U.S. patent application number 13/728059 was filed with the patent office on 2013-06-20 for fully human antibodies specific to cadm1.
This patent application is currently assigned to OXFORD BIOTHERAPEUTICS LTD. The applicant listed for this patent is Medarex, Inc., Oxford BioTherapeutics Ltd. Invention is credited to Bingliang CHEN, Haichun HUANG, Heidi N. LEBLANC, Erika MEADDOUGH, Chin PAN, Chetana RAO-NAIK, Jonathan Alexander TERRETT.
Application Number | 20130156788 13/728059 |
Document ID | / |
Family ID | 42144944 |
Filed Date | 2013-06-20 |
United States Patent
Application |
20130156788 |
Kind Code |
A1 |
TERRETT; Jonathan Alexander ;
et al. |
June 20, 2013 |
FULLY HUMAN ANTIBODIES SPECIFIC TO CADM1
Abstract
The present disclosure provides isolated monoclonal antibodies,
particularly human monoclonal antibodies, more particularly
engineered antibodies resulting in increased binding to Fc
receptors and/or increased potency for ADCC or immunoconjugates,
which specifically bind to CADM1 with high affinity. Nucleic acid
molecules encoding CADM1 antibodies, expression vectors, host cells
and methods for expressing the CADM1 antibodies are also provided.
Bispecific molecules and pharmaceutical compositions comprising the
CADM1 antibodies are also provided. Methods for detecting CADM1, as
well as methods for treating various cancers, including lung cancer
and pancreatic cancer, are disclosed.
Inventors: |
TERRETT; Jonathan Alexander;
(Sunnyvale, CA) ; LEBLANC; Heidi N.; (Mountain
View, CA) ; HUANG; Haichun; (Fremont, CA) ;
MEADDOUGH; Erika; (Gilroy, CA) ; PAN; Chin;
(Los Altos, CA) ; CHEN; Bingliang; (Alameda,
CA) ; RAO-NAIK; Chetana; (Walnut Creek, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Medarex, Inc.;
Oxford BioTherapeutics Ltd; |
Princeton
Oxon |
NJ |
US
GB |
|
|
Assignee: |
OXFORD BIOTHERAPEUTICS LTD
Oxon
NJ
MEDAREX, INC.
Princeton
|
Family ID: |
42144944 |
Appl. No.: |
13/728059 |
Filed: |
December 27, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13254752 |
Sep 2, 2011 |
8420084 |
|
|
PCT/US2010/026315 |
Mar 5, 2010 |
|
|
|
13728059 |
|
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|
61209471 |
Mar 5, 2009 |
|
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61209390 |
Mar 5, 2009 |
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Current U.S.
Class: |
424/156.1 ;
530/388.85; 530/391.7 |
Current CPC
Class: |
C07K 16/3023 20130101;
A61P 35/02 20180101; C07K 2317/77 20130101; A61K 33/24 20130101;
A61K 39/39558 20130101; C07K 16/3007 20130101; C07K 2317/24
20130101; C07K 2317/732 20130101; A61K 2039/505 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61K 33/24 20130101; C07K
2317/73 20130101; C07K 2317/41 20130101; A61K 39/39558 20130101;
A61P 35/00 20180101 |
Class at
Publication: |
424/156.1 ;
530/388.85; 530/391.7 |
International
Class: |
C07K 16/30 20060101
C07K016/30 |
Claims
1. An isolated monoclonal antibody, or an antigen-binding portion
thereof, which binds to an epitope on human CADM1 recognized by a
second antibody, wherein the second antibody comprises heavy and
light chain variable regions comprising the amino acid sequences
set forth in SEQ ID NOs:19 and 22, respectively.
2. An isolated monoclonal antibody, or an antigen-binding portion
thereof, which binds to an epitope on human CADM1 recognized by a
second antibody, wherein the second antibody comprises heavy and
light chain variable regions comprising the amino acid sequences
set forth in SEQ ID NOs:20 and 23, respectively.
3. An isolated monoclonal antibody, or an antigen-binding portion
thereof, which binds to an epitope on human CADM1 recognized by a
second antibody, wherein the second antibody comprises heavy and
light chain variable regions comprising the amino acid sequences
set forth in SEQ ID NOs:21 and 24, respectively.
4. The monoclonal antibody, or an antigen-binding portion thereof,
of claim 1, which is conjugated to a therapeutic agent.
5. The monoclonal antibody, or an antigen-binding portion thereof,
of claim 2, which is conjugated to a therapeutic agent.
6. The monoclonal antibody, or an antigen-binding portion thereof,
of claim 3, which is conjugated to a therapeutic agent.
7. A composition comprising the monoclonal antibody, or an
antigen-binding portion thereof, of claim 1 and a pharmaceutically
acceptable carrier.
8. A composition comprising the monoclonal antibody, or an
antigen-binding portion thereof, of claim 2 and a pharmaceutically
acceptable carrier.
9. A composition comprising the monoclonal antibody, or an
antigen-binding portion thereof, of claim 3 and a pharmaceutically
acceptable carrier.
10. A method for treating a disease associated with target cells
expressing CADM1 comprising administering to a subject the
monoclonal antibody, or an antigen-binding portion thereof, of
claim 1 in an amount effective to treat the disease.
11. A method for treating a disease associated with target cells
expressing CADM1 comprising administering to a subject the
monoclonal antibody, or an antigen-binding portion thereof, of
claim 2 in an amount effective to treat the disease.
12. A method for treating a disease associated with target cells
expressing CADM1 comprising administering to a subject the
monoclonal antibody, or an antigen-binding portion thereof, of
claim 3 in an amount effective to treat the disease.
13. The method of claim 10, wherein the disease is a human cancer
selected from the group consisting of: small cell lung cancer,
adult T-cell leukemia, non-small cell lung cancer, including
squamous carcinomas and adenocarcinomas, melanoma, breast cancer,
colorectal cancer, ovarian cancer, prostate cancer, neuroendocrine
cancers, including those of lung, adrenal, pituitary, GI-tract,
kidney, liver, including hepatocellular carcinomas, pancreas,
including insulinomas and glucagonomas, glioblastomas, and
carcinoid tumors, including those of the pancreas, lung, GI tract,
liver, and kidney.
14. The method of claim 11, wherein the disease is a human cancer
selected from the group consisting of: small cell lung cancer,
adult T-cell leukemia, non-small cell lung cancer, including
squamous carcinomas and adenocarcinomas, melanoma, breast cancer,
colorectal cancer, ovarian cancer, prostate cancer, neuroendocrine
cancers, including those of lung, adrenal, pituitary, GI-tract,
kidney, liver, including hepatocellular carcinomas, pancreas,
including insulinomas and glucagonomas, glioblastomas, and
carcinoid tumors, including those of the pancreas, lung, GI tract,
liver, and kidney.
15. The method of claim 12, wherein the disease is a human cancer
selected from the group consisting of: small cell lung cancer,
adult T-cell leukemia, non-small cell lung cancer, including
squamous carcinomas and adenocarcinomas, melanoma, breast cancer,
colorectal cancer, ovarian cancer, prostate cancer, neuroendocrine
cancers, including those of lung, adrenal, pituitary, GI-tract,
kidney, liver, including hepatocellular carcinomas, pancreas,
including insulinomas and glucagonomas, glioblastomas, and
carcinoid tumors, including those of the pancreas, lung, GI tract,
liver, and kidney.
16. An isolated monoclonal antibody, or antigen binding portion
thereof, comprising a heavy chain variable region and a light chain
variable region, wherein: the heavy chain variable region comprises
an amino acid sequence that is at least 80% homologous to an amino
acid sequence selected from the group consisting of SEQ ID NOs: 19,
20, and 21; the light chain variable region comprises an amino acid
sequence that is at least 80% homologous to an amino acid sequence
selected from the group consisting of SEQ ID NOs: 22, 23 and 24;
and the antibody binds to human CADM1 with a EC.sub.50 of 10 nM or
less.
17. The monoclonal antibody, or an antigen-binding portion thereof,
of claim 16, which is conjugated to a therapeutic agent.
18. A composition comprising the monoclonal antibody, or an
antigen-binding portion thereof, of claim 16 and a pharmaceutically
acceptable carrier.
19. A method for treating a disease associated with target cells
expressing CADM1 comprising administering to a subject the
monoclonal antibody, or an antigen-binding portion thereof, of
claim 16 in an amount effective to treat the disease.
20. The method of claim 19, wherein the disease is a human cancer
selected from the group consisting of: small cell lung cancer,
adult T-cell leukemia, non-small cell lung cancer, including
squamous carcinomas and adenocarcinomas, melanoma, breast cancer,
colorectal cancer, ovarian cancer, prostate cancer, neuroendocrine
cancers, including those of lung, adrenal, pituitary, GI-tract,
kidney, liver, including hepatocellular carcinomas, pancreas,
including insulinomas and glucagonomas, glioblastomas, and
carcinoid tumors, including those of the pancreas, lung, GI tract,
liver, and kidney.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/254,752, filed Sep. 2, 2011, which is a 371 of International
application No. PCT/US2010/026315, filed Mar. 5, 2010, which claims
the benefit of U.S. provisional application No. 61/209,471, filed
Mar. 5, 2009, and U.S. provisional application No. 61/209,390,
filed Mar. 5, 2009, both of which are incorporated by reference
herein in their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the fields of
immunology and molecular biology. More specifically, provided
herein are antibodies, particularly engineered antibodies resulting
in increased binding to Fc receptors and/or increased potency for
ADCC and immunoconjugates, and other therapeutic proteins directed
against immunoglobulin-like adhesion molecule CADM1, nucleic acids
encoding such antibodies and therapeutic proteins, methods for
preparing inventive monoclonal antibodies and other therapeutic
proteins, and methods for the treatment of diseases, such as
cancers mediated by CADM1 expression/activity and/or associated
with abnormal expression/activity of ligands therefore.
BACKGROUND OF THE INVENTION
[0003] Cell adhesion molecules are generally identified as
cadherins, integrins, selectins, or as members of the
Immunoglobulin (Ig) superfamily. The immunoglobulin superfamily
molecules include cell surface antigen receptors, co-receptors and
co-stimulatory molecules of the immune system, molecules involved
in antigen presentation to lymphocytes, cell adhesion molecules and
some cytokine receptors. Ig superfamily cell adhesion molecules
constitute over 100 molecules in vertebrates, and include NCAMs
(neural cell adhesion molecules), L1 family CAMs, ICAMS
(intracellular cell adhesion molecules), VCAMS (vascular cell
adhesion molecules), SIGLECs (sialic acid binding Ig-like lectins,
including CD22 and CD83), nectins, CD2, CD48.
[0004] The immunoglobulin superfamily molecule CADM1 was initially
characterized by multiple research groups; as a result the molecule
is identified by many names in scientific literature including cell
adhesion molecule 1, synaptic cell adhesion molecule (synCAM),
spermatogenic immunoglobulin superfamily molecule (sgIGSF), IGSF4,
BL2, ST17, NECL2, RA175, and CADM1A. Initially reported to further
act as a tumor suppressor, it is also known as TSLC1 (Murakami et
al., Nature Genetics 27(4):427 (2001)).
[0005] Unlike cadherins and integrins, which require divalent
cations such as Ca.sup.+2 or Mg.sup.+2 for adhesive activities, Ig
superfamily molecules are typically Ca.sup.+2 or Mg.sup.+2
independent. The CADM1 structure is characterized as having an
extracellular domain with three immunoglobulin-like motifs, a
single hydrophobic membrane-spanning .alpha. helix and an
intracellular domain that binds actin fibers via DAL-1, and a short
C-terminal cytoplasmic tail containing a PDZ-binding motif. Two
CADM1 isoforms are known, NM.sub.--014333 and NM.sub.--001098517,
the latter having a 27 amino acid deletion. Analysis indicates that
the amino acid sequences corresponding to the cytoplasmic domain of
CADM1 are identical in five mammals and highly conserved in
vertebrates, suggesting an important role of CADM1 in normal
cell-cell interaction. The mouse CADM1 orthologue (AAQ023810) shows
97% identity to the human CADM1. (Fukami et al., Gene 295:7-12
(2002)).
[0006] CADM1 is expressed in nerves and mast cells (Ito et al. J
Pharmacol Sci.; 102(1):1-5 (2006)), pulmonary alveolar cells (Ito
et al. Histol Histopathol. 18(4):1321-9 (2003)), pancreatic
secretory cells (Shingai et al. J Biol Chem.; 278(37):35421-7
(2003)), (Wakayama et al., Blood; 101(7):2601-8 (2003)). It is also
associated with hepatocellular carcinoma (HCC). CADM1 appears to be
expressed in fetal and cirrhotic adult bile duct cells, but is
absent from disease-free adult bile ducts (Ito et al., Hepatology;
45(3):684-94 (2007)). It is further reported to be associated with
glioblastomas and lung cancer.
[0007] Two mechanisms resulting in CADM1 inactivation have been
identified: through promoter methylation, and through loss of
heterozygosity at the gene locus. Methylation of the CADM1 promoter
reportedly results in loss of CADM1 expression in tumors, including
lung esophageal, pancreatic, breast, and prostate cancers,
particularly in tumors with aggressive behavior. (Murakami et al.,
Mol Cancer. 4:28 (2005)).
SUMMARY OF THE INVENTION
[0008] The present invention addresses these and other related
needs by providing antibodies, particularly engineered antibodies
resulting in increased binding to Fc receptors and/or increased
potency for ADCC and immunoconjugates, directed against CADM1
therefore, nucleic acids encoding such antibodies and therapeutic
proteins, methods for preparing anti-CADM1 monoclonal antibodies
and other therapeutic proteins, and methods for the treatment of
diseases, such as CADM1 mediated disorders, e.g., human cancers,
including small cell lung cancer, adult T-cell leukemia, non-small
cell lung cancer (including squamous carcinomas and
adenocarcinomas), melanoma, breast cancer, colorectal cancer,
ovarian cancer, prostate cancer, neuroendocrine cancers including
those of lung, adrenal, pituitary, GI-tract, kidney, liver
(including hepatocellular carcinomas), pancreas (including
insulinomas and glucagonomas), glioblastomas, and carcinoid tumors
including those of pancreas, lung, GI tract, liver, or kidney.
[0009] Thus, the present invention provides isolated monoclonal
antibodies, in particular murine, chimeric, humanized, and
fully-human monoclonal antibodies, that bind to one or more bone
morphogenic protein and receptors therefore and that exhibit one or
more desirable functional property. Such properties include, for
example, high affinity specific binding to human CADM1. Also
provided are methods for treating a variety of CADM1-mediated
diseases using the antibodies, proteins, and compositions of the
present invention.
[0010] The present invention provides an isolated monoclonal
antibody, or an antigen-binding portion thereof, an antibody
fragment, or an antibody mimetic which binds an epitope on human
CADM1 recognized by an antibody comprising a heavy chain variable
region comprising the amino acid sequence set forth in SEQ ID NO:
19, 20, or 21 and a light chain variable region comprising the
amino acid sequence set forth in SEQ ID NO: 22, 23, or 24. In some
embodiments the isolated antibody is a full-length antibody of an
IgG1, IgG2, IgG3, or IgG4 isotype.
[0011] In some embodiments, the antibody of the present invention
is selected from the group consisting of: a whole antibody, an
antibody fragment, a humanized antibody, a single chain antibody,
an immunoconjugate, an engineered antibody resulting in increased
binding to Fc receptors and/or increased potency for ADCC, and a
bispecific antibody. In a preferred embodiment, the antibody of the
present invention is an immunoconjugate or an engineered antibody
resulting in increased binding to Fc receptors and/or increased
potency for ADCC. The antibody fragment may be selected from the
group consisting of: a UniBody, a domain antibody, and a Nanobody.
In some embodiments, the immunoconjugates of the invention comprise
a therapeutic agent. In another aspect of the invention, the
therapeutic agent is a cytotoxin or a radioactive isotope.
[0012] In some embodiments, the antibody of the present invention
is selected from the group consisting of: an Affibody, a DARPin, an
Anticalin, an Avimer, a Versabody, and a Duocalin.
[0013] In some aspects of the invention, the antibody binds to
human CADM1 with an EC.sub.50 of <50 Nm, <10 Nm, or <1
Nm.
[0014] In alternative embodiments, compositions of the present
invention comprise an isolated antibody or antigen-binding portion
and a pharmaceutically acceptable carrier.
[0015] In other aspects, the antibody of the present invention is a
composition comprising the isolated antibody or antigen-binding
portion thereof of claim 1 and a pharmaceutically acceptable
carrier.
[0016] In some embodiments, the invention comprises an isolated
nucleic acid molecule encoding the heavy or light chain of the
isolated antibody or antigen-binding portion which binds an epitope
on human CADM1. Other aspects of the invention comprise expression
vectors comprising such nucleic acid molecules, and host cells
comprising such.
[0017] In some embodiments, the present invention provides a method
for preparing an anti-CADM1 antibody, said method comprising the
steps of: obtaining a host cell that contains one or more nucleic
acid molecules encoding the antibody of the invention; growing the
host cell in a host cell culture; providing host cell culture
conditions wherein the one or more nucleic acid molecules are
expressed; and recovering the antibody from the host cell or from
the host cell culture.
[0018] In other embodiments, the invention is directed to methods
for treating or preventing a disease associated with target cells
expressing CADM1, said method comprising the step of administering
to a subject an anti-CADM1 antibody, or antigen-binding portion
thereof, in an amount effective to treat or prevent the disease. In
some aspects, the disease treated or prevented by the antibodies or
antigen-binding portion thereof of the invention, is selected from
the group consisting of: human cancers. In some embodiments, the
disease treated or prevented by the antibodies of the present
invention is a cancer selected from the group consisting of: small
cell lung cancer, adult T-cell leukemia, non-small cell lung cancer
(including squamous carcinomas and adenocarcinomas), melanoma,
breast cancer, colorectal cancer, ovarian cancer, prostate cancer,
neuroendocrine cancers including those of lung, adrenal, pituitary,
GI-tract, kidney, liver (including hepatocellular carcinomas),
pancreas (including insulinomas and glucagonomas), glioblastomas,
and carcinoid tumors including those of pancreas, lung, GI tract,
liver, or kidney.
