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THAN ONE VOLUME.
THIS IS VOLUME 1 OF 2
NOTE: For additional volumes please contact the Canadian Patent Office.
CA 02615761 2008-01-17
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A CA6 ANTIGEN-SPECIFIC CYTOTOXIC CONJUGATE AND METHODS
OF USING THE SAME
FIELD OF THE INVENTION
[01] The present invention is directed to a murine anti-CA6 glycotope
monoclonal
antibody, and humanized or resurfaced versions thereof. The present invention
is also
directed to epitope-binding fragments of the anti-CA6 glycotope monoclonal
antibody, as well as to epitope-binding fragments of humanized or resurfaced
versions
of the anti-CA6 glycotope monoclonal antibody.
[02] The present invention is further directed to cytotoxic conjugates
comprising a
cell binding agent and a cytotoxic agent, therapeutic compositions comprising
the
conjugate, methods for using the conjugates in the inhibition of cell growth
and the
treatment of disease, and a kit comprising the cytotoxic conjugate. In
particular, the
cell binding agent is a monoclonal antibody, or epitope-binding fragment
thereof, that
recognizes and binds the CA6 glycotope or a humanized or resurfaced version
thereof.
BACKGROUND OF THE INVENTION
[03] There have been numerous attempts to develop anti-cancer therapeutic
agents
that specifically destroy target cancer cells without harming surrounding, non-
cancerous cells and tissue. Such therapeutic agents have the potential to
vastly
improve the treatment of cancer in human patients.
[04] One promising approach has been to link cell binding agents, such as
monoclonal
antibodies, with cytotoxic drugs (Sela et al, in Immunoconjugates 189-216 (C.
Vogel, ed.
1987); Ghose et al, in Targeted Drugs 1-22 (E. Goldberg, ed. 1983); Diener et
al, in
Antibody mediated delivery systems 1-23 (J. Rodwell, ed. 1988); Pietersz et
al, in
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Antibody mediated delivery systems 25-53 (J. Rodwell, ed. 1988); Bumol et al,
in
Antibody mediated delivery systems 55-79 (J. Rodwell, ed. 1988). Depending on
the
selection of the cell binding agent, these cytotoxic conjugates can be
designed to
recognize and bind only specific types of cancerous cells, based on the
expression profile
of molecules expressed on the surface of such cells.
[05] Cytotoxic drugs such as methotrexate, daunorubicin, doxorubicin,
vincristine,
vinblastine, melphalan, mitomycin C, and chlorambucil have been used in such
cytotoxic conjugates, linked to a variety of murine monoclonal antibodies. In
some
cases, the drug molecules were linked to the antibody molecules through an
intermediary carrier molecule such as serum albumin (Gamett et al, 46 Cancer
Res.
2407-2412 (1986); Ohkawa et al 23 Cancer Immunol. Immunother. 81-86 (1986);
Endo et al, 47 Cancer Res. 1076-1080 (1980)), dextran (Hurwitz et al, 2 Appl.
Biochem. 25-35 (1980); Manabi et al, 34 Biochem. Pharmacol. 289-291 (1985);
Dillman et al, 46 Cancer Res. 4886-4891 (1986); Shoval et al, 85 Proc. Natl.
Acad.
Sci. 8276-8280 (1988)), or polyglutamic acid (Tsukada et al, 73 J. Natl. Canc.
Inst.
721-729 (1984); Kato et al 27 J. Med. Chem. 1602-1607 (1984); Tsukada et al,
52 Br.
J. Cancer 111-116 (1985)).
[06] As an example of one specific conjugate that has shown some promise, is
the
conjugate of the C242 antibody, directed against CanAg, an antigen expressed
on
colorectal and pancreatic tumors, and the maytansine derivative DMI (Liu et
al., Proc
Natl Acad Sci USA, 93: 8618-8623 (1996)). In vitro evaluation of this
conjugate
indicated that its binding affinity towards CanAg expressed on the cell
surface was
high with an apparent Kd value of 3 x 10-11 M, and its cytotoxic potency for
CanAg-
positive cells was high with an IC50 of 6 x 10"11 M. This cytotoxicity was
antigen-
dependent since it was blocked by an excess of non-conjugated antibody, and
since
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antigen-negative cells were more than 100-fold less sensitive to the
conjugate. Other
examples of antibody-DM1 conjugates with both high affinity towards respective
target cells and high antigen-selective cytotoxicity include those of huN901,
a
humanized version of antibody against human CD56; huMy9-6, a humanized version
of antibody against human CD33; huC242, a humanized version of antibody
against
the CanAg Mucl epitope; huJ591, a deimmunized antibody against PSMA;
trastuzumab, a humanized antibody against Her2/neu; and bivatuzumab, a
humanized
antibody against CD44v6.
[07] The development of additional cytotoxic conjugates that specifically
recognize
particular types of cancerous cells will be important in the continuing
improvement of
methods used to treat patients with cancer.
[08] To that end, the present invention is directed to the development of
antibodies
that recognize and bind molecules/receptors expressed on the surface of
cancerous
cells, and to the development of novel cytotoxic conjugates comprising cell
binding
agents, such as antibodies, and cytotoxic agents that specifically target the
molecules/receptors expressed on the surface of cancerous cells.
[09] More specifically, the present invention is directed to the
characterization of a
novel CA6 sialoglycotope on the Mucl mucin receptor expressed by cancerous
cells,
and to the provision of antibodies, preferably humanized antibodies, that
recognize
the novel CA6 sialoglycotope of the Mucl mucin and that may be used to inhibit
the
growth of a cell expressing the CA6 glycotope in the context of a cytotoxic
agent.
SUMMARY OF THE INVENTION
[10] The present invention includes antibodies that specifically recognize and
bind
a novel CA6 sialoglycotope of the Mucl mucin receptor, or an epitope-binding
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fragment thereof. In another embodiment, the present invention includes a
humanized
antibody, or an epitope-binding fragment thereof, that recognizes the novel
CA6
sialoglycotope ("the CA6 glycotope") of the Mucl mucin receptor.
[11] In preferred embodiments, the present invention includes the murine anti-
CA6
monoclonal antibody DS6 ("the DS6 antibody"), and resurfaced or humanized
versions of the DS6 antibody wherein surface-exposed residues of the antibody,
or its
epitope-binding fragments, are replaced in both light and heavy chains to more
closely resemble known human antibody surfaces. The humanized antibodies and
epitope-binding fragments thereof of the present invention have improved
properties
in that they are much less immunogenic (or completely non-immunogenic) in
human
subjects to which they are administered than fully murine versions. Thus, the
humanized DS6 antibodies and epitope-binding fragments thereof of the present
invention specifically recognize a novel sialoglycotope on the Mucl mucin
receptor,
i.e., the CA6 glycotope, while not being immunogenic to a human. The humanized
antibodies and epitope-binding fragments thereof can be conjugated to a drug,
such as
a maytansinoid, to form a prodrug having specific cytotoxicity towards antigen-
expressing cells by targeting the drug to the Mucl CA6 sialoglycotope.
Cytotoxic
conjugates comprising such antibodies and small, highly toxic drugs (e.g.,
maytansinoids, taxanes, and CC-1065 analogs) can thus be used as a therapeutic
for
treatment of tumors, such as breast and ovarian tumors.
[12] The humanized versions of the DS6 antibody of the present invention are
fully
characterized herein with respect to their respective amino acid sequences of
both
light and heavy chain variable regions, the DNA sequences of the genes for the
light
and heavy chain variable regions, the identification of the CDRs, the
identification of
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their surface amino acids, and disclosure of a means for their expression in
recombinant form.
[13] In one embodiment, there is provided a humanized DS6 antibody or an
epitope-binding fragment thereof having a heavy chain including CDRs having
amino
acid sequences represented by SEQ ID NOS:1-3:
SYNMH (SEQID NO:1),
YIYPGNGATNYNQKFKG (SEQIDNO:2),
GDSVPFAY(SEQIDNO:3),
and having a light chain that comprises CDRs having amino acid sequences
represented by SEQ ID NOS:4-6:
S A H S S V S F M H (SEQ ID NO:4),
STSSLAS (SEQIDNO:5),
QQRSSFPLT (SEQIDNO:6),
[14] Also provided are humanized DS6 antibodies and epitope-binding fragments
thereof having a light chain variable region that has an amino acid sequence
that
shares at least 90% sequence identity with an amino acid sequence represented
by
SEQ ID NO:7 or SEQ ID NO: 8:
QIVLTQSPAIMSASPGEKVTITCSAHSSVSFMHWFQQKPGTSPKLWIYS
TSSLASGVPARFGGSGSGTSYSLTISRMEAEDAATYYCQQRSSFPLTFG
AGTKLELKR (SEQ ID NO:7)
EIVLTQSPATMSASPGERVTITCSAHSSVSFMHWFQQKPGTSPKLWIYS
TS SLAS GVPARFGGS GS GTSYS LTIS SMEAEDAATYYCQ QRS SFPLTFG
AGTKLELKR (SEQ ID NO:8)
[15] Similarly, there are provided humanized DS6 antibodies and epitope-
binding
fragments thereof having a heavy chain variable region that has an amino acid
CA 02615761 2008-01-17
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sequence that shares at least 90% sequence identity with an amino acid
sequence
represented by SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO: 11:
QAYLQQS GAELVRS GAS V KMS CKAS GYTFT SYNMHW VKQTP GQGLE
W IGYIYP GNGATNYNQKFKGKAT LTADP S S S TAYMQIS S LT S ED S AV Y
FCARGDSVPFAYWGQGTLVTVSA (SEQ ID NO:9)
QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLE
WIGYIYPGNGATNYNQKFQGKATLTADTS S STAYMQIS SLTSED SAVY
FCARGDSVPFAYWGQGTLVTVSA (SEQ ID NO:10)
QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLE
WIGYIYP GNGATNYNQKFQGKATLTADP S S STAYMQIS SLTSED SAVY
FCARGDSVPFAYWGQGTLVTVSA (SEQ ID NO: 11)
[16] In another embodiment, humanized DS6 antibodies and epitope-binding
fragments thereof are provided having a humanized or resurfaced light chain
variable
region having an amino acid sequence corresponding to SEQ ID NO: 8
ENLTQSPATMSASPGERVTITCSAHSSVSFMHWFQQKPGTSPKLWIYS
TSSLASGVPARFGGSGSGTSYSLTISSMEAEDAATYYCQQRSSFPLTFG
AGTKLELKR. (SEQ ID NO:8)
[17] Similarly, humanized DS6 antibodies and epitope-binding fragments thereof
are provided having a humanized or resurfaced heavy chain variable region
having an
amino acid sequence corresponding to SEQ ID NO:10 or SEQ ID NO: 11,
respectively:
QAQLVQSGAEV VKPGAS VKMSCKASGYTFTSYNMHWVKQTPGQGLE
WIGYIYPGNGATNYNQKFQGKATLTADTS S STAYMQIS SLTSEDSAVY
FCARGDSVPFAYWGQGTLVTVSA. (SEQ ID NO:10)
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QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLE
WIGYIYPGNGATNYNQKFQGKATLTADPS S STAYMQIS SLTSEDSAVY
FCARGDSVPFAYWGQGTLVTVSA. (SEQ ID NO:11)
[18] The humanized DS6 antibodies and epitope-binding fragments thereof of the
present invention can also include substitution in light and/or heavy chain
amino acid
residues at one or more positions defined by the starred residues in Table 1
which
represent the murine surface framework residues found within 5 Angstroms of a
CDR
requiring change to a human residue. For example, the first amino acid residue
Q in
the murine sequence (SEQ ID NO:7) has been replaced by E (SEQ ID NO:8) to
humanize the antibody. However, because of the proximity of this residue to a
CDR,
a back mutation to the murine residue Q may be required to maintain antibody
affinity.
Table 1
muDS6 framework residues proximal to a
CDR (Kabat numbering)
Light chain Heavy chain
Q1* 1
V3 K64*
T5 P73*
P40 S74
G57
A60
S67
E81
[19] This is further shown in Table 2 where muDS6 variable region surface
residues are shown aligned with the three most homologous human variable
region
surface residues. The amino acid residues in Table 1 correspond to the
underlined
amino acid residues in Table 2.
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Table 2
Top 3 Most Homologous Human Antibody Surfaces
Antibody Light Chain SEQ ID NO:
muDS6 QVTAIPKPGGASREK SEQIDNO:12
28E4 EVTATPRPGGASSEK SEQIDNO:13
HAZcPB EVTGTPRPGGDSREK SEQIDNO:14
SSaPB EVTGTPRPGGDSREK SEQIDNO:15
Antibody Heavy Chain SEQ ID NO:
muDS6 Q Y Q A L R S K K P G Q Q K K G P S S S E Q S SEQID NO:16
28E4 QQVAVKPKKPGQQKQGTSSSEQS SEQIDNO:17
HAZcPB - QVAVKPKKPGQQKQGESSSEQS SEQIDNO:18
SSaPB - QVAVKPKKPGQQKQGESSSEQS SEQID NO:19
[20] The present invention further provides cytotoxic conjugates comprising
(1) a
cell binding agent that recognizes and binds the CA6 glycotope, and (2) a
cytotoxic
agent. In the cytotoxic conjugates, the cell binding agent has a high affinity
for the
CA6 glycotope and the cytotoxic agent has a high degree of cytotoxicity for
cells
expressing the CA6 glycotope, such that the cytotoxic conjugates of the
present
invention form effective killing agents.
[21] In a preferred embodiment, the cell binding agent is an anti-CA6 antibody
or
an epitope-binding fragment thereof, more preferably a humanized anti-CA6
antibody
or an epitope-binding fragment thereof, wherein a cytotoxic agent is
covalently
attached, directly or via a cleavable or non-cleavable linker, to the antibody
or
epitope-binding fragment thereof. In more preferred embodiments, the cell
binding
agent is the humanized DS6 antibody or an epitope-binding fragment thereof,
and the
cytotoxic agent is a taxol, a maytansinoid, CC-1065 or a CC-1065 analog.
[22] In preferred embodiments of the invention, the cell binding agent is a
humanized anti-CA6 antibody and the cytotoxic agent is a cytotoxic drug such
as a
maytansinoid or a taxane.
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[23] More preferably, the cell binding agent is the humanized anti-CA6
antibody
DS6 and the cytotoxic agent is a maytansine compound, such as DM1 or DM4.
[24] The present invention also includes a method for inhibiting the growth of
a
cell expressing the CA6 glycotope. In preferred embodiments, the method for
inhibiting growth of the cell expressing the CA6 glycotope takes place in vivo
and
results in the death of the cell, although in vitro and ex vivo applications
are also
included.
[25] The present invention also provides a therapeutic composition comprising
the
cytotoxic conjugate, and a pharmaceutically acceptable carrier or excipient.
[26] The present invention further includes a method of treating a subject
having
cancer using the therapeutic composition. In preferred embodiments, the
cytotoxic
conjugate comprises an anti-CA6 antibody and a cytotoxic agent. In more
preferred
embodiments, the cytotoxic conjugate comprises a humanized DS6 antibody-DM1
conjugate, humanized DS6 antibody-DM4 or a humanized DS6 antibody-taxane
conjugate, and the conjugate is administered along with a pharmaceutically
acceptable
carrier or excipient.
[27] The present invention also includes a kit comprising an anti-CA6 antibody-
cytotoxic agent conjugate and instructions for use. In preferred embodiments,
the
anti-CA6 antibody is the humanized DS6 antibody, the cytotoxic agent is a
maytansine compound, such as DM1 or DM4, or a taxane, and the instructions are
for
using the conjugates in the treatment of a subject having cancer. The kit may
also
include components necessary for the preparation of a pharmaceutically
acceptable
formulation, such a diluent if the conjugate is in a lyophilized state or
concentrated
form, and for the administration of the formulation.
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[28] The present invention also includes derivatives of antibodies that
specifically
bind and recognize the CA6 glycotope. In preferred embodiments, the antibody
derivatives are prepared by resurfacing or humanizing antibodies that bind the
CA6
glycotope, wherein the derivatives have decreased immunogenicity toward the
host.
[29] The present invention further provides for humanized antibodies or
fragments
thereof that are further labeled for use in research or diagnostic
applications. In
preferred embodiments, the label is a radiolabel, a fluorophore, a
chromophore, an
imaging agent or a metal ion.
[30] A method for diagnosis is also provided in which said labeled humanized
antibodies or epitope-binding fragments thereof are administered to a subject
suspected of having a cancer, and the distribution of the label within the
body of the
subject is measured or monitored.
[31] The present invention also provides methods for the treatment of a
subject
having a cancer by administering a humanized antibody conjugate of the present
invention, either alone or in combination with other cytotoxic or therapeutic
agents.
The cancer can be one or more of, for example, breast cancer, colon cancer,
ovarian
carcinoma, endometrial cancer, osteosarcoma, cervical cancer, prostate cancer,
lung
cancer, synovial carcinoma, pancreatic cancer, a sarcoma or a carcinoma in
which
CA6 is expressed or other cancer yet to be determined in which CA6 glycotope
is
expressed predominantly.
[32] Unless otherwise stated, all references and patents cited herein are
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[33] Figure 1 shows the results of studies performed to determine the ability
of the
DS6 antibody to bind the surface of selected cancer cell lines. The
fluorescence of
CA 02615761 2008-01-17
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cell lines incubated with the DS6 primary antibody and FITC conjugated anti-
mouse
IgG(H+L) secondary antibodies was measured by flow cytometry. The DS6 antibody
bound Caov-3 (Figure lA) and T-47D (Figure 1B) cells with an apparent Kd of
1.848
nM and 2.586 nM respectively. Antigen negative cell lines, SK-OV-3 (Figure 1C)
and Co1o205 (Figure 1 D) demonstrated no antigen specific binding.
[34] Figure 2 shows the results of dot blot analysis of epitope expression.
Caov-3
(Figure 2A & Figure 2B), SKMEL28 (Figure 2C), and Co1o205 (Figure 2D) cell
lysates were individually spotted onto nitrocellulose membranes and then
incubated
individually with pronase, proteinase K, neuraminidase or periodic acid. The
membranes were then immunoblotted with the DS6 antibody (Figure 2A), the CM1
antibody (Figure 2B), the R24 antibody (Figure 2C), or the C242 antibody
(Figure
2D).
[35] Figure 3 shows the results of a dot blot analysis of DS6 antigen
expression.
Caov-3 cell lysates were individually spotted onto PVDF membranes and then
incubated in the presence of trifluoromethanesulfonic acid (TFMSA). The
membranes were then immunoblotted with the CM1 antibody (1 & 2) or the DS6
antibody (3 & 4).
[36] Figure 4 shows the results of glycotope analysis of the DS6 antigen. Caov-
3
lysates pretreated with N-glycanase ("N-gly"), 0-glycanase ("O-gly"), and/or
sialidase ("S") were spotted onto nitrocellulose and then immunoblotted with
the DS6
antibody or the CM1 antibody (Muc-1 VNTR).
[37] Figure 5 shows the results of western blot analysis of the DS6 antigen.
Cell
lysates were immunoprecipitated ("IP") and immunoblotted with the DS6
antibody.
The antigen corresponds to a >250 kDa protein band observed in antigen-
positive
Caov-3 (Figure 5A and Figure 5B) and T47D (Figure 5C) cells. Antigen negative
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SK-OV-3 (Figure 5D) and Co1o205 (Figure 5E) cell lines do not exhibit this
band.
After immunopreciptation, the Protein G beads of the Caov-3 cell lysates were
incubated with (Figure 5A) neuraminidase ("N") or (Figure 5B) periodic acid
("PA").
Antibody ("a"), pre-IP ("Lys") and post-1P flow-through ("FT") lysate controls
were
run on the same gel. Caov-3 immunoprecipitates were also incubated with N-
glycanase ("N-gly"), 0-glycanase ("O-gly"), and/or sialidase ("S") (see Figure
5F),
where the blot was alternatively probed with biotinylated-DS6 and strepavidin-
HRP.
[38] Figure 6 shows the results of immunoprecipitations and/or immunoblots of
the
DS6 antibody and the CM1 antibody on Caov-3 (Figure 6A) and HeLa (Figure 6B)
cell lysates. Overlapping CM1 and DS6 western blot signals signify that the
DS6
antigen is on the Mucl protein. In HeLa lysates, the Mucl doublet results from
Mucl
expression directed by distinct alleles differing in their number of tandem
repeats.
