Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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DELIVERY SYSTEM FOR CYTOTOXIC DRUGS BY BISPECIFIC ANTIBODY
PRETARGETING
RELATED APPLICATIONS
[01] This application claims priority to U.S. Patent Application Serial Nos.
12/949,536,
filed 11/18/2010, 12/915, 515, filed 10/29/2010; 12/871,345, filed 8/30/2010;
12/869,823,
filed 8/27/2010; 12/754,740, filed 4/6/2010; 12/752,649, filed 4/1/2010;
12/731,781, filed
3/25/2010; 12/644,146, filed 12/22/2009.
[02] This application also claims the benefit of U.S. Provisional Patent
Application
61/267,998, filed December 9, 2009.
FIELD OF THE INVENTION
[03] The present invention relates to therapeutic conjugates with improved
ability to target
diseases, such as cancer, infectious disease, autoimmune disease, immune
dysfunction (e.g.,
graft versus host disease, organ transplant rejection), cardiovascular
disease, metabolic
disease and neurologic (e.g., neurodegenerative) disease. Preferably, the
delivery system
comprises a pretargeting method in which bispecific antibodies have one or
more binding
sites for a disease associated antigen and one or more binding sites for a
hapten on a
targetable construct. The targetable construct may comprise therapeutic
agents, such as
cytotoxic drugs, and/or diagnostic agents, such as radionuclides. More
preferably, the
cytotoxic drug may be SN-38. Most preferably, the bispecific antibody is made
by the dock-
and-lock (DNL) technique.
BACKGROUND OF THE INVENTION
[04] Monoclonal antibodies have been used for the targeted delivery of toxic
agents to
cancer and other diseased cells. However, immunoconjugates of antibodies and
toxic agents
have had mixed success in the therapy of cancer or autoimmune disease, and
little application
in other diseases, such as infectious disease. The toxic agent is most
commonly a
chemotherapy drug, although particle-emitting radionuclides, or bacterial or
plant toxins have
also been conjugated to antibodies, especially for the therapy of cancer
(Sharkey and
Goldenberg, 2006, CA Cancer J Clin 56:226-243) and with radioimmunoconjugates
for the
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, .
preclinical therapy of certain infectious diseises (Dadachova and Casadevall,
2006, Q J Nucl
Med Mol Imaging 50:193-204).
1051 Camptothecin (CPT) and its derivatives are a class of potent antitumor
agents.
Irinotecan (also referred to as CPT-11) and topotecan are CPT analogs that are
approved
cancer therapeutics (Iyer and Ratain, 1998, Cancer Chemother Phamacol 42:S31-
S43). CPTs
act by inhibiting topoisomerase I (Hsiang et al., 1985, J Biol Chem 260:14873-
78). Although
potent cytotoxic agents, therapeutic use of camptothecins is limited by its
relative insolubility
in aqueous solution and high systemic toxicity, which limits the effective
dosage that may be
delivered to targeted disease cells. A need exists in the field for more
effective targeted
delivery methods for camptothecins and other therapeutic agents.
SUMMARY OF THE INVENTION
1061 The present invention resolves an unfulfilled need in the art by
providing improved
methods and compositions for targeted delivery of therapeutic agents. In
preferred
embodiments, the methods and compositions comprise pretargeting with novel
bispecific
antibody constructs, which contain at least one binding site for a disease
associated antigen,
such as a tumor-associated antigen, a B-cell associated antigen, or a pathogen-
associated
antigen, and at least one binding site for a hapten on a targetable construct.
The targetable
construct serves as a carrier for therapeutic or diagnostic agents.
1071 More preferably, the bispecific antibody constructs are prepared by the
dock-and-lock
(DNL) technique (see, e.g., U.S. Patent Nos. 7,550,143; 7,521,056; 7,534,866;
7,527,787 and
7,666,400, the Examples section). The DNL
technique utilizes the specific binding interactions occurring between a
dimerization and
docking domain (DDD moiety) from protein kinase A, and an anchoring domain (AD
moiety) from any of a number of known A-kinase anchoring proteins (AKAPs). The
DDD
moieties spontaneously form dimers which then bind to an AD moiety. By
attaching
appropriate effector moieties, such as antibodies or fragments thereof, to AD
and DDD
moieties, the DNL technique allows the specific covalent formation of any
desired targeted
delivery complex. Where the effector moiety is a protein or peptide, the AD
and DDD
moieties may be incorporated into fusion proteins conjugated to the effector
moieties.
[081 Antibodies or other targeting molecules of use may bind to any disease-
associated
antigen known in the art. Where the disease state is cancer, for example, many
antigens
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expressed by or otherwise associated with tumor cells are known in the art,
including but not
limited to, carbonic anhydrase IX, alpha-fetoprotein, a-actinin-4, A3, antigen
specific for
A33 antibody, ART-4, B7, Ba 733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-1, CASP-
8/mõ CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15,
CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33,
CD37, CD38, CD40, CD4OL, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e,
CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154,
CDC27, CDK-4/m, CDKN2A, CXCR4, CXCR7, CXCL12, HIF-la, colon-specific antigen-p
(CSAp), CEA (CEACAM5), CEACAM6, c-met, DAM, EGFR, EGFRvIII, EGP-1, EGP-2,
ELF2-M, Ep-CAM, Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gp100,
GROB, HLA-
DR, HM1.24, human chorionic gonadotropin (HCG) and its subunits, HER2/neu,
HMGB-1,
hypoxia inducible factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-y, IFN-a,
IFN-P, IL-
2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-
17, IL-18, IL-
25, insulin-like growth factor-1 (IGF-1), KC4-antigen, KS-1-antigen, KS1-4, Le-
Y,
LDR/FUT, macrophage migration inhibitory factor (MIF), MAGE, MAGE-3, MART-1,
MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2,
MUC3, MUC4, MUC5, MUM-1/2, MUM-3, NCA66, NCA95, NCA90, pancreatic cancer
mucin, placental growth factor, p53, PLAGL2, prostatic acid phosphatase, PSA,
PRAME,
PSMA, P1GF, ILGF, ILGF-1R, IL-6, IL-25, RS5, RANTES, T101, SAGE, S100,
survivin,
survivin-2B, TAC, TAG-72, tenascin, TRAIL receptors, TNF-a, Tn antigen,
Thomson-
Friedenreich antigens, tumor necrosis antigens, VEGFR, ED-B fibronectin, WT-1,
17-1A-
antigen, complement factors C3, C3a, C3b, C5a, C5, an angiogenesis marker, bc1-
2, bc1-6,
Kras, cMET, an oncogene marker and an oncogene product (see, e.g., Sensi et
al., Clin
Cancer Res 2006, 12:5023-32; Parmiani et al., J Immunol 2007, 178:1975-79;
Novellino et
al. Cancer Immunol Immunother 2005, 54:187-207).
[09] Exemplary antibodies that may be utilized include, but are not limited
to, hR1 (anti-
IGF-1R, U.S. Patent Application Serial No. 12/722,645, filed 3/12/10), hPAM4
(anti-mucin,
U.S. Patent No. 7,282,567), hA20 (anti-CD20, U.S. Patent No. 7,251,164), hAl9
(anti-CD19,
U.S. Patent No. 7,109,304), hIMMU31 (anti-AFP, U.S. Patent No. 7,300,655),
hLL1 (anti-
CD74, U.S. Patent No. 7,312,318), hLL2 (anti-CD22, U.S. Patent No. 7,074,403),
hMu-9
(anti-CSAp, U.S. Patent No. 7,387,773), hL243 (anti-FILA-DR, U.S. Patent No.
7,612,180),
hMN-14 (anti-CEACAM5, U.S. Patent No. 6,676,924), hMN-15 (anti-CEACAM6, U.S.
Patent No. 7,541,440), hRS7 (anti-EGP-1, U.S. Patent No. 7,238,785), hMN-3
(anti-
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CEACAM6, U.S. Patent Application Serial No. 7,541,440), Ab124 and Ab125 (anti-
CXCR4,
U.S. Patent No. 7,138,496). See the Examples section.
10101 An antibody or antigen-binding fragment of use may be chimeric,
humanized or
human. The use of chimeric antibodies is preferred to the parent murine
antibodies because
they possess human antibody constant region sequences and therefore do not
elicit as strong a
human anti-mouse antibody (HAMA) response as murine antibodies. The use of
humanized
antibodies is even more preferred, in order to further reduce the possibility
of inducing a
MAMA reaction. As discussed below, techniques for humanization of murine
antibodies by
replacing murine framework and constant region sequences with corresponding
human
antibody framework and constant region sequences are well known in the art and
have been
applied to numerous murine anti-cancer antibodies. Antibody humanization may
also involve
the substitution of one or more human framework amino acid residues with the
corresponding
residues from the parent murine framework region sequences. As also discussed
below,
techniques for production of human antibodies are also well known.
[0111 Various embodiments may concern use of the subject methods and
compositions to
treat a disease, including but not limited to non-Hodgkin's lymphomas, B-cell
acute and
chronic lymphoid leukemias, Burkitt lymphoma, Hodgkin's lymphoma, hairy cell
leukemia,
acute and chronic myeloid leukemias, 1-cell lymphomas and leukemias, multiple
myeloma,
glioma, Waldenstrom's macroglobulinemia, carcinomas, melanomas, sarcomas,
gliomas, and
skin cancers. The carcinomas may be selected from the group consisting of
carcinomas of the
oral cavity, gastrointestinal tract, colon, stomach, pulmonary tract, lung,
breast, ovary,
prostate, uterus, endometrium, cervix, urinary bladder, pancreas, bone, liver,
gall bladder,
kidney, skin, and testes.
[012] In addition, the subject methods and compositions may be used to treat
an
autoimmune disease, for example acute idiopathic thrombocytopenic purpura,
chronic
idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea,
myasthenia
gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever,
polyglandular
syndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonlein purpura,
post-
streptococcal nephritis, erythema nodosum, Takayasu's arteritis, Addison's
disease,
rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis,
erythema multiforme,
IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture's
syndrome,
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thromboangitis obliterans, Sjogren's syndrome, primary biliary cirrhosis,
Hashimoto's
thyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis,
polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris, Wegener's
granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes
dorsalis, giant
cell arteritis/polymyalgia, pernicious anemia, rapidly progressive
glomerulonephritis,
psoriasis, or fibrosing alveolitis.
[013] In certain embodiments, disease therapy may be enhanced by combination
therapy
with one or more other therapeutic agents. Known therapeutic agents of use
include toxins,
immunomodulators (such as cytokines, lymphokines, chemokines, growth factors
and tumor
necrosis factors), hormones, hormone antagonists, enzymes, oligonucleotides
(such as siRNA
or RNAi), photoactive therapeutic agents, anti-angiogenic agents and pro-
apoptotic agents.
The therapeutic agents may be delivered by conjugation to the same or
different antibodies or
other targeting molecules or may be administered in unconjugated form. Other
therapeutic
agents may be administered before, concurrently with or after the bispecific
antibody and
targetable construct.
[014] In a preferred embodiment, the therapeutic agent is a cytotoxic agent,
such as a drug
or a toxin. Also preferred, the drug is selected from the group consisting of
nitrogen
mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas,
gemcitabine, triazenes,
folic acid analogs, anthracyclines, taxanes, COX-2 inhibitors, pyrimidine
analogs, purine
analogs, antibiotics, enzyme inhibitors, epipodophyllotoxins, platinum
coordination
complexes, vinca alkaloids, substituted ureas, methyl hydrazine derivatives,
adrenocortical
suppressants, hormone antagonists, endostatin, taxols, camptothecins, SN-38,
doxorubicins
and their analogs, antimetabolites, alkylating agents, antimitotics, anti-
angiogenic agents,
tyrosine kinase inhibitors, mTOR inhibitors, heat shock protein (HSP90)
inhibitors,
proteosome inhibitors, HDAC inhibitors, pro-apoptotic agents, methotrexate,
CPT-11, and a
combination thereof
[015] In another preferred embodiment, the therapeutic agent is a toxin
selected from the
group consisting of ricin, abrin, alpha toxin, saporin, ribonuclease (RNase),
DNase I,
Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria
toxin,
Pseudomonas exotoxin, and Pseudomonas endotoxin and combinations thereof. Or
an
immunomodulator selected from the group consisting of a cytokine, a stem cell
growth
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factor, a lymphotoxin, a hematopoietic factor, a colony stimulating factor
(CSF), an
interferon (IFN), erythropoietin, thrombopoietin and a combinations thereof.
[016] In other preferred embodiments, the therapeutic agent is a radionuclide
selected from
212-
B1 21313i, 2"At, 62CU, 67CU, 90Y, 125j, 131/, 32p, 33p,
the group consisting of 111In, 1 77Lu,
47Sc, iliAg, 67Ga, 142pr, 153sm, 161Tb, 166Dy, 166110, 186Re, 188Re, 189Re,
212pb, 223Ra,
225Ac, "Fe, 75Se, 77As, 89Sr, 99Mo, iosRh, 109pd, 143pr, 149pm, 169Er, 1941r,
198Au, 'Au,
and 211Pb. Also preferred are radionuclides that substantially decay with
Auger-emitting
particles. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111,
Sb-119,
1-125, Ho-161, Os-189m and Ir-192. Decay energies of useful beta-particle-
emitting
nuclides are preferably <1,000 keV, more preferably <100 keV, and most
preferably <70
keV. Also preferred are radionuclides that substantially decay with generation
of alpha-
particles. Such radionuclides include, but are not limited to: Dy-152, At-211,
Bi-212, Ra-
223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221, At-217, Bi-213 and Fm-255. Decay
energies
of useful alpha-particle-emitting radionuclides are preferably 2,000-10,000
keV, more
preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV. Additional
potential
radioisotopes of use include 11C, 13N, 150, 75Br, 198Au, 224A, 126-,
1331, 77Br, 113m1,
"Ru, 97Ru, 1 3Ru, losRu, 107Hg, 203Hg, 121mTe, 122mTe, 125mTe, 165Tm, 167Tm,
168Tm,
197pt, 109pd, 105Rh, 142pr, 143pr, 161Tb, 166H0, 'Au, 57CO, 58CO, 5ICr, 59Fe,
755e, 201Ti,
225Ac, 76Br, 169Yb, and the like. In other embodiments the therapeutic agent
is a photoactive
therapeutic agent selected from the group consisting of chromogens and dyes.
[017] Alternatively, the therapeutic agent is an enzyme selected from the
group consisting
of malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase,
yeast alcohol
dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate
isomerase,
horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase,
beta-
galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate
dehydrogenase,
glucoamylase and acetylcholinesterase. Such enzymes may be used, for example,
in
combination with prodrugs that are administered in relatively non-toxic form
and converted
at the target site by the enzyme into a cytotoxic agent. In other
alternatives, a drug may be
converted into less toxic form by endogenous enzymes in the subject but may be
reconverted
into a cytotoxic form by the therapeutic enzyme.
[018] The disclosed methods and compositions may thus be applied for treatment
of
diseases and conditions for which targeting moieties are of use to deliver
cytotoxic agents.
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Such diseases or conditions may be characterized by the presence of a target
molecule or
target cell that is insufficiently affected when unconjugated, or naked,
targeting moieties are
used, such as in the immunotherapy of cancer or of infection with pathogenic
organisms. (For
methods of making immunoconjugates of antibodies with isotopes, drugs, and
toxins for use
in disease therapies, see, e.g., U.S. Patent Nos. 4,699,784; 4,824,659;
5,525,338; 5,677,427;
5,697,902; 5,716,595; 6,071,490; 6,187,284; 6,306,393; 6,548,275; 6,653,104;
6,962,702;
7,033,572; 7,147,856; 7,259,240 and U.S. Patent Appin. Publ. Nos. 20050175582
(now
abandoned); 20050136001; 20040166115 (now abandoned); 20040043030 (now
abandoned);
20030068322 (now abandoned) and 20030026764 (now abandoned), the Examples
section).
[019] Camptothecin (CPT) and its analogs and derivatives are preferred
chemotherapeutic
moieties, although the invention is not so limited. Other chemotherapeutic
moieties that are
within the scope of the invention are taxanes (e.g, baceatin III, taxol),
calicheamicin,
epothilones, anthracycline drugs (e.g., doxorubiein (DOX), epirubicin,
morpholinodoxorubicin (morpholino-DOX), cyanomorpholino-doxorubicin
(cyanomorpholino-DOX), and 2-pyrrolinodoxorubiein (2-PDOX); see Priebe W
(ed.), ACS
symposium series 574, published by American Chemical Society, Washington D.C.,
1995
(332pp) and Nagy et al., Proc. Natl. Acad. Sci. USA 93:2464-2469, 1996),
benzoquinoid
ansamycins exemplified by geldanamycin (DeBoer et al., Journal of Antibiotics
23:442-447,
1970; Neckers etal., Invest. New Drugs /7:361-373, 1999), and the like.
[020] In certain embodiments involving treatment of cancer, the drug
conjugates may be
used in combination with surgery, radiation therapy, chemotherapy,
immunotherapy with
naked antibodies, radioimmunotherapy, inummomodulators, vaccines, and the
like. Similar
combinations are preferred in the treatment of other diseases amenable to
targeting moieties,
such as autoimmune diseases. For example, eamptothecin conjugates can be
combined with
TNF inhibitors, B-cell antibodies, interferons, interleukins, and other
effective agents for the
treatment of autoimmune diseases, such as rheumatoid arthritis, systemic lupus
erythematosis, Sjogren's syndrome, multiple sclerosis, vasculitis, as well as
type-I diabetes
(juvenile diabetes). These combination therapies can allow lower doses of each
therapeutic
to be given in such combinations, thus reducing certain severe side effects,
and potentially
reducing the courses of therapy required. In viral diseases, the drug
conjugates can be
combined with other therapeutic drugs, immunomodulators, naked antibodies, or
vaccines
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(e.g., antibodies against hepatitis, HIV, or papilloma viruses, or vaccines
based on
immunogens of these viruses). Antibodies and antigen-based vaccines against
these and other
viral pathogens are known in the art and, in some cases, already in commercial
use.
[020A] The present invention as claimed relates to a method of conjugating a
dendron to a
peptide, wherein the peptide comprises a cysteine residue, the method
comprising:
a) obtaining a dendron comprising an amino focal functional group that is
conjugated to a
BOC (tert-butyloxycarbonyl) moiety; b) removing the BOC moiety from the amino
focal
functional group; c) derivatizing the amino focal functional group with a
maleimide
compound; and d) reacting the maleimide with the cysteine residue on the
peptide to
conjugate the dendron to the peptide.
BRIEF DESCRIPTION OF THE FIGURES
[021] FIG. 1. Synthesis of IMP 453.
[022] FIG. 2. Activation of SN-38 for peptide conjugation.
[023] FIG. 3. Dendron carrier for SN-38.
[024] FIG. 4. Synthesis of azido-SN-38 for attachment to dendron.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[025] Unless otherwise specified, "a" or "an" means one or more.
[026] As used herein, "about" means plus or minus 10%. For example, "about
100" would
include any number between 90 and 110.
[027] An antibody, as described herein, refers to a full-length (i.e.,
naturally occurring or
formed by normal immunoglobulin gene fragment recombinatorial processes)
immunoglobulin molecule (e.g., an IgG antibody) or an immunologically active
(i.e., specifically binding) portion of an immunoglobulin molecule, like an
antibody fragment.
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,
[028] An antibody fragment is a portion of an antibody such as F(ab')2, Fab',
Fab, Fv, sFy
and the like. Regardless of structure, an antibody fragment binds with the
same antigen that is
recognized by the full-length antibody. The term "antibody fragment" also
includes isolated
fragments consisting of the variable regions of antibodies, such as the "Fv"
fragments
consisting of the variable regions of the heavy and light chains and
recombinant single chain
polypeptide molecules in which light and heavy variable regions are connected
by a peptide
linker ("scFv proteins").
[029] A chimeric antibody is a recombinant protein that contains the variable
domains
including the complementarity determining regions (CDRs) of an antibody
derived from
one species, preferably a rodent antibody, while the constant domains of the
antibody
molecule
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are derived from those of a human antibody. For veterinary applications, the
constant
domains of the chimeric antibody may be derived from that of other species,
such as a cat or
dog.
[030] A humanized antibody is a recombinant protein in which the CDRs from an
antibody
from one species; e.g., a rodent antibody, are transferred from the heavy and
light variable
chains of the rodent antibody into human heavy and light variable domains
(e.g., framework
region sequences). The constant domains of the antibody molecule are derived
from those of
a human antibody. In certain embodiments, a limited number of framework region
amino
acid residues from the parent (rodent) antibody may be substituted into the
human antibody
framework region sequences.
[031] A human antibody is, e.g., an antibody obtained from transgenic mice
that have been
"engineered" to produce specific human antibodies in response to antigenic
challenge. In this
technique, elements of the human heavy and light chain loci are introduced
into strains of
mice derived from embryonic stem cell lines that contain targeted disruptions
of the
endogenous murine heavy chain and light chain loci. The transgenic mice can
synthesize
human antibodies specific for particular antigens, and the mice can be used to
produce human
antibody-secreting hybridomas. Methods for obtaining human antibodies from
transgenic
mice are described by Green et al., Nature Genet. 7:13 (1994), Lonberg et al.,
Nature 368:856
(1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully human antibody
also can be
constructed by genetic or chromosomal transfection methods, as well as phage
display
technology, all of which are known in the art. See for example, McCafferty et
al., Nature
348:552-553 (1990) for the production of human antibodies and fragments
thereof in vitro,
from immunoglobulin variable domain gene repertoires from unimmunized donors.
In this
technique, antibody variable domain genes are cloned in-frame into either a
major or minor
coat protein gene of a filamentous bacteriophage, 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. In this way, the phage mimics some of the properties of the B-
cell. Phage display
can be perfolined in a variety of formats, for review, see e.g. Johnson and
Chiswell, Current
Opinion in Structural Biology 3:5564-571 (1993). Human antibodies may also be
generated
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by in vitro activated B-cells. See U.S. Pat. Nos. 5,567,610 and 5,229,275, the
Examples
section.
[032] A therapeutic agent is a compound, molecule or atom which is
administered
separately, concurrently or sequentially with an antibody moiety or conjugated
to an antibody
moiety, i.e., antibody or antibody fragment, or a subfragment, and is useful
in the treatment
of a disease. Examples of therapeutic agents include antibodies, antibody
fragments, drugs,
toxins, nucleases, hormones, immunomodulators, pro-apoptotic agents, anti-
angiogenic
agents, boron compounds, photoactive agents or dyes and radioisotopes.
Therapeutic agents
of use are described in more detail below.
[0331 An immunoconjugate is an antibody, antibody fragment or fusion protein
conjugated
to at least one therapeutic and/or diagnostic agent.
[034] CPT is abbreviation for camptothecin, and as used in the present
application CPT
represents camptothecin itself or an analog or derivative of camptothecin. The
structures of
camptothecin and some of its analogs, with the numbering indicated and the
rings labeled
with letters A-E, are shown below.
CPT: RI ---- R2 = R3 = H
R3 R2 10-Hydroxy-CPT: R1= OH; R2 = R3 = H
7
C N CPT-11: RI ; R2 = ethyl; 1i = H
o
E 0
SN-38: R1= OH; R2 = ethyl; = H
OH
(1) Topotecan: I OH; R2 = H; R3 = CH2-N(CH3)2
[0351 In a preferred embodiment, the chemotherapeutic moiety is selected from
the group
consisting of doxorubicin (DOX), epirubicin, morpholinodoxorubicin (morpholino-
DOX),
cyanomorpholino-doxorubicin (cyanomorpholino-DOX), 2-pyrrolino-doxorubicin (2-
PDOX), CPT, 10-hydroxy camptothecin, SN-38, topotecan, lurtotecan, 9-
aminocamptothecin, 9-nitrocamptothecin, taxanes, geldanamycin, ansamycins, and
epothilones. In a more preferred embodiment, the chemotherapeutic moiety is SN-
38.
