquid phase synthesis method (E. Schr6der and K. Lubke (1965) "The
Peptides", volume 1, pp 76-136,
Academic Press) which is well known in the field of peptide chemistry. Linker
intermediates may be
assembled with any combination or sequence of reactions including Spacer,
Stretcher, and Amino Acid units.
The Spacer, Stretcher, and Amino Acid units may employ reactive functional
groups which are electrophilic,
nucleophilic, or free radical in nature. Reactive functional groups include,
but are not limited to carboxyls,
hydroxyls, para-nitrophenylcarbonate, isothiocyanate, and leaving groups, such
as 0-mesyl, 0-tosyl, -Cl, -Br,
-I; or maleimide.
[0570] For example, a charged substituent such as sulfonate (-S03-) or
ammonium, may increase
water solubility of the reagent and facilitate the coupling reaction of the
linker reagent with the antibody or the
drug moiety, or facilitate the coupling reaction of Ab-L (antibody-linker
intermediate) with D, or D-L (drug-
linker intermediate) with Ab, depending on the synthetic route employed to
prepare the ADC.
Linker reagents
[0571] Conjugates of the antibody and auristatin may be made using a variety
of bifunctional linker
reagents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)
cyclohexane-l-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives
of imidoesters (such as
dimethyl adipimidate HCI), active esters (such as disuccinimidyl suberate),
aldehydes (such as glutaraldehyde),
bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-
diazonium derivatives (such as bis-
(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-
diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
[0572] The antibody drug conjugates may also be prepared with linker reagents:
BMPEO, BMPS,
EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMPB, SMPH, sulfo-EMCS,
sulfo-
GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB
(succinimidyl-(4-
vinylsulfone)benzoate), and including bis-maleimide reagents: DTME, BMB, BMDB,
BMH, BMOE, 1,8-bis-
maleimidodiethyleneglycol (BM(PEO)2), and 1,11-bis-maleimidotriethyleneglycol
(BM(PEO)3), which are
commercially available from Pierce Biotechnology, Inc., ThermoScientific,
Rockford, IL, and other reagent
suppliers. Bis-maleimide reagents allow the attachment of the thiol group of a
cysteine engineered antibody to
a thiol-containing drug moiety, label, or linker intermediate, in a sequential
or concurrent fashion. Other
functional groups besides maleimide, which are reactive with a thiol group of
a cysteine engineered antibody,
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drug moiety, label, or linker intermediate include iodoacetamide,
bromoacetamide, vinyl pyridine, disulfide,
pyridyl disulfide, isocyanate, and isothiocyanate.
0
O \ O O
t NO,-_~O-**-~ N O tQo~O
BM(PEO)2 BM(PEO)3
[0573] Useful linker reagents can also be obtained via other commercial
sources, such as Molecular
Biosciences Inc.(Boulder, CO), or synthesized in accordance with procedures
described in Toki et al (2002) J.
Org. Chem. 67:1866-1872; Walker, M.A. (1995) J. Org. Chem. 60:5352-5355;
Frisch et al (1996)
Bioconjugate Chem. 7:180-186; US 6214345; WO 02/0 8 8 1 72; US 2003130189;
US2003096743; WO
03/026577; WO 03/043583; and WO 04/032828.
[0574] Stretchers of formula (IIIa) can be introduced into a Linker by
reacting the following linker
reagents with the N-terminus of an Amino Acid unit:
O O
T
N-(CH2)n-C(O)-O-N
O 0
where n is an integer ranging from 1-10 and T is -H or -SO3Na;
O O
N ~ ~ (CH2)n-C(O)-O-N
O
where n is an integer ranging from 0-3;
O O O
fo O-N O
0
O
O O-N
N
O H O O
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0 0
ON ; and
VN, O
O O O
O
O
bAOH
" v O
[0575] Stretcher units of can be introduced into a Linker by reacting the
following bifunctional
reagents with the N-terminus of an Amino Acid unit:
O O O O O O O
O \ / O-N I~N \ / O-N X`AO-N
O H
O O O
O O O O O
Br---ANFi--AO-N ~---yNH O_N
O
O O
where X is Br or I.
[0576] Stretcher units of formula can also be introduced into a Linker by
reacting the following
bifunctional reagents with the N-terminus of an Amino Acid unit:
o O
N S-S O-N
O
O O
O-N
N S-S NH .
O O
O O O
Boc-NH-NH2 C O-N Boc-NH-NH2~~N
O O
[0577] An exemplary valine-citrulline (val-cit or vc) dipeptide linker reagent
having a maleimide
Stretcher and a para-aminobenzylcarbamoyl (PAB) self-immolative Spacer has the
structure:
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O
O
H3C CH3 O O
H NO2
NN
Fmoc-N
H O
NH
H2N~0
[0578] An exemplary phe-lys(Mtr, mono-4-methoxytrityl) dipeptide linker
reagent having a
maleimide Stretcher unit and a PAB self-immolative Spacer unit can be prepared
according to Dubowchik, et
al. (1997) Tetrahedron Letters, 38:5257-60, and has the structure:
/ OH
Ph O ~
N~H \
Fmoc-N
H O
HN-Mtr
Preparation of cysteine engineered anti-CD79b antibody-drug conjugates
[0579] The ADC of Formula I may be prepared by several routes, employing
organic chemistry
reactions, conditions, and reagents known to those skilled in the art,
including: (1) reaction of a cysteine group
of a cysteine engineered antibody with a linker reagent, to form antibody-
linker intermediate Ab-L, via a
covalent bond, followed by reaction with an activated drug moiety D; and (2)
reaction of a nucleophilic group
of a drug moiety with a linker reagent, to form drug-linker intermediate D-L,
via a covalent bond, followed by
reaction with a cysteine group of a cysteine engineered antibody. Conjugation
methods (1) and (2) may be
employed with a variety of cysteine engineered antibodies, drug moieties, and
linkers to prepare the antibody-
drug conjugates of Formula I.
[0580] Antibody cysteine thiol groups are nucleophilic and capable of reacting
to form covalent
bonds with electrophilic groups on linker reagents and drug-linker
intermediates including: (i) active esters
such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl
and benzyl halides, such as
haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups; and
(iv) disulfides, including
pyridyl disulfides, via sulfide exchange. Nucleophilic groups on a drug moiety
include, but are not limited to:
amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone,
hydrazine carboxylate, and
arylhydrazide groups capable of reacting to form covalent bonds with
electrophilic groups on linker moieties
and linker reagents.
[0581] Cysteine engineered antibodies may be made reactive for conjugation
with linker reagents
by treatment with a reducing agent such as DTT (Cleland's reagent,
dithiothreitol) or TCEP (tris(2-
carboxyethyl)phosphine hydrochloride; Getz et al (1999) Anal. Biochem. Vol
273:73-80; Soltec Ventures,
Beverly, MA), followed by reoxidation to reform interchain and intrachain
disulfide bonds (Example 5). For
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example, full length, cysteine engineered monoclonal antibodies (ThioMabs)
expressed in CHO cells are
reduced with about a 50 fold molar excess of TCEP for 3 hrs at 37 C to reduce
disulfide bonds in cysteine
adducts which may form between the newly introduced cysteine residues and the
cysteine present in the
culture media. The reduced ThioMab is diluted and loaded onto HiTrap S column
in 10 mM sodium acetate,
pH 5, and eluted with PBS containing 0.3M sodium chloride. Disulfide bonds
were reestablished between
cysteine residues present in the parent Mab with dilute (200 nM) aqueous
copper sulfate (CuS04) at room
temperature, overnight. Alternatively, dehydroascorbic acid (DHAA) is an
effective oxidant to reestablish the
intrachain disulfide groups of the cysteine engineered antibody after
reductive cleavage of the cysteine adducts.
Other oxidants, i.e. oxidizing agents, and oxidizing conditions, which are
known in the art may be used.
Ambient air oxidation is also effective. This mild, partial reoxidation step
forms intrachain disulfides
efficiently with high fidelity and preserves the thiol groups of the newly
introduced cysteine residues. An
approximate 10 fold excess of drug-linker intermediate, e.g. MC-vc-PAB-MMAE,
was added, mixed, and let
stand for about an hour at room temperature to effect conjugation and form the
anti-CD79b antibody-drug
conjugate. The conjugation mixture was gel filtered and loaded and eluted
through a HiTrap S column to
remove excess drug-linker intermediate and other impurities.
[0582] Figure 16 shows the general process to prepare a cysteine engineered
antibody expressed
from cell culture for conjugation. When the cell culture media contains
cysteine, disulfide adducts can form
between the newly introduced cysteine amino acid and cysteine from media.
These cysteine adducts, depicted
as a circle in the exemplary ThioMab (left) in Figure 16, must be reduced to
generate cysteine engineered
antibodies reactive for conjugation. Cysteine adducts, presumably along with
various interchain disulfide
bonds, are reductively cleaved to give a reduced form of the antibody with
reducing agents such as TCEP.
The interchain disulfide bonds between paired cysteine residues are reformed
under partial oxidation
conditions with copper sulfate, DHAA, or exposure to ambient oxygen. The newly
introduced, engineered,
and unpaired cysteine residues remain available for reaction with linker
reagents or drug-linker intermediates
to form the antibody conjugates of the invention. The ThioMabs expressed in
mammalian cell lines result in
externally conjugated Cys adduct to an engineered Cys through -S-S- bond
formation. Hence the purified
ThioMabs are treated with the reduction and reoxidation procedures as
described in Example 5 to produce
reactive ThioMabs. These ThioMabs are used to conjugate with maleimide
containing cytotoxic drugs,
fluorophores, and other labels.
10. Immunoliposomes
[0583] The anti-CD79b antibodies disclosed herein may also be formulated as
immunoliposomes.
A "liposome" is a small vesicle composed of various types of lipids,
phospholipids and/or surfactant which is
useful for delivery of a drug to a mammal. The components of the liposome are
commonly arranged in a
bilayer formation, similar to the lipid arrangement of biological membranes.
Liposomes containing the
antibody are prepared by methods known in the art, such as described in
Epstein et al., Proc. Natl. Acad. Sci.
USA 82:3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA 77:4030 (1980);
U.S. Pat. Nos. 4,485,045 and
4,544,545; and W097/38731 published October 23, 1997. Liposomes with enhanced
circulation time are
disclosed in U.S. Patent No. 5,013,556.
[0584] Particularly useful liposomes can be generated by the reverse phase
evaporation method
with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-
derivatized
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phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of
defined pore size to yield
liposomes with the desired diameter. Fab' fragments of the antibody of the
present invention can be
conjugated to the liposomes as described in Martin et al., J. Biol. Chem.
257:286-288 (1982) via a disulfide
interchange reaction. A chemotherapeutic agent is optionally contained within
the liposome. See Gabizon et
al., J. National Cancer Inst. 81(19):1484 (1989).
B. Certain Methods of Making Antibodies
1. Screening for Anti-CD79b Antibodies With the Desired Properties
[0585] Techniques for generating antibodies that bind to CD79b polypeptides
have been described
above. One may further select antibodies with certain biological
characteristics, as desired.
[0586] The growth inhibitory effects of an anti-CD79b antibody of the
invention may be assessed
by methods known in the art, e.g., using cells which express a CD79b
polypeptide either endogenously or
following transfection with the CD79b gene. For example, appropriate tumor
cell lines and CD79b-
transfected cells may be treated with an anti-CD79b monoclonal antibody of the
invention at various
concentrations for a few days (e.g., 2-7) days and stained with crystal violet
or MTT or analyzed by some
other colorimetric assay. Another method of measuring proliferation would be
by comparing 3H-thymidine
uptake by the cells treated in the presence or absence an anti-CD79b antibody
of the invention. After
treatment, the cells are harvested and the amount of radioactivity
incorporated into the DNA quantitated in a
scintillation counter. Appropriate positive controls include treatment of a
selected cell line with a growth
inhibitory antibody known to inhibit growth of that cell line. Growth
inhibition of tumor cells in vivo can be
determined in various ways known in the art. The tumor cell may be one that
overexpresses a CD79b
polypeptide. The anti-CD79b antibody will inhibit cell proliferation of a
CD79b-expressing tumor cell in vitro
or in vivo by about 25-100% compared to the untreated tumor cell, more
preferably, by about 30-100%, and
even more preferably by about 50-100% or 70-100%, in one embodiment, at an
antibody concentration of
about 0.5 to 30 g/ml. Growth inhibition can be measured at an antibody
concentration of about 0.5 to 30
g/ml or about 0.5 nM to 200 nM in cell culture, where the growth inhibition is
determined 1-10 days after
exposure of the tumor cells to the antibody. The antibody is growth inhibitory
in vivo if administration of the
anti-CD79b antibody at about 1 g/kg to about 100 mg/kg body weight results in
reduction in tumor size or
reduction of tumor cell proliferation within about 5 days to 3 months from the
first administration of the
antibody, preferably within about 5 to 30 days.
[0587] To select for an anti-CD79b antibody which induces cell death, loss of
membrane integrity
as indicated by, e.g., propidium iodide (PI), trypan blue or 7AAD uptake may
be assessed relative to control.
A PI uptake assay can be performed in the absence of complement and immune
effector cells. CD79b
polypeptide-expressing tumor cells are incubated with medium alone or medium
containing the appropriate
anti-CD79b antibody (e.g, at about l0 g/ml). The cells are incubated for a 3
day time period. Following each
treatment, cells are washed and aliquoted into 35 mm strainer-capped 12 x 75
tubes (lml per tube, 3 tubes per
treatment group) for removal of cell clumps. Tubes then receive PI (l0 g/ml).
Samples may be analyzed
using a FACSCAN flow cytometer and FACSCONVERT Ce1lQuest software (Becton
Dickinson). Those
anti-CD79b antibodies that induce statistically significant levels of cell
death as determined by PI uptake may
be selected as cell death-inducing anti-CD79b antibodies.
[0588] To screen for antibodies which bind to an epitope on a CD79b
polypeptide bound by an
antibody of interest, a routine cross-blocking assay such as that described in
Antibodies, A Laboratory Manual,
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Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be
performed. This assay can be used
to determine if a test antibody binds the same site or epitope as a known anti-
CD79b antibody. Alternatively,
or additionally, epitope mapping can be performed by methods known in the art
. For example, the antibody
sequence can be mutagenized such as by alanine scanning, to identify contact
residues. The mutant antibody
is initially tested for binding with polyclonal antibody to ensure proper
folding. In a different method,
peptides corresponding to different regions of a CD79b polypeptide can be used
in competition assays with the
test antibodies or with a test antibody and an antibody with a characterized
or known epitope.
2. Certain Library Screening Methods
[0589] Anti-CD79b antibodies of the invention can be made by using
combinatorial libraries to
screen for antibodies with the desired activity or activities. For example, a
variety of methods are known in
the art for generating phage display libraries and screening such libraries
for antibodies possessing the desired
binding characteristics. Such methods are described generally in Hoogenboom et
al. (2001) in Methods in
Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ), and
in certain embodiments, in
Lee et al. (2004) J. Mol. Biol. 340:1073-1093.
[0590] In principle, synthetic antibody clones are selected by screening phage
libraries containing
phage that display various fragments of antibody variable region (Fv) fused to
phage coat protein. Such phage
libraries are panned by affinity chromatography against the desired antigen.
Clones expressing Fv fragments
capable of binding to the desired antigen are adsorbed to the antigen and thus
separated from the non-binding
clones in the library. The binding clones are then eluted from the antigen,
and can be further enriched by
additional cycles of antigen adsorption/elution. Any of the anti-CD79b
antibodies of the invention can be
obtained by designing a suitable antigen screening procedure to select for the
phage clone of interest followed
by construction of a full length anti-CD79b antibody clone using the Fv
sequences from the phage clone of
interest and suitable constant region (Fc) sequences described in Kabat et
al., Sequences of Proteins of
Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD
(1991), vols. 1-3.
[0591] In certain embodiments, the antigen-binding domain of an antibody is
formed from two
variable (V) regions of about 110 amino acids, one each from the light (VL)
and heavy (VH) chains, that both
present three hypervariable loops (HVRs) or complementarity-determining
regions (CDRs). Variable domains
can be displayed functionally on phage, either as single-chain Fv (scFv)
fragments, in which VH and VL are
covalently linked through a short, flexible peptide, or as Fab fragments, in
which they are each fused to a
constant domain and interact non-covalently, as described in Winter et al.,
Ann. Rev. Immunol., 12: 433-455
(1994). As used herein, scFv encoding phage clones and Fab encoding phage
clones are collectively referred
to as "Fv phage clones" or "Fv clones."
[0592] Repertoires of VH and VL genes can be separately cloned by polymerase
chain reaction
(PCR) and recombined randomly in phage libraries, which can then be searched
for antigen-binding clones as
described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Libraries
from immunized sources
provide high-affinity antibodies to the immunogen without the requirement of
constructing hybridomas.
Alternatively, the naive repertoire can be cloned to provide a single source
of human antibodies to a wide
range of non-self and also self antigens without any immunization as described
by Griffiths et al., EMBO J,
12: 725-734 (1993). Finally, naive libraries can also be made synthetically by
cloning the unrearranged V-
gene segments from stem cells, and using PCR primers containing random
sequence to encode the highly
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variable CDR3 regions and to accomplish rearrangement in vitro as described by
Hoogenboom and Winter, J.
Mol. Biol., 227: 381-388 (1992).
[0593] In certain embodiments, filamentous phage is used to display antibody
fragments by fusion
to the minor coat protein pIII. The antibody fragments can be displayed as
single chain Fv fragments, in
which VH and VL domains are connected on the same polypeptide chain by a
flexible polypeptide spacer, e.g.
as described by Marks et al., J. Mol. Biol., 222: 581-597 (1991), or as Fab
fragments, in which one chain is
fused to pIII and the other is secreted into the bacterial host cell periplasm
where assembly of a Fab-coat
protein structure which becomes displayed on the phage surface by displacing
some of the wild type coat
proteins, e.g. as described in Hoogenboom et al., Nucl. Acids Res., 19: 4133-
4137 (1991).
[0594] In general, nucleic acids encoding antibody gene fragments are obtained
from immune cells
harvested from humans or animals. If a library biased in favor of anti-CD79b
clones is desired, the subject is
immunized with CD79b to generate an antibody response, and spleen cells and/or
circulating B cells other
peripheral blood lymphocytes (PBLs) are recovered for library construction. In
a preferred embodiment, a
human antibody gene fragment library biased in favor of anti-CD79b clones is
obtained by generating an anti-
CD79b antibody response in transgenic mice carrying a functional human
immunoglobulin gene array (and
lacking a functional endogenous antibody production system) such that CD79b
immunization gives rise to B
cells producing human antibodies against CD79b. The generation of human
antibody-producing transgenic
mice is described below.
[0595] Additional enrichment for anti-CD79b reactive cell populations can be
obtained by using a
suitable screening procedure to isolate B cells expressing CD79b-specific
membrane bound antibody, e.g., by
cell separation using CD79b affinity chromatography or adsorption of cells to
fluorochrome-labeled CD79b
followed by flow-activated cell sorting (FACS).
[0596] Alternatively, the use of spleen cells and/or B cells or other PBLs
from an unimmunized
donor provides a better representation of the possible antibody repertoire,
and also permits the construction of
an antibody library using any animal (human or non-human) species in which
CD79b is not antigenic. For
libraries incorporating in vitro antibody gene construction, stem cells are
harvested from the subject to provide
nucleic acids encoding unrearranged antibody gene segments. The immune cells
of interest can be obtained
from a variety of animal species, such as human, mouse, rat, lagomorpha,
luprine, canine, feline, porcine,
bovine, equine, and avian species, etc.
[0597] Nucleic acid encoding antibody variable gene segments (including VH and
VL segments)
are recovered from the cells of interest and amplified. In the case of
rearranged VH and VL gene libraries, the
desired DNA can be obtained by isolating genomic DNA or mRNA from lymphocytes
followed by
polymerase chain reaction (PCR) with primers matching the 5' and 3' ends of
rearranged VH and VL genes as
described in Orlandi et al., Proc. Natl. Acad. Sci. (USA), 86: 3833-3837
(1989), thereby making diverse V
gene repertoires for expression. The V genes can be amplified from cDNA and
genomic DNA, with back
primers at the 5' end of the exon encoding the mature V-domain and forward
primers based within the J-
segment as described in Orlandi et al. (1989) and in Ward et al., Nature, 341:
544-546 (1989). However, for
amplifying from cDNA, back primers can also be based in the leader exon as
described in Jones et al.,
Biotechnol., 9: 88-89 (1991), and forward primers within the constant region
as described in Sastry et al., Proc.
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Natl. Acad. Sci. (USA), 86: 5728-5732 (1989). To maximize complementarity,
degeneracy can be
incorporated in the primers as described in Orlandi et al. (1989) or Sastry et
al. (1989). In certain
embodiments, library diversity is maximized by using PCR primers targeted to
each V-gene family in order to
amplify all available VH and VL arrangements present in the immune cell
nucleic acid sample, e.g. as
described in the method of Marks et al., J. Mol. Biol., 222: 581-597 (1991) or
as described in the method of
Orum et al., Nucleic Acids Res., 21: 4491-4498 (1993). For cloning of the
amplified DNA into expression
vectors, rare restriction sites can be introduced within the PCR primer as a
tag at one end as described in
Orlandi et al. (1989), or by further PCR amplification with a tagged primer as
described in Clackson et al.,
Nature, 352: 624-628 (1991).
[0598] Repertoires of synthetically rearranged V genes can be derived in vitro
from V gene
segments. Most of the human VH-gene segments have been cloned and sequenced
(reported in Tomlinson et
al., J. Mol. Biol., 227: 776-798 (1992)), and mapped (reported in Matsuda et
al., Nature Genet., 3: 88-94
(1993); these cloned segments (including all the major conformations of the H1
and H21oop) can be used to
generate diverse VH gene repertoires with PCR primers encoding H3 loops of
diverse sequence and length as
described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). VH
repertoires can also be made
with all the sequence diversity focused in a long H3 loop of a single length
as described in Barbas et al., Proc.
Natl. Acad. Sci. USA, 89: 4457-4461 (1992). Human VK and Va, segments have
been cloned and sequenced
(reported in Williams and Winter, Eur. J. Immunol., 23: 1456-1461 (1993)) and
can be used to make synthetic
light chain repertoires. Synthetic V gene repertoires, based on a range of VH
and VL folds, and L3 and H3
lengths, will encode antibodies of considerable structural diversity.
Following amplification of V-gene
encoding DNAs, germline V-gene segments can be rearranged in vitro according
to the methods of
Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
[0599] Repertoires of antibody fragments can be constructed by combining VH
and VL gene
repertoires together in several ways. Each repertoire can be created in
different vectors, and the vectors
recombined in vitro, e.g., as described in Hogrefe et al., Gene, 128: 119-126
(1993), or in vivo by
combinatorial infection, e.g., the loxP system described in Waterhouse et al.,
Nucl. Acids Res., 21: 2265-2266
(1993). The in vivo recombination approach exploits the two-chain nature of
Fab fragments to overcome the
limit on library size imposed by E. coli transformation efficiency. Naive VH
and VL repertoires are cloned
separately, one into a phagemid and the other into a phage vector. The two
libraries are then combined by
phage infection of phagemid-containing bacteria so that each cell contains a
different combination and the
library size is limited only by the number of cells present (about 1012
clones). Both vectors contain in vivo
recombination signals so that the VH and VL genes are recombined onto a single
replicon and are co-
packaged into phage virions. These huge libraries provide large numbers of
diverse antibodies of good
affinity (Kd i of about 10-8 M).
[0600] Alternatively, the repertoires may be cloned sequentially into the same
vector, e.g. as
described in Barbas et al., Proc. Natl. Acad. Sci. USA, 88: 7978-7982 (1991),
or assembled together by PCR
and then cloned, e.g. as described in Clackson et al., Nature, 352: 624-628
(1991). PCR assembly can also be
used to join VH and VL DNAs with DNA encoding a flexible peptide spacer to
form single chain Fv (scFv)
repertoires. In yet another technique, "in cell PCR assembly" is used to
combine VH and VL genes within
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lymphocytes by PCR and then clone repertoires of linked genes as described in
Embleton et al., Nucl. Acids
Res., 20: 3831-3837 (1992).
[0601] The antibodies produced by naive libraries (either natural or
synthetic) can be of moderate
affinity (Kd i of about 106 to 10' M-i), but affinity maturation can also be
mimicked in vitro by constructing
and reselecting from secondary libraries as described in Winter et al. (1994),
supra. For example, mutation
can be introduced at random in vitro by using error-prone polymerase (reported
in Leung et al., Technique, 1:
11-15 (1989)) in the method of Hawkins et al., J. Mol. Biol., 226: 889-896
(1992) or in the method of Gram et
al., Proc. Natl. Acad. Sci USA, 89: 3576-3580 (1992). Additionally, affinity
maturation can be performed by
randomly mutating one or more CDRs, e.g. using PCR with primers carrying
random sequence spanning the
CDR of interest, in selected individual Fv clones and screening for higher
affinity clones. WO 9607754
(published 14 March 1996) described a method for inducing mutagenesis in a
complementarity determining
region of an immunoglobulin light chain to create a library of light chain
genes. Another effective approach is
to recombine the VH or VL domains selected by phage display with repertoires
of naturally occurring V
domain variants obtained from unimmunized donors and screen for higher
affinity in several rounds of chain
reshuffling as described in Marks et al., Biotechnol., 10: 779-783 (1992).
This technique allows the
production of antibodies and antibody fragments with affinities of about 10-9
M or less.
[0602] Screening of the libraries can be accomplished by various techniques
known in the art. For
example, CD79b can be used to coat the wells of adsorption plates, expressed
on host cells affixed to
adsorption plates or used in cell sorting, or conjugated to biotin for capture
with streptavidin-coated beads, or
used in any other method for panning phage display libraries.