[39] Figure 7 shows a DS6 antibody sandwich ELISA design (Figure 7A) and a
standard curve (Figure 7B). The standard curve was generated using known
concentrations of commercially available CA15-3 standards (where 1 CA15-3 unit
=
1 DS6 unit).
[40] Figure 8 shows quantitative ELISA standard curves. The standard curves of
the detection antibody (streptavidin-HRP / biotin-DS6) signal (Figure 8C) were
determined using known concentrations of biotin-DS6 either captured by plated
goat
anti-mouse IgG (Figure 8A) or bound directly onto the ELISA plate (Figure 8B).
[41] Figure 9 shows the cDNA and amino acid sequences of the light chain
(Figure
9A) and heavy chain (Figure 9B) variable region for the murine DS6 antibody.
The
three CDRs in each sequence are underlined (Kabat definitions).
[42] Figure 10 shows the light (Figure l0A) and heavy chain (Figure lOB) CDRs
of the murine DS6 antibody determined by Kabat definitions. The AbM modeling
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software produces a slightly different definition for the heavy chain CDRs
(Figure
lOC).
[43] Figure 11 shows the light chain ("muDS6LC") (residues 1-95 of SEQ ID
NO:7) and heavy chain ("muDS6HC") (residues 1-98 of SEQ ID NO:9) amino acid
sequences for the murine DS6 antibody aligned with the germline sequences for
the
IgVxap4 (SEQ ID NO:23) and IgVh J558.41 (SEQ ID NO:24) genes. Grey indicates
sequence divergence.
[44] Figure 12 shows the ten light chain and heavy chain antibody sequences
most
homologous to the murine DS6 (muDS6) light chain ("muDS6LC") and heavy chain
("muDS6HC") sequences that have solved structure files in the Brookhaven
database.
Sequences are aligned in order of most to least homologous. .
[45] Figure 13 shows surface accessibility data and calculations to predict
which
framework residues of the murine DS6 antibody light chain variable region are
surface accessible. The positions with 25-35% average surface accessibility
are
marked (*??*) and were subjected to the second round analysis. DS6 antibody
light
chain variable region (Figure 13A) and heavy chain variable region (Figure
13B).
[46] Figure 14 shows the prDS6 vl-O mammalian expression plasmid map. This
plasmid was used to build and express the recombinant chimeric and humanized
DS6
antibodies.
[47] Figure 15 shows amino acid sequences of murine ("muDS6") and humanized
("huDS6") (1.01 & 1.21) DS6 antibody light chain (Figure 15A) and heavy chain
(Figure 15B) variable domains.
[48] Figure 16 shows the cDNA and amino acid sequences of the light chain
variable region for the humanized DS6 antibody ("huDS6") (1.01 and 1.21).
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[49] Figure 17 shows the cDNA and amino acid sequences of the heavy chain
variable region for the humanized DS6 antibody ("huDS6") 1.01 (Figure 17A) and
1.21 (Figure 17B).
[50] Figure 18 shows flow cytometry binding curves of murine DS6 (muDS6)
chimeric DS6 (chDS6), and human DS6 version 1.01 (huDS6 v1.01) and version
huDS6 version 1.21 (huDS6 vl.21) from an assay performed on KB cells. The
avidities of the murine, chimeric, and human vl.01 and vl.21 DS6 antibodies
=
(muDS6 = 0.82 nM, chDS6 = 0.69 nM, huDS6v1.01 = 0.82 nM and huDS6vl.21
0.85 nM) are comparable, indicating that resurfacing has not diminished the
avidity.
[51] Figure 19 shows the results of a competition binding assay of muDS6,
chDS6,
huDS6 v1.01 and huDS6 vl.21 antibodies with biotinylated muDS6. Varying
concentrations of naked muDS6, chDS6, huDS6v1.01 and huDS6vl.21were
combined with 2 nM of biotin-muDS6 and the streptavidin-DTAF secondary. The
IC50's (muDS6 = 1.9 nM, chDS6 = 1.7 nM, huDS6vl.01 = 3.0 nM, and huDS6vl.21
= 1.9 nM) of all antibodies are similar indicating that humanization has not
reduced
the avidity.
[52] Figure 20 shows the results of a determination of the binding affinity of
un-
conjugated DS6 antibody versus a DS6 antibody-DM1 conjugate. The results
demonstrated that DM1 conjugation does not adversely affect the binding
affinity of
the antibody. The apparent Kd of the DS6 antibody-DM1 conjugate (3.902 nM)
("DS6-DM1") was slightly greater than the naked antibody (2.020 nM) ("DS6").
[53] Figure 21 shows the results of an indirect cell viability assay using the
DS6
antibody in the presence or absence of the anti-mouse IgG (H+L) DMl conjugate
(2 Ab-DM1). Antigen-positive Caov-3 cells were killed in a DS6 antibody-
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dependent manner (IC50 = 424.9 pM) only in the presence of the secondary
conjugate
("DS6 + 2 Ab-DM1").
[54] Figure 22 shows the results of a complement-dependent cytotoxicity (CDC)
assay of the muDS6 antibody. The results demonstrate that there is no CDC
mediated
effect of the DS6 antibody or on HPAC (Figure 22A) and ZR-75-1 (Figure 22B)
cells.
[55] Figure 23 shows the results of an in vitro cytotoxicity assay of a DS6
antibody-DM1 conjugate versus free maytansine. In a clonogenic assay, DS6
antigen-positive ovarian (Figure 23A), breast (Figure 23B), cervical (Figure
23C), and
pancreatic (Figure 23D) cancer cell lines were tested for cytotoxicity of
continuous
exposure to a DS6 antibody-DM1 conjugate (left panels). These cell lines were
similarly tested for maytansine sensitivity by a 72h exposure to free
maytansine (right
panels). The ovarian cancer cell lines tested were OVCAR5, TOV-21 G, Caov-4
and
Caov-3. The breast cancer cell lines tested were T47D, BT-20 and BT-483. The
cervical cancer cell lines tested were KB, HeLa and WISH. The pancreatic
cancer
cell lines tested were HPAC, Hs766T and HPAF-II.
[56] Figure 24 shows the results of an in vitro cytotoxicity assay of a DS6
antibody-DM1 conjugate. In a MTT cell viability assay, human ovarian (Figure
24A,
Figure 24B & Figure 24C), breast (Figure 24D & Figure 24E), cervical (Figure
24F &
Figure 24G), and pancreatic (Figure 24H & Figure 241) cancer cells were killed
in a
DS6 antibody-DM1 conjugate-dependent manner. Naked DS6 did not adversely
affect the growth of these cells, indicating that DM1 conjugation is required
for the
cytotoxicity.
[57] Figure 25A shows the results of an in vivo anti-tumor efficacy study of a
DS6
antibody-DM1 conjugate on established subcutaneous KB tumor xenografts. Tumor
cells were inoculated on day 0, and the first treatment was given on day 6.
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Immunoconjugate treatments continued daily for a total of 5 doses. PBS control
animals were euthanized once tumor volumes exceeded 1500 mm3. The conjugate
was given at a dose of 150 or 225 g/kg DM1, corresponding to antibody
concentrations of 5.7 and 8.5 mg/kg respectively. The body weights (Figure
25B) of
the mice were monitored during the course of the study.
[58] Figure 26 shows the results of an antitumor efficacy study of a DS6
antibody-
DM1 conjugate on established subcutaneous tumor xenografts. OVCAR5 (Figure
26A and Figure 26B), TOV-21G (Figure 26C and Figure 26D), HPAC (Figure 26E
and Figure 26F), and HeLa (Figure 26G and Figure 26H) cells were inoculated on
day
0, and immunoconjugate treatments were given on day 6 and 13. PBS control
animals
were euthanized once tumor volumes exceeded 1000 mm3. The conjugate was given
at a dose of 600 g/kg DM1, corresponding to an antibody concentration 27.7
mg/kg.
Tumor volume (Figure 26A, Figure 26C, Figure 26E, and Figure 26G) and body
weight (Figure 26B, Figure 26D, Figure 26F, and Figure 26H) of the mice were
monitored during the course of the study.
[59] Figure 27 shows the results of an in vivo efficacy study of a muDS6
antibody-
DM1 conjugate on intraperitoneal OVCAR5 tumors. Tumor cells were injected
intraperitoneally on day 0, and immunoconjugate treatments were given on day 6
and
13. Animals were euthanized once body weight loss exceeded 20%.
[60] Figure 28 shows the flow cytometry binding curve from a study of the
binding
affinity of naked and taxane-conjugated DS6 antibody on HeLa cells. Taxane
(MM1-
202)-conjugation does not adversely affect the binding affinity of the
antibody. The
apparent Kd of the DS6-MM1-202 conjugate (1.24 nM) was slightly greater than
the
naked DS6 antibody (620 pM).
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[61] Figure 29 shows in vitro binding and potency of humanized DS6 version
1.01
antibody conjugate. Conjugation of huDS6v1.01 with DM4 has little effect on
the
avidity of huDS6v1.01 for KB cells (Figure 29A). huDS6v1.01-DM4 shows potent
in vitro cytotoxicity toward DS6-expressing WISH cells with an IC50 of 0.44 nM
(Figure 29B).
[62] Figure 30 shows the results of an in vivo efficacy study with huDS6v1.01-
DM4 conjugate in an HPAC pancreatic cancer model. huDS6vl.01-DM4 showed
potent anti-tumor activity whereas the B4-DM4 control conjugate whose target
is not
expressed in the HPAC model had essentially no activity (Figure 30A). The
administered dose of 200 g/kg was not toxic to the animals as indicated by
the lack
of weight loss (Figure 30B).
DETAILED DESCRIPTION OF THE INVENTION
[63] The present invention provides, among other features, anti-CA6 monoclonal
antibodies, anti-CA6 humanized antibodies, and fragments of the anti-CA6
antibodies. Each of the antibodies and antibody fragments of the present
invention
are designed to specifically recognize and bind the CA6 glycotope on the
surface of a
cell. CA6 is known to be expressed by many human tumors: 95% of serous ovarian
carcinomas, 50% of endometrioid ovarian carcinomas, 50% of the neoplasms of
the
uterine cervix, 69% of the neoplasms of the endometrius, 80% of neoplasms of
the
vulva, 60% of breast carcinomas, 67% pancreatic tumors, and 48% of tumors of
the
urothelium, but is rarely expressed by normal human tissue.
[64] A report by Kearse et al., Int. J. Cancer 88(6):866-872 (2000)
misidentified
the protein on which the CA6 epitope is found as an 80 kDa protein having an N-
linked carbohydrate containing the CA6 epitope when they used a hybridoma
supernatant to characterize it. Using purified DS6 we have since demonstrated
that
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the CA6 epitope is found on an 0-linked carbohydrate of a greater than 250 kDa
non-
disulfide-linked glycoprotein. Furthermore, the glycoprotein was identified as
the
mucin, Mucl. Because different Muc1 alleles have varying numbers of tandem
repeats in the variable number tandem repeat (VNTR) domain cells often express
two
distinct Muc1 proteins of different size (Taylor-Papadimitriou, Biochim.
Biophys.
Acta 1455(2-3):301-13 (1999). Because of differences in the number of repeats
in the
VNTR domain as well as differences in glycosylation the molecular weight of
Mucl
varies from cell line to cell line.
[65] The susceptibility of CA6 immunoreactivity to periodic acid indicates CA6
is
a carbohydrate epitope "glycotope." The additional susceptibility of CA6
immunoreactivity to treatment with neuraminidase from Vibrio cholerae
indicates that
the CA6 epitope is a sialic acid dependent glycotope, thus a "sialoglycotope."
[66] Details of the characterization of CA6 can be found in the Example 2 (see
below). Additional details on CA6 may be found in WO 02/16401; Wennerberg et
al., Am. J Pathol. 143(4):1050-1054 (1993); Smith et al., Human Antibodies
9:61-65
(1999); Kearse et al., Int. J. Cancer 88(6):866-872 (2000); Smith et al., Int.
J.
Gynecol. Pathol. 20(3):260-6 (2001); and Smith et al., Appl. Immunohistochem.
Mol.
Morphol. 10(2):152-8 (2002).
[67] The present invention also includes cytotoxic conjugates comprising two
primary components. The first component is a cell binding agent that
recognizes and
binds the CA6 glycotope. The cell binding agent should recognize the CA6
sialoglycotope on Muc 1 with a high degree of specificity so that the
cytotoxic
conjugates recognize and bind only the cells for which they are intended. A
high
degree of specificity will allow the conjugates to act in a targeted fashion
with little
side-effects resulting from non-specific binding.
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[68] In another embodiment, the cell binding agent of the present invention
also
recognizes the CA6 glycotope with a high degree of affinity so that the
conjugates
will be in contact with the target cell for a sufficient period of time to
allow the
cytotoxic drug portion of the conjugate to act on the cell, and/or to allow
the
conjugates sufficient time in which to be internalized by the cell.
[69] In a preferred embodiment, the cytotoxic conjugates comprise an anti-CA6
antibody as the cell binding agent, more preferably the murine DS6 anti-CA6
monoclonal antibody. In a more preferred embodiment, the cytotoxic conjugates
comprises a humanized DS6 antibody or an epitope-binding fragment thereof. The
DS6 antibody is able to recognize CA6 with a high degree of specificity and
directs
the cytotoxic agent to an abnormal cell or a tissue, such as cancer cells, in
a targeted
fashion.
[70] The second component of the cytotoxic conjugates of the present invention
is
a cytotoxic agent. In preferred embodiments, the cytotoxic agent is a taxol, a
maytansinoid such as DM1 or DM4, CC-1065 or a CC-1065 analog. In preferred
embodiments, the cell binding agents of the present invention are covalently
attached,
directly or via a cleavable or non-cleavable linker, to the cytotoxic agent.
[711 The cell binding agents, cytotoxic agents, and linkers are discussed in
more
detail below.
Cell Binding A2ents
[72] The effectiveness of the compounds of the present invention as
therapeutic
agents depends on the careful selection of an appropriate cell binding agent.
Cell
binding agents may be of any kind presently known, or that become known and
includes peptides and non-peptides. The cell binding agent may be any compound
that
can bind a cell, either in a specific or non-specific manner. Generally, these
can be
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antibodies (especially monoclonal antibodies), lymphokines, hormones, growth
factors, vitamins, nutrient-transport molecules (such as transferrin), or any
other cell
binding molecule or substance.
[73] More specific examples of cell binding agents that can be used include:
(a) polyclonal antibodies;
(b) monoclonal antibodies;
(c) fragments of antibodies such as Fab, Fab', and F(ab')2, Fv (Parham, J.
Immunol. 131:2895-2902 (1983); Spring et al. J. Immunol. 113:470-478 (1974);
Nisonoff et al. Arch. Biochem. Biophys. 89:230-244 (1960));
(d) interferons (e.g. .alpha., .beta., .gamma.);
(e) lymphokines such as IL-2, IL-3, IL-4, IL-6;
(f) hormones such as insulin, TRH (thyrotropin releasing hormone), MSH
(melanocyte-stimulating hormone), steroid hormones, such as androgens and
estrogens;
(g) growth factors and colony-stimulating factors such as EGF, TGF-alpha,
FGF, VEGF, G-CSF, M-CSF and GM-CSF (Burgess, Immunology Today 5:155-158
(1984));
(h) transferrin (O'Keefe et al. J. Biol. Chem. 260:932-937 (1985)); and
(i) vitamins, such as folate.
Antibodies
[74] Selection of the appropriate cell binding agent is a matter of choice
that
depends upon the particular cell population that is to be targeted, but in
general,
antibodies are preferred if an appropriate one is available or can be
prepared, more
preferably a monoclonal antibody.
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[75] Monoclonal antibody techniques allow for the production of extremely
specific cell binding agents in the form of specific monoclonal antibodies.
Particularly well known in the art are techniques for creating monoclonal
antibodies
produced by immunizing mice, rats, hamsters or any other mammal with the
antigen
of interest such as the intact target cell, antigens isolated from the target
cell, whole
virus, attenuated whole virus, and viral proteins such as viral coat proteins.
Sensitized
human cells can also be used. Another method of creating monoclonal antibodies
is
the use of phage libraries of scFv (single chain variable region),
specifically human
scFv (see e.g., Griffiths et al., U.S. Patent Nos. 5,885,793 and 5,969,108;
McCafferty
et al., WO 92/01047; Liming et al., WO 99/06587).
[76] A typical antibody is comprised of two identical heavy chains and two
identical light chains that are joined by disulfide bonds. The variable region
is a
portion of the antibody heavy chains and light chains that differs in sequence
among
antibodies and that cooperates in the binding and specificity of each
particular
antibody for its antigen. Variability is not usually evenly distributed
throughout
antibody variable regions. It is typically concentrated within three segments
of a
variable region called complementarity-determining regions (CDRs) or
hypervariable
regions, both in the light chain and the heavy chain variable regions. The
more highly
conserved portions of the variable regions are called the framework regions.
The
variable regions of heavy and light chains comprise four framework regions,
largely
adopting a beta-sheet configuration, with each framework region connected by
the
three CDRs, which form loops connecting the beta-sheet structure, and in some
cases
forming part of the beta-sheet structure. The CDRs in each chain are held in
close
proximity by the framework regions and, with the CDRs from the other chain,
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contribute to the formation of the antigen binding site of antibodies (E. A.
Kabat et al.
Sequences of Proteins of Immunological Interest, Fifth Edition, 1991, NIH).
[77] The constant region is a portion of the heavy chain. While not involved
directly in binding an antibody to an antigen, it does exhibit various
effector
functions, such as participation of the antibody in antibody-dependent
cellular
toxicity.
[78] A suitable monoclonal antibody for use in the present invention includes
the
murine DS6 monoclonal antibody (U.S. Patent No. 6,596,503; ATCC deposit number
PTA-4449).
Humanized or Resurfaced DS6 Antibodies
[79] Preferably, a humanized anti-CA6 antibody is used as the cell binding
agent of
the present invention. A preferred embodiment of such a humanized antibody is
a
humanized DS6 antibody, or an epitope-binding fragment thereof.
[80] The goal of humanization is a reduction in the immunogenicity of a
xenogenic
antibody, such as a murine antibody, for introduction into a human, while
maintaining
the full antigen binding affinity and specificity of the antibody.
[81] Humanized antibodies may be produced using several technologies such as
resurfacing and CDR grafting. As used herein, the resurfacing technology uses
a
combination of molecular modeling, statistical analysis and mutagenesis to
alter the
non-CDR surfaces of antibody variable regions to resemble the surfaces of
known
antibodies of the target host.
[82] Strategies and methods for the resurfacing of antibodies, and other
methods
for reducing immunogenicity of antibodies within a different host, are
disclosed in US
Patent 5,639,641 (Pedersen et al.), which is hereby incorporated in its
entirety by
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reference. Briefly, in a preferred method, (1) position alignments of a pool
of
antibody heavy and light chain variable regions is generated to give a set of
heavy and
light chain variable region framework surface exposed positions wherein the
alignment positions for all variable regions are at least about 98% identical;
(2) a set
of heavy and light chain variable region framework surface exposed amino acid
residues is defined for a rodent antibody (or fragment thereof); (3) a set of
heavy and
light chain variable region framework surface exposed amino acid residues that
is
most closely identical to the set of rodent surface exposed amino acid
residues is
identified; (4) the set of heavy and light chain variable region framework
surface
exposed amino acid residues defined in step (2) is substituted with the set of
heavy
and light chain variable region framework surface exposed amino acid residues
identified in step (3), except for those amino acid residues that are within 5
A of any
atom of any residue of the complementarity-determining regions of the rodent
antibody; and (5) the humanized rodent antibody having binding specificity is
produced.
[83] Antibodies can be humanized using a variety of other techniques including
CDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat. Nos. 5,530,101; and
5,585,089), veneering or resurfacing (EP 0 592 106; EP 0 519 596; Padlan E.
A.,
1991, Molecular Immunology 28(4/5):489-498; Studnicka G. M. et al., 1994,
Protein
Engineering 7(6):805-814; Roguska M.A. et al., 1994, PNAS 91:969-973), and
chain
shuffling (U.S. Pat. No. 5,565,332). Human antibodies can be made by a variety
of
methods known in the art including phage display methods. See also U.S. Pat.
Nos.
4,444,887, 4,716,111, 5,545,806, and 5,814,318; and international patent
application
publication numbers WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654,
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WO 96/34096, WO 96/33735, and WO 91/10741 (said references incorporated by
reference in their entireties).
[84] In preferred embodiment, the present invention provides humanized
antibodies
or fragments thereof that recognizes a novel sialoglycotope (the CA6
glycotope) on
the Mucl mucin. In another embodiment, the humanized antibodies or epitope-
binding fragments thereof have the additional ability to inhibit growth of a
cell
expressing the CA6 glycotope.