Targetable Constructs
[036] In certain embodiments, the moiety labeled with one or more diagnostic
and/or
therapeutic agents may comprise a peptide or other targetable construct.
Labeled peptides (or
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proteins) may be selected to bind directly to a targeted cell, tissue,
pathogenic organism or
other target. In other embodiments, labeled peptides may be selected to bind
indirectly, for
example using a bispecific antibody with one or more binding sites for a
targetable construct
peptide and one or more binding sites for a target antigen associated with a
disease or
condition. Bispecific antibodies may be used, for example, in a pretargeting
technique
wherein the antibody may be administered first to a subject. Sufficient time
may be allowed
for the bispecific antibody to bind to a target antigen and for unbound
antibody to clear from
circulation. Then a targetable construct, such as a labeled peptide, may be
administered to
the subject and allowed to bind to the bispecific antibody and localize at the
diseased cell or
tissue.
[037] Such targetable constructs can be of diverse structure and are selected
not only for the
availability of an antibody or fragment that binds with high affinity to the
targetable
construct, but also for rapid in vivo clearance when used within the pre-
targeting method and
bispecific antibodies or multispecific antibodies. Hydrophobic agents are best
at eliciting
strong immune responses, whereas hydrophilic agents are preferred for rapid in
vivo
clearance. Thus, a balance between hydrophobic and hydrophilic character is
established.
This may be accomplished, in part, by using hydrophilic chelating agents to
offset the
inherent hydrophobicity of many organic moieties. Also, subunits of the
targetable construct
may be chosen which have opposite solution properties, for example, peptides,
which contain
amino acids, some of which are hydrophobic and some of which are hydrophilic.
Aside from
peptides, carbohydrates may also be used.
[038] Peptides having as few as two amino acid residues, preferably two to ten
residues,
may be used and may also be coupled to other moieties, such as chelating
agents. The linker
should be a low molecular weight conjugate, preferably having a molecular
weight of less
than 50,000 daltons, and advantageously less than about 20,000 daltons, 10,000
daltons or
5,000 daltons. More usually, the targetable construct peptide will have four
or more residues,
such as the peptide DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH2 (SEQ ID NO:81), wherein
DOTA is 1,4,7,10-tetraazacyclododecane1,4,7,10-tetraacetic acid and HSG is the
histamine
succinyl glycyl group. Alternatively, DOTA may be replaced by NOTA (1,4,7-
triaza-
cyclononane-1,4,7-triacetic acid), TETA (p-bromoacetamido-benzyl-
tetraethylaminetetraacetic acid), NETA ([2-(4,7-
biscarboxymethyl[1,4,7]triazacyclononan-l-
yl-ethyl]-2-carbonylmethyl-amino]acetic acid), DTPA or other known chelating
moieties.
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[039] The targetable construct may also comprise unnatural amino acids, e.g.,
D-amino
acids, in the backbone structure to increase the stability of the peptide in
vivo. In alternative
embodiments, other backbone structures such as those constructed from non-
natural amino
acids or peptoids may be used.
[040] The peptides used as targetable constructs are conveniently synthesized
on an
automated peptide synthesizer using a solid-phase support and standard
techniques of
repetitive orthogonal deprotection and coupling. Free amino groups in the
peptide, that are to
be used later for conjugation of chelating moieties or other agents, are
advantageously
blocked with standard protecting groups such as a Boc group, while N-terminal
residues may
be acetylated to increase serum stability. Such protecting groups are well
known to the skilled
artisan. See Greene and Wuts Protective Groups in Organic Synthesis, 1999
(John Wiley and
Sons, N.Y.). When the peptides are prepared for later use within the
bispecific antibody
system, they are advantageously cleaved from the resins to generate the
corresponding C-
terminal amides, in order to inhibit in vivo carboxypeptidase activity.
Exemplary methods of
peptide synthesis are disclosed in the Examples below.
[041] Where pretargeting with bispecific antibodies is used, the antibody will
contain a first
binding site for an antigen produced by or associated with a target tissue and
a second
binding site for a hapten on the targetable construct. Exemplary haptens
include, but are not
limited to, HSG and In-DTPA. Antibodies raised to the HSG hapten are known
(e.g. 679
antibody) and can be easily incorporated into the appropriate bispecific
antibody (see, e.g.,
U.S. Patent Nos. 6,962,702; 7,138,103 and 7,300,644, with
respect to the Examples sections). However, other haptens and antibodies that
bind to them
are known in the art and may be used, such as ln-DTPA and the 734 antibody
(e.g., U.S.
Patent No.7,534,431, see the Examples section).
[042] In alternative embodiments, the specificity of the click chemistry
reaction may be
used as a substitute for the antibody-hapten binding interaction used in
pretargeting with
bispecific antibodies. As discussed below, the specific reactivity of e.g.,
cyclooctyne
moieties for azide moieties or alkyne moieties for nitrone moieties may be
used in an in vivo
cycloaddition reaction. An antibody or other targeting molecule is activated
by incorporation
of a substituted cyclooctyne, an azide or a nitrone moiety. A targetable
construct is labeled
with one or more diagnostic or therapeutic agents and a complementary reactive
moiety. I.e.,
where the targeting molecule comprises a cyclooctyne, the targetable construct
will comprise
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an azide; where the targeting molecule comprises a nitrone, the targetable
construct will
comprise an alkyne, etc. The activated targeting molecule is administered to a
subject and
allowed to localize to a targeted cell, tissue or pathogen, as disclosed for
pretargeting
protocols. The reactive labeled targetable construct is then administered.
Because the
cyclooctyne, nitrone or azide on the targetable construct is unreactive with
endogenous
biomolecules and highly reactive with the complementary moiety on the
targeting molecule,
the specificity of the binding interaction results in the highly specific
binding of the targetable
construct to the tissue-localized targeting molecule.
[043] The skilled artisan will realize that although the majority of
targetable constructs
disclosed in the Examples below are peptides, other types of molecules may be
used as
targetable constructs. For example, polymeric molecules, such as polyethylene
glycol (PEG),
may be easily derivatized with functional groups to bind diagnostic or
therapeutic agents.
Following attachment of an appropriate reactive group, such as a substituted
cyclooctyne, a
nitrone or an azide, the labeled polymer may be utilized for delivery of
diagnostic or
therapeutic agents. Many examples of such carrier molecules are known in the
art and may
be utilized, including but not limited to polymers, nanoparticles,
microspheres, liposomes and
micelles.
Antibodies
Target Antigens
[044] Targeting antibodies of use may be specific to or selective for a
variety of cell surface
or disease-associated antigens. Exemplary target antigens of use for imaging
or treating
various diseases, conditions, syndromes or disorders, such as a malignant
disease, a
cardiovascular disease, an infectious disease, an inflammatory disease, an
autoimmune
disease, a metabolic (e.g., endocrine) disease, or a neurological (e.g.,
neurodegenerative)
disease, such as Alzheimer's, may include carbonic anhydrase IX, CCCL19,
CCCL21, CSAp,
CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, IGF-
1R, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40,
CD4OL, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74,
CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, CXCR4, CXCR7,
CXCL12, HIF-la, AFP, PSMA, CEACAM5, CEACAM6, c-met, B7, ED-B of fibronectin,
Factor H, FHL-1, Flt-3, folate receptor, GROB, HMGB-1, hypoxia inducible
factor (HIF),
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HM1.24, insulin-like growth factor-I (ILGF-1), IFN-a, IFN-J3, IL-2, IL-4R,
IL-6R,
IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25,
IP-10,
MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5,
NCA-95, NCA-90, Ia, HM1.24, EGP-1, EGP-2, HLA-DR, tenascin, Le(y), RANTES,
T101,
TAC, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, INF-
a, TRAIL
receptor (R1 and R2), VEGFR, EGFR, PIGF, complement factors C3, C3a, C3b, C5a,
C5,
PLAGL2, and an oncogene product.
[045] In certain embodiments, such as treating tumors, antibodies of use may
target tumor-
associated antigens. These antigenic markers may be substances produced by a
tumor or may
be substances which accumulate at a tumor site, on tumor cell surfaces or
within tumor cells.
Among such tumor-associated markers are those disclosed by Herberman,
"Immunodiagnosis
of Cancer", in Fleisher ed., "The Clinical Biochemistry of Cancer", page 347
(American
Association of Clinical Chemists, 1979) and in U.S. Pat. Nos. 4,150,149;
4,361,544; and
4,444,744, see the Examples section.
Reports on tumor associated antigens (TAAs) include Mizukami et al., (2005,
Nature Med.
11:992-97); Hatfield et al., (2005, Curr. Cancer Drug Targets 5:229-48);
Vallbohmer et al.
(2005, J. Clin. Oncol. 23:3536-44); and Ren et al. (2005, Ann. Surg. 242:55-
63),
with respect to the TAAs identified.
[046] Tumor-associated markers have been categorized by Herbennan, supra, in a
number
of categories including oncofetal antigens, placental antigens, oncogenic or
tumor virus
associated antigens, tissue associated antigens, organ associated antigens,
ectopic hormones
and normal antigens or variants thereof. Occasionally, a sub-unit of a tumor-
associated
marker is advantageously used to raise antibodies having higher tumor-
specificity, e.g., the
beta-subunit of human chorionic gonadotropin (HCG) or the gamma region of
carcinoembryonic antigen (CEA), which stimulate the production of antibodies
having a
greatly reduced cross-reactivity to non-tumor substances as disclosed in U.S.
Pat. Nos.
4,361,644 and 4,444,744.
[047] Another marker of interest is transmembrane activator and CAML-
interactor (TACI).
See Yu et al. Nat. Immunol. 1:252-256 (2000). Briefly, TACI is a marker for B-
cell
malignancies (e.g., lymphoma). TACI and B-cell maturation antigen (BCMA) are
bound by
the tumor necrosis factor homolog - a proliferation-inducing ligand (APRIL).
APRIL
stimulates in vitro proliferation of primary B and 1-cells and increases
spleen weight due to
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accumulation of B-cells in vivo. APRIL also competes with TALL-I (also called
BLyS or
BAFF) for receptor binding. Soluble BCMA and TACI specifically prevent binding
of
APRIL and block APRIL-stimulated proliferation of primary B-cells. BCMA-Fc
also inhibits
production of antibodies against keyhole limpet hemocyanin and Pneumovax in
mice,
indicating that APRIL and/or TALL-I signaling via BCMA and/or TACI are
required for
generation of humoral immunity. Thus, APRIL-TALL-I and BCMA-TACI foul' a two
ligand-two receptor pathway involved in stimulation of B and T-cell function.
[048] Where the disease involves a lymphoma, leukemia or autoimmune disorder,
targeted
antigens may be selected from the group consisting of CD4, CD5, CD8, CD14,
CD15, CD19,
CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD4OL, CD46, CD52,
CD54, CD67, CD74, CD79a, CD80, CD126, CD138, CD154, CXCR4, B7, MUC1, Ia,
HM1.24, HLA-DR, tenascin, VEGF, P1GF, ED-B fibronectin, an oncogene, an
oncogene
product (e.g., c-met or PLAGL2), CD66a-d, necrosis antigens, IL-2, T101, TAG,
IL-6, MIF,
TRAIL-R1 (DR4) and TRAIL-R2 (DR5).
Methods for Raising Antibodies
[049] MAbs can be isolated and purified from hybridoma cultures by a variety
of well-
established techniques. Such isolation techniques include affinity
chromatography with
Protein-A or Protein-G Sepharose, size-exclusion chromatography, and ion-
exchange
chromatography. See, for example, Coligan at pages 2.7.1-2.7.12 and pages
2.9.1-2.9.3.
Also, see Baines et al., "Purification of Immunoglobulin G (IgG)," in METHODS
IN
MOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992). After
the initial raising of antibodies to the immunogen, the antibodies can be
sequenced and
subsequently prepared by recombinant techniques. Humanization and
chimerization of
murine antibodies and antibody fragments are well known to those skilled in
the art, as
discussed below.
Chimeric Antibodies
[050] A chimeric antibody is a recombinant protein in which the variable
regions of a
human antibody have been replaced by the variable regions of, for example, a
mouse
antibody, including the complementarity-determining regions (CDRs) of the
mouse antibody.
Chimeric antibodies exhibit decreased immunogenicity and increased stability
when
administered to a subject. General techniques for cloning murine
immunoglobulin variable
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domains are disclosed, for example, in Orlandi etal., Proc. Nat'l Acad Sci.
USA 6: 3833
(1989). Techniques for constructing chimeric antibodies are well known to
those of skill in
the art. As an example, Leung etal., Hybridoma /3:469 (1994), produced an LL2
chimera
by combining DNA sequences encoding the Võ and VH domains of murine LL2, an
anti-
CD22 monoclonal antibody, with respective human K. and IgGi constant region
domains.
Humanized Antibodies
[051] Techniques for producing humanized MAbs are well known in the art (see,
e.g., Jones
etal., Nature 321: 522 (1986), Riechmann et cd., Nature 332: 323 (1988),
Verhoeyen etal.,
Science 239: 1534 (1988), Carter et al., Proc. Nat'l Acad. Sci. USA 89: 4285
(1992), Sandhu,
Crit Rev. Biotech. 12: 437 (1992), and Singer etal., I Immun. 150: 2844
(1993)). A
chimeric or murine monoclonal antibody may be humanized by transferring the
mouse CDRs
from the heavy and light variable chains of the mouse immunoglobulin into the
corresponding variable domains of a human antibody. The mouse framework
regions (FR) in
the chimeric monoclonal antibody are also replaced with human FR sequences. As
simply
transferring mouse CDRs into human FRs often results in a reduction or even
loss of antibody
affinity, additional modification might be required in order to restore the
original affinity of the
murine antibody. This can be accomplished by the replacement of one or more
human residues
in the FR regions with their murine counterparts to obtain an antibody that
possesses good
binding affinity to its epitope. See, for example, Tempest etal.,
Biotechnology 9:266 (1991) and
Verhoeyen etal., Science 239: 1534 (1988). Preferred residues for substitution
include FR
residues that are located within 1, 2, or 3 Angstroms of a CDR residue side
chain, that are
located adjacent to a CDR sequence, or that are predicted to interact with a
CDR residue.
Human Antibodies
[0521 Methods for producing fully human antibodies using either combinatorial
approaches
or transgenic animals transfolined with human immunoglobulin loci are known in
the art
(e.g., Mancini et al., 2004, New Microbiol. 27:315-28; Conrad and Scheller,
2005, Comb.
Chem. High Throughput Screen. 8:117-26; Brekke and Loset, 2003, Curr. Opin.
Pharmacol.
3:544-50). A fully human antibody also can be constructed by genetic or
chromosomal
transfection methods, as well as phage display technology, all of which are
known in the art.
See for example, McCafferty etal., Nature 348:552-553 (1990). Such fully human
antibodies are expected to exhibit even fewer side effects than chimeric or
humanized
antibodies and to function in vivo as essentially endogenous human antibodies.
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[053] In one alternative, the phage display technique may be used to generate
human
antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. MoL Res. 4:126-40).
Human antibodies
may be generated from normal humans or from humans that exhibit a particular
disease state,
such as cancer (Dantas-Barbosa et al., 2005). The advantage to constructing
human
antibodies from a diseased individual is that the circulating antibody
repertoire may be biased
towards antibodies against disease-associated antigens.
[054] In one non-limiting example of this methodology, Dantas-Barbosa et al.
(2005)
constructed a phage display library of human Fab antibody fragments from
osteosarcoma
patients. Generally, total RNA was obtained from circulating blood lymphocytes
(Id).
Recombinant Fab were cloned from the , y and lc chain antibody repertoires
and inserted
into a phage display library (Id.). RNAs were converted to cDNAs and used to
make Fab
cDNA libraries using specific primers against the heavy and light chain
itnmunoglobulin
sequences (Marks et al., 1991, J. MoL Biol. 222:581-97). Library construction
was
performed according to Andris-Widhopf et al. (2000, In: Phage Display
Laboratory Manual,
Barbas et al. (eds), 1 edition, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY
pp. 9.1 to 9.22). The final Fab fragments were digested with restriction
endonucleases and
inserted into the bacteriophage genome to make the phage display library. Such
libraries may
be screened by standard phage display methods, as known in. the art. Phage
display can be
performed in a variety of formats, for their review, see e.g. Johnson and
Chiswell, Current
Opinion in Structural Biology 3:5564-571 (1993).
[055] Human antibodies may also be generated by in vitro activated B-cells.
See U.S.
Patent Nos. 5,567,610 and 5,229,275. The
skilled artisan will realize that these techniques are exemplary and any known
method for
making and screening human antibodies or antibody fragments may be utilized.
[056] In another alternative, transgenic animals that have been genetically
engineered to
produce human antibodies may be used to generate antibodies against
essentially any
immunogenic target, using standard immunization protocols. Methods for
obtaining human
antibodies from transgenic mice are disclosed by Green et al., Nature Genet.
7:13 (1994),
Lonberg et aL, Nature 368:856 (1994), and Taylor et al., Int. Immun. 6:579
(1994). A non-
limiting example of such a system is the XenoMousee (e.g., Green et al., 1999,
J. Immunol.
Methods 23 1 : 11-23) from Abgenix (Fremont, CA). In the
XenoMousee and similar animals, the mouse antibody genes have been inactivated
and
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replaced by functional human antibody genes, while the remainder of the mouse
immune
system remains intact.
[057] The XenoMouse was transformed with germline-configured YACs (yeast
artificial
chromosomes) that contained portions of the human IgH and Igkappa loci,
including the
majority of the variable region sequences, along with accessory genes and
regulatory
sequences. The human variable region repertoire may be used to generate
antibody
producing B-cells, which may be processed into hybridomas by known techniques.
A
XenoMouse immunized with a target antigen will produce human antibodies by
the normal
immune response, which may be harvested and/or produced by standard techniques
discussed
above. A variety of strains of XenoMouse are available, each of which is
capable of
producing a different class of antibody. Transgenically produced human
antibodies have
been shown to have therapeutic potential, while retaining the pharmacokinetic
properties of
normal human antibodies (Green et al., 1999). The skilled artisan will realize
that the
claimed compositions and methods are not limited to use of the XenoMouse
system but
may utilize any transgenic animal that has been genetically engineered to
produce human
antibodies.
Known Antibodies
[058] The skilled artisan will realize that the targeting molecules of use may
incorporate any
antibody or fragment known in the art that has binding specificity for a
target antigen
associated with a disease state or condition. Such known antibodies include,
but are not
limited to, hR1 (anti-IGF-1R, U.S. Patent Application Serial No. 12/772,645,
filed 3/12/10)
hPAM4 (anti-pancreatic cancer mucin, U.S. Patent No. 7,282,567), hA20 (anti-
CD20, U.S.
Patent No. 7,251,164), hAl 9 (anti-CD19, U.S. Patent No. 7,109,304), hIMMU31
(anti-AFP,
U.S. Patent No. 7,300,655), hLL1 (anti-CD74, U.S. Patent No. 7,312,318), 1ILL2
(anti-CD22,
U.S. Patent No. 7,074,403), hMu-9 (anti-CSAp, U.S. Patent No. 7,387,773),
hL243 (anti-
HLA-DR, U.S. Patent No. 7,612,180), hMN-14 (anti-CEACAM5, U.S. Patent No.
6,676,924), hMN-15 (anti-CEACAM6, U.S. Patent No. 7,662,378, U.S. Patent
Application
Serial No. 12/846,062, filed 7/29/10), hRS7 (anti-EGP-1, U.S. Patent No.
7,238,785) and
hMN-3 (anti-CEACAM6, U.S. Patent No. 7,541,440), Ab124 and Ab125 (anti-CXCR4,
U.S.
Patent No. 7,138,496), see the Examples section.
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[059] Candidate anti-HIV antibodies include the anti-envelope antibody
described by
Johansson et al. (AIDS. 2006 Oct 3;20(15):1911-5), as well as the anti-HIV
antibodies
described and sold by Polymun (Vienna, Austria), also described in U.S. Patent
5,831,034,
U.S. patent 5,911,989, and Vcelar et al., AIDS 2007; 21(16):2161-2170 and Joos
eta!,
Antimicrob. Agents Chemother. 2006; 50(5):1773-9.
[060] In one embodiment, a pharmaceutical composition of the present invention
may be
used to treat a subject having a metabolic disease, such amyloidosis, or a
neurodegenerative
disease, such as Alzheimer's disease. Bapineuzumab is in clinical trials for
Alzheimer's
disease therapy. Other antibodies proposed for therapy of Alzheimer's disease
include Alz 50
(Ksiezak-Reding et al., 1987, J Biol Chem 263:7943-47), gantenerumab, and
solanezumab.
Infliximab, an anti-TNF-ct antibody, has been reported to reduce amyloid
plaques and
improve cognition. Anti-CD3 antibodies have been proposed for therapy of type
1 diabetes
(Cernea et al., 2010, Diabetes Metab Rev 26:602-05). In addition, a
pharmaceutical
composition of the present invention may be used to treat a subject having an
immune-
dysregulatory disorder such as graft-versus-host disease or organ transplant
rejection.
[061] In a preferred embodiment, diseases that may be treated using the
claimed
compositions and methods include cardiovascular diseases, such as fibrin
clots,
atherosclerosis, myocardial ischemia and infarction. Antibodies to fibrin
(e.g., scFv(59D8);
T2G Is; MH1) are known and in clinical trials as imaging agents for disclosing
said clots and
pulmonary emboli, while anti-granulocyte antibodies, such as MN-3, MN-15, anti-
NCA95,
and anti-CD15 antibodies, can target myocardial infarcts and myocardial
ischemia. (See,
e.g., U.S. Patent Nos. 5,487,892; 5,632,968; 6,294,173; 7,541,440, the
Examples section).
Anti-macrophage, anti-low-density lipoprotein (LDL)
and anti-CD74 (e.g., hLL1) antibodies can be used to target atherosclerotic
plaques.
Abciximab (anti-glycoprotein fib/Ma) has been approved for adjuvant use for
prevention of
restenosis in percutaneous coronary interventions and the treatment of
unstable angina
(Waldmann et al., 2000, Hematol 1:394-408). Anti-CD3 antibodies have been
reported to
reduce development and progression of atherosclerosis (Steffens et al., 2006,
Circulation
114:1977-84). Antibodies against oxidized LDL induced a regression of
established
atherosclerosis in a mouse model (Ginsberg, 2007, J Am Coll Cardiol 52:2319-
21). Anti-
ICAM-1 antibody was shown to reduce ischemic cell damage after cerebral artery
occlusion
in rats (Zhang et al., 1994, Neurology 44:1747-51). Commercially available
monoclonal
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=
antibodies to leukocyte antigens are represented by: OKT anti-T-cell
monoclonal antibodies
(available from Ortho Pharmaceutical Company) which bind to normal T-
lymphocytes; the
monoclonal antibodies produced by the hybridomas having the ATCC accession
numbers
HB44, 11B55, HB12, HB78 and HB2; G7E11, W8E7, NKP15 and G022 (Becton
Dickinson);
NEN9.4 (New England Nuclear); and FMCII (Sera Labs). A description of
antibodies against
fibrin and platelet antigens is contained in Knight, Semin. Nucl. Med., 20:52-
67 (1990).
[062] Where bispecific antibodies are used, the second MAb may be selected
from any
anti-hapten antibody known in the art, including but not limited to h679 (U.S.
Patent No.
7,429,381) and 734 (U.S. Patent Nos. 7,429,381; 7,563,439; 7,666,415; and
7,534,431), see
the Examples section.