[0603] The phage library samples are contacted with immobilized CD79b under
conditions suitable
for binding at least a portion of the phage particles with the adsorbent.
Normally, the conditions, including pH,
ionic strength, temperature and the like are selected to mimic physiological
conditions. The phages bound to
the solid phase are washed and then eluted by acid, e.g. as described in
Barbas et al., Proc. Natl. Acad. Sci
USA, 88: 7978-7982 (1991), or by alkali, e.g. as described in Marks et al., J.
Mol. Biol., 222: 581-597 (1991),
or by CD79b antigen competition, e.g. in a procedure similar to the antigen
competition method of Clackson et
al., Nature, 352: 624-628 (1991). Phages can be enriched 20-1,000-fold in a
single round of selection.
Moreover, the enriched phages can be grown in bacterial culture and subjected
to further rounds of selection.
[0604] The efficiency of selection depends on many factors, including the
kinetics of dissociation
during washing, and whether multiple antibody fragments on a single phage can
simultaneously engage with
antigen. Antibodies with fast dissociation kinetics (and weak binding
affinities) can be retained by use of
short washes, multivalent phage display and high coating density of antigen in
solid phase. The high density
not only stabilizes the phage through multivalent interactions, but favors
rebinding of phage that has
dissociated. The selection of antibodies with slow dissociation kinetics (and
good binding affinities) can be
promoted by use of long washes and monovalent phage display as described in
Bass et al., Proteins, 8: 309-
314 (1990) and in WO 92/09690, and a low coating density of antigen as
described in Marks et al., Biotechnol.,
10: 779-783 (1992).
[0605] It is possible to select between phage antibodies of different
affinities, even with affinities
that differ slightly, for CD79b. However, random mutation of a selected
antibody (e.g. as performed in some
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affinity maturation techniques) is likely to give rise to many mutants, most
binding to antigen, and a few with
higher affinity. With limiting CD79b, rare high affinity phage could be
competed out. To retain all higher
affinity mutants, phages can be incubated with excess biotinylated CD79b, but
with the biotinylated CD79b at
a concentration of lower molarity than the target molar affinity constant for
CD79b. The high affinity-binding
phages can then be captured by streptavidin-coated paramagnetic beads. Such
"equilibrium capture" allows
the antibodies to be selected according to their affinities of binding, with
sensitivity that permits isolation of
mutant clones with as little as two-fold higher affinity from a great excess
of phages with lower affinity.
Conditions used in washing phages bound to a solid phase can also be
manipulated to discriminate on the basis
of dissociation kinetics.
[0606] Anti-CD79b clones may be selected based on activity. In certain
embodiments, the
invention provides anti-CD79b antibodies that bind to living cells that
naturally express CD79b. In one
embodiment, the invention provides anti-CD79b antibodies that block the
binding between a CD79b ligand
and CD79b, but do not block the binding between a CD79b ligand and a second
protein. Fv clones
corresponding to such anti-CD79b antibodies can be selected by (1) isolating
anti-CD79b clones from a phage
library as described above, and optionally amplifying the isolated population
of phage clones by growing up
the population in a suitable bacterial host; (2) selecting CD79b and a second
protein against which blocking
and non-blocking activity, respectively, is desired; (3) adsorbing the anti-
CD79b phage clones to immobilized
CD79b; (4) using an excess of the second protein to elute any undesired clones
that recognize CD79b-binding
determinants which overlap or are shared with the binding determinants of the
second protein; and (5) eluting
the clones which remain adsorbed following step (4). Optionally, clones with
the desired blocking/non-
blocking properties can be further enriched by repeating the selection
procedures described herein one or more
times.
[0607] DNA encoding hybridoma-derived monoclonal antibodies or phage display
Fv clones of the
invention is readily isolated and sequenced using conventional procedures
(e.g. by using oligonucleotide
primers designed to specifically amplify the heavy and light chain coding
regions of interest from hybridoma
or phage DNA template). Once isolated, the DNA can be placed into expression
vectors, which are then
transfected into host cells such as E. coli cells, simian COS cells, Chinese
hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein, to obtain
the synthesis of the desired
monoclonal antibodies in the recombinant host cells. Review articles on
recombinant expression in bacteria of
antibody-encoding DNA include Skerra et al., Curr. Opinion in Immunol., 5: 256
(1993) and Pluckthun,
Immunol. Revs, 130: 151 (1992).
[0608] DNA encoding the Fv clones of the invention can be combined with known
DNA sequences
encoding heavy chain and/or light chain constant regions (e.g. the appropriate
DNA sequences can be obtained
from Kabat et al., supra) to form clones encoding full or partial length heavy
and/or light chains. It will be
appreciated that constant regions of any isotype can be used for this purpose,
including IgG, IgM, IgA, IgD,
and IgE constant regions, and that such constant regions can be obtained from
any human or animal species.
An Fv clone derived from the variable domain DNA of one animal (such as human)
species and then fused to
constant region DNA of another animal species to form coding sequence(s) for
"hybrid," full length heavy
chain and/or light chain is included in the definition of "chimeric" and
"hybrid" antibody as used herein. In
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certain embodiments, an Fv clone derived from human variable DNA is fused to
human constant region DNA
to form coding sequence(s) for full- or partial-length human heavy and/or
light chains.
[0609] DNA encoding anti-CD79b antibody derived from a hybridoma can also be
modified, for
example, by substituting the coding sequence for human heavy- and light-chain
constant domains in place of
homologous murine sequences derived from the hybridoma clone (e.g. as in the
method of Morrison et al.,
Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). DNA encoding a hybridoma-
or Fv clone-derived
antibody or fragment can be further modified by covalently joining to the
immunoglobulin coding sequence all
or part of the coding sequence for a non-immunoglobulin polypeptide. In this
manner, "chimeric" or "hybrid"
antibodies are prepared that have the binding specificity of the Fv clone or
hybridoma clone-derived
antibodies of the invention.
C. Antibody Dependent Enzyme Mediated Prodrug TherMy (ADEPT)
[0610] The antibodies of the present invention may also be used in ADEPT by
conjugating the
antibody to a prodrug-activating enzyme which converts a prodrug (e.g., a
peptidyl chemotherapeutic agent,
see W081/01145) to an active anti-cancer drug. See, for example, WO 88/07378
and U.S. Patent No.
4,975,278.
[0611] The enzyme component of the immunoconjugate useful for ADEPT includes
any enzyme
capable of acting on a prodrug in such a way so as to covert it into its more
active, cytotoxic form.
[0612] Enzymes that are useful in the method of this invention include, but
are not limited to,
alkaline phosphatase useful for converting phosphate-containing prodrugs into
free drugs; arylsulfatase useful
for converting sulfate-containing prodrugs into free drugs; cytosine deaminase
useful for converting non-toxic
5-fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases, such as
serratia protease, thermolysin,
subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L),
that are useful for converting
peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases, useful
for converting prodrugs that
contain D-amino acid substituents; carbohydrate-cleaving enzymes such as (3-
galactosidase and neuraminidase
useful for converting glycosylated prodrugs into free drugs; (3-lactamase
useful for converting drugs
derivatized with (3-lactams into free drugs; and penicillin amidases, such as
penicillin V amidase or penicillin
G amidase, useful for converting drugs derivatized at their amine nitrogens
with phenoxyacetyl or
phenylacetyl groups, respectively, into free drugs. Alternatively, antibodies
with enzymatic activity, also
known in the art as "abzymes", can be used to convert the prodrugs of the
invention into free active drugs (see,
e.g., Massey, Nature 328:457-458 (1987)). Antibody-abzyme conjugates can be
prepared as described herein
for delivery of the abzyme to a tumor cell population.
[0613] The enzymes of this invention can be covalently bound to the anti-CD79b
antibodies by
techniques well known in the art such as the use of the heterobifunctional
crosslinking reagents discussed
above. Alternatively, fusion proteins comprising at least the antigen binding
region of an antibody of the
invention linked to at least a functionally active portion of an enzyme of the
invention can be constructed
using recombinant DNA techniques well known in the art (see, e.g., Neuberger
et al., Nature 312:604-608
(1984).
D. Anti-CD79b Antibody
[0614] In addition to the anti-CD79b antibodies described herein, it is
contemplated that anti-
CD79b antibody variants can be prepared. Anti-CD79b antibody variants can be
prepared by introducing
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appropriate nucleotide changes into the encoding DNA, and/or by synthesis of
the desired antibody or
polypeptide. Those skilled in the art will appreciate that amino acid changes
may alter post-translational
processes of the anti-CD79b antibody, such as changing the number or position
of glycosylation sites or
altering the membrane anchoring characteristics.
[0615] Variations in the anti-CD79b antibodies described herein, can be made,
for example, using
any of the techniques and guideLines for conservative and non-conservative
mutations set forth, for instance, in
U.S. Patent No. 5,364,934. Variations may be a substitution, deletion or
insertion of one or more codons
encoding the antibody or polypeptide that results in a change in the amino
acid sequence as compared with the
native sequence antibody or polypeptide. Optionally the variation is by
substitution of at least one amino acid
with any other amino acid in one or more of the domains of the anti-CD79b
antibody. Guidance in
determining which amino acid residue may be inserted, substituted or deleted
without adversely affecting the
desired activity may be found by comparing the sequence of the anti-CD79b
antibody with that of homologous
known protein molecules and minimizing the number of amino acid sequence
changes made in regions of high
homology. Amino acid substitutions can be the result of replacing one amino
acid with another amino acid
having similar structural and/or chemical properties, such as the replacement
of a leucine with a serine, i.e.,
conservative amino acid replacements. Insertions or deletions may optionally
be in the range of about 1 to 5
amino acids. The variation allowed may be determined by systematically making
insertions, deletions or
substitutions of amino acids in the sequence and testing the resulting
variants for activity exhibited by the full-
length or mature native sequence.
[0616] Anti-CD79b antibody fragments are provided herein. Such fragments may
be truncated at
the N-terminus or C-terminus, or may lack internal residues, for example, when
compared with a full length
native antibody or protein. Certain fragments lack amino acid residues that
are not essential for a desired
biological activity of the anti-CD79b antibody.
[0617] Anti-CD79b antibody fragments may be prepared by any of a number of
conventional
techniques. Desired peptide fragments may be chemically synthesized. An
alternative approach involves
generating antibody or polypeptide fragments by enzymatic digestion, e.g., by
treating the protein with an
enzyme known to cleave proteins at sites defined by particular amino acid
residues, or by digesting the DNA
with suitable restriction enzymes and isolating the desired fragment. Yet
another suitable technique involves
isolating and amplifying a DNA fragment encoding a desired antibody or
polypeptide fragment, by
polymerase chain reaction (PCR). Oligonucleotides that define the desired
termini of the DNA fragment are
employed at the 5' and 3' primers in the PCR. Preferably, anti-CD79b antibody
fragments share at least one
biological and/or immunological activity with the native anti-CD79b antibody
disclosed herein.
[0618] In particular embodiments, conservative substitutions of interest are
shown in Table 6 under
the heading of preferred substitutions. If such substitutions result in a
change in biological activity, then more
substantial changes, denominated exemplary substitutions in Table 6, or as
further described below in
reference to amino acid classes, are introduced and the products screened.
Table 6
Original Exemplary Preferred
Residue Substitutions Substitutions
Ala (A) val; leu; ile val
Arg (R) lys; gln; asn lys
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Asn (N) gln; his; lys; arg gln
Asp (D) glu glu
Cys (C) ser ser
Gln (Q) asn asn
Glu (E) asp asp
Gly (G) pro; ala ala
His (H) asn; gln; lys; arg arg
Ile (I) leu; val; met; ala; phe;
norleucine leu
Leu (L) norleucine; ile; val;
met; ala; phe ile
Lys (K) arg; gln; asn arg
Met (M) leu; phe; ile leu
Phe (F) leu; val; ile; ala; tyr leu
Pro (P) ala ala
Ser (S) thr thr
Thr (T) ser ser
Trp (W) tyr; phe tyr
Tyr (Y) trp; phe; thr; ser phe
Val (V) ile; leu; met; phe;
ala; norleucine leu
[0619] Substantial modifications in function or immunological identity of the
anti-CD79b antibody
are accomplished by selecting substitutions that differ significantly in their
effect on maintaining (a) the
structure of the polypeptide backbone in the area of the substitution, for
example, as a sheet or helical
conformation, (b) the charge or hydrophobicity of the molecule at the target
site, or (c) the bulk of the side
chain. Naturally occurring residues are divided into groups based on common
side-chain properties:
(1) hydrophobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu;
(4) basic: asn, gln, his, lys, arg;
(5) residues that influence chain orientation: gly, pro; and
(6) aromatic: trp, tyr, phe.
[0620] Non-conservative substitutions will entail exchanging a member of one
of these classes for
another class. Such substituted residues also may be introduced into the
conservative substitution sites or,
more preferably, into the remaining (non-conserved) sites.
[0621] The variations can be made using methods known in the art such as
oligonucleotide-
mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis.
Site-directed mutagenesis
[Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids
Res., 10:6487 (1987)], cassette
mutagenesis [Wells et al., Gene, 34:315 (1985)], restriction selection
mutagenesis [Wells et al., Philos. Trans.
R. Soc. London SerA, 317:415 (1986)] or other known techniques can be
performed on the cloned DNA to
produce the anti-CD79b antibody variant DNA.
[0622] Scanning amino acid analysis can also be employed to identify one or
more amino acids
along a contiguous sequence. Among the preferred scanning amino acids are
relatively small, neutral amino
acids. Such amino acids include alanine, glycine, serine, and cysteine.
Alanine is typically a preferred
scanning amino acid among this group because it eliminates the side-chain
beyond the beta-carbon and is less
likely to alter the main-chain conformation of the variant [Cunningham and
Wells, Science, 244:1081-1085
(1989)]. Alanine is also typically preferred because it is the most common
amino acid. Further, it is
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frequently found in both buried and exposed positions [Creighton, The
Proteins, (W.H. Freeman & Co., N.Y.);
Chothia, J. Mol. Biol., 150:1 (1976)]. If alanine substitution does not yield
adequate amounts of variant, an
isoteric amino acid can be used.
[0623] Any cysteine residue not involved in maintaining the proper
conformation of the anti-CD79b
antibody also may be substituted, generally with serine, to improve the
oxidative stability of the molecule and
prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to
the anti-CD79b antibody to
improve its stability (particularly where the antibody is an antibody fragment
such as an Fv fragment).
[0624] A particularly preferred type of substitutional variant involves
substituting one or more
hypervariable region residues of a parent antibody (e.g., a humanized or human
antibody). Generally, the
resulting variant(s) selected for further development will have improved
biological properties relative to the
parent antibody from which they are generated. A convenient way for generating
such substitutional variants
involves affinity maturation using phage display. Briefly, several
hypervariable region sites (e.g., 6-7 sites)
are mutated to generate all possible amino substitutions at each site. The
antibody variants thus generated are
displayed in a monovalent fashion from filamentous phage particles as fusions
to the gene III product of M13
packaged within each particle. The phage-displayed variants are then screened
for their biological activity
(e.g., binding affinity) as herein disclosed. In order to identify candidate
hypervariable region sites for
modification, alanine scanning mutagenesis can be performed to identify
hypervariable region residues
contributing significantly to antigen binding. Alternatively, or additionally,
it may be beneficial to analyze a
crystal structure of the antigen-antibody complex to identify contact points
between the antibody and CD79b
polypeptide. Such contact residues and neighboring residues are candidates for
substitution according to the
techniques elaborated herein. Once such variants are generated, the panel of
variants is subjected to screening
as described herein and antibodies with superior properties in one or more
relevant assays may be selected for
further development.
[0625] Nucleic acid molecules encoding amino acid sequence variants of the
anti-CD79b antibody
are prepared by a variety of methods known in the art. These methods include,
but are not limited to, isolation
from a natural source (in the case of naturally occurring amino acid sequence
variants) or preparation by
oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and
cassette mutagenesis of an
earlier prepared variant or a non-variant version of the anti-CD79b antibody.
E. Modifications of Anti-CD79b Antibodies
[0626] Covalent modifications of anti-CD79b antibodies are included within the
scope of this
invention. One type of covalent modification includes reacting targeted amino
acid residues of an anti-CD79b
antibody with an organic derivatizing agent that is capable of reacting with
selected side chains or the N- or C-
terminal residues of the anti-CD79b antibody. Derivatization with bifunctional
agents is useful, for instance,
for crosslinking anti-CD79b antibody to a water-insoluble support matrix or
surface for use in the method for
purifying anti-CD79b antibodies, and vice-versa. Commonly used crosslinking
agents include, e.g., 1,1-
bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters,
for example, esters with 4-
azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl
esters such as 3,3'-
dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-
maleimido- 1, 8 -octane and agents
such as methyl-3-[(p-azidophenyl)dithio]propioimidate.
[0627] Other modifications include deamidation of glutaminyl and asparaginyl
residues to the
corresponding glutamyl and aspartyl residues, respectively, hydroxylation of
proline and lysine,
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phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation
of the a-amino groups of lysine,
arginine, and histidine side chains [T.E. Creighton, Proteins: Structure and
Molecular Properties, W.H.
Freeman & Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminal
amine, and amidation of any
C-terminal carboxyl group.
[0628] Another type of covalent modification of the anti-CD79b antibody
included within the scope
of this invention comprises altering the native glycosylation pattern of the
antibody or polypeptide. "Altering
the native glycosylation pattern" is intended for purposes herein to mean
deleting one or more carbohydrate
moieties found in native sequence anti-CD79b antibody (either by removing the
underlying glycosylation site
or by deleting the glycosylation by chemical and/or enzymatic means), and/or
adding one or more
glycosylation sites that are not present in the native sequence anti-CD79b
antibody. In addition, the phrase
includes qualitative changes in the glycosylation of the native proteins,
involving a change in the nature and
proportions of the various carbohydrate moieties present.
[0629] Glycosylation of antibodies and other polypeptides is typically either
N-linked or 0-linked.
N-linked refers to the attachment of the carbohydrate moiety to the side chain
of an asparagine residue. The
tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X
is any amino acid except
proline, are the recognition sequences for enzymatic attachment of the
carbohydrate moiety to the asparagine
side chain. Thus, the presence of either of these tripeptide sequences in a
polypeptide creates a potential
glycosylation site. 0-linked glycosylation refers to the attachment of one of
the sugars N-aceylgalactosamine,
galactose, or xylose to a hydroxyamino acid, most commonly serine or
threonine, although 5-hydroxyproline
or 5-hydroxylysine may also be used.
[0630] Addition of glycosylation sites to the anti-CD79b antibody is
conveniently accomplished by
altering the amino acid sequence such that it contains one or more of the
above-described tripeptide sequences
(for N-linked glycosylation sites). The alteration may also be made by the
addition of, or substitution by, one
or more serine or threonine residues to the sequence of the original anti-
CD79b antibody (for 0-linked
glycosylation sites). The anti-CD79b antibody amino acid sequence may
optionally be altered through
changes at the DNA level, particularly by mutating the DNA encoding the anti-
CD79b antibody at preselected
bases such that codons are generated that will translate into the desired
amino acids.
[0631] Another means of increasing the number of carbohydrate moieties on the
anti-CD79b
antibody is by chemical or enzymatic coupling of glycosides to the
polypeptide. Such methods are described
in the art, e.g., in WO 87/05330 published 11 September 1987, and in Aplin and
Wriston, CRC Crit. Rev.
Biochem., pp. 259-306 (1981).
[0632] Removal of carbohydrate moieties present on the anti-CD79b antibody may
be
accomplished chemically or enzymatically or by mutational substitution of
codons encoding for amino acid
residues that serve as targets for glycosylation. Chemical deglycosylation
techniques are known in the art and
described, for instance, by Hakimuddin, et al., Arch. Biochem. Biophys.,
259:52 (1987) and by Edge et al.,
Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on
polypeptides can be
achieved by the use of a variety of endo- and exo-glycosidases as described by
Thotakura et al., Meth.
Enzymol., 138:350 (1987).
[0633] Another type of covalent modification of anti-CD79b antibody comprises
linking the
antibody to one of a variety of nonproteinaceous polymers, e.g., polyethylene
glycol (PEG), polypropylene
glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos.
4,640,835; 4,496,689; 4,301,144;
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4,670,417; 4,791,192 or 4,179,337. The antibody also may be entrapped in
microcapsules prepared, for
example, by coacervation techniques or by interfacial polymerization (for
example, hydroxymethylcellulose or
gelatin-microcapsules and poly-(methylmethacylate) microcapsules,
respectively), in colloidal drug delivery
systems (for example, liposomes, albumin microspheres, microemulsions, nano-
particles and nanocapsules),
or in macroemulsions. Such techniques are disclosed in Remington's
Pharmaceutical Sciences, 16th edition,
Oslo, A., Ed., (1980).
[0634] The anti-CD79b antibody of the present invention may also be modified
in a way to form
chimeric molecules comprising an anti-CD79b antibody fused to another,
heterologous polypeptide or amino
acid sequence.
[0635] In one embodiment, such a chimeric molecule comprises a fusion of the
anti-CD79b
antibody with a tag polypeptide which provides an epitope to which an anti-tag
antibody can selectively bind.
The epitope tag is generally placed at the amino- or carboxyl- terminus of the
anti-CD79b antibody. The
presence of such epitope-tagged forms of the anti-CD79b antibody can be
detected using an antibody against
the tag polypeptide. Also, provision of the epitope tag enables the anti-CD79b
antibody to be readily purified
by affinity purification using an anti-tag antibody or another type of
affinity matrix that binds to the epitope
tag. Various tag polypeptides and their respective antibodies are well known
in the art. Examples include
poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the
flu HA tag polypeptide and its
antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc
tag and the 8F9, 3C7, 6E10,
G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and Cellular
Biology, 5:3610-3616 (1985)]; and
the Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et
al., Protein Engineering,
3(6):547-553 (1990)]. Other tag polypeptides include the Flag-peptide [Hopp et
al., BioTechnology, 6:1204-
1210 (1988)]; the KT3 epitope peptide [Martin et al., Science, 255:192-194
(1992)]; an a-tubulin epitope
peptide [Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7
gene 10 protein peptide tag
[Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)].
[0636] In an alternative embodiment, the chimeric molecule may comprise a
fusion of the anti-
CD79b antibody with an immunoglobulin or a particular region of an
immunoglobulin. For a bivalent form of
the chimeric molecule (also referred to as an "immunoadhesin"), such a fusion
could be to the Fc region of an
IgG molecule. The Ig fusions preferably include the substitution of a soluble
(transmembrane domain deleted
or inactivated) form of an anti-CD79b antibody in place of at least one
variable region within an Ig molecule.
In a particularly preferred embodiment, the immunoglobulin fusion includes the
hinge, CH2 and CH3, or the
hinge, CH1, CH2 and CH3 regions of an IgGl molecule. For the production of
immunoglobulin fusions see
also US Patent No. 5,428,130 issued June 27, 1995.
F. Preparation of Anti-CD79b Antibodies
[0637] The description below relates primarily to production of anti-CD79b
antibodies by culturing
cells transformed or transfected with a vector containing anti-CD79b antibody-
encoding nucleic acid. It is, of
course, contemplated that alternative methods, which are well known in the
art, may be employed to prepare
anti-CD79b antibodies. For instance, the appropriate amino acid sequence, or
portions thereof, may be
produced by direct peptide synthesis using solid-phase techniques [see, e.g.,
Stewart et al., Solid-Phase
Peptide Synthesis, W.H. Freeman Co., San Francisco, CA (1969); Merrifield, J.
Am. Chem. Soc., 85:2149-
2154 (1963)]. In vitro protein synthesis may be performed using manual
techniques or by automation.
Automated synthesis may be accomplished, for instance, using an Applied
Biosystems Peptide Synthesizer
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(Foster City, CA) using manufacturer's instructions. Various portions of the
anti-CD79b antibody may be
chemically synthesized separately and combined using chemical or enzymatic
methods to produce the desired
anti-CD79b antibody.
1. Isolation of DNA Encoding Anti-CD79b Antibody
[0638] DNA encoding anti-CD79b antibody may be obtained from a cDNA library
prepared from
tissue believed to possess the anti-CD79b antibody mRNA and to express it at a
detectable level. Accordingly,
human anti-CD79b antibody DNA can be conveniently obtained from a cDNA library
prepared from human
tissue. The anti-CD79b antibody-encoding gene may also be obtained from a
genomic library or by known
synthetic procedures (e.g., automated nucleic acid synthesis).
[0639] Libraries can be screened with probes (such as oligonucleotides of at
least about 20-80
bases) designed to identify the gene of interest or the protein encoded by it.
Screening the cDNA or genomic
library with the selected probe may be conducted using standard procedures,
such as described in Sambrook et
al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor
Laboratory Press, 1989). An
alternative means to isolate the gene encoding anti-CD79b antibody is to use
PCR methodology [Sambrook et
al., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring
Harbor Laboratory Press,
1995)].
[0640] Techniques for screening a cDNA library are well known in the art. The
oligonucleotide
sequences selected as probes should be of sufficient length and sufficiently
unambiguous that false positives
are minimized. The oligonucleotide is preferably labeled such that it can be
detected upon hybridization to
DNA in the library being screened. Methods of labeling are well known in the
art, and include the use of
radiolabels like 32P-labeled ATP, biotinylation or enzyme labeling.
Hybridization conditions, including
moderate stringency and high stringency, are provided in Sambrook et al., supr
[0641] Sequences identified in such library screening methods can be compared
and aligned to
other known sequences deposited and available in public databases such as
GenBank or other private sequence
databases. Sequence identity (at either the amino acid or nucleotide level)
within defined regions of the
molecule or across the full-length sequence can be determined using methods
known in the art and as
described herein.