[85] In more preferred embodiments, there are provided resurfaced or humanized
versions of the DS6 antibody wherein surface-exposed residues of the antibody
or its
fragments are replaced in both light and heavy chains to more closely resemble
known human antibody surfaces. The humanized DS6 antibodies or epitope-binding
fragments thereof of the present invention have improved properties. For
example,
humanized DS6 antibodies or epitope-binding fragments thereof specifically
recognize a novel sialoglycotope (the CA6 glycotope) on the Mucl mucin. More
preferably, the humanized DS6 antibodies or epitope-binding fragments thereof
have
the additional ability to inhibit growth of a cell expressing the CA6
glycotope. The
humanized antibody or an epitope-binding fragment thereof can be conjugated to
a
drug, such as a maytansinoid, to form a prodrug having specific cytotoxicity
towards
antigen-expressing cells by targeting the drug to the novel Mucl
sialoglycotope, CA6.
Cytotoxic conjugates comprising such antibodies and a small, highly toxic drug
(e.g.,
maytansinoids, taxanes, and CC-1065 analogs) can be used as a therapeutic for
treatment of tumors, such as breast and ovarian tumors.
[86] The humanized versions of the DS6 antibody are also fully characterized
herein with respect to their respective amino acid sequences of both light and
heavy
chain variable regions, the DNA sequences of the genes for the light and heavy
chain
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variable regions, the identification of the CDRs, the identification of their
surface
amino acids, and disclosure of a means for their expression in recombinant
form.
[87] In one embodiment, there is provided a humanized antibody or epitope-
binding fragment thereof having a heavy chain including CDRs having amino acid
sequences represented by SEQ ID NOs: 1-3:
SYNMH (SEQIDNO:1)
YIYPGNGATNYNQKFKG (SEQIDNO:2)
G D S V P F A Y (SEQ ID NO:3)
[88] When the heavy chain CDRs are determined by the AbM modeling software
they are represented by SEQ ID NOs:20-22:
GYTFTSYNMH (SEQIDNO:20)
YIYPGNGATN(SEQIDNO:21)
G D S V P F A Y (SEQ ID NO:22)
[89] In the same embodiment, the humanized antibody or epitope-binding
fragment
thereof has a light chain that comprises CDRs having amino acid sequences
represented by SEQ ID NOS:4-6:
S A H S S V S F M H (SEQ ID NO:4)
STSSLAS (SEQID NO:5)
QQRSSFPLT (SEQIDNO:6)..
[90] Also provided are humanized antibodies and epitope-binding fragments
thereof having a light chain variable region that has an amino acid sequence
that
shares at least 90% sequence identity with an amino acid sequence represented
by
SEQ ID NO:7 or SEQ ID NO:8:
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QIVLTQSPAIMSASPGEKVTITCSAHSSVSFMHWFQQKPGTSPKLWIYS
TSSLASGVPARFGGSGSGTSYSLTISRMEAEDAATYYCQQRSSFPLTFG
AGTKLELKR. (SEQ ID NO:7)
EIVLTQSPATMSASPGERVTITCSAHSSVSFMHWFQQKPGTSPKLWIYS
TSSLASGVPARFGGSGSGTSYSLTISSMEAEDAATYYCQQRSSFPLTFG
AGTKLELKR. (SEQ ID NO:8)
[91] Similarly, there are provided humanized antibodies and epitope-binding
fragments thereof having a heavy chain variable region that has an amino acid
sequence that shares at least 90% sequence identity with an amino acid
sequence
represented by SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11:
QAYLQQSGAELVRSGAS VKMSCKASGYTFTSYNMHW VKQTPGQGLE
WIGYIYP GNGATNYNQKFKGKATLTADPS S STAYMQIS SLTSEDSAVY
FCARGDSVPFAYWGQGTLVTVSA. (SEQ ID NO:9)
QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLE
WIGYIYPGNGATNYNQKFQGKATLTADTS S STAYMQIS SLTSEDSAVY
FCARGDSVPFAYWGQGTLVTVSA. (SEQ IDNO:l0)
QAQLVQSGAEV VKPGAS VKMSCKASGYTFTSYNMHW VKQTPGQGLE
WIGYIYPGNGATNYNQKFQGKATLTADPSS STAYMQISSLTSEDSAVY
FCARGDSVPFAYWGQGTLVTVSA. (SEQ ID NO:11)
[92] In another embodiment, humanized antibodies and epitope-binding fragments
thereof are provided having a humanized or resurfaced light chain variable
region
having an amino acid sequence corresponding to SEQ ID NO: 8
EIVLTQSPATMSASPGERVTITCSAHSSVSFMHWFQQKPGTSPKLWIYS
TSSLASGVPARFGGSGSGTSYSLTISSMEAEDAATYYCQQRSSFPLTFG
AGTKLELKR. (SEQ ID NO:8)
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[93] Similarly, humanized antibodies and epitope-binding fragments thereof are
provided having a humanized or resurfaced heavy chain variable region having
an
amino acid sequence corresponding to SEQ ID NO:10 or SEQ ID NO:11:
QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLE
WIGYIYPGNGATNYNQKFQGKATLTADTS S STAYMQIS SLTSEDSAVY
FCARGDSVPFAYWGQGTLVTVSA. (SEQ IDNO:10)
QAQLV QSGAEV VKPGAS VKMSCKAS GYTFTSYNIVII IW VKQTPGQGLE
WIGYIYPGNGATNYNQKFQGKATLTADPS S STAYMQIS SLTSEDSAVY
FCARGDSVPFAYWGQGTLVTVSA. (SEQ ID NO:11)
[94] The humanized antibodies and epitope-binding fragments thereof of the
present invention can also include versions of light and/or heavy chain
variable
regions in which human surface amino acid residues in proximity to the CDRs
are
replaced by the corresponding muDS6 surface residues at one or more positions
defined by the residues in Table 1 (Kabat numbering) marked with an asterisk
in order
to retain the binding affinity and specificity of muDS6.
Table 1
muDS6 framework residues proximal to a
CDR (Kabat numbering)
Light chain Heavy chain
1* Q1
V3 K64*
T5 P73*
P40 S74
G57
A60
S67
E81
[95] The primary amino acid and DNA sequences of the DS6 antibody light and
heavy chains, and of humanized versions thereof, are disclosed herein.
However, the
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scope of the present invention is not limited to antibodies and fragments
comprising
these sequences. Instead, all antibodies and fragments that specifically bind
to CA6 as
a unique tumor-specific glycotope on the Muc 1 receptor are included in the
present
invention. Preferably, the antibodies and fragments that specifically bind to
CA6 also
antagonize the biological activity of the receptor. More preferably, such
antibodies
further are substantially devoid of agonist activity. Thus, antibodies and
antibody
fragments of the present invention may differ from the DS6 antibody or the
humanized derivatives thereof, in the amino acid sequences of their scaffold,
CDRs,
and/or light chain and heavy chain, and still fall within the scope of the
present
invention.
[96] The CDRs of the DS6 antibody are identified by modeling and their
molecular
structures have been predicted. Again, while the CDRs are important for
epitope
recognition, they are not essential to the antibodies and fragments of the
invention.
Accordingly, antibodies and fragments are provided that have improved
properties
produced by, for example, affinity maturation of an antibody of the present
invention.
[97] The mouse light chain IgVK ap4 germline gene and heavy chain IgVh J558.41
germline gene from which DS6 was likely derived are shown in FIG. 11 aligned
with
the sequence of the DS6 antibody. The comparison identifies probable somatic
mutations in the DS6 antibody, including several in the CDRs.
[98] The sequence of the heavy chain and light chain variable region of the
DS6
antibody, and the sequences of the CDRs of the DS6 antibody were not
previously
known and are set forth in Figures 9A and 9B. Such information can be used to
produce humanized versions of the DS6 antibody.
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Antibody Fragments
[99] The antibodies of the present invention include both the full length
antibodies
discussed above, as well as epitope-binding fragments. As used herein,
"antibody
fragments" include any portion of an antibody that retains the ability to bind
to the
epitope recognized by the full length antibody, generally termed "epitope-
binding
fragments." Examples of antibody fragments include, but are not limited to,
Fab,
Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies,
disulfide-
linked Fvs (dsFv) and fragments comprising either a VL or VH region. Epitope-
binding fragments, including single-chain antibodies, may comprise the
variable
region(s) alone or in combination with the entirety or a portion of the
following: hinge
region, CH1, CH2, and CH3 domains.
[100] Such fragments may contain one or both Fab fragments or the F(ab')2
fragment. Preferably, the antibody fragments contain all six CDRs of the whole
antibody, although fragments containing fewer than all of such regions, such
as three,
four or five CDRs, are also functional. Further, the fragments may be or may
combine members of any one of the following immunoglobulin classes: IgG, IgM,
IgA, IgD, or IgE, and the subclasses thereof.
[101] Fab and F(ab')2 fragments may be produced by proteolytic cleavage, using
enzymes such as papain (Fab fragments) or pepsin (F(ab')2 fragments).
[102] The single-chain FVs (scFvs) fragments are epitope-binding fragments
that
contain at least one fragment of an antibody heavy chain variable region (VH)
linked
to at least one fragment of an antibody light chain variable region (VL). The
linker
may be a short, flexible peptide selected to assure that the proper three-
dimensional
folding of the (VL) and (VH) regions occurs once they are linked so as to
maintain the
target molecule binding-specificity of the whole antibody from which the
single-chain
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antibody fragment is derived. The carboxyl terminus of the (VL) or (VH)
sequence
may be covalently linked by a linker to the amino acid terminus of a
complementary
(VL) or (VH) sequence.
[103] Single-chain antibody fragments of the present invention contain amino
acid
sequences having at least one of the variable or complementarity determining
regions
(CDRs) of the whole antibodies described in this specification, but are
lacking some
or all of the constant domains of those antibodies. These constant domains are
not
necessary for antigen binding, but constitute a major portion of the structure
of whole
antibodies. Single-chain antibody fragments may therefore overcome some of the
problems associated with the use of antibodies containing a part or all of a
constant
domain. For example, single-chain antibody fragments tend to be free of
undesired
interactions between biological molecules and the heavy-chain constant region,
or
other unwanted biological activity. Additionally, single-chain antibody
fragments are
considerably smaller than whole antibodies and may therefore have greater
capillary
permeability than whole antibodies, allowing single-chain antibody fragments
to
localize and bind to target antigen-binding sites more efficiently. Also,
antibody
fragments can be produced on a relatively large scale in prokaryotic cells,
thus
facilitating their production. Furthermore, the relatively small size of
single-chain
antibody fragments makes them less likely to provoke an immune response in a
recipient than whole antibodies.
[104] Single-chain antibody fragments may be generated by molecular cloning,
antibody phage display library or similar techniques well known to the skilled
artisan.
These proteins may be produced, for example, in eukaryotic cells or
prokaryotic cells,
including bacteria. The epitope-binding fragments of the present invention can
also
be generated using various phage display methods known in the art. In phage
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methods, functional antibody domains are displayed on the surface of phage
particles
which carry the polynucleotide sequences encoding them. In particular, such
phage
can be utilized to display epitope-binding domains expressed from a repertoire
or
combinatorial antibody library (e.g., human or murine). Phage expressing an
epitope-
binding domain that binds the antigen of interest can be selected or
identified with
antigen, e.g., using labeled antigen bound or captured to a solid surface or
bead.
Phage used in these methods are typically filamentous phage including fd and
M13
binding domains expressed from phage with Fab, Fv or disulfide-stabilized Fv
antibody domains recombinantly fused to either the phage gene III or gene VIII
protein..
[105] Examples of phage display methods that can be used to make the epitope-
binding fragments of the present invention include those disclosed in Brinkman
et al.,
1995, J. Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods
184:177-186; Kettleborough et al., 1994, Eur. J. Immunol. 24:952-958; Persic
et al.,
1997, Gene 187:9-18; Burton et al., 1994, Advances in Immunology 57:191-280;
PCT
application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737;
WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S.
Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;
5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743
and
5,969,108; each of which is incorporated herein by reference in its entirety.
[106] After phage selection, the regions of the phage encoding the fragments
can be
isolated and used to generate the epitope-binding fragments through expression
in a
chosen host, including mammalian cells, insect cells, plant cells, yeast, and
bacteria,
using recombinant DNA technology, e.g., as described in detail below. For
example,
techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also
be
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employed using methods known in the art such as those disclosed in PCT
publication
WO 92/22324; Mullinax et al., 1992, BioTechniques 12(6):864-869; Sawai et al.,
1995, AJRI 34:26-34; and Better et al., 1988, Science 240:1041-1043; said
references
incorporated by reference in their entireties. Examples of techniques which
can be
used to produce single-chain Fvs and antibodies include those described in
U.S. Pat.
Nos. 4,946,778 and 5,258,498; Huston et al., 1991, Methods in Enzymology
203:46-
88; Shu et al., 1993, PNAS 90:7995-7999; Skerra et al., 1988, Science 240:1038-
1040.
Functional Equivalents
[107] Also included within the scope of the invention are functional
equivalents of
the anti-CA6 antibody and the humanized anti-CA6 antibody. The term
"functional
equivalents" includes antibodies with homologous sequences, chimeric
antibodies,
artificial antibodies and modified antibodies, for example, wherein each
functional
equivalent is defined by its ability to bind to CA6. The skilled artisan will
understand
that there is an overlap in the group of molecules termed "antibody fragments"
and
the group termed "functional equivalents." Methods of producing functional
equivalents are disclosed, for example, in PCT Application WO 93/21319,
European
Patent Application No. 239,400; PCT Application WO 89/09622; European Patent
Application 338,745; and European Patent Application EP 332,424, which are
incorporated in their respective entireties by reference.
[108] Antibodies with homologous sequences are those antibodies with amino
acid
sequences that have sequence homology with amino acid sequence of an anti-CA6
antibody and a humanized anti-CA6 antibody of the present invention.
Preferably
homology is with the amino acid sequence of the variable regions of the anti-
CA6
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antibody and humanized anti-CA6 antibody of the present invention. "Sequence
homology" as applied to an amino acid sequence herein is defined as a sequence
with
at least about 90%, 91%, 92%, 93%, or 94% sequence homology, and more
preferably
at least about 95%, 96%, 97%, 98%, or 99% sequence homology to another amino
acid sequence, as determined, for example, by the FASTA search method in
accordance with Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85, 2444-2448
(1988).
[109] As used herein, a chimeric antibody is one in which different portions
of an
antibody are derived from different animal species. For example, an antibody
having
a variable region derived from a murine monoclonal antibody paired with a
human
immunoglobulin constant region. Methods for producing chimeric antibodies are
known in the art. See, e.g., Morrison, 1985, Science 229:1202; Oi et al.,
1986,
BioTechniques 4:214; Gillies et al., 1989, J. Immunol. Methods 125:191-202;
U.S.
Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein
by
reference in their entireties.
[110] Humanized forms of chimeric antibodies are made by substituting the
complementarity determining regions of, for example, a mouse antibody, into a
human framework domain, e.g., see PCT Pub. No. W092/22653. Humanized chimeric
antibodies preferably have constant regions and variable regions other than
the
complementarity determining regions (CDRs) derived substantially or
exclusively
from the corresponding human antibody regions and CDRs derived substantially
or
exclusively from a mammal other than a human.
[111] Artificial antibodies include scFv fragments, diabodies, triabodies,
tetrabodies
and mru (see reviews by Winter, G. and Milstein, C., 1991, Nature 349: 293-
299;
Hudson, P.J., 1999, Current Opinion in Immunology 11: 548-557), each of which
has
33
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antigen-binding ability. In the single chain Fv fragment (scFv), the VH and VL
domains of an antibody are linked by a flexible peptide. Typically, this
linker peptide
is about 15 amino acid residues long. If the linker is much smaller, for
example 5
amino acids, diabodies are formed, which are bivalent scFv dimers. If the
linker is
reduced to less than three amino acid residues, trimeric and tetrameric
structures are
formed that are called triabodies and tetrabodies. The smallest binding unit
of an
antibody is a CDR, typically the CDR2 of the heavy chain which has sufficient
specific recognition and binding that it can be used separately. Such a
fragment is
called a molecular recognition unit or mru. Several such mrus can be linked
together
with short linker peptides, therefore forming an artificial binding protein
with higher
avidity than a single mru.
[112] The functional equivalents of the present application also include
modified
antibodies, e.g., antibodies modified by the covalent attachment of any type
of
molecule to the antibody. For example, modified antibodies include antibodies
that
have been modified, e.g., by glycosylation, acetylation, pegylation,
phosphorylation,
amidation, derivatization by known protecting/blocking groups, proteolytic
cleavage,
linkage to a cellular ligand or other protein, etc. The covalent attachment
does not
prevent the antibody from generating an anti-idiotypic response. These
modifications
may be carried out by known techniques, including, but not limited to,
specific
chemical cleavage, acetylation, formylation, metabolic synthesis of
tunicamycin, etc.
Additionally, the modified antibodies may contain one or more non-classical
amino
acids.
[113] Functional equivalents may be produced by interchanging different CDRs
on
different chains within different frameworks. Thus, for example, different
classes of
antibody are possible for a given set of CDRs by substitution of different
heavy
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chains, whereby, for example, IgGl-4, IgM, IgAl-2, IgD, IgE antibody types and
isotypes may be produced. Similarly, artificial antibodies within the scope of
the
invention may be produced by embedding a given set of CDRs within an entirely
synthetic framework.
[114] Functional equivalents may be readily produced by mutation, deletion
and/or
insertion within the variable and/or constant region sequences that flank a
particular
set of CDRs, using a wide variety of methods known in the art.
[115] The antibody fragments and functional equivalents of the present
invention
encompass those molecules with a detectable degree of binding to CA6, when
compared to the DS6 antibody. A detectable degree of binding includes all
values in
the range of at least 10-100%, preferably at least 50%, 60% or 70%, more
preferably
at least 75%, 80%, 85%, 90%, 95% or 99% the binding ability of the murine DS6
antibody to CA6.
Improved Antibodies
[116] The CDRs are of primary importance for epitope recognition and antibody
binding. However, changes may be made to the residues that comprise the CDRs
without interfering with the ability of the antibody to recognize and bind its
cognate
epitope. For example, changes that do not affect epitope recognition, yet
increase the
binding affinity of the antibody for the epitope may be made.
[117] Thus, also included in the scope of the present invention are improved
versions of both the murine and humanized antibodies, which also specifically
recognize and bind CA6, preferably with increased affinity.
[118] Several studies have surveyed the effects of introducing one or more
amino
acid changes at various positions in the sequence of an antibody, based on the
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knowledge of the primary antibody sequence, on its properties such as binding
and
level of expression (Yang, W. P. et al., 1995, J. Mol. Biol., 254, 392-403;
Rader, C. et
al., 1998, Proc. Natl. Acad. Sci. USA, 95, 8910-8915; Vaughan, T. J. et al.,
1998,
Nature Biotechnology, 16, 535-539).
[119] In these studies, equivalents of the primary antibody have been
generated by
changing the sequences of the heavy and light chain genes in the CDR1, CDR2,
CDR3, or framework regions, using methods such as oligonucleotide-mediated
site-
directed mutagenesis, cassette mutagenesis, error-prone PCR, DNA shuffling, or
mutator-strains of E. coli (Vaughan, T. J. et al., 1998, Nature Biotechnology,
16, 535-
539; Adey, N. B. et al., 1996, Chapter 16, pp. 277-291, in "Phage Display of
Peptides
and Proteins", Eds. Kay, B. K. et al., Academic Press). These methods of
changing
the sequence of the primary antibody have resulted in improved affinities of
the
secondary antibodies (Gram, H. et al., 1992, Proc. Natl. Acad. Sci. USA, 89,
3576-
3580; Boder, E. T. et al., 2000, Proc. Natl. Acad. Sci. USA, 97, 10701-10705;
Davies,
J. and Riechmann, L., 1996, Immunotechnolgy, 2, 169-179; Thompson, J. et al.,
1996,
J. Mol. Biol., 256, 77-88; Short, M. K. et al., 2002, J. Biol. Chem., 277,
16365-16370;
Furukawa, K. et al., 2001, J. Biol. Chem., 276, 27622-27628).
[120] By a similar directed strategy of changing one or more amino acid
residues of
the antibody, the antibody sequences described in this invention can be used
to
develop anti-CA6 antibodies with improved functions, including improved
affinity for
CA6.