[063] Various other antibodies of use are known in the art (e.g., U.S. Patent
Nos. 5,686,072;
5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104; 6,730.300; 6,899,864;
6,926,893;
6,962,702; 7,074,403; 7,230,084; 7,238,785; 7,238,786; 7,256,004; 7,282,567;
7,300,655;
7,312,318; 7,585,491; 7,612,180; 7,642,239 and U.S. Patent Application Publ.
No.
20060193865). Such known antibodies are of use for
detection and/or treating a variety of disease states or conditions (e.g., hMN-
14 or TF2 (CEA-
expressing carcinomas), hA20 or TF-4 (lymphoma), hPAM4 or TF-10 (pancreatic
cancer),
RS7 (lung, breast, ovarian, prostatic cancers), hMN-15 or liMN3
(inflammation), anti-gp120
and/or anti-gp41 (HIV), anti-platelet and anti-thrombin (blood clots), anti-
myosin (cardiac
necrosis), anti-CXCR4 (cancer and inflammatory disease)).
10641 Antibodies of use may be commercially obtained from a wide variety of
known
sources. For example, a variety of antibody secreting hybridoma lines are
available from the
American Type Culture Collection (ATCC, Manassas, VA). A large number of
antibodies
against various disease targets, including but not limited to tumor-associated
antigens, have
been deposited at the ATCC and/or have published variable region sequences and
are
available for use in the claimed methods and compositions. See, e.g., U.S.
Patent Nos.
7,312,318; 7,282,567; 7,151,164; 7,074,403; 7,060,802; 7,056,509; 7,049,060;
7,045,132;
7,041,803; 7,041,802; 7,041,293; 7,038,018; 7,037,498; 7,012,133; 7,001,598;
6,998,468;
6,994,976; 6,994,852; 6,989,241; 6,974,863; 6,965,018; 6,964,854; 6,962,981;
6,962,813;
6,956,107; 6,951,924; 6,949,244; 6,946,129; 6,943,020; 6,939,547; 6,921,645;
6,921,645;
6,921,533; 6,919,433; 6,919,078; 6,916,475; 6,905,681; 6,899,879; 6,893,625;
6,887,468;
6,887,466; 6,884,594; 6,881,405; 6,878,812; 6,875,580; 6,872,568; 6,867,006;
6,864,062;
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6,861,511; 6,861,227; 6,861,226; 6,838,282; 6,835,549; 6,835,370; 6,824,780;
6,824,778;
6,812,206; 6,793,924; 6,783,758; 6,770,450; 6,767,711; 6,764,688; 6,764,681;
6,764,679;
6,743,898; 6,733,981; 6,730,307; 6,720,15; 6,716,966; 6,709,653; 6,693,176;
6,692,908;
6,689,607; 6,689,362; 6,689,355; 6,682,737; 6,682,736; 6,682,734; 6,673,344;
6,653,104;
6,652,852; 6,635,482; 6,630,144; 6,610,833; 6,610,294; 6,605,441; 6,605,279;
6,596,852;
6,592,868; 6,576,745; 6,572;856; 6,566,076; 6,562,618; 6,545,130; 6,544,749;
6,534,058;
6,528,625; 6,528,269; 6,521,227; 6,518,404; 6,511,665; 6,491,915; 6,488,930;
6,482,598;
6,482,408; 6,479,247; 6,468,531; 6,468,529; 6,465,173; 6,461,823; 6,458,356;
6,455,044;
6,455,040, 6,451,310; 6,444,206' 6,441,143; 6,432,404; 6,432,402; 6,419,928;
6,413,726;
6,406,694; 6,403,770; 6,403,091; 6,395,276; 6,395,274; 6,387,350; 6,383,759;
6,383,484;
6,376,654; 6,372,215; 6,359,126; 6,355,481; 6,355,444; 6,355,245; 6,355,244;
6,346,246;
6,344,198; 6,340,571; 6,340,459; 6,331,175; 6,306,393; 6,254,868; 6,187,287;
6,183,744;
6,129,914; 6,120,767; 6,096,289; 6,077,499; 5,922,302; 5,874,540; 5,814,440;
5,798,229;
5,789,554; 5,776,456; 5,736,119; 5,716,595; 5,677,136; 5,587,459; 5,443,953,
5,525,338.
These are exemplary only and a wide variety of other antibodies and their
hybridomas are
known in the art. The skilled artisan will realize that antibody sequences or
antibody-
secreting hybridomas against almost any disease-associated antigen may be
obtained by a
simple search of the ATCC, NCBI and/or USPTO databases for antibodies against
a selected
disease-associated target of interest. The antigen binding domains of the
cloned antibodies
may be amplified, excised, ligated into an expression vector, transfected into
an adapted host
cell and used for protein production, using standard techniques well known in
the art.
Antibody Fragments
[0651 Antibody fragments which recognize specific epitopes can be generated by
known
techniques. The antibody fragments are antigen binding portions of an
antibody, such as F(a1:02,
Fab', F(ab)2, Fab, Fv, sFy and the like. F(ab')2fragments can be produced by
pepsin digestion
of the antibody molecule and Fab' fragments can be generated by reducing
disulfide bridges
of the F(ab')2fragments. Alternatively, Fab' expression libraries can be
constructed (Huse et
al., 1989, Science, 246:1274-1281) to allow rapid and easy identification of
monoclonal Fab'
fragments with the desired specificity. An antibody fragment can be prepared
by proteolytic
hydrolysis of the full length antibody or by expression in E. coli or another
host of the DNA
coding for the fragment. These methods are described, for example, by
Goldenberg, U.S.
Patent Nos. 4,036,945 and 4,331,647.
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,52392-94
Also, see Nisonoff et al., Arch Biochem.
Biophys. 89: 230 (1960); Porter, Biochem. J. 73: 119 (1959), Edelman et al.,
in METHODS
IN ENZYMOLOGY VOL. 1, page 422 (Academic Press 1967), and Coligan at pages
2.8.1-
2.8.10 and 2.10.-2.10.4.
[066] A single chain Fv molecule (scFv) comprises a VL domain and a VH domain.
The VL
and VH domains associate to form a target binding site. These two domains are
further
covalently linked by a peptide linker (L). Methods for making scFv molecules
and designing
suitable peptide linkers are described in US Patent No. 4,704,692, US Patent
No. 4,946,778,
R. Raag and M. Whitlow, "Single Chain Fvs." FASEB Vol 9:73-80 (1995) and R.E.
Bird and
B.W. Walker, "Single Chain Antibody Variable Regions," TIBTECH, Vol 9: 132-137
(1991).
[067] An scFv library with a large repertoire can be constructed by isolating
V-genes from
non-immunized human donors using PCR primers corresponding to all known VH,
Vkappa and
V80 gene families. See, e.g., Vaughn et al., Nat. Biotechnol., 14: 309-314
(1996). Following
amplification, the Vkappa and Viambda pools are combined to form one pool.
These fragments
are ligated into a phagemid vector. The scFv linker is then ligated into the
phagemid
upstream of the VL fragment. The VH and linker-VL fragments are amplified and
assembled
on the JH region. The resulting VH -linker-VL fragments are ligated into a
phagemid vector.
The phagemid library can be panned for binding to the selected antigen.
[068] Other antibody fragments, for example single domain antibody fragments,
are known
in the art and may be used in the claimed constructs. Single domain antibodies
(VHH) may
be obtained, for example, from camels, alpacas or llamas by standard
immunization
techniques. (See, e.g., Muyldermans et al., TIBS 26:230-235, 2001; Yau et al.,
3 Immunol
Methods 281:161-75, 2003; Maass et al., J Immunol Methods 324:13-25, 2007).
The VHH
may have potent antigen-binding capacity and can interact with novel epitopes
that are
inaccessible to conventional VH-VL pairs. (Muyldermans et al., 2001) Alpaca
serum IgG
contains about 50% camelid heavy chain only IgG antibodies (Cabs) (Maass et
al., 2007).
Alpacas may be immunized with known antigens and VHHs can be isolated that
bind to and
neutralize the target antigen (Maass et al., 2007). PCR primers that amplify
virtually all
alpaca VHH coding sequences have been identified and may be used to construct
alpaca
VHH phage display libraries, which can be used for antibody fragment isolation
by standard
biopanning techniques well known in the art (Maass et al., 2007). These and
other known
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antigen-binding antibody fragments may be utilized in the claimed methods and
compositions.
General techniques for antibody cloning and production
[069] Various techniques, such as production of chimeric or humanized
antibodies, may involve
procedures of antibody cloning and construction. The antigen-binding Vic
(variable light chain)
and VH (variable heavy chain) sequences for an antibody of interest may be
obtained by a
variety of molecular cloning procedures, such as RT-PCR, 5'-RACE, and cDNA
library
screening. The V genes of a MAb from a cell that expresses a murine MAb can be
cloned by
PCR amplification and sequenced. To confirm their authenticity, the cloned VL
and VH genes
can be expressed in cell culture as a chimeric Ab as described by Orlandi et
al., (Proc. Natl.
Acad. Sci., USA, 86: 3833 (1989)). Based on the V gene sequences, a humanized
MAb can
then be designed and constructed as described by Leung et al. (Mot Inununot,
32: 1413 (1995)).
[070] cDNA can be prepared from any known hybridoma line or transfected cell
line producing
a murine MAb by general molecular cloning techniques (Sambrook et al.,
Molecular Cloning,
A laboratory manual, 2'd Ed (1989)). The Vx sequence for the MAb may be
amplified using the
primers VKlBACK and VK1FOR (Orlandi etal., 1989) or the extended primer set
described by
Leung etal. (BioTechniques, 15: 286 (1993)). The VH sequences can be amplified
using the
primer pair VH1BACK/VH1FOR (Orlandi etal., 1989) or the primers annealing to
the constant
region of murine IgG described by Leung et al. (Hybridoma, 13:469 (1994)).
Humanized V
genes can be constructed by a combination of long oligonucleotide template
syntheses and PCR
amplification as described by Leung et al. (Mol. Immunot , 32: 1413 (1995)).
[071] PCR products for Vic can be subcloned into a staging vector, such as a
pBR327-based
staging vector, VKpBR, that contains an Ig promoter, a signal peptide sequence
and convenient
restriction sites. PCR products for VH can be subcloned into a similar staging
vector, such as the
pBluescript-based VHpBS. Expression cassettes containing the VIC and VH
sequences together
with the promoter and signal peptide sequences can be excised from VKpBR and
VHpBS and
ligated into appropriate expression vectors, such as pKh and pG1g,
respectively (Leung et al.,
Hybridoma, 13:469 (1994)). The expression vectors can be co-transfected into
an appropriate
cell and supernatant fluids monitored for production of a chimeric, humanized
or human MAb.
Alternatively, the Vic and VH expression cassettes can be excised and
subcloned into a single
expression vector, such as pdHL2, as described by Gillies etal.
Immunot Methods 125:191
(1989) and also shown in Losman et al., Cancer, 80:2660 (1997)).
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[072] In an alternative embodiment, expression vectors may be transfected into
host cells that
have been pre-adapted for transfection, growth and expression in serum-free
medium.
Exemplary cell lines that may be used include the Sp/EEE, Sp/ESF and Sp/ESF-X
cell lines
(see, e.g., U.S. Patent Nos. 7,531,327; 7,537,930 and 7,608,425; the Examples
section).
These exemplary cell lines are based on the
Sp2/0 myeloma cell line, transfected with a mutant Bcl-EEE gene, exposed to
methotrexate
to amplify transfected gene sequences and pre-adapted to serum-free cell line
for protein
expression.
Bispecific and Multispecilie Antibodies
[073] In certain embodiments, the techniques and compositions for therapeutic
agent
delivery disclosed herein may be used with bispecific or multispecific
antibodies as the
targeting moieties. Numerous methods to produce bispecific or multispecific
antibodies are
known, as disclosed, for example, in U.S. Patent No. 7,405,320, see the
Examples section.
Bispecific antibodies can be produced by the
quadroma method, which involves the fusion of two different hybridomas, each
producing a
monoclonal antibody recognizing a different antigenic site (Milstein and
Cuello, Nature,
1983; 305:537-540).
[074] Another method for producing bispecific antibodies uses
heterobifunctional cross-
linkers to chemically tether two different monoclonal antibodies (Staerz, et
al. Nature. 1985;
314:628-631; Perez, et al. Nature. 1985; 316:354-356). Bispecific antibodies
can also be
produced by reduction of each of two parental monoclonal antibodies to the
respective half
molecules, which are then mixed and allowed to reoxidize to obtain the hybrid
structure
(Staerz and Bevan. Proc Natl Acad Sci U S A. 1986; 83:1453-1457). Another
alternative
involves chemically cross-linking two or three separately purified Fab'
fragments using
appropriate linkers. (See, e.g.,
European Patent Application 0453082).
[075] Other methods include improving the efficiency of generating hybrid
hybridomas by
gene transfer of distinct selectable markers via retrovirus-derived shuttle
vectors into
respective parental hybridomas, which are fused subsequently (DeMonte, et al.
Proc Natl
Acad Sci U S A. 1990, 87:2941-2945); or transfection of a hybridoma cell line
with
expression plasmids containing the heavy and light chain genes of a different
antibody.
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[076] Cognate VH and VL domains can be joined with a peptide linker of
appropriate
composition and length (usually consisting of more than 12 amino acid
residues) to form a
single-chain Fv (scFv) with binding activity. Methods of manufacturing scFvs
are disclosed
in U.S. Pat. No. 4,946,778 and U.S. Pat. No. 5,132,405 see the Examples
section.
Reduction of the peptide linker length to less than
12 amino acid residues prevents pairing of VH and VL domains on the same chain
and forces
pairing of VH and VL domains with complementary domains on other chains,
resulting in the
formation of functional multimers. Polypeptide chains of VH and VL domains
that are joined
with linkers between 3 and 12 amino acid residues form predominantly dimers
(termed
diabodies). With linkers between 0 and 2 amino acid residues, trimers (termed
triabody) and
tetramers (termed tetrabody) are favored, but the exact patterns of
oligomerization appear to
depend on the composition as well as the orientation of V-domains (VH-linker-
VL or Vt.-
linker-VH), in addition to the linker length.
[077] These techniques for producing multispecific or bispecific antibodies
exhibit various
difficulties in terms of low yield, necessity for purification, low stability
or the labor-
intensiveness of the technique. More recently, a technique known as "dock and
lock" (DNL)
has been utilized to produce combinations of virtually any desired antibodies,
antibody
fragments and other effector molecules (see, e.g., U.S. Patent Nos. 7,550,143;
7,521,056;
7,534,866; 7,527,787 and USSN 11/925,408, the Examples section).
The technique utilizes complementary protein binding
domains, referred to as anchoring domains (AD) and dimerization and docking
domains
(DDD), which bind to each other and allow the assembly of complex structures,
ranging from
dimers, trimers, tetramers, quintamers and hexamers. These form stable
complexes in high
yield without requirement for extensive purification. The DNL technique allows
the
assembly of monospecific, bispecific or multispecific antibodies. Any of the
techniques
known in the art for making bispecific or multispecific antibodies may be
utilized in the
practice of the presently claimed methods.
10781 In various embodiments, a conjugate as disclosed herein may be part of a
composite,
multispecific antibody. Such antibodies may contain two or more different
antigen binding
sites, with differing specificities. The multispecific composite may bind to
different epitopes
of the same antigen, or alternatively may bind to two different antigens.
Dock-and-Lock (DNL)
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[079] In preferred embodiments, bispecific or multispecific antibodies or
other constructs
may be produced using the dock-and-lock technology (see, e.g., U.S. Patent
Nos. 7,550,143;
7,521,056; 7,534,866; 7,527,787 and 7,666,400, the Examples section).
The DNL method exploits specific protein/protein interactions that
occur between the regulatory (R) subunits of cAMP-dependent protein kinase
(PKA) and the
anchoring domain (AD) of A-kinase anchoring proteins (AKAPs) (Baillie et al.,
FEBS
Letters. 2005; 579: 3264. Wong and Scott, Nat. Rev. Mol. Cell Biol. 2004; 5:
959). PKA,
which plays a central role in one of the best studied signal transduction
pathways triggered by
the binding of the second messenger cAMP to the R subunits, was first isolated
from rabbit
skeletal muscle in 1968 (Walsh etal., J. Biol. Chem. 1968;243:3763). The
structure of the
holoenzyme consists of two catalytic subunits held in an inactive form by the
R subunits
(Taylor, J. Biol. Chem. 1989;264:8443). Isozymes of PKA are found with two
types of R
subunits (RI and Rh), and each type has a and (3 isoforms (Scott, Phannacol.
Ther.
1991;50:123). The R subunits have been isolated only as stable dimers and the
dimerization
domain has been shown to consist of the first 44 amino-terminal residues
(Newlon et al., Nat.
Struct. Biol. 1999; 6:222). Binding of cAMP to the R subunits leads to the
release of active
catalytic subunits for a broad spectrum of serine/threonine kinase activities,
which are
oriented toward selected substrates through the compartmentalization of PKA
via its docking
with AKAPs (Scott et al., J. Biol. Chem. 1990;265;21561)
[080] Since the first AKAP, microtubule-associated protein-2, was
characterized in 1984
(Lohmann etal., Proc. Natl. Acad. Sci USA. 1984; 81:6723), more than 50 AKAPs
that
localize to various sub-cellular sites, including plasma membrane, actin
cytoskeleton,
nucleus, mitochondria, and endoplasmic reticulum, have been identified with
diverse
structures in species ranging from yeast to humans (Wong and Scott, Nat. Rev.
Mol. Cell
Biol. 2004;5:959). The AD of AKAPs for PKA is an amphipathic helix of 14-18
residues
(Carr etal., J. Biol. Chem. 1991;266:14188). The amino acid sequences of the
AD are quite
varied among individual AKAPs, with the binding affinities reported for Rh
dimers ranging
from 2 to 90 nM (Alto etal., Proc. Natl. Acad. Sci. USA. 2003;100:4445). AKAPs
will only
bind to dimeric R subunits. For human Rlla, the AD binds to a hydrophobic
surface formed
by the 23 amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999;
6:216). Thus,
the dimerization domain and AKAP binding domain of human R.% are both located
within
the same N-terminal 44 amino acid sequence (Newlon etal., Nat. Struct. Biol.
1999;6:222;
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Newlon et al., EMBO J. 2001;20:1651), which is termed the DDD herein.
[081] We have developed a platform technology to utilize the DDD of human Rlla
and the
AD of AKAP as an excellent pair of linker modules for docking any two
entities, referred to
hereafter as A and B, into a noncovalent complex, which could be further
locked into a stably
tethered structure through the introduction of cysteine residues into both the
DDD and AD at
strategic positions to facilitate the formation of disulfide bonds. The
general methodology of
the "dock-and-lock" approach is as follows. Entity A is constructed by linking
a DDD
sequence to a precursor of A, resulting in a first component hereafter
referred to as a.
Because the DDD sequence would effect the spontaneous formation of a dimer, A
would thus
be composed of a2. Entity B is constructed by linking an AD sequence to a
precursor of B,
resulting in a second component hereafter referred to as b. The dimeric motif
of DDD
contained in a2 will create a docking site for binding to the AD sequence
contained in b, thus
facilitating a ready association of a2 and b to foun a binary, trimeric
complex composed of
a2b. This binding event is made irreversible with a subsequent reaction to
covalently secure
the two entities via disulfide bridges, which occurs very efficiently based on
the principle of
effective local concentration because the initial binding interactions should
bring the reactive
thiol groups placed onto both the DDD and AD into proximity (Chmura et al.,
Proc. Natl.
Acad. Sci. USA. 2001;98:8480) to ligate site-specifically. Using various
combinations of
linkers, adaptor modules and precursors, a wide variety of DNL constructs of
different
stoichiometry may be produced and used, including but not limited to dimeric,
trimeric,
tetrameric, pentameric and hexameric DNL constructs (see, e.g., U.S. Nos.
7,550,143;
7,521,056; 7,534,866; 7,527,787 and 7,666,400.)
[082] By attaching the DDD and AD away from the functional groups of the two
precursors, such site-specific ligations are also expected to preserve the
original activities of
the two precursors. This approach is modular in nature and potentially can be
applied to link,
site-specifically and covalently, a wide range of substances, including
peptides, proteins,
antibodies, antibody fragments, and other effector moieties with a wide range
of activities.
Utilizing the fusion protein method of constructing AD and DDD conjugated
effectors
described in the Examples below, virtually any protein or peptide may be
incorporated into a
DNL construct. However, the technique is not limiting and other methods of
conjugation
may be utilized.
[083] A variety of methods are known for making fusion proteins, including
nucleic acid
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synthesis, hybridization and/or amplification to produce a synthetic double-
stranded nucleic
acid encoding a fusion protein of interest. Such double-stranded nucleic acids
may be
inserted into expression vectors for fusion protein production by standard
molecular biology
techniques (see, e.g. Sambrook et al., Molecular Cloning, A laboratory manual,
2' Ed, 1989).
In such preferred embodiments, the AD and/or DDD moiety may be attached to
either the N-
terminal or C-terminal end of an effector protein or peptide. However, the
skilled artisan will
realize that the site of attachment of an AD or DDD moiety to an effector
moiety may vary,
depending on the chemical nature of the effector moiety and the part(s) of the
effector moiety
involved in its physiological activity. Site-specific attachment of a variety
of effector moieties
may be perfatined using techniques known in the art, such as the use of
bivalent cross-linking
reagents and/or other chemical conjugation techniques.
[084] In other alternative embodiments, click chemistry reactions may be used
to produce an
AD or DDD peptide conjugated to an effector moiety, or even to covalently
attach the AD and
DDD moiety to each other to provide an irreversible covalent bond to stabilize
the DNL
complex.
Pre-Targeting
[085] Bispecific or multispecific antibodies may be utilized in pre-targeting
techniques.
Pre-targeting is a multistep process originally developed to resolve the slow
blood clearance
of directly targeting antibodies, which contributes to undesirable toxicity to
noimal tissues
such as bone marrow. With pre-targeting, a radionuclide or other therapeutic
agent is
attached to a small delivery molecule (targetable construct) that is cleared
within minutes
from the blood. A pre-targeting bispecific or multispecific antibody, which
has binding sites
for the targetable construct as well as a target antigen, is administered
first, free antibody is
allowed to clear from circulation and then the targetable construct is
administered.
[086] Pre-targeting methods are disclosed, for example, in Goodwin et al.,
U.S. Pat. No.
4,863,713; Goodwin et al., J. Nucl. Med. 29:226, 1988; Hnatowich et al., J.
Nucl. Med.
28:1294, 1987; Oehr et al., J. Nucl. Med. 29:728, 1988; Klibanov et al., J.
Nucl. Med.
29:1951, 1988; Sinitsyn et al., J. Nucl. Med. 30:66, 1989; Kalofonos et al.,
J. Nucl. Med.
31:1791, 1990; Schechter et al., Int. J. Cancer 48:167, 1991; Paganelli et
al., Cancer Res.
51:5960, 1991; Paganelli et al., Nucl. Med. Commun. 12:211, 1991; U.S. Pat.
No. 5,256,395;
Stickney et al., Cancer Res. 51:6650, 1991; Yuan et al., Cancer Res. 51:3119,
1991; U.S. Pat.
Nos. 6,077,499; 7,011,812; 7,300,644; 7,074,405; 6,962,702; 7,387,772;
7,052,872;
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7,138,103; 6,090,381; 6,472,511; 6,962,702; and 6,962,702.
[087] A pre-targeting method of treating or diagnosing a disease or disorder
in a subject
may be provided by: (1) administering to the subject a bispecific antibody or
antibody
fragment; (2) optionally administering to the subject a clearing composition,
and allowing the
composition to clear the antibody from circulation; and (3) administering to
the subject the
targetable construct, containing one or more chelated or chemically bound
therapeutic or
diagnostic agents.