[0019] In other embodiments, the invention is directed to an
anti-CADM1 antibody, or antigen-binding portion thereof, for use in
treating or preventing a disease associated with target cells
expressing CADM1. In some aspects, the disease treated or prevented
by the antibody or antigen-binding portion thereof of the
invention, is human cancer. In some embodiments, the disease
treated or prevented by the antibodies of the present invention is
a cancer selected from the group consisting of: small cell lung
cancer, adult T-cell leukemia, non-small cell lung cancer
(including squamous carcinomas and adenocarcinomas), melanoma,
breast cancer, colorectal cancer, ovarian cancer, prostate cancer,
neuroendocrine cancers including those of lung, adrenal, pituitary,
GI-tract, kidney, liver (including hepatocellular carcinomas),
pancreas (including insulinomas and glucagonomas), glioblastomas,
and carcinoid tumors including those of pancreas, lung, GI tract,
liver, or kidney.
[0020] In other embodiments, the invention is directed to the use
of an anti-CADM1 antibody, or antigen-binding portion thereof, for
the manufacture of a medicament for use in treating or preventing a
disease associated with target cells expressing CADM1. In some
aspects, the disease treated or prevented by the medicament of the
invention, is human cancer. In some embodiments, the disease
treated or prevented by the medicament of the invention is a cancer
selected from the group consisting of: small cell lung cancer,
adult T-cell leukemia, non-small cell lung cancer (including
squamous carcinomas and adenocarcinomas), melanoma, breast cancer,
colorectal cancer, ovarian cancer, prostate cancer, neuroendocrine
cancers including those of lung, adrenal, pituitary, GI-tract,
kidney, liver (including hepatocellular carcinomas), pancreas
(including insulinomas and glucagonomas), glioblastomas, and
carcinoid tumors including those of pancreas, lung, GI tract,
liver, or kidney.
[0021] In other embodiments, the present invention is an isolated
monoclonal antibody or an antigen binding portion thereof, an
antibody fragment, or an antibody mimetic which binds an epitope on
human CADM1 recognized by an antibody comprising a heavy chain
variable region and a light chain variable region selected from the
group consisting of the heavy chain variable region amino acid
sequence set forth in SEQ ID NO:19 and the light chain variable
region amino acid sequence set forth in SEQ ID NO:22; the heavy
chain variable region amino acid sequence set forth in SEQ ID NO:20
and the light chain variable region amino acid sequence set forth
in SEQ ID NO:23; the heavy chain variable region amino acid
sequence set forth in SEQ ID NO:21 and the light chain variable
region amino acid sequence set forth in SEQ ID NO:24. In further
aspects, the antibody is selected from the group consisting of: a
whole antibody, an antibody fragment, a humanized antibody, a
single chain antibody, an immunoconjugate, an engineered antibody
resulting in increased binding to Fc receptors and/or increased
potency for ADCC, and a bispecific antibody. In a preferred aspect,
the antibody is an immunoconjugate or an engineered antibody
resulting in increased binding to Fc receptors and/or increased
potency for ADCC. In further aspects of the invention, the antibody
fragment is selected from the group consisting of: a UniBody, a
domain antibody, and a Nanobody. In some embodiments, the antibody
mimetic is selected from the group consisting of: an Affibody, a
DARPin, an Anticalin, an Avimer, a Versabody, and a Duocalin. In
further embodiments, the composition comprises the isolated
antibody or antigen binding portion thereof and a pharmaceutically
acceptable carrier.
[0022] In some embodiments, the present invention is an isolated
nucleic acid molecule encoding the heavy or light chain of the
isolated antibody or antigen binding portion thereof of antibody of
the invention, and in further aspects may include an expression
vector comprising such nucleic acids, and host cells comprising
such expression vectors.
[0023] Another embodiment of the present invention is a hybridoma
expressing the antibody or antigen binding portion thereof of any
one of antibodies of the invention.
[0024] Other aspects of the invention are directed to methods of
making the antibodies of the invention, comprising the steps of:
immunizing a transgenic animal comprising human immunoglobulin
genes with a CADM1 peptide; recovering B-cells from said transgenic
animal; making hybridomas from said B-cells; selecting hybridomas
that express antibodies that bind CADM1; and recovering said
antibodies that bind CADM1 from said selected hybridomas.
[0025] In other embodiments, the method of making anti-CADM1
antibodies, comprises the steps of:
[0026] immunizing a transgenic animal comprising human
immunoglobulin genes with a CADM1 peptide;
[0027] recovering mRNA from the B cells of said transgenic animal;
converting said mRNA to cDNA;
[0028] expressing said cDNA in phages such that anti-CADM1
antibodies encoded by said cDNA are presented on the surface of
said phages;
[0029] selecting phages that present anti-CADM1 antibodies;
[0030] recovering nucleic acid molecules from said selected phages
that encode said anti-CADM1 immunoglobulins;
[0031] expressing said recovered nucleic acid molecules in a host
cell; and
[0032] recovering antibodies from said host cell that bind
CADM1.
[0033] In some aspects of the invention, the isolated monoclonal
antibody, or an antigen binding portion thereof, binds an epitope
on the CADM1 polypeptide having an amino acid sequence of SEQ ID
NOS: 43 or 44 recognized by an antibody comprising a heavy chain
variable region comprising the amino acid sequence set forth in SEQ
ID NOS: 19, 20, or 21 and a light chain variable region comprising
the amino acid sequence set forth in SEQ ID NOS: 22, 23, or 24.
[0034] Other features and advantages of the instant invention will
be apparent from the following detailed description and examples
which should not be construed as limiting. The contents of all
references, Genbank entries, patents and published patent
applications cited throughout this application are expressly
incorporated herein by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1A shows the nucleotide sequence (SEQ ID NO:25) and
amino acid sequence (SEQ ID NO:19) of the heavy chain variable
region of the PTA021_A1 human monoclonal antibody. The CDR1 (SEQ ID
NO:1), CDR2 (SEQ ID NO:4) and CDR3 (SEQ ID NO:7) regions are
delineated and the V, D and J germline derivations are
indicated.
[0036] FIG. 1B shows the nucleotide sequence (SEQ ID NO:28) and
amino acid sequence (SEQ ID NO:22) of the light chain variable
region of the PTA021_A1 human monoclonal antibody. The CDR1 (SEQ ID
NO:10), CDR2 (SEQ ID NO:13) and CDR3 (SEQ ID NO:16) regions are
delineated and the V and J germline derivations are indicated.
[0037] FIG. 2A shows the nucleotide sequence (SEQ ID NO:26) and
amino acid sequence (SEQ ID NO:20) of the heavy chain variable
region of the PTA021_A2 human monoclonal antibody. The CDR1 (SEQ ID
NO:2), CDR2 (SEQ ID NO:5) and CDR3 (SEQ ID NO:8) regions are
delineated and the V and J germline derivations are indicated.
[0038] FIG. 2B shows the nucleotide sequence (SEQ ID NO:29) and
amino acid sequence (SEQ ID NO:23) of the light chain variable
region of the PTA021_A2 human monoclonal antibody. The CDR1 (SEQ ID
NO:11), CDR2 (SEQ ID NO:14) and CDR3 (SEQ ID NO:17) regions are
delineated and the V and J germline derivations are indicated.
[0039] FIG. 3A shows the nucleotide sequence (SEQ ID NO:27) and
amino acid sequence (SEQ ID NO:21) of the heavy chain variable
region of the PTA021_A3 human monoclonal antibody. The CDR1 (SEQ ID
NO:3), CDR2 (SEQ ID NO:6) and CDR3 (SEQ ID NO:9) regions are
delineated and the V, D and J germline derivations are
indicated.
[0040] FIG. 3B shows the nucleotide sequence (SEQ ID NO:30) and
amino acid sequence (SEQ ID NO:24) of the light chain variable
region of the PTA021_A3 human monoclonal antibody. The CDR1 (SEQ ID
NO:12), CDR2 (SEQ ID NO:15) and CDR3 (SEQ ID NO:18) regions are
delineated and the V and J germline derivations are indicated.
[0041] FIG. 4 shows the alignment of the amino acid sequence of the
heavy chain variable region of PTA021_A1 (SEQ ID NO:19) with the
human germline V.sub.H 2-05 amino acid sequence (SEQ ID NO:31) and
the human germline J.sub.H JH5b amino acid sequence (SEQ ID
NO:37).
[0042] FIG. 5 shows the alignment of the amino acid sequence of the
heavy chain variable region of PTA021_A2 (SEQ ID NO:20) with the
human germline V.sub.H 2-05 amino acid sequences (SEQ ID NO:32) and
the human germline J.sub.H 5b amino acid sequence (SEQ ID
NO:38).
[0043] FIG. 6 shows the alignment of the amino acid sequence of the
heavy chain variable region of PTA021_A3 (SEQ ID NO:21) with the
human germline V.sub.H 2-05 amino acid sequences (SEQ ID NO:33) and
the human germline J.sub.H 5b amino acid sequence (SEQ ID
NO:39).
[0044] FIG. 7 shows the alignment of the amino acid sequence of the
light chain variable region of PTA021_A1 (SEQ ID NO:22) with the
human germline V.sub.K L15 amino acid sequence (SEQ ID NO:34) and
the human germline J.sub.K 4 amino acid sequence (SEQ ID
NO:40).
[0045] FIG. 8 shows the alignment of the amino acid sequence of the
light chain variable region of PTA021_A2 (SEQ ID NO:23) with the
human germline V.sub.k L15 amino acid sequence (SEQ ID NO:35) and
the human germline J.sub.K 4 amino acid sequence (SEQ ID
NO:41).
[0046] FIG. 9 shows the alignment of the amino acid sequence of the
light chain variable region of PTA021_A3 (SEQ ID NO:24) with the
human germline V.sub.k L15 amino acid sequence (SEQ ID NO:36) and
the human germline J.sub.K 4 amino acid sequence (SEQ ID
NO:42).
[0047] FIG. 10 shows the results of FACS analysis on PTA021_A1,
PTA021_A2 and PTA021_A3 in NCI-H69 small cell lung cancer
cells.
[0048] FIGS. 11A and 11B show the results of FACS analysis on
PTA021_A1, PTA021_A2 and PTA021_A3 in NCI_H69 and DMS79 small cell
lung cancer cells respectively.
[0049] FIG. 12 shows the results of FACS analysis on PTA021_A3 in
SKOV3 ovarian cancer cells.
[0050] FIG. 13 shows the results of FACS analysis on PTA021_A3 in
A549 non-small cell lung cancer cells.
[0051] FIG. 14 shows the results of FACS analysis on PTA021_A3 in
786-O renal cell carcinoma cells and SkMel28 melanoma cells.
[0052] FIG. 15 shows the results of FACS analysis on PTA021_A3 and
the non-fucosylated version of PTA021_A3 (nf) in NCI_H69 and DMS79
small cell lung cancer cells.
[0053] FIGS. 16A and 16B show antibody dependant cellular
cytotoxicity (ADCC) mediated by PTA021_A1, PTA021_A2, and PTA021_A3
with the DMS79 and NCI-H69 cell lines, respectively.
[0054] FIGS. 17A and 17B show the results of Hum-ZAP assays on
PTA021_A1, PTA021_A2, and PTA021_A3 in DMS79 and NCI_H69 cells
respectively.
[0055] FIG. 18 presents a graph showing the effect of treatment
using fucosylated or non-fucosylated PTA021_A3 antibodies on HepG2
tumor size in a mouse xenograft model.
[0056] FIG. 19 presents a graph showing the effect of treatment
with a PTA021_A3-Formula M conjugate on HepG2 tumor size in a mouse
xenograft model.
[0057] FIG. 20A presents a graph showing the effect of treatment
with dosages of 0.3 umol/kg of a PTA021_A3-Formula M conjugate on
DMS79 tumor size in a mouse xenograft model. FIG. 20B presents a
graph showing the effect of treatment with dosages of 0.1 and 0.03
umol/kg of a PTA021_A3-Formula M conjugate on DMS79 tumor size in a
mouse xenograft model. The study completed on day 60.
[0058] FIGS. 21, 22 and 23 present graphs showing ADCC activity of
PTA021_A3 and non-fucosylated PTA021_A3 upon HepG2, 786-O and DMS79
cells, respectively.
[0059] FIG. 24 presents a graph showing the effect of treatment
with PTA021_A3 antibody alone or in combination with cisplatin on
DMS79 tumor size in a mouse xenograft model.
[0060] FIGS. 25A and 25B show the Body Weight data for male monkeys
and female monkeys respectively in an exploratory toxicology study
on PTA021_A3 and the non-fucosylated version of PTA021_A3(NF) in
cynomolgus macaques.
[0061] FIGS. 26A and 26B show the Glucose data for male monkeys and
female monkeys respectively in an exploratory toxicology study on
PTA021_A3 and the non-fucosylated version of PTA021_A3(NF) in
cynomolgus macaques.
[0062] FIGS. 27A and 27B show the Alanine Transaminase data for
male monkeys and female monkeys respectively in an exploratory
toxicology study on PTA021_A3 and the non-fucosylated version of
PTA021_A3(NF) in cynomolgus macaques.
[0063] FIGS. 28A and 28B show the Aspartate Transaminase data for
male monkeys and female monkeys respectively in an exploratory
toxicology study on PTA021_A3 and the non-fucosylated version of
PTA021_A3(NF) in cynomolgus macaques.
[0064] FIGS. 29A and 29B show the Alkaline Phosphatase data for
male monkeys and female monkeys respectively in an exploratory
toxicology study on PTA021_A3 and the non-fucosylated version of
PTA021_A3(NF) in cynomolgus macaques.
[0065] FIGS. 30A and 30B show the Lactate Dehydrogenase data for
male monkeys and female monkeys respectively in an exploratory
toxicology study on PTA021_A3 and the non-fucosylated version of
PTA021_A3(NF) in cynomolgus macaques.
[0066] FIGS. 31A and 31B show the RBC data for male monkeys and
female monkeys respectively in an exploratory toxicology study on
PTA021_A3 and the non-fucosylated version of PTA021_A3(NF) in
cynomolgus macaques.
[0067] FIGS. 32A and 32B show the WBC data for male monkeys and
female monkeys respectively in an exploratory toxicology study on
PTA021_A3 and the non-fucosylated version of PTA021_A3(NF) in
cynomolgus macaques.
DETAILED DESCRIPTION OF THE INVENTION
[0068] The present invention relates to isolated monoclonal
antibodies, particularly human monoclonal antibodies, more
particularly immunoconjugates and engineered antibodies resulting
in increased binding to Fc receptors and/or increased potency for
ADCC, which bind specifically to CADM1 with high affinity. In
certain embodiments, the antibodies of the invention are derived
from particular heavy and light chain germline sequences and/or
comprise particular structural features such as CDR regions
comprising particular amino acid sequences. The invention provides
isolated antibodies, engineered antibodies resulting in increased
binding to Fc receptors and/or increased potency for ADCC,
immunoconjugates, bispecific molecules, affibodies, domain
antibodies, nanobodies, and unibodies, methods of making said
molecules, and pharmaceutical compositions comprising said
molecules and a pharmaceutical carrier. The invention also relates
to methods of using the molecules, such as to detect CADM1, as well
as to treat diseases associated with expression of CADM1, such as
CADM1 expressed on tumors, including those tumors of small cell
lung cancer, and neuroendocrine pancreatic cancer, lung carcinoids,
and gastro-intestinal carcinoids.
[0069] In order that the present invention may be more readily
understood, certain terms are first defined. Additional definitions
are set forth throughout the detailed description.
[0070] The terms "CADM1", "IGSF4", "immunoglobulin superfamily,
member 4D", "cell adhesion molecule 1", "tumor suppressor in lung
cancer 1", "TSLC1", "BL2", "ST17", "synaptic cell adhesion
molecule", "syncam 1", "nectin-like protein 2" and "NECL2" are used
interchangeably, and include variants, isoforms and species
homologs of human CADM1, including CADM1 isoform 1 (Genbank
Accession Nos. NM.sub.--014333), and 2 (Genbank Accession Nos.
NM.sub.--001098517). The two isoforms have also been identified as
OGTA025a and OGTA025b in PCT Application (OBT Docket No.:
OGL-P121PCT), which is incorporated herein by reference in its
entirety. Human antibodies of this disclosure may, in certain
cases, cross-react with CADM1 from species other than human. In
certain embodiments, the antibodies may be completely specific for
one or more human CADM1 and may not exhibit species or other types
of non-human cross-reactivity. The complete amino acid sequence of
an exemplary human CADM1 has Genbank accession number
NM.sub.--014333.
[0071] The term "immune response" refers to the action of, for
example, lymphocytes, antigen presenting cells, phagocytic cells,
granulocytes, and soluble macromolecules produced by the above
cells or the liver (including antibodies, cytokines, and
complement) that results in selective damage to, destruction of, or
elimination from the human body of invading pathogens, cells or
tissues infected with pathogens, cancerous cells, or, in cases of
autoimmunity or pathological inflammation, normal human cells or
tissues.
[0072] A "signal transduction pathway" refers to the biochemical
relationship between various of signal transduction molecules that
play a role in the transmission of a signal from one portion of a
cell to another portion of a cell. As used herein, the phrase "cell
surface receptor" includes, for example, molecules and complexes of
molecules capable of receiving a signal and the transmission of
such a signal across the plasma membrane of a cell. An example of a
"cell surface receptor" of the present invention is the CADM1
receptor.