[121] Improved antibodies also include those antibodies having improved
characteristics that are prepared by the standard techniques of animal
immunization,
hybridoma formation and selection for antibodies with specific characteristics
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Cytotoxic Agents
[122] The cytotoxic agent used in the cytotoxic conjugate of the present
invention
may be any compound that results in the death of a cell, or induces cell
death, or in
some manner decreases cell viability. Preferred cytotoxic agents include, for
example, maytansinoids and maytansinoid analogs, taxoids, CC-1065 and CC-1065
analogs, dolastatin and dolastatin analogs, defined below. These cytotoxic
agents are
conjugated to the antibodies, antibodies fragments, functional equivalents,
improved
antibodies and their analogs as disclosed herein
[123] The cytotoxic conjugates may be prepared by in vitro methods. In order
to
link a drug or prodrug to the antibody, a linking group is used. Suitable
linking
groups are well known in the art and include disulfide groups, thioether
groups, acid
labile groups, photolabile groups, peptidase labile groups and esterase labile
groups.
Preferred linking groups are disulfide groups and thioether groups. For
example,
conjugates can be constructed using a disulfide exchange reaction or by
forming a
thioether bond between the antibody and the drug or prodrug.
Maytansinoids
[124] Among the cytotoxic agents that maybe used in the present invention to
form
a cytotoxic conjugate, are maytansinoids and maytansinoid analogs. Examples of
suitable maytansin6ids include maytansinol and maytansinol analogs.
Maytansinoids
are drugs that inhibit microtubule formation and that are highly toxic to
mammalian
cells.
[125] Examples of suitable maytansinol analogues include those having a
modified
aromatic ring and those having modifications at other positions. Such suitable
maytansinoids are disclosed in U.S. Patent Nos. 4,424,219; 4,256,746;
4,294,757;
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4,307,016; 4,313,946; 4,315,929; 4,331,598; 4,361,650; 4,362,663; 4,364,866;
4,450,254; 4,322,348; 4,371,533; 6,333,410; 5,475,092; 5,585,499; and
5,846,545.
[126] Specific examples of suitable analogues of maytansinol having a modified
aromatic ring include:
[127] (1) C-19-dechloro (U.S. Pat. No. 4,256,746) (prepared by LAH reduction
of
ansamytocin P2);
[128] (2) C-20-hydroxy (or C-20-demethyl) +/-C-19-dechloro (U.S. Pat. Nos.
4,361,650 and 4,307,016) (prepared by demethylation using Streptomyces or
Actinomyces or dechlorination using LAH); and
[129] (3) C-20-demethoxy, C-20-acyloxy (-OCOR), +/-dechloro (U.S. Pat. No.
4,294,757) (prepared by acylation using acyl chlorides).
[130] Specific examples of suitable analogues of maytansinol having
modifications
of other positions include:
[131] (1) C-9-SH (U.S. Pat. No. 4,424,219) (prepared by the reaction of
maytansinol
with H2S or P2S5);
[132] (2) C-14-alkoxymethyl (demethoxy/CH2OR) (U.S. Pat. No. 4,331,598);
[133] (3) C-14-hydroxymethyl or acyloxymethyl (CH2OH or CHZOAc) (U.S. Pat.
No. 4,450,254) (prepared from Nocardia);
[134] (4) C-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866) (prepared by the
conversion of maytansinol by Streptomyces);
[135] (5) C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated from
Trewia nudiflora);
[136] (6) C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared
by
the demethylation of maytansinol by Streptomyces); and
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[137] (7) 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by the titanium
trichloride/LAH reduction of maytansinol).
[138] In a preferred embodiment, the cytotoxic conjugates of the present
invention
utilize the thiol-containing maytansinoid (DM1), formally termed N2'-deacetyl-
N2'-(3-
mercapto-l-oxopropyl)-maytansine, as the cytotoxic agent. DMI is represented
by
the following structural formula (I):
O
O
i SH
CI O O
MeO N O
0
NH O
OH
Me0 (I)
[139] In another preferred embodiment, the cytotoxic conjugates of the present
invention utilize the thiol-containing maytansinoid N2'-deacetyl-N-2'(4-methyl-
4-
mercapto-l-oxopentyl)-maytansine as the cytotoxic agent. DM4 is represented by
the
following structural formula (II):
O
O
N SH
Me0 ~O
nO
e(II)
[140] In further embodiments of the invention, other maytansines, including
thiol
and disulfide-containing maytansinoids bearing a mono or di-alkyl substitution
on the
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carbon atom bearing the sulfur atom, may be used. These include a maytansinoid
having, at C-3, C-14 hydroxymethyl, C-15 hydroxy, or C-20 desmethyl, an
acylated
amino acid side chain with an acyl group bearing a hindered sulfhydryl group,
wherein the carbon atom of the acyl group bearing the thiol functionality has
one or
two substituents, said substituents being CH3, C2H5, linear or branched alkyl
or
alkenyl having from 1 to 10 carbon atoms, cyclic alkyl or alkenyl having from
3 to 10
carbon atoms, phenyl, substituted phenyl, or heterocyclic aromatic or
heterocycloalkyl
radical, and further wherein one of the substituents can be H, and wherein the
acyl
group has a linear chain length of at least three carbon atoms between the
carbonyl
functionality and the sulfur atom.
[141] Such additional maytansines include compounds represented by
formula.(III):
O
N
cl O
\
MeO N O
O
NH O
OH
(III)
MeO
wherein:
Y' represents
(CR7CR8)1(CR9=CR1o)pC=CqAr(CR5CR6)mDõ(CR11=CR12)r(C=C)sBt(CR3CR4)õCRIRz
SZ,
wherein:
Rl and R2 are each independently CH3, C2H5i linear alkyl or alkenyl having
from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3
to 10
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carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or
heterocycloalkyl
radical, and in addition R2 can be H;
A, B, D are cycloalkyl or cycloalkenyl having 3 -10 carbon atoms, simple or
substituted aryl or heterocyclic aromatic or heterocycloalkyl radical;
R3, R4, R5, R6, R7, R8, R9, Rl t, and R12 are each independently H, CH3, C2H5,
linear
alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or
alkenyl
having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic
aromatic
or heterocycloalkyl radical;
1, m, n, o, p, q, r, s, and t are each independently 0 or an integer of from 1
to 5,
provided that at least two of 1, m, n, o, p, q, r, s and t are not zero at any
one time; and
Z is H, SR or -COR, wherein R is linear alkyl or alkenyl having from 1 to 10
carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon
atoms,
or simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl
radical.
[142] Preferred embodiments of formula (III) include compounds of formula
(III)
wherein:
Rl isH,RzismethylandZisH.
Rl and R2 are methyl and Z is H.
RI is H, R2 is methyl, and Z is -SCH3.
Rl and R2 are methyl, and Z is -SCH3.
[143] Such additional maytansines also include compounds represented by
formula
(IV-L), (IV-D), or (W-D,L):
H3C,, H O H3C ,~.'H O H3C H j
~'~~ O ~ O
/O NY N Y / N Y
May I May O I May O
O
(IV-L) (IV-D)
(IV-D,L)
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wherein:
Y represents (CR7CR$),(CR5CR6)m(CR3CR4)õCR1R2SZ,
wherein:
Rl and R2 are each independently CH3, C2H5, linear alkyl or alkenyl having
from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3
to 10
carbon atoms, phenyl, substituted phenyl, or heterocyclic aromatic or
heterocycloalkyl
radical, and in addition R2 can be H;
R3, R4, R5, R6, R7 and R8 are each independently H, CH3, C2H5, linear alkyl or
alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl
having
from 3 to 10 carbon atoms, phenyl, substituted phenyl, or heterocyclic
aromatic or
heterocycloalkyl radical;
1, m and n are each independently an integer of from 1 to 5, and in addition n
can be 0;
Z is H, SR or -COR wherein R is linear or branched alkyl or alkenyl having
from I to 10 carbon atoms, cyclic alkyl or alkenyl having from 3 to 10 carbon
atoms,
or simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl
radical; and
May represents a maytansinoid which bears the side chain at C-3, C-14
hydroxymethyl, C-15 hydroxy or C-20 desmethyl.
[144] Preferred embodiments of formulas (IV-L), (IV-D) and (IV-D,L) include
compounds of formulas (IV-L), (IV-D) and (IV-D,L) wherein:
RI is H, R2 is methyl, R5, R6, R7, and R8 are each H,1 and m are each 1, n is
0,
and Z is H.
Rl and R2 are methyl, R5, R6, R7, R8 are each H,1 and m are 1, n is 0, and Z
is
H.
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Rl is H, R2 is methyl, R5, R6, R7, and R8 are each H,1 and m are each 1, n is
0,
and Z is -SCH3.
Rl and R2 are methyl, R5, R6, R7, R8 are each H,1 and m are 1, n is 0, and Z
is
-SCH3.
[145] Preferably the cytotoxic agent is represented by formula (IV-L).
[146] Such additional maytansines also include compounds represented by
formula
(V):
O
"K
N Y
cl O O
Me0 N O
O 'I-," "'~ ---~
NH O
OH
Me0 (V)
wherein:
Y represents (CR7CR$)1(CR5CR6)m(CR3CR4)nCR1R2SZ,
wherein:
Rl and R2 are each independently CH3, C2H5, linear alkyl or alkenyl having
from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3
to 10
carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or
heterocycloalkyl
radical, and in addition R2 can be H;
R3, R4, R5, R6, R7 and R$ are each independently H, CH3, C2H5, linear alkyl or
alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl
having
43
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from 3 to 10 carbon atoms, phenyl, substituted phenyl, or heterocyclic
aromatic or
heterocycloalkyl radical;
1, m and n are each independently an integer of from 1 to 5, and in addition n
can be 0; and
Z is H, SR or -COR, wherein R is linear alkyl or alkenyl having from 1 to 10
carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon
atoms,
or simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl
radical.
[147] Preferred embodiments of formula (V) include compounds of formula (V)
wherein:
R1 is H, R2 is methyl, R5, R6, R7, and R8 are each H; 1 and m are each 1; n is
0; and Z is H.
Rl and R2 are methyl; R5, R6, R7, R8 are each H,1 and m are 1; n is 0; and Z
is
H.
Rl is H, R2 is methyl, R5, R6, R7, and R8 are each H,1 and m are each 1, n is
0,
and Z is -SCH3.
Rl and R2 are methyl, R5, R6, R7, R8 are each H, 1 and m are 1, n is 0, and Z
is
-SCH3.
[148] Such additional maytansines further include compounds represented by
formula (VI-L), (VI-D), or (VI-D,L):
O O
H3C H D H3C H ~ O HC H~
May N Y2 May i Y2 May i Y2
0 O O
(VI-L) (VI-D) (VI-D, L)
wherein:
Y2 represents (CR7CRg)1(CR5CRb)n,(CR3CR4)õCR1R2SZ2,
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wherein:
Rl and R2 are each independently CH3, C2H5, linear alkyl or alkenyl having
from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3
to 10
carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or
heterocycloalkyl
radical, and in addition R2 can be H;
R3, R4, R5, R6, R7 and R8 are each independently H, CH3, C2H5, linear cyclic
alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or
alkenyl
having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic
aromatic
or heterocycloalkyl radical;
1, m and n are each independently an integer of from 1 to 5, and in addition n
can be 0;
Z2 is SR or COR, wherein R is linear alkyl or alkenyl having from 1 to 10
carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon
atoms,
or simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl
radical; and
May is a maytansinoid.
[149] Such additional maytansines also include compounds represented by
formula
(VII):
O
O ~
N Y2'
CI O I
MeO N
O
NH O
OH
MeO (VII)
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wherein:
Y2' represents
(CR7CR8)1(CR9=CRlo)p(C=C)yA,(CR5CR6)mD,,(CR11=CR12)r(C=C)sBt(CR3CR4)nCRI
R2SZ2,
wherein:
Rl and R2 are each independently CH3, C2H5, linear branched or alkyl or
alkenyl having from 1 to 10 carbon atoms, cyclic alkyl or alkenyl having from
3 to 10
carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or
heterocycloalkyl
radical, and in addition R2 can be H;
A, B, and D each independently is cycloalkyl or cycloalkenyl having 3 to 10
carbon atoms, simple or substituted aryl, or heterocyclic aromatic or
heterocycloalkyl
radical;
R3, R4, R5, R6, R7, R8, R9, R11, and R12 are each independently H, CH3, C2H5,
linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic
alkyl or
alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or
heterocyclic
aromatic or heterocycloalkyl radical;
1, m, n, o, p, q, r, s, and t are each independently 0 or an integer of from 1
to 5,
provided that at least two of 1, m, n, o, p, q, r, s and t are not zero at any
one time; and
Z2 is SR or -COR, wherein R is linear alkyl or alkenyl having from 1 to 10
carbon atoms, branched or cyclic alkyl or alkenyl having from 3 - 10 carbon
atoms, or
simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl
radical.
[150] Preferred embodiments of formula (VII) include compounds of formula
(VII)
wherein: R1 is H and R2 is methyl.
[151] The above-mentioned maytansinoids can be conjugated to anti-CA6 antibody
DS6, or a homologue or fragment thereof, wherein the antibody is linked to the
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maytansinoid using the thiol or disulfide functionality that is present on the
acyl group
of an acylated amino acid side chain found at C-3, C-14 hydroxymethyl, C-15
hydroxy or C-20 desmethyl of the maytansinoid, and wherein the acyl group of
the
acylated amino acid side chain has its thiol or disulfide functionality
located at a
carbon atom that has one or two substituents, said substituents being CH3,
C2H5i
linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic
alkyl or
alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or
heterocyclic
aromatic or heterocycloalkyl radical, and in addition one of the substituents
can be H,
and wherein the acyl group has a linear chain length of at least three carbon
atoms
between the carbonyl functionality and the sulfur atom.
[152] A preferred conjugate of the present invention is the one that comprises
the
anti- anti-CA6 antibody DS6, or a homologue or fragment thereof, conjugated to
a
maytansinoid of formula (VIII):
O
i Yl'
CI O
Me0 N O
O
NH O
OH
Me0 (VIII)
wherein:
Yl' represents
(CR7CR$)1(CR9=CR10)p(C=C)yAr(CR5CR6)mDu(CR11=CR12)r(C=C)sBt(CR3CR4)nCRI Rz
S-,
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wherein:
A, B, and D, each independently is cycloalkyl or cycloalkenyl having 3 -10
carbon atoms, simple or substituted aryl, or heterocyclic aromatic or
heterocycloalkyl
radical;
R3, R4, R5, R6, R7, Rg; R9, R, i, and R12 are each independently H, CH3, C2H5,
linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic
alkyl or
alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or
heterocyclic
aromatic or heterocycloalkyl radical; and
l, m, n, o, p, q, r, s, and t are each independently 0 or an integer of from 1
to 5,
provided that at least two of 1, m, n, o, p, q, r, s and t are non-not zero at
any one time.
[153] Preferably, Rl is H and R2 is methyl or Ri and R2 are methyl.
[154] An even more preferred conjugate of the present invention is the one
that
comprises the anti-CA6 antibody DS6, or a homologue or fragment thereof,
conjugated to a maytansinoid of formula (IX-L), (IX-D), or (IX-D,L):
H3C~~ H D H3C H O ~ H3C H p
.,~~
nnayO N ~ Y, May Di Y, May I X i Y,
o D o
IX-L IX-D IX-D,L
wherein:
Y1 represents (CR7CR8)i(CR5CR5)m(CR3CR4)nCR1R2S-,
wherein:
R, and R2 are each independently CH3, C2H5, linear alkyl or alkenyl having
from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3
to 10
carbon atoms, phenyl, substituted phenyl, heterocyclic aromatic or
heterocycloalkyl
radical, and in addition R2 can be H;
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R3, R4, R5, R6, R7 and R8 are each independently H, CH3, C2H5, linear alkyl or
alkenyl having from I to 10 carbon atoms, branched or cyclic alkyl or alkenyl
having
from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic
or
heterocycloalkyl radical;
1, m and n are each independently an integer of from 1 to 5, and in addition n
can be 0; and
May represents a maytansinol which bears_the side chain at C-3, C-14
hydroxymethyl, C-15 hydroxy or C-20 desmethyl.
[155] Preferred embodiments of formulas (IX-L), (IX-D) and (IX-D,L) include
compounds of formulas (IX-L), (IX-D) and (IX-D,L) wherein:
R, is H and R2 is methyl or R, and R2 are methyl,
R1 is H, R2 is methyl, R5, R6, R7 and R8 are each H; 1 and m are each 1; n is
0,
R, and R2 are methyl; R5, R6, R7 and R8 are each H; 1 and m are 1; n is 0.
[156] Preferably the cytotoxic agent is represented by formula (IX-L).
[1571 An further preferred conjugate of the present invention is the one that
comprises the anti-CA6 antibody DS6, or a homologue or fragment thereof,
conjugated to a maytansinoid of formula (X):
O
O
N Y,
CI O
O
N O
NH O
OH
MeO (X)
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wherein the substituents are as defined for formula (IX) above.
[158] Especially preferred are any of the above-described compounds, wherein
R, is
H, R2 is methyl, R5, R6, R7 and R8 are each H, 1 and m are each 1, and n is 0.
[159] Further especially preferred are any of the above-described compounds,
wherein Rl and R2 are methyl, R5, R6, R7, R$ are each H, 1 and m are 1, and n
is 0
[160] Further, the L-aminoacyl stereoisomer is preferred.
[161] Each of the maytansinoids taught in pending U.S. patent application
number
10/849,136, filed May 20, 2004, may also be used in the cytotoxic conjugate of
the
present invention. The entire disclosure of U.S. patent application number
10/849,136 is incorporated herein by reference.
Disulfide-containin lg inkinggroups
[162] In order to link the maytansinoid to a cell binding agent, such as the
DS6
antibody, the maytansinoid comprises a linking moiety. The linking moiety
contains '
a chemical bond that allows for the release of fully active maytansinoids at a
particular site. Suitable chemical bonds are well known in the art and include
disulfide bonds, acid labile bonds, photolabile bonds, peptidase labile bonds
and
esterase labile bonds. Preferred are disulfide bonds.
[163] The linking moiety also comprises a reactive chemical group. In a
preferred
embodiment, the reactive chemical group can be covalently bound to the
maytansinoid via a disulfide bond linking moiety.
[164] Particularly preferred reactive chemical groups are N-succinimidyl
esters and
N-sulfosuccinimidyl esters.
[165] Particularly preferred maytansinoids comprising a linking moiety that
contains
a reactive chemical group are C-3 esters of maytansinol and its analogs where
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linking moiety contains a disulfide bond and the chemical reactive group
comprises a
N-succinimidyl or N-sulfosuccinimidyl ester.
[166] Many positions on maytansinoids can serve as the position to chemically
link
the linking moiety. For example, the C-3 position having a hydroxyl group, the
C-14
position modified with hydroxymethyl, the C-15 position modified with hydroxy
and
the C-20 position having a hydroxy group are all expected to be useful.
However the
C-3 position is preferred and the C-3 position of maytansinol is especially
preferred.
[167] While the synthesis of esters of maytansinol having a linking moiety is
described in terms of disulfide bond-containing linking moieties, one of skill
in the art
will understand that linking moieties with other chemical bonds (as described
above)
can also be used with the present invention, as can other maytansinoids.
Specific
examples of other chemical bonds include acid labile bonds, photolabile bonds,
peptidase labile bonds and esterase labile bonds. The disclosure of U.S.
Patent No.
5,208,020, incorporated herein, teaches the production of maytansinoids
bearing such
bonds.
[168] The synthesis of maytansinoids and maytansinoid derivatives having a
disulfide moiety that bears a reactive group is described in U.S. Patent Nos.
6,
441,163 and 6,333,410, and U.S. Application No. 10/161,651, each of which is
herein
incorporated by reference.
[169] The reactive group-containing maytansinoids, such as DM1, are reacted
with
an antibody, such as the DS6 antibody, to produce cytotoxic conjugates. These
conjugates may be purified by HPLC or by gel-filtration.
[170] Several excellent schemes for producing such antibody-maytansinoid
conjugates are provided in U.S. Patent No. 6,333,410, and U.S. Application
Nos.
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09/867,598, 10/161,651 and 10/024,290, each of which is incorporated herein in
its
entirety.
[171] In general, a solution of an antibody in aqueous buffer may be incubated
with
a molar excess of maytansinoids having a disulfide moiety that bears a
reactive group.
The reaction mixture can be quenched by addition of excess amine (such as
ethanolamine, taurine, etc.). The maytansinoid-antibody conjugate may then be
purified by gel-filtration.
[172] The number of maytansinoid molecules bound per antibody molecule can be
determined by measuring spectrophotometrically the ratio of the absorbance at
252
nm and 280 nm. An average of 1-10 maytansinoid molecules/antibody molecule is
preferred.