[0642] Nucleic acid having protein coding sequence may be obtained by
screening selected cDNA
or genomic libraries using the deduced amino acid sequence disclosed herein
for the first time, and, if
necessary, using conventional primer extension procedures as described in
Sambrook et al., supr, to detect
precursors and processing intermediates of mRNA that may not have been reverse-
transcribed into cDNA.
2. Selection and Transformation of Host Cells
[0643] Host cells are transfected or transformed with expression or cloning
vectors described herein
for anti-CD79b antibody production and cultured in conventional nutrient media
modified as appropriate for
inducing promoters, selecting transformants, or amplifying the genes encoding
the desired sequences. The
culture conditions, such as media, temperature, pH and the like, can be
selected by the skilled artisan without
undue experimentation. In general, principles, protocols, and practical
techniques for maximizing the
productivity of cell cultures can be found in Mammalian Cell Biotechnology: a
Practical Approach, M. Butler,
ed. (IRL Press, 1991) and Sambrook et al., supr.
[0644] Methods of eukaryotic cell transfection and prokaryotic cell
transformation, which means
introduction of DNA into the host so that the DNA is replicable, either as an
extrachromosomal or by
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chromosomal integrant, are known to the ordinarily skilled artisan, for
example, CaClz, CaPO4, liposome-
mediated, polyethylene-gycol/DMSO and electroporation. Depending on the host
cell used, transformation is
performed using standard techniques appropriate to such cells. The calcium
treatment employing calcium
chloride, as described in Sambrook et al., supr, or electroporation is
generally used for prokaryotes. Infection
with Agrobacterium tumefaciens is used for transformation of certain plant
cells, as described by Shaw et al.,
Gene, 23:315 (1983) and WO 89/05859 published 29 June 1989. For mammalian
cells without such cell walls,
the calcium phosphate precipitation method of Graham and van der Eb, Virology,
52:456-457 (1978) can be
employed. General aspects of mammalian cell host system transfections have
been described in U.S. Patent
No. 4,399,216. Transformations into yeast are typically carried out according
to the method of Van Solingen
et al., J. Bact., 130:946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci.
(USA), 76:3829 (1979). However, other
methods for introducing DNA into cells, such as by nuclear microinjection,
electroporation, bacterial
protoplast fusion with intact cells, or polycations, e.g., polybrene,
polyornithine, may also be used. For
various techniques for transforming mammalian cells, see Keown et al., Methods
in Enzymology, 185:527-
537 (1990) and Mansour et al., Nature, 336:348-352 (1988).
[0645] Suitable host cells for cloning or expressing the DNA in the vectors
herein include
prokaryote, yeast, or higher eukaryote cells.
a. Prokaryotic Host Cells
[0646] Suitable prokaryotes include but are not limited to archaebacteria and
eubacteria, such as
Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such
as E. coli. Various E. coli
strains are publicly available, such as E. coli K12 strain MM294 (ATCC
31,446); E. coli X1776 (ATCC
31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC 53,635). Other
suitable prokaryotic host
cells include Enterobacteriaceae such as Escherichia, e.g., E. coli,
Enterobacter, Erwinia, Klebsiella, Proteus,
Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans,
and Shigella, as well as Bacilli
such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41P disclosed
in DD 266,710 published 12 April
1989), Pseudomonas such as P. aeruginosa, Rhizobia, Vitreoscilla, Paracoccus
and Streptomyces. These
examples are illustrative rather than limiting. Strain W3110 is one
particularly preferred host or parent host
because it is a common host strain for recombinant DNA product fermentations.
Preferably, the host cell
secretes minimal amounts of proteolytic enzymes. For example, strain W3110
(Bachmann, Cellular and
Molecular Biology, vol. 2 (Washington, D.C.: American Society for
Microbiology, 1987), pp. 1190-1219;
ATCC Deposit No. 27,325) may be modified to effect a genetic mutation in the
genes encoding proteins
endogenous to the host, with examples of such hosts including E. coli W3110
strain 1A2, which has the
complete genotype tonA ; E. coli W3110 strain 9E4, which has the complete
genotype tonA ptr3; E. coli
W3110 strain 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3
phoA E15 (argF-lac)169
degP ompT kan'; E. coli W3110 strain 37D6, which has the complete genotype
tonA ptr3 phoA E15 (argF-
lac)169 degP ompT rbs7 ilvG kanY; E. coli W3110 strain 40B4, which is strain
37D6 with a non-kanamycin
resistant degP deletion mutation; E. coli W3110 strain 33D3 having genotype
W3110 AfhuA (AtonA) ptr3 lac
Iq lacL8 AompTA(mnpc fepE) degP41 kanR (U.S. Pat. No. 5,639,635) and an E.
coli strain having mutant
periplasmic protease disclosed in U.S. Patent No. 4,946,783 issued 7 August
1990. Other strains and
derivatives thereof, such as E. coli 294 (ATCC 31,446), E. coli B, E. coli~,
1776 (ATCC 31,537) and E. coli
RV308(ATCC 31,608) are also suitable. These examples are illustrative rather
than limiting. Methods for
constructing derivatives of any of the above-mentioned bacteria having defined
genotypes are known in the art
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and described in, for example, Bass et al., Proteins, 8:309-314 (1990). It is
generally necessary to select the
appropriate bacteria taking into consideration replicability of the replicon
in the cells of a bacterium. For
example, E. coli, Serratia, or Salmonella species can be suitably used as the
host when well known plasmids
such as pBR322, pBR325, pACYC177, or pKN410 are used to supply the replicon.
Typically the host cell
should secrete minimal amounts of proteolytic enzymes, and additional protease
inhibitors may desirably be
incorporated in the cell culture. Alternatively, in vitro methods of cloning,
e.g., PCR or other nucleic acid
polymerase reactions, are suitable.
[0647] Full length antibody, antibody fragments, and antibody fusion proteins
can be produced in
bacteria, in particular when glycosylation and Fc effector function are not
needed, such as when the
therapeutic antibody is conjugated to a cytotoxic agent (e.g., a toxin) and
the immunoconjugate by itself shows
effectiveness in tumor cell destruction. Full length antibodies have greater
half life in circulation. Production
in E. coli is faster and more cost efficient. For expression of antibody
fragments and polypeptides in bacteria,
see, e.g., U.S. 5,648,237 (Carter et. al.), U.S. 5,789,199 (Joly et al.), and
U.S. 5,840,523 (Simmons et al.)
which describes translation initiation regio (TIR) and signal sequences for
optimizing expression and secretion,
these patents incorporated herein by reference. After expression, the antibody
is isolated from the E. coli cell
paste in a soluble fraction and can be purified through, e.g., a protein A or
G column depending on the isotype.
Final purification can be carried out similar to the process for purifying
antibody expressed e.g,, in CHO cells.
b. Eukaryotic Host Cells
[0648] In addition to prokaryotes, eukaryotic microbes such as filamentous
fungi or yeast are
suitable cloning or expression hosts for anti-CD79b antibody-encoding vectors.
Saccharomyces cerevisiae is
a commonly used lower eukaryotic host microorganism. Others include
Schizosaccharomyces pombe (Beach
and Nurse, Nature, 290: 140 [1981]; EP 139,383 published 2 May 1985);
Kluyveromyces hosts (U.S. Patent
No. 4,943,529; Fleer et al., Bio/Technology, 9:968-975 (1991)) such as, e.g.,
K. lactis (MW98-8C, CBS683,
CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742 [1983]), K.
fragilis (ATCC 12,424), K. bulgaricus
(ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K.
drosophilarum (ATCC 36,906;
Van den Berg et al., Bio/Technology, 8:135 (1990)), K. thennotolerans, and K.
marxianus; yarrowia (EP
402,226); Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic
Microbiol., 28:265-278 [1988]); Candida;
Trichoderma reesia (EP 244,234); Neurospora crassa (Case et al., Proc. Natl.
Acad. Sci. USA, 76:5259-5263
[1979]); Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538
published 31 October 1990); and
filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO
91/00357 published 10 January
1991), and Aspergillus hosts such as A. nidulans (Ballance et al., Biochem.
Biophys. Res. Commun., 112:284-
289 [1983]; Tilburn et al., Gene, 26:205-221 [1983]; Yelton et al., Proc.
Natl. Acad. Sci. USA, 81: 1470-1474
[1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479 [1985]).
Methylotropic yeasts are suitable herein
and include, but are not limited to, yeast capable of growth on methanol
selected from the genera consisting of
Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and
Rhodotorula. A list of specific
species that are exemplary of this class of yeasts may be found in C. Anthony,
The Biochemistry of
Meth, l~phs, 269 (1982).
[0649] Suitable host cells for the expression of glycosylated anti-CD79b
antibody are derived from
multicellular organisms. Examples of invertebrate cells include insect cells
such as Drosophila S2 and
Spodoptera Sf9, as well as plant cells, such as cell cultures of cotton, corn,
potato, soybean, petunia, tomato,
and tobacco. Numerous baculoviral strains and variants and corresponding
permissive insect host cells from
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hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito),
Aedes albopictus (mosquito),
Drosophila melanogaster (fruitfly), and Bombyx mori have been identified. A
variety of viral strains for
transfection are publicly available, e.g., the L-1 variant of Autographa
californica NPV and the Bm-5 strain of
Bombyx mori NPV, and such viruses may be used as the virus herein according to
the present invention,
particularly for transfection of Spodoptera fi-ugiperda cells.
[0650] However, interest has been greatest in vertebrate cells, and
propagation of vertebrate cells in
culture (tissue culture) has become a routine procedure. Examples of useful
mammalian host cell lines are
monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human
embryonic kidney line
(293 or 293 cells subcloned for growth in suspension culture, Graham et al.,
J. Gen Virol. 36:59 (1977)); baby
hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR
(CHO, Urlaub et al., Proc.
Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol.
Reprod. 23:243-251 (1980));
monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-
76, ATCC CRL-
1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells
(MDCK, ATCC CCL 34);
buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC
CCL 75); human liver
cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI
cells (Mather et al.,
Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human
hepatoma line (Hep G2).
[0651] Host cells are transformed with the above-described expression or
cloning vectors for anti-
CD79b antibody production and cultured in conventional nutrient media modified
as appropriate for inducing
promoters, selecting transformants, or amplifying the genes encoding the
desired sequences.
3. Selection and Use of a Replicable Vector
[0652] For recombinant production of an antibody of the invention, the nucleic
acid (e.g., cDNA or
genomic DNA) encoding it is isolated and inserted into a replicable vector for
further cloning (amplification of
the DNA) or for expression. DNA encoding the antibody is readily isolated and
sequenced using conventional
procedures (e.g., by using oligonucleotide probes that are capable of binding
specifically to genes encoding
the heavy and light chains of the antibody). Many vectors are available. The
choice of vector depends in part
on the host cell to be used. Generally, preferred host cells are of either
prokaryotic or eukaryotic (generally
mammalian) origin.
[0653] The vector may, for example, be in the form of a plasmid, cosmid, viral
particle, or phage.
The appropriate nucleic acid sequence may be inserted into the vector by a
variety of procedures. In general,
DNA is inserted into an appropriate restriction endonuclease site(s) using
techniques known in the art. Vector
components generally include, but are not limited to, one or more of a signal
sequence, an origin of replication,
one or more marker genes, an enhancer element, a promoter, and a transcription
termination sequence.
Construction of suitable vectors containing one or more of these components
employs standard ligation
techniques which are known to the skilled artisan.
[0654] The CD79b may be produced recombinantly not only directly, but also as
a fusion
polypeptide with a heterologous polypeptide, which may be a signal sequence or
other polypeptide having a
specific cleavage site at the N-terminus of the mature protein or polypeptide.
In general, the signal sequence
may be a component of the vector, or it may be a part of the anti-CD79b
antibody-encoding DNA that is
inserted into the vector. The signal sequence may be a prokaryotic signal
sequence selected, for example,
from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable
enterotoxin II leaders. For yeast
secretion the signal sequence may be, e.g., the yeast invertase leader, alpha
factor leader (including
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Saccharomyces and Kluyveromyces a-factor leaders, the latter described in U.S.
Patent No. 5,010,182), or acid
phosphatase leader, the C. albicans glucoamylase leader (EP 362,179 published
4 April 1990), or the signal
described in WO 90/13646 published 15 November 1990. In mammalian cell
expression, mammalian signal
sequences may be used to direct secretion of the protein, such as signal
sequences from secreted polypeptides
of the same or related species, as well as viral secretory leaders.
a. Prokaryotic Host Cells
[0655] Polynucleotide sequences encoding polypeptide components of the
antibody of the invention
can be obtained using standard recombinant techniques. Desired polynucleotide
sequences may be isolated
and sequenced from antibody producing cells such as hybridoma cells.
Alternatively, polynucleotides can be
synthesized using nucleotide synthesizer or PCR techniques. Once obtained,
sequences encoding the
polypeptides are inserted into a recombinant vector capable of replicating and
expressing heterologous
polynucleotides in prokaryotic hosts. Many vectors that are available and
known in the art can be used for the
purpose of the present invention. Selection of an appropriate vector will
depend mainly on the size of the
nucleic acids to be inserted into the vector and the particular host cell to
be transformed with the vector. Each
vector contains various components, depending on its function (amplification
or expression of heterologous
polynucleotide, or both) and its compatibility with the particular host cell
in which it resides.
[0656] In general, plasmid vectors containing replicon and control sequences
which are derived
from species compatible with the host cell are used in connection with these
hosts. Both expression and
cloning vectors contain a nucleic acid sequence that enables the vector to
replicate in one or more selected
host cells, as well as marking sequences which are capable of providing
phenotypic selection in transformed
cells. Such sequences are well known for a variety of bacteria, yeast, and
viruses. The origin of replication
from the plasmid pBR322, which contains genes encoding ampicillin (Amp) and
tetracycline (Tet) resistance
and thus provides easy means for identifying transformed cells, is suitable
for most Gram-negative bacteria,
the 2 plasmid origin is suitable for yeast, and various viral origins (SV40,
polyoma, adenovirus, VSV or
BPV) are useful for cloning vectors in mammalian cells. pBR322, its
derivatives, or other microbial plasmids
or bacteriophage may also contain, or be modified to contain, promoters which
can be used by the microbial
organism for expression of endogenous proteins. Examples of pBR322 derivatives
used for expression of
particular antibodies are described in detail in Carter et al., U.S. Patent
No. 5,648,237.
[0657] In addition, phage vectors containing replicon and control sequences
that are compatible
with the host microorganism can be used as transforming vectors in connection
with these hosts. For example,
bacteriophage such as kGEM.TM.-11 may be utilized in making a recombinant
vector which can be used to
transform susceptible host cells such as E. coli LE392.
[0658] The expression vector of the invention may comprise two or more
promoter-cistron pairs,
encoding each of the polypeptide components. A promoter is an untranslated
regulatory sequence located
upstream (5') to a cistron that modulates its expression. Prokaryotic
promoters typically fall into two classes,
inducible and constitutive. Inducible promoter is a promoter that initiates
increased levels of transcription of
the cistron under its control in response to changes in the culture condition,
e.g. the presence or absence of a
nutrient or a change in temperature.
[0659] A large number of promoters recognized by a variety of potential host
cells are well known.
The selected promoter can be operably linked to cistron DNA encoding the light
or heavy chain by removing
the promoter from the source DNA via restriction enzyme digestion and
inserting the isolated promoter
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sequence into the vector of the invention. Both the native promoter sequence
and many heterologous
promoters may be used to direct amplification and/or expression of the target
genes. In some embodiments,
heterologous promoters are utilized, as they generally permit greater
transcription and higher yields of
expressed target gene as compared to the native target polypeptide promoter.
[0660] Promoters recognized by a variety of potential host cells are well
known. Promoters suitable
for use with prokaryotic hosts include the PhoA promoter, the 0-galactamase
and lactose promoter systems
[Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544
(1979)], alkaline phosphatase, a
tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980);
EP 36,776] and hybrid
promoters such as the tac [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25
(1983)] or the trc promoter.
Promoters for use in bacterial systems also will contain a Shine-Dalgarno
(S.D.) sequence operably linked to
the DNA encoding anti-CD79b antibody. However, other promoters that are
functional in bacteria (such as
other known bacterial or phage promoters) are suitable as well. Their
nucleotide sequences have been
published, thereby enabling a skilled worker operably to ligate them to
cistrons encoding the target light and
heavy chains (Siebenlist et al. (1980) Ce1120: 269) using linkers or adaptors
to supply any required restriction
sites.
[0661] In one aspect of the invention, each cistron within the recombinant
vector comprises a
secretion signal sequence component that directs translocation of the
expressed polypeptides across a
membrane. In general, the signal sequence may be a component of the vector, or
it may be a part of the target
polypeptide DNA that is inserted into the vector. The signal sequence selected
for the purpose of this
invention should be one that is recognized and processed (i.e. cleaved by a
signal peptidase) by the host cell.
For prokaryotic host cells that do not recognize and process the signal
sequences native to the heterologous
polypeptides, the signal sequence is substituted by a prokaryotic signal
sequence selected, for example, from
the group consisting of the alkaline phosphatase, penicillinase, Ipp, or heat-
stable enterotoxin II (STII) leaders,
LamB, PhoE, Pe1B, OmpA and MBP. In one embodiment of the invention, the signal
sequences used in both
cistrons of the expression system are STII signal sequences or variants
thereof.
[0662] In another aspect, the production of the immunoglobulins according to
the invention can
occur in the cytoplasm of the host cell, and therefore does not require the
presence of secretion signal
sequences within each cistron. In that regard, immunoglobulin light and heavy
chains are expressed, folded
and assembled to form functional immunoglobulins within the cytoplasm. Certain
host strains (e.g., the E. coli
trxB- strains) provide cytoplasm conditions that are favorable for disulfide
bond formation, thereby permitting
proper folding and assembly of expressed protein subunits. Proba and Pluckthun
Gene, 159:203 (1995).
[0663] The present invention provides an expression system in which the
quantitative ratio of
expressed polypeptide components can be modulated in order to maximize the
yield of secreted and properly
assembled antibodies of the invention. Such modulation is accomplished at
least in part by simultaneously
modulating translational strengths for the polypeptide components.
[0664] One technique for modulating translational strength is disclosed in
Simmons et al., U.S. Pat.
No. 5,840,523. It utilizes variants of the translational initiation region
(TIR) within a cistron. For a given TIR,
a series of amino acid or nucleic acid sequence variants can be created with a
range of translational strengths,
thereby providing a convenient means by which to adjust this factor for the
desired expression level of the
specific chain. TIR variants can be generated by conventional mutagenesis
techniques that result in codon
changes which can alter the amino acid sequence, although silent changes in
the nucleotide sequence are
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preferred. Alterations in the TIR can include, for example, alterations in the
number or spacing of Shine-
Dalgarno sequences, along with alterations in the signal sequence. One method
for generating mutant signal
sequences is the generation of a "codon bank" at the beginning of a coding
sequence that does not change the
amino acid sequence of the signal sequence (i.e., the changes are silent).
This can be accomplished by
changing the third nucleotide position of each codon; additionally, some amino
acids, such as leucine, serine,
and arginine, have multiple first and second positions that can add complexity
in making the bank. This
method of mutagenesis is described in detail in Yansura et al. (1992) METHODS:
A Companion to Methods in
Enzymol. 4:151-158.
[0665] Preferably, a set of vectors is generated with a range of TIR strengths
for each cistron
therein. This limited set provides a comparison of expression levels of each
chain as well as the yield of the
desired antibody products under various TIR strength combinations. TIR
strengths can be determined by
quantifying the expression level of a reporter gene as described in detail in
Simmons et al. U.S. Pat. No. 5,
840,523. Based on the translational strength comparison, the desired
individual TIRs are selected to be
combined in the expression vector constructs of the invention.
b. Eukaryotic Host Cells
[0666] The vector components generally include, but are not limited to, one or
more of the
following: a signal sequence, an origin of replication, one or more marker
genes, an enhancer element, a
promoter, and a transcription termination sequence.
(1) Signal sequence component
[0667] A vector for use in a eukaryotic host cell may also contain a signal
sequence or other
polypeptide having a specific cleavage site at the N-terminus of the mature
protein or polypeptide of interest.
The heterologous signal sequence selected preferably is one that is recognized
and processed (i.e., cleaved by
a signal peptidase) by the host cell. In mammalian cell expression, mammalian
signal sequences as well as
viral secretory leaders, for example, the herpes simplex gD signal, are
available.
[0668] The DNA for such precursor region is ligated in reading frame to DNA
encoding the
antibody.
(2) Origin of replication
[0669] Generally, an origin of replication component is not needed for
mammalian expression
vectors. For example, the SV40 origin may typically be used only because it
contains the early promoter.
(3) Selection gene component
[0670] Expression and cloning vectors will typically contain a selection gene,
also termed a
selectable marker. Typical selection genes encode proteins that (a) confer
resistance to antibiotics or other
toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)
complement auxotrophic deficiencies, or
(c) supply critical nutrients not available from complex media, e.g., the gene
encoding D-alanine racemase for
Bacilli.
[0671] One example of a selection scheme utilizes a drug to arrest growth of a
host cell. Those
cells that are successfully transformed with a heterologous gene produce a
protein conferring drug resistance
and thus survive the selection regimen. Examples of such dominant selection
use the drugs neomycin,
mycophenolic acid and hygromycin.
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[0672] An example of suitable selectable markers for mammalian cells are those
that enable the
identification of cells competent to take up the anti-CD79b antibody-encoding
nucleic acid, such as DHFR or
thymidine kinase, metallothionein-I and -II, preferably primate
metallothionein genes, adenosine deaminase,
ornithine decarboxylase, etc. An appropriate host cell when wild-type DHFR is
employed is the CHO cell line
deficient in DHFR activity (e.g., ATCC CRL-9096), prepared and propagated as
described by Urlaub et al.,
Proc. Natl. Acad. Sci. USA, 77:4216 (1980). For example, cells transformed
with the DHFR selection gene
are first identified by culturing all of the transformants in a culture medium
that contains methotrexate (Mtx),
a competitive antagonist of DHFR. Alternatively, host cells (particularly wild-
type hosts that contain
endogenous DHFR) transformed or co-transformed with DNA sequences encoding an
antibody, wild-type
DHFR protein, and another selectable marker such as aminoglycoside 3'-
phosphotransferase (APH) can be
selected by cell growth in medium containing a selection agent for the
selectable marker such as an
aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S.
Patent No. 4,965,199.
[0673] A suitable selection gene for use in yeast is the tzp1 gene present in
the yeast plasmid YRp7
[Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141
(1979); Tschemper et al., Gene,
10:157 (1980)]. The tzpl gene provides a selection marker for a mutant strain
of yeast lacking the ability to
grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 [Jones, Genetics,
85:12 (1977)].
(4) Promoter Component
[0674] Expression and cloning vectors usually contain a promoter operably
linked to the anti-
CD79b antibody- encoding nucleic acid sequence to direct mRNA synthesis.
Promoters recognized by a
variety of potential host cells are well known.
[0675] Virtually alleukaryotic genes have an AT-rich region located
approximately 25 to 30 bases
upstream from the site where transcription is initiated. Another sequence
found 70 to 80 bases upstream from
the start of transcription of many genes is a CNCAAT region where N may be any
nucleotide. At the 3' end of
most eukaryotic genes is an AATAAA sequence that may be the signal for
addition of the poly A tail to the 3'
end of the coding sequence. All of these sequences are suitably inserted into
eukaryotic expression vectors.
[0676] Examples of suitable promoting sequences for use with yeast hosts
include the promoters for
3-phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem., 255:2073 (1980)]
or other glycolytic enzymes
[Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry,
17:4900 (1978)], such as enolase,
glyceraldehyde-3 -phosphate dehydrogenase, hexokinase, pyruvate decarboxylase,
phosphofructokinase,
glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase,
triosephosphate isomerase,
phosphoglucose isomerase, and glucokinase.
[0677] Other yeast promoters, which are inducible promoters having the
additional advantage of
transcription controlled by growth conditions, are the promoter regions for
alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase, degradative enzymes associated with
nitrogen metabolism,
metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes
responsible for maltose and
galactose utilization. Suitable vectors and promoters for use in yeast
expression are further described in EP
73,657.
[0678] Anti-CD79b antibody transcription from vectors in mammalian host cells
is controlled, for
example, by promoters obtained from the genomes of viruses such as polyoma
virus, fowlpox virus (UK
2,211,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine
papilloma virus, avian sarcoma
virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40
(SV40), from heterologous
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mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter,
and from heat-shock
promoters, provided such promoters are compatible with the host cell systems.
[0679] The early and late promoters of the SV40 virus are conveniently
obtained as an SV40
restriction fragment that also contains the SV40 viral origin of replication.
The immediate early promoter of
the human cytomegalovirus is conveniently obtained as a HindIII E restriction
fragment. A system for
expressing DNA in mammalian hosts using the bovine papilloma virus as a vector
is disclosed in U.S. Patent
No. 4,419,446. A modification of this system is described in U.S. Patent No.
4,601,978. See also Reyes et al.,
Nature 297:598-601 (1982) on expression of human 0-interferon cDNA in mouse
cells under the control of a
thymidine kinase promoter from herpes simplex virus. Alternatively, the Rous
Sarcoma Virus long terminal
repeat can be used as the promoter.
(5) Enhancer Element Component
[0680] Transcription of a DNA encoding the anti-CD79b antibody by higher
eukaryotes may be
increased by inserting an enhancer sequence into the vector. Enhancers are cis-
acting elements of DNA,
usually about from 10 to 300 bp, that act on a promoter to increase its
transcription. Many enhancer sequences
are now known from mammalian genes (globin, elastase, albumin, a-fetoprotein,
and insulin). Typically,
however, one will use an enhancer from a eukaryotic cell virus. Examples
include the SV40 enhancer on the
late side of the replication origin (bp 100-270), the cytomegalovirus early
promoter enhancer, the polyoma
enhancer on the late side of the replication origin, and adenovirus enhancers.