[0073] The term "antibody" as referred to herein includes whole
antibodies and any antigen binding fragment (i.e., "antigen-binding
portion") or single chains thereof. An "antibody" refers to a
glycoprotein which may comprise at least two heavy (H) chains and
two light (L) chains inter-connected by disulfide bonds, or an
antigen binding portion thereof. Each heavy chain is comprised of a
heavy chain variable region (abbreviated herein as V.sub.H) and a
heavy chain constant region. The heavy chain constant region is
comprised of three domains, C.sub.H1, C.sub.H2 and C.sub.H3. Each
light chain is comprised of a light chain variable region
(abbreviated herein as V.sub.L or V.sub.K) and a light chain
constant region. The light chain constant region is comprised of
one domain, C.sub.L. The V.sub.H and V.sub.L/V.sub.K regions can be
further subdivided into regions of hypervariability, termed
complementarity determining regions (CDR), interspersed with
regions that are more conserved, termed framework regions (FR).
Each V.sub.H and V.sub.L/V.sub.K is composed of three CDRs and four
FRs, arranged from amino-terminus to carboxy-terminus in the
following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable
regions of the heavy and light chains contain a binding domain that
interacts with an antigen. The constant regions of the antibodies
may mediate the binding of the immunoglobulin to host tissues or
factors, including various cells of the immune system (e.g.,
effector cells) and the first component (Clq) of the classical
complement system.
[0074] The term "antigen-binding portion" of an antibody (or simply
"antibody portion"), as used herein, refers to one or more
fragments of an antibody that retain the ability to specifically
bind to an antigen (e.g., CADM1). It has been shown that the
antigen-binding function of an antibody can be performed by
fragments of a full-length antibody. Examples of binding fragments
encompassed within the term "antigen-binding portion" of an
antibody include (i) a Fab fragment, a monovalent fragment
consisting of the V.sub.L/V.sub.K, V.sub.H, C.sub.L and C.sub.H1
domains; (ii) a F(ab').sub.2 fragment, a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the
hinge region; (iii) a Fab' fragment, which is essentially an Fab
with part of the hinge region (see, FUNDAMENTAL IMMUNOLOGY (Paul
ed., 3.sup.rd ed. 1993); (iv) a Fd fragment consisting of the
V.sub.H and C.sub.H1 domains; (v) a Fv fragment consisting of the
V.sub.L and V.sub.H domains of a single arm of an antibody, (vi) a
dAb fragment (Ward et al., (1989) Nature 341:544-546), which
consists of a V.sub.H domain; (vii) an isolated complementarity
determining region (CDR); and (viii) a nanobody, a heavy chain
variable region containing a single variable domain and two
constant domains. Furthermore, although the two domains of the Fv
fragment, V.sub.L/V.sub.K and V.sub.H, are coded for by separate
genes, they can be joined, using recombinant methods, by a
synthetic linker that enables them to be made as a single protein
chain in which the V.sub.L/V.sub.K and V.sub.H regions pair to form
monovalent molecules (known as single chain Fv (scFv); see e.g.,
Bird et al. (1988) Science 242:423-426; and Huston et al. (1988)
Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain
antibodies are also intended to be encompassed within the term
"antigen-binding portion" of an antibody. These antibody fragments
are obtained using conventional techniques known to those with
skill in the art, and the fragments are screened for utility in the
same manner as are intact antibodies.
[0075] An "isolated antibody," as used herein, is intended to refer
to an antibody that is substantially free of other antibodies
having different antigenic specificities (e.g., an isolated
antibody that specifically binds CADM1 is substantially free of
antibodies that specifically bind antigens other than CADM1). An
isolated antibody that specifically binds CADM1 may, however, have
cross-reactivity to other antigens, such as CADM1 molecules from
other species. Moreover, an isolated antibody may be substantially
free of other cellular material and/or chemicals.
[0076] The terms "monoclonal antibody" or "monoclonal antibody
composition" as used herein refer to a preparation of antibody
molecules of single molecular composition. A monoclonal antibody
composition displays a single binding specificity and affinity for
a particular epitope.
[0077] The term "human antibody," as used herein, is intended to
include antibodies having variable regions in which both the
framework and CDR regions are derived from human germline
immunoglobulin sequences. Furthermore, if the antibody contains a
constant region, the constant region also is derived from human
germline immunoglobulin sequences. The human antibodies of the
invention may include amino acid residues not encoded by human
germline immunoglobulin sequences (e.g., mutations introduced by
random or site-specific mutagenesis in vitro or by somatic mutation
in vivo). However, the term "human antibody," as used herein, is
not intended to include antibodies in which CDR sequences derived
from the germline of another mammalian species, such as a mouse,
have been grafted onto human framework sequences.
[0078] The term "human monoclonal antibody" refers to antibodies
displaying a single binding specificity which have variable regions
in which both the framework and CDR regions are derived from human
germline immunoglobulin sequences. In one embodiment, the human
monoclonal antibodies are produced by a hybridoma which includes a
B cell obtained from a transgenic nonhuman animal, e.g., a
transgenic mouse, having a genome comprising a human heavy chain
transgene and a light chain transgene fused to an immortalized
cell.
[0079] The term "recombinant human antibody," as used herein,
includes all human antibodies that are prepared, expressed, created
or isolated by recombinant means, such as (a) antibodies isolated
from an animal (e.g., a mouse) that is transgenic or
transchromosomal for human immunoglobulin genes or a hybridoma
prepared therefrom (described further below), (b) antibodies
isolated from a host cell transformed to express the human
antibody, e.g., from a transfectoma, (c) antibodies isolated from a
recombinant, combinatorial human antibody library, and (d)
antibodies prepared, expressed, created or isolated by any other
means that involve splicing of human immunoglobulin gene sequences
to other DNA sequences. Such recombinant human antibodies have
variable regions in which the framework and CDR regions are derived
from human germline immunoglobulin sequences. In certain
embodiments, however, such recombinant human antibodies can be
subjected to in vitro mutagenesis (or, when an animal transgenic
for human Ig sequences is used, in vivo somatic mutagenesis) and
thus the amino acid sequences of the V.sub.H and V.sub.L/V.sub.K
regions of the recombinant antibodies are sequences that, while
derived from and related to human germline V.sub.H and
V.sub.L/V.sub.K sequences, may not naturally exist within the human
antibody germline repertoire in vivo.
[0080] As used herein, "isotype" refers to the antibody class
(e.g., IgM or IgGl) that is encoded by the heavy chain constant
region genes.
[0081] The phrases "an antibody recognizing an antigen" and "an
antibody specific for an antigen" are used interchangeably herein
with the term "an antibody which binds specifically to an
antigen."
[0082] The term "human antibody derivatives" refers to any modified
form of the human antibody, e.g., a conjugate of the antibody and
another agent or antibody.
[0083] The term "humanized antibody" is intended to refer to
antibodies in which CDR sequences derived from the germline of
another mammalian species, such as a mouse, have been grafted onto
human framework sequences. Additional framework region
modifications may be made within the human framework sequences.
[0084] The term "chimeric antibody" is intended to refer to
antibodies in which the variable region sequences are derived from
one species and the constant region sequences are derived from
another species, such as an antibody in which the variable region
sequences are derived from a mouse antibody and the constant region
sequences are derived from a human antibody.
[0085] As used herein, an antibody that "specifically binds to
human CADM1" is intended to refer to an antibody that binds to
human CADM1 with an EC.sub.50 of 50 Nm or less, 10 Nm or less, or
more preferably 1 Nm or less.
[0086] The term "does not substantially bind" to a protein or
cells, as used herein, means does not bind or does not bind with a
high affinity to the protein or cells, i.e. binds to the protein or
cells with a K.sub.D of 1.times.10.sup.-6 M or more, more
preferably 1.times.10.sup.-5 M or more, more preferably
1.times.10.sup.-4 M or more, more preferably 1.times.10.sup.-3 M or
more, even more preferably 1.times.10.sup.-2 M or more.
[0087] The term "EC.sub.50" as used herein, is intended to refer to
the potency of a compound by quantifying the concentration that
leads to 50% maximal response/effect.
[0088] The term "K.sub.assoc" or "K.sub.a," as used herein, is
intended to refer to the association rate of a particular
antibody-antigen interaction, whereas the term "K.sub.dis" or
"K.sub.d," as used herein, is intended to refer to the dissociation
rate of a particular antibody-antigen interaction. The term
"K.sub.D," as used herein, is intended to refer to the dissociation
constant, which is obtained from the ratio of K.sub.d to K.sub.a
(i.e., K.sub.d/K.sub.a) and is expressed as a molar concentration
(M). K.sub.D values for antibodies can be determined using methods
well established in the art. A preferred method for determining the
K.sub.D of an antibody is by using surface plasmon resonance,
preferably using a biosensor system such as a Biacore.RTM.
system.
[0089] As used herein, the term "high affinity" for an IgG antibody
refers to an antibody having a K.sub.D of 1.times.10.sup.-7 M or
less, more preferably 5.times.10.sup.-8 M or less, even more
preferably 1.times.10.sup.-8 M or less, even more preferably
5.times.10.sup.-9 M or less and even more preferably
1.times.10.sup.-9 M or less for a target antigen. However, "high
affinity" binding can vary for other antibody isotypes. For
example, "high affinity" binding for an IgM isotype refers to an
antibody having a K.sub.D of 10.sup.-6 M or less, more preferably
10.sup.-7 M or less, even more preferably 10.sup.-8 M or less.
[0090] The term "epitope" or "antigenic determinant" refers to a
site on an antigen to which an immunoglobulin or antibody
specifically binds. Epitopes can be formed both from contiguous
amino acids or noncontiguous amino acids juxtaposed by tertiary
folding of a protein. Epitopes formed from contiguous amino acids
are typically retained on exposure to denaturing solvents, whereas
epitopes formed by tertiary folding are typically lost on treatment
with denaturing solvents. An epitope typically includes at least 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique
spatial conformation. Methods of determining spatial conformation
of epitopes include techniques in the art and those described
herein, for example, x-ray crystallography and 2-dimensional
nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in
Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed.
(1996)).
[0091] Accordingly, also encompassed by the present invention are
antibodies that bind to (i.e., recognize) the same epitope as the
antibodies described herein (i.e., PTA021_A1, PTA021_A2 and
PTA021_A3). Antibodies that bind to the same epitope can be
identified by their ability to cross-compete with (i.e.,
competitively inhibit binding of) a reference antibody to a target
antigen in a statistically significant manner. Competitive
inhibition can occur, for example, if the antibodies bind to
identical or structurally similar epitopes (e.g., overlapping
epitopes), or spatially proximal epitopes which, when bound, causes
steric hindrance between the antibodies.
[0092] Competitive inhibition can be determined using routine
assays in which the immunoglobulin under test inhibits specific
binding of a reference antibody to a common antigen. Numerous types
of competitive binding assays are known, for example: solid phase
direct or indirect radioimmunoassay (RIA), solid phase direct or
indirect enzyme immunoassay (EIA), sandwich competition assay (see
Stahli et al., Methods in Enzymology 9:242 (1983)); solid phase
direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614
(1986)); solid phase direct labeled assay, solid phase direct
labeled sandwich assay (see Harlow and Lane, Antibodies: A
Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase
direct label RIA using I-125 label (see Morel et al., Mol. Immunol.
25(1):7 (1988)); solid phase direct biotin-avidin EIA (Cheung et
al., Virology 176:546 (1990)); and direct labeled RIA. (Moldenhauer
et al., Scand. J. Immunol. 32:77 (1990)). Typically, such an assay
involves the use of purified antigen bound to a solid surface or
cells bearing either of these, an unlabeled test immunoglobulin and
a labeled reference immunoglobulin. Competitive inhibition is
measured by determining the amount of label bound to the solid
surface or cells in the presence of the test immunoglobulin.
Usually the test immunoglobulin is present in excess. Usually, when
a competing antibody is present in excess, it will inhibit specific
binding of a reference antibody to a common antigen by at least
50-55%, 55-60%, 60-65%, 65-70% 70-75% or more.
[0093] Other techniques include, for example, epitope mapping
methods, such as x-ray analyses of crystals of antigen:antibody
complexes which provides atomic resolution of the epitope. Other
methods monitor the binding of the antibody to antigen fragments or
mutated variations of the antigen where loss of binding due to a
modification of an amino acid residue within the antigen sequence
is often considered an indication of an epitope component. In
addition, computational combinatorial methods for epitope mapping
can also be used. These methods rely on the ability of the antibody
of interest to affinity isolate specific short peptides from
combinatorial phage display peptide libraries. The peptides are
then regarded as leads for the definition of the epitope
corresponding to the antibody used to screen the peptide library.
For epitope mapping, computational algorithms have also been
developed which have been shown to map conformational discontinuous
epitopes.
[0094] As used herein, the term "subject" includes any human or
nonhuman animal. The term "nonhuman animal" includes all
vertebrates, e.g., mammals and non-mammals, such as nonhuman
primates, sheep, dogs, cats, horses, cows, chickens, amphibians,
reptiles, etc.
[0095] Various aspects of the invention are described in further
detail in the following subsections.
Anti-CADM1 Antibodies
[0096] The antibodies of the invention are characterized by
particular functional features or properties of the antibodies. For
example, the antibodies bind specifically to human CADM1.
Preferably, an antibody of the invention binds to CADM1 with high
affinity, for example with a K.sub.D of 8.times.10.sup.-7 M or
less, even more typically 1.times.10.sup.-8 M or less. The
anti-CADM1 antibodies of the invention preferably exhibit one or
more of the following characteristics:
[0097] binds to human CADM1 with a EC.sub.50 of 50 Nm or less, 10
Nm or less, or more preferably 1 Nm or less;
[0098] binds to human cells expressing CADM1.
[0099] In one embodiment, the antibodies preferably bind to an
antigenic epitope present in CADM1, which epitope is not present in
other proteins. The antibodies typically bind CADM1 but does not
bind to other proteins, or binds to proteins with a low affinity,
such as a K.sub.D of 1.times.10.sup.-6 M or more, more preferably
1.times.10.sup.-5 M or more, more preferably 1.times.10.sup.-4 M or
more, more preferably 1.times.10.sup.-3 M or more, even more
preferably 1.times.10.sup.-2 M or more. Preferably, the antibodies
do not bind to related proteins, for example, the antibodies do not
substantially bind to ICAMs, VCAMs, or other cell adhesion
molecules. In one embodiment, the antibody may be internalized into
a cell expressing CADM1. Standard assays to evaluate antibody
internalization are known in the art, including, for example, a
HumZap internalization assay.
[0100] Standard assays to evaluate the binding ability of the
antibodies toward CADM1 are known in the art, including for
example, ELISAs, Western blots, RIAs, and flow cytometry analysis.
Suitable assays are described in detail in the Examples. The
binding kinetics (e.g., binding affinity) of the antibodies also
can be assessed by standard assays known in the art, such as by
Biacore.RTM. system analysis. To assess binding to Raji or Daudi B
cell tumor cells, Raji (ATCC Deposit No. CCL-86) or Daudi (ATCC
Deposit No. CCL-213) cells can be obtained from publicly available
sources, such as the American Type Culture Collection, and used in
standard assays, such as flow cytometric analysis.
Monoclonal Antibodies PTA021_A1, PTA021_A2, PTA021_A3
[0101] Preferred antibodies of the invention are the human
monoclonal antibodies PTA021_A1, PTA021_A2, and PTA021_A3, isolated
and structurally characterized as described in Examples 1, 2, and
3. The V.sub.H amino acid sequences of PTA021_A1, PTA021_A2, and
PTA021_A3 are shown in SEQ ID NOs:19, 20, and 21, respectively. The
V.sub.K amino acid sequences of PTA021_A1, PTA021_A2, and PTA021_A3
are shown in SEQ ID NOs:22, 23, and 24, respectively.
[0102] Given that each of these antibodies can bind to CADM1, the
V.sub.H and V.sub.K sequences can be "mixed and matched" to create
other anti-CADM1 binding molecules of the invention. CADM1 binding
of such "mixed and matched" antibodies can be tested using the
binding assays described above and in the Examples (e.g., ELISAs).
Preferably, when V.sub.H and V.sub.K chains are mixed and matched,
a V.sub.H sequence from a particular V.sub.H/V.sub.K pairing is
replaced with a structurally similar V.sub.H sequence. Likewise,
preferably a V.sub.L sequence from a particular V.sub.H/V.sub.K
pairing is replaced with a structurally similar V.sub.K
sequence.
[0103] Accordingly, in one aspect, the invention provides an
isolated monoclonal antibody, or antigen binding portion thereof
comprising:
a heavy chain variable region comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs:19, 20, and 21;
and a light chain variable region comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs:22, 23, and 24;
wherein the antibody specifically binds CADM1, preferably human
CADM1.
[0104] Preferred heavy and light chain combinations include:
a heavy chain variable region comprising the amino acid sequence of
SEQ ID NO:19 and a light chain variable region comprising the amino
acid sequence of SEQ ID NO:22; or a heavy chain variable region
comprising the amino acid sequence of SEQ ID NO:20; and a light
chain variable region comprising the amino acid sequence of SEQ ID
NO:23, or a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO:21; and a light chain variable region
comprising the amino acid sequence of SEQ ID NO:24.
[0105] In another aspect, the invention provides antibodies that
comprise the heavy chain and light chain CDR1s, CDR2s and CDR3s of
PTA021_A1, PTA021_A2, and PTA021_A3, or combinations thereof. The
amino acid sequences of the V.sub.H CDR1s of PTA021_A1, PTA021_A2,
and PTA021_A3 are shown in SEQ ID NOs: 1, 2, and 3. The amino acid
sequences of the V.sub.H CDR2s of PTA021_A1, PTA021_A2, and
PTA021_A3 are shown in SEQ ID NOs: 4, 5, and 6. The amino acid
sequences of the V.sub.H CDR3s of PTA021_A1, PTA021_A2, and
PTA021_A3 are shown in SEQ ID NOs:7, 8, and 9. The amino acid
sequences of the V.sub.K CDR1s of PTA021_A1, PTA021_A2, and
PTA021_A3 are shown in SEQ ID NOs:10, 11, and 12. The amino acid
sequences of the V.sub.K CDR2s of PTA021_A1, PTA021_A2, and
PTA021_A3 are shown in SEQ ID NOs:13, 14, and 15. The amino acid
sequences of the V.sub.K CDR3s of PTA021_A1, PTA021_A2, and
PTA021_A3 are shown in SEQ ID NOs:16, 17, and 18. The CDR regions
are delineated using the Kabat system (Kabat, E. A., et al. (1991)
Sequences of Proteins of Immunological Interest, Fifth Edition,
U.S. Department of Health and Human Services, NIH Publication No.