[173] Conjugates of antibodies with maytansinoid drugs can be evaluated for
their
ability to suppress proliferation of various unwanted cell lines in vitro. For
example,
cell lines such as the human epidermoid carcinoma line A-43 1, the human small
cell
lung cancer cell line SW2, the human breast tumor line SKBR3 and the Burkitt's
lymphoma line Namalwa can easily be used for the assessment of cytotoxicity of
these compounds. Cells to be evaluated can be exposed to the compounds for 24
hours and the surviving fractions of cells measured in direct assays by known
methods. IC50 values can then be calculated from the results of the assays.
PEG-containin linking inking rgoups
[174] Maytansinoids may also be linked to cell binding agents using PEG
linking
groups, as set forth in U.S. Application No. 10/024,290. These PEG linking
groups
are soluble both in water and in non-aqueous solvents, and can be used to join
one or
more cytotoxic agents to a cell binding agent. Exemplary PEG linking groups
include
hetero-bifunctional PEG linkers that bind to cytotoxic agents and cell binding
agents
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at opposite ends of the linkers through a functional sulfhydryl or disulfide
group at
one end, and an active ester at the other end.
[175] As a general example of the synthesis of a cytotoxic conjugate using a
PEG
linking group, reference is again made to U.S. Application No. 10/024,290 for
specific details. Synthesis begins with the reaction of one or more cytotoxic
agents
bearing a reactive PEG moiety with a cell-binding agent, resulting in
displacement of
the terminal active ester of each reactive PEG moiety by an amino acid residue
of the
cell binding agent, to yield a cytotoxic conjugate comprising one or more
cytotoxic
agents covalently bonded to a cell binding agent through a PEG linking group.
Taxanes
11761 The cytotoxic agent used in the cytotoxic conjugates according to the
present
invention may also be a taxane or derivative thereof.
[177] Taxanes are a family of compounds that includes paclitaxel (Taxol), a
cytotoxic natural product, and docetaxel (Taxotere), a semi-synthetic
derivative, two
compounds that are widely used in the treatment of cancer. Taxanes are mitotic
spindle poisons that inhibit the depolymerization of tubulin, resulting in
cell death.
While docetaxel and paclitaxel are useful agents in the treatment of cancer,
their
antitumor activity is limited because of their non-specific toxicity towards
normal
cells. Further, compounds like paclitaxel and docetaxel themselves are not
sufficiently potent to be used in conjugates of cell binding agents.
[178] A preferred taxane for use in the preparation of cytotoxic conjugates is
the
taxane of formula (XI):
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I 0
O S.S--~AO O OH
NH O
_ 0~~O
' H= O
OH OH p OAc
O
MeO & OMe
(XI)
[179] Methods for synthesizing taxanes that may be used in the cytotoxic
conjugates
of the present invention, along with methods for conjugating the taxanes to
cell
binding agents such as antibodies, are described in detail in U.S. Patent Nos.
5,416,064, 5,475,092, 6,340,701, 6,372,738 and 6,436,931, and in U.S.
Application
Nos. 10/024,290, 10/144,042, 10/207,814, 10/210,112 and 10/369,563.
CC-1065 Analogues
[180] The cytotoxic agent used in the cytotoxic conjugates according to the
present
invention may also be CC-1065 or a derivative thereof.
[181] CC-1065 is a potent anti-tumor antibiotic isolated from the culture
broth of
Streptomyces zelensis. CC-1065 is about 1000-fold more potent in vitro than
are
commonly used anti-cancer drugs, such as doxorubicin, methotrexate and
vincristine
(B.K. Bhuyan et al., Cancer Res., 42, 3532-3537 (1982)). CC-1065 and its
analogs
are disclosed in U.S. Patent Nos. 6,372,738, 6,340,701, 5,846,545 and
5,585,499.
[182] The cytotoxic potency of CC-1065 has been correlated with its alkylating
activity and its DNA-binding or DNA-intercalating activity. These two
activities
reside in separate parts of the molecule. Thus, the alkylating activity is
contained in
the cyclopropapyrroloindole (CPI) subunit and the DNA-binding activity resides
in
the two pyrroloindole subunits.
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[183] Although CC-1065 has certain attractive features as a cytotoxic agent,
it has
limitations in therapeutic use. Administration of CC-1065 to mice caused a
delayed
hepatotoxicity leading to mortality on day 50 after a single intravenous dose
of 12.5
g/kg {V. L. Reynolds et al., J. Antibiotics, XXIX, 319-334 (1986)}. This has
spurred efforts to develop analogs that do not cause delayed toxicity, and the
synthesis
of simpler analogs modeled on CC-1065 has been described {M.A. Warpehoski et
al.,
J. Med. Chem., 31, 590-603 (1988)}.
[1841 In another series of analogs, the CPI moiety was replaced by a
cyclopropabenzindole (CBI) moiety {D.L. Boger et al., J. Org. Chem., 55, 5823-
5833,
(1990), D.L. Boger et al., BioOrg. Med. Chem. Lett., 1, 115-120 (1991)}. These
compounds maintain the high in vitro potency of the parental drug, without
causing
delayed toxicity in mice. Like CC-1065, these compounds are alkylating agents
that
bind to the minor groove of DNA in a covalent manner to cause cell death.
However,
clinical evaluation of the most promising analogs, Adozelesin and Carzelesin,
has led
to disappointing results {B.F. Foster et al., Investigational New Drugs, 13,
321-326
(1996); I. Wolff et al., Clin. Cancer Res., 2,1717-1723 (1996)}. These drugs
display
poor therapeutic effects because of their high systemic toxicity.
[185] The therapeutic efficacy of CC-1065 analogs can be greatly improved by
changing the in vivo distribution through targeted delivery to the tumor site,
resulting
in lower toxicity to non-targeted tissues, and thus, lower systemic toxicity.
In order to
achieve this goal, conjugates of analogs and derivatives of CC-1065 with cell-
binding
agents that specifically target tumor cells have been described {US Patents;
5,475,092; 5,585,499; 5,846,545}. These conjugates typically display high
target-
specific cytotoxicity in vitro, and exceptional anti-tumor activity in human
tumor
xenograft models in mice {R.V. J. Chari et al., Cancer Res., 55, 4079-4084
(1995)}.
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[186] Methods for synthesizing CC-1065 analogs that may be used in the
cytotoxic
conjugates of the present invention, along with methods for conjugating the
analogs to
cell binding agents such as antibodies, are described in detail in U.S. Patent
Nos.
5,475,092, 5,846,545, 5,585,499, 6,534,660 and 6,586,618 and in U.S.
Application
Nos. 10/116,053 and 10/265,452.
Other Drugs
[187] Drugs such as methotrexate, daunorubicin, doxorubicin, vincristine,
vinblastine, melphalan, mitomycin C, chlorambucil, calicheamicin, tubulysin
and
tubulysin analogs, duocarmycin and duocarmycin analogs, dolastatin and
dolastatin
analogs are also suitable for the preparation of conjugates of the present
invention.
The drug molecules can also be linked to the antibody molecules through an
intermediary carrier molecule such as serum albumin. Doxarubicin and
Danorubicin
compounds, as described, for example, in U.S. Serial No. 09/740991, may also
be
useful cytotoxic agents.
Therapeutic Composition
[1881 The present invention also provides a therapeutic composition
comprising:
(a) an effective amount of one or more cytotoxic conjugate, and
(b) a pharmaceutically acceptable carrier.
[189] Similarly, the present invention provides a method for inhibiting the
growth of
selected cell populations comprising contacting target cells, or tissue
containing target
cells, with an effective amount of a cytotoxic conjugate, or therapeutic agent
comprising a cytotoxic conjugate, either alone or in combination with other
cytotoxic
or therapeutic agents.
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[190] The present invention also comprises a method for treating a subject
having
cancer using the therapeutic composition of the present invention.
[191] Cytotoxic conjugates can be evaluated for in vitro potency and
specificity by
methods previously described (see, e.g., R.V.J. Chari et al, Cancer Res.
55:4079-4084
(1995)).. Anti-tumor activity can be evaluated in human tumor xenograft models
in
mice by methods also previously described (see, e.g., Liu et al, Proc. Nati.
Acad. Sci.
93:8618-8623 (1996)).
[192] Suitable pharmaceutically-acceptable carriers are well known and can be
determined by those of ordinary skill in the art as the clinical situation
warrants. As
used herein, carriers include diluents and excipients.
[193] Examples of suitable carriers, diluents and/or excipients include: (1)
Dulbecco's phosphate buffered saline, pH - 7.4, containing or not containing
about 1
mg/ml to 25 mg/ml human serum albumin, (2) 0.9% saline (0.9% w/v sodium
chloride (NaCI)), and (3) 5% (w/v) dextrose; and may also contain an
antioxidant
such as tryptamine and a stabilizing agerit such as Tween 20.
[194] The method for inhibiting the growth of selected cell populations can be
practiced in vitro, in vivo, or ex vivo. As used herein, inhibiting growth
means
slowing the growth of a cell, decreasing cell viability, causing the death of
a cell,
lysing a cell and inducing cell death, whether over a short or long period of
time.
[195] Examples of in vitro uses include treatments of autologous bone marrow
prior
to their transplant into the same patient in order to kill diseased or
malignant cells;
treatments of bone marrow prior to its transplantation in order to kill
competent T
cells and prevent graft-versus-host-disease (GVHD); treatments of cell
cultures in
order to kill all cells except for desired variants that do not express the
target antigen;
or to kill variants that express undesired antigen.
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[196] The conditions of non-clinical in vitro use are readily determined by
one of
ordinary skill in the art.
[197] Examples of clinical ex vivo use are to remove tumor cells or lymphoid
cells
from bone marrow prior to autologous transplantation in cancer treatment or in
treatment of autoimmune disease, or to remove T cells and other lymphoid cells
from
autologous or allogeneic bone marrow or tissue prior to transplant in order to
prevent
graft versus host disease (GVHD). Treatment can be carried out as follows.
Bone
marrow is harvested from the patient or other individual and then incubated in
medium containing serum to which is added the cytotoxic agent of the
invention.
Concentrations range from about 10 M to 1 pM, for about 30 minutes to about
48
hours at about 37 C. The exact conditions of concentration and time of
incubation,
i.e., the dose, are readily determined by one of ordinary skill in the art.
After
incubation the bone marrow cells are washed with medium containing serum and
returned to the patient by i.v. infusion according to known methods. In
circumstances
where the patient receives other treatment such as a course of ablative
chemotherapy
or total-body irradiation between the time of harvest of the marrow and
reinfusion of
the treated cells, the treated marrow cells are stored frozen in liquid
nitrogen using
standard medical.equipment.
[198] For clinical in vivo use, the cytotoxic conjugate of the invention will
be
supplied as solutions that are tested for sterility and for endotoxin levels.
Examples of
suitable protocols of cytotoxic conjugate administration are as follows.
Conjugates
are given weekly for 4 weeks as an i.v. bolus each week. Bolus doses are given
in 50
to 100 ml of normal saline to which 5 to 10 ml of human serum albumin can be
added. Dosages will be 10 g to 100 mg per administration, i.v. (range of 100
ng to 1
mg/kg per day). More preferably, dosages will range from 50 g to 30 mg. Most
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preferably, dosages will range from I mg to 20 mg. After four weeks of
treatment,
the patient can continue to receive treatment on a weekly basis. Specific
clinical
protocols with regard to route of administration, excipients, diluents,
dosages, times,
etc., can be determined by one of ordinary skill in the art as the clinical
situation
warrants.
[199] Examples of medical conditions that can be treated according to the in
vivo or
ex vivo methods of killing selected cell populations include malignancy of any
type
including, for example, cancer of the lung, breast, colon, prostate, kidney,
pancreas,
ovary, cervix and lymphatic organs, osteosarcoma, synovial carcinoma, a
sarcoma or
a carcinoma in which CA6 is expressed, and other cancers yet to be determined
in
which CA6 glycotope is expressed predominantly; autoimmune diseases, such as
systemic lupus, rheumatoid arthritis, and multiple sclerosis; graft
rejections, such as
renal transplant rejection, liver transplant rejection, lung transplant
rejection, cardiac
transplant rejection, and bone marrow transplant rejection; graft versus host
disease;
viral infections, such as mV infection, HIV infection, AIDS, etc.; and
parasite
infections, such as giardiasis, amoebiasis, schistosomiasis, and others as
determined
by one of ordinary skill in the art.
Kit
[200] The present invention also includes kits, e.g., comprising a described
cytotoxic
conjugate and instructions for the use of the cytotoxic conjugate for killing
of
particular cell types. The instructions may include directions for using the
cytotoxic
conjugates in vitro, in vivo or ex vivo.
[201] Typically, the kit will have a compartment containing the cytotoxic
conjugate.
The cytotoxic conjugate may be in a lyophilized form, liquid form, or other
form
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amendable to being included in a kit. The kit may also contain additional
elements
needed to practice the method described on the instructions in the kit, such a
sterilized
solution for reconstituting a lyophilized powder, additional agents for
combining with
the cytotoxic conjugate prior to administering to a patient, and tools that
aid in
administering the conjugate to a patient.
Additional Embodiments
[202] The present invention further provides for monoclonal antibodies,
humanized
antibodies and epitope-binding fragments thereof that are further labeled for
use in
research or diagnostic applications. In preferred embodiments, the label is a
radiolabel, a fluorophore, a chromophore, an imaging agent or a metal ion.
[203] A method for diagnosis is also provided in which said labeled antibodies
or
epitope-binding fragments thereof are administered to a subject suspected of
having a
cancer, and the distribution of the label within the body of the subject is
measured or
monitored.
Examples
[204] The broad scope of this invention is best understood with reference to
the
following examples, which are not intended to limit the invention to specific
embodiments.
Example 1: Identification of antigen positive and negative cell lines by flow
c3~ometry bindingassays
[205] Flow cytometric analysis was used to localize the DS6 epitope, CA6, to
the
cell surface. Human cell lines were obtained from the American Type Culture
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Collection (ATCC) with the exception of OVCAR5 (Kearse et al., Int. J. Cancer
88(6):866-872 (2000)), OVCAR8 and IGROV 1 cells (M. Seiden, Massachusetts
General Hospital). All cells were grown in RPMI 1640 supplemented with 4 mM L-
glutamine, 50 U/ml penicillin, 50 g/mi streptomycin (Cambrex Bio Science,
Rockland, ME) and 10% v/v fetal bovine serum (Atlas Biologicals, Fort Collins,
CO),
referred hereafter as culture media. Cells were maintained in a 37 C, 5% COZ
humidified incubator.
[206] Cells (1-2 x 10-5 cells/well) were incubated, on ice for 3-4 h, with
serially
diluted concentrations of the DS6 antibody prepared in FACS buffer (2% goat
serum,
RPMI) into 96-well plates. The cells were spun down in a table top centrifuge
at
1500 rpm for 5 min at 4 C. After removing the media, the wells were then
refilled
with 150 l of FACS buffer. The wash step was then repeated. FITC-labeled goat
anti-mouse IgG (Jackson Immunoresearch) was diluted 1:100 to FACS buffer and
incubated with the cells for 1 h on ice. The plate was covered in foil to
prevent
photobleaching of the signal. After two washes, the cells were fixed with 1%
formaldehyde and analyzed on a flow cytometer.
[207] Predominantly, CA6 epitope was found in cell lines of ovarian; breast,
cervical, and pancreatic origin (Table 3) as predicted from the tumor
immunohistochemistry. However, some cell lines of other tumor types exhibited
limited CA6 expression. The DS6 antibody binds with an apparent KD of 135.6 pM
(in PC-3 cells, Table 3). The maximum mean fluorescence (Table 3) of the
binding
curves (Figure 1) in the antigen positive cell lines are suggestive of the
relative
antigen density.
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Table 3
Cell Line Apparent Cell Line Apparent
Tissue Antigen MMF* Kd (M) Tissue Antigen MMF* Kd (M)
HL-60 Blood - Caov-3 Ovary + 465.20 5.478x 10"09
Jurkat Blood - Caov-4 Ovary + 149.00 4.043 x 10-09
Namalwa Blood - ES-2 Ovary -
U-937 Blood - IGROVI Ovary -
T98G Brain + 35.94 1.775 x 10-10 OV-90 Ovary -
BT-20 Breast + 232.20 9.142 x 10-10 OVCAR-3 Ovary -
BT-474 Breast - OVCAR5 Ovary + 97.10 1.473 x] 0-09
BT-483 Breast + 1911.00 1.366 x 10"08 OVCAR8 Ovary -
BT-549 Breast + 71.39 1.046 x]0-09 PA-1 Ovary -
CAMA-1 Breast + 12.46 2.330 x 10-09 SK-OV-3 Ovary -
MCF-7 Breast + 81.41 2.890 x 10"09 SW 626 Ovary -
MDA-MB-157 Breast + 8.635 1.972 x 100 TOV-1 12D Ovary -
MDA-MB-231 Breast + 31.85 1.460 x 10-09 TOV-21G Ovary + 87.79 3.067 x 10-10
MDA-MB-468 Breast + 71.58 8.127 x 10-10 AsPC-1 Pancreas -
SK-BR-3 Breast - BxPC-3 Pancreas + 79.99 5.263 x 10"09
T-47D Breast + 559.58 3.424 x 10"09 HPAC Pancreas + 2228.00 2.348 x 10-08
ZR-75-1 Breast + 811.67 4.299 x 10"09 HPAF-1I Pancreas + 266.50 2.811 x 10" 9
HeLa Cervix + 242.50 6.938 x 10-10 Hs766T Pancreas + 182.90 2.319 x 10"09
KB Cervix + 119.56 1.110 x 10"09 MIAPaCa2 Pancreas -
WISH Cervix + 1133.55 2.380 x 10"09 MPanc96 Pancreas -
Colo205 Colon - SU.86.86 Pancreas + 36.86 1.043 x 10"09
DLD-1 Colon - SW1990 Pancreas + 36.17 3.679 x 10"10
HCT-8 Colon - PC-3 Prostate + 24.81 1.356 x 100
HT-29 Colon - A375 Skin -
Caki-I Kidney - SKMEL28 Skin -
A549 Lung - KLE Uterus -
SW2 Lung -
average maximum relative mean fluorescence
Example 2: Characterization of DS6 Epitope
[208] The properties of the DS6 antigen, CA6, were analyzed by immunoblotting
the dot blots of CA6-positive cell lysates (Caov-3) that were digested with
proteolytic
(pronase and proteinase K) and/or glycolytic (neuraminidase and periodic acid)
treatments. For positive controls, other antibodies recognizing a variety of
epitope
types were tested on lysates of antigen positive cell lines (Caov-3 and CM1;
Co1o205
and C242; SKMEL28 and R24). CM1 is an antibody recognizing a protein epitope
of
the variable number tandem repeat domain (VNTR) of Muc-1 and thus, provides a
control for a protein epitope. C242 binds to a novel colorectal cancer
specific sialic
acid-dependent glycotope on Muc-1 (CanAg) which provides a control for a
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glycotope on a protein. R24 binds to the GD3 ganglioside that is specific for
melanoma and thus provides a control for a glycotope on a non-protein
scaffold.
[2091 Caov-3, Co1o205, and SKMEL28 cells were plated in 15 cm tissue culture
plates. Culture media (30 mL/plate) was refreshed the day before lysis. A
modified
RIPA buffer (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 5 mM EDTA, 1% NP40,
0.25% sodium deoxycholate), protease inhibitors (PMSF, Pepstatin A, Leupeptin,
and
Aprotinin), and PBS were pre-chilled on ice. After the culture media was
aspirated
from the plates, the cells were washed twice with 10 ml of chilled PBS. All of
the
subsequent steps were conducted on ice and/or in a 4 C cold room. After the
last
wash of PBS was aspirated, the cells were lysed in 1-2 mL of lysis buffer
(RIPA
buffer with freshly added protease inhibitors to.a final concentration of 1 mM
PMSF,
1 M Pepstatin A, 10 g/ml Leupeptin, and 2 g/ml Aprotinin). The lysates were
scraped off of the plates using a cell lifter and triturated by pipetting the
suspensions
up and down (5-10 times) with an 18G needle. The lysates were rotated for 10
min
and then centrifuged in a microcentrifuge at maximum (13K rpm) for 10 min. The
pellets were discarded and the supernatants were then assayed using a Bradford
Protein assay kit (Biorad).
[210] The lysates (2 l) were pipetted directly onto dry 0.2 m nitrocellulose
membranes. The spots were allowed to air dry for approximately 30 min. The
membrane was sectioned into pieces that each contained a single spot. Spots
were
incubated in the presence of pronase (1 mg/ml enzyme, 50 mM Tris pH 7.5, 5 mM
CaC12), proteinase K(1 mg/ml enzyme, 50 mM Tris pH 7.5, 5 mM CaC12),
neuraminidase (20 mU/ml enzyme, 50 mM sodium acetate pH 5, 5 mM CaC12, 100
g/ml BSA) or periodic acid (20 mM, 0.5M sodium acetate pH 5) for lh at 37 C.