See also Yaniv, Nature 297:17-
18 (1982) on enhancing elements for activation of eukaryotic promoters. The
enhancer may be spliced into
the vector at a position 5' or 3' to the anti-CD79b antibody coding sequence,
but is preferably located at a site
5' from the promoter.
(6) Transcription Termination Component
[0681] Expression vectors used in eukaryotic host cells (yeast, fungi, insect,
plant, animal, human,
or nucleated cells from other multicellular organisms) will also contain
sequences necessary for the
termination of transcription and for stabilizing the mRNA. Such sequences are
commonly available from the
5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or
cDNAs. These regions contain
nucleotide segments transcribed as polyadenylated fragments in the
untranslated portion of the mRNA
encoding anti-CD79b antibody. One useful transcription termination component
is the bovine growth
hormone polyadenylation region. See W094/11026 and the expression vector
disclosed therein.
[0682] Still other methods, vectors, and host cells suitable for adaptation to
the synthesis of anti-
CD79b antibody in recombinant vertebrate cell culture are described in Gething
et al., Nature, 293:620-625
(1981); Mantei et al., Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.
4. Culturing the Host Cells
[0683] The host cells used to produce the anti-CD79b antibody of this
invention may be cultured in
a variety of media.
a. Prokaryotic Host Cells
[0684] Prokaryotic cells used to produce the polypeptides of the invention are
grown in media
known in the art and suitable for culture of the selected host cells. Examples
of suitable media include luria
broth (LB) plus necessary nutrient supplements. In some embodiments, the media
also contains a selection
agent, chosen based on the construction of the expression vector, to
selectively permit growth of prokaryotic
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cells containing the expression vector. For example, ampicillin is added to
media for growth of cells
expressing ampicillin resistant gene.
[0685] Any necessary supplements besides carbon, nitrogen, and inorganic
phosphate sources may
also be included at appropriate concentrations introduced alone or as a
mixture with another supplement or
medium such as a complex nitrogen source. Optionally the culture medium may
contain one or more reducing
agents selected from the group consisting of glutathione, cysteine, cystamine,
thioglycollate, dithioerythritol
and dithiothreitol.
[0686] The prokaryotic host cells are cultured at suitable temperatures. For
E. coli growth, for
example, the preferred temperature ranges from about 20 C to about 39 C, more
preferably from about 25 C
to about 37 C, even more preferably at about 30 C. The pH of the medium may be
any pH ranging from
about 5 to about 9, depending mainly on the host organism. For E. coli, the pH
is preferably from about 6.8 to
about 7.4, and more preferably about 7Ø
[0687] If an inducible promoter is used in the expression vector of the
invention, protein expression
is induced under conditions suitable for the activation of the promoter. In
one aspect of the invention, PhoA
promoters are used for controlling transcription of the polypeptides.
Accordingly, the transformed host cells
are cultured in a phosphate-limiting medium for induction. Preferably, the
phosphate-limiting medium is the
C.R.A.P medium (see, e.g., Simmons et al., J. Inmunol. Methods (2002), 263:133-
147). A variety of other
inducers may be used, according to the vector construct employed, as is known
in the art.
[0688] In one embodiment, the expressed polypeptides of the present invention
are secreted into
and recovered from the periplasm of the host cells. Protein recovery typically
involves disrupting the
microorganism, generally by such means as osmotic shock, sonication or lysis.
Once cells are disrupted, cell
debris or whole cells may be removed by centrifugation or filtration. The
proteins may be further purified, for
example, by affinity resin chromatography. Alternatively, proteins can be
transported into the culture media
and isolated therein. Cells may be removed from the culture and the culture
supernatant being filtered and
concentrated for further purification of the proteins produced. The expressed
polypeptides can be further
isolated and identified using commonly known methods such as polyacrylamide
gel electrophoresis (PAGE)
and Western blot assay.
[0689] In one aspect of the invention, antibody production is conducted in
large quantity by a
fermentation process. Various large-scale fed-batch fermentation procedures
are available for production of
recombinant proteins. Large-scale fermentations have at least 1000 liters of
capacity, preferably about 1,000
to 100,000 liters of capacity. These fermentors use agitator impellers to
distribute oxygen and nutrients,
especially glucose (the preferred carbon/energy source). Small scale
fermentation refers generally to
fermentation in a fermentor that is no more than approximately 100 liters in
volumetric capacity, and can
range from about 1 liter to about 100 liters.
[0690] In a fermentation process, induction of protein expression is typically
initiated after the cells
have been grown under suitable conditions to a desired density, e.g., an OD550
of about 180-220, at which
stage the cells are in the early stationary phase. A variety of inducers may
be used, according to the vector
construct employed, as is known in the art and described above. Cells may be
grown for shorter periods prior
to induction. Cells are usually induced for about 12-50 hours, although longer
or shorter induction time may
be used.
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[0691] To improve the production yield and quality of the polypeptides of the
invention, various
fermentation conditions can be modified. For example, to improve the proper
assembly and folding of the
secreted antibody polypeptides, additional vectors overexpressing chaperone
proteins, such as Dsb proteins
(DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (a peptidylprolyl cis,trans-
isomerase with chaperone
activity) can be used to co-transform the host prokaryotic cells. The
chaperone proteins have been
demonstrated to facilitate the proper folding and solubility of heterologous
proteins produced in bacterial host
cells. Chen et al. (1999) JBio Chem 274:19601-19605; Georgiou et al., U.S.
Patent No. 6,083,715; Georgiou
et al., U.S. Patent No. 6,027,888; Bothmann and Pluckthun (2000) J Biol. Chem.
275:17100-17105; Ramm
and Pluckthun (2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol.
Microbiol. 39:199-210.
[0692] To minimize proteolysis of expressed heterologous proteins (especially
those that are
proteolytically sensitive), certain host strains deficient for proteolytic
enzymes can be used for the present
invention. For example, host cell strains may be modified to effect genetic
mutation(s) in the genes encoding
known bacterial proteases such as Protease III, OmpT, DegP, Tsp, Protease I,
Protease Mi, Protease V,
Protease VI and combinations thereof. Some E. coli protease-deficient strains
are available and described in,
for example, Joly et al. (1998), supra; Georgiou et al., U.S. Patent No.
5,264,365; Georgiou et al., U.S. Patent
No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996).
[0693] In one embodiment, E. coli strains deficient for proteolytic enzymes
and transformed with
plasmids overexpressing one or more chaperone proteins are used as host cells
in the expression system of the
invention.
b. Eukaryotic Host Cells
[0694] Commercially available media such as Ham's F10 (Sigma), Minimal
Essential Medium
((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium
((DMEM), Sigma) are
suitable for culturing the host cells. In addition, any of the media described
in Ham et al., Meth. Enz. 58:44
(1979), Barnes et al., Anal. Biochem.102:255 (1980), U.S. Pat. Nos. 4,767,704;
4,657,866; 4,927,762;
4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Patent Re. 30,985
may be used as culture
media for the host cells. Any of these media may be supplemented as necessary
with hormones and/or other
growth factors (such as insulin, transferrin, or epidermal growth factor),
salts (such as sodium chloride,
calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such
as adenosine and
thymidine), antibiotics (such as GENTAMYCINTM drug), trace elements (defined
as inorganic compounds
usually present at final concentrations in the micromolar range), and glucose
or an equivalent energy source.
Any other necessary supplements may also be included at appropriate
concentrations that would be known to
those skilled in the art. The culture conditions, such as temperature, pH, and
the like, are those previously
used with the host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
5. Detecting Gene Amplification/Expression
[0695] Gene amplification and/or expression may be measured in a sample
directly, for example, by
conventional Southern blotting, Northern blotting to quantitate the
transcription of mRNA [Thomas, Proc.
Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in
situ hybridization, using an
appropriately labeled probe, based on the sequences provided herein.
Alternatively, antibodies may be
employed that can recognize specific duplexes, including DNA duplexes, RNA
duplexes, and DNA-RNA
hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled
and the assay may be
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carried out where the duplex is bound to a surface, so that upon the formation
of duplex on the surface, the
presence of antibody bound to the duplex can be detected.
[0696] Gene expression, alternatively, may be measured by immunological
methods, such as
immunohistochemical staining of cells or tissue sections and assay of cell
culture or body fluids, to quantitate
directly the expression of gene product. Antibodies useful for
immunohistochemical staining and/or assay of
sample fluids may be either monoclonal or polyclonal, and may be prepared in
any mammal. Conveniently,
the antibodies may be prepared against a native sequence CD79b polypeptide or
against a synthetic peptide
based on the DNA sequences provided herein or against exogenous sequence fused
to CD79b DNA and
encoding a specific antibody epitope.
6. Purification of Anti-CD79b Antibody
[0697] Forms of anti-CD79b antibody may be recovered from culture medium or
from host cell
lysates. If membrane-bound, it can be released from the membrane using a
suitable detergent solution (e.g.
Triton-X 100) or by enzymatic cleavage. Cells employed in expression of anti-
CD79b antibody can be
disrupted by various physical or chemical means, such as freeze-thaw cycling,
sonication, mechanical
disruption, or cell lysing agents.
[0698] It may be desired to purify anti-CD79b antibody from recombinant cell
proteins or
polypeptides. The following procedures are exemplary of suitable purification
procedures: by fractionation on
an ion-exchange column; ethanol precipitation; reverse phase HPLC;
chromatography on silica or on a cation-
exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate
precipitation; gel filtration
using, for example, Sephadex G-75; protein A Sepharose columns to remove
contaminants such as IgG; and
metal chelating columns to bind epitope-tagged forms of the anti-CD79b
antibody. Various methods of
protein purification may be employed and such methods are known in the art and
described for example in
Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification:
Principles and Practice,
Springer-Verlag, New York (1982). The purification step(s) selected will
depend, for example, on the nature
of the production process used and the particular anti-CD79b antibody
produced.
[0699] When using recombinant techniques, the antibody can be produced
intracellularly, in the
periplasmic space, or directly secreted into the medium. If the antibody is
produced intracellularly, as a first
step, the particulate debris, either host cells or lysed fragments, are
removed, for example, by centrifugation or
ultrafiltration. Carter et al., Bio/Technology 10:163-167 (1992) describe a
procedure for isolating antibodies
which are secreted to the periplasmic space of E. coli. Briefly, cell paste is
thawed in the presence of sodium
acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30
min. Cell debris can be
removed by centrifugation. Where the antibody is secreted into the medium,
supernatants from such
expression systems are generally first concentrated using a commercially
available protein concentration filter,
for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease
inhibitor such as PMSF may be
included in any of the foregoing steps to inhibit proteolysis and antibiotics
may be included to prevent the
growth of adventitious contaminants.
[0700] The antibody composition prepared from the cells can be purified using,
for example,
hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity
chromatography, with affinity
chromatography being the preferred purification technique. The suitability of
protein A as an affinity ligand
depends on the species and isotype of any immunoglobulin Fc domain that is
present in the antibody. Protein
A can be used to purify antibodies that are based on human yl, y2 or y4 heavy
chains (Lindmark et al., J.
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Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse
isotypes and for human y3 (Guss
et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is
attached is most often agarose,
but other matrices are available. Mechanically stable matrices such as
controlled pore glass or
poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing
times than can be achieved with
agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABXTMresin
(J. T. Baker, Phillipsburg,
NJ) is useful for purification. Other techniques for protein purification such
as fractionation on an ion-
exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on
silica, chromatography on
heparin SEPHAROSETM chromatography on an anion or cation exchange resin (such
as a polyaspartic acid
column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are
also available depending on
the antibody to be recovered.
[0701] Following any preliminary purification step(s), the mixture comprising
the antibody of
interest and contaminants may be subjected to low pH hydrophobic interaction
chromatography using an
elution buffer at a pH between about 2.5-4.5, preferably performed at low salt
concentrations (e.g., from about
0-0.25M salt).
G. Pharmaceutical Formulations
[0702] The antibody-drug conjugates (ADC) of the invention may be administered
by any route
appropriate to the condition to be treated. The ADC will typically be
administered parenterally, i.e. infusion,
subcutaneous, intramuscular, intravenous, intradermal, intrathecal and
epidural.
[0703] For treating these cancers, in one embodiment, the antibody-drug
conjugate is administered
via intravenous infusion. The dosage administered via infusion is in the range
of about 1 g/m2 to about
10,000 g/m2 per dose, generally one dose per week for a total of one, two,
three or four doses. Alternatively,
the dosage range is of about 1 g/m2 to about 1000 g/m2 , about 1 g/m2 to
about 800 g/m2, about 1 g/m2
to about 600 g/m2 , about 1 g/m2 to about 400 g/m2 , about 10 g/m2 to
about 500 g/m2, about 10 g/m2 to
about 300 g/m2 , about 10 g/m2 to about 200 g/m2 , and about 1 g/m2 to
about 200 g/m2 . The dose may
be administered once per day, once per week, multiple times per week, but less
than once per day, multiple
times per month but less than once per day, multiple times per month but less
than once per week, once per
month or intermittently to relieve or alleviate symptoms of the disease.
Administration may continue at any of
the disclosed intervals until remission of the tumor or symptoms of the
lymphoma, leukemia being treated.
Administration may continue after remission or relief of symptoms is achieved
where such remission or relief
is prolonged by such continued administration.
[0704] The invention also provides a method of alleviating an autoimmune
disease, comprising
administering to a patient suffering from the autoimmune disease, a
therapeutically effective amount of a
humanized 2F2 antibody-drug conjugate of any one of the preceding embodiments.
In preferred embodiments
the antibody is administered intravenously or subcutaneously. The antibody-
drug conjugate is administered
intravenously at a dosage in the range of about 1 g/m2 to about 100 mg/ m2
per dose and in a specific
embodiment, the dosage is 1 g/m2 to about 500 g/m2 . The dose may be
administered once per day, once per
week, multiple times per week, but less than once per day, multiple times per
month but less than once per day,
multiple times per month but less than once per week, once per month or
intermittently to relieve or alleviate
symptoms of the disease. Administration may continue at any of the disclosed
intervals until relief from or
alleviation of symptoms of the autoimmune disease being treated.
Administration may continue after relief
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from or alleviation of symptoms is achieved where such alleviation or relief
is prolong by such continued
administration.
[0705] The invention also provides a method of treating a B cell disorder
comprising administering
to a patient suffering from a B cell disorder, such as a B cell proliferative
disorder (including without
limitation lymphoma and leukemia) or an autoimmune disease, a therapeutically
effective amount of a
humanized 2F2 antibody of any one of the preceding embodiments, which antibody
is not conjugated to a
cytotoxic molecule or a detectable molecule. The anatibody will typically be
administered in a dosage range
of about 1 g/m2 to about 1000 mg/m2.
[0706] In one aspect, the invention further provides pharmaceutical
formulations comprising at least
one anti-CD79b antibody of the invention and/or at least one immunoconjugate
thereof and/or at least one
anti-CD79b antibody-drug conjugate of the invention. In some embodiments, a
pharmaceutical formulation
comprises (1) an antibody of the invention and/or an immunoconjugate thereof,
and (2) a pharmaceutically
acceptable carrier. In some embodiments, a pharmaceutical formulation
comprises (1) an antibody of the
invention and/or an immunoconjugate thereof, and optionally, (2) at least one
additional therapeutic agent.
Additional therapeutic agents include, but are not limited to, those described
below. The ADC will typically
be administered parenterally, i.e. infusion, subcutaneous, intramuscular,
intravenous, intradermal, intrathecal
and epidural.
[0707] Therapeutic formulations comprising an anti-CD79b antibody or CD79b
immunoconjugate
used in accordance with the present invention are prepared for storage by
mixing the antibody or
immunoconjugate, having the desired degree of purity with optional
pharmaceutically acceptable carriers,
excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition,
Osol, A. Ed. (1980)), in the form
of lyophilized formulations or aqueous solutions. Acceptable carriers,
excipients, or stabilizers are nontoxic to
recipients at the dosages and concentrations employed, and include buffers
such as acetate, Tris, phosphate,
citrate, and other organic acids; antioxidants including ascorbic acid and
methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride,
benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular
weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers
such as polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or
lysine; monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrins;
chelating agents such as EDTA; tonicifiers such as trehalose and sodium
chloride; sugars such as sucrose,
mannitol, trehalose or sorbitol; surfactant such as polysorbate; salt-forming
counter-ions such as sodium;
metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants
such as TWEEN ,
PLURONICS or polyethylene glycol (PEG). Pharmaceutical formulations to be
used for in vivo
administration are generally sterile. This is readily accomplished by
filtration through sterile filtration
membranes.
[0708] The formulations herein may also contain more than one active compound
as necessary for
the particular indication being treated, preferably those with complementary
activities that do not adversely
affect each other. For example, in addition to an anti-CD79b antibody, it may
be desirable to include in the
one formulation, an additional antibody, e.g., a second anti-CD79b antibody
which binds a different epitope
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on the CD79b polypeptide, or an antibody to some other target such as a growth
factor that affects the growth
of the particular cancer. Alternatively, or additionally, the composition may
further comprise a
chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent,
anti-hormonal agent, and/or
cardioprotectant. Such molecules are suitably present in combination in
amounts that are effective for the
purpose intended.
[0709] The active ingredients may also be entrapped in microcapsules prepared,
for example, by
coacervation techniques or by interfacial polymerization, for example,
hydroxymethylcellulose or gelatin-
microcapsules and poly-(methylmethacylate) microcapsules, respectively, in
colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions, nano-particles
and nanocapsules) or in
macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical
Sciences, 16th edition, Osol,
A. Ed. (1980).
[0710] Sustained-release preparations may be prepared. Suitable examples of
sustained-release
preparations include semi-permeable matrices of solid hydrophobic polymers
containing the antibody, which
matrices are in the form of shaped articles, e.g., films, or microcapsules.
Examples of sustained-release
matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-
methacrylate), or
poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-
glutamic acid and y ethyl-L-
glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-
glycolic acid copolymers such as the
LUPRON DEPOT (injectable microspheres composed of lactic acid-glycolic acid
copolymer and leuprolide
acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic
acid-glycolic acid enable release of molecules for over 100 days, certain
hydrogels release proteins for shorter
time periods. When encapsulated immunoglobulins remain in the body for a long
time, they may denature or
aggregate as a result of exposure to moisture at 37 C, resulting in a loss of
biological activity and possible
changes in immunogenicity. Rational strategies can be devised for
stabilization depending on the mechanism
involved. For example, if the aggregation mechanism is discovered to be
intermolecular S-S bond formation
through thio-disulfide interchange, stabilization may be achieved by modifying
sulfhydryl residues,
lyophilizing from acidic solutions, controlling moisture content, using
appropriate additives, and developing
specific polymer matrix compositions.
[0711 ] An antibody may be formulated in any suitable form for delivery to a
target cell/tissue. For
example, antibodies may be formulated as immunoliposomes. A "liposome" is a
small vesicle composed of
various types of lipids, phospholipids and/or surfactant which is useful for
delivery of a drug to a mammal.
The components of the liposome are commonly arranged in a bilayer formation,
similar to the lipid
arrangement of biological membranes. Liposomes containing the antibody are
prepared by methods known in
the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA
82:3688 (1985); Hwang et al., Proc.
Natl Acad. Sci. USA 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545;
and W097/38731 published
October 23, 1997. Liposomes with enhanced circulation time are disclosed in
U.S. Patent No. 5,013,556.
[0712] Particularly useful liposomes can be generated by the reverse phase
evaporation method
with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-
derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of
defined pore size to yield
liposomes with the desired diameter. Fab' fragments of the antibody of the
present invention can be
conjugated to the liposomes as described in Martin et al., J. Biol. Chem.
257:286-288 (1982) via a disulfide
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interchange reaction. A chemotherapeutic agent is optionally contained within
the liposome. See Gabizon et
a1.,J. National Cancer Inst. 81(19):1484 (1989).
[0713] The formulations to be used for in vivo administration must be sterile.
This is readily
accomplished by filtration through sterile filtration membranes.
H. Treatment with Anti-CD79b Antibodies
[0714] To determine CD79b expression in the cancer, various detection assays
are available. In one
embodiment, CD79b polypeptide overexpression may be analyzed by
immunohistochemistry (IHC). Parrafin
embedded tissue sections from a tumor biopsy may be subjected to the IHC assay
and accorded a CD79b
protein staining intensity criteria as follows:
Score 0 - no staining is observed or membrane staining is observed in less
than 10% of tumor cells.
Score 1+ - a faint/barely perceptible membrane staining is detected in more
than 10% of the tumor
cells. The cells are only stained in part of their membrane.
Score 2+ - a weak to moderate complete membrane staining is observed in more
than 10% of the
tumor cells.
Score 3+ - a moderate to strong complete membrane staining is observed in more
than 10% of the
tumor cells.
[0715] Those tumors with 0 or 1+ scores for CD79b polypeptide expression may
be characterized
as not overexpressing CD79b, whereas those tumors with 2+ or 3+ scores may be
characterized as
overexpressing CD79b.
[0716] Alternatively, or additionally, FISH assays such as the INFORM (sold
by Ventana,
Arizona) or PATHVISION (Vysis, Illinois) may be carried out on formalin-
fixed, paraffin-embedded tumor
tissue to determine the extent (if any) of CD79b overexpression in the tumor.
[0717] CD79b overexpression or amplification may be evaluated using an in vivo
detection assay,
e.g., by administering a molecule (such as an antibody) which binds the
molecule to be detected and is tagged
with a detectable label (e.g., a radioactive isotope or a fluorescent label)
and externally scanning the patient for
localization of the label.
[0718] As described above, the anti-CD79b antibodies of the invention have
various non-
therapeutic applications. The anti-CD79b antibodies of the present invention
can be useful for staging of
CD79b polypeptide-expressing cancers (e.g., in radioimaging). The antibodies
are also useful for purification
or immunoprecipitation of CD79b polypeptide from cells, for detection and
quantitation of CD79b
polypeptide in vitro, e.g., in an ELISA or a Western blot, to kill and
eliminate CD79b-expressing cells from a
population of mixed cells as a step in the purification of other cells.
[0719] Currently, depending on the stage of the cancer, cancer treatment
involves one or a
combination of the following therapies: surgery to remove the cancerous
tissue, radiation therapy, and
chemotherapy. Anti-CD79b antibody therapy may be especially desirable in
elderly patients who do not
tolerate the toxicity and side effects of chemotherapy well and in metastatic
disease where radiation therapy
has limited usefulness. The tumor targeting anti-CD79b antibodies of the
invention are useful to alleviate
CD79b-expressing cancers upon initial diagnosis of the disease or during
relapse. For therapeutic applications,
the anti-CD79b antibody can be used alone, or in combination therapy with,
e.g., hormones, antiangiogens, or
radiolabelled compounds, or with surgery, cryotherapy, and/or radiotherapy.
Anti-CD79b antibody treatment
can be administered in conjunction with other forms of conventional therapy,
either consecutively with, pre- or
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post-conventional therapy. Chemotherapeutic drugs such as TAXOTERE
(docetaxel), TAXOL (palictaxel),
estramustine and mitoxantrone are used in treating cancer, in particular, in
good risk patients. In the present
method of the invention for treating or alleviating cancer, the cancer patient
can be administered anti-CD79b
antibody in conjunction with treatment with the one or more of the preceding
chemotherapeutic agents. In
particular, combination therapy with palictaxel and modified derivatives (see,
e.g., EP0600517) is
contemplated. The anti-CD79b antibody will be administered with a
therapeutically effective dose of the
chemotherapeutic agent. In another embodiment, the anti-CD79b antibody is
administered in conjunction
with chemotherapy to enhance the activity and efficacy of the chemotherapeutic
agent, e.g., paclitaxel. The
Physicians' Desk Reference (PDR) discloses dosages of these agents that have
been used in treatment of
various cancers. The dosing regimen and dosages of these aforementioned
chemotherapeutic drugs that are
therapeutically effective will depend on the particular cancer being treated,
the extent of the disease and other
factors familiar to the physician of skill in the art and can be determined by
the physician.
[0720] In one particular embodiment, a conjugate comprising an anti-CD79b
antibody conjugated
with a cytotoxic agent is administered to the patient. Preferably, the
immunoconjugate bound to the CD79b
protein is internalized by the cell, resulting in increased therapeutic
efficacy of the immunoconjugate in killing
the cancer cell to which it binds. In a preferred embodiment, the cytotoxic
agent targets or interferes with the
nucleic acid in the cancer cell. Examples of such cytotoxic agents are
described above and include
maytansinoids, calicheamicins, ribonucleases and DNA endonucleases.
[0721] The anti-CD79b antibodies or toxin conjugates thereof are administered
to a human patient,
in accord with known methods, such as intravenous administration, e.g.,, as a
bolus or by continuous infusion
over a period of time, by intramuscular, intraperitoneal, intracerobrospinal,
subcutaneous, intra-articular,
intrasynovial, intrathecal, oral, topical, or inhalation routes. Intravenous
or subcutaneous administration of the
antibody is preferred.
[0722] Other therapeutic regimens may be combined with the administration of
the anti-CD79b
antibody. The combined administration includes co-administration, using
separate formulations or a single
pharmaceutical formulation, and consecutive administration in either order,
wherein preferably there is a time
period while both (or all) active agents simultaneously exert their biological
activities. Preferably such
combined therapy results in a synergistic therapeutic effect.
[0723] It may also be desirable to combine administration of the anti-CD79b
antibody or antibodies,
with administration of an antibody directed against another tumor antigen
associated with the particular cancer.