91-3242).
[0106] Given that each of these antibodies can bind to CADM1 and
that antigen-binding specificity is provided primarily by the CDR1,
CDR2, and CDR3 regions, the V.sub.H CDR1, CDR2, and CDR3 sequences
and V.sub.K CDR1, CDR2, and CDR3 sequences can be "mixed and
matched" (i.e., CDRs from different antibodies can be mixed and
match, although each antibody must contain a V.sub.H CDR1, CDR2,
and CDR3 and a V.sub.K CDR1, CDR2, and CDR3) to create other
anti-CADM1 binding molecules of the invention. CADM1 binding of
such "mixed and matched" antibodies can be tested using the binding
assays described above and in the Examples (e.g., ELISAs,
Biacore.RTM. analysis). Preferably, when V.sub.H CDR sequences are
mixed and matched, the CDR1, CDR2 and/or CDR3 sequence from a
particular V.sub.H sequence is replaced with a structurally similar
CDR sequence(s). Likewise, when V.sub.K CDR sequences are mixed and
matched, the CDR1, CDR2 and/or CDR3 sequence from a particular
V.sub.k sequence preferably is replaced with a structurally similar
CDR sequence(s). It will be readily apparent to the ordinarily
skilled artisan that novel V.sub.H and V.sub.K sequences can be
created by substituting one or more V.sub.H and/or V.sub.L/V.sub.K
CDR region sequences with structurally similar sequences from the
CDR sequences disclosed herein for monoclonal antibodies PTA021_A1,
PTA021_A2 and PTA021_A3.
[0107] Accordingly, in another aspect, the invention provides an
isolated monoclonal antibody, or antigen binding portion thereof
comprising:
a heavy chain variable region CDR1 comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs:1, 2, and
3; a heavy chain variable region CDR2 comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs:4, 5, and
6; a heavy chain variable region CDR3 comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs:7, 8, and
9; a light chain variable region CDR1 comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs:10, 11,
and 12; a light chain variable region CDR2 comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs:13, 14,
and 15; and a light chain variable region CDR3 comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs:16,
17, and 18; wherein the antibody specifically binds CADM1,
preferably human CADM1.
[0108] In a preferred embodiment, the antibody comprises:
a heavy chain variable region CDR1 comprising SEQ ID NO:1; a heavy
chain variable region CDR2 comprising SEQ ID NO: 4; a heavy chain
variable region CDR3 comprising SEQ ID NO: 7; a light chain
variable region CDR1 comprising SEQ ID NO: 10; a light chain
variable region CDR2 comprising SEQ ID NO: 13; and a light chain
variable region CDR3 comprising SEQ ID NO: 16.
[0109] In another preferred embodiment, the antibody comprises:
a heavy chain variable region CDR1 comprising SEQ ID NO: 2; a heavy
chain variable region CDR2 comprising SEQ ID NO: 5; a heavy chain
variable region CDR3 comprising SEQ ID NO: 8; a light chain
variable region CDR1 comprising SEQ ID NO: 11; a light chain
variable region CDR2 comprising SEQ ID NO: 14; and a light chain
variable region CDR3 comprising SEQ ID NO: 17.
[0110] In another preferred embodiment, the antibody comprises:
a heavy chain variable region CDR1 comprising SEQ ID NO: 3; a heavy
chain variable region CDR2 comprising SEQ ID NO:6; a heavy chain
variable region CDR3 comprising SEQ ID NO:9; a light chain variable
region CDR1 comprising SEQ ID NO:12; a light chain variable region
CDR2 comprising SEQ ID NO: 15; and a light chain variable region
CDR3 comprising SEQ ID NO: 18.
[0111] It is well known in the art that the CDR3 domain,
independently from the CDR1 and/or CDR2 domain(s), alone can
determine the binding specificity of an antibody for a cognate
antigen and that multiple antibodies can predictably be generated
having the same binding specificity based on a common CDR3
sequence. See, for example, Klimka et al., British J. of Cancer
83(2):252-260 (2000) (describing the production of a humanized
anti-CD30 antibody using only the heavy chain variable domain CDR3
of murine anti-CD30 antibody Ki-4); Beiboer et al., J. Mol. Biol.
296:833-849 (2000) (describing recombinant epithelial
glycoprotein-2 (EGP-2) antibodies using only the heavy chain CDR3
sequence of the parental murine MOC-31 anti-EGP-2 antibody); Rader
et al., Proc. Natl. Acad. Sci. U.S.A. 95:8910-8915 (1998)
(describing a panel of humanized anti-integrin
.alpha..sub.v.beta..sub.3 antibodies using a heavy and light chain
variable CDR3 domain of a murine anti-integrin
.alpha..sub.v.beta..sub.3 antibody LM609 wherein each member
antibody comprises a distinct sequence outside the CDR3 domain and
capable of binding the same epitope as the parent murine antibody
with affinities as high or higher than the parent murine antibody);
Barbas et al., J. Am. Chem. Soc. 116:2161-2162 (1994) (disclosing
that the CDR3 domain provides the most significant contribution to
antigen binding); Barbas et al., Proc. Natl. Acad. Sci. U.S.A.
92:2529-2533 (1995) (describing the grafting of heavy chain CDR3
sequences of three Fabs (SI-1, SI-40, and SI-32) against human
placental DNA onto the heavy chain of an anti-tetanus toxoid Fab
thereby replacing the existing heavy chain CDR3 and demonstrating
that the CDR3 domain alone conferred binding specificity); and
Ditzel et al., J. Immunol. 157:739-749 (1996) (describing grafting
studies wherein transfer of only the heavy chain CDR3 of a parent
polyspecific Fab LNA3 to a heavy chain of a monospecific IgG
tetanus toxoid-binding Fab p313 antibody was sufficient to retain
binding specificity of the parent Fab). Each of these references is
hereby incorporated by reference in its entirety.
[0112] Accordingly, the present invention provides monoclonal
antibodies comprising one or more heavy and/or light chain CDR3
domains from an antibody derived from a human or non-human animal,
wherein the monoclonal antibody is capable of specifically binding
to CADM1. Within certain aspects, the present invention provides
monoclonal antibodies comprising one or more heavy and/or light
chain CDR3 domain from a non-human antibody, such as a mouse or rat
antibody, wherein the monoclonal antibody is capable of
specifically binding to CADM1. Within some embodiments, such
inventive antibodies comprising one or more heavy and/or light
chain CDR3 domain from a non-human antibody (a) are capable of
competing for binding with; (b) retain the functional
characteristics; (c) bind to the same epitope; and/or (d) have a
similar binding affinity as the corresponding parental non-human
antibody.
[0113] Within other aspects, the present invention provides
monoclonal antibodies comprising one or more heavy and/or light
chain CDR3 domain from a human antibody, such as, for example, a
human antibody obtained from a non-human animal, wherein the human
antibody is capable of specifically binding to CADM1. Within other
aspects, the present invention provides monoclonal antibodies
comprising one or more heavy and/or light chain CDR3 domain from a
first human antibody, such as, for example, a human antibody
obtained from a non-human animal, wherein the first human antibody
is capable of specifically binding to CADM1 and wherein the CDR3
domain from the first human antibody replaces a CDR3 domain in a
human antibody that is lacking binding specificity for CADM1 to
generate a second human antibody that is capable of specifically
binding to CADM1. Within some embodiments, such inventive
antibodies comprising one or more heavy and/or light chain CDR3
domain from the first human antibody (a) are capable of competing
for binding with; (b) retain the functional characteristics; (c)
bind to the same epitope; and/or (d) have a similar binding
affinity as the corresponding parental first human antibody. In
preferred embodiments, the first human antibody is PTA021_A1,
PTA021_A2 or PTA021_A3.
Antibodies Having Particular Germline Sequences
[0114] In certain embodiments, an antibody of the invention
comprises a heavy chain variable region from a particular germline
heavy chain immunoglobulin gene and/or a light chain variable
region from a particular germline light chain immunoglobulin
gene.
[0115] For example, in a preferred embodiment, the invention
provides an isolated monoclonal antibody, or an antigen-binding
portion thereof, comprising a heavy chain variable region that is
the product of or derived from a human V.sub.H 2-05 gene, wherein
the antibody specifically binds CADM1. In yet another preferred
embodiment, the invention provides an isolated monoclonal antibody,
or an antigen-binding portion thereof, comprising a light chain
variable region that is the product of or derived from a human
V.sub.K L15 gene, wherein the antibody specifically binds CADM1. In
yet another preferred embodiment, the invention provides an
isolated monoclonal antibody, or antigen-binding portion thereof,
wherein the antibody:
comprises a heavy chain variable region that is the product of or
derived from a human V.sub.H 2-05 gene (which genes encode the
amino acid sequences set forth in SEQ ID NOs: 31, 32, or 33,
respectively); comprises a light chain variable region that is the
product of or derived from a human V.sub.K L15 gene (which genes
encode the amino acid sequences set forth in SEQ ID NOs: 34, 35, or
36, respectively); and specifically binds to CADM1, preferably
human CADM1. Examples of an antibody having V.sub.H and V.sub.K of
V.sub.H 2-05 and V.sub.K L15, respectively, are PTA021_A1,
PTA021_A2, and PTA021_A3.
[0116] As used herein, a human antibody comprises heavy or light
chain variable regions that is "the product of" or "derived from" a
particular germline sequence if the variable regions of the
antibody are obtained from a system that uses human germline
immunoglobulin genes. Such systems include immunizing a transgenic
mouse carrying human immunoglobulin genes with the antigen of
interest or screening a human immunoglobulin gene library displayed
on phage with the antigen of interest. A human antibody that is
"the product of" or "derived from" a human germline immunoglobulin
sequence can be identified as such by comparing the amino acid
sequence of the human antibody to the amino acid sequences of human
germline immunoglobulins and selecting the human germline
immunoglobulin sequence that is closest in sequence (i.e., greatest
% identity) to the sequence of the human antibody. A human antibody
that is "the product of" or "derived from" a particular human
germline immunoglobulin sequence may contain amino acid differences
as compared to the germline sequence, due to, for example,
naturally-occurring somatic mutations or intentional introduction
of site-directed mutation. However, a selected human antibody
typically is at least 90% identical in amino acids sequence to an
amino acid sequence encoded by a human germline immunoglobulin gene
and contains amino acid residues that identify the human antibody
as being human when compared to the germline immunoglobulin amino
acid sequences of other species (e.g., murine germline sequences).
In certain cases, a human antibody may be at least 95%, or even at
least 96%, 97%, 98%, or 99% identical in amino acid sequence to the
amino acid sequence encoded by the germline immunoglobulin gene.
Typically, a human antibody derived from a particular human
germline sequence will display no more than 10 amino acid
differences from the amino acid sequence encoded by the human
germline immunoglobulin gene. In certain cases, the human antibody
may display no more than 5, or even no more than 4, 3, 2, or 1
amino acid difference from the amino acid sequence encoded by the
germline immunoglobulin gene.
Homologous Antibodies
[0117] In yet another embodiment, an antibody of the invention
comprises heavy and light chain variable regions comprising amino
acid sequences that are homologous to the amino acid sequences of
the preferred antibodies described herein, and wherein the
antibodies retain the desired functional properties of the
anti-CADM1 antibodies of the invention.
[0118] For example, the invention provides an isolated monoclonal
antibody, or antigen binding portion thereof, comprising a heavy
chain variable region and a light chain variable region,
wherein:
[0119] the heavy chain variable region comprises an amino acid
sequence that is at least 80% homologous to an amino acid sequence
selected from the group consisting of SEQ ID NOs:19, 20, and
21;
[0120] the light chain variable region comprises an amino acid
sequence that is at least 80% homologous to an amino acid sequence
selected from the group consisting of SEQ ID NOs:22, 23, and 24;
and
[0121] the antibody binds to human CADM1 with a EC.sub.50 of 10 Nm
or less.
[0122] The antibody may also bind to CHO cells transfected with
human CADM1.
[0123] In various embodiments, the antibody can be, for example, a
human antibody, a humanized antibody or a chimeric antibody.
[0124] In other embodiments, the V.sub.H and/or V.sub.K amino acid
sequences may be 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to
the sequences set forth above. An antibody having V.sub.H and
V.sub.K regions having high (i.e., 80% or greater) homology to the
V.sub.H and V.sub.K regions of the sequences set forth above, can
be obtained by mutagenesis (e.g., site-directed or PCR-mediated
mutagenesis) of nucleic acid molecules encoding SEQ ID NOs:25, 26,
27, 28, 29, and 30, followed by testing of the encoded altered
antibody for retained function using the functional assays
described herein.
[0125] As used herein, the percent homology between two amino acid
sequences is equivalent to the percent identity between the two
sequences. The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences (i.e., % homology=# of identical positions/total # of
positions.times.100), taking into account the number of gaps, and
the length of each gap, which need to be introduced for optimal
alignment of the two sequences. The comparison of sequences and
determination of percent identity between two sequences can be
accomplished using a mathematical algorithm, as described in the
non-limiting examples below.
[0126] The percent identity between two amino acid sequences can be
determined using the algorithm of E. Meyers and W. Miller (Comput.
Appl. Biosci., 4:11-17 (1988)) which has been incorporated into the
ALIGN program (version 2.0), using a PAM120 weight residue table, a
gap length penalty of 12 and a gap penalty of 4. In addition, the
percent identity between two amino acid sequences can be determined
using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970))
algorithm which has been incorporated into the GAP program in the
GCG software package (available at http://www.gcg.com), using
either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of
16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or
6.
[0127] Additionally or alternatively, the protein sequences of the
present invention can further be used as a "query sequence" to
perform a search against public databases to, for example, identify
related sequences. Such searches can be performed using the XBLAST
program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.
215:403-10. BLAST protein searches can be performed with the XBLAST
program, score=50, wordlength=3 to obtain amino acid sequences
homologous to the antibody molecules of the invention. To obtain
gapped alignments for comparison purposes, Gapped BLAST can be
utilized as described in Altschul et al., (1997) Nucleic Acids Res.
25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs,
the default parameters of the respective programs (e.g., XBLAST and
NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
Antibodies with Conservative Modifications
[0128] In certain embodiments, an antibody of the invention
comprises a heavy chain variable region comprising CDR1, CDR2 and
CDR3 sequences and a light chain variable region comprising CDR1,
CDR2 and CDR3 sequences, wherein one or more of these CDR sequences
comprise specified amino acid sequences based on the preferred
antibodies described herein (e.g., PTA021_A1, PTA021_A2 or
PTA021_A3), or conservative modifications thereof, and wherein the
antibodies retain the desired functional properties of the
anti-CADM1 antibodies of the invention. Accordingly, the invention
provides an isolated monoclonal antibody, or antigen binding
portion thereof, comprising a heavy chain variable region
comprising CDR1, CDR2, and CDR3 sequences and a light chain
variable region comprising CDR1, CDR2, and CDR3 sequences,
wherein:
the heavy chain variable region CDR3 sequence comprises an amino
acid sequence selected from the group consisting of amino acid
sequences of SEQ ID NOs:7, 8, and 9, and conservative modifications
thereof; the light chain variable region CDR3 sequence comprises an
amino acid sequence selected from the group consisting of amino
acid sequence of SEQ ID NOs:16, 17, and 18, and conservative
modifications thereof; and the antibody binds to human CADM1 with a
EC.sub.50 of 1 nM or less.
[0129] The antibody may also bind to CHO cells transfected with
human CADM1.
[0130] In a preferred embodiment, the heavy chain variable region
CDR2 sequence comprises an amino acid sequence selected from the
group consisting of amino acid sequences of SEQ ID NOs:4, 5, and 6,
and conservative modifications thereof; and the light chain
variable region CDR2 sequence comprises an amino acid sequence
selected from the group consisting of amino acid sequences of SEQ
ID NOs:13, 14, and 15, and conservative modifications thereof. In
another preferred embodiment, the heavy chain variable region CDR1
sequence comprises an amino acid sequence selected from the group
consisting of amino acid sequences of SEQ ID NOs:1, 2, and 3, and
conservative modifications thereof; and the light chain variable
region CDR1 sequence comprises an amino acid sequence selected from
the group consisting of amino acid sequences of SEQ ID NOs:10, 11,
and 12, and conservative modifications thereof.
[0131] In various embodiments, the antibody can be, for example,
human antibodies, humanized antibodies or chimeric antibodies.
[0132] As used herein, the term "conservative sequence
modifications" is intended to refer to amino acid modifications
that do not significantly affect or alter the binding
characteristics of the antibody containing the amino acid sequence.
Such conservative modifications include amino acid substitutions,
additions and deletions. Modifications can be introduced into an
antibody of the invention by standard techniques known in the art,
such as site-directed mutagenesis and PCR-mediated mutagenesis.
Conservative amino acid substitutions are ones in which the amino
acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine, tryptophan),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one
or more amino acid residues within the CDR regions of an antibody
of the invention can be replaced with other amino acid residues
from the same side chain family and the altered antibody can be
tested for retained function using the functional assays described
herein.