Reagents were purchased from Roche (enzymes) and VWR (periodic acid).
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Membranes were washed (5 min) in T-TBS wash buffer (0.1% Tween 20, 1X TBS),
blocked in blocking buffer (3% BSA, T-TBS) for 2 h at room temperature, and
incubated overnight with 2 g/ml of primary antibody (i.e. DS6, CMI, C242,
R24) in
blocking buffer at 4 C. The membranes were washed three times for 5 min in T-
TBS
and then incubated in HRP-conjugated goat anti-mouse (or human) IgG secondary
antibody (Jackson Immunoresearch; 1:2000 dilution in blocking buffer) for I h
at
room temperature. The immunoblots were washed three times and developed using
an ECL system (Amersham).
[211] The immunoblots (Figure 2) of the digested control lysates showed that
the
CMl signal was destroyed by the proteolytic treatments while the signals of
the
glycolytic digests were unaffected as would be expected for an antibody
recognizing a
protein epitope. The C242 signal was destroyed by either the proteolytic or
glycolytic
treatments as would be expected for an antibody recognizing a glycotope found
on a
protein. The R24 signal, unaffected by the proteolytic treatments, was
abolished with
neuraminidase or periodate treatments as expected for an antibody recognizing
a
ganglioside. The DS6 immunoblot of the digested Caov-3 lysate dot blots showed
signal loss upon treatment with either the proteolytic and glycolytic
compounds.
Thus, like C242, DS6 binds to a carbohydrate epitope on a proteinaceous core.
Furthermore, the signal in the DS6 immunoblot was sensitive to neuraminidase
treatment. Therefore, CA6, like CanAg, is a sialic acid-dependent glycotope.
[2121 In order to confirm the carbohydrate nature of CA6, Caov-3 lysate was
spotted
onto PVDF membrane and treated with the chemical deglycosylating agent,
trifluoromethane sulfonic acid (TFMSA), under nitrogen at ambient temperature
for 5
minutes. The blot was washed with T-TBS and immunoblotted with either CM1 or
DS6 (Figure 3). The DS6 signal was destroyed upon the acid treatment providing
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further evidence that CA6 is a glycotope. The enhancement of the CM1 signal
upon
TFSMA treatment indicates that the acid treatment did not affect the protein
on the
filter and suggests that the glycolytic treatment unmasked the protein epitope
recognized by CM1.
[213] To further elucidate the structure of the carbohydrate on which CA6
resides,
dot blots were digested with N-glycanase, 0-glycanase, and/or sialidase
(Figure 4).
Caov-3 cell lysates (100 g, 30 l) were incubated at 100 C for 5 min with 2.5
l of
denaturation buffer (Glyko) containing SDS and [3-mercaptoethanol. The
denatured
lysates were then digested with 1 l of N-glycanase, 0-glycanase, and/or
Sialidase A
(Glyko) at 37 C for I h. The digested lysates were then spotted (2 l) onto
nitrocellulose and immunoblotted as described above.
[214] N-glycanase had no apparent effect on the DS6 immunoblot signals.
However, samples digested with sialidase produced no signal. Because 0-
glycanase
cannot digest sialyated 0-linked carbohydrates without pretreatment with
sialidase,
the DS6 signal of samples processed with 0-glycanase alone would not be
affected.
N-glycanase, in contrast, does not require pretreatment with any glycosidic
enzymes
for activity. The fact that N-glycanase treatment does not affect the DS6
signal
suggests that the CA6 epitope is most likely present on sialyated 0-linked
carbohydrate chains.
Example 3: Elucidation of the antigen on which the CA6 epitope is found
[215] To identify the antigen on which the CA6 sialoglycotope is found, DS6
immunoprecipitates were analyzed by SDS-PAGE and Western blotting. Cell lysate
supernatants (1 mL/sample; 3-5 mg protein) were pre-cleared with Protein G
beads
(30 l), equilibrated with 1 ml of RIPA buffer, for 1-2 h, with rotation, at 4
C. All of
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the subsequent steps were conducted on ice and/or in a 4 C cold room. The pre-
cleared beads were spun down briefly (2-3 s) in a microcentrifuge. The pre-
cleared
supernatants were transferred to fresh tubes and incubated overnight with 2 g
of
DS6, with rotation. Fresh, equilibrated Protein G beads (30 l) were added to
the
lysates and incubated for lh, with rotation. The bead-lysate suspensions were
briefly
spun down in a microcentrifuge and samples of the post-immunoprecipitation
lysates
were optionally taken. The beads were washed 5-10 times with 1 mL of RIPA
buffer.
[216] Immunoprecipitated DS6 samples were then digested with 30 l
neuraminidase (20 mU neuraminidase (Roche), 50 mM sodium acetate pH 5, 5 mM
CaC12, 100 g/ml BSA) or 30 l periodic acid (20 mM periodic acid (VWR), 0.5M
sodium acetate pH 5) for lh at 37 C. They were then resuspended in 30 1 of 2X
sample loading buffer (containing (3-mercaptoethanol). The beads were boiled
for 5
min and the loading buffer supernatants were loaded onto 4-12% or 4-20% Tris-
Glycine gels (Invitrogen). The gels were run in Laemmli electrophoresis
running
buffer at 125 V for 1.5 h. The gel samples were transferred, overnight at 20
mA, onto
0.2 m nitrocellulose membranes (Invitrogen) using a Mini Trans-blot transfer
apparatus (Biorad). Membranes were immunoblotted with DS6 as described above
in
Example 2.
[217] Alternatively, the immunoprecipitated beads were first denatured and
then
enzymatically digested with N-glycanase, 0-glycanase and/or sialidase A
(Glyko).
The beads were resuspended in 27 l incubation buffer and 2 l denaturation
solution
(Glyko) and incubated at 100 C for 5 minutes. After cooling to room
temperature,
detergent solution (2 l) was added and the samples were incubated with 1 l
of N-
glycanase, O-glycanase, and/or Sialidase A at 37 C for 4 h. After adding 5X
sample
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loading buffer (7 l), the samples were boiled for 5 min. The samples were
subjected
to SDS-PAGE and immunoblotted as described above.
[218] DS6 immunoprecipitates a >250 kDa protein band that cari be seen in
antigen
positive cell lysates (Figure 5A, B, and C). In some cell lines (i.e. T-47D),
a doublet
is observed. The >250 kDa band was abolished in Caov-3 immunoprecipitates that
were treated with neuraminidase or periodic acid (Figure 5 A and B) suggesting
that
the CA6 epitope resides on the >250 kDa band. The >250 kDa band was also shown
to be insensitive to N-glycanase treatment of immunoprecipitates consistent
with CA6
residing on an 0-linked carbohydrate (Figure 5F). Further supporting that the
250
kDa band is the CA6 antigen is the fact that DS6 immunoprecipitates no such
band
from DS6 antigen negative cells (Figure 5D and E).
[219] Several lines of evidence suggested that the CA6 antigen was Mucl.
Because
of the high molecular weight and the sensitivity to 0-linked carbohydrate-
specific
glycolytic enzymes, it seemed likely that the CA6 antigen Was a mucin. Mucin
overexpression is well characterized in tumors particularly of the breast and
ovary,
consistent with the major tumor reactivities of DS6. Furthermore, CA6, like
CanAg
(a sialoglycotope on Muc 1), is not susceptible to perchloric acid
precipitation
suggesting the CA6 antigen is heavily 0-glycosylated. The observation that in
some
DS6 expressing cell lines, DS6 immunoprecipitated a doublet of >250 kDa
suggested
that the CA6 was Mucl. A hallmark of Mucl in humans is the presence of two
distinct Mucl alleles differing in number of tandem repeats resulting in the
expression
of two Mucl proteins of different molecular weights.
[220] To test whether CA6 is found on Mucl, DS6 immunoprecipitates from Caov-3
lystate were subjected to SDS-PAGE and immunoblotted with either DS6 or a Mucl
VNTR antibody, CM1. As can be seen in Figure 6A, CM1 reacts strongly with.the
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>250 kDa band immunoprecipitated by DS6. In Figure 6B, DS6 and CM1
immunoprecipitates from HeLa cell lysate show the same >250 kDa doublet when
immunoblotted with either DS6 or CM1. These results indicate that the CA6
epitope
is indeed located on the Muc-1 protein. The DS6 doublet seen in HeLa (and T-
47D)
cells can be explained by the fact that Muc-1 expression is directed by
distinct alleles
having differing number of tandem repeats.
[221] Although CM1 and DS6 bind to the same Muc-1 protein, they are distinct
epitopes. Chemical deglycosylation of Caov-3 lysate dot blots by
trifluoromethane
sulfonic acid (TFMSA) abolished the DS6 signal (Figure 3). However, this same
treatment enhanced the CMI signal. Deglycosylation may have revealed hidden
epitopes for the CM1 antibody. Furthermore, a comparison of the flow cytometry
binding results of DS6 and CM1 (Table 4) demonstrates that the CA6 epitope
does
not exist on every cell expressing Mucl. It is interesting to note that the
CA6 epitope
is not expressed on Co1o205 (Table 3), a cell line known to express high
levels of the
Mucl CanAg sialoglycotope.
Table 4
DS6 CMl
Apparent Apparent
Cell Line MMF* Kd (M) MMF* Kd (M)
BT549 71.39 1.046 x 10-09 187.90 6.056 x 10-09
DS6 positive & CaOV3 465.20 5.478 x 10-09 1031.00 7.479 x 10-09 CM1 positive
HeLa 242.50 6.938 x 10-10 334.80 2.907 x 10-09
KB 119.56 1.110 x 10 09 338.00 5.345 x 10 9
MCF7 81.41 2.890 x 10-09 1023.00 8.694 x 10-09
DS6 negative & KLE 27.48 - 561.70 8.156 x 10-09
CM1 positive OVCAR3 21.19 192.50 5.949 x 10 09
SKOV3 17.53 - 49.41 6.246 x 10-09
MMF = maximum mean relative fluorescence
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Example 4: Ouantitative analysis of shed CA6 epitope
[222] Because the CA6 epitope resides on Mucl, a molecule known to be shed
into
the blood stream in many cancer patients, a quantitative approach was
undertaken in
order to determine whether such levels would be prohibitive for DS6 antibody
therapy. Binding of circulating antibody to antigen is thought to lead to
rapid
clearance of immune complexes from the blood. If a significant portion of the
administered antibody dose is rapidly removed from circulation the amount
reaching
the tumor is likely to be diminished resulting in decreased anti-tumor
activity of an
antibody therapeutic. When the antibody is conjugated to a highly potent
cytotoxic
compound the rapid clearance of conjugate could potentially increase non-
specific
toxicity. Thus, in the case of antibody-small drug conjugates such as DS6-DM1,
high
levels of shed antigen might be expected to both reduce the anti-tumor effect
and
increase the dose-limiting toxicity.
[223] Recent clinical trials of antibody therapeutics have yielded information
as to
the impact of shed antigen concentration on pharmacokinetics. For example, in
clinical trials with trastuzumab (Herceptin), an antibody used for the
treatment of
her2/neti-expressing metastatic breast cancer, the pharmacokinetics of
trastuzumab
clearance was shown to be unaltered when the shed Her2/neu level was less than
500
ng/mL (Pegram et al., J. Clin. Oncol. 16(8):2659-71 (1998). Assuming a
molecular
weight of shed Her2/neu of 110,000 Daltons, a molar concentration shed
Her2/neu
below 4.5 nM appears to have little influence on the pharmacokinetics.
[224] In another example, a clinical trial with cantuzumab mertansine (huC242-
DM 1) indicated that there was no correlation with pretreatment shed CanAg
(C242
epitope) levels and pharmacokinetics of antibody clearance (Tolcher et al., J.
Clin.
Oncol. 21(2):211-22 (2003). The CanAg epitope, similar to the CA6 epitope
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recognized by DS6, is a unique tumor-specific 0-linked sialoglycotope on Muc1.
However, the heterogeneous nature of the CanAg epitope makes it difficult to
quantify in molar terms. In the general population Mucl alleles vary in length
depending upon the number of tandem repeats in the variable number tandem
repeat
(VNTR) domain. Several sites for 0-linked glycosylation occur in each tandem
repeat. Adding to the complexity of CanAg expression is the cell-to-cell
variation in
inherent glycosyl transferase activity. Thus a wide range of CanAg epitopes
per
Mucl molecule are possible even in a single patient. Moreover, the ratio of
CanAg
epitope per Mucl molecule will be different across a population of patients.
For this
reason, shed CanAg in serum samples is measured by sandwich ELISA where shed
Mucl with CanAg epitope is captured by C242 and detected by a biotinylated
C242/Streptavidin HRP system. The shed CanAg is quantified in standardized
units
(U) proportional to the number of epitopes per ml of serum rather than by a
molar
concentration of Mucl. By analogy, a similar situation occurs for the
quantification
of shed CA6 epitopes. In contrast, for trastuzumab there is only one epitope
per shed
her2/neu molecule vastly simplifying the quantification of shed antigen.
[225] In order to relate CA6 shed epitope levels to those found in clinical
trials with
trastuzumab and cantuzumab mertansine, a method for obtaining molar
concentrations
of complex shed epitopes such as sialoglycotopes on Mucl was developed. First,
a
simple sandwich ELISA assay for DS6 was established. A representation of the
assay
is shown in Figure 7A. DS6 was used to capture Mucl having CA6 epitope.
Because
each Mucl molecule has multiple CA6 epitopes, biotinylated DS6 was also used
as
the tracer antibody. Biotinylated DS6 bound to captured CA6 was detected by
Streptavidin-HRP using ABTS as the substrate. CA6 epitope was captured from
ovarian cancer patient serum or from standards which come from a commercially
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available Mucl test kit (CA15-3) used to monitor shed Mucl in breast cancer
patients.
DS6 units/ml were arbitrarily set equal to CA15-3 standards units/ml.
[226] In Figure 7B is shown the results of the DS6 sandwich ELISA in which
CA15-
3 standards were used. The curve generated is very similar to that obtained
with .
CA15-3 standards in the CA15-3 assay. In order to convert DS6 unit/ml to a
molar
concentration of CA6 a standard curve for biotinylated DS6 which converts
signal to
picograms of DS6 is required. Assuming a one-to-one stoichiometry between CA6
epitope and biotinylated DS6 antibody and a molecular weight of 160,000
Daltons for
biotinylated DS6 the moles of CA6 captured per volume of sample added can be
computed.
[227] In Figure 8A and B are representations of two alternative means of
generating
a standard curve for biotinylated DS6. In Figure 8A, Goat anti-mouse IgG
polyclonal
antibody is used to capture biotinylated DS6 which is in turn detected in a
manner
identical to that used in the sandwich ELISA assay shown in Figure 7. In the
method
shown in Figure 8B biotinylated DS6 is plated directly onto the ELISA plate
and
detected as in Figure 8A. As seen in Figure 8C the biotinylated DS6 standard
curves
generated by each method are in good agreement.
[228] In Table 5 the analysis of ovarian cancer patient serum samples for
various
shed antigens is shown. CA125 ELISA is generally used to monitor the treatment
of
ovarian cancer patients by measuring shed CA125 units/ml. The CA125 status was
provided with the serum samples. CA15-3 ELISA is generally used to monitor the
treatment of breast cancer patients by measuring the units/ml of shed Mucl
using
capture and detections antibodies recognizing epitopes distinct from that
recognized
by DS6. In Table 5, CA15-3 is measured in ovarian cancer patients serum
samples.
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Table 5
Serum CA125' CA15-3' DS62 DS6' DS6'
No. (U/ml) (U/ml). (U/ml) (pM) (pM)
4 72.80 117.72 29.79 52.13 188.94
3651.90 98.19 567.02 654.44 >2560.00
6 930.50 87.08 504.15 667.56 2505.00
7 76.00 72.70 135.65 246.94 778.25
8 32.50 18.44 39.96 65.19 239.88
9 551.70 292.39 >975.61 1512.31 >2560.00
90.00 42.40 49.48 85.19 305.88
11 200.50 60.58 92.32 152.38 526.75
12 283.00 35.67 83.65 135.81 485.06
13 197.50 20.61 35.92 61.06 216.25
14 100.60 6.13 12.39 23.19 88.06
34.60 59.18 199.85 286.63 1228.56
17 196.40 56.75 66.53 130.44 405.88
18 16.90 30.45 34.43 60.81 223.69
19 22.00 .263.93 118.98 191.69 728.94
22 110.70 21.44 16.46 29.94 111.38
~ determined by commercial ELISA kit
2 determined by conunercial CA15-3 standards (1 CA15-3 U 1 DS6 U)
3 goat anti-mouse IgG & biotin-DS6 standard curve
4 biotin-DS6 standard curve
[229] For the CA15-3 values reported in Table 5, a commercially available CA15-
3
Enzyme Immuno Assay kit from CanAg Diagnostics was used. For the DS6 units/ml
a standard curve was generated using the CA15-3 standards (from the CA15-3
Enzyme Immuno Assay kit from CanAg Diagnostics) in the DS6 sandwich ELISA.
DS6 units/ml were arbitrarily set equal to CA15-3 units/ml. In the last two
columns
picomolar (pM) shed CA6 was calculated using the biotinylated DS6 standard
curves
shown in Figure 8C.
[230] For the quantitative analysis of CanAg levels, CanAg serum levels were
those
reported for patients participating in a cantuzumab mertansine clinical trial
prior to
treatment (Tolcher et al., J. Clin. Oncol. 21(2):211-22 (2003). An ELISA assay
analogous to the one described for DS6 was used to make a CanAg standard curve
using CanAg standards. C242 was used to capture the CanAg standards. Detection
of
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captured CanAg was achieved using biotinylated C242 tracer followed by
development with streptavidin-HRP using ABTS as substrate. A biotinylated-C242
standard curve was constructed as done for biotinylated DS6 allowing for the
conversion of units/ml to a molar concentration of circulating CanAg epitopes.
In
Table 6 CanAg levels from cantuzumab mertansine clinical trial patients are
reported
along with the corresponding calculated molar concentrations of circulating
CanAg.
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Table 6
CanAg' CanAgZ CanAg3
(U/ml) (pM) (pM)
31240 19185.7 34592.8
8687 3535 9619.3
7456 4579 8256.2
3686 2263.7 4081.6
1447 888.7 1602.3
1262 775 1397.4
718 441 795.1
547 335.9 605.7
394 242 436.3
381 234 421.9
329 202.1 364.3
322 197.8 356.6
306 187 338.8
284 174.4 314.5
247 151.7 273.5
242 148.6 268
229 140.6 253.6
227 139.4 251.4
184 113 203.7
120 73:7 132.9
107 65.7 118.5
100 61.4 110.7
81 49.7 89.7
81 49.7 89.7
67 41.1 74.2
53 32.5 58.7
45 27.6 49.8
43 26.4 47.6
39 24 43.2
36 22.1 39.9
24 14.7 26.6
18 11.1 19.9
17 10.4 18.8
<10 6.1 11.3
<10 6.1 11.3
<10 6.1 11.3
<10 6.1 11.3
' pretreatment levels of circulating CanAg measured by sandwich ELISA
2 goat anti-mouse IgG & biotin-C242 standard curve
3 biotin-C242 standard curve
[231] A comparison of the pM levels of shed CA6 in ovarian cancer patients
with
those calculated for shed CanAg in CanAg-positive cancer patients shows that
in
general shed CA6 levels are similar to shed CanAg levels. Furthermore, only 2
out of
16 ovarian cancer patients serum samples potentially have CA6 levels greater
than
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4.5nM, (serum samples 5 and 9 for which the signal was out of the range of the
standard curve), the level above which altered herceptin pharmacokinetics was
observed in clinical trials with Her2/neu-positive breast cancer patients.
CanAg levels
above 4.5nM were only seen in 3 out of 37 clinical trial patients. In this
clinical trial
there was no correlation with shed CanAg levels and more rapid clearance of
cantuzumab mertansine. However, the patient with the highest CanAg level
(31240
U/ml) was only sampled for 8 hours post-transfusion. These results indicate
that
certain epitopes of Mucl, such as CA6 and CanAg, while shed in cancer
patients, are
not shed at levels prohibitive for antibody therapeutic treatment.
Example 6: Cloning Murine DS6 Antibody Variable Regions.
[232] Murine monoclonal antibodies such as DS6 have limited utility in a
clinical
setting because they are recognized as foreigri by the human immune system.