[0724] In another embodiment, the therapeutic treatment methods of the present
invention involves
the combined administration of an anti-CD79b antibody (or antibodies), and one
or more chemotherapeutic
agents or growth inhibitory agents, including co-administration of cocktails
of different chemotherapeutic
agents, or other cytotoxic agent(s) or other therapeutic agent(s) which also
inhibits tumor growth.
Chemotherapeutic agents include estramustine phosphate, prednimustine,
cisplatin, 5-fluorouracil, melphalan,
cyclophosphamide, hydroxyurea and hydroxyureataxanes (such as paclitaxel and
doxetaxel) and/or
anthracycline antibiotics. Preparation and dosing schedules for such
chemotherapeutic agents may be used
according to manufacturers' instructions or as determined empirically by the
skilled practitioner. Preparation
and dosing schedules for such chemotherapy are also described in Chemotherapy
Service Ed., M.C. Perry,
Williams & Wilkins, Baltimore, MD (1992). The antibody may be combined with an
anti-hormonal
compound; e.g., an anti-estrogen compound such as tamoxifen; an anti-
progesterone such as onapristone (see,
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EP 616 812); or an anti-androgen such as flutamide, in dosages known for such
molecules. Where the cancer
to be treated is androgen independent cancer, the patient may previously have
been subjected to anti-androgen
therapy and, after the cancer becomes androgen independent, the anti-CD79b
antibody (and optionally other
agents as described herein) may be administered to the patient.
[0725] Sometimes, it may be beneficial to also co-administer a
cardioprotectant (to prevent or
reduce myocardial dysfunction associated with the therapy) or one or more
cytokines to the patient. In
addition to the above therapeutic regimes, the patient may be subjected to
surgical removal of cancer cells
and/or radiation therapy (e.g. external beam irradiation or therapy with a
radioactive labeled agent, such as an
antibody), before, simultaneously with, or post antibody therapy. Suitable
dosages for any of the above co-
administered agents are those presently used and may be lowered due to the
combined action (synergy) of the
agent and anti-CD79b antibody.
[0726] The antibody composition of the invention will be formulated, dosed,
and administered in a
fashion consistent with good medical practice. Factors for consideration in
this context include the particular
disorder being treated, the particular mammal being treated, the clinical
condition of the individual patient, the
cause of the disorder, the site of delivery of the agent, the method of
administration, the scheduling of
administration, and other factors known to medical practitioners. The antibody
need not be, but is optionally
formulated with one or more agents currently used to prevent or treat the
disorder in question. The effective
amount of such other agents depends on the amount of antibodies of the
invention present in the formulation,
the type of disorder or treatment, and other factors discussed above. These
are generally used in the same
dosages and with administration routes as used hereinbefore or about from 1 to
99% of the heretofore
employed dosages.
[0727] For the prevention or treatment of disease, the dosage and mode of
administration will be
chosen by the physician according to known criteria. The appropriate dosage of
antibody will depend on the
type of disease to be treated, as defined above, the severity and course of
the disease, whether the antibody is
administered for preventive or therapeutic purposes, previous therapy, the
patient's clinical history and
response to the antibody, and the discretion of the attending physician. The
antibody is suitably administered
to the patient at one time or over a series of treatments. Preferably, the
antibody is administered by
intravenous infusion or by subcutaneous injections. Depending on the type and
severity of the disease, about 1
g/kg to about 50 mg/kg body weight (e.g., about 0.1-15mg/kg/dose) of antibody
can be an initial candidate
dosage for administration to the patient, whether, for example, by one or more
separate administrations, or by
continuous infusion. A dosing regimen can comprise administering an initial
loading dose of about 4 mg/kg,
followed by a weekly maintenance dose of about 2 mg/kg of the anti-CD79b
antibody. However, other dosage
regimens may be useful. A typical daily dosage might range from about 1 g/kg
to 100 mg/kg or more,
depending on the factors mentioned above. For repeated administrations over
several days or longer,
depending on the condition, the treatment is sustained until a desired
suppression of disease symptoms occurs.
The progress of this therapy can be readily monitored by conventional methods
and assays and based on
criteria known to the physician or other persons of skill in the art.
[0728] Aside from administration of the antibody protein to the patient, the
present application
contemplates administration of the antibody by gene therapy. Such
administration of nucleic acid encoding
the antibody is encompassed by the expression "administering a therapeutically
effective amount of an
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antibody". See, for example, W096/07321 published March 14, 1996 concerning
the use of gene therapy to
generate intracellular antibodies.
[0729] There are two major approaches to getting the nucleic acid (optionally
contained in a vector)
into the patient's cells; in vivo and ex vivo. For in vivo delivery the
nucleic acid is injected directly into the
patient, usually at the site where the antibody is required. For ex vivo
treatment, the patient's cells are removed,
the nucleic acid is introduced into these isolated cells and the modified
cells are administered to the patient
either directly or, for example, encapsulated within porous membranes which
are implanted into the patient
(see, e.g., U.S. Patent Nos. 4,892,538 and 5,283,187). There are a variety of
techniques available for
introducing nucleic acids into viable cells. The techniques vary depending
upon whether the nucleic acid is
transferred into cultured cells in vitro, or in vivo in the cells of the
intended host. Techniques suitable for the
transfer of nucleic acid into mammalian cells in vitro include the use of
liposomes, electroporation,
microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation
method, etc. A commonly
used vector for ex vivo delivery of the gene is a retroviral vector.
[0730] The currently preferred in vivo nucleic acid transfer techniques
include transfection with
viral vectors (such as adenovirus, Herpes simplex I virus, or adeno-associated
virus) and lipid-based systems
(useful lipids for lipid-mediated transfer of the gene are DOTMA, DOPE and DC-
Chol, for example). For
review of the currently known gene marking and gene therapy protocols see
Anderson et al., Science 256:808-
813 (1992). See also WO 93/25673 and the references cited therein.
[0731] The anti-CD79b antibodies of the invention can be in the different
forms encompassed by
the definition of "antibody" herein. Thus, the antibodies include full length
or intact antibody, antibody
fragments, native sequence antibody or amino acid variants, humanized,
chimeric or fusion antibodies,
immunoconjugates, and functional fragments thereof. In fusion antibodies an
antibody sequence is fused to a
heterologous polypeptide sequence. The antibodies can be modified in the Fc
region to provide desired
effector functions. As discussed in more detail in the sections herein, with
the appropriate Fc regions, the
naked antibody bound on the cell surface can induce cytotoxicity, e.g., via
antibody-dependent cellular
cytotoxicity (ADCC) or by recruiting complement in complement dependent
cytotoxicity, or some other
mechanism. Alternatively, where it is desirable to eliminate or reduce
effector function, so as to minimize
side effects or therapeutic complications, certain other Fc regions may be
used.
[0732] In one embodiment, the antibody competes for binding or bind
substantially to, the same
epitope as the antibodies of the invention. Antibodies having the biological
characteristics of the present anti-
CD79b antibodies of the invention are also contemplated, specifically
including the in vivo tumor targeting
and any cell proliferation inhibition or cytotoxic characteristics.
[0733] Methods of producing the above antibodies are described in detail
herein.
[0734] The present anti-CD79b antibodies are useful for treating a CD79b-
expressing cancer or
alleviating one or more symptoms of the cancer in a mammal. Such a cancer
includes, but is not limited to,
hematopoietic cancers or blood-related cancers, such as lymphoma, leukemia,
myeloma or lymphoid
malignancies, but also cancers of the spleen and cancers of the lymph nodes.
More particular examples of
such B-cell associated cancers, including for example, high, intermediate and
low grade lymphomas
(including B cell lymphomas such as, for example, mucosa-associated-lymphoid
tissue B cell lymphoma and
non-Hodgkin's lymphoma, mantle cell lymphoma, Burkitt's lymphoma, small
lymphocytic lymphoma,
marginal zone lymphoma, diffuse large cell lymphoma, follicular lymphoma, and
Hodgkin's lymphoma and T
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cell lymphomas) and leukemias (including secondary leukemia, chronic
lymphocytic leukemia, such as B cell
leukemia (CD5+ B lymphocytes), myeloid leukemia, such as acute myeloid
leukemia, chronic myeloid
leukemia, lymphoid leukemia, such as acute lymphoblastic leukemia and
myelodysplasia), and other
hematological and/or B cell- or T-cell-associated cancers. The cancers
encompass metastatic cancers of any
of the preceding. The antibody is able to bind to at least a portion of the
cancer cells that express CD79b
polypeptide in the mammal. In a preferred embodiment, the antibody is
effective to destroy or kill CD79b-
expressing tumor cells or inhibit the growth of such tumor cells, in vitro or
in vivo, upon binding to CD79b
polypeptide on the cell. Such an antibody includes a naked anti-CD79b antibody
(not conjugated to any
agent). Naked antibodies that have cytotoxic or cell growth inhibition
properties can be further harnessed with
a cytotoxic agent to render them even more potent in tumor cell destruction.
Cytotoxic properties can be
conferred to an anti-CD79b antibody by, e.g., conjugating the antibody with a
cytotoxic agent, to form an
immunoconjugate as described herein. The cytotoxic agent or a growth
inhibitory agent is preferably a small
molecule. Toxins such as calicheamicin or a maytansinoid and analogs or
derivatives thereof, are preferable.
[0735] The invention provides a composition comprising an anti-CD79b antibody
of the invention,
and a carrier. For the purposes of treating cancer, compositions can be
administered to the patient in need of
such treatment, wherein the composition can comprise one or more anti-CD79b
antibodies present as an
immunoconjugate or as the naked antibody. In a further embodiment, the
compositions can comprise these
antibodies in combination with other therapeutic agents such as cytotoxic or
growth inhibitory agents,
including chemotherapeutic agents. The invention also provides formulations
comprising an anti-CD79b
antibody of the invention, and a carrier. In one embodiment, the formulation
is a therapeutic formulation
comprising a pharmaceutically acceptable carrier.
[0736] Another aspect of the invention is isolated nucleic acids encoding the
anti-CD79b antibodies.
Nucleic acids encoding both the H and L chains and especially the
hypervariable region residues, chains which
encode the native sequence antibody as well as variants, modifications and
humanized versions of the
antibody, are encompassed.
[0737] The invention also provides methods useful for treating a CD79b
polypeptide-expressing
cancer or alleviating one or more symptoms of the cancer in a mammal,
comprising administering a
therapeutically effective amount of an anti-CD79b antibody to the mammal. The
antibody therapeutic
compositions can be administered short term (acute) or chronic, or
intermittent as directed by physician. Also
provided are methods of inhibiting the growth of, and killing a CD79b
polypeptide-expressing cell.
[0738] The invention also provides kits and articles of manufacture comprising
at least one anti-
CD79b antibody. Kits containing anti-CD79b antibodies find use, e.g., for
CD79b cell killing assays, for
purification or immunoprecipitation of CD79b polypeptide from cells. For
example, for isolation and
purification of CD79b, the kit can contain an anti-CD79b antibody coupled to
beads (e.g., sepharose beads).
Kits can be provided which contain the antibodies for detection and
quantitation of CD79b in vitro, e.g., in an
ELISA or a Western blot. Such antibody useful for detection may be provided
with a label such as a
fluorescent or radiolabel.
1. Antibody-Drug Conjugate Treatments
[0739] It is contemplated that the antibody-drug conjugates (ADC) of the
present invention may be
used to treat various diseases or disorders, e.g. characterized by the
overexpression of a tumor antigen.
Exemplary conditions or hyperproliferative disorders include benign or
malignant tumors; leukemia and
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lymphoid malignancies. Others include neuronal, glial, astrocytal,
hypothalamic, glandular, macrophagal,
epithelial, stromal, blastocoelic, inflammatory, angiogenic and immunologic,
including autoimmune, disorders.
[0740] The ADC compounds which are identified in the animal models and cell-
based assays can
be further tested in tumor-bearing higher primates and human clinical trials.
Human clinical trials can be
designed to test the efficacy of the anti-CD79b monoclonal antibody or
immunoconjugate of the invention in
patients experiencing a B cell proliferative disorder including without
limitation lymphoma, non-Hodgkins
lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent
NHL, refractory NHL,
refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic
lymphoma, leukemia, hairy
cell leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle cell
lymphoma. The clinical trial may
be designed to evaluate the efficacy of an ADC in combinations with known
therapeutic regimens, such as
radiation and/or chemotherapy involving known chemotherapeutic and/or
cytotoxic agents.
[0741] Generally, the disease or disorder to be treated is a
hyperproliferative disease such as a B
cell proliferative disorder and/or a B cell cancer. Examples of cancer to be
treated herein include, but are not
limited to, B cell proliferative disorder is selected from lymphoma, non-
Hodgkins lymphoma (NHL),
aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory
NHL, refractory indolent NHL,
chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia,
hairy cell leukemia (HCL),
acute lymphocytic leukemia (ALL), and mantle cell lymphoma.
[0742] The cancer may comprise CD79b-expressing cells, such that the ADC of
the present
invention are able to bind to the cancer cells. To determine CD79b expression
in the cancer, various
diagnostic/prognostic assays are available. In one embodiment, CD79b
overexpression may be analyzed by
IHC. Parrafin-embedded tissue sections from a tumor biopsy may be subjected to
the IHC assay and accorded
a CD79b protein staining intensity criteria with respect to the degree of
staining and in what proportion of
tumor cells examined.
[0743] For the prevention or treatment of disease, the appropriate dosage of
an ADC will depend on
the type of disease to be treated, as defined above, the severity and course
of the disease, whether the molecule
is administered for preventive or therapeutic purposes, previous therapy, the
patient's clinical history and
response to the antibody, and the discretion of the attending physician. The
molecule is suitably administered
to the patient at one time or over a series of treatments. Depending on the
type and severity of the disease,
about 1 g/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of molecule is an initial
candidate dosage for administration to
the patient, whether, for example, by one or more separate administrations, or
by continuous infusion. A
typical daily dosage might range from about 1 g/kg to 100 mg/kg or more,
depending on the factors
mentioned above. An exemplary dosage of ADC to be administered to a patient is
in the range of about 0.1 to
about 10 mg/kg of patient weight.
[0744] For repeated administrations over several days or longer, depending on
the condition, the
treatment is sustained until a desired suppression of disease symptoms occurs.
An exemplary dosing regimen
comprises administering an initial loading dose of about 4 mg/kg, followed by
a weekly maintenance dose of
about 2 mg/kg of an anti-ErbB2 antibody. Other dosage regimens may be useful.
The progress of this therapy
is easily monitored by conventional techniques and assays.
J. Combination Therapy
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[0745] An antibody-drug conjugate (ADC) of the invention may be combined in a
pharmaceutical
combination formulation, or dosing regimen as combination therapy, with a
second compound having anti-
cancer properties. The second compound of the pharmaceutical combination
formulation or dosing regimen
preferably has complementary activities to the ADC of the combination such
that they do not adversely affect
each other.
[0746] The second compound may be a chemotherapeutic agent, cytotoxic agent,
cytokine, growth
inhibitory agent, anti-hormonal agent, and/or cardioprotectant. Such molecules
are suitably present in
combination in amounts that are effective for the purpose intended. A
pharmaceutical composition containing
an ADC of the invention may also have a therapeutically effective amount of a
chemotherapeutic agent such
as a tubuLin-forming inhibitor, a topoisomerase inhibitor, or a DNA binder.
[0747] In one aspect, the first compound is an anti-CD79b ADC of the invention
and the second
compound is an anti-CD20 antibody (either a naked antibody or an ADC). In one
embodiment the second
compound is an anti-CD20 antibody rituximab (Rituxan ) or 2H7 (Genentech,
Inc., South San Francisco, CA).
Another antibodies useful for combined immunotherapy with anti-CD79b ADCs of
the invention includes
without limitation, anti-VEGF (e.g, Avastin ).
[0748] Other therapeutic regimens may be combined with the administration of
an anticancer agent
identified in accordance with this invention, including without limitation
radiation therapy and/or bone
marrow and peripheral blood transplants, and/or a cytotoxic agent, a
chemotherapeutic agent, or a growth
inhibitory agent. In one of such embodiments, a chemotherapeutic agent is an
agent or a combination of
agents such as, for example, cyclophosphamide, hydroxydaunorubicin,
adriamycin, doxorubincin, vincristine
(OncovinTM), prednisolone, CHOP, CVP, or COP, or immunotherapeutics such as
anti-CD20 (e.g., Rituxan )
or anti-VEGF (e.g., Avastin ). [0749] The combination therapy may be
administered as a simultaneous
or sequential regimen. When administered sequentially, the combination may be
administered in two or more
administrations. The combined administration includes coadministration, using
separate formulations or a
single pharmaceutical formulation, and consecutive administration in either
order, wherein preferably there is
a time period while both (or all) active agents simultaneously exert their
biological activities.
[0750] In one embodiment, treatment with an ADC involves the combined
administration of an
anticancer agent identified herein, and one or more chemotherapeutic agents or
growth inhibitory agents,
including coadministration of cocktails of different chemotherapeutic agents.
Chemotherapeutic agents
include taxanes (such as paclitaxel and docetaxel) and/or anthracycline
antibiotics. Preparation and dosing
schedules for such chemotherapeutic agents may be used according to
manufacturer's instructions or as
determined empirically by the skilled practitioner. Preparation and dosing
schedules for such chemotherapy
are also described in "Chemotherapy Service", (1992) Ed., M.C. Perry, Williams
& Wilkins, Baltimore, Md.
[0751] Suitable dosages for any of the above coadministered agents are those
presently used and
may be lowered due to the combined action (synergy) of the newly identified
agent and other
chemotherapeutic agents or treatments.
[0752] The combination therapy may provide "synergy" and prove "synergistic",
i.e. the effect
achieved when the active ingredients used together is greater than the sum of
the effects that results from using
the compounds separately. A synergistic effect may be attained when the active
ingredients are: (1) co-
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formulated and administered or delivered simultaneously in a combined, unit
dosage formulation; (2)
delivered by alternation or in parallel as separate formulations; or (3) by
some other regimen. When delivered
in alternation therapy, a synergistic effect may be attained when the
compounds are administered or delivered
sequentially, e.g. by different injections in separate syringes. In general,
during alternation therapy, an
effective dosage of each active ingredient is administered sequentially, i.e.
serially, whereas in combination
therapy, effective dosages of two or more active ingredients are administered
together.
K. Articles of Manufacture and Kits
[0753] Another embodiment of the invention is an article of manufacture
containing materials
useful for the treatment, prevention and/or diagnosis of CD79b-expressing
cancer. The article of manufacture
comprises a container and a label or package insert on or associated with the
container. Suitable containers
include, for example, bottles, vials, syringes, etc. The containers may be
formed from a variety of materials
such as glass or plastic. The container holds a composition which is effective
for treating, preventing and/or
diagnosing the cancer condition and may have a sterile access port (for
example the container may be an
intravenous solution bag or a vial having a stopper pierceable by a hypodermic
injection needle). At least one
active agent in the composition is an anti-CD79b antibody of the invention.
The label or package insert
indicates that the composition is used for treating cancer. The label or
package insert will further comprise
instructions for administering the antibody composition to the cancer patient.
Additionally, the article of
manufacture may further comprise a second container comprising a
pharmaceutically-acceptable buffer, such
as bacteriostatic water for injection (BWFI), phosphate-buffered saline,
Ringer's solution and dextrose solution.
It may further include other materials desirable from a commercial and user
standpoint, including other buffers,
diluents, filters, needles, and syringes.
[0754] Kits are also provided that are useful for various purposes , e.g., for
CD79b-expressing cell
killing assays, for purification or immunoprecipitation of CD79b polypeptide
from cells. For isolation and
purification of CD79b polypeptide, the kit can contain an anti-CD79b antibody
coupled to beads (e.g.,
sepharose beads). Kits can be provided which contain the antibodies for
detection and quantitation of CD79b
polypeptide in vitro, e.g., in an ELISA or a Western blot. As with the article
of manufacture, the kit comprises
a container and a label or package insert on or associated with the container.
The container holds a
composition comprising at least one anti-CD79b antibody of the invention.
Additional containers may be
included that contain, e.g., diluents and buffers, control antibodies. The
label or package insert may provide a
description of the composition as well as instructions for the intended in
vitro or detection use.
L. Uses for CD79b Polypeptides
[0755] This invention encompasses methods of screening compounds to identify
those that mimic the
CD79b polypeptide (agonists) or prevent the effect of the CD79b polypeptide
(antagonists). Screening assays for
antagonist drug candidates are designed to identify compounds that bind or
complex with the CD79b polypeptides
encoded by the genes identified herein, or otherwise interfere with the
interaction of the encoded polypeptides with
other cellular proteins, including e.g., inhibiting the expression of CD79b
polypeptide from cells. Such screening
assays will include assays amenable to high-throughput screening of chemical
libraries, making them particularly
suitable for identifying small molecule drug candidates.
[0756] The assays can be performed in a variety of formats, including protein-
protein binding assays,
biochemical screening assays, immunoassays, and cell-based assays, which are
well characterized in the art.
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[0757] All assays for antagonists are common in that they call for contacting
the drug candidate with a
CD79b polypeptide encoded by a nucleic acid identified herein under conditions
and for a time sufficient to allow
these two components to interact.
[0758] In binding assays, the interaction is binding and the complex formed
can be isolated or detected in
the reaction mixture. In a particular embodiment, the CD79b polypeptide
encoded by the gene identified herein or
the drug candidate is immobilized on a solid phase, e.g., on a microtiter
plate, by covalent or non-covalent
attachments. Non-covalent attachment generally is accomplished by coating the
solid surface with a solution of the
CD79b polypeptide and drying. Alternatively, an immobilized antibody, e.g., a
monoclonal antibody, specific for
the CD79b polypeptide to be immobilized can be used to anchor it to a solid
surface. The assay is performed by
adding the non-immobilized component, which may be labeled by a detectable
label, to the immobilized component,
e.g., the coated surface containing the anchored component. When the reaction
is complete, the non-reacted
components are removed, e.g., by washing, and complexes anchored on the solid
surface are detected. When the
originally non-immobilized component carries a detectable label, the detection
of label immobilized on the surface
indicates that complexing occurred. Where the originally non-immobilized
component does not carry a label,
complexing can be detected, for example, by using a labeled antibody
specifically binding the immobilized complex.
[0759] If the candidate compound interacts with but does not bind to a
particular CD79b polypeptide
encoded by a gene identified herein, its interaction with that polypeptide can
be assayed by methods well known for
detecting protein-protein interactions. Such assays include traditional
approaches, such as, e.g., cross-linking, co-
immunoprecipitation, and co-purification through gradients or chromatographic
columns. In addition, protein-
protein interactions can be monitored by using a yeast-based genetic system
described by Fields and co-workers
(Fields and Song, Nature (London), 340:245-246 (1989); Chien et al., Proc.
Natl. Acad. Sci. USA, 88:9578-9582
(1991)) as disclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89:
5789-5793 (1991). Many
transcriptional activators, such as yeast GAL4, consist of two physically
discrete modular domains, one acting as
the DNA-binding domain, the other one functioning as the transcription-
activation domain. The yeast expression
system described in the foregoing publications (generally referred to as the
"two-hybrid system") takes advantage of
this property, and employs two hybrid proteins, one in which the target
protein is fused to the DNA-binding domain
of GAL4, and another, in which candidate activating proteins are fused to the
activation domain. The expression of
a GAL1-lacZ reporter gene under control of a GAL4-activated promoter depends
on reconstitution of GAL4
activity via protein-protein interaction. Colonies containing interacting
polypeptides are detected with a
chromogenic substrate for (3-galactosidase. A complete kit (MATCHMAKERTm) for
identifying protein-protein
interactions between two specific proteins using the two-hybrid technique is
commercially available from Clontech.
This system can also be extended to map protein domains involved in specific
protein interactions as well as to
pinpoint amino acid residues that are crucial for these interactions.
[0760] Compounds that interfere with the interaction of a gene encoding a
CD79b polypeptide identified
herein and other intra- or extracellular components can be tested as follows:
usually a reaction mixture is prepared
containing the product of the gene and the intra- or extracellular component
under conditions and for a time
allowing for the interaction and binding of the two products. To test the
ability of a candidate compound to inhibit
binding, the reaction is run in the absence and in the presence of the test
compound. In addition, a placebo may be
added to a third reaction mixture, to serve as positive control. The binding
(complex formation) between the test
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compound and the intra- or extracellular component present in the mixture is
monitored as described hereinabove.
The formation of a complex in the control reaction(s) but not in the reaction
mixture containing the test compound
indicates that the test compound interferes with the interaction of the test
compound and its reaction partner.
[0761] To assay for antagonists, the CD79b polypeptide may be added to a cell
along with the compound
to be screened for a particular activity and the ability of the compound to
inhibit the activity of interest in the
presence of the CD79b polypeptide indicates that the compound is an antagonist
to the CD79b polypeptide.
Alternatively, antagonists may be detected by combining the CD79b polypeptide
and a potential antagonist with
membrane-bound CD79b polypeptide receptors or recombinant receptors under
appropriate conditions for a
competitive inhibition assay. The CD79b polypeptide can be labeled, such as by
radioactivity, such that the number
of CD79b polypeptide molecules bound to the receptor can be used to determine
the effectiveness of the potential
antagonist. The gene encoding the receptor can be identified by numerous
methods known to those of skill in the
art, for example, ligand panning and FACS sorting. Coligan et al., Current
Protocols in Immun., 1(2): Chapter 5
(1991). Preferably, expression cloning is employed wherein polyadenylated RNA
is prepared from a cell
responsive to the CD79b polypeptide and a cDNA library created from this RNA
is divided into pools and used to
transfect COS cells or other cells that are not responsive to the CD79b
polypeptide. Transfected cells that are
grown on glass slides are exposed to labeled CD79b polypeptide. The CD79b
polypeptide can be labeled by a
variety of means including iodination or inclusion of a recognition site for a
site-specific protein kinase. Following
fixation and incubation, the slides are subjected to autoradiographic
analysis. Positive pools are identified and sub-
pools are prepared and re-transfected using an interactive sub-pooling and re-
screening process, eventually yielding
a single clone that encodes the putative receptor.