[0133] The heavy chain CDR1 sequence of SEQ ID NO:1, 2, or 3 may
comprise one or more conservative sequence modification, such as
one, two, three, four, five or more amino acid substitutions,
additions or deletions; the light chain CDR1 sequence of SEQ ID
NO:10, 11, or 12 may comprise one or more conservative sequence
modification, such as one, two, three, four, five or more amino
acid substitutions, additions or deletions; the heavy chain CDR2
sequence shown in SEQ ID NO:4, 5, or 6 may comprise one or more
conservative sequence modification, such as one, two, three, four,
five or more amino acid substitutions, additions or deletions; the
light chain CDR2 sequence shown in SEQ ID NO:13, 14, or 15 may
comprise one or more conservative sequence modification, such as
one, two, three, four, five or more amino acid substitutions,
additions or deletions; the heavy chain CDR3 sequence shown in SEQ
ID NO:7, 8, or 9 may comprise one or more conservative sequence
modification, such as one, two, three, four, five or more amino
acid substitutions, additions or deletions; and/or the light chain
CDR3 sequence shown in SEQ ID NO:16, 17, or 18 may comprise one or
more conservative sequence modification, such as one, two, three,
four, five or more amino acid substitutions, additions or
deletions.
Antibodies that Bind to the Same Epitope as Anti-CADM1 Antibodies
of the Invention
[0134] In another embodiment, the invention provides antibodies
that bind to the same epitope on human CADM1 as any of the CADM1
monoclonal antibodies of the invention (i.e., antibodies that have
the ability to cross-compete for binding to CADM1 with any of the
monoclonal antibodies of the invention). In preferred embodiments,
the reference antibody for studies on cross-competition for binding
can be the monoclonal antibody PTA021_A1 (having V.sub.H and
V.sub.K sequences as shown in SEQ ID NOs:19 and 22, respectively),
the monoclonal antibody PTA021_A2 (having V.sub.H and V.sub.K
sequences as shown in SEQ ID NOs:20 and 23 respectively), or the
monoclonal antibody PTA021_A3 (having V.sub.H and V.sub.K sequences
as shown in SEQ ID NOs:21 and 24 respectively). Such
cross-competing antibodies can be identified based on their ability
to cross-compete with PTA021_A1, PTA021_A2 or PTA021_A3 in standard
CADM1 binding assays. For example, BIAcore analysis, ELISA assays
or flow cytometry may be used to demonstrate cross-competing for
binding with the antibodies of the current invention. The ability
of a test antibody to inhibit the binding of, for example,
PTA021_A1, PTA021_A2 or PTA021_A3, to human CADM1 demonstrates that
the test antibody can compete with PTA021_A1, PTA021_A2 or
PTA021_A3 for binding to human CADM1 and thus binds to the same
epitope on human CADM1 as PTA021_A1, PTA021_A2 or PTA021_A3. In a
preferred embodiment, the antibody that binds to the same epitope
on human CADM1 as PTA021_A1, PTA021_A2 or PTA021_A3 is a human
monoclonal antibody. Such human monoclonal antibodies can be
prepared and isolated as described in the Examples.
Engineered and Modified Antibodies
[0135] An antibody of the invention further can be prepared using
an antibody having one or more of the V.sub.H and/or V.sub.L
sequences disclosed herein can be used as starting material to
engineer a modified antibody, which modified antibody may have
altered properties as compared to the starting antibody. An
antibody can be engineered by modifying one or more amino acids
within one or both variable regions (i.e., V.sub.H and/or V.sub.L),
for example within one or more CDR regions and/or within one or
more framework regions. Additionally or alternatively, an antibody
can be engineered by modifying residues within the constant
region(s), for example to alter the effector function(s) of the
antibody.
[0136] In certain embodiments, CDR grafting can be used to engineer
variable regions of antibodies. Antibodies interact with target
antigens predominantly through amino acid residues that are located
in the six heavy and light chain complementarity determining
regions (CDRs). For this reason, the amino acid sequences within
CDRs are more diverse between individual antibodies than sequences
outside of CDRs. Because CDR sequences are responsible for most
antibody-antigen interactions, it is possible to express
recombinant antibodies that mimic the properties of specific
naturally occurring antibodies by constructing expression vectors
that include CDR sequences from the specific naturally occurring
antibody grafted onto framework sequences from a different antibody
with different properties (see, e.g., Riechmann, L. et al. (1998)
Nature 332:323-327; Jones, P. et al. (1986) Nature 321:522-525;
Queen, C. et al. (1989) Proc. Natl. Acad. See. U.S.A.
86:10029-10033; U.S. Pat. No. 5,225,539 to Winter, and U.S. Pat.
Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et
al.)
[0137] Accordingly, another embodiment of the invention pertains to
an isolated monoclonal antibody, or antigen binding portion
thereof, comprising a heavy chain variable region comprising CDR1,
CDR2, and CDR3 sequences comprising an amino acid sequence selected
from the group consisting of SEQ ID NOs:1, 2, and 3, SEQ ID NOs:4,
5, and 6, and SEQ ID NOs:7, 8, and 9, respectively, and a light
chain variable region comprising CDR1, CDR2, and CDR3 sequences
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs:10, 11, and 12, SEQ ID NOs:13, 14, and 15,
and SEQ ID NOs:16, 17, and 18, respectively. Thus, such antibodies
contain the V.sub.H and V.sub.K CDR sequences of monoclonal
antibodies PTA021_A1, PTA021_A2 or PTA021_A3 yet may contain
different framework sequences from these antibodies.
[0138] Such framework sequences can be obtained from public DNA
databases or published references that include germline antibody
gene sequences. For example, germline DNA sequences for human heavy
and light chain variable region genes can be found in the "VBase"
human germline sequence database (available on the Internet at
www.mrc-cpe.cam.ac.uk/vbase), as well as in Kabat, E. A., et al.
(1991) Sequences of Proteins of Immunological Interest, Fifth
Edition, U.S. Department of Health and Human Services, NIH
Publication No. 91-3242; Tomlinson, I. M., et al. (1992) "The
Repertoire of Human Germline V.sub.H Sequences Reveals about Fifty
Groups of V.sub.H Segments with Different Hypervariable Loops" J.
Mol. Biol. 227:776-798; and Cox, J. P. L. et al. (1994) "A
Directory of Human Germ-line V.sub.H Segments Reveals a Strong Bias
in their Usage" Eur. J. Immunol. 24:827-836; the contents of each
of which are expressly incorporated herein by reference. As another
example, the germline DNA sequences for human heavy and light chain
variable region genes can be found in the Genbank database. For
example, the following heavy chain germline sequences found in the
HCo7 HuMAb mouse are available in the accompanying Genbank
accession numbers: 1-69 (NG.sub.--0010109, NT.sub.--024637 and
BC070333), 3-33 (NG.sub.--0010109 and NT.sub.--024637) and 3-7
(NG.sub.--0010109 and NT.sub.--024637). As another example, the
following heavy chain germline sequences found in the HCo12 HuMAb
mouse are available in the accompanying Genbank accession numbers:
1-69 (NG.sub.--0010109, NT.sub.--024637 and BC070333), 5-51
(NG.sub.--0010109 and NT.sub.--024637), 4-34 (NG.sub.--0010109 and
NT.sub.--024637), 3-30.3 (CAJ556644) and 3-23 (AJ406678).
[0139] Antibody protein sequences are compared against a compiled
protein sequence database using one of the sequence similarity
searching methods called the Gapped BLAST (Altschul et al. (1997)
Nucleic Acids Research 25:3389-3402), which is well known to those
skilled in the art. BLAST is a heuristic algorithm in that a
statistically significant alignment between the antibody sequence
and the database sequence is likely to contain high-scoring segment
pairs (HSP) of aligned words. Segment pairs whose scores cannot be
improved by extension or trimming is called a hit. Briefly, the
nucleotide sequences of VBASE origin
(http://vbase.mrc-cpe.cam.ac.uk/vbase1/list2.php) are translated
and the region between and including FR1 through FR3 framework
region is retained. The database sequences have an average length
of 98 residues. Duplicate sequences which are exact matches over
the entire length of the protein are removed. A BLAST search for
proteins using the program blastp with default, standard parameters
except the low complexity filter, which is turned off, and the
substitution matrix of BLOSUM62, filters for top 5 hits yielding
sequence matches. The nucleotide sequences are translated in all
six frames and the frame with no stop codons in the matching
segment of the database sequence is considered the potential hit.
This is in turn confirmed using the BLAST program tblastx, which
translates the antibody sequence in all six frames and compares
those translations to the VBASE nucleotide sequences dynamically
translated in all six frames.
[0140] The identities are exact amino acid matches between the
antibody sequence and the protein database over the entire length
of the sequence. The positives (identities+substitution match) are
not identical but amino acid substitutions guided by the BLOSUM62
substitution matrix. If the antibody sequence matches two of the
database sequences with same identity, the hit with most positives
would be decided to be the matching sequence hit.
[0141] Preferred framework sequences for use in the antibodies of
the invention are those that are structurally similar to the
framework sequences used by selected antibodies of the invention,
e.g., similar to the V.sub.H 2-05 framework sequences (SEQ ID
NO:31) and/or the V.sub.K L15 framework sequences (SEQ ID NO:34)
used by preferred monoclonal antibodies of the invention. The
V.sub.H CDR1, CDR2, and CDR3 sequences, and the V.sub.K CDR1, CDR2,
and CDR3 sequences, can be grafted onto framework regions that have
the identical sequence as that found in the germline immunoglobulin
gene from which the framework sequence derive, or the CDR sequences
can be grafted onto framework regions that contain one or more
mutations as compared to the germline sequences. For example, it
has been found that in certain instances it is beneficial to mutate
residues within the framework regions to maintain or enhance the
antigen binding ability of the antibody (see e.g., U.S. Pat. Nos.
5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).
[0142] Another type of variable region modification is to mutate
amino acid residues within the V.sub.H and/or V.sub.K CDR1, CDR2
and/or CDR3 regions to thereby improve one or more binding
properties (e.g., affinity) of the antibody of interest.
Site-directed mutagenesis or PCR-mediated mutagenesis can be
performed to introduce the mutation(s) and the effect on antibody
binding, or other functional property of interest, can be evaluated
in in vitro or in vivo assays as described herein and provided in
the Examples. Preferably conservative modifications (as discussed
above) are introduced. The mutations may be amino acid
substitutions, additions or deletions, but are preferably
substitutions. Moreover, typically no more than one, two, three,
four or five residues within a CDR region are altered.
[0143] Accordingly, in another embodiment, the instant disclosure
provides isolated anti-CADM1 monoclonal antibodies, or antigen
binding portions thereof, comprising a heavy chain variable region
comprising: (a) a V.sub.H CDR1 region comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs:1, 2, and
3, or an amino acid sequence having one, two, three, four or five
amino acid substitutions, deletions or additions as compared to SEQ
ID NOs:1, 2, and 3; (b) a V.sub.H CDR2 region comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs:4,
5, and 6, or an amino acid sequence having one, two, three, four or
five amino acid substitutions, deletions or additions as compared
to SEQ ID NOs:4, 5, and 6; (c) a V.sub.H CDR3 region comprising an
amino acid sequence selected from the group consisting of SEQ ID
NOs:7, 8, and 9, or an amino acid sequence having one, two, three,
four or five amino acid substitutions, deletions or additions as
compared to SEQ ID NOs:7, 8, and 9; (d) a V.sub.K CDR1 region
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs:10, 11, and 12, or an amino acid sequence
having one, two, three, four or five amino acid substitutions,
deletions or additions as compared to SEQ ID NOs:10, 11, and 12;
(e) a V.sub.K CDR2 region comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs:13, 14, and 15, or
an amino acid sequence having one, two, three, four or five amino
acid substitutions, deletions or additions as compared to SEQ ID
NOs:13, 14, and 15; and (f) a V.sub.K CDR3 region comprising an
amino acid sequence selected from the group consisting of SEQ ID
NOs:16, 17, and 18, or an amino acid sequence having one, two,
three, four or five amino acid substitutions, deletions or
additions as compared to SEQ ID NOs:16, 17, and 18.
[0144] Engineered antibodies of the invention include those in
which modifications have been made to framework residues within
V.sub.H and/or V.sub.K, e.g. to improve the properties of the
antibody. Typically such framework modifications are made to
decrease the immunogenicity of the antibody. For example, one
approach is to "backmutate" one or more framework residues to the
corresponding germline sequence. More specifically, an antibody
that has undergone somatic mutation may contain framework residues
that differ from the germline sequence from which the antibody is
derived. Such residues can be identified by comparing the antibody
framework sequences to the germline sequences from which the
antibody is derived. For example, for PTA021_A1, using the Kabat
numbering system, amino acid residue #30 (within FR3) of V.sub.H is
an asparagine (SEQ ID NO:19) whereas this residue in the
corresponding V.sub.H 2-05 germline sequence is a serine (SEQ ID
NO:31). To return the framework region sequences to their germline
configuration, the somatic mutations can be "backmutated" to the
germline sequence by, for example, site-directed mutagenesis or
PCR-mediated mutagenesis (e.g., residue #30 of the V.sub.H of
PTA021_A1 can be "backmutated" from asparagine to serine).
[0145] As another example, for PTA021_A1, amino acid residue #54 of
V.sub.H is an aspartic acid (SEQ ID NO:19) whereas this residue in
the corresponding V.sub.H 2-05 germline sequence is an asparagine
(SEQ ID NO:31). To return the framework region sequences to their
germline configuration, for example, residue #54 of the V.sub.H of
PTA021_A1 can be "backmutated" from aspartic acid to asparagine.
Such "backmutated" antibodies are also intended to be encompassed
by the invention.
[0146] As another example, for PTA021_A1, amino acid residue #50 of
V.sub.K is a glycine (SEQ ID NO:22) whereas this residue in the
corresponding V.sub.K L15 germline sequence is an alanine (SEQ ID
NO:34). To return the framework region sequences to their germline
configuration, for example, residue #50 of the V.sub.K of PTA021_A1
can be "backmutated" from glycine to alanine. Such "backmutated"
antibodies are also intended to be encompassed by the
invention.
[0147] As another example, for PTA021_A1, amino acid residue #77 of
V.sub.K is an asparagine (SEQ ID NO:22) whereas this residue in the
corresponding V.sub.K L15 germline sequence is a serine (SEQ ID
NO:34). To return the framework region sequences to their germline
configuration, for example, residue #77 of the V.sub.K of PTA021_A1
can be "backmutated" from asparagine to serine. Such "backmutated"
antibodies are also intended to be encompassed by the
invention.
[0148] As another example, for PTA021_A2, amino acid residue #54 of
V.sub.H is an aspartic acid (SEQ ID NO:20) whereas this residue in
the corresponding V.sub.H 2-05 germline sequence is an asparagine
(SEQ ID NO:32). To return the framework region sequences to their
germline configuration, for example, residue #54 of the V.sub.H of
PTA021_A2 can be "backmutated" from aspartic acid to asparagine.
Such "backmutated" antibodies are also intended to be encompassed
by the invention.
[0149] As yet another example, for PTA021_A2, amino acid residue
#89 of V.sub.H is an isoleucine (SEQ ID NO:20) whereas this residue
in the corresponding V.sub.H 2-05 germline sequence is a threonine
(SEQ ID NO:32). To return the framework region sequences to their
germline configuration, for example, residue #89 within FR1 of the
V.sub.H of PTA021_A2 can be "backmutated" from isoleucine to
threonine. Such "backmutated" antibodies are also intended to be
encompassed by the invention.
[0150] As another example, for PTA021_A3, amino acid residue #54 of
V.sub.H is an aspartic acid (SEQ ID NO:21) whereas this residue in
the corresponding V.sub.H 2-05 germline sequence is an asparagine
(SEQ ID NO:33). To return the framework region sequences to their
germline configuration, for example, residue #54 of the V.sub.H of
PTA021_A3 can be "backmutated" from aspartic acid to asparagine.
Such "backmutated" antibodies are also intended to be encompassed
by the invention.
[0151] As another example, for PTA021_A3, amino acid residue #77 of
V.sub.K is an asparagine (SEQ ID NO:24) whereas this residue in the
corresponding V.sub.K L15 germline sequence is a serine (SEQ ID
NO:36). To return the framework region sequences to their germline
configuration, for example, residue #77 of the V.sub.K of PTA021_A1
can be "backmutated" from asparagine to serine. Such "backmutated"
antibodies are also intended to be encompassed by the
invention.
[0152] Another type of framework modification involves mutating one
or more residues within the framework region, or even within one or
more CDR regions, to remove T cell epitopes to thereby reduce the
potential immunogenicity of the antibody. This approach is also
referred to as "deimmunization" and is described in further detail
in U.S. Patent Publication No. 20030153043 by Carr et al.
[0153] In addition or alternative to modifications made within the
framework or CDR regions, antibodies of the invention may be
engineered to include modifications within the Fc region, typically
to alter one or more functional properties of the antibody, such as
serum half-life, complement fixation, Fc receptor binding, and/or
antigen-dependent cellular cytotoxicity. Furthermore, an antibody
of the invention may be chemically modified (e.g., one or more
chemical moieties can be attached to the antibody) or be modified
to alter its glycosylation, again to alter one or more functional
properties of the antibody. Each of these embodiments is described
in further detail below. The numbering of residues in the Fc region
is that of the EU index of Kabat.
[0154] In one embodiment, the hinge region of CH1 is modified such
that the number of cysteine residues in the hinge region is
altered, e.g., increased or decreased. This approach is described
further in U.S. Pat. No. 5,677,425 by Bodmer et al. The number of
cysteine residues in the hinge region of CH1 is altered to, for
example, facilitate assembly of the light and heavy chains or to
increase or decrease the stability of the antibody.
[0155] In another embodiment, the Fc hinge region of an antibody is
mutated to decrease the biological half life of the antibody. More
specifically, one or more amino acid mutations are introduced into
the CH2-CH3 domain interface region of the Fc-hinge fragment such
that the antibody has impaired Staphylococcal protein A (SpA)
binding relative to native Fc-hinge domain SpA binding. This
approach is described in further detail in U.S. Pat. No. 6,165,745
by Ward et al.