Immunoconjugates
1088] In preferred embodiments, a therapeutic or diagnostic agent may be
covalently
attached to an antibody or antibody fragment to form an itnmunoconjugate.
Carrier moieties
may be attached, for example to reduced SH groups and/or to carbohydrate side
chains. A
carrier moiety can be attached at the hinge region of a reduced antibody
component via
disulfide bond formation. Alternatively, such agents can be attached using a
heterobifunctional cross-linker, such as N-succinyl 3-(2-
pyridyldithio)propionate (SPDP).
Yu et aL, Mt. J Cancer 56: 244 (1994). General techniques for such conjugation
are well-
known in the art. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION
AND CROSS-LINKING (CRC Press 1991); Upeslacis etal., "Modification of
Antibodies by
Chemical Methods," in MONOCLONAL ANTIBODIES: PRINCIPLES AND
APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc. 1995);
Price,
"Production and Characterization of Synthetic Peptide-Derived Antibodies," in
MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL
APPLICATION, Ritter et al. (eds.), pages 60-84 (Cambridge University Press
1995).
Alternatively, the carrier moiety can be conjugated via a carbohydrate moiety
in the Fe region
of the antibody.
[089] Methods for conjugating functional groups to antibodies via an antibody
carbohydrate
moiety are well-known to those of skill in the art. See, for example, Shih et
al., Int. J. Cancer
41: 832 (1988); Shih et al., Mt. J. Cancer 46: 1101(1990); and Shih et al.,
U.S. Patent No.
5,057,313, the Examples section. The general
method involves reacting an antibody having an oxidized carbohydrate portion
with a carrier
polymer that has at least one free amine function. This reaction results in an
initial Schiff
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base (imine) linkage, which can be stabilized by reduction to a secondary
amine to form the
final conjugate.
[090j The Fe region may be absent if the antibody component of the
irnmunoconjugate is an
antibody fragment. However, it is possible to introduce a carbohydrate moiety
into the light
chain variable region of a full length antibody or antibody fragment. See, for
example, Leung
et al., J. Immunol. 154: 5919 (1995); U.S. Patent Nos. 5,443,953 and
6,254,868, the
Examples section. The engineered carbohydrate
moiety is used to attach the therapeutic or diagnostic agent.
[0911 An alternative method for attaching carrier moieties to a targeting
molecule involves
use of click chemistry reactions. The click chemistry approach was originally
conceived as a
method to rapidly generate complex substances by joining small subunits
together in a
modular fashion. (See, e.g., Kolb et al., 2004, Angew Chem IM Ed 40:3004-31;
Evans, 2007,
Aust J Chem 60:384-95.) Various forms of click chemistry reaction are known in
the art,
such as the Huisgen 1,3-dipolar cycloaddition copper catalyzed reaction (Tomoe
et al., 2002,
J Organic Chem 67:3057-64), which is often referred to as the "click
reaction." Other
alternatives include cycloaddition reactions such as the Diels-Alder,
nucleophilic substitution
reactions (especially to small strained rings like epoxy and aziridine
compounds), carbonyl
chemistry formation of urea compounds and reactions involving carbon-carbon
double bonds,
such as alkynes in thiol-yne reactions.
[0921 The azide alkyne Huisgen cycloaddition reaction uses a copper catalyst
in the
presence of a reducing agent to catalyze the reaction of a terminal alkyne
group attached to a
first molecule. In the presence of a second molecule comprising an azide
moiety, the azide
reacts with the activated alkyne to form a 1,4-disubstituted 1,2,3-triazole.
The copper
catalyzed reaction occurs at room temperature and is sufficiently specific
that purification of
the reaction product is often not required. (Rostovstev et al., 2002, Angew
Chem Int Ed
41:2596; Tomoe et al., 2002, .1 Org Chem 67:3057.) The azide and alkyne
functional groups
are largely inert towards biomolecules in aqueous medium, allowing the
reaction to occur in
complex solutions. The triazole formed is chemically stable and is not subject
to enzymatic
cleavage, making the click chemistry product highly stable in biological
systems. Although
the copper catalyst is toxic to living cells, the copper-based click chemistry
reaction may be
used in vitro for immunoconjugate formation.
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[093] A copper-free click reaction has been proposed for covalent modification
of
biomolecules. (See, e.g., Agard et al., 2004, J Am Chem Soc 126:15046-47.) The
copper-
free reaction uses ring strain in place of the copper catalyst to promote a [3
+ 2] azide-alkyne
cycloaddition reaction (Id.) For example, cyclooctyne is an 8-carbon ring
structure
comprising an internal alkyne bond. The closed ring structure induces a
substantial bond
angle deformation of the acetylene, which is highly reactive with azide groups
to form a
triazole. Thus, cyclooctyne derivatives may be used for copper-free click
reactions (Id.)
[094] Another type of copper-free click reaction was reported by Ning et al.
(2010, Angew
Chem Int Ed 49:3065-68), involving strain-promoted alkyne-nitrone
cycloaddition. To
address the slow rate of the original cyclooctyne reaction, electron-
withdrawing groups are
attached adjacent to the triple bond (Id.) Examples of such substituted
cyclooctynes include
difluorinated cyclooctynes, 4-dibenzocyclooctynol and azacyclooctyne (Id.) An
alternative
copper-free reaction involved strain-promoted akyne-nitrone cycloaddition to
give N-
alkylated isoxazolines (Id.) The reaction was reported to have exceptionally
fast reaction
kinetics and was used in a one-pot three-step protocol for site-specific
modification of
peptides and proteins (Id.) Nitrones were prepared by the condensation of
appropriate
aldehydes with N-methylhydroxylamine and the cycloaddition reaction took place
in a
mixture of acetonitrile and water (Id.) These and other known click chemistry
reactions may
be used to attach carrier moieties to antibodies in vitro.
[095] Agard et al. (2004, J Am Chem Soc 126:15046-47) demonstrated that a
recombinant
glycoprotein expressed in CHO cells in the presence of peracetylated N-
azidoacetylmannosamine resulted in the bioincorporation of the corresponding N-
azidoacetyl
sialic acid in the carbohydrates of the glycoprotein. The azido-derivatized
glycoprotein
reacted specifically with a biotinylated cyclooctyne to form a biotinylated
glycoprotein, while
control glycoprotein without the azido moiety remained unlabeled (Id.)
Laughlin et al. (2008,
Science 320:664-667) used a similar technique to metabolically label cell-
surface glycans in
zebrafish embryos incubated with peracetylated N-azidoacetylgalactosamine. The
azido-
derivatized glycans reacted with difluorinated cyclooctyne (DIFO) reagents to
allow
visualization of glycans in vivo.
[096] The Diels-Alder reaction has also been used for in vivo labeling of
molecules. Rossin
et al. (2010, Angew Chem Int Ed 49:3375-78) reported a 52% yield in vivo
between a tumor-
localized anti-TAG72 (CC49) antibody carrying a trans-cyclooctene (TCO)
reactive moiety
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and an 111In-labeled tetrazine DOTA derivative. The TCO-labeled CC49 antibody
was
administered to mice bearing colon cancer xenografts, followed 1 day later by
injection of
"In-labeled tetrazine probe (Id.) The reaction of radiolabeled probe with
tumor localized
antibody resulted in pronounced radioactivity localization in the tumor, as
demonstrated by
SPECT imaging of live mice three hours after injection of radiolabeled probe,
with a tumor-
to-muscle ratio of 13:1 (Id.) The results confirmed the in vivo chemical
reaction of the TCO
and tetrazine-labeled molecules.
10971 Antibody labeling techniques using biological incorporation of labeling
moieties are
further disclosed in U.S. Patent No. 6,953,675(see the Examples section).
Such "landscaped" antibodies were prepared to have reactive ketone
groups on glycosylated sites. The method involved expressing cells transfected
with an
expression vector encoding an antibody with one or more N-glycosylation sites
in the CHI or
VK domain in culture medium comprising a ketone derivative of a saccharide or
saccharide
precursor. Ketone-derivatized saccharides or precursors included N-levulinoyl
mannosamine
and N-levulinoyl fucose. The landscaped antibodies were subsequently reacted
with agents
comprising a ketone-reactive moiety, such as hydrazide, hydrazine,
hydroxylamino or
thiosemicarbazide groups, to form a labeled targeting molecule. Exemplary
agents attached
to the landscaped antibodies included chelating agents like DTPA, large drug
molecules such
as doxorubicin-dextran, and acyl-hydrazide containing peptides. The
landscaping technique is
not limited to producing antibodies comprising ketone moieties, but may be
used instead to
introduce a click chemistry reactive group, such as a nitrone, an azide or a
cyclooctyne, onto
an antibody or other biological molecule.
[098] Modifications of click chemistry reactions are suitable for use in vitro
or in vivo.
Reactive targeting molecule may be formed either by either chemical
conjugation or by
biological incorporation. The targeting molecule, such as an antibody or
antibody fragment,
may be activated with an azido moiety, a substituted cyclooctyne or alkyne
group, or a
nitrone moiety. Where the targeting molecule comprises an azido or nitrone
group, the
corresponding targetable construct will comprise a substituted cyclooctyne or
alkyne group,
and vice versa. Such activated molecules may be made by metabolic
incorporation in living
cells, as discussed above. Alternatively, methods of chemical conjugation of
such moieties to
biomolecules are well known in the art, and any such known method may be
utilized.
Therapeutic and Diagnostic Agents
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[099] In certain embodiments, the targeting molecules or targetable constructs
disclosed herein
may be attached to one or more therapeutic and/or diagnostic agents.
Therapeutic agent are
preferably selected from the group consisting of a radionuclide, an
immunomodulator, an anti-
angiogenic agent, a cytokine, a chemokine, a growth factor, a hormone, a drug,
a prodrug, an
enzyme, an oligonucleotide, a pro-apoptotic agent, an interference RNA, a
photoactive
therapeutic agent, a cytotoxic agent, which may be a chemotherapeutic agent or
a toxin, and a
combination thereof. The drugs of use may possess a pharmaceutical property
selected from the
group consisting of antimitotic, antikinase, alkylating, antimetabolite,
antibiotic, alkaloid, anti-
angiogenic, pro-apoptotic agents and combinations thereof.
101001 Exemplary drugs of use include, but are not limited to, 5-fluorouracil,
aplidin,
azaribine, anastrozole, anthracyclines, bendamustine, bleomycin, bortezomib,
bryostatin-1,
busulfan, calicheamycin, camptothecin, carboplatin, 10-hydroxycamptothecin,
carmustine,
celebrex, chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan (CPT-
11), SN-38,
carboplatin, cladribine, camptothecans, cyclophosphamide, cytarabine,
dacarbazine,
docetaxel, dactinomycin, daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine
(2P-DOX),
cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicin glucuronide,
estramustine, epipodophyllotoxin, estrogen receptor binding agents, etoposide
(VP16),
etoposide glucuronide, etoposide phosphate, floxuridine (FUdR), 3',5'-0-
dioleoyl-FudR
(FUdR-d0), fludarabine, flutamide, farnesyl-protein transferase inhibitors,
gemcitabine,
hydroxyurea, idarubicin, ifosfamide, L-asparaginase, lenolidamide, leucovorin,
lomustine,
mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate,
mitoxantrone, mithramycin, mitomycin, mitotane, navelbine, nitrosourea,
plicomycin,
procarbazine, paclitaxel, pentostatin, PSI-341, raloxifene, semustine,
streptozocin,
tamoxifen, taxol, temazolomide (an aqueous form of DTIC), transplatinum,
thalidomide,
thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vinorelbine,
vinblastine,
vincristine and vinca alkaloids.
[0101] Toxins of use may include ricin, abrin, alpha toxin, saporin,
ribonuclease (RNase),
e.g., onconase, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral
protein, gelonin,
diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.
[0102] Immunomodulators of use may be selected from a cytokine, a stem cell
growth factor, a
lymphotoxin, an hematopoietic factor, a colony stimulating factor (CSF), an
interferon (IFN),
erythropoietin, thrombopoietin and a combination thereof Specifically useful
are
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lymphotoxins such as tumor necrosis factor (TNF), hematopoietic factors, such
as interleukin
(IL), colony stimulating factor, such as granulocyte-colony stimulating factor
(G-CSF) or
granulocyte macrophage-colony stimulating factor (GM-CSF), interferon, such as
interferons-a, -f3 or -y, and stem cell growth factor, such as that designated
"Si factor".
Included among the cytokines are growth hormones such as human growth hormone,
N-
methionyl human growth hormone, and bovine growth hormone; parathyroid
hotmone;
thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones
such as follicle
stimulating homione (FSH), thyroid stimulating hormone (TSH), and luteinizing
hormone
(LH); hepatic growth factor; prostaglandin, fibroblast growth factor;
prolactin; placental
lactogen, OB protein; tumor necrosis factor-a and - B; mullerian-inhibiting
substance; mouse
gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth
factor;
integrin; thrombopoietin (TP0); nerve growth factors such as NGF-B; platelet-
growth factor;
transforming growth factors (TGFs) such as TGF- a and TGF- B; insulin-like
growth factor-I
and -II; erythropoietin (EPO); osteoinductive factors; interferons such as
interferon-a, -13, and
-y; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);
interleukins (ILs)
such as IL-1, IL-la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-
11, IL-12; IL-13,
IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand or FLT-3,
angiostatin,
thrombospondin, endostatin, tumor necrosis factor and LT.
[0103] Chemokines of use include RANTES, MCAF, MIP1-alpha, MIP1-Beta and IP-
10.
[0104] Radioactive isotopes useful for treating diseased tissue include, but
are not limited to-
IIIIn, r77Lu, 212Bi, 213Bi, 211Ar, 62cti, 67c.u, 90y, 125/5 131/, 32/35 33/3,
475c, 111Ag5 67Ga5
142pr, 1535m, 161Tb, 166Dy, r66}10, 186Re, 188Re, 189Re, 212pb, 223Ra, 225Ac,
59Fe, 75Se,
77As, "Sr, 99Mo, 105Rh, 109pd, 143pr, 149pm, 169Er, 194/r, 198Aa, 199Au, and
211Pb. The
therapeutic radionuclide preferably has a decay-energy in the range of 20 to
6,000 keV,
preferably in the ranges 60 to 200 keV for an Auger emitter, 100-2,500 keV for
a beta
emitter, and 4,000-6,000 keV for an alpha emitter. Maximum decay energies of
useful beta-
particle-emitting nuclides are preferably 20-5,000 keV, more preferably 100-
4,000 keV, and
most preferably 500-2,500 keV. Also preferred are radionuclides that
substantially decay
with Auger-emitting particles. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-
103m, Pt-
109, In-111, Sb-119, 1-125, Ho-161, Os-189m and Ir-192. Decay energies of
useful beta-
particle-emitting nuclides are preferably <1,000 keV, more preferably <100
keV, and most
preferably <70 keV. Also preferred are radionuclides that substantially decay
with generation
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of alpha-particles. Such radionuclides include, but are not limited to: Dy-
152, At-211, Bi-
212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221, At-217, Bi-213 and Fm-
255. Decay
energies of useful alpha-particle-emitting radionuclides are preferably 2,000-
10,000 keV,
more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.
Additional
potential radioisotopes of use include 11C, 13N, 150, 75Br, 198Au, 224Ac, 126-
,
1 1331, 77Br,
"3mIn, "Ru, 97Ru, 1 3Ru, 1 osRu, Io7Hg, 203Hg, 121mTe, 122mTe, 125mTe, 165Tna,
167Tm,
168Ttn, 197pt, 109pd, 105Rh, 142pr, 143pr, 161Tb, 166H0, I99AU, 57CO, 58CO,
51Cr, 59Fe, 75Se,
201T1, 225Ac, 76Br, 169<Yr+D ,
and the like.
[0105] Therapeutic agents may include a photoactive agent or dye. Fluorescent
compositions, such as fluorochrome, and other chromogens, or dyes, such as
porphyrins
sensitive to visible light, have been used to detect and to treat lesions by
directing the suitable
light to the lesion. In therapy, this has been termed photoradiation,
phototherapy, or
photodynamic therapy. See Joni et al. (eds.), PHOTODYNAMIC THERAPY OF TUMORS
AND OTHER DISEASES (Libreria Progetto 1985); van den Bergh, Chem. Britain
(1986),
22:430. Moreover, monoclonal antibodies have been coupled with photoactivated
dyes for
achieving phototherapy. See Mew et al., J. Immunol. (1983),130:1473; idem.,
Cancer Res.
(1985), 45:4380; Oseroff et al., Proc. Natl. Acad. Sci. USA (1986), 83:8744;
idem.,.
Photochem. Photobiol. (1987), 46:83; Hasan et al., Prog. Clin. Biol. Res.
(1989), 288:471;
Tatsuta et al., Lasers Surg. Med. (1989), 9:422; Pelegrin et al., Cancer
(1991), 67:2529.
[0106] Corticosteroid hormones can increase the effectiveness of other
chemotherapy agents,
and consequently, they are frequently used in combination treatments.
Prednisone and
dexamethasone are examples of corticosteroid hormones. .
[0107] In certain embodiments, anti-angiogenic agents, such as angiostatin,
baculostatin,
canstatin, maspin, anti-placenta growth factor (P1GF) peptides and antibodies,
anti-vascular
growth factor antibodies (such as anti-VEGF and anti-P1GF), anti-Flk-1
antibodies, anti-Flt-1
antibodies and peptides, anti-Kras antibodies, anti-cMET antibodies, anti-MIF
(macrophage
migration-inhibitory factor) antibodies, laminin peptides, fibronectin
peptides, plasminogen
activator inhibitors, tissue metalloproteinase inhibitors, interferons,
interleukin-12, IP-10,
Gro-I3, thrombospondin, 2-methoxyoestradiol, proliferin-related protein,
carboxiamidotriazole, CM101, Marimastat, pentosan polysulphate, angiopoietin-
2,
interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment, Linomide,
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thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel,
accutin, angiostatin,
cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4 or minocycline
may be of use.
[01081 The therapeutic agent may comprise and oligonucleotide, such as a
siRNA. The
skilled artisan will realize that any siRNA or interference RNA species may be
attached to a
targetable construct for delivery to a targeted tissue. Many siRNA species
against a wide
variety of targets are known in the art, and any such known siRNA may be
utilized in the
claimed methods and compositions.
[01091 Known siRNA species of potential use include those specific for IKK-
gamma (U.S.
Patent 7,022,828); VEGF, Flt-1 and Flk-1/KDR (U.S. Patent 7,148,342); Bc12 and
EGFR
(U.S. Patent 7,541,453); CDC20 (U.S. Patent 7,550,572); transducin (beta)-like
3 (U.S.
Patent 7,576,196); KRAS (U.S. Patent 7,576,197); carbonic anhydrase II (U.S.
Patent
7,579,457); complement component 3 (U.S. Patent 7,582,746); interleukin-1
receptor-
associated kinase 4 (IRAK4) (U.S. Patent 7,592,443); survivin (U.S. Patent
7,608,7070);
superoxide dismutase 1 (U.S. Patent 7,632,938); MET proto-oncogene (U.S.
Patent
7,632,939); amyloid beta precursor protein (APP) (U.S. Patent 7,635,771); IGF-
1R (U.S.
Patent 7,638,621); ICAM1 (U.S. Patent 7,642,349); complement factor B (U.S.
Patent
7,696,344); p53 (7,781,575), and apolipoprotein B (7,795,421), see the
Examples section.
[0110] Additional siRNA species are available from known commercial sources,
such as
Sigma-Aldrich (St Louis, MO), Invitrogen (Carlsbad, CA), Santa Cruz
Biotechnology (Santa
Cruz, CA), Ambion (Austin, TX), Dharmacon (Thermo Scientific, Lafayette, CO),
Promega
(Madison, WI), Mirus Bio (Madison, WI) and Qiagen (Valencia, CA), among many
others.
Other publicly available sources of siRNA species include the siRNAdb database
at the
Stockholm Bioinformatics Centre, the MIT/ICBP siRNA Database, the RNAi
Consortium
shRNA Library at the Broad Institute, and the Probe database at NCBI. For
example, there
are 30,852 siRNA species in the NCBI Probe database. The skilled artisan will
realize that
for any gene of interest, either a siRNA species has already been designed, or
one may
readily be designed using publicly available software tools. Any such siRNA
species may be
delivered using the subject DNL complexes.
[0111] Exemplary siRNA species known in the art are listed in Table 1.
Although siRNA is
delivered as a double-stranded molecule, for simplicity only the sense strand
sequences are
shown in Table 1.
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Table 1. Exemplary siRNA Sequences
Target Sequence SEQ ID NO
VEGF R2 AATGCGGCGGTGGTGACAGTA SEQ ID NO:1
VEGF R2 AAGCTCAGCACACAGAAAGAC SEQ ID NO:2
CXCR4 UAAAAUCUUCCUGCCCACCdTdT SEQ ID NO:3
CXCR4 GGAAGCUGUUGGCUGAAAAdTdT SEQ ID NO:4
PPARC1 AAGACCAGCCUCUUUGCCCAG SEQ ID NO:5
Dynamin 2 GGACCAGGCAGAAAACGAG SEQ ID NO:6
Catenin CUAUCAGGAUGACGCGG SEQ ID NO:7
ElA binding protein UGACACAGGCAGGCUUGACUU SEQ ID NO:8
Plasminogen GGTGAAGAAGGGCGTCCAA SEQ ID NO:9
activator
K-ras GATCCGTTGGAGCTGTTGGCGTAGTT SEQ ID NO:10
CAAGAGACTCGCCAACAGCTCCAACT
TTTGGAAA
Sortilin 1 AGGTGGTGTTAACAGCAGAG SEQ ID NO:11
Apolipoprotein E AAGGTGGAGCAAGCGGTGGAG SEQ ID NO:12
Apolipoprotein E AAGGAGTTGAAGGCCGACAAA SEQ ID NO:13
Bcl-X UAUGGAGCUGCAGAGGAUGdTdT SEQ ID NO:14
Raf-1 TTTGAATATCTGTGCTGAGAACACA SEQ ID NO:15
GTTCTCAGCACAGATATTCTTTTT
Heat shock AATGAGAAAAGCAAAAGGTGCCCTGTCTC SEQ ID NO:16
transcription factor 2
IGFBP3 AAUCAUCAUCAAGAAAGGGCA SEQ ID NO:17
Thioredoxin AUGACUGUCAGGAUGUUGCdTdT SEQ ID NO:18
CD44 GAACGAAUCCUGAAGACAUCU SEQ ID NO:19
MMP14 AAGCCTGGCTACAGCAATATGCCTGTCTC SEQ ID NO:20
MAPKAPK2 UGACCAUCACCGAGUUUAUdTdT SEQ ID NO:21
FGFR1 AAGTCGGACGCAACAGAGAAA SEQ ID NO:22
ERBB2 CUACCUUUCUACGGACGUGdTdT SEQ ID NO:23
BCL2L1 CTGCCTAAGGCGGATTTGAAT SEQ ID NO:24
ABL1 TTAUUCCUUCUUCGGGAAGUC SEQ ID NO:25
CEACAM1 AACCTTCTGGAACCCGCCCAC SEQ ID NO:26
CD9 GAGCATCTTCGAGCAAGAA SEQ ID NO:27
CD151 CATGTGGCACCGTTTGCCT SEQ ID NO:28
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Caspase 8 AACTACCAGAAAGGTATACCT SEQ ID N0:29
BRCA 1 UCACAGUGUCCUUUAUGUAdTdT SEQ ID NO:30
p53 GCAUGAACCGGAGGCCCAUTT SEQ ID N0:31
CEACAM6 CCGGACAGTTCCATGTATA SEQ ID NO:32
[0112] The skilled artisan will realize that Table 1 represents a very small
sampling of the
total number of siRNA species known in the art, and that any such known siRNA
may be
utilized in the claimed methods and compositions.