[0762] As an alternative approach for receptor identification, labeled CD79b
polypeptide can be
photoaffinity-linked with cell membrane or extract preparations that express
the receptor molecule. Cross-linked
material is resolved by PAGE and exposed to X-ray film. The labeled complex
containing the receptor can be
excised, resolved into peptide fragments, and subjected to protein micro-
sequencing. The amino acid sequence
obtained from micro- sequencing would be used to design a set of degenerate
oligonucleotide probes to screen a
cDNA library to identify the gene encoding the putative receptor.
[0763] In another assay for antagonists, mammalian cells or a membrane
preparation expressing the
receptor would be incubated with labeled CD79b polypeptide in the presence of
the candidate compound. The
ability of the compound to enhance or block this interaction could then be
measured.
[0764] More specific examples of potential antagonists include an
oligonucleotide that binds to the
fusions of immunoglobulin with CD79b polypeptide, and, in particular,
antibodies including, without limitation,
poly- and monoclonal antibodies and antibody fragments, single-chain
antibodies, anti-idiotypic antibodies, and
chimeric or humanized versions of such antibodies or fragments, as well as
human antibodies and antibody
fragments. Alternatively, a potential antagonist may be a closely related
protein, for example, a mutated form of the
CD79b polypeptide that recognizes the receptor but imparts no effect, thereby
competitively inhibiting the action of
the CD79b polypeptide.
[0765] Antibodies specifically binding a CD79b polypeptide identified herein,
as well as other molecules
identified by the screening assays disclosed hereinbefore, can be administered
for the treatment of various disorders,
including cancer, in the form of pharmaceutical compositions.
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[0766] If the CD79b polypeptide is intracellular and whole antibodies are used
as inhibitors, internalizing
antibodies are preferred. However, lipofections or liposomes can also be used
to deliver the antibody, or an
antibody fragment, into cells. Where antibody fragments are used, the smallest
inhibitory fragment that specifically
binds to the binding domain of the target protein is preferred. For example,
based upon the variable-region
sequences of an antibody, peptide molecules can be designed that retain the
ability to bind the target protein
sequence. Such peptides can be synthesized chemically and/or produced by
recombinant DNA technology. See,
e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993).
[0767] The formulation herein may also contain more than one active compound
as necessary for the
particular indication being treated, preferably those with complementary
activities that do not adversely affect each
other. Alternatively, or in addition, the composition may comprise an agent
that enhances its function, such as, for
example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-
inhibitory agent. Such molecules are
suitably present in combination in amounts that are effective for the purpose
intended.
M. Antibody Derivatives
[0768] The antibodies of the present invention can be further modified to
contain additional
nonproteinaceous moieties that are known in the art and readily available.
Preferably, the moieties suitable for
derivatization of the antibody are water soluble polymers. Non-limiting
examples of water soluble polymers
include, but are not limited to, polyethylene glycol (PEG), copolymers of
ethylene glycol/propylene glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone,
poly-1, 3-dioxolane, poly- 1,3,6-trioxane,
ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or
random copolymers), and dextran
or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol
homopolymers, prolypropylene
oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol),
polyvinyl alcohol, and mixtures
thereof. Polyethylene glycol propionaldehyde may have advantages in
manufacturing due to its stability in water.
The polymer may be of any molecular weight, and may be branched or unbranched.
The number of polymers
attached to the antibody may vary, and if more than one polymers are attached,
they can be the same or different
molecules. In general, the number and/or type of polymers used for
derivatization can be determined based on
considerations including, but not limited to, the particular properties or
functions of the antibody to be improved,
whether the antibody derivative will be used in a therapy under defined
conditions, etc.
N. Method of Screening
[0769] Yet another embodiment of the present invention is directed to a method
of determining the
presence of a CD79b polypeptide in a sample suspected of containing the CD79b
polypeptide, wherein the method
comprises exposing the sample to an antibody drug conjugate thereof, that
binds to the CD79b polypeptide and
determining binding of the antibody drug conjugate thereof, to the CD79b
polypeptide in the sample, wherein the
presence of such binding is indicative of the presence of the CD79b
polypeptide in the sample. Optionally, the
sample may contain cells (which may be cancer cells) suspected of expressing
the CD79b polypeptide. The
antibody drug conjugate thereof, employed in the method may optionally be
detectably labeled, attached to a solid
support, or the like.
[0770] Another embodiment of the present invention is directed to a method of
diagnosing the presence
of a tumor in a mammal, wherein the method comprises (a) contacting a test
sample comprising tissue cells
obtained from the mammal with an antibody drug conjugate thereof, that binds
to a CD79b polypeptide and (b)
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detecting the formation of a complex between the antibody drug conjugate
thereof, and the CD79b polypeptide in
the test sample, wherein the formation of a complex is indicative of the
presence of a tumor in the mammal.
Optionally, the antibody drug conjugate thereof, is detectably labeled,
attached to a solid support, or the like, and/or
the test sample of tissue cells is obtained from an individual suspected of
having a cancerous tumor.
IV. Further Methods of Using Anti-CD79b Antibodies and ImmunoconL ates
A. Diagnostic Methods and Methods of Detection
[0771] In one aspect, anti-CD79b antibodies and immunoconjugates of the
invention are useful for
detecting the presence of CD79b in a biological sample. The term "detecting"
as used herein encompasses
quantitative or qualitative detection. In certain embodiments, a biological
sample comprises a cell or tissue. In
certain embodiments, such tissues include normal and/or cancerous tissues that
express CD79b at higher levels
relative to other tissues, for example, B cells and/or B cell associated
tissues.
[0772] In one aspect, the invention provides a method of detecting the
presence of CD79b in a biological
sample. In certain embodiments, the method comprises contacting the biological
sample with an anti-CD79b
antibody under conditions permissive for binding of the anti-CD79b antibody to
CD79b, and detecting whether a
complex is formed between the anti-CD79b antibody and CD79b.
[0773] In one aspect, the invention provides a method of diagnosing a disorder
associated with increased
expression of CD79b. In certain embodiments, the method comprises contacting a
test cell with an anti-CD79b
antibody; determining the level of expression (either quantitatively or
qualitatively) of CD79b by the test cell by
detecting binding of the anti-CD79b antibody to CD79b; and comparing the level
of expression of CD79b by the
test cell with the level of expression of CD79b by a control cell (e.g., a
normal cell of the same tissue origin as the
test cell or a cell that expresses CD79b at levels comparable to such a normal
cell), wherein a higher level of
expression of CD79b by the test cell as compared to the control cell indicates
the presence of a disorder associated
with increased expression of CD79b. In certain embodiments, the test cell is
obtained from an individual suspected
of having a disorder associated with increased expression of CD79b. In certain
embodiments, the disorder is a cell
proliferative disorder, such as a cancer or a tumor.
[0774] Exemplary cell proliferative disorders that may be diagnosed using an
antibody of the invention
include a B cell disorder and/or a B cell proliferative disorder including,
but not limited to, lymphoma, non-
Hodgkins lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed
indolent NHL, refractory NHL,
refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic
lymphoma, leukemia, hairy cell
leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle cell lymphoma.
[0775] In certain embodiments, a method of diagnosis or detection, such as
those described above,
comprises detecting binding of an anti-CD79b antibody to CD79b expressed on
the surface of a cell or in a
membrane preparation obtained from a cell expressing CD79b on its surface. In
certain embodiments, the method
comprises contacting a cell with an anti-CD79b antibody under conditions
permissive for binding of the anti-CD79b
antibody to CD79b, and detecting whether a complex is formed between the anti-
CD79b antibody and CD79b on
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the cell surface. An exemplary assay for detecting binding of an anti-CD79b
antibody to CD79b expressed on the
surface of a cell is a "FACS" assay.
[0776] Certain other methods can be used to detect binding of anti-CD79b
antibodies to CD79b. Such
methods include, but are not limited to, antigen-binding assays that are well
known in the art, such as western blots,
radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich"
immunoassays,
immunoprecipitation assays, fluorescent immunoassays, protein A immunoassays,
and immunohistochemistry
(IHC).
[0777] In certain embodiments, anti-CD79b antibodies are labeled. Labels
include, but are not limited to,
labels or moieties that are detected directly (such as fluorescent,
chromophoric, electron-dense, chemiluminescent,
and radioactive labels), as well as moieties, such as enzymes or ligands, that
are detected indirectly, e.g., through an
enzymatic reaction or molecular interaction. Exemplary labels include, but are
not limited to, the radioisotopes 32P,
14C 125I, 3H, and13'I, fluorophores such as rare earth chelates or fluorescein
and its derivatives, rhodamine and its
derivatives, dansyl, umbelliferone, luceriferases, e.g., firefly luciferase
and bacterial luciferase (U.S. Pat. No.
4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase
(HRP), alkaline phosphatase, (3-
galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose
oxidase, galactose oxidase, and glucose-
6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine
oxidase, coupled with an enzyme
that employs hydrogen peroxide to oxidize a dye precursor such as HRP,
lactoperoxidase, or microperoxidase,
biotin/avidin, spin labels, bacteriophage labels, stable free radicals, and
the like.
[0778] In certain embodiments, anti-CD79b antibodies are immobilized on an
insoluble matrix.
Immobilization entails separating the anti-CD79b antibody from any CD79b that
remains free in solution. This
conventionally is accomplished by either insolubilizing the anti-CD79b
antibody before the assay procedure, as by
adsorption to a water-insoluble matrix or surface (Bennich et al.., U.S.
3,720,760), or by covalent coupling (for
example, using glutaraldehyde cross-linking), or by insolubilizing the anti-
CD79b antibody after formation of a
complex between the anti-CD79b antibody and CD79b, e.g., by
immunoprecipitation.
[0779] Any of the above embodiments of diagnosis or detection may be carried
out using an
immunoconjugate of the invention in place of or in addition to an anti-CD79b
antibody.
B. Therapeutic Methods
[0780] An antibody or immunoconjugate of the invention may be used in, for
example, in vitro, ex vivo,
and in vivo therapeutic methods. In one aspect, the invention provides methods
for inhibiting cell growth or
proliferation, either in vivo or in vitro, the method comprising exposing a
cell to an anti-CD79b antibody or
immunoconjugate thereof under conditions permissive for binding of the
immunoconjugate to CD79b. "Inhibiting
cell growth or proliferation" means decreasing a cell's growth or
proliferation by at least 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, 95%, or 100%, and includes inducing cell death. In
certain embodiments, the cell is a
tumor cell. In certain embodiments, the cell is a B cell. In certain
embodiments, the cell is a xenograft, e.g., as
exemplified herein.
[0781] In one aspect, an antibody or immunoconjugate of the invention is used
to treat or prevent a B cell
proliferative disorder. In certain embodiments, the cell proliferative
disorder is associated with increased
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expression and/or activity of CD79b. For example, in certain embodiments, the
B cell proliferative disorder is
associated with increased expression of CD79b on the surface of a B cell. In
certain embodiments, the B cell
proliferative disorder is a tumor or a cancer. Examples of B cell
proliferative disorders to be treated by the
antibodies or immunoconjugates of the invention include, but are not limited
to, lymphoma, non-Hodgkins
lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent
NHL, refractory NHL, refractory
indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma,
leukemia, hairy cell leukemia
(HCL), acute lymphocytic leukemia (ALL), and mantle cell lymphoma.
[0782] In one aspect, the invention provides methods for treating a B cell
proliferative disorder
comprising administering to an individual an effective amount of an anti-CD79b
antibody or immunoconjugate
thereof. In certain embodiments, a method for treating a B cell proliferative
disorder comprises administering to an
individual an effective amount of a pharmaceutical formulation comprising an
anti-CD79b antibody or anti-CD79b
immunoconjugate and, optionally, at least one additional therapeutic agent,
such as those provided below. In
certain embodiments, a method for treating a cell proliferative disorder
comprises administering to an individual an
effective amount of a pharmaceutical formulation comprising 1) an
immunoconjugate comprising an anti-CD79b
antibody and a cytotoxic agent; and optionally, 2) at least one additional
therapeutic agent, such as those provided
below.
[0783] In one aspect, at least some of the antibodies or immunoconjugates of
the invention can bind
CD79b from species other than human. Accordingly, antibodies or
immunoconjugates of the invention can be used
to bind CD79b, e.g., in a cell culture containing CD79b, in humans, or in
other mammals having a CD79b with
which an antibody or immunoconjugate of the invention cross-reacts (e.g.
chimpanzee, baboon, marmoset,
cynomolgus and rhesus monkeys, pig or mouse). In one embodiment, an anti-CD79b
antibody or immunoconjugate
can be used for targeting CD79b on B cells by contacting the antibody or
immunoconjugate with CD79b to form an
antibody or immunoconjugate-antigen complex such that a conjugated cytotoxin
of the immunoconjugate accesses
the interior of the cell. In one embodiment, the CD79b is human CD79b.
[0784] In one embodiment, an anti-CD79b antibody or immunoconjugate can be
used in a method for
binding CD79b in an individual suffering from a disorder associated with
increased CD79b expression and/or
activity, the method comprising administering to the individual the antibody
or immunoconjugate such that CD79b
in the individual is bound. In one embodiment, the bound antibody or
immunoconjugate is internalized into the B
cell expressing CD79b. In one embodiment, the CD79b is human CD79b, and the
individual is a human individual.
Alternatively, the individual can be a mammal expressing CD79b to which an
anti-CD79b antibody binds. Still
further the individual can be a mammal into which CD79b has been introduced
(e.g., by administration of CD79b or
by expression of a transgene encoding CD79b).
[0785] An anti-CD79b antibody or immunoconjugate can be administered to a
human for therapeutic
purposes. Moreover, an anti-CD79b antibody or immunoconjugate can be
administered to a non-human mammal
expressing CD79b with which the antibody cross-reacts (e.g., a primate, pig,
rat, or mouse) for veterinary purposes
or as an animal model of human disease. Regarding the latter, such animal
models may be useful for evaluating the
therapeutic efficacy of antibodies or immunoconjugates of the invention (e.g.,
testing of dosages and time courses
of administration).
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[0786] Antibodies or immunoconjugates of the invention can be used either
alone or in combination with
other compositions in a therapy. For instance, an antibody or immunoconjugate
of the invention may be co-
administered with at least one additional therapeutic agent and/or adjuvant.
In certain embodiments, an additional
therapeutic agent is a cytotoxic agent, a chemotherapeutic agent, or a growth
inhibitory agent. In one of such
embodiments, a chemotherapeutic agent is an agent or a combination of agents
such as, for example,
cyclophosphamide, hydroxydaunorubicin, adriamycin, doxorubincin, vincristine
(OncovinTM), prednisolone, CHOP,
CVP, or COP, or immunotherapeutics such as anti-CD20 (e.g., Rituxan ) or anti-
VEGF (e.g., Avastin ), wherein
the combination therapy is useful in the treatment of cancers and/or B cell
disorders such as B cell proliferative
disorders including lymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL,
relapsed aggressive NHL,
relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic
lymphocytic leukemia (CLL), small
lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic
leukemia (ALL), and mantle cell
lymphoma.
[0787] Such combination therapies noted above encompass combined
administration (where two or more
therapeutic agents are included in the same or separate formulations), and
separate administration, in which case,
administration of the antibody or immunoconjugate of the invention can occur
prior to, simultaneously, and/or
following, administration of the additional therapeutic agent and/or adjuvant.
Antibodies or immunoconjugates of
the invention can also be used in combination with radiation therapy.
[0788] An antibody or immunoconjugate of the invention (and any additional
therapeutic agent or
adjuvant) can be administered by any suitable means, including parenteral,
subcutaneous, intraperitoneal,
intrapulmonary, and intranasal, and, if desired for local treatment,
intralesional administration. Parenteral infusions
include intramuscular, intravenous, intraarterial, intraperitoneal, or
subcutaneous administration. In addition, the
antibody or immunoconjugate is suitably administered by pulse infusion,
particularly with declining doses of the
antibody or immunoconjugate. Dosing can be by any suitable route, e.g. by
injections, such as intravenous or
subcutaneous injections, depending in part on whether the administration is
brief or chronic.
[0789] Antibodies or immunoconjugates of the invention would be formulated,
dosed, and administered
in a fashion consistent with good medical practice. Factors for consideration
in this context include the particular
disorder being treated, the particular mammal being treated, the clinical
condition of the individual patient, the
cause of the disorder, the site of delivery of the agent, the method of
administration, the scheduling of
administration, and other factors known to medical practitioners. The antibody
or immunoconjugate need not be,
but is optionally formulated with one or more agents currently used to prevent
or treat the disorder in question. The
effective amount of such other agents depends on the amount of antibody or
immunoconjugate present in the
formulation, the type of disorder or treatment, and other factors discussed
above. These are generally used in the
same dosages and with administration routes as described herein, or about from
1 to 99% of the dosages described
herein, or in any dosage and by any route that is empirically/clinically
determined to be appropriate.
[0790] For the prevention or treatment of disease, the appropriate dosage of
an antibody or
immunoconjugate of the invention (when used alone or in combination with one
or more other additional
therapeutic agents, such as chemotherapeutic agents) will depend on the type
of disease to be treated, the type of
antibody or immunoconjugate, the severity and course of the disease, whether
the antibody or immunoconjugate is
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administered for preventive or therapeutic purposes, previous therapy, the
patient's clinical history and response to
the antibody or immunoconjugate, and the discretion of the attending
physician. The antibody or immunoconjugate
is suitably administered to the patient at one time or over a series of
treatments. Depending on the type and severity
of the disease, about 1 g/kg to 100 mg/kg (e.g. 0.lmg/kg-20mg/kg) of antibody
or immunoconjugate can be an
initial candidate dosage for administration to the patient, whether, for
example, by one or more separate
administrations, or by continuous infusion. One typical daily dosage might
range from about 1 g/kg to 100 mg/kg
or more, depending on the factors mentioned above. For repeated
administrations over several days or longer,
depending on the condition, the treatment would generally be sustained until a
desired suppression of disease
symptoms occurs. One exemplary dosage of the antibody or immunoconjugate would
be in the range from about
0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0
mg/kg, 4.0 mg/kg or 10 mg/kg (or
any combination thereof) of antibody or immunoconjugate may be administered to
the patient. Such doses may be
administered intermittently, e.g. every week or every three weeks (e.g. such
that the patient receives from about two
to about twenty, or e.g. about six doses of the antibody or immunoconjugate).
An initial higher loading dose,
followed by one or more lower doses may be administered. An exemplary dosing
regimen comprises administering
an initial loading dose of about 4 mg/kg, followed by a weekly maintenance
dose of about 2 mg/kg of the antibody.
However, other dosage regimens may be useful. The progress of this therapy is
easily monitored by conventional
techniques and assays.
C. Activi , AssEs
[0791] Anti-CD79b antibodies and immunoconjugates of the invention may be
characterized for their
physical/chemical properties and/or biological activities by various assays
known in the art.
1. Activi , assEs
[0792] In one aspect, assays are provided for identifying anti-CD79b
antibodies or immunoconjugates
thereof having biological activity. Biological activity may include, e.g., the
ability to inhibit cell growth or
proliferation (e.g., "cell killing" activity), or the ability to induce cell
death, including programmed cell death
(apoptosis). Antibodies or immunoconjugates having such biological activity in
vivo and/or in vitro are also
provided.
[0793] In certain embodiments, an anti-CD79b antibody or immunoconjugate
thereof is tested for its
ability to inhibit cell growth or proliferation in vitro. Assays for
inhibition of cell growth or proliferation are well
known in the art. Certain assays for cell proliferation, exemplified by the
"cell killing" assays described herein,
measure cell viability. One such assay is the Ce1lTiter-G1oTM Luminescent Cell
Viability Assay, which is
commercially available from Promega (Madison, WI). That assay determines the
number of viable cells in culture
based on quantitation of ATP present, which is an indication of metabolically
active cells. See Crouch et al (1993)
J. Immunol. Meth. 160:81-88, US Pat. No. 6602677. The assay may be conducted
in 96- or 384-well format,
making it amenable to automated high-throughput screening (HTS). See Cree et
al (1995) AntiCancer Drugs 6:398-
404. The assay procedure involves adding a single reagent (Ce1lTiter-Glo
Reagent) directly to cultured cells. This
results in cell lysis and generation of a luminescent signal produced by a
luciferase reaction. The luminescent
signal is proportional to the amount of ATP present, which is directly
proportional to the number of viable cells
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present in culture. Data can be recorded by luminometer or CCD camera imaging
device. The luminescence output
is expressed as relative light units (RLU).
[0794] Another assay for cell proliferation is the "MTT" assay, a colorimetric
assay that measures the
oxidation of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to
formazan by mitochondrial reductase.
Like the Ce1lTiter-GloTm assay, this assay indicates the number of
metabolically active cells present in a cell culture.
See, e.g., Mosmann (1983) J. Immunol. Meth. 65:55-63, and Zhang et al. (2005)
Cancer Res. 65:3877-3882.
[0795] In one aspect, an anti-CD79b antibody is tested for its ability to
induce cell death in vitro. Assays
for induction of cell death are well known in the art. In some embodiments,
such assays measure, e.g., loss of
membrane integrity as indicated by uptake of propidium iodide (PI), trypan
blue (see Moore et al. (1995)
Cytotechnology, 17:1-11), or 7AAD. In an exemplary PI uptake assay, cells are
cultured in Dulbecco's Modified
Eagle Medium (D-MEM):Ham's F-12 (50:50) supplemented with 10% heat-inactivated
FBS (Hyclone) and 2 mM
L-glutamine. Thus, the assay is performed in the absence of complement and
immune effector cells. Cells are
seeded at a density of 3 x 106 per dish in 100 x 20 mm dishes and allowed to
attach overnight. The medium is
removed and replaced with fresh medium alone or medium containing various
concentrations of the antibody or
immunoconjugate. The cells are incubated for a 3-day time period. Following
treatment, monolayers are washed
with PBS and detached by trypsinization. Cells are then centrifuged at 1200
rpm for 5 minutes at 4 C, the pellet
resuspended in 3 ml cold Ca2+ binding buffer (10 m1V1 Hepes, pH 7.4, 140 m1V1
NaCl, 2.5 mM CaClz) and aliquoted
into 35 mm strainer-capped 12 x 75 mm tubes (1 ml per tube, 3 tubes per
treatment group) for removal of cell
clumps. Tubes then receive PI (10 g/ml). Samples are analyzed using a
FACSCANTM flow cytometer and
FACSCONVERTTM Ce1lQuest software (Becton Dickinson). Antibodies or
immunoconjugates which induce
statistically significant levels of cell death as determined by PI uptake are
thus identified.
[0796] In one aspect, an anti-CD79b antibody or immunoconjugate is tested for
its ability to induce
apoptosis (programmed cell death) in vitro. An exemplary assay for antibodies
or immunconjugates that induce
apoptosis is an annexin binding assay. In an exemplary annexin binding assay,
cells are cultured and seeded in
dishes as discussed in the preceding paragraph. The medium is removed and
replaced with fresh medium alone or
medium containing 0.001 to 10 g/ml of the antibody or immunoconjugate.
Following a three-day incubation
period, monolayers are washed with PBS and detached by trypsinization. Cells
are then centrifuged, resuspended in
Ca2+ binding buffer, and aliquoted into tubes as discussed in the preceding
paragraph. Tubes then receive labeled
annexin (e.g. annexin V-FITC) (1 g/ml). Samples are analyzed using a
FACSCANTM flow cytometer and
FACSCONVERTTM Ce1lQuest software (BD Biosciences). Antibodies or
immunoconjugates that induce
statistically significant levels of annexin binding relative to control are
thus identified. Another exemplary assay for
antibodies or immunconjugates that induce apoptosis is a histone DNA ELISA
colorimetric assay for detecting
internucleosomal degradation of genomic DNA. Such an assay can be performed
using, e.g., the Cell Death
Detection ELISA kit (Roche, Palo Alto, CA).
[0797] Cells for use in any of the above in vitro assays include cells or cell
lines that naturally express
CD79b or that have been engineered to express CD79b. Such cells include tumor
cells that overexpress CD79b
relative to normal cells of the same tissue origin. Such cells also include
cell lines (including tumor cell lines) that
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express CD79b and cell lines that do not normally express CD79b but have been
transfected with nucleic acid
encoding CD79b.
[0798] In one aspect, an anti-CD79b antibody or immunoconjugate thereof is
tested for its ability to
inhibit cell growth or proliferation in vivo. In certain embodiments, an anti-
CD79b antibody or immunoconjugate
thereof is tested for its ability to inhibit tumor growth in vivo. In vivo
model systems, such as xenograft models,
can be used for such testing. In an exemplary xenograft system, human tumor
cells are introduced into a suitably
immunocompromised non-human animal, e.g., a SCID mouse. An antibody or
immunoconjugate of the invention
is administered to the animal. The ability of the antibody or immunoconjugate
to inhibit or decrease tumor growth
is measured. In certain embodiments of the above xenograft system, the human
tumor cells are tumor cells from a
human patient. Such cells useful for preparing xenograft models include human
leukemia and lymphoma cell lines,
which include without limitation the BJAB-luc cells (an EBV-negative Burkitt's
lymphoma cell line transfected
with the luciferase reporter gene), Ramos cells (ATCC, Manassas, VA, CRL-
1923), SuDHL-4 cells (DSMZ,
Braunschweig, Germany, AAC 495), DoHH2 cells (see Kluin-Neilemans, H.C. et
al., Leukemia 5:221-224 (1991),
and Kluin-Neilemans, H.C. et al., Leukemia 8:1385-1391 (1994)), Granta-519
cells (see Jadayel, D.M. et al,
Leukemia 11(1):64-72 (1997)). In certain embodiments, the human tumor cells
are introduced into a suitably
immunocompromised non-human animal by subcutaneous injection or by
transplantation into a suitable site, such
as a mammary fat pad.