Patients
quickly develop human anti-mouse antibodies (HAMA) resulting in rapid
clearance of
murine antibodies. For this reason, the variable region of murine DS6 (muDS6)
was
resurfaced to produce humanized DS6 (huDS6) antibodies.
[233] The murine DS6 antibody variable regions were cloned by RT-PCR. Total
RNA was purified from a confluent T175 flask of DS6 hybridoma cells using the
Qiagen RNeasy miniprep kit. RNA concentrations were determined by UV
spectrophotometry and RT reactions were done with 4-5 g total RNA using the
Gibco Superscript II kit and random hexamer primers.
[234] PCR reactions were performed with degenerate primers based on those
described in Wang Z et al., Jlmmunol Methods. Jan 13;233(1-2):167-77 (2000).
The
RT reaction mix was used directly for degenerate PCR reactions. The 3' light
chain
primer, HindKL,
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(TATAGAGCTCAAGCTTGGATGGTGGGAAGATGGATACAGTTGGTGC)
(SEQ ID NO:25)
and 3' heavy chain primer, BamIgGl,
(GGAGGATCCATAGACAGATGGGGGTGTCGTTTTGGC) (SEQ ID NO:26)
were used, and for the 5' end PCR primers were Sac1MK
(GGGAGCTCGAYATTGTGMTSACMCARWCTMCA) (SEQ ID NO:27) for the
light chain and an equal mix of EcoR1MH1
(CTTCCGGAATTCSARGTNMAGCTGSAGSAGTC) (SEQ ID NO:28) and
EcoR1MH2 (CTTCCGGAATTCSARGTNMAGCTGSAGSAGTCWGG) (SEQ ID
NO:29) for the heavy chain (mixed bases: H = A+T+C, S = G+C, Y = C+T, K
G+T, M = A+C, R = A+G, W = A+T, V = A+C+G, N = A+T+G+C).
[235] PCR reactions were standard except they were supplemented with 10%
DMSO (50 l reaction mixes contained final concentrations of 1X reaction buffer
(ROCHE), 2mM each dNTP, 1mM each primer, 2 l RT reaction, 5 l DMSO, and
0.5 l Taq (ROCHE)). The PCR reactions were performed on an MJ research
thermocycler using a program adapted from Wang Z et al., (Jlmmunol Methods.
Jan
13;233(1-2):167-77 (2000)): 1) 94 C, 3 min; 2) 94 C, 15 sec; 3) 45 C, 1
min; 4) 72
C, 2 min; 5) cycle back to step #2 29 times; 6) finish with a final extension
step at 72
C for 10 min. The PCR products were cloned into pBluescript II SK+
(Stratagene)
using restriction enzymes created by the PCR primers. Seqwright sequencing
services
sequenced the heavy and light chain clones.
[236] In order to confirm the 5' end cDNA sequences, additional PCR and
cloning
was done. The DS6 light chain and heavy chain cDNA sequences, determined from
the degenerate PCR clones, were plugged into the NCBI's Blast search website
and
murine antibody sequences with signal sequence submitted were saved. PCR
primers
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were designed from these signal peptides using conserved stretches among the
related
DNA sequences. EcoRI restriction sites were added to the leader sequence
primers
(Table 7) and these were used in RT-PCR reactions as described above.
Table 7
DS6 Signal Sequence Degenerate Primers
Name Sequence
Heavy Chain - DS6HClead ttttgaattcaataactacaggtgtccact - SEQ ID NO:30
Light Chain - KTILCIead ttttgagctccagattttcagcttcctgct - SEQ ID NO:31
[237] Several individual light and heavy chain clones were sequenced to
identify
and avoid possible polymerase generated sequence errors. Only a single
sequence
was obtained for both the light chain and heavy chain RT-PCR clones. These
sequences were sufficient to design primers that could amplify the murine DS6
light
and heavy chain sequences extending into the signal sequence. The subsequent
clones from these follow-up PCR reactions confirmed the 5' end sequences of
the
variable region that had been altered by the original degenerate primers. The
cumulative results from the various cDNA clones provided the final murine DS6
light
and heavy chain sequences presented in Figure 9. Using Kabat and AbM
definitions,
the three light chain and heavy chain CDRs were identified (Figures 9 and 10).
A
search of the NCBI IgBlast database indicates that the muDS6 antibody light
chain
variable region most likely derives from the murine IgVK ap4 germline gene
while the
heavy chain variable region most likely derives from the murine IgVh J558.41
germline gene (Figure 11).
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Example 7: Determination of the Variable Region Surface Residues of DS6
Antibody
[238] The antibody resurfacing techniques described by Pedersen et al. (1994)
and
Roguska et al. (1996) begin by predicting the surface residues of the murine
antibody
variable sequences. A surface residue is defined as an amino acid that has at
least
30% of its total surface area accessible to a water molecule. In the absence
of a
solved structure to find the surface residues for muDS6, we aligned the ten
antibodies
with the most homologous sequences in the set of 127 antibody structure files
(Figure
12). The solvent accessibility for each Kabat position was averaged for these
aligned
sequences (Figures 13A and B).
[239] Surface positions with average accessibilities of between 25% and 35%
were
subjected to a second round of analysis by comparing a subset of antibodies
containing two identical residues flanking on either side (Figures 13A and B).
After
the second round analysis, the 21 predicted surface residues for the muDS6
heavy
chain were increased to 23, adding Tyr3 and Lys23 to the list of residues with
predicted surface accessibility greater than 30%. In most of our resurfaced
antibodies
the Kabat definition of the heavy chain CDR1 is used, but for DS6 the AbM
definition
was inadvertently used during the calculations so the heavy chain residue T28
was not
defined as a framework surface residue as it might otherwise have been. The
number
of light chain surface positions was reduced from 16 to 15 because the
predicted
surface accessibility of A1a80 was reduced from 30.5% to 27.8% in the second
round
analysis. Together, the muDS6 heavy and light chain variable sequences have 38
predicted surface accessible framework residues.
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Example 8: Human antibody selection
[240] The surface positions of the murine DS6 variable region were compared to
the
corresponding positions in human antibody sequences in the Kabat database
(Johnson
G, Wu TT. Nucleic Acids Res. Jan 1;29(1):205-6 ( 2001)). The antibody database
management software SR (Searle, 1998) was used to extract and align the
surface
residues from natural heavy and light chain human aritibody pairs. The human
antibody variable region surface with the most identical surface residues,
with special
consideration given to positions that come within 5 A of a CDR, was chosen to
replace the murine DS6 antibody variable region surface residues.
Example 9: Expression vector for chimeric and humanized antibodies
[2411 The light and heavy chain paired sequences were cloned into a single
mammalian expression vector. The PCR primers for the human variable sequences
created restriction sites that allowed the human signal sequence to be added
in the
pBluescriptIl cloning vector. The variable sequences could then be cloned into
the
mammalian expression plasmid with EcoRl and BsiWI or HindIIl and Apal for the
light chain or heavy chain respectively (Figure 14). The light chain variable
sequences were cloned in-frame onto the human IgKappa constant region and the
heavy chain variable sequences were cloned into the human IgGammal constant
region sequence. In the final expression plasmids, human CMV promoters drive
the
expression of both the light and heavy chain cDNA sequences.
Example 10: Identification of Residues That May Ne atg ively Affect DS6
Activity
[242] In most of the humanizations to date a molecular model of the subject
antibody has been built to identify residues proximal to a CDR as potential
problem
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residues. With an expanding number of resurfaced antibodies to work from,
historical
experience is at least as effective at predicting problems as building a
model, so no
molecular model was built for DS6. Instead, the murine DS6 surface residues
were
compared with those of previously resurfaced antibodies and residues with low
to
high risk for affecting the antibody's binding activity were identified.
[243] Similar sets of residues are repeatedly identified as being within 5A of
a CDR
in both the available solved antibody structures and the molecular models from
previous humanizations. Using this data, Table 1 gives the murine DS6 residues
that
are likely proximal to and possibly within 5A of a CDR. Many of these
positions
have also been changed in previous humanizations, but only heavy chain
position 74
has ever resulted in a loss of binding activity. The murine residue was
retained in this
position in both huC242 and huB4 in order to conserve the binding activity of
the
murine antibody. On the other hand, this same position was changed to the
corresponding human residue in humanized 6.2G5C6 without loss of activity
(6.2G5C6 is the anti-IGF1-R antibody often referred to simply as anti-C6).
While any
of the residues in Table 1 could present a problem in the humanized antibody,
the
heavy chain residue P73 will be of particular concern due to previous
experiences in
this position.
Example 11: Selection of the Most Homologous Human Surface
[244] Candidate human antibody surfaces for resurfacing muDS6 were pulled from
the Kabat antibody sequence database using SR software. This software provides
an
interface to search only specified residue positions against the antibody
database. To
preserve the natural pairs, the surface residues of both the light and heavy
chains were
compared together. The most homologous human surfaces from the Kabat database
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were aligned in order of rank of sequence identity. The top 3 surfaces as
aligned by
the SR Kabat database software are given in Table 2. The surfaces were then
compared to identify which human surfaces would require the least changes to
the
residues identified in Table 1. The anti-Rh(D) antibody, 28E4 (Boucher et al,
1997),
requires the least number of surface residue changes (11 total) and only 3 of
these
residues are included in the list of potential problem residues. Since the
28E4
antibody provides the most homologous human surface, it is the best candidate
to
resurface muDS6.
Example 12: Construction of the DNA Sequences for Humanized DS6 Antibodies
[245] The 11 surface residue changes for DS6 were made using PCR mutagenesis.
PCR mutagenesis was performed on the murine DS6 variable region cDNA clones to
build the resurfaced, human DS6 gene. Humanization primer sets were designed
to
make the amino acid chariges required for resurfaced DS6, shown below in Table
8.
Table 8
Primer Name Primer Sequence
DS6HCapa cgatgggcccttggtggaggctgcagagacagtgaccaga SEQ ID NO:32
DS6LCBsi ttttcgtacgtttcagctccagcttggt SEQ ID NO:33
DS6HC5end caggtgtacactcccaggcttatctccagcagtct SEQ ID NO:34
huC6HCApa c atgggcccttg t gaggcggcagagacagt accaga SEQ ID NO:35
ds6lc5et caggtgtacactccgagattgttetcacccagtctccagcaacc SEQ ID NO:36
atgtctgcatct
ds6LCrl8 ggcactgca gttatggt accctctcccctggaga SEQ ID NO:37
ds61cs77f caatcagcagcat gaggct aaga SEQ ID NO:38
ds6lcs77r gcctccatgctgctgattgtgaga SEQ ID NO:39
DS6HCvvkp caggtgtacactcccaggctcagctcgtgcagtctggggctg SEQ ID NO:40
aggtggtgaa cccggg cctcagt
DS6HCt ttgactgcagacacatcctccagcaca SEQ ID NO:41
ds6hcQT gtgtctgcagtcaatgtggccttgccctggaacttctgat SEQ ID NO:42
huDS6HCapa cgatgggcccttggtggaggcggcaga acagtgacaaga SEQ ID NO:43
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[246] PCR reactions were standard except they were supplemented with 10%
DMSO (50 l reaction mixes contained final concentrations of 1X reaction
buffer
(ROCHE), 2 mM each dNTP, 1 mM each primer, 100 ng template, 5 1 DMSO, and
0.5 l Taq (ROCHE)). They were run on an MJ Research thermocycler with the
following program: 1) 94 C, 1 min; 2) 94 C, 15 sec; 3) 55 C, 1 min; 4) 72
C, 1
min; 5) cycle back to step #2 29 times; 6) finish with a final extension step
at 72 C
for 4 min. The PCR products were digested with their corresponding restriction
enzymes and cloned into the pBluescript cloning vectors. Clories were
sequenced to
confirm the amino acid changes.
[247] Since changing heavy chain residue P73 has caused problems in the past,
two
versions of the heavy chain were built, one with the human 28E4 T73 and one
retaining the murine P73. The other 10 surface residue were changed from
murine
to the human 28E4 residue in both versions of humanized DS6 (Table 2).
Sticking
with the usual naming method, the most human is version 1.0 since it has all I
1
human surface residues. The heavy chain version retaining the murine P73 is
named
version 1.2 in case further versions are required so version 1.1 is reserved
for a
version containing the maximum number of murine residues. The amino acid
sequences of the two humanized versions are shown aligned with the murine DS6
amino acid sequence in Figure 15A and B. Both of the humanized DS6 antibody
genes were cloned into the antibody expression plasmid (Figure 14) for
transient and
stable transfections. The cDNA and amino acid sequences of the humanized
versions
1.01 and 1.21 are light chain variable region are the same and shown in Figure
16.
The heavy chain cDNA and amino acid sequences of the humanized versions 1.01
and
1.21 are shown in Figure 17A and B.
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Example 13: Expression and purification of huDS6 in CHO cells and affinity
measurements
[248] To determine whether the humanized DS6 versions retained the binding
affinity of muDS6 it was necessary to express and purify the antibodies. CHO
cells
were stably transfected with a chimeric version of DS6 (chDS6) having the
human
constant region and the murine variable region, or with huDS6v1.01 or
huDS6vl.21.
[249] CHODG44 cells (4.32 x 106 cells/plate) were seeded in 15 cm plates in
non-
selective media (Alpha MEM + nucleotides (Gibco), supplemented with 4 mM L-
glutamine, 50 U/ml penicillin, 50 g/mi streptomycin, and 10% v/v FBS) and
placed
in a 37 C, 5% CO2 humidified incubator. The following day, the cells were
transfected with the chDS6 expression plasmid using a modified version of the
Qiagen recommended protocol for Polyfect Transfection. The non-selective media
was aspirated from the cells. The plates were washed with 7 ml of pre-warmed
(37 C) PBS and replenished with 20 ml of non-selective media. The plasmid DNA
(11 g) was diluted into 800 l of Hybridoma SFM (Gibco). Then, 70 l of
Polyfect
(Qiagen) was added to the DNA/SFM mixture. The Polyfect mixture was then
gently
vortexed for several seconds and incubated for 10 min at ambient temperature.
Non-
selective media (2.7 ml) was added to the mixture. This final mixture was
incubated
with the plated cells for 24 h.
[250] The transfection mixture/media was removed from the plates and the cells
were then trypsinized and counted. The cells were then plated in selective
media
(Alpha MEM -nucleotides, supplemented with 4 mM L-glutamine, 50 U/ml
penicillin, 50 g/mi streptomycin, 10% v/v FBS, 1.25 mg/ml G418) in 96 well
plates
(250 l/well) at various densities (1800, 600, 200, and 67 cells/well). The
cells were
incubated for 2-3 weeks, supplementing media if necessary. Wells were screened
for
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antibody production levels using a quantitative ELISA. An Immulon 2HB 96 well
plate was coated with goat anti-human IgG F(ab)2 antibody (Jackson
Immunoresearch; 1 g/well in 100 l 50 mM sodium carbonate buffer pH 9.6) and
incubated for 1.5 h at ambient temperature, with rocking. All subsequent steps
were
conducted at ambient temperature. The wells were washed twice with T-TBS (0.1%
Tween-20, TBS) and blocked with 200 l of blocking buffer (1% BSA, T-TBS) for
1
h. The wells were washed twice with T-TBS. In a separate plate, the antibody
standard, EM164 (100 ng/ml), and culture supernatants were serially diluted
(1:2 or
1:3) in blocking buffer. These dilutions (100 l) were transferred to the
ELISA plate
and incubated for 1 h. The wells were washed 3 times with T-TBS and incubated
with 100 l of goat anti-human IgG Fc-AP (Jackson ImmunoResearch) diluted
1:3000
in blocking buffer for 45 min. After 5 washes with T-TBS, the wells were
developed
using 100 l of PNPP development reagent (10 mg/ml PNPP (p-Nitrophenyl
Phosphate, Disodium Salt; Pierce), 0.1 M diethanolamine pH 10.3 buffer) for 25
min.
The absorbance at 405 nm was measured in an ELISA plate reader. Absorbance
readings (of the culture supernatant) in the linear portion of the standard
curve were
used to determine the antibody levels.
[251] The highest producing clones, identified by the ELISA, were then
subcloned,
expanded, and frozen cell stocks were prepared.
[252] For expression of huDS6v1.01 and huDS6vl.21, DG44 CHO cells (Dr.
Lawrence Chasin, Columbia University, NY) were cultured in Alpha MEM with
ribonucleosides and deoxyribonucleosides (Gibco catalog #12571, Grand Island,
New
York). The medium was supplemented with 10% fetal bovine serum (HyClone
catalog # SH30071.03, Logan, UT), 1% gentamicin (Mediatech catalog# 30-005-CR,
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Herndon, VA), and 2mM L-glutamine (L-glut) (BioWhittaker catalog#17-605E,
Walkersville, MD). This formulation was termed CHO Complete Medium.
[253] DG44 CHO cells (5x 106) were transfected with 50 g of huDS6 plasmid
DNA. Prior to transfection, cells were removed from flasks with trypsin (Gibco
catalog # 15090-046, Grand Island, NY) and washed two times with
unsupplemented
Alpha MEM lacking ribonucleosides and deoxyribonucleosides (Gibco catalog
#12561, Grand Island, NY). This was termed Wash Medium. Cells were mixed with
plasmid DNA in 0.4 cm gap electrode cuvettes (BioRad catalog # 1652088,
Hercules,
PA). They were placed on ice fortwo minutes, and then pulsed at 1,000 F and
260
volts in a BioRad electroporation apparatus. Following electroporation, cells
were
incubated on ice for two minutes. The cells were then plated in five 24 well
plates
(Costar catalog # 3524) in CHO Complete Medium and were maintained in a 37 C
incubator with 5% CO2. After 48 hours, the medium was removed from the wells.
Wells were rinsed once with Wash Medium and fed with Alpha MEM without
ribonucleosides and deoxyribonucleosides (Gibco catalog #12561, Grand Island,
NY)
supplemented with 1% gentamicin, 2 mM L-glut, 10% dialyzed fetal bovine serum
(Gibco catalog # 26400-044, Grand Island, NY), and 1.25 mg/mL geneticin (G418)
(Gibco catalog # 11811, Grand Island, NY). This complete formulation was
termed
Selection Medium. Clones were incubated in Selection Medium for approximately
two weeks at which time they were screened for antibody production by
Quantitative
ELISA. The highest producing clones were then subcloned, expanded, and frozen
cell stocks were prepared.
[254] To produce sufficient amount of antibody to purify, cells were expanded
onto
15 cm plates (- 1 x 106 cells/plate) with 30 ml of selective media
supplemented with
Ultra Low IgG FBS (Gibco) and incubated for 1 week. Culture supernatants were
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collected into 250 ml conical tubes, spun down in a tabletop centrifuge (2000
rpm, 5
min, 4 C), and then sterile-filtered through a 0.2 m filter apparatus.
12551 For purification of DS6, pellets of NaOH were added to the filtered
culture
supernatants to a final pH of 8Ø A Hi Trap rProtein A column (Amersham) was
equilibrated with 20-50 column volumes of binding buffer. The supernatant was
loaded onto the column using a peristaltic pump. Then, the column was washed
with
50 column volumes of binding buffer. The bound antibody was eluted off of the
column using elution buffer (100 mM acetic acid, 50 mM NaCl, pH 3) into tubes
set
in a fraction collector. The eluted antibody was neutralized using
neutralization
buffer (2 M K2HPO4, pH 10.0) and dialyzed overnight in PBS. The dialyzed
antibody
was filtered through a 0.2 m syringe filter. The absorbance at 280 nm was
measured
to determine the final protein concentration..
[256] The affinity of the purified huIgG was compared with muDS6 by flow
cytometry. In the first set of experiments direct binding to a CA6-expressing
cell line,
KB, was measured. As shown in Figure 18 the muDS6, chDS6, huDS6vl.01 and
huDS6 v 1.21 show very similar affinities with apparent Kds of 0.82 nM, 0.69
nM,
0.82 and 0.85 nM, respectively, suggesting that resurfacing has not disrupted
the
CDRs. To confirm that the huDS6 versions retain the affinity of muDS6,
competitive
binding experiments were conducted. The advantage of this format is that the
same
detection system is used for both murine and human antibodies; that is biotin-
muDS6/streptavidin-DTAF. The results of the competition binding assay
comparing
the ability of muDS6, chDS6, huDS6v1.01 and huDS6vl.21 to compete with biotin-
DS6 is shown in Figure 19. The apparent EC50 are 1.9 nM, 1.7 nM, 3.0 and 1.9
nM
for muDS6, chDS6, huDS6 v1.01, and huDS6 vl.21, respectively. These results
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indicate that resurfacing of muDS6 to produce a humanized DS6 causes little
reduction in binding affinity.