[0113] Diagnostic agents are preferably selected from the group consisting of
a radionuclide, a
radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label,
a
chemiluminescent label, an ultrasound contrast agent and a photoactive agent.
Such
diagnostic agents are well known and any such known diagnostic agent may be
used. Non-
limiting examples of diagnostic agents may include a radionuclide such as 18F,
52Fe, 110/n,
1"bl, 177LU, 52Fe, 62cu, 64cti, 67cu, 67Ga, 68Ga, 86y, , 90-
Y 89Zr, 94mTc, 94Tc, 99mTc, 1201, 123/, 1241,
1251, 1311, 154-158Gd, 3213, 11C, 13N, 150, 186Re, 188Re, 51mn, 52m- -,
Mn 55Co, 72As, 75Br, 76Br, 82mRb,
835r, or other gamma-, beta-, or positron-emitters.
[0114] Paramagnetic ions of use may include chromium (III), manganese (II),
iron (III), iron
(II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III),
ytterbium (III),
gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium
(III) or erbium (III).
Metal contrast agents may include lanthanum (III), gold (III), lead (II) or
bismuth (III).
[0115] Ultrasound contrast agents may comprise liposomes, such as gas filled
liposomes.
Radiopaque diagnostic agents may be selected from compounds, barium compounds,
gallium
compounds, and thallium compounds. A wide variety of fluorescent labels are
known in the
art, including but not limited to fluorescein isothiocyanate, rhodamine,
phycoerytherin,
phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
Chemiluminescent labels
of use may include luminol, isoluminol, an aromatic acridinium ester, an
imidazole, an
acridinium salt or an oxalate ester.
Therapeutic Treatment
[0116] In another aspect, the invention relates to a method of treating a
subject, comprising
administering a therapeutically effective amount of a therapeutic conjugate as
described
herein to a subject. Diseases that may be treated with the therapeutic
conjugates described
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,52392-94
herein include, but are not limited to B-cell malignancies (e.g., non-
Hodgkin's lymphoma and
chronic lymphocytic leukemia using, for example LL2 antibody; see U.S. Patent
No.
6,183,744), adenocarcinomas of endodermally-derived digestive system
epithelia, cancers
such as breast cancer and non-small cell lung cancer, and other carcinomas,
sarcomas, glial
tumors, myeloid leukemias, etc. In particular, antibodies against an antigen,
e.g., an oncofetal
antigen, produced by or associated with a malignant solid tumor or
hematopoietic neoplasm,
e.g., a gastrointestinal, lung, breast, prostate, ovarian, testicular, brain
or lymphatic tumor, a
sarcoma or a melanoma, are advantageously used. Such therapeutics can be given
once or
repeatedly, depending on the disease state and tolerability of the conjugate,
and can also be
used optimally in combination with other therapeutic modalities, such as
surgery, external
radiation, radioimmunotherapy, immunotherapy, chemotherapy, antisense therapy,
interference RNA therapy, gene therapy, and the like. Each combination will be
adapted to
the tumor type, stage, patient condition and prior therapy, and other factors
considered by the
managing physician.
[0117] As used herein, the term "subject" refers to any animal (i.e.,
vertebrates and
invertebrates) including, but not limited to mammals, including humans. It is
not intended
that the term be limited to a particular age or sex. Thus, adult and newborn
subjects, as well
as fetuses, whether male or female, are encompassed by the term.
[0118] In a preferred embodiment, therapeutic conjugates comprising the Mu-9
antibody can
be used to treat colorectal, as well as pancreatic and ovarian cancers as
disclosed in U.S.
Patent Nos. 6,962,702 and7,387,772, see the Examples section.
In addition, therapeutic conjugates comprising the PAM4 antibody can be used
to
treat pancreatic cancer, as disclosed in U.S. Patent Nos. 7,238,786 and
7,282,567, see the
Examples section.
[0119] In another preferred embodiment, therapeutic conjugates comprising the
RS7
antibody (binding to epithelial glycoprotein-1 [EGP-1] antigen) can be used to
treat
carcinomas such as carcinomas of the lung, stomach, urinary bladder, breast,
ovary, uterus,
and prostate, as disclosed in U.S. Patent No. 7,238,785, see the Examples
section.
101201 In another preferred embodiment, therapeutic conjugates comprising the
anti-AFP
antibody can be used to treat hepatocellular carcinoma, germ cell tumors, and
other AFP-
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producing tumors using humanized, chimeric and human antibody forms, as
disclosed in U.S.
Patent No. 7,300,655, see the Examples section.
101211 In another preferred embodiment, therapeutic conjugates comprising anti-
tenascin
antibodies can be used to treat hematopoietic and solid tumors and conjugates
comprising
antibodies to tenascin can be used to treat solid tumors, preferably brain
cancers like
glioblastomas.
[0122] In a preferred embodiment, the antibodies that are used in the
treatment of human
disease are human or humanized (CDR-grafted) versions of antibodies; although
murine and
chimeric versions of antibodies can be used. Same species IgG molecules as
delivery agents
are mostly preferred to minimize immune responses. This is particularly
important when
considering repeat treatments. For humans, a human or humanized IgG antibody
is less
likely to generate an anti-IgG immune response from patients. Antibodies such
as hLL1 and
hLL2 rapidly internalize after binding to internalizing antigen on target
cells, which means
that the chemotherapeutic drug being carried is rapidly internalized into
cells as well.
However, antibodies that have slower rates of internalization can also be used
to effect
selective therapy.
[0123] In another preferred embodiment, the therapeutic conjugates can be used
against
pathogens, since antibodies against pathogens are known. For example,
antibodies and
antibody fragments which specifically bind markers produced by or associated
with
infectious lesions, including viral, bacterial, fungal and parasitic
infections, for example
caused by pathogens such as bacteria, rickettsia, mycoplasma, protozoa, fungi,
and viruses,
and antigens and products associated with such microorganisms have been
disclosed, inter
alia, in Hansen et al., U.S. Pat. No. 3,927,193 and Goldenberg U.S. Pat. Nos.
4,331,647,
4,348,376, 4,361,544, 4,468,457, 4,444,744, 4,818,709 and 4,624,846, see the
Examples
section, and in Reichert and Dewitz, cited above. In a
preferred embodiment, the pathogens are selected from the group consisting of
HIV virus,
Mycobacterium tuberculosis, Streptococcus agalactiae, methicillin-resistant
Staphylococcus
aureus, Legionella pneumophila, Streptococcus pyogenes, Escherichia coli,
Neisseria
gonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans,
Histoplasma capsulatum, Hemophilus influenzae B, Treponema pallidum, Lyme
disease
spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abort us,
rabies virus,
influenza virus, cytomegalovirus, herpes simplex virus I, herpes simplex virus
II, human
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serum parvo-like virus, respiratory syncytial virus, varicella-zoster virus,
hepatitis B virus,
hepatitis C virus, measles virus, adenovirus, human T-cell leukemia viruses,
Epstein-Barr
virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis
virus,
lymphocytic choriomeningitis virus, wart virus, blue tongue virus, Sendai
virus, feline
leukemia virus, Reovirus, polio virus, simian virus 40, mouse mammary tumor
virus, dengue
virus, rubella virus, West Nile virus, Plasmodium falciparum, Plasmodium
vivax,
Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma
rhodesiense,
Trypanosoma brucei, Schistosoma mansoni, Schistosoma japonicum, Babesia bovis,
Eimeria
tenella, Onchocerca volvulus, Leishmania tropica, Trichinella spiralis,
Theileria parva,
Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus,
Mesocestoides
corti, Mycoplasma art hritidis, M hyorhinis, M orale, M arginini, Acholeplasma
laidlcrwii,
Al. salivarium and M pneumoniae, as disclosed in U.S. Patent No. 6,440,416,
see the
Examples section.
101241 In a more preferred embodiment, drug conjugates of the present
invention comprising
anti-gp120 and other such anti-HIV antibodies can be used as therapeutics for
HIV in AIDS
patients; and drug conjugates of antibodies to Mycobacterium tuberculosis are
suitable as
therapeutics for drug-refractive tuberculosis. Fusion proteins of anti-gpl 20
antibody (anti
HIV antibody) and a toxin, such as Pseudomonas exotoxin, have been examined
for antiviral
properties (Van Oigen et al., J Drug Target, 5:75-91, 1998). Attempts at
treating HIV
infection in AIDS patients failed, possibly due to insufficient efficacy or
unacceptable host
toxicity. The drug conjugates of the present invention advantageously lack
such toxic side
effects of protein toxins, and are therefore advantageously used in treating
HIV infection in
AIDS patients. These drug conjugates can be given alone or in combination with
other
antibiotics or therapeutic agents that are effective in such patients when
given alone.
Candidate anti-HIV antibodies include the anti-envelope antibody described by
Johansson et
al. (AIDS. 2006 Oct 3;20(15):1911-5), as well as the anti-HIV antibodies
described and sold
by Polymun (Vienna, Austria), also described in U.S. Patent 5,831,034, U.S.
Patent
5,911,989, and Vcelar et at., AIDS 2007; 21(16):2161-2170 and Joos etal.,
Antimicrob.
Agens Chemother. 2006; 50(5):1773-9. A preferred
targeting agent for HIV is various combinations of these antibodies in order
to overcome
resistance.
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[0125] In another preferred embodiment, diseases that may be treated using the
therapeutic
conjugates of the preferred embodiments of the present invention include, but
are not limited
to immune dysregulation disease and related autoimmune diseases, including
Class III
autoimmune diseases such as immune-mediated thrombocytopenias, such as acute
idiopathic
thrombocytopenic purpura and chronic idiopathic thrombocytopenic purpura,
dermatomyositis, Sjogren's syndrome, multiple sclerosis, Sydenham's chorea,
myasthenia
gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever,
polyglandular
syndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonlein purpura,
post-
streptococcal nephritis, erythema nodosum, Takayasu's arteritis, Addison's
disease,
rheumatoid arthritis, sarcoidosis, ulcerative colitis, erythema multiforme,
IgA nephropathy,
polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome,
thromboangitis
obliterans, Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's
thyroiditis,
thyrotoxicosis, scleroderma, chronic active hepatitis, rheumatoid arthritis,
polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris, Wegener's
granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes
dorsalis, giant
cell arteritis/polymyalgia, pernicious anemia, rapidly progressive
glomerulonephritis and
fibrosing alveolitis, and also juvenile diabetes, as disclosed in U.S.
Provisional Application
Serial No. 60/360,259, filed March 1, 2002 (now expired). Typical antibodies
useful in these
diseases include, but are not limited to, those reactive with HLA-DR antigens,
B-cell and
plasma-cell antigens (e.g., CD19, CD20, CD21, CD22, CD23, CD4, CD5, CD8, CD14,
CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD4OL,
CD46, CD52, CD54, CD74, CD80, CD126, CD138, B7, MUC1, Ia, HM1.24, and HLA-DR),
IL-6, IL-17. Since many of these autoimmune diseases are affected by
autoantibodies made
by aberrant B-cell populations, depletion of these B-cells by therapeutic
conjugates involving
such antibodies-therapeutic agent conjugates described herein is a preferred
method of
autoimmune disease therapy, especially when B-cell antibodies are combined, in
certain
circumstances, with HLA-DR antibodies and/or T-cell antibodies (including
those which
target IL-2 as an antigen, such as anti-TAC antibody). In a preferred
embodiment, the anti-B-
cell, anti-T-cell, or anti-macrophage or other such antibodies of use in the
treatment of
patients with autoimmune diseases also can be conjugated to result in more
effective
therapeutics to control the host responses involved in said autoimmune
diseases, and can be
given alone or in combination with other therapeutic agents, such as TNF
inhibitors or 'INF
antibodies, unconjugated B- or T-cell antibodies, and the like.
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101261 In a preferred embodiment, a more effective incorporation into cells
and pathogens
can be accomplished by using multivalent, multispecific or multivalent,
monospecific
antibodies. Examples of such bivalent and bispecific antibodies are found in
U.S. Patent Nos.
7,387,772; 7,300,655; 7,238,785; and 7,282,567. see the Examples section.
These multivalent or multispecific antibodies are
particularly preferred in the targeting of cancers and infectious organisms
(pathogens), which
express multiple antigen targets and even multiple epitopes of the same
antigen target, but
which often evade antibody targeting and sufficient binding for immunotherapy
because of
insufficient expression or availability of a single antigen target on the cell
or pathogen. By
targeting multiple antigens or epitopes, said antibodies show a higher binding
and residence
time on the target, thus affording a higher saturation with the drug being
targeted in this
invention.
Methods of Administration
[0127j The subject molecules labeled with diagnostic or therapeutic agents may
be
formulated to obtain compositions that include one or more pharmaceutically
suitable
excipients, one or more additional ingredients, or some combination of these.
These can be
accomplished by known methods to prepare pharmaceutically useful dosages,
whereby the
active ingredients (i.e., the labeled molecules) are combined in a mixture
with one or more
pharmaceutically suitable excipients. Sterile phosphate-buffered saline is one
example of a
pharmaceutically suitable excipient. Other suitable excipients are well known
to those in the
art. See, e.g., Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUG
DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Germaro (ed.),
REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing
Company 1990).
101281 The preferred route for administration of the compositions described
herein is
parenteral injection. Injection may be intravenous, intraarterial,
intralymphatic, intrathecal,
or intracavitary (i.e., parenterally). In parenteral administration, the
compositions will be
formulated in a unit dosage injectable form such as a solution, suspension or
emulsion, in
association with a pharmaceutically acceptable excipient. Such excipients are
inherently
nontoxic and nontherapeutic. Examples of such excipients are saline, Ringer's
solution,
dextrose solution and Hank's solution. Nonaqueous excipients such as fixed
oils and ethyl
oleate may also be used. A preferred excipient is 5% dextrose in saline. The
excipient may
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contain minor amounts of additives such as substances that enhance isotonicity
and chemical
stability, including buffers and preservatives. Other methods of
administration, including
oral administration, are also contemplated.
[0129] Formulated compositions comprising labeled molecules can be used for
intravenous
administration via, for example, bolus injection or continuous infusion.
Compositions for
injection can be presented in unit dosage form, e.g., in ampoules or in multi-
dose containers,
with an added preservative. Compositions can also take such foans as
suspensions, solutions
or emulsions in oily or aqueous vehicles, and can contain formulatory agents
such as
suspending, stabilizing and/or dispersing agents. Alternatively, the
compositions can be in
powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-
free water, before
use.
=
[0130] The compositions may be administered in solution. The pH of the
solution should be
in the range of pH 5 to 9.5, preferably pH 6.5 to 7.5. The formulation thereof
should be in a
solution having a suitable pharmaceutically acceptable buffer such as
phosphate, TRIS
(hydroxymethyl) aminomethane-HC1 or citrate and the like. Buffer
concentrations should be
in the range of 1 to 100 mM. The formulated solution may also contain a salt,
such as
sodium chloride or potassium chloride in a concentration of 50 to 150 mM. An
effective
amount of a stabilizing agent such as mannitol, trehalose, sorbitol, glycerol,
albumin, a
globulin, a detergent, a gelatin, a protamine or a salt of protamine may also
be included. The
compositions may be administered to a mammal subcutaneously, intravenously,
intramuscularly or by other parenteral routes. Moreover, the administration
may be by
continuous infusion or by single or multiple boluses.
[01311 Where bispecific antibodies are administered, for example in a
pretargeting technique,
the dosage of an administered antibody for humans will vary depending upon
such factors as
the patient's age, weight, height, sex, general medical condition and previous
medical history.
Typically, it is desirable to provide the recipient with a dosage of
bispecific antibody that is
in the range of from about 1 mg to 200 mg as a single intravenous infusion,
although a lower
or higher dosage also may be administered as circumstances dictate. Typically,
it is desirable
to provide the recipient with a dosage that is in the range of from about 10
mg per square
meter of body surface area or 17 to 18 mg of the antibody for the typical
adult, although a
lower or higher dosage also may be administered as circumstances dictate.
Examples of
dosages of bispecific antibodies that may be administered to a human subject
are 1 to 200
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mg, more preferably 1 to 70 mg, most preferably 1 to 20 mg, although higher or
lower doses
may be used. Dosages of therapeutic bispecific antibodies may be higher, such
as 1 to 200, 1
to 100, 100 to 1000, 100 to 500, 200 to 750 mg or any range in between.
[0132] In general, the dosage of labeled molecule(s) to administer will vary
depending upon
such factors as the patient's age, weight, height, sex, general medical
condition and previous
medical history. Preferably, a saturating dose of the labeled molecules is
administered to a
patient. For administration of radiolabeled molecules, the dosage may be
measured by
millicuries.
[0133] In preferred embodiments, the labeled peptides, proteins and/or
antibodies are of use
for therapy of cancer. Examples of cancers include, but are not limited to,
carcinoma,
lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More
particular
examples of such cancers are noted below and include: squamous cell cancer
(e.g. epithelial
squamous cell cancer), lung cancer including small-cell lung cancer, non-small
cell lung
cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer
of the
peritoneum, hepatocellular cancer, gastric or stomach cancer including
gastrointestinal
cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer,
liver cancer, bladder
cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal
cancer, endometrial
cancer or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer,
prostate
cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma,
penile carcinoma,
as well as head and neck cancer. The term "cancer" includes primary malignant
cells or
tumors (e.g., those whose cells have not migrated to sites in the subject's
body other than the
site of the original malignancy or tumor) and secondary malignant cells or
tumors (e.g., those
arising from metastasis, the migration of malignant cells or tumor cells to
secondary sites that
are different from the site of the original tumor).
[0134] Other examples of cancers or malignancies include, but are not limited
to: Acute
Childhood Lymphoblastic Leukemia, Acute Lymphoblastic Leukemia, Acute
Lymphocytic
Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, Adult (Primary)
Hepatocellular Cancer, Adult (Primary) Liver Cancer, Adult Acute Lymphocytic
Leukemia,
Adult Acute Myeloid Leukemia, Adult Hodgkin's Disease, Adult Hodgkin's
Lymphoma,
Adult Lymphocytic Leukemia, Adult Non-Hodgkin's Lymphoma, Adult Primary Liver
Cancer, Adult Soft Tissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related
Malignancies,
Anal Cancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain
Stem
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Glioma, Brain Tumors, Breast Cancer, Cancer of the Renal Pelvis and Ureter,
Central
Nervous System (Primary) Lymphoma, Central Nervous System Lymphoma, Cerebellar
Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Childhood (Primary)
Hepatocellular
Cancer, Childhood (Primary) Liver Cancer, Childhood Acute Lymphoblastic
Leukemia,
Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, Childhood
Cerebellar
Astrocytoma, Childhood Cerebral Astrocytoma, Childhood Extracranial Germ Cell
Tumors,
Childhood Hodgkin's Disease, Childhood Hodgkin's Lymphoma, Childhood
Hypothalamic
and Visual Pathway Glioma, Childhood Lymphoblastic Leukemia, Childhood
Medulloblastoma, Childhood Non-Hodgkin's Lymphoma, Childhood Pineal and
Supratentorial Primitive Neuroectodermal Tumors, Childhood Primary Liver
Cancer,
Childhood Rhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood Visual
Pathway
and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, Chronic Myelogenous
Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, Endocrine Pancreas Islet
Cell
Carcinoma, Endometrial Cancer, Ependymoma, Epithelial Cancer, Esophageal
Cancer,
Ewing's Sarcoma and Related Tumors, Exocrine Pancreatic Cancer, Extracranial
Germ Cell
Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye
Cancer, Female
Breast Cancer, Gaucher's Disease, Gallbladder Cancer, Gastric Cancer,
Gastrointestinal
Carcinoid Tumor, Gastrointestinal Tumors, Germ Cell Tumors, Gestational
Trophoblastic
Tumor, Hairy Cell Leukemia, Head and Neck Cancer, Hepatocellular Cancer,
Hodgkin's
Disease, Hodgkin's Lymphoma, Hypergammaglobulinemia, Hypopharyngeal Cancer,
Intestinal Cancers, Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell
Pancreatic Cancer,
Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and Oral Cavity Cancer,
Liver
Cancer, Lung Cancer, Lymphoproliferative Disorders, Macroglobulinemia, Male
Breast
Cancer, Malignant Mesothelioma, Malignant Thymoma, Medulloblastoma, Melanoma,
Mesothelioma, Metastatic Occult Primary Squamous Neck Cancer, Metastatic
Primary
Squamous Neck Cancer, Metastatic Squamous Neck Cancer, Multiple Myeloma,
Multiple
Myeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, Myelogenous Leukemia,
Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavity and Paranasal
Sinus Cancer,
Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin's Lymphoma During Pregnancy,
Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Occult Primary Metastatic
Squamous Neck Cancer, Oropharyngeal Cancer, Osteo-/Malignant Fibrous Sarcoma,
Osteosarcoma/Malignant Fibrous Histiocytoma, Osteosarcoma/Malignant Fibrous
Histiocytoma of Bone, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor,
Ovarian Low
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Malignant Potential Tumor, Pancreatic Cancer, Paraproteinemias, Purpura,
Parathyroid
Cancer, Penile Cancer, Pheochromocytoma, Pituitary Tumor, Plasma Cell
Neoplasm/Multiple Myeloma, Primary Central Nervous System Lymphoma, Primary
Liver
Cancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvis and
Ureter Cancer,
Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoidosis Sarcomas,
Sezary
Syndrome, Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft
Tissue
Sarcoma, Squamous Neck Cancer, Stomach Cancer, Supratentorial Primitive
Neuroectodeimal and Pineal Tumors, T-Cell Lymphoma, Testicular Cancer,
Thymoma,
Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,
Transitional Renal
Pelvis and Ureter Cancer, Trophoblastic Tumors, Ureter and Renal Pelvis Cell
Cancer,
Urethral Cancer, Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Visual
Pathway and
Hypothalamic Glioma, Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms'
Tumor,
and any other hyperproliferative disease, besides neoplasia, located in an
organ system listed
above.
[0135] The methods and compositions described and claimed herein may be used
to detect or
treat malignant or premalignant conditions. Such uses are indicated in
conditions known or
suspected of preceding progression to neoplasia or cancer, in particular,
where non-neoplastic
cell growth consisting of hyperplasia, metaplasia, or most particularly,
dysplasia has occurred
(for review of such abnormal growth conditions, see Robbins and Angell, Basic
Pathology,
2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976)).
[0136] Dysplasia is frequently a forerunner of cancer, and is found mainly in
the epithelia. It
is the most disorderly form of non-neoplastic cell growth, involving a loss in
individual cell
uniformity and in the architectural orientation of cells. Dysplasia
characteristically occurs
where there exists chronic irritation or inflammation. Dysplastic disorders
which can be
detected include, but are not limited to, anhidrotic ectodermal dysplasia,
anterofacial
dysplasia, asphyxiating thoracic dysplasia, atriodigital dysplasia,
bronchopulmonary
dysplasia, cerebral dysplasia, cervical dysplasia, chondroectodermal
dysplasia, cleidocranial
dysplasia, congenital ectodennal dysplasia, craniodiaphysial dysplasia,
craniocarpotarsal
dysplasia, craniometaphysial dysplasia, dentin dysplasia, diaphysial
dysplasia, ectodennal
dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia, dysplasia
epiphysialis
hemimelia, dysplasia epiphysialis multiplex, dysplasia epiphysialis punctata,
epithelial
dysplasia, faciodigitogenital dysplasia, familial fibrous dysplasia of jaws,
familial white
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folded dysplasia, fibromuscular dysplasia, fibrous dysplasia of bone, florid
osseous dysplasia,
hereditary renal-retinal dysplasia, hidrotic ectodermal dysplasia,
hypohidrotic ectodermal
dysplasia, lymphopenic thymic dysplasia, mammary dysplasia, mandibulofacial
dysplasia,
metaphysial dysplasia, Mondini dysplasia, monostotic fibrous dysplasia,
mucoepithelial
dysplasia, multiple epiphysial dysplasia, oculoauriculovertebral dysplasia,
oculodentodigital
dysplasia, oculovertebral dysplasia, odontogenic dysplasia,
opthalmomandibulomelic
dysplasia, periapical cemental dysplasia, polyostotic fibrous dysplasia,
pseudoachondroplastic spondyloepiphysial dysplasia, retinal dysplasia, septo-
optic dysplasia,
spondyloepiphysial dysplasia, and ventriculoradial dysplasia.