2. Binding assays and other assEs
[0799] In one aspect, an anti-CD79b antibody is tested for its antigen binding
activity. For example, in
certain embodiments, an anti-CD79b antibody is tested for its ability to bind
to CD79b expressed on the surface of a
cell. A FACS assay may be used for such testing.
[0800] In one aspect, competition assays may be used to identify a monoclonal
antibody that competes
with murine 2F2 antibody and/or humanized 2F2.D7 antibody for binding to
CD79b. In certain embodiments, such
a competing antibody binds to the same epitope (e.g., a linear or a
conformational epitope) that is bound by murine
2F2 antibody and/or humanized 2F2.D7 antibody. Exemplary competition assays
include, but are not limited to,
routine assays such as those provided in Harlow and Lane (1988) Antibodies: A
Laboratory Manual ch. 14 (Cold
Spring Harbor Laboratory, Cold Spring Harbor, NY). Detailed exemplary methods
for mapping an epitope to
which an antibody binds are provided in Morris (1996) "Epitope Mapping
Protocols," in Methods in Molecular
Biology vol. 66 (Humana Press, Totowa, NJ). Two antibodies are said to bind to
the same epitope if each blocks
binding of the other by 50% or more.
[0801] In an exemplary competition assay, immobilized CD79b is incubated in a
solution comprising a
first labeled antibody that binds to CD79b (e.g., murine 2F2 antibody and/or
humanized 2F2.D7 antibody) and a
second unlabeled antibody that is being tested for its ability to compete with
the first antibody for binding to CD79b.
The second antibody may be present in a hybridoma supernatant. As a control,
immobilized CD79b is incubated in
a solution comprising the first labeled antibody but not the second unlabeled
antibody. After incubation under
conditions permissive for binding of the first antibody to CD79b, excess
unbound antibody is removed, and the
amount of label associated with immobilized CD79b is measured. If the amount
of label associated with
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immobilized CD79b is substantially reduced in the test sample relative to the
control sample, then that indicates that
the second antibody is competing with the first antibody for binding to CD79b.
In certain embodiments,
immobilized CD79b is present on the surface of a cell or in a membrane
preparation obtained from a cell expressing
CD79b on its surface.
[0802] In one aspect, purified anti-CD79b antibodies can be further
characterized by a series of assays
including, but not limited to, N-terminal sequencing, amino acid analysis, non-
denaturing size exclusion high
pressure liquid chromatography (HPLC), mass spectrometry, ion exchange
chromatography and papain digestion.
[0803] In one embodiment, the invention contemplates an altered antibody that
possesses some but not
all effector functions, which make it a desirable candidate for many
applications in which the half life of the
antibody in vivo is important yet certain effector functions (such as
complement and ADCC) are unnecessary or
deleterious. In certain embodiments, the Fc activities of the antibody are
measured to ensure that only the desired
properties are maintained. In vitro and/or in vivo cytotoxicity assays can be
conducted to confirm the
reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor
(FcR) binding assays can be
conducted to ensure that the antibody lacks FcyR binding (hence likely lacking
ADCC activity), but retains FcRn
binding ability. The primary cells for mediating ADCC, NK cells, express
FcyRIII only, whereas monocytes
express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is
summarized in Table 3 on page 464
of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). An example of an in
vitro assay to assess ADCC
activity of a molecule of interest is described in U.S. Patent No. 5,500,362
or 5,821,337. Useful effector cells for
such assays include peripheral blood mononuclear cells (PBMC) and Natural
Killer (NK) cells. Alternatively, or
additionally, ADCC activity of the molecule of interest may be assessed in
vivo, e.g., in a animal model such as that
disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998). Clq binding assays
may also be carried out to confirm
that the antibody is unable to bind Cl q and hence lacks CDC activity. To
assess complement activation, a CDC
assay, e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods
202:163 (1996), may be performed. FcRn
binding and in vivo clearance/half life determinations can also be performed
using methods known in the art.
[0804] The following examples are offered for illustrative purposes only, and
are not intended to limit the
scope of the present invention in any way.
[0805] All patent and literature references cited in the present specification
are hereby incorporated by
reference in their entirety.
EXAMPLES
[0806] Commercially available reagents referred to in the examples were used
according to
manufacturer's instructions unless otherwise indicated. Antibodies used in the
examples include commercially
available antibodies. The source of those cells identified in the following
examples, and throughout the
specification, by ATCC accession numbers, is the American Type Culture
Collection, Manassas, VA.
EXAMPLE 1: Generation of Humanized anti-CD79b Antibody
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[0807] Residue numbers are according to Kabat (Kabat et al., Sequences
ofproteins of immunological
interest, 5th Ed., Public Health Service, National Institutes of Health,
Bethesda, MD (1991)). Single letter amino
acid abbreviations are used. DNA degeneracies are represented using the IUB
code (N = A/C/G/T, D = A/G/T, V
A/C/G, B= C/G/T, H= A/C/T, K = G/T, M = A/C, R = A/G, S = G/C, W= A/T, Y =
C/T).
[0808] Chimeric 2F2 antibody (herein referred to as "ch2F2") was generated as
previously described in
US Application No. 11/462,336, filed August 3, 2006.
A. Humanized Anti-CD79b Antibody Graft
[0809] A humanized anti-CD79b antibody was generated. The VL and VH domains
from murine 2F2
antibody (mu2F2) were aligned with the human consensus VL kappa I (huKI) and
human subgroup III consensus
VH (huIII) domains. To make the HVR graft, the acceptor VH framework, which
differs from the human subgroup
III consensus VH domain at 3 positions: R71A, N73T, and L78A (Carter et al.,
Proc. Natl. Acad. Sci. USA 89:4285
(1992)) was used. Hypervariable regions from the murine 2F2 (mu2F2) antibody
were engineered into the acceptor
human consensus framework to generate a direct HVR-graft of 2F2 (herein
referred to as "2F2 graft" or "2F2-
grafted `humanized' antibody" or "hu2F2 graft"). In the VL domain the
following regions were grafted to the
human consensus acceptor: positions 24-34 (L1), 50-56 (L2) and 89-97 (L3)
(Figure 7). In the VH domain,
positions 26-35 (H1), 49-65 (H2) and 93-102 (H3) were grafted (Figures 8A and
8B). MacCallum et al.
(MacCallum et al., J. Mol. Biol., 262: 732-745 (1996)) analyzed antibody and
antigen complex crystal structures
and found positions 49, 93 and 94 of the heavy chain are part of the contact
region and are thus included in the
definition of HVR-H2 and HVR-H3 when humanizing antibodies.
[0810] The direct-graft variant (2F2-graft) was generated by Kunkel
mutagenesis, as both the Fab
displayed on phage and as an IgG, using a separate oligonucleotide for each
hypervariable region. Correct clones
were assessed by DNA sequencing.
B. Humanized Anti-CD79b Antibody Graft Variants
[0811] Anti-CD79b antibody graft variants which included mutational diversity
in the hypervariable
regions of the 2F2-grafted "humanized" antibody were generated using phage
libraries. The anti-CD79b antibody
graft variants included multiple position variations in the HVRs (Figure 9).
C. Phage Selection
[0812] For phage selection, huCD79beCd (2 g/ml) was immobilized in PBS on
MaxiSorp microtiter
plates (Nunc) overnight at 4 C. Plates were blocked for at least 1 h using
Casein Blocker (Pierce). Phage were
harvested from the culture supernatant and suspended in PBS containing 0.5 %
BSA and 0.05 % Tween 20
(PBSBT). Following addition of the phage library and phage selection for 2 h,
microtiter wells were washed
extensively with PBS containing 0.05 % Tween 20 (PBST) to remove unbound phage
and bound phage were eluted
by incubating the wells with 100 m1V1 HCI for 30 min. Selection stringency may
be increased during successive
rounds of selection by increasing the number of washes with PBST or by
incubating with soluble huCD79beCd for
increasing time periods prior to elution.
[0813] Eluted phage were neutralized with 1 M Tris, pH 8 and amplified using
XL1-Blue cells and
M13/K07 helper phage and grown overnight at 37 C in 2YT, 50 g/ml
carbenacillin. The titers of phage eluted
from a target containing well were compared to titers of phage recovered from
a non-target containing well to
assess enrichment.
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D. Fab Production and IgG Production
[0814] To express Fab protein for affinity measurements, a stop codon was
introduced between the heavy
chain and g3 in the phage display vector. Clones were transformed into E. coli
34B8 cells and grown in Complete
C.R.A.P. media at 30 C (Presta et al. Cancer Res. 57: 4593-4599 (1997)). Cells
were harvested by centrifugation,
suspended in PBS, 100 M PMSF, 100 M benzamidine, 2.4 mM EDTA and broken open
using a microfluidizer.
Fab was purified with Protein G affinity chromatography.
[0815] For screening purposes, IgG variants were initially produced in 293
cells. Vectors coding for VL
and VH (25 g) were transfected into 293 cells using the FuGene system. 500 l
of FuGene was mixed with 4.5 ml
of DMEM media containing no FBS and incubated at room temperature for 5 min.
Each chain (25 g) was added
to this mixture and incubated at room temperature for 20 min and then
transferred to a flask for transfection
overnight at 37 C in 5% COz. The following day the media containing the
transfection mixture was removed and
replaced with 23 ml PSO4 media with 0.1 ml/L trace elements and 10 mg/L
insulin. Cells were incubated for an
additional 5 days after which the media was harvested at 1000 rpm for 5 min
and sterile filtered using a 0.22 m
low protein-binding filter. Samples could be stored at 4 C after addition of
2.5 m10.1 % PMSF for every 125 ml of
media.
E. Affinity Determination (Biacore Analysis)
[0816] For affinity determination of the 2F2-grafted "humanized" antibody
variants, the extracellular
domain of human CD79b (huCD79be d) was expressed in CHO cells alone or as a Fc
fusion (huCD79be d-Fc) and
purified by conventional means. In addition, a 16 amino acid peptide
(ARSEDRYRNPKGSACK) (SEQ ID NO:
78) containing the epitope for 2F2 was synthesized by conventional means.
[0817] Characterization of the epitope for 2F2 antibody (labeled as "test
peptide" in Figure 14) was
previously disclosed in US Application No. 11/462,336, filed August 3, 2006.
The epitope for 2F2 antibody was
located in the extracellular peptide region distal to the transmembrane domain
and was present in the full-length and
truncated forms of human CD79b (Cragg, Blood, 100(9): 3068-76 (2002)), which
have been described in normal
and malignant B cells (Hashimoto, S. et al., Mol. Immunol., 32(9): 651-9
(1995); Alfarano et al., Blood, 93(7):
2327-35 (1999)). The truncated form of CD79b lacks the entire extracellular Ig-
like domain (the extracellular Ig-
like domain that is not present in the spliced truncated form of CD79b is
boxed in Figure 14).
[0818] Binding of Fab and IgG variants of ch2F2, the 2F2-grafted "humanized"
antibody (hu2F2
graft) or 2F2-grafted "humanized" antibody variant 7 (hu2F2.D7) to immobilized
huCD79be d or CD79b-Fc or the
16 amino acid peptide containing the epitope for 2F2 was measured by surface
plasma resonance. Affinity
determinations were performed by surface plasmon resonance using a BIAcoreTM-
2000. The antigen, huCD79beCd or
huCD79b-Fc was immobilized (approximately 50 - 200 RU) in 10 m1VI sodium
acetate pH 4.8 on a CM5 sensor
chip. In experiments that measured binding to the 16 amino acid peptide
(ARSEDRYRNPKGSACK) (SEQ ID NO:
78) containing the epitope (amino acids 1-11 of SEQ ID NO: 78) for 2F2, the
biotinylated peptide was captured
(approximately 20 RU) on a streptavidin coated sensor chip. Purified 2F2-
grafted "humanized" antibody variant (as
Fab or IgG) (a 2-fold serial dilution of 0.5 to 1000 nM in PBST) was injected
at a flow rate of 30 L/min. Each
sample was analyzed with 4-minute association and 10-minute disassociation.
After each injection the chip was
regenerated using 10 m1VI Glycine pH 1.7.
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[0819] Binding response was corrected by subtracting a control flow cell from
2F2-grafted
"humanized" antibody variant (as Fab or IgG) flow cells. A 1:1 Languir model
of simultaneous fitting of koõ and
koff was used for kinetics analysis.
F. Binding Analysis (FACS Analysis)
[0820] To further determine binding of the 2F2-grafted "humanized" antibody
variant 7 (hu2F2.D7)
arising from the SR libraries to BJAB cells, binding of labeled hu2F2.D7 (IgG
variants) antibodies to BJAB cells
was analyzed using FACS analysis.
[0821] For FACS analysis, monoclonal antibodies ch2F2 and 2F2.D7 were labeled
with Zenon
Alexa Fluor 488 Human IgG Labeling Kit (Invitrogen, Carlsbad, California),
according to manufacturer's
instructions. BJAB cells (1 x 106 in 100 1 volume) were stained with 1 g
each of labeled antibodies, hIgGl
Isotype, ch2F2 or 2F2.D7.
G. Affinity Determination (Scatchard Analysis)
[0822] To further determine binding of the IgG variants of the 2F2-graft
"humanized" antibody
variant 7 (hu2F2.D7) having changes in HVR-L3, binding of iodinated anti-CD79b
(with the same epitope as
ch2F2) to BJAB cells expressing human CD79b and cynomologous CD79b using
competition with unlabeled
ch2F2 was analyzed and Scatchard analysis was performed.
[0823] For Scatchard analysis, 0.5 nM 1121 labeled anti-human CD79b with the
same epitope as ch2F2
or 0.5 nM Ii2s labeled 2F2-graft "humanized" antibody variant 7 (hu2F2.D7) was
competed against unlabeled
ch2F2 or hu2F2.D7, respectively, ranging from 50 to 0.02 nM (12 step 1:2
serial dilution) in the presence of a
transfected BJAB line stably expressing cynomologous-CD79b and endogenous
human-CD79b. After a four hour
incubation at 4 C, cells were washed and cell pellet counts were read by a
gamma counter (1470 WIZARD
Automatic Gamma Counter; Perkin Elmer, Walthem, MA). All points were done in
triplicate and counted for 10
minutes. The average CPM was used for Kd calculation using the New Ligand
(Genentech, South San Francisco,
CA) program.
Results and Discussion
A. Results of Generation of Humanized anti-CD79b Antibody
[0824] The human acceptor framework used for the generation of humanized anti-
CD79b comprises the
consensus human kappa I VL domain and a variant of the human subgroup III
consensus VH domain. The variant
VH domain has 3 changes from the human consensus: R71A, N73T and L78A. The VL
and VH domains of murine
2F2 (mu2F2) were aligned with the human kappa I and subgroup III domains; each
HVR was identified and then
grafted into the human acceptor framework to generate a HVR-graft that could
be displayed as a Fab on phage
(Figures 7 and 8).
[0825] Phage displaying the 2F2-graft as a Fab did not bind to immobilized
huCD79be,d (data not shown).
In addition, the 2F2-graft as a Fab did not bind huCD79be,d and the 2F2-graft
as a Fab or as an Ig did not bind
huCD79be,d-Fc (Figure 10, NB = no binding) as determined by Biacore analysis.
1. CDR Repair
[0826] 2F2-grafted "humanized" antibody variants that were able to bind to
immobilized huCD79beCd
with the following sequence changes were identified.
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[0827] Only sequence changes targetting HVRs in L3 was observed in the
libraries containing multiple
position changes and are shown in Figure 9 (for L3 mutation: W89F and Y96F
(2F2.D7 mutation) (SEQ ID NO:
18).
[0828] Select clones were reformatted as Fab for analysis by FACS and as IgG
for further analysis by
Biacore and Scatchard.
a. Affinity Determination (Biacore Analysis)
[0829] As shown in Figure 10, showing Biacore analysis, this CDR-repair
approach identified sequence
changes in HVR-L3 (hu2F2.D7) that restore the affinity of the 2F2-grafted
"humanized" antibody. The surface
plasmon resonance assays showed that the changes in L3 (hu2F2.D7) had similar
affinity (Figure 10) as ch2F2
when binding to immobilized huCD79be,d or the 16 amino acid peptide (SEQ ID
NO: 78) containing the epitope
(amino acids 1-11 of SEQ ID NO: 78) for 2F2 as determinedby Biacore analysis.
b. Affinity Determination (Scatchard Analysis)
[0830] As assessed by Scatchard analysis, this CDR-repair approach identified
sequence changes that
improved the affinity of the 2F2-grafted "humanized" antibody. Specifically,
the cell binding assays showed that
the affinity of ch2F2 and 2F2-grafted "humanized" antibody variant 7
(hu2F2.D7) (reformatted as IgG) for binding
BJAB cells stably expressing cynomologous CD79b and endogenous human CD79b was
with Kd values of 1 nM
(ch2F2; Kd = 0.99 0.23 nM) and 2 nM (hu2F2.D7; Kd = 2.0 0.53 nM),
respectively (data not shown), as
determined by Scatchard analysis.
c. Binding Determination (FACS Analysis)
[0831] As assessed by FACS analysis, this CDR-repair approach identified
sequence changes that
improved the binding of the 2F2-grafted "humanized" antibody (hu2F2 graft) to
BJAB cells (data not shown).
Specifically, FACS analysis of monoclonal hu2F2.D7 (IgG variants) identified
from the phage libraries to BJAB
cells showed binding of the hu2F2.D7 variant to BJAB cells (data not shown).
B. Discussion of humanization of 2F2 antibodies
[0832] Starting from a graft of the 6 murine 2F2 HVRs (defined as positions 24-
34 (L1), 50-56 (L2), 89-
97 (L3), 26-35 (H1), 49-65 (H2) and 93-102 (H3)) into the human consensus
Kappa I VL and subgroup III VH
(containing A7 1, T73 and A78), CDR repair was used to identify changes in
HVRs 1-6 that improve binding
affinity. HVR sequence changes identified in Figure 101ed to humanized
variants of 2F2 with affinities similar to
ch2F2.
EXAMPLE 2: Generation ofAnti-CD79b Antibody DruQ Conjugates (ADCs)
[0833] To test the efficacy of IgG variants of 2F2-grafted "humanized"
antibody variants, the 2F2-grafted
"humanized" antibody variants were conjugated to drugs, such as DM1. The
variants conjugated to DM1 included
the variants having changes in HVR-L3.
[0834] The drugs used for generation of antibody drug conjugates (ADCs) for
anti-CD79b antibodies
included maytansinoid DM1 and may include dolastatinlO derivatives
monomethylauristatin E (MMAE) and
monomethylauristatin F (MMAF). (See US2005/0276812; US 2005/0238649; Doronina
et al., Bioconjug. Chem.,
17:114-123 (2006)), DM1, MMAE and MMAF are mitotic inhibitors that are at
least 100 fold more cytotoxic than
the vinca alkaloid mitotic inhibitors used in chemotherapeutic treatments of
NHLs (Doronina et al., Bioconjug.
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Chem., 17:114-123 (2006); Doronina et al., Nat. Biotechnol., 21: 778-784
(2003); Erickson et al., Cancer Res., 66:
4426-4433 (2006), all of which are herein incorporated by reference in their
entirety). Linkers useful for generation
of the ADCs are BMPEO, SPP or SMCC (also referred to herein as "MCC") for DM1
and MC or MC-vc-PAB for
MMAE and MMAF. For DM1, the antibodies were linked to the thio group of DM1
and through the s-amino
group of lysine using the linker reagent SMCC. Alternatively, for DM1, the
antibodies may be linked to DM1
through the e-amino group of lysine using the SPP linker. SPP (N-succinimidyl
4-(2'-pyridldithio) pentanoate)
reacts with the epsilon amino group of lysines to leave a reactive 2-pyridyl
disulfide linker on the protein. With SPP
linkers, upon reaction with a free sulthydral (e.g. DM1), the pyridyl group is
displaced, leaving the DM1 attached
via a reducible disulfide bond. DM1 attached via a SPP linker is released
under reducing conditions (i.e., for
example, within cells) while DM1 attached via the SMCC linker is resistant to
cleavage in reducing conditions.
Further, SMCC-DM1 ADCs induce cell toxicity if the ADC is internalized and
targeted to the lysosome causing the
release of lysine-N-DM1, which is an effective anti-mitotic agent inside the
cell, and when released from the cell,
lysine-N8-DM1 is non-toxic (Erickson et al., Cancer Res., 66: 4426-4433
(2006)) For MMAE and MMAF, the
antibodies may be linked to MMAE or MMAF through the cysteine by
maleimidocaproyl-valine-citruline (vc)-p-
aminobenzyloxycarbonyl (MC-vc-PAB). For MMAF, the antibodies may be
alternatively linked to MMAF through
the cysteine by maleeimidocaproyl (MC) linker. The MC-vc-PAB linker is
cleavable by intercellular proteases
such as cathepsin B and when cleaved, releases free drug (Doronina et al.,
Nat. Biotechnol., 21: 778-784 (2003))
while the MC linker is resistant to cleavage by intracellular proteases.
[0835] Antibody drug conjugates (ADCs) for anti-CD79b, using SMCC-DM1, were
generated similar to
the procedure described in US2005/0276812. Anti-CD79b purified antibodies were
buffer-exchanged into a
solution containing 50 mM potassium phosphate and 2mM EDTA, pH 7Ø SMCC
(Pierce Biotechnology,
Rockford, IL) was dissolved in dimethylacetamide (DMA) and added to the
antibody solution to make a final
SMCC/Ab molar ratio of 10:1. The reaction was allowed to proceed for three
hours at room temperature with
mixing. The SMCC-modified antibody was subsequently purified on a GE
Healthcare HiTrap desalting column (G-
25) equilibrated in 35 m1V1 sodium citrate with 150 mM NaCl and 2 mM EDTA, pH
6Ø DM1, dissolved in DMA,
was added to the SMCC antibody preparation to give a molar ratio of DM1 to
antibody of 10:1. The reaction was
allowed to proceed for 4-20 hrs at room temperature with mixing. The DM1-
modified antibody solution was
diafiltered with 20 volumes of PBS to remove unreacted DM1, sterile filtered,
and stored at 4 degrees C. Typically,
a 40-60% yield of antibody was achieved through this process. The preparation
was usually >95% monomeric as
assessed by gel filtration and laser light scattering. Since DM1 has an
absorption maximum at 252 nm, the amount
of drug bound to the antibody could be determined by differential absorption
measurements at 252 and 280 nm.
Typically, the drug to antibody ratio was 3 to 4.
[0836] Antibody drug conjugates (ADCs) for anti-CD79b antibodies described
herein using SPP-DM1
linkers may be generated similar to the procedure described in US
2005/0276812. Anti-CD79b purified antibodies
are buffer-exchanged into a solution containing 50 mM potassium phosphate and
2 mM EDTA, pH 7.0 SPP
(Immunogen) was dissolved in DMA and added to the antibody solution to make a
final SPP/Ab molar ratio of
approximately 10:1, the exact ratio depending upon the desired drug loading of
the antibody. A 10:1 ratio will
usually result in a drug to antibody ratio of approximately 3-4. The SPP is
allowed to react for 3-4 hours at room
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temperature with mixing. The SPP-modified antibody is subsequently purified on
a GE Healthcare HiTrap desalting
column (G-25) equilibrated in 35 mM sodium citrate with 150 mM NaCl and 2 m1V1
EDTA, pH 6.0 or phosphate
buffered saline, pH 7.4. DM1 is dissolved in DMA and added to the SPP antibody
preparation to give a molar ratio
of DM1 to antibody of 10:1, which results in a 3-4 fold molar excess over the
available SPP linkers on the antibody.
The reaction with DM1 is allowed to proceed for 4-20 hrs at room temperature
with mixing. The DM1-modified
antibody solution is diafiltered with 20 volumes of PBS to remove unreacted
DM1, sterile filtered, and stored at 4
degrees C. Typically, yields of antibody of 40-60% or greater are achieved
with this process. The antibody-drug
conjugate is usually >95% monomeric as assessed by gel filtration and laser
light scattering. The amount of bound
drug is determined by differential absorption measurements at 252 and 280 nm
as described for the preparation of
SMCC-DM1 conjugates (described above).
[0837] Antibody drug conjugates (ADC) for anti-CD79b antibodies described
herein using MC-MMAF,
MC-MMAE, MC-val-cit (vc)-PAB-MMAE or MC-val-cit (vc)-PAB-MMAF drug linkers may
also be generated
similar to the procedure described in US 2005/0238649. Purified anti-CD79b
antibody is dissolved in 500 mM
sodium borate and 500 m1V1 sodium chloride at pH 8.0 and further treated with
an excess of 100 MM dithiothreitol
(DTT). After incubation at 37 degrees C for about 30 minutes, the buffer is
exchanged by elution over Sephadex
G25 resin and eluted with PBS with 1 mM DTPA. The thiol/Ab value is checked by
determining the reduced
antibody concentration from the absorbance at 280 nm of the solution and the
thiol concentration by reaction with
DTNB (Aldrich, Milwaukee, WI) and determination of the absorbance at 412 nm.
The reduced antibody is
dissolved in PBS was chilled on ice. The drug linker, for example, MC-val-cit
(vc)-PAB-MMAE, in DMSO, is
dissolved in acetonitrile and water, and added to the chilled reduced antibody
in PBS. After an hour incubation, an
excess of maleimide is added to quench the reaction and cap any unreacted
antibody thiol groups. The reaction
mixture is concentrated by centrifugal ultrafiltration and the antibody drug
conjugate, is purified and desalted by
elution through G25 resin in PBS, filtered through 0.2 m filters under
sterile conditions, and frozen for storage.