Example 14: Preparation of muDS6-DM1 cytotoxic conjugate
[257] The muDS6 antibody (8 mg/ml) was modified using 8-fold molar excess of N-
succinimidyl-4-(2-pyridyldithio) pentanoate (SPP) to introduce dithiopyridyl
groups.
The reaction was camed out in 95% v/v Buffer A (50 mM KPi, 50 mM NaCl, 2 mM
EDTA, pH 6.5) and 5% v/v DMA for 2 h at room temperature. The slightly turgid
reaction mixture was gel-filtered through a NAP or Sephadex G25 column
(equilibrated in Buffer A). The degree of modification was determined by
measuring
the absorbance of the antibody at 280 nm and the DTT released 2-
mercaptopyridine
(Spy) at 280 and 343 nm. Modified muDS6 was then conjugated at 2.5 mg Ab/mL
using a 1.7-fold molar excess of N2'-deacetyl-N-2'(3-mercapto-l-oxopropyl)-
maytansine (L-DMI) over SPy. The reaction was carried out in Buffer A (97%
v/v)
with DMA (3% v/v). The reaction was incubated at room temperature overnight
for
-20 h. The opaque reaction mixture was centrifuged (1162 x g, 10 min) and the
supernatant was then gel-filtered through a NAP-25 or S300 (Tandem 3, 3x 26/10
desalting columns, G25 medium) column equilibrated in Buffer B(1X PBS pH 6.5):
The pellet was discarded. The conjugate was sterile-filtered using a 0.22 m
Millex-
GV filter and was dialyzed in Buffer B with a Slide-A-Lyzer. The number of DMl
molecules linked per molecule of muDS6 was determined by measuring the
absorbance at both 252 nm and 280 nm of the filtered material. The DMl/Ab
ratio
was found to be 4.36 and the step yield of conjugated MUDS6 was 55%. The
conjugated antibody concentration was 1.32 mg/mL. The purified conjugate was
biochemically characterized by size exclusion chromography (SEC) and found to
be
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92% monomer. Analysis of DM1 in the purified conjugated indicated that 99% was
covalently bound to antibody. In Figure 20, flow cytometric binding of the
muDS6-
DM1 conjugate and unmodified muDS6 to Caov-3 cells shows that conjugation of
muDS6 results in only a slight loss of affinity.
Example 15: In vitro Cytotoxicity of muDS6-DM 1
[258] As a naked antibody, muDS6 has shown no proliferative or growth
inhibitive
activity in cell cultures (Figure 21) However, when muDS6 is incubated with
cells in
the presence of a DMl conjugate to Goat anti-mouse IgG heavy and light chain,
muDS6 is very effective at targeting and delivering this conjugate to the cell
resulting
in indirect cytotoxicity (Figure 21). To further test the inherent activity of
naked
muDS6, a complement-dependent cytotoxicity (CDC) assay using muDS6 was
conducted. HPAC and ZR-75-1 cells (25000 cells/well) were plated in 96 well
plates,
in the presence of 5% human or rabbit serum and various dilutions of muDS6, in
200
l of RHBP media (RPMI-1640, 0.1% BSA, 20mM HEPES (pH 7.2-7.4), 100 U/ml
penicillin and 100 ug/mi streptomycin). The cells were incubated for 2 h at 37
C.
Then Alamar Blue (10% of final concentration) reagent (Biosource) was added to
the
supernatant. The cells were incubated for 5-24 hrs before measuring
fluorescence.
Murine DS6 had no effect in a complement-dependent cytotoxicity (CDC) assay
(Figure 22) This suggests that the therapeutic application of muDS6 would
require
the conjugation of a toxic effector molecule.
[259] The cytotoxicity of maytansinoid conjugated muDS6 antibody was examined
using 2 different assay formats in various DS6 positive cell lines. Clonogenic
assays
were conducted where cells (1000-2500 cells/well) were plated on 6-well plates
in 2
ml of conjugate diluted in culture media. The cells were continuously exposed
to the
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conjugate at several concentrations, generally between 3 x 10-" M to 3 x 10-9
M, and
were incubated in a 37 C, 6% CO2 humidified chamber for 5-9 days. The wells
were
washed with PBS and the colonies were stained with a 1% w/v crystal violet/10
% v/v
formaldehyde/PBS solution. Unbound stain was then washed thoroughly from the
wells with distilled water, and the plates were allowed to dry. The colonies
were
counted using a Leica StereoZoom 4 dissecting microscope.
[260] Plating efficiency (PE) was calculated as the number of colonies/number
of
cells plated. Surviving fraction was calculated as PE of treated cells/PE of
non-
treated cells. The IC50 concentration was determined by graphing the surviving
fraction of cells vs. the molar concentration of the conjugate. In a
clonogenic assay
(Figure 23), muDS6-DM1 was effective in killing Caov-3 cells with an estimated
IC5o
of 800 pM. Antigen negative cells, A375, were only slightly affected by the
conjugate at a concentration of 3 x 10-9 M, the highest concentration of muDS6-
DM1
tested, demonstrating that the cell killing activity of the conjugate is
directed
specifically toward antigen-expressing cells. However, despite apparent
sensitivity to
maytansine, many of the other DS6 positive cell lines were not particularly
sensitive
to the immunoconjugate. All of the cervical cell lines (HeLa, KB, and WISH)
were
sensitive to the conjugate whereas only a select number of the ovarian and
breast cell
lines showed any cytotoxic affects. None of the pancreatic cell lines appeared
to have
been affected.
[261] In the MTT assay, cells were seeded in 96-we11 plates at a density of
1000-
5000 cells/well. The cells were plated with serial dilutions of either naked
muDS6 or
muDS6-DM1 immunoconjugate in 200 l of culture media. The samples were run in
triplicate. The cells and antibody/conjugate mixtures were then incubated for
2-7 d, at
which time cell viability was assessed by an MTT ([3(4,5-dimethylthiazol-2-yl)-
2,5-
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diphenyl tetrazolium bromide)] assay. MTT (50 g/well) was added to the
culture
supernatant and allowed to incubate for 3-4 h at 37 C. The media was removed
and
the MTT formazan was solubilized in DMSO (175 p1/well). The absorbance at 540-
545 nm was measured. In a MTT cell viability assay (Figure 24C), the
irnmunoconjugate was able to effectively kill Caov-3 cells with an estimated
IC50 of
1.61 nM. The wells with the highest concentrations of conjugate contained no
viable
cells as compared to naked antibody which had no effect (Figures 21 and 24).
[262] The results of the MTT assays on the other cell lines were slightly
different
(Figure 24A, B, and D-I). In many cases, although some cytotoxicity was seen,
the
conjugate was unable to completely kill the entire cell population (with the
exception
of WISH cells). BT-20, OVCAR5, and HPAC cells were particularly resistant: in
the
highest conjugate concentration (32 nM) wells, over 50% of the cells were
still viable.
Example 16: In vivo Conjugate Anti-tumor Activity.
[263] To demonstrate the in vivo activity of the muDS6-DM1 conjugate, human
tumor xenografts were established in SCID mice. A subcutaneous model of the
human cervical carcinoma cell-line, KB, was developed. KB cells were grown in
vitro, collected, and 5 x 106 cells in a 100 L of serum free medium were
injected
under the right shoulder of each mouse and allowed to grow for 6 days to an
average
tumor volume of 144 125 mm3 at which time drug treatment was initiated. Mice
were given either PBS, conjugate at 150 g/kg DM1, or conjugate at 225 g/kg
DM1
(2 mice per group) intravenously every day for 5 days. Toxic responses were
monitored daily during the treatment. Tumor volumes (Figure 25A) and
corresponding body weights (Figure 25B) were monitored throughout the study.
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[264] The KB tumors treated with PBS control grew rapidly with a doubling time
of
about 4 days. In contrast, both groups of mice treated with conjugate exhibit
complete tumor regression 14 days and 18 days after treatment initiation for
the 225
g/kg and 150 g/kg dose groups, respectively. At the 150 g/kg dose the tumor
delay was approximately 70 days. Treatment at 225 g/kg resulted in cures as
there
was no evidence of tumor recurrence at the termination of the study on day
120. As
seen in Figure 25B the mice in the 150 g/kg group showed no weight loss
indicating
that the dose was well tolerated. At the higher dose the mice experience only
a
temporary 3% reduction in body weight. During the 5-day treatment course, mice
exhibited no visible signs of toxicity. Taken together, this study
demonstrates that
muDS6-DM1 treatment can cure mice of KB xenograft tumors at a non-toxic dose.
[265] muDS6-DM1 activity was further tested on a panel of subcutaneous
xenograft
models (see Figure 26). The tumor cell-lines used to make xenografts displayed
a
range of in vitro maytansine sensitivities and CA6 epitope densities (Table 9
below).
OVCAR5 cells and TOV-21G are ovarian tumor cell lines; HPAC is a pancreatic
tumor cell line; HeLa is a cervical tumor cell line. OVCAR5 and TOV-21 G cells
have low surface CA6 expression; HeLa cells have an intermediate level of
surface
CA6 expression; HPAC cells have a high CA6 density of surface expression. TOV-
21G and HPAC cells are maytansine sensitive; OVCAR5 and HeLa cells are 2-7-
fold
less maytansine sensitive.
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Table 9
Clonogenic Assay Clonogenic MTT Assay
Cell MMFt Apparent Maytansine IC50 Assay Conjugate Conjugate EC50
Line Kd ~) (M) ICso (M) (M)
BT-20 232.20 9.14 x 10"10 3.50 x 10-10 > 3.00 x 109 1.44 x 10-08
BT-483 1911.00 1.37 x 10-08 1.50 x 10"10 1.00 x 10"I0 N/A
Caov-3 465.20 5.48 x 10-09 3.20 x 10-" 8.00 x 10"10 1.61 x 10'09
Caov-4 149.00 4.04 x 10-09 6.00 x 10-10 > 3.00 x 10"09 N/A
HeLa 242.50 6.94 x 10-10 1.00 x 10"'0 1.80 x 10'09 N/A
HPAC 2228.00 2.35 x 10-08 5.50 x 10-" 1.80 x 109 1.84 x 10-09
HPAF-II 266.50 2.81 x 109 6.00 x 10-10 > 3.00 x 10- 9 1.00 x 10"08
Hs766T 182.90 2.32 x 10-09 > 3.00 x 10"09 > 3.00 x 10-09 > 3.20 x 10-08
KB 119.56 1.11 x 10-10 3.00 x 10-11 1.40 x 10"09 3.01 x 10-09
OVCAR5 97.10 1.47 x 10-09 3.20 x 100 > 3.00 x 10- 9 8.46 x 10" '
T-47D 559.58 3.42 x 10-09 1.20 x 10-10 > 3.00 x 10"09 N/A
TOV-21G 87.79 3.07 x 10-10 4.80 x 10"" 2.00 x 10'09 6.88 x 10-09
WISH 1133.55 2.38 x 10-09 9.00 x 10-11 4.60 x 10-10 6.69 x 10-'0
ZR-75-1 811.67 4.30 x 10-09 1.00 x 10-10 N/A 9.45 x 10-10
average maximum relative mean fluorescence
[266] The 4 cell-lines were grown in vitro, collected, and 1 x 107 cells in a
100 L of
serum free medium were injected under the right shoulder of each mouse (6 mice
per
model) and allowed to grow for 6 days to an average tumor volume of 57.6 6.7
and
90.2 13.4 mm3 for the test and control groups respectively of OVCAR5, 147.1
29.6
and 176.2 18.9 mm3 for the test and control groups respectively of HPAC,
194.3
37.2 and 201.7 71.7 mm3 for the test and control groups respectively of HeLa,
and
96.6 22.8 and 155.6 13.4 mm3 for the test and control groups respectively of
TOV-
21 G, at which time drug treatment was initiated. For each model three control
mice
were treated with two weekly doses of PBS and three test mice were treated
with two
weekly doses of conjugate (600 g/kg DMl) intravenously. Toxic responses were
monitored daily during the treatment and tumor volumes and body weights were
monitored throughout the study. The conjugate efficacy for the various models
is
shown graphically in Figure 26A, C, E, and G and the corresponding body
weights
are plotted in Figure 26B, D, F, and H. OVCAR5, TOV-21 G, and HPAC cell-lines
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form aggressive tumors as can be seen in the PBS controls for each model. The
HeLa
model had about a 3 week lag period before beginning exponential growth. In
all
models, DS6-DMI conjugate treatment resulted in a complete tumor regression in
all
mice. For the TOV-21G, HPAC, and HeLa models the mice remain tumor-free at day
61. In the OVCAR5 model tumors recurred at about day 45 after tumor
inoculation.
Thus, muDS6-DMl treatment in this model results in a tumor growth delay of
approximately 34 days. The growth delay is significant as OVCAR5 cells are
less
maytansine-sensitive and have low CA6 epitope expression. In models where
either
the CA6 epitope density is higher or the model has greater maytansine
sensitivity, the
tumor regression is more robust. It is important to note that only 2 doses
were
administered. Clearly the dosing schedule used in this study was not toxic to
the mice
as no weight loss was observed. It is likely that cures could be achieved with
additional or higher conjugate doses.
[267] Human ovarian cancer is largely a disease of the peritoneum. OVCAR5
cells
grow aggressively as an intraperitoneal (IP) model in SCID mice forming tumor
nodules and producing ascites in a manner similar to human disease. To
demonstrate
activity in an IP model, muDS6-DM1 was used to treat mice bearing OVCAR5 IP
tumors (Figure 27). OVCAR5 cells were grown in vitro, harvested and 1 x 107
cells
in 100 L of serum free medium were injected intraperitoneally. Tumors were
allowed to grow for 6 days at which time treatment was initiated. Mice were
treated
weekly for 2 weeks with either PBS or DS6-DM1 conjugate at a dose of 600 g/kg
DM1 and monitored for weight loss resulting from peritoneal disease. By day
28, the
PBS group of mice had lost greater than 20% body weight and were euthanized.
The
treated group was sacrificed at day 45 after exceeding 20% body weight loss.
This
study demonstrates that muDS6-DMI is able to delay tumor growth in the
aggressive
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OVCAR5 IP model despite the fact that OVCAR5 cells are less sensitive to
maytansine and have few CA6 epitopes per cell. Because the dosing schedule
used
elicited no visible signs of toxicity, it is likely that additional and higher
doses could
be used to achieve further tumor growth delay or cures.
Example 17: Synthesis and Characterization of DS6-SPP-MMl-202 Taxoid Cytotoxic
Conjugate
[268] muDS6 was modified with the N-sulfosuccinimidyl 4-nitro-2-pyridyl-
pentanoate (SSNPP) linker. To 50 mg of muDS6 Ab in 90% Buffer A, 10% DMA
was added 10 equivalents of SSNPP in DMA. The final concentration of Ab was 8
mg/ml. The reaction was stirred for 4 hours at room temperature, then purified
by G25
chromatography. The extent of antibody modification was measured
spectophotometrically using the absorbance at 280 nm (antibody) and 325
(linker) and
found to have 3.82 linkers/antibody. Recovery of the antibody was 43.3 mg
giving an
87% yield. Conjugation of muDS6-nitroSPP was conjugated with Taxoid MM1-202
(1812 P.16). Conjugation was carried out on a 42 mg scale in 90% Buffer A, 10%
DM1. The taxoid was added in 4 aliquots of 0.43 eq/Linker (each aliquot) over
a
period of about 20 hours. By this time the reaction had turned noticeably
cloudy.
After G25 purification the resulting conjugate, recovered in about 64% yield
had
about 4.3 taxoids/Ab and about 1 equivalent of unreacted linker left. To
quench
unreacted linker, 1 equivalent of cysteine/unreacted linker was added to the
conjugate
with stirring overnight. A definite yellowish tinge was noticeable upon
cysteine
addition indicating release of thiopyridine. The reaction solution was then
dialyzed in
Buffer B/0.01 % Tween 20 followed by further dialysis in Buffer B alone over
several
days. The final conjugate had 2.86 drugs/antibody. The antibody recovery was
14.7
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mg, giving a 35% yield overall. Conjugate was further biochemically
characterized
by SEC and found to have 89% monomer, 10.5% dimer and 0.5% higher molecular
weight aggregate.
[2691 The results of a flow cytometry analysis comparing the binding of muDS6-
SPP-MM1-202 taxoid versus muDS6 antibody on HeLa cells is shown in Figure 28.
The results indicate that muDS6 retains binding activity when conjugated to a
taxane.
Example 18: In vitro and in vivo activity of humanized DS6 conjugate
[2701 A huDS6v1.01-SPDB-DM4 conjugate was constructed. This conjugate is
similar to the muDS6-SPP-DM1 conjugate described in Example 14 except that the
linker/maytansine drug part of the conjugate differs in the structure around
the
disulfide bond; the muDS6-SPP-DMl conjugate has one methyl group hindrance on
the disulfide carbon on the antibody side of the linker while the SPDB-DM4
conjugate has two methyl group hindrance on the disulfide carbon on the
maytansine
side of the linker.
[2711 The huDS6v1.01 antibody (8 mg/ml) was modified using 8-fold molar excess
of N-succinimidyl-4-(2-pyridyldithio) butanoate (SPDB) to introduce
dithiopyridyl
groups. The reaction was carried out in 95% v/v Buffer A (50 mM KPi, 50 mM
NaCI, 2 mM EDTA, pH 6.5) and 5% v/v ethanol for 1.5 h at room temperature. The
reaction mixture was gel-filtered through a 15m1 Sephadex G25 column
(equilibrated
in Buffer A). The degree of modification was determined by measuring the
absorbance of the antibody at 280 nm and the DTT released 2-mercaptopyridine
(Spy)
at 280 and 343 nm. Modified DS6 was then conjugated at 1.8 mg Ab/mL using a
1.7-
fold molar excess of Nz'-deacetyl-N-2'(4-methyl-4-mercapto-l-oxopentyl)-
maytansine (L-DM4) over SPy. The reaction was camed out in Buffer A (97% v/v)
CA 02615761 2008-01-17
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with DMA (3% v/v). The reaction was incubated at room temperature overnight
for
-20 h. In contrast to the conjugation of muDS6, the reaction mixture was clear
and
immediately underwent gel-filtration through a NAP15m1 G25 column equilibrated
in
Citrate buffer (20 mM citrate, 135 mM NaCI, pH 5.5). The conjugate was sterile-
filtered using a 0.22 m Millex-GV filter. The number of DM4 molecules linked
per
molecule of DS6 was determined by measuring the absorbance at both 252 nm and
280 nm of the filtered material. The DM4/Ab ratio was found to be 3.2 and the
step
yield of conjugated DS6 was 69%. The conjugated antibody concentration was
1.51
mg/mL. The purified conjugate was biochemically characterized by size
exclusion
chromotography (SEC) and found to be 92.5% monomer. Analysis of DM4 in the
purified conjugated indicated that >99% was covalently bound to antibody.
[272] In Figure 29A, flow cytometric binding of the huDS6vl.01-DM4 conjugate
and unmodified DS6 to KB cells shows that conjugation of huDS6v1.01 results in
essentially no loss of affinity. The binding was conducted essentially as
described for
Figure 20 except that KB cells were used rather than CaOv-3 cells. In vitro
cytotoxicity of huDS6v1.01 was tested essentially as described in Figure 24G.
huDS6vl.01 killed WISH cells with an IC50 of 4.4 x 10-10 M whereas
unconjugated
huDS6vl.0l showed no cytotoxic activity.
[273] The in vivo activity of huDS6v1.01-DM4 was tested on the HPAC pancreatic
model. HPAC cells were inoculated on day 0, and immunoconjugate treatments
were
given on day 13. PBS control animals were euthanized once tumor volumes
exceeded
1000 mm3. The conjugate was given at a dose of either 200 g/kg or 600 g/kg
DM4,
corresponding to an antibody concentration 15 mg/kg and 45 mg/kg,
respectively.
Tumor volume (Figure 30A) and body weight (Figure 30B) of the mice were
monitored during the course of the study. The huDS6v1.01-DM4 showed potent
anti-
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tumor activity at 200 g/kg DM4 with all mice achieving complete tumor
regression.
The control B4-DM4 conjugate recognizing an antigen not expressed on HPAC
xenografts had essentially no activity at 200 g/kg. The lack of body weight
loss
(Figure 30B) of the mice indicates that the treatment with 200 g/kg conjugate
is
below the maximum tolerated dose. This result demonstrates that a humanized
version of DS6 is able to mediate targeted delivery of a maytansinoid drug
resulting in
potent anti-tumor activity.
97
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