[0156] In another embodiment, the antibody is modified to increase
its biological half life. Various approaches are possible. For
example, one or more of the following mutations can be introduced:
T252L, T254S, T256F, as described in U.S. Pat. No. 6,277,375 to
Ward. Alternatively, to increase the biological half life, the
antibody can be altered within the CH1 or C.sub.L region to contain
a salvage receptor binding epitope taken from two loops of a CH2
domain of an Fc region of an IgG, as described in U.S. Pat. Nos.
5,869,046 and 6,121,022 by Presta et al.
[0157] In another embodiment, the antibody is produced as a UniBody
as described in WO/2007/059782 which is incorporated herein by
reference in its entirety.
[0158] In yet other embodiments, the Fc region is altered by
replacing at least one amino acid residue with a different amino
acid residue to alter the effector function(s) of the antibody. For
example, one or more amino acids selected from amino acid residues
234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a
different amino acid residue such that the antibody has an altered
affinity for an effector ligand but retains the antigen-binding
ability of the parent antibody. The effector ligand to which
affinity is altered can be, for example, an Fc receptor or the C1
component of complement. This approach is described in further
detail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et
al.
[0159] In another example, one or more amino acids selected from
amino acid residues 329, 331 and 322 can be replaced with a
different amino acid residue such that the antibody has altered C1q
binding and/or reduced or abolished complement dependent
cytotoxicity (CDC). This approach is described in further detail in
U.S. Pat. No. 6,194,551 by Idusogie et al.
[0160] In another example, one or more amino acid residues within
amino acid positions 231 and 239 are altered to thereby alter the
ability of the antibody to fix complement. This approach is
described further in PCT Publication WO 94/29351 by Bodmer et
al.
[0161] In yet another example, the Fc region is modified to
increase the ability of the antibody to mediate antibody dependent
cellular cytotoxicity (ADCC) and/or to increase the affinity of the
antibody for an Fc.gamma. receptor by modifying one or more amino
acids at the following positions: 238, 239, 248, 249, 252, 254,
255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283,
285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305,
307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333,
334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398,
414, 416, 419, 430, 434, 435, 437, 438 or 439. This approach is
described further in PCT Publication WO 00/42072 by Presta.
Moreover, the binding sites on human IgG1 for Fc.gamma.R1,
Fc.gamma.RII, Fc.gamma.RIII and FcRn have been mapped and variants
with improved binding have been described (see Shields, R. L. et
al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at
positions 256, 290, 298, 333, 334 and 339 were shown to improve
binding to Fc.gamma.RIII. Additionally, the following combination
mutants were shown to improve Fc.gamma.RIII binding: T256A/S298A,
S298A/E333A, S298A/K224A and S298A/E333A/K334A.
[0162] In still another embodiment, the glycosylation of an
antibody is modified. For example, an aglycoslated antibody can be
made (i.e., the antibody lacks glycosylation). Glycosylation can be
altered to, for example, increase the affinity of the antibody for
antigen. Such carbohydrate modifications can be accomplished by,
for example, altering one or more sites of glycosylation within the
antibody sequence. For example, one or more amino acid
substitutions can be made that result in elimination of one or more
variable region framework glycosylation sites to thereby eliminate
glycosylation at that site. Such aglycosylation may increase the
affinity of the antibody for antigen. Such an approach is described
in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 by Co
et al.
[0163] Additionally or alternatively, an antibody can be made that
has an altered type of glycosylation, such as a hypofucosylated
antibody having reduced amounts of fucosyl residues or an antibody
having increased bisecting GlcNac structures. Such altered
glycosylation patterns have been demonstrated to increase the ADCC
ability of antibodies. Such carbohydrate modifications can be
accomplished by, for example, expressing the antibody in a host
cell with altered glycosylation machinery. Cells with altered
glycosylation machinery have been described in the art and can be
used as host cells in which to express recombinant antibodies of
the invention to thereby produce an antibody with altered
glycosylation. For example, the cell lines Ms704, Ms705, and Ms709
lack the fucosyltransferase gene, FUT8 (alpha (1,6)
fucosyltransferase), such that antibodies expressed in the Ms704,
Ms705, and Ms709 cell lines lack fucose on their carbohydrates. The
Ms704, Ms705, and Ms709 FUT8.sup.-/- cell lines were created by the
targeted disruption of the FUT8 gene in CHO/DG44 cells using two
replacement vectors (see U.S. Patent Publication No. 20040110704 by
Yamane et al. and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng
87:614-22). As another example, EP 1,176,195 by Hanai et al.
describes a cell line with a functionally disrupted FUT8 gene,
which encodes a fucosyl transferase, such that antibodies expressed
in such a cell line exhibit hypofucosylation by reducing or
eliminating the alpha 1,6 bond-related enzyme. Hanai et al. also
describe cell lines which have a low enzyme activity for adding
fucose to the N-acetylglucosamine that binds to the Fc region of
the antibody or does not have the enzyme activity, for example the
rat myeloma cell line YB2/0 (ATCC CRL 1662). PCT Publication WO
03/035835 by Presta describes a variant CHO cell line, Lec13 cells,
with reduced ability to attach fucose to Asn(297)-linked
carbohydrates, also resulting in hypofucosylation of antibodies
expressed in that host cell (see also Shields, R. L. et al. (2002)
J. Biol. Chem. 277:26733-26740). PCT Publication WO 99/54342 by
Umana et al. describes cell lines engineered to express
glycoprotein-modifying glycosyl transferases (e.g.,
beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that
antibodies expressed in the engineered cell lines exhibit increased
bisecting GlcNac structures which results in increased ADCC
activity of the antibodies (see also Umana et al. (1999) Nat.
Biotech. 17:176-180). Alternatively, the fucose residues of the
antibody may be cleaved off using a fucosidase enzyme. For example,
the fucosidase alpha-L-fucosidase removes fucosyl residues from
antibodies (Tarentino, A. L. et al. (1975) Biochem.
14:5516-23).
[0164] Another modification of the antibodies herein that is
contemplated by the invention is pegylation. An antibody can be
pegylated to, for example, increase the biological (e.g., serum)
half life of the antibody. To pegylate an antibody, the antibody,
or fragment thereof, typically is reacted with polyethylene glycol
(PEG), such as a reactive ester or aldehyde derivative of PEG,
under conditions in which one or more PEG groups become attached to
the antibody or antibody fragment. Preferably, the pegylation is
carried out via an acylation reaction or an alkylation reaction
with a reactive PEG molecule (or an analogous reactive
water-soluble polymer). As used herein, the term "polyethylene
glycol" is intended to encompass any of the forms of PEG that have
been used to derivatize other proteins, such as mono (C1-C10)
alkoxy- or aryloxy-polyethylene glycol or polyethylene
glycol-maleimide. In certain embodiments, the antibody to be
pegylated is an aglycosylated antibody. Methods for pegylating
proteins are known in the art and can be applied to the antibodies
of the invention. See for example, EP 0 154 316 by Nishimura et al.
and EP 0 401 384 by Ishikawa et al.
Antibody Physical Properties
[0165] The antibodies of the present invention may be further
characterized by the various physical properties of the anti-CADM1
antibodies. Various assays may be used to detect and/or
differentiate different classes of antibodies based on these
physical properties.
[0166] In some embodiments, antibodies of the present invention may
contain one or more glycosylation sites in either the light or
heavy chain variable region. The presence of one or more
glycosylation sites in the variable region may result in increased
immunogenicity of the antibody or an alteration of the pK of the
antibody due to altered antigen binding (Marshall et al (1972) Annu
Rev Biochem 41:673-702; Gala F A and Morrison S L (2004) J Immunol
172:5489-94; Wallick et al (1988) J Exp Med 168:1099-109; Spiro RG
(2002) Glycobiology 12:43 R-56R; Parekh et al (1985) Nature
316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706).
Glycosylation has been known to occur at motifs containing an
N-X-S/T sequence. Variable region glycosylation may be tested using
a Glycoblot assay, which cleaves the antibody to produce a Fab, and
then tests for glycosylation using an assay that measures periodate
oxidation and Schiff base formation. Alternatively, variable region
glycosylation may be tested using Dionex light chromatography
(Dionex-LC), which cleaves saccharides from a Fab into
monosaccharides and analyzes the individual saccharide content. In
some instances, it is preferred to have an anti-CADM1 antibody that
does not contain variable region glycosylation. This can be
achieved either by selecting antibodies that do not contain the
glycosylation motif in the variable region or by mutating residues
within the glycosylation motif using standard techniques well known
in the art.
[0167] As an example of glycosylation, for PTA021_A1, using the
Kabat numbering system, amino acid residue #30 (within FR3) of
V.sub.H is an asparagine (SEQ ID NO:19). This is a potential
glycosylation site and this residue can be mutated to a
glutamine.
[0168] In a preferred embodiment, the antibodies of the present
invention do not contain asparagine isomerism sites. A deamidation
or isoaspartic acid effect may occur on N-G or D-G sequences,
respectively. The deamidation or isoaspartic acid effect results in
the creation of isoaspartic acid which decreases the stability of
an antibody by creating a kinked structure off a side chain carboxy
terminus rather than the main chain. The creation of isoaspartic
acid can be measured using an iso-quant assay, which uses a
reverse-phase HPLC to test for isoaspartic acid.
[0169] Each antibody will have a unique isoelectric point (pI), but
generally antibodies will fall in the pH range of between 6 and
9.5. The pI for an IgG1 antibody typically falls within the pH
range of 7-9.5 and the pI for an IgG4 antibody typically falls
within the pH range of 6-8. Antibodies may have a pI that is
outside this range. Although the effects are generally unknown,
there is speculation that antibodies with a pI outside the normal
range may have some unfolding and instability under in vivo
conditions. The isoelectric point may be tested using a capillary
isoelectric focusing assay, which creates a pH gradient and may
utilize laser focusing for increased accuracy (Janini et al (2002)
Electrophoresis 23:1605-11; Ma et al. (2001) Chromatographia
53:S75-89; Hunt et al (1998) J Chromatogr A 800:355-67). In some
instances, it is preferred to have an anti-CADM1 antibody that
contains a pI value that falls in the normal range. This can be
achieved either by selecting antibodies with a pI in the normal
range, or by mutating charged surface residues using standard
techniques well known in the art.
[0170] Each antibody will have a melting temperature that is
indicative of thermal stability (Krishnamurthy R and Manning M C
(2002) Curr Pharm Biotechnol 3:361-71). A higher thermal stability
indicates greater overall antibody stability in vivo. The melting
point of an antibody may be measure using techniques such as
differential scanning calorimetry (Chen et al (2003) Pharm Res
20:1952-60; Ghirlando et al (1999) Immunol Lett 68:47-52). T.sub.M1
indicates the temperature of the initial unfolding of the antibody.
T.sub.M2 indicates the temperature of complete unfolding of the
antibody. Generally, it is preferred that the T.sub.M1 of an
antibody of the present invention is greater than 60.degree. C.,
preferably greater than 65.degree. C., even more preferably greater
than 70.degree. C. Alternatively, the thermal stability of an
antibody may be measure using circular dichroism (Murray et al.
(2002) J. Chromatogr Sci 40:343-9).
[0171] In a preferred embodiment, antibodies are selected that do
not rapidly degrade. Fragmentation of an anti-CADM1 antibody may be
measured using capillary electrophoresis (CE) and MALDI-MS, as is
well understood in the art (Alexander A J and Hughes D E (1995)
Anal Chem 67:3626-32).
[0172] In another preferred embodiment, antibodies are selected
that have minimal aggregation effects. Aggregation may lead to
triggering of an unwanted immune response and/or altered or
unfavorable pharmacokinetic properties. Generally, antibodies are
acceptable with aggregation of 25% or less, preferably 20% or less,
even more preferably 15% or less, even more preferably 10% or less
and even more preferably 5% or less. Aggregation may be measured by
several techniques well known in the art, including size-exclusion
column (SEC) high performance liquid chromatography (HPLC), and
light scattering to identify monomers, dimers, trimers or
multimers.
Methods of Engineering Antibodies
[0173] As discussed above, the anti-CADM1 antibodies having V.sub.H
and V.sub.K sequences disclosed herein can be used to create new
anti-CADM1 antibodies by modifying the V.sub.H and/or V.sub.K
sequences, or the constant region(s) attached thereto. Thus, in
another aspect of the invention, the structural features of an
anti-CADM1 antibody of the invention, e.g. PTA021_A1, PTA021_A2, or
PTA021_A3, are used to create structurally related anti-CADM1
antibodies that retain at least one functional property of the
antibodies of the invention, such as binding to human CADM1. For
example, one or more CDR regions of PTA021_A1, PTA021_A2, or
PTA021_A3, or mutations thereof, can be combined recombinantly with
known framework regions and/or other CDRs to create additional,
recombinantly-engineered, anti-CADM1 antibodies of the invention,
as discussed above. Other types of modifications include those
described in the previous section. The starting material for the
engineering method is one or more of the V.sub.H and/or V.sub.K
sequences provided herein, or one or more CDR regions thereof. To
create the engineered antibody, it is not necessary to actually
prepare (i.e., express as a protein) an antibody having one or more
of the V.sub.H and/or V.sub.K sequences provided herein, or one or
more CDR regions thereof. Rather, the information contained in the
sequence(s) is used as the starting material to create a "second
generation" sequence(s) derived from the original sequence(s) and
then the "second generation" sequence(s) is prepared and expressed
as a protein.
[0174] Accordingly, in another embodiment, the invention provides a
method for preparing an anti-CADM1 antibody comprising:
providing: (i) a heavy chain variable region antibody sequence
comprising a CDR1 sequence selected from the group consisting of
SEQ ID NOs:1, 2, and 3, a CDR2 sequence selected from the group
consisting of SEQ ID NOs:4, 5, and 6, and/or a CDR3 sequence
selected from the group consisting of SEQ ID NOs:7, 8, and 9;
and/or (ii) a light chain variable region antibody sequence
comprising a CDR1 sequence selected from the group consisting of
SEQ ID NOs:10, 11, and 12, a CDR2 sequence selected from the group
consisting of SEQ ID NOs:13, 14, and 15, and/or a CDR3 sequence
selected from the group consisting of SEQ ID NOs:16, 17, and 18;
altering at least one amino acid residue within the heavy chain
variable region antibody sequence and/or the light chain variable
region antibody sequence to create at least one altered antibody
sequence; and expressing the altered antibody sequence as a
protein.
[0175] Standard molecular biology techniques can be used to prepare
and express the altered antibody sequence.
[0176] Preferably, the antibody encoded by the altered antibody
sequence(s) is one that retains one, some or all of the functional
properties of the anti-CADM1 antibodies described herein, which
functional properties include, but are not limited to:
binds to human CADM1 with a K.sub.D of 1.times.10.sup.-7 M or less;
binds to human CHO cells transfected with CADM1.
[0177] The functional properties of the altered antibodies can be
assessed using standard assays available in the art and/or
described herein, such as those set forth in the Examples (e.g.,
flow cytometry, binding assays).
[0178] In certain embodiments of the methods of engineering
antibodies of the invention, mutations can be introduced randomly
or selectively along all or part of an anti-CADM1 antibody coding
sequence and the resulting modified anti-CADM1 antibodies can be
screened for binding activity and/or other functional properties as
described herein. Mutational methods have been described in the
art. For example, PCT Publication WO 02/092780 by Short describes
methods for creating and screening antibody mutations using
saturation mutagenesis, synthetic ligation assembly, or a
combination thereof. Alternatively, PCT Publication WO 03/074679 by
Lazar et al. describes methods of using computational screening
methods to optimize physiochemical properties of antibodies.
Nucleic Acid Molecules Encoding Antibodies of the Invention
[0179] Another aspect of the invention pertains to nucleic acid
molecules that encode the antibodies of the invention. The nucleic
acids may be present in whole cells, in a cell lysate, or in a
partially purified or substantially pure form. A nucleic acid is
"isolated" or "rendered substantially pure" when purified away from
other cellular components or other contaminants, e.g., other
cellular nucleic acids or proteins, by standard techniques,
including alkaline/SDS treatment, CsCl banding, column
chromatography, agarose gel electrophoresis and others well known
in the art. See, F. Ausubel, et al., ed. (1987) Current Protocols
in Molecular Biology, Greene Publishing and Wiley Interscience, New
York. A nucleic acid of the invention can be, for example, DNA or
RNA and may or may not contain intronic sequences. In a preferred
embodiment, the nucleic acid is a cDNA molecule.
[0180] Nucleic acids of the invention can be obtained using
standard molecular biology techniques. For antibodies expressed by
hybridomas (e.g., hybridomas prepared from transgenic mice carrying
human immunoglobulin genes as described further below), cDNAs
encoding the light and heavy chains of the antibody made by the
hybridoma can be obtained by standard PCR amplification or cDNA
cloning techniques. For antibodies obtained from an immunoglobulin
gene library (e.g., using phage display techniques), nucleic acids
encoding the antibody can be recovered from the library.
[0181] Preferred nucleic acids molecules of the invention are those
encoding the V.sub.H and V.sub.L sequences of the PTA021_A1,
PTA021_A2, or PTA021_A3 monoclonal antibodies. DNA sequences
encoding the V.sub.H sequences of PTA021_A1, PTA021_A2, and
PTA021_A3 are shown in SEQ ID NOs: 25, 26, and 27, respectively.
DNA sequences encoding the V.sub.K sequences of PTA021_A1,
PTA021_A2, and PTA021_A3 are shown in SEQ ID NOs: 28, 29, and 30,
respectively.
[0182] Other preferred nucleic acids of the invention are nucleic
acids having at least 80% sequence identity, such as at least 85%,
at least 90%, at least 95%, at least 98% or at least 99% sequence
identity, with one of the sequences shown in SEQ ID NOs: 25, 26,
27, 28, 29, and 30, which nucleic acids encode an antibody of the
invention, or an antigen-binding portion thereof.