[0838] Antibody drug conjugates (using anti-CD79b antibodies describedherein)
were diluted at 2 x 10
g/ml in assay medium. Conjugates were linked with crosslinkers SMCC
(alternative disulfide linker may be used
for SPP to maytansinoid DM1 toxin) (See US 2005/0276812 and US 2005/0238649).
Further, conjugates may be
linked with MC-valine-citrulline (vc)-PAB or MC to dolastatinlO derivatives,
monomethylauristatin E (MMAE)
toxin or monomethylauristatin F (MMAF) toxin (See US Application Nos.
11/141,344, filed May 31, 2005 and US
Application No. 10/983,340, filed November 5, 2004). Negative controls
included HERCEPTIN (trastuzumab)
(anti-HER2) based conjugates (SMCC-DM1 or SPP-DM1 or MC-vc-MMAE or MC-vc-
MMAF). Positive controls
may include free L-DM1 equivalent to the conjugate loading dose. Samples were
vortexed to ensure homogenous
mixture prior to dilution.
[0839] Anti-CD79b antibodies for drug conjugation included 2F2 chimeric
antibodies (described in
Example 1A) and antibodies further described herein (see Example 1), including
hu2F2.D7.
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EXAMPLE 3: In Vivo Tumor Cell Killing Assay
A. Xenografts
[0840] To test the efficacy of IgG variants of 2F2-grafted "humanized"
antibody variants having changes
in HVR-L3 (hu2F2.D7), the hu2F2.D7 variant was conjugated to DM1 and the
effect of the conjugated variant on
tumors in mice were analyzed.
[0841] Specifically, the ability of the antibodies to regress tumors in
multiple xenograft models,
including RAMOS cells, BJAB cells (Burkitt's lymphoma cell line that contain
the t(2;8)(p112;q24) (IGK-MYC)
translocation, a mutated p53 gene and are Epstein-Barr virus (EBV) negative)
(Drexler, H.G., The Leukemia-
Lymphoma Cell Line Facts Book, San Diego: Academic Press, 2001)), Granta 519
cells (mantle cell lymphoma cell
line that contains the t(11;14)(q13;q32) (BCL1-IGH) translocation that results
in the over-expression of cyclin Dl
(BCL1), contains P161NK4B and P16INK4A deletions and are EBV positive)
(Drexler, H.G., The Leukemia-
Lymphoma Cell Line Facts Book, San Diego: Academic Press, 2001)), U698M cells
(lymphoblastic
lymphosarcoma B cell line; (Drexler, H.G., The Leukemia-Lymphoma Cell Line
Facts Book, San Diego: Academic
Press, 2001) and DoHH2 cells (follicular lymphoma cell line that contains the
translocation characteristic of
follicular lymphoma t(14;18)(q32;q21) that results in the over-expression of
Bcl-2 driven by the Ig heavy chain,
contains the P161NK4A deletion, contains the t(8;14)(q24;q32) (IGH-MYC)
translocation and are EBV negative)
(Drexler, H.G., The Leukemia-Lymphoma Cell Line Facts Book, San Diego:
Academic Press, 2001)), may be
examined.
[0842] For analysis of efficacy of 2F2-grafted "humanized" antibody variants,
female CB17 ICR SCID
mice (6-8 weeks of age from Charles Rivers Laboratories; Hollister, CA) were
inoculated subcutaneously with 2 X
10' BJAB-luciferase cells or Granta-519 cells via injection into the flanks of
CB 17 ICR SCID mice and the
xenograft tumors were allowed to grow to an average of 200 mm2. Day 0 refers
to the day the tumors were an
average of 200 mm2 and when the first/or only dose of treatment was
administered, unless indicated specifically
below. Tumor volume was calculated based on two dimensions, measured using
calipers, and was expressed in
mm3 according to the formula: V= 0.5a X b2, where a and b are the long and the
short diameters of the tumor,
respectively. Data collected from each experimental group were expressed as
mean + SE. Groups of 10 mice were
treated with a single intravenous (i.v.) dose of between 50 and 210 g of
antibody-linked drug/m2 mouse
(corresponding to -1-4 mg/kg of mouse) with 2F2-grafted "humanized" antibody
variants or control antibody-drug
conjugates. Tumors were measured either once or twice a week throughout the
experiment. Body weights of mice
were measured either once or twice a week throughout the experiment. Mice were
euthanized before tumor
volumes reached 3000 mm3 or when tumors showed signs of impending ulceration.
All animal protocols were
approved by an Institutional Animal Care and Use Committee (IACUC).
[0843] Linkers between the antibody and the toxin that were used were
thioether crosslinker SMCC for
DM1. Additional linkers may include disulfide linker SPP or thioether
crosslinker SMCC for DM1 or MC or MC-
valine-citrulline(vc)-PAB or (a valine-citrulline (vc)) dipeptide linker
reagent) having a maleimide component and
a para-aminobenzylcarbamoyl (PAB) self-immolative component for
monomethylauristatin E (MMAE) or
monomethylauristan F (MMAF). Toxins used were DM1. Additional toxins may
include MMAE or MMAF.
[0844] CD79b antibodies for this experiment included chimeric 2F2 (ch2F2)
antibodies as described in
US Application No. 11/462,336, filed August 3, 2006 (see Example 1A) as well
as 2F2-grafted "humanized"
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antibody variants described herein (see Example 1). Additional antibodies may
include 2F2 antibodies generated
from hybridomas deposited with the ATCC as PTA-7712 on July 11, 2006.
[0845] Negative controls included HERCEPTIN (trastuzumab) (anti-HER2) based
conjugates
(SMCC-DM1).
B. Results
1. BJAB-Luciferase Xenografts
[0846] In a 36 day time course, 2F2-grafted "humanized" antibody variant 7
(hu2F2.D7 variant)
(reformatted as IgG) (and chimeric anti-CD79b antibody (ch2F2) conjugated to
DM1 (hu2F2.D7-SMCC-DM1 and
ch2F2-SMCC-DM1, respectively), showed inhibition of tumor growth in SCID mice
with BJAB-luciferase tumors
compared to negative control, HERCEPTIN (trastuzumab)-SMCC-DM1 (anti-HER2-
SMCC-DM1). ADCs were
administered in a single dose (as indicated in Table 7) at day 0 for all ADCs
and controls. Specifically, the
hu2F2.D7-SMCC-DM1 antibodies (reformatted as IgG) and ch2F2-SMCC-DM1
significantly inhibited tumor
growth (Figure 19). Further, in Table 7, the number of mice out of the total
number tested showing PR = Partial
Regression (where the tumor volume at any time after administration dropped
below 50% of the tumor volume
measured at day 0) or CR = Complete Remission (where the tumor volume at any
time after administration dropped
to 0 mm3) are indicated.
Table 7: BJAB-Luc Xenorg a (20 million cells/mouse) in SCID Mice
Antibody Administered (Treatment) PR CR Dose Ab Dose Drug - Drug Ratio
(mg/kg) DM1 (Drug/Ab)
( g/m)
Control anti-HER2-SMCC-DM1 0/10 0/10 2 100 3.3
ch2F2-SMCC-DM1 0/10 1/10 2.3 100 3
ch2F2-SMCC-DM1 0/10 0/10 1.2 50 3
hu2F2.D7-SMCC-DM1 1/10 1/10 2.9 100 2.3
hu2F2.D7-SMCC-DM1 1/10 0/10 1.5 50 2.3
2. Granta-519 (Human Mantle Cell Lymphoma) Xenografts
[0847] In a 14 day time course, 2F2-grafted "humanized" antibody variant 7
(hu2F2.D7 variant)
(reformatted as IgG) (hu2F2.D7-SMCC-DM1), showed inhibition of tumor growth in
SCID mice with Granta-519
tumors compared to negative control, HERCEPTIN (trastuzumab)-SMCC-DM1 (anti-
HER2-SMCC-DM1).
ADCs were administered in a single dose (as indicated in Table 8) at day 0 for
all ADCs and controls. Specifically,
the hu2F2.D7-SMCC-DM1 antibodies (reformatted as IgG) significantly inhibited
tumor growth (Figure 20A).
[0848] Further, treatment with hu2F2.D7-SMCC-DM1 and control HERCEPTIN
(trastuzumab)-
SMCC-DM1 (anti-HER2-SMCC-DM1) did not result in a decrease in percent body
weight of the mice (Figure
20B). Even further, in Table 8, the number of mice out of the total number of
ten mice tested showing PR = Partial
Regression (where the tumor volume at any time after administration dropped
below 50% of the tumor volume
measured at day 0) or CR = Complete Remission (where the tumor volume at any
time after administration dropped
to 0 mm3) are indicated.
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Table 8: Granta-519 Xenograft (20 million cells/mouse) in SCID Mice
Antibody Administered (Treatment) PR CR Dose Ab Dose Drug - Drug Ratio
(mg/kg) DM1 (Drug/Ab)
( g/m)
Control anti-HER2-SMCC-DM1 0/10 0/10 4 206 3.4
hu2F2.D7-SMCC-DM1 0/10 0/10 4 166 2.8
[0849] In light of the ability of 2F2-grafted "humanized" antibody ADCs to
significantly inhibit tumor
progression in xenografts, CD79b molecules may be excellent targets for
therapy of tumors in mammals, including
B-cell associated cancers, such as lymphomas (i.e. Non-Hodgkin's Lymphoma),
leukemias (i.e. chronic
lymphocytic leukemia), and other cancers of hematopoietic cells. Further, 2F2-
grafted "humanized" ADCs are
useful for reducing in vivo tumor growth of tumors, including B-cell
associated cancers, such as lymphomas (i.e.
Non-Hodgkin's Lymphoma), leukemias (i.e. chronic lymphocytic leukemia), and
other cancers of hematopoietic
cells.
EXAMPLE 4: CD79b Antibody Colocalization
[0850] To determine where 2F2-grafted "humanized" antibodies and antibody
variants are delivered
upon internalization into the cell, colocalization studies of the anti-CD79b
antibodies internalized into B-cell lines
may be assessed in Ramos cell lines. LAMP-1 is a marker for late endosomes and
lysosomes (Kleijmeer et al.,
Journal of Cell Biology, 139(3): 639-649 (1997); Hunziker et al., Bioessays,
18:379-389 (1996); Mellman et al.,
Annu. Rev. Dev. Biology, 12:575-625 (1996)), including MHC class II
compartments (MIICs), which is a late
endosome/lysome-like compartment. HLA-DM is a marker for MIICs.
[0851] Ramos cells are incubated for 3 hours at 37 C with 1 g/ml 2F2-grafted
"humanized"
antibodies and antibody variants, FcR block (Miltenyi) and 25 g/ml A1exa647-
Transferrin (Molecular Probes) in
complete carbonate-free medium (Gibco) with the presence of 10 g/ml leupeptin
(Roche) and 5 M pepstatin
(Roche) to inhibit lysosmal degradation. Cells are then washed twice, fixed
with 3% paraformaldehyde (Electron
Microscopy Sciences) for 20 minutes at room temperature, quenched with 50 mM
NH4C1 (Sigma), permeabilized
with 0.4% Saponin/2% FBS/1% BSA for 20 minutes and then incubated with 1 g/ml
Cy3 anti-mouse (Jackson
Immunoresearch) for 20 minutes. The reaction is then blocked for 20 minutes
with mouse IgG (Molecular Probes),
followed by a 30 minute incubation with Image-iT FX Signal Enhancer (Molecular
Probes). Cells are finally
incubated with Zenon A1exa488-labeled mouse anti-LAMP1 (BD Pharmingen), a
marker for both lysosomes and
MIIC (a lysosome-like compartment that is part of the MHC class II pathway),
for 20 minutes, and post-fixed with
3% PFA. Cells are resuspended in 20 1 saponin buffer and allowed to adhere to
poly-lysine (Sigma) coated slides
prior to mounting a coverglass with DAPI-containing VectaShield (Vector
Laboratories). For immunofluorescence
of the MIIC or lysosomes, cells are fixed, permeabilized and enhanced as
above, then co-stained with Zenon labeled
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A1exa555-HLA-DM (BD Pharmingen) and A1exa488-Lampl in the presence of excess
mouse IgG as per the
manufacturer's instructions (Molecular Probes).
[0852] Accordingly, colocalization of 2F2-grafted "humanized" antibodies or
antibody variants with
MIIC or lysosomes of B-cell lines as assessed by immunofluorescence may
indicate the molecules as excellent
agents for therapy of tumors in mammals, including B-cell associated cancers,
such as lymphomas (i.e. Non-
Hodgkin's Lymphoma), leukemias (i.e. chronic lymphocytic leukemia), and other
cancers of hematopoietic cells.
EXAMPLE 5: Preparation of Cysteine Engineered Anti-CD79b Antibodies
[0853] Preparation of cysteine engineered anti-CD79b antibodies was performed
as disclosed herein.
[0854] DNA encoding the ch2F2 antibody (light chain, SEQ ID NO: 4, Figure 4;
and heavy chain, SEQ
ID NO: 5, Figure 5), may be mutagenized by methods disclosed herein to modify
the light chain and heavy chain.
[0855] DNA encoding the hu2F2.D7 antibody (heavy chain (SEQ ID NO: 90) and
light chain (SEQ ID
NO: 89), Figure 13) was mutagenized by methods disclosed herein to modify the
heavy chain. DNA encoding the
hu2F2.D7 antibody (heavy chain, (SEQ ID NO: 90) Figure 13), may also be
mutagenized by methods disclosed
herein to modify the Fc region of the heavy chain.
[0856] In the preparation of the cysteine engineered anti-CD79b antibodies,
DNA encoding the light
chain was mutagenized to substitute cysteine for valine at Kabat position 205
in the light chain (sequential position
210) as shown in Figure 18 (light chain SEQ ID NO: 88 of hu2F2.D7 thioMAb).
DNA encoding the heavy chain
was mutagenized to substitute cysteine for alanine at EU position 118 in the
heavy chain (sequential position 118;
Kabat number 114) as shown in Figure 17 (heavy chain SEQ ID NO: 85 of hu2F2.D7
thioMAb). The Fc region of
anti-CD79b antibodies may be mutagenized to substitute cysteine for serine at
EU position 400 in the heavy chain
Fc region (sequential position 400; Kabat number 396) as shown in Table 2-3.
A. Preparation of cysteine engineered anti-CD79b antibodies for conjugation by
reduction and
reoxidation
[0857] Full length, cysteine engineered anti-CD79b monoclonal antibodies
(ThioMabs) expressed in
CHO cells and purified on a protein A affinity chromatography followed by a
size exclusion chromatography. The
purified antibodies are reconstituted in 500mM sodium borate and 500 mM sodium
chloride at about pH 8.0 and
reduced with about a 50-100 fold molar excess of 1 m1V1 TCEP (tris(2-
carboxyethyl)phosphine hydrochloride; Getz
et al (1999) Anal. Biochem. Vo1273:73-80; Soltec Ventures, Beverly, MA) for
about 1-2 hrs at 37 C. The reduced
ThioMab is diluted and loaded onto a HiTrap S column in 10 mM sodium acetate,
pH 5, and eluted with PBS
containing 0.3M sodium chloride. The eluted reduced ThioMab is treated with 2
mM dehydroascorbic acid (dhAA)
at pH 7 for 3 hours, or 2 mM aqueous copper sulfate (CuS04) at room
temperature overnight. Ambient air
oxidation may also be effective. The buffer is exchanged by elution over
Sephadex G25 resin and eluted with PBS
with 1mM DTPA. The thiol/Ab value is estimated by determining the reduced
antibody concentration from the
absorbance at 280 nm of the solution and the thiol concentration by reaction
with DTNB (Aldrich, Milwaukee, WI)
and determination of the absorbance at 412 nm.
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EXAMPLE 6: Preparation of Cysteine Engineered Anti-CD79b Antibody Drug ConL ag
tes by Conjugation of
Cysteine Engineered Anti-CD79b Antibodies And Drug-Linker Intermediates
[0858] After the reduction and reoxidation procedures of Example 5, the
cysteine engineered anti-CD79b
antibody is reconstituted in PBS (phosphate buffered saline) buffer and
chilled on ice. About 1.5 molar equivalents
relative to engineered cysteines per antibody of an auristatin drug linker
intermediate, such as MC-MMAE
(maleimidocaproyl-monomethyl auristatin E), MC-MMAF, MC-val-cit-PAB-MMAE, or
MC-val-cit-PAB-MMAF,
with a thiol-reactive functional group such as maleimido, is dissolved in
DMSO, diluted in acetonitrile and water,
and added to the chilled reduced, reoxidized antibody in PBS. After about one
hour, an excess of maleimide is
added to quench the reaction and cap any unreacted antibody thiol groups. The
reaction mixture is concentrated by
centrifugal ultrafiltration and the cysteine engineered anti-CD79b antibody
drug conjugate is purified and desalted
by elution through G25 resin in PBS, filtered through 0.2 m filters under
sterile conditions, and frozen for storage.
[0859] Preparation of hu2F2.D7-HC(A118C) thioMAb-BMPEO-DM1 may be performed as
follows.
The free cysteine on hu2F2.D7-HC(A118C) thioMAb is modified by the bis-
maleimido reagent BM(PEO)3 (Pierce
Chemical), leaving an unreacted maleimido group on the surface of the
antibody. This is accomplished by
dissolving BM(PEO)3 in a 50% ethanoUwater mixture to a concentration of 10
m1V1 and adding a tenfold molar
excess of BM(PEO)3 to a solution containing hu2F2.D7-HC(A118C) thioMAb in
phosphate buffered saline at a
concentration of approximately 1.6 mg/ml (10 micromolar) and allowing it to
react for 1 hour. Excess BM(PEO)3
is removed by gel filtration (HiTrap column, Pharmacia) in 30 mM citrate, pH 6
with 150 mM NaCl buffer. An
approximate 10 fold molar excess DM1 dissolved in dimethyl acetamide (DMA) is
added to the hu2F2.D7-
HC(A118C) thioMAb-BMPEO intermediate. Dimethylformamide (DMF) may also be
employed to dissolve the
drug moiety reagent. The reaction mixture is allowed to react overnight before
gel filtration or dialysis into PBS to
remove unreacted drug. Gel filtration on S200 columns in PBS is used to remove
high molecular weight aggregates
and furnish purified hu2F2.D7-HC(A118C) thioMAb-BMPEO-DM1.
[0860] By the same protocols, thio control hu-anti-HER2-HC(A118C)-BMPEO-DM1,
thio control hu-
anti-HER2 -HC(A 1 18C)-MC-MMAF, thio control hu-anti-HER2-HC(A 1 18C)-MCvcPAB-
MMAE and thio control
anti-CD22-HC(A118C)-MC-MMAF may be generated.
[0861] By the procedures above, cysteine engineered anti-CD79b antibody drug
conjugates (TDCs), for
example but not limited by the following, may be prepared and tested:
1. thio hu2F2.D7-HC(A118C)-MC-MMAF by conjugation of A118C thio hu2F2.D7-
HC(A118C) and
MC-MMAF;
2. thio hu2F2.D7-HC(A118C)-BMPEO-DM1 by conjugation of A118C thio hu2F2.D7-
HC(A118C) and
BMPEO-DM1;
3. thio hu2F2.D7-HC(A118C)-MCvcPAB-MMAE by conjugation of A118C thio hu2F2.D7-
HC(A118C)
and MC-val-cit-PAB-MMAE;
4. thio ch2F2-HC(A118C)-MC-MMAF by conjugation of thio ch2F2-HC(A118C) and MC-
MMAF; and
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5. thio ch2F2-LC(V205C)-MC-MMAF by conjugation of thio ch2F2-LC(V205C) and MC-
MMAF.
EXAMPLE 7: Characterization of Binding Affini ,t~Cysteine Engineered ThioMAb
Drug Conjugates to Cell
Surface Antigen
[0862] The binding affinity of thio hu2F2.D7 drug conjugates and thio ch2F2
drug conjugates to CD79b
expressed on BJAB-luciferase cells is determined by FACS analysis.
[0863] Briefly, approximately 1x106 cells in 100 l are contacted with varying
amounts (1.0 ug, 01. ug or
0.01 ug of Ab per million cells of BJAB-luciferase cells) of one of, but not
limited to, the following anti-CD79b
thioMAb drug conjugates or naked (unconjugated Ab as a control): (1) thio
ch2F2-LC(V205C)-MC-MMAF or (2)
thio ch2F2-HC(A118C)-MC-MMAF; (3) thio hu2F2.D7-HC(A118C)-MCvcPAB-MMAE, (4)
thio hu2F2.D7-
HC(A118C)-BMPEO-DM1, or (5) thio hu2F2.D7-HC(A118C)-MC-MMAF. PE conjugated
mouse anti-human Ig
is used as the secondary detecting antibody (BD Cat#555787).
[0864] Anti-CD79b antibody bound to the cell surface is detected using PE
conjugated mouse anti-
human Ig.
EXAMPLE 8: Assay for In Vitro Cell Proliferation Reduction by Anti-CD79b
ThioMab Drug Conjugates
[0865] The in vitro potency of anti-CD79b ThioMAb-drug conjugates (including
but not limited to thio
hu2F2.D7-HC(A118C)-MCMMAF, thio hu2F2.D7-HC(A118C)-MCvcPAB-MMAE and thio
hu2F2.D7-
HC(A118C)-BMPEO-DM1), is measured by a cell proliferation assay (for example
in BJAB-luciferase, Granta-519,
WSU-DLCL2 cells). The Ce1lTiter-Glo Luminescent Cell Viability Assay is a
commercially available (Promega
Corp., Madison, WI), homogeneous assay method based on the recombinant
expression of Coleoptera luciferase
(US 5583024; US 5674713; US 5700670). This cell proliferation assay determines
the number of viable cells in
culture based on quantitation of the ATP present, an indicator of
metabolically active cells (Crouch et al., J.
bnmunol. Metho., 160: 81-88 (1993); US 6602677). The Ce1lTiter-Glo Assay is
conducted in 96 well format,
making it amenable to automated high-throughput screening (HTS) (Cree et al.,
AntiCancer Drugs, 6:398-404
(1995)). The homogeneous assay procedure involves adding the single reagent
(The Ce1lTiter-Glo Reagent)
directly to cells cultured in serum-supplemented medium.
[0866] The homogeneous "add-mix-measure" format results in cell lysis and
generation of a luminescent
signal proportional to the amount of ATP present. The substrate, Beetle
Luciferin, is oxidatively decarboxylated by
recombinant firefly luciferase with concimatant conversion of ATP to AMP and
generation of photons. Viable cells
are reflected in relative luminescence units (RLU). Data can be recorded by
luminometer or CCD camera imaging
device. The luminescence output is presented as RLU, measured over time. %RLU
is normalized RLU percentage
compared to a "non-drug-conjugate" control. Alternatively, photons from
luminescence can be counted in a
scintillation counter in the presence of a scintillant. The light units can be
represented then as CPS (counts per
second).
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[0867] Efficacy of thioMAb-drug conjugates are measured by a cell
proliferation assay employing the
following protocol, adapted from Ce1lTiter Glo Luminescent Cell Viability
Assay, Promega Corp. Technical
bulletin TB288; Mendoza et al., Cancer Res., 62: 5485-5488 (2002)):
1. An aliquot of 40 l of cell culture containing about 3000 BJAB, Granta-519
or WSU-DLCL2
cells in medium is deposited in each well of a 384-well, opaque-walled plate.
2. TDC (ThioMab Drug Conjugate) (10 l) is added to quadruplicate experimental
wells to final
concentration of 10000, 3333, 1111, 370, 123, 41, 13.7, 4.6 or 1.5 ng/mL, with
"non-drug conjugate" control wells
receiving medium alone, and incubated for 3 days.
3. The plates are equilibrated to room temperature for approximately 30
minutes.
4. Ce1lTiter-Glo Reagent (50 1) is added.
5. The contents are mixed for 2 minutes on an orbital shaker to induce cell
lysis.
6. The plate is incubated at room temperature for 10 minutes to stabilize the
luminescence signal.
7. Luminescence is recorded and reported in graphs as %RLU (relative
luminescence units). Data
from cells incubated with drug-conjugate-free medium are plotted at 0.51
ng/ml.
Media: BJAB, Granta-519 and WSU-DLCL2 cells grow in RPMI1640/10%FBS/2mM
glutamine.
EXAMPLE 9: Assay for Inhibition of In Vivo Tumor Growth by Anti-CD79b ThioMab
Drug Conjugates
[0868] In a similar study, using the same xenograft study protocol as
disclosed in the Example 3 (see
above), varying the drug conjugates and doses administered, the efficacy of
thioMAb drug conjugates in reducing
B-cell tumor volume in xenograft models, for example, Granta-519 xenografts
(Human Mantle Cell Lymphoma),
DOHH2 (Follicular Lympohoma) xenografts, WSU-DLCL2 (Diffuse Large Cell
Lymphoma) xenografts or BJAB
(Burkitt's Lymphoma) xenografts, is studied.
[0869] The foregoing written specification is considered to be sufficient to
enable one skilled in the
art to practice the invention. The present invention is not to be limited in
scope by the construct deposited, since the
deposited embodiment is intended as a single illustration of certain aspects
of the invention and any constructs that
are functionally equivalent are within the scope of this invention. The
deposit of material herein does not constitute
an admission that the written description herein contained is inadequate to
enable the practice of any aspect of the
invention, including the best mode thereof, nor is it to be construed as
limiting the scope of the claims to the specific
illustrations that it represents. Indeed, various modifications of the
invention in addition to those shown and
described herein will become apparent to those skilled in the art from the
foregoing description and fall within the
scope of the appended claims.
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