[0137] Additional pre-neoplastic disorders which can be detected and/or
treated include, but
are not limited to, benign dysproliferative disorders (e.g., benign tumors,
fibrocystic
conditions, tissue hypertrophy, intestinal polyps, colon polyps, and
esophageal dysplasia),
leukoplakia, keratoses, Bowen's disease, Farmer's Skin, solar cheilitis, and
solar keratosis.
[0138] Additional hyperproliferative diseases, disorders, and/or conditions
include, but are
not limited to, progression, and/or metastases of malignancies and related
disorders such as
leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute
myelocytic
leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic,
and
erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic
(granulocytic) leukemia
and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g.,
Hodgkin's disease
and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia,
heavy
chain disease, and solid tumors including, but not limited to, sarcomas and
carcinomas such
as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma,
chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,
leiomyosarcoma,
rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian
cancer,
prostate cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland
carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary
adenocarcinomas,
cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell
carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma,
Wilm's
tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung
carcinoma, bladder
carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma,
craniopharyngioma,
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ependymoma, pinealoma, emangioblastoma, acoustic neuroma, oligodendroglioma,
menangioma, melanoma, neuroblastoma, and retinoblastoma.
[0139] The exemplary conditions listed above that may be treated are not
limiting. The
skilled artisan will be aware that antibodies, antibody fragments or targeting
peptides are
known for a wide variety of conditions, such as autoimmune disease, graft-
versus-host-
disease, organ transplant rejection, cardiovascular disease, neurodegenerative
disease,
metabolic disease, cancer, infectious disease and hyperproliferative disease.
[0140] Exemplary autoimmune diseases include acute idiopathic thrombocytopenic
purpura,
chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's
chorea,
myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic
fever,
polyglandular syndromes, bullous pemphigoid, juvenile diabetes mellitus,
Henoch-Schonlein
purpura, post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis,
Addison's
disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative
colitis, erythema
multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis,
Goodpasture's
syndrome, thromboangitis obliterans, Sjogren's syndrome, primary biliary
cirrhosis,
Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic active
hepatitis,
polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris, Wegener's
granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes
dorsalis, giant
cell arteritis/polymyalgia, pernicious anemia, rapidly progressive
glomerulonephritis,
psoriasis and fibrosing alveolitis.
Kits
[0141] Various embodiments may concern kits containing components suitable for
treating
diseased tissue in a patient. Exemplary kits may contain at least one
conjugated antibody or
other targeting moiety as described herein. If the composition containing
components for
administration is not formulated for delivery via the alimentary canal, such
as by oral
delivery, a device capable of delivering the kit components through some other
route may be
included. One type of device, for applications such as parenteral delivery, is
a syringe that is
used to inject the composition into the body of a subject. Inhalation devices
may also be used.
[0142] The kit components may be packaged together or separated into two or
more
containers. In some embodiments, the containers may be vials that contain
sterile,
lyophilized formulations of a composition that are suitable for
reconstitution. A kit may also
contain one or more buffers suitable for reconstitution and/or dilution of
other reagents. Other
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containers that may be used include, but are not limited to, a pouch, tray,
box, tube, or the
like. Kit components may be packaged and maintained sterilely within the
containers.
Another component that can be included is instructions to a person using a kit
for its use.
EXAMPLES
[0143] Various embodiments of the present invention are illustrated by the
following
examples, without limiting the scope thereof.
Example 1. Preparation of Dock-and-Lock (DNL) Constructs
DDD and AD Fusion Proteins
[0144] The DNL technique can be used to make dimers, trimers, tetramers,
hexamers, etc.
comprising virtually any antibody, antibody fragment, or other effector
moiety. For certain
preferred embodiments, the antibodies and antibody fragments may be produced
as fusion
proteins comprising either a dimerization and docking domain (DDD) or
anchoring domain
(AD) sequence. However, the skilled artisan will realize that other methods of
conjugation
exist, such as chemical cross-linking, click chemistry reaction, etc.
[0145] The technique is not limiting and any protein or peptide of use may be
produced as an
AD or DDD fusion protein for incorporation into a DNL construct. Where
chemical cross-
linking is utilized, the AD and DDD conjugates may comprise any molecule that
may be
cross-linked to an AD or DDD sequence using any cross-linking technique known
in the art.
In certain exemplary embodiments, a dendrimer or other polymeric moiety such
as
polyethylene glycol (PEG) may be incorporated into a DNL construct, as
described in further
detail below.
[0146] For different types of DNL constructs, different AD or DDD sequences
may be
utilized. Exemplary DDD and AD sequences are provided below.
DDD1: SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID
NO:33)
DDD2: CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID
NO :34)
AD1: QIEYLAKQIVDNAIQQA (SEQ ID NO:35)
AD2: CGQIEYLAKQIVDNAIQQAGC (SEQ ID NO:36)
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[0147] The skilled artisan will realize that DDD1 and DDD2 comprise the DDD
sequence of
the human Ma fonn of protein kinase A. However, in alternative embodiments,
the DDD
and AD moieties may be based on the DDD sequence of the human RIcc form of
protein
kinase A and a corresponding AKAP sequence, as exemplified in DDD3, DDD3C and
AD3
below.
DDD3
SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK (SEQ ID
NO :37)
DDD3C
MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK
(SEQ ID NO:38)
AD3
CGFEELAWKIAKMIWSDVFQQGC (SEQ ID NO:39)
Expression Vectors
[0148] The plasmid vector pdHL2 has been used to produce a number of
antibodies and
antibody-based constructs. See Gillies etal., J Immunol Methods (1989),
125:191-202;
Losman et al., Cancer (Phila) (1997), 80:2660-6. The di-cistronic mammalian
expression
vector directs the synthesis of the heavy and light chains of IgG. The vector
sequences are
mostly identical for many different IgG-pdHL2 constructs, with the only
differences existing
in the variable domain (VH and VL) sequences. Using molecular biology tools
known to
those skilled in the art, these IgG expression vectors can be converted into
Fab-DDD or Fab-
AD expression vectors. To generate Fab-DDD expression vectors, the coding
sequences for
the hinge, CH2 and CH3 domains of the heavy chain are replaced with a sequence
encoding
the first 4 residues of the hinge, a 14 residue Gly-Ser linker and the first
44 residues of human
RIIcc (referred to as DDD1). To generate Fab-AD expression vectors, the
sequences for the
hinge, CH2 and CH3 domains of IgG are replaced with a sequence encoding the
first 4
residues of the hinge, a 15 residue Gly-Ser linker and a 17 residue synthetic
AD called
AKAP-IS (referred to as AD1), which was generated using bioinformatics and
peptide array
technology and shown to bind Rlla dimers with a very high affinity (0.4 nM).
See Alto, et
al. Proc. Natl. Acad. Sci., U.S.A (2003), 100:4445-50.
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[0149] Two shuttle vectors were designed to facilitate the conversion of IgG-
pdHL2 vectors
to either Fab-DDD1 or Fab-AD1 expression vectors, as described below.
Preparation of CH1
[0150] The CH1 domain was amplified by PCR using the pdHL2 plasmid vector as a
template. The left PCR primer consisted of the upstream (5') end of the CH1
domain and a
SacII restriction endonuclease site, which is 5' of the CH1 coding sequence.
The right primer
consisted of the sequence coding for the first 4 residues of the hinge (PKSC)
followed by four
glycines and a serine, with the final two codons (GS) comprising a Bam HI
restriction site.
The 410 bp PCR amplimer was cloned into the PGEMT PCR cloning vector
(PROMEGA , Inc.) and clones were screened for inserts in the T7 (5')
orientation.
[0151] A duplex oligonucleotide was synthesized to code for the amino acid
sequence of
DDD1 preceded by 11 residues of the linker peptide, with the first two codons
comprising a
BamHI restriction site. A stop codon and an EagI restriction site are appended
to the 3'end.
The encoded polypeptide sequence is shown below.
GSGGGGSGGGGSHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA
(SEQ ID NO:40)
[0152] Two oligonucleotides, designated RIIA1-44 top and RIIA1-44 bottom,
which overlap
by 30 base pairs on their 3' ends, were synthesized and combined to comprise
the central 154
base pairs of the 174 bp DDD1 sequence. The oligonucleotides were annealed and
subjected
to a primer extension reaction with Taq polymerase. Following primer
extension, the duplex
was amplified by PCR. The amplimer was cloned into PGEMT and screened for
inserts in
the T7 (5') orientation.
[0153] A duplex oligonucleotide was synthesized to code for the amino acid
sequence of
AD1 preceded by 11 residues of the linker peptide with the first two codons
comprising a
BamHI restriction site. A stop codon and an EagI restriction site are appended
to the 3'end.
The encoded polypeptide sequence is shown below.
GSGGGGSGGGGSQIEYLAKQIVDNAIQQA (SEQ ID NO:41)
[0154] Two complimentary overlapping oligonucleotides encoding the above
peptide
sequence, designated AKAP-IS Top and AKAP-IS Bottom, were synthesized and
annealed.
The duplex was amplified by PCR. The amplimer was cloned into the PGEMT
vector and
screened for inserts in the T7 (5') orientation.
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Ligating DDDI with CH1
[0155] A 190 bp fragment encoding the DDD1 sequence was excised from PGEMT
with
BamHI and NotI restriction enzymes and then ligated into the same sites in CH1-
PGEMT
to generate the shuttle vector CH1-DDD1-PGEMT .
Ligating AD1 with CH1
[0156] A 110 bp fragment containing the AD1 sequence was excised from PGEMT
with
BamHI and NotI and then ligated into the same sites in CH1-PGEMT to generate
the
shuttle vector CH1-AD1-PGEMT .
Cloning CH1-DDD1 or CH1-AD1 into pdHL2-based vectors
[0157] With this modular design either CH1-DDD1 or CH1-AD1 can be incorporated
into
any IgG construct in the pdHL2 vector. The entire heavy chain constant domain
is replaced
with one of the above constructs by removing the SacII/EagI restriction
fragment (CH1-CH3)
from pdHL2 and replacing it with the SacII/EagI fragment of CH1-DDD1 or CH1-
AD1,
which is excised from the respective pGemT shuttle vector.
Construction of h679-Fd-AD]-pdHL2
[0158] h679-Fd-AD1-pdHL2 is an expression vector for production of h679 Fab
with AD1
coupled to the carboxyl terminal end of the CH1 domain of the Fd via a
flexible Gly/Ser
peptide spacer composed of 14 amino acid residues. A pdHL2-based vector
containing the
variable domains of h679 was converted to h679-Fd-AD1-pdHL2 by replacement of
the
SacII/EagI fragment with the CH1-AD1 fragment, which was excised from the CH1-
AD1-
SV3 shuttle vector with SacII and EagI.
Construction of C-DDD1-Fd-hMN-14-pdHL2
[0159] C-DDD1-Fd-hMN-14-pdHL2 is an expression vector for production of a
stable dimer
that comprises two copies of a fusion protein C-DDD1-Fab-hMN-14, in which DDD1
is
linked to hMN-14 Fab at the carboxyl terminus of CH1 via a flexible peptide
spacer. The
plasmid vector hMN-14(I)-pdHL2, which has been used to produce hMN-14 IgG, was
converted to C-DDD1-Fd-hMN-14-pdHL2 by digestion with SacII and EagI
restriction
endonucleases to remove the CH1-CH3 domains and insertion of the CH1-DDD1
fragment,
which was excised from the CH1-DDD1-SV3 shuttle vector with SacII and EagI.
[0160] The same technique has been utilized to produce plasmids for Fab
expression of a
wide variety of known antibodies, such as hLL1, hLL2, hPAM4, hRl, hRS7, hMN-
14, hMN-
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15, hA19, hA20 and many others. Generally, the antibody variable region coding
sequences
were present in a pdHL2 expression vector and the expression vector was
converted for
production of an AD- or DDD-fusion protein as described above. The AD- and DDD-
fusion
proteins comprising a Fab fragment of any of such antibodies may be combined,
in an
approximate ratio of two DDD-fusion proteins per one AD-fusion protein, to
generate a
trimeric DNL construct comprising two Fab fragments of a first antibody and
one Fab
fragment of a second antibody.
Construction of N-DDD1-Fd-hAIN-14-pdHL2
[0161] N-DDD1-Fd-hMN-14-pdHL2 is an expression vector for production of a
stable dimer
that comprises two copies of a fusion protein N-DDD1-Fab-hMN-14, in which DDD1
is
linked to hMN-14 Fab at the amino terminus of VH via a flexible peptide
spacer. The
expression vector was engineered as follows. The DDD1 domain was amplified by
PCR.
[0162] As a result of the PCR, an NcoI restriction site and the coding
sequence for part of the
linker containing a BamHI restriction were appended to the 5' and 3' ends,
respectively. The
170 bp PCR amplimer was cloned into the pGemT vector and clones were screened
for
inserts in the T7 (5') orientation. The 194 bp insert was excised from the
pGemT vector with
NcoI and Sall restriction enzymes and cloned into the SV3 shuttle vector,
which was
prepared by digestion with those same enzymes, to generate the intettnediate
vector DDD1-
SV3.
[0163] The hMN-14 Fd sequence was amplified by PCR. As a result of the PCR, a
BamHI
restriction site and the coding sequence for part of the linker were appended
to the 5' end of
the amplimer. A stop codon and EagI restriction site was appended to the 3'
end. The 1043
bp amplimer was cloned into pGemT. The hMN-14-Fd insert was excised from pGemT
with
BamHI and EagI restriction enzymes and then ligated with DDD1-SV3 vector,
which was
prepared by digestion with those same enzymes, to generate the construct N-
DDD1-hMN-
14Fd-SV3.
[0164] The N-DDD1-hMN-14 Fd sequence was excised with XhoI and EagI
restriction
enzymes and the 1.28 kb insert fragment was ligated with a vector fragment
that was
prepared by digestion of C-hMN-14-pdHL2 with those same enzymes. The final
expression
vector was N-DDD1-Fd-hMN-14-pDHL2. The N-linked Fab fragment exhibited similar
DNL complex fointation and antigen binding characteristics as the C-linked Fab
fragment
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(not shown).
C-DDD2-Fd-hMN-1 4-pdHL 2
[0165] C-DDD2-Fd-hMN-14-pdHL2 is an expression vector for production of C-DDD2-
Fab-
hMN-14, which possesses a dimerization and docking domain sequence of DDD2
appended
to the carboxyl terminus of the Fd of hMN-14 via a 14 amino acid residue
Gly/Ser peptide
linker. The fusion protein secreted is composed of two identical copies of hMN-
14 Fab held
together by non-covalent interaction of the DDD2 domains.
[0166] The expression vector was engineered as follows. Two overlapping,
complimentary
oligonucleotides, which comprise the coding sequence for part of the linker
peptide and
residues 1-13 of DDD2, were made synthetically. The oligonucleotides were
annealed and
phosphorylated with T4 PNK, resulting in overhangs on the 5' and 3' ends that
are compatible
for ligation with DNA digested with the restriction endonucleases BamHI and
PstI,
respectively.
[0167] The duplex DNA was ligated with the shuttle vector CH1-DDD1-PGEMT ,
which
was prepared by digestion with BamHI and PstI, to generate the shuttle vector
CH1-DDD2-
PGEMT . A 507 bp fragment was excised from CH1-DDD2-PGEMT with SacII and EagI
and ligated with the IgG expression vector hMN-14(I)-pdHL2, which was prepared
by
digestion with SacII and EagI. The final expression construct was designated C-
DDD2-Fd-
hMN-14-pdHL2. Similar techniques have been utilized to generated DDD2-fusion
proteins
of the Fab fragments of a number of different humanized antibodies.
h679-Fd-AD2-pdHL2
[0168] h679-Fab-AD2, was designed to pair as B to C-DDD2-Fab-hMN-14 as A. h679-
Fd-
AD2-pdHL2 is an expression vector for the production of h679-Fab-AD2, which
possesses
an anchoring domain sequence of AD2 appended to the carboxyl terminal end of
the CH1
domain via a 14 amino acid residue Gly/Ser peptide linker. AD2 has one
cysteine residue
preceding and another one following the anchor domain sequence of AD1.
[0169] The expression vector was engineered as follows. Two overlapping,
complimentary
oligonucleotides (AD2 Top and AD2 Bottom), which comprise the coding sequence
for AD2
and part of the linker sequence, were made synthetically. The oligonucleotides
were
annealed and phosphorylated with T4 PNK, resulting in overhangs on the 5' and
3' ends that
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are compatible for ligation with DNA digested with the restriction
endonucleases BamHI and
SpeI, respectively.
[0170] The duplex DNA was ligated into the shuttle vector Cl1-AD1-PGEMT ,
which was
prepared by digestion with BamHI and SpeI, to generate the shuttle vector CH1-
AD2-
PGEMT . A 429 base pair fragment containing CH1 and AD2 coding sequences was
excised from the shuttle vector with Sacll and EagI restriction enzymes and
ligated into
h679-pdHL2 vector that prepared by digestion with those same enzymes. The
final
expression vector is h679-Fd-AD2-pdHL2.
Example 2. Generation of TF1 DNL Construct
[0171] A large scale preparation of a DNL construct, referred to as TF1, was
carried out as
follows. N-DDD2-Fab-hMN-14 (Protein L-purified) and h679-Fab-AD2 (IMP-291-
purified)
were first mixed in roughly stoichiometric concentrations in 1mM EDTA, PBS, pH
7.4.
Before the addition of TCEP, SE-HPLC did not show any evidence of a2b
formation (not
shown). Instead there were peaks representing a4 (7.97 min; 200 kDa), a2 (8.91
mm; 100
kDa) and B (10.01 min; 50 kDa). Addition of 5 mM TCEP rapidly resulted in the
formation
of the a2b complex as demonstrated by a new peak at 8.43 min, consistent with
a 150 kDa
protein (not shown). Apparently there was excess B in this experiment as a
peak attributed to
h679-Fab-AD2 (9.72 min) was still evident yet no apparent peak corresponding
to either a2 or
a4 was observed. After reduction for one hour, the TCEP was removed by
overnight dialysis
against several changes of PBS. The resulting solution was brought to 10% DMSO
and held
overnight at room temperature.
[0172] When analyzed by SE-HPLC, the peak representing a2b appeared to be
sharper with a
slight reduction of the retention time by 0.1 min to 8.31 min (not shown),
which, based on
our previous findings, indicates an increase in binding affinity. The complex
was further
purified by IMP-291 affinity chromatography to remove the kappa chain
contaminants. As
expected, the excess h679-AD2 was co-purified and later removed by preparative
SE-HPLC
(not shown).
[0173] TF1 is a highly stable complex. When TF1 was tested for binding to an
HSG (IMP-
239) sensorchip, there was no apparent decrease of the observed response at
the end of
sample injection. In contrast, when a solution containing an equimolar mixture
of both C-
DDD1-Fab-hMN-14 and h679-Fab-AD1 was tested under similar conditions, the
observed
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increase in response units was accompanied by a detectable drop during and
immediately
after sample injection, indicating that the initially formed a2b structure was
unstable.
Moreover, whereas subsequent injection of WI2 gave a substantial increase in
response units
for TF1, no increase was evident for the C-DDD1/AD1 mixture.
[0174] The additional increase of response units resulting from the binding of
WI2 to TF1
immobilized on the sensorchip corresponds to two fully functional binding
sites, each
contributed by one subunit of N-DDD2-Fab-hMN-14. This was confiimed by the
ability of
TF1 to bind two Fab fragments of WI2 (not shown). When a mixture containing
h679-AD2
and N-DDD1-hMN14, which had been reduced and oxidized exactly as TF1, was
analyzed
by BIAcore, there was little additional binding of WI2 (not shown), indicating
that a
disulfide-stabilized a2b complex such as TF1 could only form through the
interaction of
DDD2 and AD2.
[0175] Two improvements to the process were implemented to reduce the time and
efficiency
of the process. First, a slight molar excess of N-DDD2-Fab-hMN-14 present as a
mixture of
a4/a2 structures was used to react with h679-Fab-AD2 so that no free h679-Fab-
AD2
remained and any a4/a2 structures not tethered to h679-Fab-AD2, as well as
light chains,
would be removed by IMP-291 affinity chromatography. Second, hydrophobic
interaction
chromatography (HIC) has replaced dialysis or diafiltration as a means to
remove TCEP
following reduction, which would not only shorten the process time but also
add a potential
viral removing step. N-DDD2-Fab-hMN-14 and 679-Fab-AD2 were mixed and reduced
with
mM TCEP for 1 hour at room temperature. The solution was brought to 0.75 M
ammonium sulfate and then loaded onto a Butyl FF HIC column. The column was
washed
with 0.75 M ammonium sulfate, 5 mM EDTA, PBS to remove TCEP. The reduced
proteins
were eluted from the HIC column with PBS and brought to 10% DMSO. Following
incubation at room temperature overnight, highly purified TF1 was isolated by
IMP-291
affinity chromatography (not shown). No additional purification steps, such as
gel filtration,
were required.
Example 3. Generation of TF2 DNL Construct
[0176] A trimeric DNL construct designated TF2 was obtained by reacting C-DDD2-
Fab-
hMN-14 with h679-Fab-AD2. A pilot batch of TF2 was generated with >90% yield
as
follows. Protein L-purified C-DDD2-Fab-hMN-14 (200 mg) was mixed with h679-Fab-
AD2
(60 mg) at a 1.4:1 molar ratio. The total protein concentration was 1.5 mg/ml
in PBS
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containing 1 mM EDTA. Subsequent steps involved TCEP reduction, HIC
chromatography,
DMSO oxidation, and IMP 291 affinity chromatography. Before the addition of
TCEP, SE-
HPLC did not show any evidence of a2b formation. Addition of 5 mM TCEP rapidly
resulted in the formation of a2b complex consistent with a 157 kDa protein
expected for the
binary structure. TF2 was purified to near homogeneity by IMP 291 affinity
chromatography
(not shown). IMP 291 is a synthetic peptide containing the HSG hapten to which
the 679 Fab
binds (Rossi et al., 2005, Clin Cancer Res 11:7122s-29s). SE-HPLC analysis of
the IMP 291
unbound fraction demonstrated the removal of a4, a2 and free kappa chains from
the product
(not shown).
[0177] The functionality of TF2 was determined by BIACOREO assay. TF2, C-DDD1-
hMN-14+h679-AD1 (used as a control sample of noncovalent a2b complex), or C-
DDD2-
hMN-14+h679-AD2 (used as a control sample of unreduced a2 and b components)
were
diluted to 1 ug/m1 (total protein) and passed over a sensorchip immobilized
with HSG. The
response for TF2 was approximately two-fold that of the two control samples,
indicating that
only the h679-Fab-AD component in the control samples would bind to and remain
on the
sensorchip. Subsequent injections of WI2 IgG, an anti-idiotype antibody for
hMN-14,
demonstrated that only TF2 had a DDD-Fab-hMN-14 component that was tightly
associated
with h679-Fab-AD as indicated by an additional signal response. The additional
increase of
response units resulting from the binding of WI2 to TF2 immobilized on the
sensorchip
corresponded to two fully functional binding sites, each contributed by one
subunit of C-
DDD2-Fab-hMN-14. This was confirmed by the ability of TF2 to bind two Fab
fragments of
WI2 (not shown).