[0183] The percent identity between two nucleic acid sequences is
the number of positions in the sequence in which the nucleotide is
identical, taking into account the number of gaps and the length of
each gap, which need to be introduced for optimal alignment of the
two sequences. The comparison of sequences and determination of
percent identity between two sequences can be accomplished using a
mathematical algorithm, such as the algorithm of Meyers and Miller
or the XBLAST program of Altschul described above.
[0184] Still further, preferred nucleic acids of the invention
comprise one or more CDR-encoding portions of the nucleic acid
sequences shown in SEQ ID NOs:25, 26, 27, 28, 29, and 30. In this
embodiment, the nucleic acid may encode the heavy chain CDR1, CDR2
and/or CDR3 sequence of PTA021_A1, PTA021_A2, or PTA021_A3 or the
light chain CDR1, CDR2 and/or CDR3 sequence of PTA021_A1,
PTA021_A2, or PTA021_A3.
[0185] Nucleic acids which have at least 80%, such as at least 85%,
at least 90%, at least 95%, at least 98% or at least 99% sequence
identity, with such a CDR-encoding portion of SEQ ID NO: 25, 26,
27, 28, 29, or 30 (VH and VK seqs) are also preferred nucleic acids
of the invention. Such nucleic acids may differ from the
corresponding portion of SEQ ID NO: 25, 26, 27, 28, 29, or 30 in a
non-CDR coding region and/or in a CDR-coding region. Where the
difference is in a CDR-coding region, the nucleic acid CDR region
encoded by the nucleic acid typically comprises one or more
conservative sequence modification as defined herein compared to
the corresponding CDR sequence of PTA021_A1, PTA021_A2, or
PTA021_A3.
[0186] Once DNA fragments encoding V.sub.H and V.sub.K segments are
obtained, these DNA fragments can be further manipulated by
standard recombinant DNA techniques, for example to convert the
variable region genes to full-length antibody chain genes, to Fab
fragment genes or to a scFv gene. In these manipulations, a
V.sub.K- or V.sub.H-encoding DNA fragment is operatively linked to
another DNA fragment encoding another protein, such as an antibody
constant region or a flexible linker. The term "operatively
linked," as used in this context, is intended to mean that the two
DNA fragments are joined such that the amino acid sequences encoded
by the two DNA fragments remain in-frame.
[0187] The isolated DNA encoding the V.sub.H region can be
converted to a full-length heavy chain gene by operatively linking
the VH-encoding DNA to another DNA molecule encoding heavy chain
constant regions (CH1, CH2 and CH3). The sequences of human heavy
chain constant region genes are known in the art (see e.g., Kabat,
E. A., et al. (1991) Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publication No. 91-3242) and DNA fragments
encompassing these regions can be obtained by standard PCR
amplification. The heavy chain constant region can be an IgG1,
IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most
preferably is an IgG1 or IgG4 constant region. For a Fab fragment
heavy chain gene, the V.sub.H-encoding DNA can be operatively
linked to another DNA molecule encoding only the heavy chain CH1
constant region.
[0188] The isolated DNA encoding the V.sub.L/V.sub.K region can be
converted to a full-length light chain gene (as well as a Fab light
chain gene) by operatively linking the V.sub.L-encoding DNA to
another DNA molecule encoding the light chain constant region, CL.
The sequences of human light chain constant region genes are known
in the art (see e.g., Kabat, E. A., et al. (1991) Sequences of
Proteins of Immunological Interest, Fifth Edition, U.S. Department
of Health and Human Services, NIH Publication No. 91-3242) and DNA
fragments encompassing these regions can be obtained by standard
PCR amplification. In preferred embodiments, the light chain
constant region can be a kappa or lambda constant region.
[0189] To create a scFv gene, the V.sub.H- and
V.sub.L/V.sub.K-encoding DNA fragments are operatively linked to
another fragment encoding a flexible linker, e.g., encoding the
amino acid sequence (Gly.sub.4-Ser).sub.3, such that the V.sub.H
and V.sub.L/V.sub.K sequences can be expressed as a contiguous
single-chain protein, with the V.sub.L/V.sub.K and V.sub.H regions
joined by the flexible linker (see e.g., Bird et al. (1988) Science
242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA
85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).
Production of Monoclonal Antibodies
[0190] Monoclonal antibodies (mAbs) of the present invention can be
produced by a variety of techniques, including conventional
monoclonal antibody methodology e.g., the standard somatic cell
hybridization technique of Kohler and Milstein (1975) Nature 256:
495. Although somatic cell hybridization procedures are preferred,
in principle, other techniques for producing monoclonal antibody
can be employed e.g., viral or oncogenic transformation of B
lymphocytes.
[0191] The preferred animal system for preparing hybridomas is the
murine system. Hybridoma production in the mouse is a very
well-established procedure. Immunization protocols and techniques
for isolation of immunized splenocytes for fusion are known in the
art. Fusion partners (e.g., murine myeloma cells) and fusion
procedures are also known.
[0192] Chimeric or humanized antibodies of the present invention
can be prepared based on the sequence of a non-human monoclonal
antibody prepared as described above. DNA encoding the heavy and
light chain immunoglobulins can be obtained from the non-human
hybridoma of interest and engineered to contain non-murine (e.g.,
human) immunoglobulin sequences using standard molecular biology
techniques. For example, to create a chimeric antibody, murine
variable regions can be linked to human constant regions using
methods known in the art (see e.g., U.S. Pat. No. 4,816,567 to
Cabilly et al.). To create a humanized antibody, murine CDR regions
can be inserted into a human framework using methods known in the
art (see e.g., U.S. Pat. No. 5,225,539 to Winter, and U.S. Pat.
Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et
al.).
[0193] In a preferred embodiment, the antibodies of the invention
are human monoclonal antibodies. Such human monoclonal antibodies
directed against CADM1 can be generated using transgenic or
transchromosomic mice carrying parts of the human immune system
rather than the mouse system. These transgenic and transchromosomic
mice include mice referred to herein as mice of the HuMAb
Mouse.RTM. and KM Mouse.RTM. strains, respectively, and are
collectively referred to herein as "human Ig mice."
[0194] The HuMAb Mouse.RTM. strain (Medarex.RTM., Inc.) contains
human immunoglobulin gene miniloci that encode unrearranged human
heavy (.mu. and .gamma.) and .kappa. light chain immunoglobulin
sequences, together with targeted mutations that inactivate the
endogenous .mu. and .kappa. chain loci (see e.g., Lonberg, et al.
(1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit
reduced expression of mouse IgM or .kappa., and in response to
immunization, the introduced human heavy and light chain transgenes
undergo class switching and somatic mutation to generate high
affinity human IgG.kappa. monoclonal antibodies (Lonberg, N. et al.
(1994), supra; reviewed in Lonberg, N. (1994) Handbook of
Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D.
(1995) Intern. Rev. Immunol. 13: 65-93, and Harding, F. and
Lonberg, N. (1995) Ann. N.Y. Acad. Sci. 764:536-546). Preparation
and use of the HuMAb Mouse.RTM., and the genomic modifications
carried by such mice, is further described in Taylor, L. et al.
(1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993)
International Immunology 5: 647-656; Tuaillon et al. (1993) Proc.
Natl. Acad. Sci. USA 90:3720-3724; Choi et al. (1993) Nature
Genetics 4:117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830;
Tuaillon et al. (1994) J. Immunol. 152:2912-2920; Taylor, L. et al.
(1994) International Immunology 6: 579-591; and Fishwild, D. et al.
(1996) Nature Biotechnology 14: 845-851, the contents of all of
which are hereby specifically incorporated by reference in their
entirety. See further, U.S. Pat. Nos. 5,545,806; 5,569,825;
5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318;
5,874,299; and 5,770,429; all to Lonberg and Kay; U.S. Pat. No.
5,545,807 to Surani et al.; PCT Publication Nos. WO 92/03918, WO
93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO 99/45962,
all to Lonberg and Kay; and PCT Publication No. WO 01/14424 to
Korman et al.
[0195] In another embodiment, human antibodies of the invention can
be raised using a mouse that carries human immunoglobulin sequences
on transgenes and transchromosomes, such as a mouse that carries a
human heavy chain transgene and a human light chain
transchromosome. This mouse is referred to herein as a mouse of the
"KM Mouse.RTM." strain, and is described in detail in PCT
Publication WO 02/43478 to Ishida et al.
[0196] Still further, alternative transgenic animal systems
expressing human immunoglobulin genes are available in the art and
can be used to raise anti-CADM1 antibodies of the invention. For
example, an alternative transgenic system referred to as the
Xenomouse (Amgen, Inc.) can be used; such mice are described in,
for example, U.S. Pat. Nos. 5,939,598; 6,075,181; 6,114,598;
6,150,584 and 6,162,963 to Kucherlapati et al.
[0197] Moreover, alternative transchromosomic animal systems
expressing human immunoglobulin genes are available in the art and
can be used to raise anti-CADM1 antibodies of the invention. For
example, mice carrying both a human heavy chain transchromosome and
a human light chain tranchromosome, referred to as "TC mice" can be
used; such mice are described in Tomizuka et al. (2000) Proc. Natl.
Acad. Sci. USA 97:722-727. Furthermore, cows carrying human heavy
and light chain transchromosomes have been described in the art
(Kuroiwa et al. (2002) Nature Biotechnology 20:889-894) and PCT
application No. WO/2002/092812 and can be used to raise anti-CADM1
antibodies of the invention.
[0198] Human monoclonal antibodies of the invention can also be
prepared using phage display methods for screening libraries of
human immunoglobulin genes. Such phage display methods for
isolating human antibodies are established in the art. See for
example: U.S. Pat. Nos. 5,223,409; 5,403,484; and 5,571,698 to
Ladner et al.; U.S. Pat. Nos. 5,427,908 and 5,580,717 to Dower et
al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 to McCafferty et al.;
and U.S. Pat. Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313;
6,582,915 and 6,593,081 to Griffiths et al.
[0199] Human monoclonal antibodies of the invention can also be
prepared using SCID mice into which human immune cells have been
reconstituted such that a human antibody response can be generated
upon immunization. Such mice are described in, for example, U.S.
Pat. Nos. 5,476,996 and 5,698,767 to Wilson et al.
Immunization of Human Ig Mice
[0200] When human Ig mice are used to raise human antibodies of the
invention, such mice can be immunized with a purified or enriched
preparation of CADM1 antigen and/or recombinant CADM1, or cells
expressing CADM1, or a CADM1 fusion protein, as described by
Lonberg, N. et al. (1994) Nature 368(6474): 856-859; Fishwild, D.
et al. (1996) Nature Biotechnology 14: 845-851; and PCT Publication
WO 98/24884 and WO 01/14424. Preferably, the mice will be 6-16
weeks of age upon the first infusion. For example, a purified or
recombinant preparation (5-50 .mu.g) of CADM1 antigen can be used
to immunize the human Ig mice intraperitoneally.
[0201] Detailed procedures to generate fully human monoclonal
antibodies to CADM1 are described in Example 1 below. Cumulative
experience with various antigens has shown that the transgenic mice
respond when initially immunized intraperitoneally (IP) with
antigen in complete Freund's adjuvant, followed by every other week
IP immunizations (up to a total of 6) with antigen in incomplete
Freund's adjuvant. However, adjuvants other than Freund's are also
found to be effective. In addition, whole cells in the absence of
adjuvant are found to be highly immunogenic. The immune response
can be monitored over the course of the immunization protocol with
plasma samples being obtained by retroorbital bleeds. The plasma
can be screened by ELISA (as described below), and mice with
sufficient titers of anti-CADM1 human immunoglobulin can be used
for fusions. Mice can be boosted intravenously with antigen 3 days
before sacrifice and removal of the spleen. It is expected that 2-3
fusions for each immunization may need to be performed. Between 6
and 24 mice are typically immunized for each antigen. In one
embodiment, mouse strains bearing an HCo7, HCo12 or HCo17 human
heavy chain transgene strains may used. Alternatively or
additionally, the KM Mouse.RTM. strain can be used. In addition,
two or more of these strains can be bred together into a single
mouse having a plurality of different human heavy chain
transgenes.
Generation of Hybridomas Producing Human Monoclonal Antibodies
[0202] To generate hybridomas producing human monoclonal antibodies
of the invention, splenocytes and/or lymph node cells from
immunized mice can be isolated and fused to an appropriate
immortalized cell line, such as a mouse myeloma cell line. The
resulting hybridomas can be screened for the production of
antigen-specific antibodies. For example, single cell suspensions
of splenic lymphocytes from immunized mice can be fused to
one-sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma
cells (ATCC, CRL 1580) with 50% PEG. Alternatively, the single cell
suspension of splenic lymphocytes from immunized mice can be fused
using an electric field based electrofusion method, using a
CytoPulse large chamber cell fusion electroporator (CytoPulse
Sciences, Inc., Glen Burnie Md.). Cells are plated at approximately
2.times.10.sup.5 in flat bottom microtiter plate, followed by a two
week incubation in selective medium containing 20% fetal Clone
Serum, 18% "653" conditioned media, 5% origen (IGEN), 4 mM
L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM
2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin,
50 mg/ml gentamycin and 1.times.HAT (Sigma; the HAT is added 24
hours after the fusion). After approximately two weeks, cells can
be cultured in medium in which the HAT is replaced with HT.
Individual wells can then be screened by ELISA for human monoclonal
IgM and IgG antibodies. Once extensive hybridoma growth occurs,
medium can be observed usually after 10-14 days. The antibody
secreting hybridomas can be replated, screened again, and if still
positive for human IgG, the monoclonal antibodies can be subcloned
at least twice by limiting dilution. The stable subclones can then
be cultured in vitro to generate small amounts of antibody in
tissue culture medium for characterization.
[0203] To purify human monoclonal antibodies, selected hybridomas
can be grown in two-liter spinner-flasks for monoclonal antibody
purification. Supernatants can be filtered and concentrated before
affinity chromatography with protein A-sepharose (Pharmacia,
Piscataway, N.J.). Eluted IgG can be checked by gel electrophoresis
and high performance liquid chromatography to ensure purity. The
buffer solution can be exchanged into PBS, and the concentration
can be determined by OD280 using 1.43 extinction coefficient. The
monoclonal antibodies can be aliquoted and stored at -80.degree.
C.
Generation of Transfectomas Producing Monoclonal Antibodies
[0204] Antibodies of the invention also can be produced in a host
cell transfectoma using, for example, a combination of recombinant
DNA techniques and gene transfection methods as is well known in
the art (e.g., Morrison, S. (1985) Science 229:1202).
[0205] For example, to express the antibodies, or antibody
fragments thereof, DNAs encoding partial or full-length light and
heavy chains, can be obtained by standard molecular biology
techniques (e.g., PCR amplification or cDNA cloning using a
hybridoma that expresses the antibody of interest) and the DNAs can
be inserted into expression vectors such that the genes are
operatively linked to transcriptional and translational control
sequences. In this context, the term "operatively linked" is
intended to mean that an antibody gene is ligated into a vector
such that transcriptional and translational control sequences
within the vector serve their intended function of regulating the
transcription and translation of the antibody gene. The expression
vector and expression control sequences are chosen to be compatible
with the expression host cell used. The antibody light chain gene
and the antibody heavy chain gene can be inserted into separate
vector or, more typically, both genes are inserted into the same
expression vector. The antibody genes are inserted into the
expression vector by standard methods (e.g., ligation of
complementary restriction sites on the antibody gene fragment and
vector, or blunt end ligation if no restriction sites are present).
The light and heavy chain variable regions of the antibodies
described herein can be used to create full-length antibody genes
of any antibody isotype by inserting them into expression vectors
already encoding heavy chain constant and light chain constant
regions of the desired isotype such that the V.sub.H segment is
operatively linked to the C.sub.H segment(s) within the vector and
the V.sub.K segment is operatively linked to the C.sub.L segment
within the vector. Additionally or alternatively, the recombinant
expression vector can encode a signal peptide that facilitates
secretion of the antibody chain from a host cell. The antibody
chain gene can be cloned into the vector such that the signal
peptide is linked in-frame to the amino terminus of the antibody
chain gene. The signal peptide can be an immunoglobulin signal
peptide or a heterologous signal peptide (i.e., a signal peptide
from a non-immunoglobulin protein).
[0206] In addition to the antibody chain genes, the recombinant
expression vectors of the invention carry regulatory sequences that
control the expression of the antibody chain genes in a host cell.
The term "regulatory sequence" is intended to include promoters,
enhancers and other expression control elements (e.g.,
polyadenylation signals) that control the transcription or
translation of the antibody chain genes. Such regulatory sequences
are described, for example, in Goeddel (Gene Expression Technology.
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990)). It will be appreciated by those skilled in the art that
the design of the expression vector, including the selection of
regulatory sequences, may depend on such factors as the choice of
the host cell to be transformed, the level of expression of protein
desired, etc. Preferred regulatory sequences for mammalian host
cell expression include viral elements that direct high levels of
protein expression in mammalian cells, such as promoters and/or
enhancers derived from cytomegalovirus (CMV), Simian Virus 40
(SV40), adenovirus, (e.g., the adenovirus major late promoter
(AdMLP) and polyoma. Alternatively, nonviral regulatory sequences
may be used, such as the ubiquitin promoter or .beta.-globin
promoter. Still further, regulatory elements composed of sequences
from different sources, such as the SR.alpha. promoter system,
which contains sequences from the SV40 early promoter and the long
terminal repeat of human T cell leukemia virus type 1 (Takebe, Y.
et al. (1988) Mol. Cell. Biol. 8:466-472).
[0207] In addition to the antibody chain genes and regulatory
sequences, the recombinant expression vectors of the invention may
carry additional sequences, such as sequences that regulate
replication of the vector in host cells (e.g., origins of
replication) and selectable marker genes. The selectable marker
gene facilitates selection of host cells into which the vector has
been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and
5,179,017, all by Axel et al.). For example, typically the
selectable marker gene confers resistance to drugs, such as G418,
hygromycin or methotrexate, on a host cell into which the vector
has been introduced. Preferred selectable marker genes include the
dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells
with methotrexate selection/amplification) and the neo gene (for
G418 selection).