Example 4. Production of TF10 Bispecific Antibody
[0178] A similar protocol was used to generate a trimeric TF10 DNL construct,
comprising
two copies of a C-DDD2-Fab-hPAM4 and one copy of C-AD2-Fab-679. The cancer-
targeting antibody component in TF10 was derived from hPAM4, a humanized anti-
pancreatic cancer mucin MAb that has been studied in detail as a radiolabeled
MAb (e.g.,
Gold et al., Clin. Cancer Res. 13: 7380-7387, 2007). The hapten-binding
component was
derived from h679, a humanized anti-histaminyl-succinyl-glycine (HSG) MAb. The
TF10
bispecific ([hPAM4]2 x h679) antibody was produced using the method disclosed
for
production of the (anti CEA)2 x anti HSG bsAb TF2, as described above. The
TF10 construct
bears two humanized PAM4 Fabs and one humanized 679 Fab.
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[0179] The two fusion proteins (hPAM4-DDD and h679-AD2) were expressed
independently in stably transfected myeloma cells. The tissue culture
supernatant fluids were
combined, resulting in a two-fold molar excess of hPAM4-DDD. The reaction
mixture was
incubated at room temperature for 24 hours under mild reducing conditions
using 1 mM
reduced glutathione. Following reduction, the DNL reaction was completed by
mild
oxidation using 2 mM oxidized glutathione. TF10 was isolated by affinity
chromatography
using IMP 291-affigel resin, which binds with high specificity to the h679
Fab.
[0180] The skilled artisan will realize that the DNL techniques disclosed
above may be used
to produce complexes comprising any combination of antibodies,
immunoconjugates, or
other effector moieties that may be attached to an AD or DDD moiety.
Example 5. Production of AD- and DDD-linked Fab and IgG Fusion Proteins From
Multiple Antibodies
[0181] Using the techniques described in the preceding Examples, the IgG and
Fab fusion
proteins shown in Table 2 were constructed and incorporated into DNL
constructs. The
fusion proteins retained the antigen-binding characteristics of the parent
antibodies and the
DNL constructs exhibited the antigen-binding activities of the incorporated
antibodies or
antibody fragments.
Example 6. Sequence variants for DNL
[0182] In certain preferred embodiments, the AD and DDD sequences incorporated
into the
DNL construct comprise the amino acid sequences of AD1, AD2, AD3, DDD1, DDD2,
DDD3 or DDD3C as discussed above. However, in alternative embodiments sequence
variants of AD and/or DDD moieties may be utilized in construction of the DNL
complexes.
For example, there are only four variants of human PKA DDD sequences,
corresponding to
the DDD moieties of PKA Ma, RIIa, RII3 and R1113. The RIIa DDD sequence is the
basis of
DDD1 and DDD2 disclosed above. The four human PKA DDD sequences are shown
below.
The DDD sequence represents residues 1-44 of RIIa, 1-44 of RIII3, 12-61 of RIa
and 13-66 of
R113. (Note that the sequence of DDD1 is modified slightly from the human PKA
Rila DDD
moiety.)
PKA Rig
SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEAK (SEQ ID
NO:42)
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Table 2. Fusion proteins comprising IgG or Fab
Fusion Protein Binding Specificity
C-AD1-Fab-h679 HSG
C-AD2-Fab-h679 HSG
C-(AD)2-Fab-h679 HSG
C-AD2-Fab-h734 Indium-DTPA
C-AD2-Fab-hA20 CD20
C-AD2-Fab-hA2OL CD20
C-AD2-Fab-hL243 HLA-DR
C-AD2-Fab-hLL2 CD22
N-AD2-Fab-hLL2 CD22
C-AD2-IgG-hMN-14 CEACAM5
C-AD2-IgG-hR1 IGF-1R
C-AD2-IgG-hRS7 EGP-1
C-AD2-IgG-hPAM4 MUC
C-A02-IgG-hLL1 CD74
C-DDD1-Fab-hMN-14 CEACAM5
C-DDD2-Fab-hMN-14 CEACAM5
C-DDD2-Fab-h679 HSG
C-DDD2-Fab-hAl9 CD19
C-DDD2-Fab-hA20 CO20
C-DDD2-Fab-hAFP AFP
C-DDD2-Fab-hL243 HLA-DR
C-DDD2-Fab-hLL1 CD74
C-DDD2-Fab-hLL2 CD22
C-DDD2-Fab-hMN-3 CEACAM6
C-DDD2-Fab-hMN-15 CEACAM6
C-DDD2-Fab-hPAM4 MUC
C-DDD2-Fab-hR1 IGF-1R
C-DDD2-Fab-hRS7 EGP-1
N-DDD2-Fab-hMN-14 CEACAM5
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PKA Kip
SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEENRQILA (SEQ
ID NO:43)
PKA Rik
SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQ (SEQ ID NO:44)
PKA
SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER (SEQ ID NO:45)
[0183] The structure-function relationships of the AD and DDD domains have
been the
subject of investigation. (See, e.g., Bums-Hamuro et al., 2005, Protein Sci
14:2982-92; Carr
et al., 2001, J Biol Chem 276:17332-38; Alto et al., 2003, Proc Natl Acad Sci
USA 100:4445-
50; Hundsrucker et al., 2006, Biochem J 396:297-306; Stokka et al., 2006,
Biochem J
400:493-99; Gold et al., 2006, Mol Cell 24:383-95; Kinderman et al., 2006, Mol
Cell 24:397-
408).
[0184] For example, Kinderman et al. (2006) examined the crystal structure of
the AD-DDD
binding interaction and concluded that the human DDD sequence contained a
number of
conserved amino acid residues that were important in either dimer formation or
AKAP
binding, underlined in SEQ ID NO:33 below. (See Figure 1 of Kinderman et al.,
2006).
The skilled artisan will realize that in designing sequence
variants of the DDD sequence, one would desirably avoid changing any of the
underlined
residues, while conservative amino acid substitutions might be made for
residues that are less
critical for dimerization and AKAP binding.
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID
NO:33)
[0185] Alto et al. (2003) performed a bioinformatic analysis of the AD
sequence of various
AKAP proteins to design an RII selective AD sequence called AKAP-IS (SEQ ID
NO:35),
with a binding constant for DDD of 0.4 nM. The AKAP-IS sequence was designed
as a
peptide antagonist of AKAP binding to PKA. Residues in the AKAP-IS sequence
where
substitutions tended to decrease binding to DDD are underlined in SEQ ID
NO:35. The
skilled artisan will realize that in designing sequence variants of the AD
sequence, one would
desirably avoid changing any of the underlined residues, while conservative
amino acid
substitutions might be made for residues that are less critical for DDD
binding.
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AKAP-IS SEQUENCE
QIEYLAKQIVDNAIQQA (SEQ ID NO:35)
[0186] Gold (2006) utilized crystallography and peptide screening to develop a
SuperAKAP-
IS sequence (SEQ ID NO:46), exhibiting a five order of magnitude higher
selectivity for the
RII isofolin of PKA compared with the RI isoform. Underlined residues indicate
the
positions of amino acid substitutions, relative to the AKAP-IS sequence, which
increased
binding to the DDD moiety of Rlla. In this sequence, the N-terminal Q residue
is numbered
as residue number 4 and the C-terminal A residue is residue number 20.
Residues where
substitutions could be made to affect the affinity for RIIa were residues 8,
11, 15, 16, 18, 19
and 20 (Gold et al., 2006). It is contemplated that in certain alternative
embodiments, the
SuperAKAP-IS sequence may be substituted for the AKAP-IS AD moiety sequence to
prepare DNL constructs. Other alternative sequences that might be substituted
for the
AKAP-IS AD sequence are shown in SEQ ID NO:47-49. Substitutions relative to
the
AKAP-IS sequence are underlined. It is anticipated that, as with the AD2
sequence shown in
SEQ ID NO:46, the AD moiety may also include the additional N-terminal
residues cysteine
and glycine and C-teiminal residues glycine and cysteine.
SuperAKAP-IS
QIEYVAKQIVDYAIHQA (SEQ ID NO:46)
Alternative AKAP sequences
QIEYKAKQIVDHAIHQA (SEQ ID NO:47)
QIEYHAKQIVDHAIHQA (SEQ ID NO:48)
QIEYVAKQIVDHAIHQA (SEQ ID NO:49)
[0187] Figure 2 of Gold et al. disclosed additional DDD-binding sequences from
a variety of
AKAP proteins, shown below.
RII-Specific AKAPs
AKAP-KL
PLEYQAGLLVQNAIQQAI (SEQ ID NO:50)
AKAP79
LLIETASSLVKNAIQLSI (SEQ ID NO:51)
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AKAP-Lbc
LIEEAASRIVDAVIEQVK (SEQ ID NO:52)
RI-Specific AKAPs
AKAPce
ALYQFADRFSELVISEAL (SEQ ID NO:53)
RIAD
LEQVANQLADQIIKEAT (SEQ ID NO:54)
PV38
FEELAWKIAKMIWSDVF (SEQ ID NO:55)
Dual-Specificity AKAPs
AKAP7
ELVRLSKRLVENAVLKAV (SEQ ID NO:56)
MAP2D
TAEEVSARIVQVVTAEAV (SEQ ID NO:57)
DAKAP1
QIKQAAFQLISQVILEAT (SEQ ID NO:58)
DAKAP2
LAWKIAKMIVSDVMQQ (SEQ ID NO:59)
[0188] Stokka et al. (2006) also developed peptide competitors of AKAP binding
to PKA,
shown in SEQ ID NO:60-62. The peptide antagonists were designated as Ht31 (SEQ
ID
NO:60), RIAD (SEQ ID NO:61) and PV-38 (SEQ ID NO:62). The Ht-31 peptide
exhibited a
greater affinity for the RII isofolin of PKA, while the RIAD and PV-38 showed
higher
affinity for RI.
Ht3 I
DLIEEAASRIVDAVIEQVKAAGAY (SEQ ID NO:60)
RIAD
LEQYANQLADQIIKEATE (SEQ ID NO:61)
PV-38
FEELAWKIAKMIWSDVFQQC (SEQ ID NO:62)
[0189] Hundsrucker et al. (2006) developed still other peptide competitors for
AKAP binding
to PKA, with a binding constant as low as 0.4 nM to the DDD of the RII form of
PKA. The
sequences of various AKAP antagonistic peptides are provided in Table 1 of
Hundsrucker et
al., reproduced in Table 3 below. AKAPIS represents a synthetic Rh I subunit-
binding
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,52392-94
peptide. All other peptides are derived from the RI-binding domains of the
indicated
AICAPs.
Table 3. AKAP Peptide sequences
Peptide Sequence
AKAPIS QIEYLAKQIVDNAIQQA (SEQ ID NO:35)
AKAPIS-P QIEYLAKQIPDNAIQQA (SEQ ID NO:63)
Ht31 KGADLIEEAASRIVDAVIEQVKAAG (SEQ ID NO:64)
Ht31-P KGADLIEEAASRIPDAPIEQVKAAG (SEQ ID NO:65)
AKAP7(5-wt-pep PEDAELVRLSKRLVENAVLKAVQQY (SEQ ID NO:66)
AKAP7o-L304T-pep PEDAELVRTSICRLVENAVLICAVQQY (SEQ ID NO:67)
AKAP7d-L308D-pep PEDAELVRLSKRDVENAVLKAVQQY (SEQ ID NO:68)
AKAP7o-P-pep PEDAELVRLSKRLPENAVLKAVQQY (SEQ ID NO:69)
AICAP7o-PP-pep PEDAELVRLSKRLPENAPLKAVQQY (SEQ ID NO:70)
AKAP7o-L314E-pep PEDAELVRLSKRLVENAVEKAVQQY (SEQ ID NO:71)
AKAP1-pep EEGLDRNEEIKRAAFQIISQVISEA (SEQ ID NO:72)
AKAP2-pep LVDDPLEYQAGLLVQNAIQQAIAEQ (SEQ ID NO:73)
AKAP5-pep QYETLLIETASSLVKNAIQLSIEQL (SEQ ID NO:74)
AKAP9-pep LEKQYQEQLEEEVAKVIVSMSIAFA (SEQ ID NO:75)
AKAP10-pep NTDEAQEELAWKIAKMIVSDIMQQA (SEQ ID NO:76)
AICAP11-pep VNLDKKAVLAEKIVAEAIEKAEREL (SEQ ID NO:77)
AKAP12-pep NGILELETKSSKLVQNIIQTAVDQF (SEQ ID NO:78)
AKAP14-pep TQDKNYEDELTQVALALVEDVINYA (SEQ ID NO:79)
Rab32-pep ETSAKDNINIEEAARFLVEKILVNH (SEQ ID NO:80)
101901 Residues that were highly conserved among the AD domains of different
AKAP
proteins are indicated below by underlining with reference to the AKAP IS
sequence (SEQ
ID NO:35). The residues are the same as observed by Alto et al. (2003), with
the addition of
the C-terminal alanine residue. (See FIG. 4 of Hundsrucker et al. (2006)).
The sequences of peptide antagonists with particularly high affinities for the
RII DDD sequence were those of AKAP-IS, AKAP78-wt-pep, AKAP745-L304T-pep and
AKAP78-L308D-pep.
AKAP-IS
QIEYLAKQIVDNAIQQA (SEQ ID NO:35)
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[0191] Can et al. (2001) examined the degree of sequence homology between
different
AKAP-binding DDD sequences from human and non-human proteins and identified
residues
in the DDD sequences that appeared to be the most highly conserved among
different DDD
moieties. These are indicated below by underlining with reference to the human
PKA RIIa
DDD sequence of SEQ ID NO:33. Residues that were particularly conserved are
further
indicated by italics. The residues overlap with, but are not identical to
those suggested by
Kinderman et al. (2006) to be important for binding to AKAP proteins. The
skilled artisan
will realize that in designing sequence variants of DDD, it would be most
preferred to avoid
changing the most conserved residues (italicized), and it would be preferred
to also avoid
changing the conserved residues (underlined), while conservative amino acid
substitutions
may be considered for residues that are neither underlined nor italicized..
SHIQIPPGLTELLOGY TV EVLRQQPPDLVEFAVEYF1RLREARA (SEQ ID NO:33)
[0192] The skilled artisan will realize that these and other amino acid
substitutions in the
antibody moiety or linker portions of the DNL constructs may be utilized to
enhance the
therapeutic and/or phaimacokinetic properties of the resulting DNL constructs.
Example 7. Antibody-Dendrimer DNL Complex
[0193] We synthesized and characterized a novel immunoconjugate, designated El-
G5/2,
which was made by the DNL method to comprise half of a generation 5 (G5) PAMAM
dendrimer (G5/2) site-specifically linked to a stabilized dimer of Fab derived
from hRS7, a
humanized antibody that is rapidly internalized upon binding to the Trop-2
antigen expressed
on various solid cancers.
Methods
[0194] E1-G5/2 was prepared by combining two self-assembling modules, AD2-G5/2
and
hRS7-Fab-DDD2, under mild redox conditions, followed by purification on a
Protein L
column. To make AD2-G5/2, we derivatized the AD2 peptide with a maleimide
group to
react with the single thiol generated from reducing a G5 PAMAM with a
cystamine core and
used reversed-phase HPLC to isolate AD2-G5/2. We produced hRS7-Fab-DDD2 as a
fusion
protein in myeloma cells, as described in the Examples above.
[0195] The molecular size, purity and composition of El-G5/2 were analyzed by
size-
exclusion HPLC, SDS-PAGE, and Western blotting. The biological functions of El
-G5/2
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were assessed by binding to an anti-idiotype antibody against hRS7, a gel
retardation assay,
and a DNase protection assay.
Results
[0196] El-G5/2 was shown by size-exclusion HPLC to consist of a major peak
(>90%)
flanked by several minor peaks. The three constituents of E1-G5/2 (Fd-DDD2,
the light
chain, and AD2-G5/2) were detected by reducing SDS-PAGE and confirmed by
Western
blotting. Anti-idiotype binding analysis revealed El-G5/2 contained a
population of
antibody-dendrimer conjugates of different size, all of which were capable of
recognizing the
anti-idiotype antibody, thus suggesting structural variability in the size of
the purchased G5
dendrimer.
Conclusion
[0197] The DNL technique can be used to build dendrimer-based nanoparticles
that are
targetable with antibodies. Such agents have improved properties as carriers
of drugs,
plasmids or siRNAs for applications in vitro and in vivo.
Example 8. Maleimide AD2 Conjugate for DNL Dendrimers
IMP 498 (SEQ ID NO:83)
0
H
0 -C(SS-tbu)GQIEYLAKQIVDNAIQQAGC(SS-tbu)NH2
H
0
[0198] The peptide IMP 498 up to and including the PEG moiety was synthesized
on a
Protein Technologies PS3 peptide synthesizer by the Fmoc method on Sieber
Amide resin
(0.1 mmol scale). The maleimide was added manually by mixing the f3-
maleimidopropionic
acid NHS ester with diisopropylethylamine and DMF with the resin for 4 hr. The
peptide was
cleaved from the resin with 15 mL TFA, 0.5 mL H20, 0.5 mL triisopropylsilane,
and 0.5 mL
thioanisole for 3 hr at room temperature. The peptide was purified by reverse
phase HPLC
using H20/CH3CN TFA buffers to obtain about 90 mg of purified product after
lyophilization.
Synthesis of Reduced G5 Dendrimer (G5/2)
[0199] The G-5 dendrimer (10% in Me0H, Dendritic Nanotechnologies), 2.03 g,
7.03 x 10-6
mol was reduced with 0.1426 TCEP.HC11:1 Me0H/H20 (¨ 4 mL) and stirred
overnight at
room temperature. The reaction mixture was purified by reverse phase HPLC on a
C-18
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column eluted with 0.1 % TFA H20/CH3CN buffers to obtain 0.0633 g of the
desired product
after lyophilization.
Synthesis of G5/2 Dendrimer-AD2 Conjugate
[0200] The G5/2 Dendrimer, 0.0469 g (3.35 x 10-6 mol) was mixed with 0.0124 g
of IMP 498
(4.4 x 10-6 mol) and dissolved in 1:1 Me0H/1M NaHCO3 and mixed for 19 hr at
room
temperature followed by treatment with 0.0751 g dithiothreitol and 0.0441 g
TCEP HCI. The
solution was mixed overnight at room temperature and purified on a C4 reverse
phase HPLC
column using 0.1 % TFA H20/CH3CN buffers to obtain 0.0033 g of material
containing the
conjugated AD2 and dendrimer as judged by gel electrophoresis and Western
blot.
Example 9. Delivery System for Cytotoxic Drugs via Bispecific Antibody
Pretargeting
[0201] As discussed above, pretargeting methods have been used with bispecific
antibodies
and targetable constructs for improved targeted delivery of therapeutic agents
with decreased
systemic toxicity. In pretargeting, the bispecific antibody (bsMAb) is
administered first to
the subject and allowed to localize to a targeted cell or tissue. Optionally,
a clearing agent
may be administered to expedite clearance of the bsMAb from circulation. After
the bsMAb
has cleared from circulation, a targetable construct is administered that
binds to the bsMAb
localized in the target tissue. The targetable construct is conjugated to one
or more
therapeutic and/or diagnostic agents. Because the targetable construct clears
very rapidly
from circulation and is typically excreted intact, primarily in the urine, the
cytotoxic
therapeutic agent spends little time in circulation and is not taken up by non-
targeted tissues,
thus reducing systemic toxicity.
[0202] The object of the present Example was to develop novel reagents for use
in
therapeutic pretargeting. These were tested in an animal model for human
colorectal cancer,
using an anti-carcinoembryonic antigen (CEACAM5) bispecific antibody. An
exemplary
cytotoxic drug used in the pretargeting study was SN-38.
[0203] A core peptide targetable construct, described in detail below (IMP
457), was
developed. The targetable construct was modified to attach SN-38 and can
attach up to 4 SN-
38 moieties per core peptide. A dendron polymer was also prepared that can
bind 8 to 16 SN-
38 moieties per polymer molecule. The targetable construct has the ability to
bind both
therapeutic radionuclides and chemotherapeutic agents for combination therapy
of diseased
tissues, such as cancer.
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[02041 An exemplary bispecific antibody used was the TF2 DNL construct,
described in the
Examples above. TF2 contains two CEACAM5-binding hMN-14 Fab moieties and one
HSG-binding h679 Fab moiety. The targetable construct contained two HSG
haptens per
peptide to allow cross-linking of two TF2 bsMAbs at the tumor surface. Cross-
linking of the
two bispecific antibodies enhances the retention of pretargeted peptide on the
tumor surface
(Barbet et al., 1999, Cancer Biother Radiopharm 14:153-66).
[0205] Preferably, the peptide-drug conjugates are designed to allow for the
slow release of
the drug, for example with a drug linkage that is stable for up to 1 day, but
then released in a
time-dependent manner. This matches the kinetics of pretargeting, where the
peptide reaches
maximum accumulation in the tumor within 1 h, and over the next few hours over
90% is
cleared from the bloodstream by urinary excretion. Unlike direct drug-antibody
conjugates
that are retained in the body for sustained periods, allowing catabolism in
the liver and other
organs, in pretargeting most of the injected product is excreted intact to
minimize systemic
side effects. But the drug-peptide conjugate localized in the tumor is slowly
released within
the tumor.
Synthesis of Targetable Construct Peptides
[0206] Peptides were synthesized by solid phase peptide synthesis using a
combination of
Aloc and Fmoc protecting groups to allow selective modification of peptide
side chains and
elongation of the peptide during peptide synthesis. IMP 402 was initially
synthesized and
used to make IMP 453, according to FIG. I. IMP 402 is also suitable for
conjugation to a
dendron drug carrier.
[0207] IMP 402 was synthesized on Sieber amide resin as follows. Aloc-D-
Lys(Fmoc)-OH
was attached to the resin. The lysine side chain Fmoc was removed and the N-
Trityl-
histaminyl-succinyl-glycyl group (Trityl-HSG-OH) was attached. The Aloc group
was
removed from the lysine and the Fmoc-D-Tyr(But)-OH was added to the peptide.
Another
Aloc-D-Lys(Fmoc)-OH was added to the peptide and the Trityl-HSG-OH group was
added to
that lysine side chain. The Aloc group was removed from the lysine and Fmoc-D-
Ala-OH,
Fmoc-D-Cys(Trt)-OH and Tri-t-butyl-DOTA-OH were added to the peptide using
standard
peptide coupling methods. The peptide was cleaved from the resin and purified
by HPLC.
Synthesis of Peptide Drug Conjugates
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[0208] The synthesis of the SN-38 precursor needed for peptide coupling is
shown in FIG. 2.
The 10 position of SN-38 was first protected with a Boc group and the 20
position was then
modified with p-nitrophenyl chloroformate to produce the 10-Boc-20-p-
nitrophenylcarbonate
SN-38 precursor. The activated SN-38 was then mixed with the peptide to
produce the Boc-
SN-38 protected conjugate, which was purified by HPLC. The Boc group was then
removed
under mild conditions to produce the desired product in 20% overall yield for
the whole
conjugation process. The resulting SN-38-conjugated peptide IMP 453 contains
one DOTA,
one SN-38 and two HSG moieties.
[0209] An initial study with "In-labeled IMP 453 showed excellent tumor
targeting to the
LS174 human colon cancer cell line (28% ID/g) (Table 4). Most of the peptide
was cleared
by urinary excretion (Table 4). Renal uptake at 3 hr was elevated (21% ID/g ),
higher than
was observed with bis-DTPA peptides (not shown), but 50% of the initial kidney
uptake was
eliminated by 24 hr. When the peptide was injected in mice that did not
receive bispecific
antibody, kidney uptake was only 9.97 %ID/g (Table 5). The higher uptake in
the kidneys of
pretargeted mice is probably due the presence of bispecific antibody in the
blood or kidney.
Modification of the peptide to contain a DTPA instead of DOTA chelating moiety
may
reduce kidney uptake, to the same range as seen with bis-DTPA peptides like
IMP 225 and
IMP 274. In the absence of TF2, there was little uptake of labeled peptide
into the tumor
(Table 5).
Table 4. "In IMP 453 biodistribution in scLS174T tumor-bearing nude mice
pretargeted
with TF2. Tissue uptake shown as %1D/g.
Tissue 3 Hr 24 Hr 48 Hr
Tumor 28.32 4.03 15.44 1.18 9.69 1.97
Liver 0.56 0.08 0.53 0.19 0.36 0.06
Spleen 0.37 0.11 0.66 0.89 0.25 0.07
Kidney 21.10 4.14 10.00 2.45 7.11 1.17
Lung 0.56 0.10 0.18 0.07 0.14 0.03
Blood 0.29 0.03 0.09 0.04 0.04 0.01
Stomach 0.41 0.33 0.20 0.12 0.07 0.01
Sm. Int. 0.68 0.45 0.22 0.09 0.12 0.03
Lg. Int. 1.23 1.41 0.23 0.05 0.16 0.07
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Table 5. "'In IMP 453 biodistribution in scLS174T tumor-bearing nude mice
without
bsMAb.
Tissue 3 Hr
Tumor 0.37 0.09
Liver 0.37 0.18
Spleen 0.22 0.07
Kidney 9.97 0.94
Lung 0.31 0.14
Blood 0.24 0.01
Stomach 0.11 0.06
Sm. Int. 0.20 0.10
Lg. Int. 0.52 0.27
DTPA Conjugated Peptide
[0210] An analog of IMP 453 is synthesized as described above, with the DOTA
group
replaced by a DTPA group. The peptide is labeled with 111In and the tumor
targeting and
clearance of the peptide is examined in LS174T tumor-bearing nude mice. The
peptide
shows targeting in vivo that is similar to the DOTA labeled peptide, but with
lower renal
uptake at 3 hours. The peptide toxicity is formulated in an acetate buffer
between pH 5-6
with an excipient added and lyophilized for therapeutic use.
Dendron Conjugation
[0211] The advantage of a dendron carrier molecule is that it is asymmetrical,
with surface
groups and a focal functional group for differential substitutions. Attachment
of the bis-HSG
peptide at the defined focal site results in site-specific placement. A PAMAM
dendron is
exemplified in FIG. 3, although other dendrons may be used with up to sixteen
surface
groups. Briefly, this involves multiple derivatizations with acetylene groups
for introducing
multiple molecules of SN-38 via azide-yne click cycloaddition, as discussed
above.
[0212] The focal functional group is transformed by `BOC' deprotection and
derivatization
to a maleimide, which is conjugated to a cysteine-containing-bis-HSG peptide
for
pretargeting. The same peptide also contains a DOTA molecule that will enable
labeling with
In-111 radiolabel for determining in vivo targeting. Dendron with either amino
group or some
other group on the surface is purchased if found to be cost effective.
Alternatively, the
dendron specified is made in-house by an iterative sequence of methacrylate
reaction and
ethylene diamine-based esterolysis, starting with mono-protected 1,6-
diaminohexane. The
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. 52392-94
BOC-protected amino group serves as the focal functional group that will
ultimately carry the
bis-HSG peptide site-selectively.
Azido-SN-38 preparation
[0213] For click chemistry reactions, such as the click chemistry addition of
SN-38 to a
targetable construct, an azido-SN-38 moiety may be prepared to react with a
cyclooctyne or
alkyne moiety on the targetable construct. An exemplary preparation is shown
in FIG. 4.
SN-38 silyl ether (intermediate 1) has been prepared in a number of small
scale reactions as
well as in one large scale reaction, using 3.43 g SN-38 with reproducibly >74%
yield. The
carbonate (intermediate 3) was prepared five times, using cross-linker as a
limiting reagent in
quantities in the range of 0.24-2.0 g, to obtain the purified carbonate in
0.33-2.63 g
(77-90%). At this stage, deblocking of silyl group was effected and the
material was purified
by a simple aqueous work-up that ensured the removal of the fluoride reagent.
The azido-SN-
38, which is intermediate 4 in FIG. 4, is used for click cycloaddition to
acetylene groups on
the dendrimer.
[0214] The click cycloaddition has been simplified from that published (Moon
et al., 2008,
Chemotherapy. Med. Chem. 51: 6916-6926) by resorting to a homogeneous reaction
in
dichloromethane using triphenylphosphine and cuprous bromide in 0.1 to 0.2
equivalents,
with attendant improvements in the quality and the yield of the product. With
the old method,
the yield was 58-82%, while with the new method, it was 86%. We believe this
new process
is amenable to easy scale-up in view of the homogeneous reaction condition.
The final
reaction in the synthetic sequence is the removal of `MMT' group using a mild
acid, such as
dichloroacetic acid, which proceeds in a high yield. The click cycloaddition
will also be
examined in aqueous reaction condition involving copper sulfate and ascorbate,
using DMSO
as cosolvent.
[0215] From the foregoing description, one skilled in the art can easily
ascertain the essential
characteristics of this invention, and without departing from the spirit and
scope thereof, can
make various changes and modifications of the invention to adapt it to various
usage and
conditions without undue experimentation.
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:A 02782398 2012-05-30
SEQUENCE LISTING IN ELECTRONIC FORM
In accordance with Section 111(1) of the Patent Rules, this
description contains a sequence listing in electronic form in ASCII
text format (file: 52392-94 Seq 18-APR-12 vl.txt).
A copy of the sequence listing in electronic form is available from
the Canadian Intellectual Property Office.
The sequences in the sequence listing in electronic form are
reproduced in the following table.
SEQUENCE TABLE
<110> IMMUNOMEDICS, INC.
<120> DELIVERY SYSTEM FOR CYTOTOXIC DRUGS BY BISPECIFIC ANTIBODY
PRETARGETING
<130> 52392-94
<140> CA national phase of PCT/US2010/059686
<141> 2010-12-09
<150> 12/949,536
<151> 2010-11-18
<150> 12/915,515
<151> 2010-10-29
<150> 12/871,345
<151> 2010-08-30
<150> 12/869,823
<151> 2010-08-27
<150> 12/754,740
<151> 2010-04-06
<150> 12/752,649
<151> 2010-04-01
<150> 12/731,781
<151> 2010-03-25
<150> 12/644,146
<151> 2009-12-22
<150> 61/267,998
<151> 2009-12-09
<160> 83
<170> PatentIn version 3.5
71a
:A 02782398 2012-05-30
<210> 1
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 1
aatgcggcgg tggtgacagt a 21
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 2
aagctcagca cacagaaaga c 21
<210> 3
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<220>
<223> Description of Combined DNA/RNA Molecule: Synthetic
oligonucleotide
<400> 3
uaaaaucuuc cugcccacct t 21
<210> 4
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<220>
<223> Description of Combined DNA/RNA Molecule: Synthetic
oligonucleotide
7 lb
:A 02782398 2012-05-30
<400> 4
ggaagcuguu ggcugaaaat t 21
<210> 5
<211> 21
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 5
aagaccagcc ucuuugccca g 21
<210> 6
<211> 19
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 6
ggaccaggca gaaaacgag 19
<210> 7
<211> 17
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 7
cuaucaggau gacgcgg 17
<210> 8
<211> 21
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 8
ugacacaggc aggcuugacu u 21
71c
:A 02782398 2012-05-30
<210> 9
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 9
ggtgaagaag ggcgtccaa 19
<210> 10
<211> 60
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 10
gatccgttgg agctgttggc gtagttcaag agactcgcca acagctccaa cttttggaaa 60
<210> 11
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 11
aggtggtgtt aacagcagag 20
<210> 12
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 12
aaggtggagc aagcggtgga g 21
<210> 13
<211> 21
<212> DNA
<213> Artificial Sequence
71d
:A 02782398 2012-05-30
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 13
aaggagttga aggccgacaa a 21
<210> 14
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<220>
<223> Description of Combined DNA/RNA Molecule: Synthetic
oligonucleotide
<400> 14
uauggagcug cagaggaugt t 21
<210> 15
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 15
tttgaatatc tgtgctgaga acacagttct cagcacagat attcttttt 49
<210> 16
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 16
aatgagaaaa gcaaaaggtg ccctgtctc 29
<210> 17
<211> 21
<212> RNA
<213> Artificial Sequence
71e
:A 02782398 2012-05-30
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 17
aaucaucauc aagaaagggc a 21
<210> 18
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<220>
<223> Description of Combined DNA/RNA Molecule: Synthetic
oligonucleotide
<400> 18
augacuguca ggauguugct t 21
<210> 19
<211> 21
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 19
gaacgaaucc ugaagacauc u 21
<210> 20
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 20
aagcctggct acagcaatat gcctgtctc 29
<210> 21
<211> 21
<212> DNA
<213> Artificial Sequence
71f
:A 02782398 2012-05-30
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<220>
<223> Description of Combined DNA/RNA Molecule: Synthetic
oligonucleotide
<400> 21
ugaccaucac cgaguuuaut t 21
<210> 22
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 22
aagtcggacg caacagagaa a 21
<210> 23
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<220>
<223> Description of Combined DNA/RNA Molecule: Synthetic
oligonucleotide
<400> 23
cuaccuuucu acggacgugt t 21
<210> 24
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 24
ctgcctaagg cggatttgaa t 21
<210> 25
<211> 21
71g
:A 02782398 2012-05-30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<220>
<223> Description of Combined DNA/RNA Molecule: Synthetic
oligonucleotide
<400> 25
ttauuccuuc uucgggaagu c 21
<210> 26
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 26
aaccttctgg aacccgccca c 21
<210> 27
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 27
gagcatcttc gagcaagaa 19
<210> 28
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 28
catgtggcac cgtttgcct 19
<210> 29
<211> 21
71h
,
:A 02782398 2012-05-30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 29
aactaccaga aaggtatacc t
21
<210> 30
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<220>
<223> Description of Combined DNA/RNA Molecule: Synthetic
oligonucleotide
<400> 30
ucacaguguc cuuuauguat t
21
<210> 31
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<220>
<223> Description of Combined DNA/RNA Molecule: Synthetic
oligonucleotide
<400> 31
gcaugaaccg gaggcccaut t
21
<210> 32
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 32
ccggacagtt ccatgtata
19
71i
:A 02782398 2012-05-30
<210> 33
<211> 44
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
polypeptide
<400> 33
Ser His Ile Gin Ile Pro Pro Gly Leu Thr Glu Leu Leu Gin Gly Tyr
1 5 10 15
Thr Val Glu Val Leu Arg Gin Gin Pro Pro Asp Leu Val Glu Phe Ala
20 25 30
Val Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala
35 40
<210> 34
<211> 45
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
polypeptide
<400> 34
Cys Gly His Ile Gin Ile Pro Pro Gly Leu Thr Glu Leu Leu Gin Gly
1 5 10 15
Tyr Thr Val Glu Val Leu Arg Gin Gin Pro Pro Asp Leu Val Glu Phe
20 25 30
Ala Val Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala
35 40 45
<210> 35
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 35
Gin Ile Glu Tyr Leu Ala Lys Gin Ile Val Asp Asn Ala Ile Gin Gin
1 5 10 15
Ala
<210> 36
<211> 21
<212> PRT
<213> Artificial Sequence
71j
:A 02782398 2012-05-30
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 36
Cys Gly Gin Ile Glu Tyr Leu Ala Lys Gin Ile Val Asp Asn Ala Ile
1 5 10 15
Gin Gin Ala Gly Cys
<210> 37
<211> 50
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
polypeptide
<400> 37
Ser Leu Arg Glu Cys Glu Leu Tyr Val Gin Lys His Asn Ile Gin Ala
1 5 10 15
Leu Leu Lys Asp Ser Ile Val Gin Leu Cys Thr Ala Arg Pro Glu Arg
20 25 30
Pro Met Ala Phe Leu Arg Glu Tyr Phe Glu Arg Leu Glu Lys Glu Glu
35 40 45
Ala Lys
<210> 38
<211> 55
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
polypeptide
<400> 38
Met Ser Cys Gly Gly Ser Leu Arg Glu Cys Glu Leu Tyr Val Gin Lys
1 5 10 15
His Asn Ile Gin Ala Leu Leu Lys Asp Ser Ile Val Gin Leu Cys Thr
20 25 30
Ala Arg Pro Glu Arg Pro Met Ala Phe Leu Arg Glu Tyr Phe Glu Arg
35 40 45
Leu Glu Lys Glu Glu Ala Lys
50 55
<210> 39
<211> 23
<212> PRT
<213> Artificial Sequence
71k
= :A 02782398 2012-05-30
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 39
Cys Gly Phe Glu Glu Leu Ala Trp Lys Ile Ala Lys Met Ile Trp Ser
1 5 10 15
Asp Val Phe Gln Gln Gly Cys
<210> 40
<211> 55
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
polypeptide
<400> 40
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His Ile Gln Ile
1 5 10 15
Pro Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr Thr Val Glu Val Leu
20 25 30
Arg Gln Gln Pro Pro Asp Leu Val Glu Phe Ala Val Glu Tyr Phe Thr
35 40 45
Arg Leu Arg Glu Ala Arg Ala
50 55
<210> 41
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 41
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile Glu Tyr
1 5 10 15
Leu Ala Lys Gln Ile Val Asp Asn Ala Ile Gln Gln Ala
20 25
<210> 42
<211> 51
<212> PRT
<213> Homo sapiens
<400> 42
Ser Leu Arg Glu Cys Glu Leu Tyr Val Gln Lys His Asn Ile Gln Ala
1 5 10 15
Leu Leu Lys Asp Val Ser Ile Val Gln Leu Cys Thr Ala Arg Pro Glu
20 25 30
711
:A 02782398 2012-05-30
Arg Pro Met Ala Phe Leu Arg Glu Tyr Phe Glu Lys Leu Glu Lys Glu
35 40 45
Glu Ala Lys
<210> 43
<211> 54
<212> PRT
<213> Homo sapiens
<400> 43
Ser Leu Lys Gly Cys Glu Leu Tyr Val Gin Leu His Gly Ile Gin Gin
1 5 10 15
Val Leu Lys Asp Cys Ile Val His Leu Cys Ile Ser Lys Pro Glu Arg
20 25 30
Pro Met Lys Phe Leu Arg Glu His Phe Glu Lys Leu Glu Lys Glu Glu
35 40 45
Asn Arg Gin Ile Leu Ala
<210> 44
<211> 44
<212> PRT
<213> Homo sapiens
<400> 44
Ser His Ile Gin Ile Pro Pro Gly Leu Thr Glu Leu Leu Gin Gly Tyr
1 5 10 15
Thr Val Glu Val Gly Gin Gin Pro Pro Asp Leu Val Asp Phe Ala Val
20 25 30
Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Arg Gin
35 40
<210> 45
<211> 44
<212> PRT
<213> Homo sapiens
<400> 45
Her Ile Glu Ile Pro Ala Gly Leu Thr Glu Leu Leu Gin Gly Phe Thr
1 5 10 15
Val Glu Val Leu Arg His Gin Pro Ala Asp Leu Leu Glu Phe Ala Leu
20 25 30
Gin His Phe Thr Arg Leu Gin Gin Glu Asn Glu Arg
35 40
<210> 46
<211> 17
<212> PRT
<213> Artificial Sequence
71m
= :A 02782398 2012-05-30
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 46
Gin Ile Glu Tyr Val Ala Lys Gin Ile Val Asp Tyr Ala Ile His Gin
1 5 10 15
Ala
<210> 47
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 47
Gin Ile Glu Tyr Lys Ala Lys Gin Ile Val Asp His Ala Ile His Gin
1 5 10 15
Ala
<210> 48
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 48
Gin Ile Glu Tyr His Ala Lys Gin Ile Val Asp His Ala Ile His Gin
1 5 10 15
Ala
<210> 49
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 49
Gin Ile Glu Tyr Val Ala Lys Gin Ile Val Asp His Ala Ile His Gin
1 5 10 15
Ala
<210> 50
<211> 18
71n
:A 02782398 2012-05-30
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 50
Pro Leu Glu Tyr Gln Ala Gly Leu Leu Val Gln Asn Ala Ile Gln Gln
1 5 10 15
Ala Ile
<210> 51
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 51
Leu Leu Ile Glu Thr Ala Ser Ser Leu Val Lys Asn Ala Ile Gln Leu
1 5 10 15
Ser Ile
<210> 52
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 52
Leu Ile Glu Glu Ala Ala Ser Arg Ile Val Asp Ala Val Ile Glu Gln
1 5 10 15
Val Lys
<210> 53
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 53
Ala Leu Tyr Gln Phe Ala Asp Arg Phe Ser Glu Leu Val Ile Ser Glu
1 5 10 15
Ala Leu
710
:A 02782398 2012-05-30
<210> 54
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 54
Leu Glu Gin Val Ala Asn Gin Leu Ala Asp Gin Ile Ile Lys Glu Ala
1 5 10 15
Thr
<210> 55
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 55
Phe Glu Glu Leu Ala Trp Lys Ile Ala Lys Met Ile Trp Ser Asp Val
1 5 10 15
Phe
<210> 56
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 56
Glu Leu Val Arg Leu Ser Lys Arg Leu Val Glu Asn Ala Val Leu Lys
1 5 10 15
Ala Val
<210> 57
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
71p
:A 02782398 2012-05-30
<400> 57
Thr Ala Glu Glu Val Ser Ala Arg Ile Val Gin Val Val Thr Ala Glu
1 5 10 15
Ala Val
<210> 58
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 58
Gin Ile Lys Gin Ala Ala Phe Gin Leu Ile Ser Gin Val Ile Leu Glu
1 5 10 15
Ala Thr
<210> 59
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 59
Leu Ala Trp Lys Ile Ala Lys Met Ile Val Ser Asp Val Met Gin Gin
1 5 10 15
<210> 60
<211> 24
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 60
Asp Leu Ile Glu Glu Ala Ala Ser Arg Ile Val Asp Ala Val Ile Glu
1 5 10 15
Gin Val Lys Ala Ala Gly Ala Tyr
<210> 61
<211> 18
<212> PRT
<213> Artificial Sequence
71q
:A 02782398 2012-05-30
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 61
Leu Glu Gln Tyr Ala Asn Gln Leu Ala Asp Gln Ile Ile Lys Glu Ala
1 5 10 15
Thr Glu
<210> 62
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 62
Phe Glu Glu Leu Ala Trp Lys Ile Ala Lys Met Ile Trp Ser Asp Val
1 5 10 15
Phe Gln Gln Cys
<210> 63
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 63
Gln Ile Glu Tyr Leu Ala Lys Gln Ile Pro Asp Asn Ala Ile Gln Gln
1 5 10 15
Ala
<210> 64
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 64
Lys Gly Ala Asp Leu Ile Glu Glu Ala Ala Ser Arg Ile Val Asp Ala
1 5 10 15
Val Ile Glu Gln Val Lys Ala Ala Gly
20 25
71r
:A 02782398 2012-05-30
<210> 65
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 65
Lys Gly Ala Asp Leu Ile Glu Glu Ala Ala Ser Arg Ile Pro Asp Ala
1 5 10 15
Pro Ile Glu Gln Val Lys Ala Ala Gly
20 25
<210> 66
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 66
Pro Glu Asp Ala Glu Leu Val Arg Leu Ser Lys Arg Leu Val Glu Asn
1 5 10 15
Ala Val Leu Lys Ala Val Gln Gln Tyr
20 25
<210> 67
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 67
Pro Glu Asp Ala Glu Leu Val Arg Thr Ser Lys Arg Leu Val Glu Asn
1 5 10 15
Ala Val Leu Lys Ala Val Gln Gln Tyr
20 25
<210> 68
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
71s
:A 02782398 2012-05-30
<400> 68
Pro Glu Asp Ala Glu Leu Val Arg Leu Ser Lys Arg Asp Val Glu Asn
1 5 10 15
Ala Val Leu Lys Ala Val Gln Gin Tyr
20 25
<210> 69
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 69
Pro Glu Asp Ala Glu Leu Val Arg Leu Ser Lys Arg Leu Pro Glu Asn
1 5 10 15
Ala Val Leu Lys Ala Val Gin Gin Tyr
20 25
<210> 70
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 70
Pro Glu Asp Ala Glu Leu Val Arg Leu Ser Lys Arg Leu Pro Glu Asn
1 5 10 15
Ala Pro Leu Lys Ala Val Gin Gin Tyr
20 25
<210> 71
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 71
Pro Glu Asp Ala Glu Leu Val Arg Leu Ser Lys Arg Leu Val Glu Asn
1 5 10 15
Ala Val Glu Lys Ala Val Gin Gin Tyr
20 25
<210> 72
<211> 25
71t
:A 02782398 2012-05-30
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 72
Glu Glu Gly Leu Asp Arg Asn Glu Glu Ile Lys Arg Ala Ala Phe Gin
1 5 10 15
Ile Ile Ser Gin Val Ile Ser Glu Ala
20 25
<210> 73
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 73
Leu Val Asp Asp Pro Leu Glu Tyr Gin Ala Gly Leu Leu Val Gin Asn
1 5 10 15
Ala Ile Gin Gin Ala Ile Ala Glu Gin
20 25
<210> 74
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 74
Gin Tyr Glu Thr Leu Leu Ile Glu Thr Ala Ser Ser Leu Val Lys Asn
1 5 10 15
Ala Ile Gin Leu Ser Ile Glu Gin Leu
20 25
<210> 75
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
71u
,
:A 02782398 2012-05-30
<400> 75
Leu Glu Lys Gin Tyr Gin Glu Gin Leu Glu Glu Glu Val Ala Lys Val
1 5 10 15
Ile Val Ser Met Ser Ile Ala Phe Ala
20 25
<210> 76
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 76
Asn Thr Asp Glu Ala Gin Glu Glu Leu Ala Trp Lys Ile Ala Lys Met
1 5 10 15
Ile Val Ser Asp Ile Met Gin Gin Ala
20 25
<210> 77
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 77
Val Asn Leu Asp Lys Lys Ala Val Leu Ala Glu Lys Ile Val Ala Glu
1 5 10 15
Ala Ile Glu Lys Ala Glu Arg Glu Leu
20 25
<210> 78
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 78
Asn Gly Ile Leu Glu Leu Glu Thr Lys Ser Ser Lys Leu Val Gin Asn
1 5 10 15
Ile Ile Gin Thr Ala Val Asp Gin Phe
20 25
<210> 79
<211> 25
71v
:A 02782398 2012-05-30
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 79
Thr Gln Asp Lys Asn Tyr Glu Asp Glu Leu Thr Gin Val Ala Leu Ala
1 5 10 15
Leu Val Glu Asp Val Ile Asn Tyr Ala
20 25
<210> 80
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 80
Glu Thr Ser Ala Lys Asp Asn Ile Asn Ile Glu Glu Ala Ala Arg Phe
1 5 10 15
Leu Val Glu Lys Ile Leu Val Asn His
20 25
<210> 81
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<220>
<223> N-term DOTA
<220>
<221> MOD RES
_
<222> (2)..(2)
<223> Lys(HSG)
<220>
<221> MOD RES
_
<222> (4)..(4)
<223> Lys(HSG)
<220>
<223> C-term amidated
71w
:A 02782398 2012-05-30
<400> 81
Phe Lys Tyr Lys
1
<210> 82
<400> 82
000
<210> 83
<211> 21
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
peptide
<400> 83
Cys Gly Gln Ile Glu Tyr Leu Ala Lys Gln Ile Val Asp Asn Ala Ile
1 5 10 15
Gln Gln Ala Gly Cys
71x