[0208] For expression of the light and heavy chains, the expression
vector(s) encoding the heavy and light chains is transfected into a
host cell by standard techniques. The various forms of the term
"transfection" are intended to encompass a wide variety of
techniques commonly used for the introduction of exogenous DNA into
a prokaryotic or eukaryotic host cell, e.g., electroporation,
calcium-phosphate precipitation, DEAE-dextran transfection and the
like. Although it is theoretically possible to express the
antibodies of the invention in either prokaryotic or eukaryotic
host cells, expression of antibodies in eukaryotic cells, and most
preferably mammalian host cells, is the most preferred because such
eukaryotic cells, and in particular mammalian cells, are more
likely than prokaryotic cells to assemble and secrete a properly
folded and immunologically active antibody. Prokaryotic expression
of antibody genes has been reported to be ineffective for
production of high yields of active antibody (Boss, M. A. and Wood,
C. R. (1985) Immunology Today 6:12-13).
[0209] Preferred mammalian host cells for expressing the
recombinant antibodies of the invention include Chinese Hamster
Ovary (CHO cells) (including dhfr.sup.- CHO cells, described in
Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220,
used with a DHFR selectable marker, e.g., as described in R. J.
Kaufman and P. A. Sharp (1982) J. Mol. Biol. 159:601-621), NSO
myeloma cells, COS cells and SP2 cells. In particular, for use with
NSO myeloma cells, another preferred expression system is the GS
gene expression system disclosed in WO 87/04462 (to Wilson), WO
89/01036 (to Bebbington) and EP 338,841 (to Bebbington). When
recombinant expression vectors encoding antibody genes are
introduced into mammalian host cells, the antibodies are produced
by culturing the host cells for a period of time sufficient to
allow for expression of the antibody in the host cells or, more
preferably, secretion of the antibody into the culture medium in
which the host cells are grown. Antibodies can be recovered from
the culture medium using standard protein purification methods.
Characterization of Antibody Binding to Antigen
[0210] Antibodies of the invention can be tested for binding to
CADM1 by, for example, standard ELISA. Briefly, microtiter plates
are coated with purified CADM1 at 0.25 .mu.g/ml in PBS, and then
blocked with 5% bovine serum albumin in PBS. Dilutions of antibody
(e.g., dilutions of plasma from CADM1-immunized mice) are added to
each well and incubated for 1-2 hours at 37.degree. C. The plates
are washed with PBS/Tween and then incubated with secondary reagent
(e.g., for human antibodies, a goat-anti-human IgG Fc-specific
polyclonal reagent) conjugated to alkaline phosphatase for 1 hour
at 37.degree. C. After washing, the plates are developed with pNPP
substrate (1 mg/ml), and analyzed at OD of 405-650. Preferably,
mice which develop the highest titers will be used for fusions.
[0211] An ELISA assay as described above can also be used to screen
for hybridomas that show positive reactivity with CADM1 immunogen.
Hybridomas that bind with high avidity to CADM1 are subcloned and
further characterized. One clone from each hybridoma, which retains
the reactivity of the parent cells (by ELISA), can be chosen for
making a 5-10 vial cell bank stored at -140.degree. C., and for
antibody purification.
[0212] To purify anti-CADM1 antibodies, selected hybridomas can be
grown in two-liter spinner-flasks for monoclonal antibody
purification. Supernatants can be filtered and concentrated before
affinity chromatography with protein A-sepharose (Pharmacia,
Piscataway, N.J.). Eluted IgG can be checked by gel electrophoresis
and high performance liquid chromatography to ensure purity. The
buffer solution can be exchanged into PBS, and the concentration
can be determined by OD280 using 1.43 extinction coefficient. The
monoclonal antibodies can be aliquoted and stored at -80.degree.
C.
[0213] To determine if the selected anti-CADM1 monoclonal
antibodies bind to unique epitopes, each antibody can be
biotinylated using commercially available reagents (Pierce,
Rockford, Ill.). Studies for competing for binding with the
antibodies of the invention using unlabeled monoclonal antibodies
and biotinylated monoclonal antibodies can be performed using CADM1
coated-ELISA plates as described above. Biotinylated mAb binding
can be detected with a strep-avidin-alkaline phosphatase probe.
[0214] To determine the isotype of purified antibodies, isotype
ELISAs can be performed using reagents specific for antibodies of a
particular isotype. For example, to determine the isotype of a
human monoclonal antibody, wells of microtiter plates can be coated
with 1 .mu.g/ml of anti-human immunoglobulin overnight at 4.degree.
C. After blocking with 1% BSA, the plates are reacted with 1
.mu.g/ml or less of test monoclonal antibodies or purified isotype
controls, at ambient temperature for one to two hours. The wells
can then be reacted with either human IgG1 or human IgM-specific
alkaline phosphatase-conjugated probes. Plates are developed and
analyzed as described above.
[0215] Anti-CADM1 human IgGs can be further tested for reactivity
with CADM1 antigen by Western blotting. Briefly, CADM1 can be
prepared and subjected to sodium dodecyl sulfate polyacrylamide gel
electrophoresis. After electrophoresis, the separated antigens are
transferred to nitrocellulose membranes, blocked with 10% fetal
calf serum, and probed with the monoclonal antibodies to be tested.
Human IgG binding can be detected using anti-human IgG alkaline
phosphatase and developed with BCIP/NBT substrate tablets (Sigma
Chem. Co., St. Louis, Mo.).
[0216] The binding specificity of an antibody of the invention may
also be determined by monitoring binding of the antibody to cells
expressing CADM1, for example by flow cytometry. Typically, a cell
line, such as a CHO cell line, may be transfected with an
expression vector encoding a transmembrane form of CADM1. In some
embodiments, a full length CADM1 molecule with an amino terminal HA
tag (SEQ ID NO:43) is expressed on the cell surface of the CHO cell
line. The transfected protein may comprise a tag, such as a
myc-tag, preferably at the N-terminus, for detection using an
antibody to the tag. Binding of an antibody of the invention to
CADM1 may be determined by incubating the transfected cells with
the antibody, and detecting bound antibody. Binding of an antibody
to the tag on the transfected protein may be used as a positive
control.
[0217] The specificity of an antibody of the invention for CADM1
may be further studied by determining whether or not the antibody
binds to other proteins, such as TSLL2/CADM1C or other members of
the immunoglobulin superfamily using the same methods by which
binding to CADM1 is determined.
Antibody-Partner Molecule Conjugates
[0218] In a preferred aspect, there is provided a conjugate
comprising an anti-CADM1 antibody according to this invention and a
partner molecule, the conjugate being represented by formula
(a)
Z[(X.sup.Z).sub.aC(X.sup.D).sub.bD].sub.m (a)
where Z is an antibody according to this invention; D is a partner
molecule; and (X.sup.Z).sub.aC(X.sup.D).sub.b are collectively
referred to as a "linker moiety" or "linker" because they link the
first two elements. Within the linker, C is a cleavable group
designed to be cleaved at the site of intended biological action of
partner molecule D; X.sup.Z and X.sup.D are referred to as spacer
moieties (or "spacers") because they space apart Z and C and C and
D, respectively; subscripts a and b are independently 0 or 1 (that
is, the presence of X.sup.Z and/or X.sup.D is optional); and
subscript m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (preferably 1, 2,
3, or 4). Each of the foregoing terms is more fully defined herein
below.
[0219] Antibody Z serves a targeting function: by binding to a
target tissue or cell where its antigen is located, it directs the
conjugate there. Preferably, the target tissue or cell is a tumor
or a cancer cell and the antigen is a tumor-associated or
tumor-specific antigen. Cleavage of group C at the target tissue or
cell releases partner molecule D to perform its intended biological
function. In some instances, the conjugate is internalized into a
target cell by endocytosis and cleavage takes place within the
target cell. In this manner, precise delivery of partner molecule D
is achieved at the site of action, reducing the dosage needed.
Also, partner molecule D is normally biologically inactive (or
significantly less active) in its conjugated state, thereby
reducing undesired toxicity against non-target tissue or cells. As
anticancer drugs are often highly toxic compounds with a low
therapeutic index, this is an important consideration.
[0220] As reflected by the subscript m, each molecule of antibody Z
can conjugate with more than one partner molecule D, depending on
the number of sites the former has available for conjugation and
the experimental conditions employed. Those skilled in the art will
appreciate that, while each molecule of antibody Z is conjugated to
an integer number of partner molecules D, a preparation of the
conjugate may analyze for a non-integer ratio of partner molecules
D to antibody Z, reflecting a statistical average.
Antibody Z
[0221] Any one of several different reactive groups on antibody Z
can be used as a conjugation site, including .epsilon.-amino groups
in lysine residues, pendant carbohydrate moieties, carboxylic acid
groups, disulfide groups, and thiol groups. Each type of reactive
group represents a trade-off, having some advantages but some
countervailing limitations. For reviews on antibody reactive groups
suitable for conjugation, see, e.g., Garnett, Adv. Drug Delivery
Rev. 53 (2001), 171-216 and Dubowchik and Walker, Pharmacology
& Therapeutics 83 (1999), 67-123, the disclosures of which are
incorporated herein by reference.
[0222] In one embodiment, antibody Z is conjugated via a lysine
.epsilon.-amino group. Most antibodies have multiple exposed lysine
.epsilon.-amino groups, which can be conjugated via amide, urea,
thiourea, or carbamate bonds using techniques known in the art,
including modification with a heterobifunctional agent (as further
described herein below). However, it is difficult to control which
and how many .epsilon.-amino groups react, leading to potential
batch-to-batch variability in conjugate preparations. Also,
conjugation may cause neutralization of a protonated
.epsilon.-amino group important for maintaining the antibody's
native conformation or may take place at a lysine near or at the
antigen binding site, neither being a desirable occurrence.
[0223] In another embodiment, antibody Z can be conjugated via a
carbohydrate side chain, as many antibodies are glycosylated. The
carbohydrate side chain can be oxidized with periodate to generate
aldehyde groups, which in turn can be reacted with amines to form
an imine group, such as in a semicarbazone, oxime, or hydrazone. If
desired, the imine group can be reduced with sodium
cyanoborohydride to produce a more stable bond. For additional
disclosures on conjugation via carbohydrate side chains, see, e.g.,
Rodwell et al., Proc. Nat'l Acad. Sci. USA 83, 2632-2636 (1986);
the disclosure of which is incorporated herein by reference. As
with lysine .epsilon.-amino groups, there are concerns regarding
location of the conjugation site(s) and stoichiometry.
[0224] In yet another embodiment, antibody Z can be conjugated via
a carboxylic acid group. In one embodiment, a terminal carboxylic
acid group is functionalized to generate a carbohydrazide, which is
then reacted with an aldehyde-bearing conjugation moiety. See Fisch
et al., Bioconjugate Chemistry 1992, 3, 147-153.
[0225] In yet another embodiment, antibody Z can be conjugated via
a disulfide group bridging the sulfur of a cysteine residue on
antibody Z and a sulfur on the other portion of the conjugate. Some
antibodies (such as the IgG isotype) lack free thiol groups but
have disulfide groups, for example in the hinge region. In such
case, free thiol groups can be generated by reduction of native
disulfide groups. The thiol groups so generated can then be used
for conjugation. See, for example, Packard et al., Biochemistry
1986, 25, 3548-3552; King et al., Cancer Res. 54, 6176-6185 (1994);
and Doronina et al., Nature Biotechnol. 21(7), 778-784 (2003); the
disclosures of which are incorporated herein by reference. Again,
there are concerns regarding the conjugation location and
stoichiometry and the possible disruption of antibody native
conformation.
[0226] In yet another preferred embodiment, antibody Z is
conjugated via the nucleophilic addition product of a thiol group
to an acceptor moiety. A preferred acceptor moiety is a maleimide
group, whose reaction with an antibody thiol group is illustrated
below:
##STR00001##
[0227] A number of methods have been developed to introduce free
thiol groups into antibodies without breaking native disulfide
bonds, which methods can be practiced with an antibody Z of this
invention. Depending on the method employed, it may be possible to
introduce a predictable number of free sulfhydryls at specific
locations. In one approach, mutated antibodies are prepared in
which a cysteine is substituted for another amino acid. See, for
example, Eigenbrot et al., US 2007/0092940 A1; Chilkoti et al.,
Bioconjugate Chem. 1994, 5, 504-507; Urnovitz et al., U.S. Pat. No.
4,698,420 (1987); Stimmel et al., J. Biol. Chem., 275 (39),
30445-30450 (2000); Bam et al., U.S. Pat. No. 7,311,902 B2 (2007);
Kuan et al., J. Biol. Chem., 269 (10), 7610-7618 (1994); Poon et
al., J. Biol. Chem., 270 (15), 8571-8577 (1995). In another
approach, an extra cysteine is added to the C-terminus. See, e.g.
Cumber et al., J. Immunol., 149, 120-126 (1992); King et al, Cancer
Res., 54, 6176-6185 (1994); Li et al., Bioconjugate Chem., 13,
985-995 (2002); Yang et al., Protein Engineering, 16, 761-770
(2003); and Olafson et al., Protein Engineering Design &
Selection, 17, 21-27 (2004). A preferred method for introducing
free cysteines is that taught by King, in U.S. Provisional
Application Ser. No. 60/957,271, filed Aug. 22, 2007, in which a
cysteine bearing amino acid sequence is added to the C-terminus of
the heavy chain of an antibody. This method introduces a known
number of cysteine residues (one per heavy chain) at a known
location remote from the antigen binding site. The disclosures of
the documents cited in this paragraph are all incorporated herein
by reference.
[0228] In yet another embodiment, lysine .epsilon.-amino groups can
be modified with heterobifunctional reagents such as
2-iminothiolane or N-succinimidyl-3-(2-pyridyldithio)propionate
(SPDP), converting an .epsilon.-amino group into a thiol or
disulfide group--creating a cysteine surrogate, as it were.
However, this method suffers from the same conjugation location and
stoichiometry limitations associated with .epsilon.-amino groups
proper.
Partner Molecule D
[0229] Partner molecule D can be a therapeutic agent or a marker.
If the former, it can be, for example, a cytotoxin, a non-cytotoxic
drug (e.g., an immunosuppressant), a radioactive agent, another
antibody, or an enzyme or active fragment thereof, such as abrin,
ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such
as tumor necrosis factor or interferon-.gamma.; or, biological
response modifiers such as, for example, lymphokines, interleukin-1
("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"),
granulocyte macrophage colony stimulating factor ("GM-CSF"),
granulocyte colony stimulating factor ("G-CSF"), or other growth
factors. If the latter, it can be any moiety that generates a
detectable signal, such as a radiolabel, a fluorescent label, or an
enzyme that catalyzes a detectable modification to a substrate.
Examples of radioactive isotopes that can be conjugated to
antibodies for use diagnostically or therapeutically include, but
are not limited to, iodine.sup.131, indium.sup.111, yttrium.sup.90
and lutetium.sup.177. Method for preparing radioimmunconjugates are
established in the art. Examples of radioimmunoconjugates are
commercially available, including Zevalin.RTM. (IDEC
Pharmaceuticals) and Bexxar.RTM. (Corixa Pharmaceuticals), and
similar methods can be used to prepare radioimmunoconjugates using
the antibodies of the invention.
[0230] A cytotoxin or cytotoxic agent includes any agent that is
detrimental to (e.g., kills) cells. Examples include taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, 1-dehydrotestosterone, glucocorticoids, procaine,
tetracaine, lidocaine, propranolol, and puromycin and analogs or
homologs thereof.
[0231] Where partner molecule D is a therapeutic agent, suitable
classes of therapeutic agents include antimetabolites, alkylating
agents, DNA minor groove binders, DNA intercalators, DNA
crosslinkers, histone deacetylase inhibitors, nuclear export
inhibitors, proteasome inhibitors, topoisomerase I or II
inhibitors, heat shock protein inhibitors, tyrosine kinase
inhibitors, antibiotics, and anti-mitotic agents. Examples of
suitable therapeutic agents include actinomycin D, anthracyclines,
anthramycin (AMC), auristatin, bleomycin, busulfan, calicheamicin,
camptothecin, carmustine, chlorambucil, cis-dichlorodiamine
platinum (II) (DDP), cisplatin, colchicin, cyclophosphamide,
cytarabine, cytochalasin B, dactinomycin, daunorubicin,
decarbazine, 1-dehydrotestosterone, dibromomannitol,
dihydroxyanthracindione, doxorubicin, emetine, epothilone, ethidium
bromide, etoposide, 5-fluorouracil, gemcitabine, glucocorticoids,
gramicidin D, imatinib, irinotecan, .beta.-lapachone, lidocaine,
lomustine, maytansine, mechlorethamine, melphalan,
6-mercaptopurine, methotrexate, mithramycin, mitomycin C,
mitoxantrone, paclitaxel, procaine, propranolol, puromycin, ricin,
streptozotocin, suberoylanilide hydroxamic acid (SAHA),
tallisomycin, tenoposide, tetracaine, thioepa, 6-thioguanine,
tubulysin, vinblastine, vincristine, and analogs, homologs or
derivatives thereof.
[0232] Preferably, partner molecule D is a cytotoxin selected from
the group consisting of auristatins (especially MMAE and MMAF),
enediyne antibiotics (especially calicheamicin and CalichDMH),
doxorubicin, maytansinoids (especially DM1 and DM4), Pseudomonas
exotoxin A (especially its truncated form), DNA minor
groove-binding alkylators (especially CC-1065 and duocarmycin
analogs), and analogs or derivatives thereof. An example of a
calicheamicin antibody conjugate is commercially available
(Mylotarg.RTM.; American Home Products). Those skilled in the art
will appreciate that the foregoing cytotoxins are mostly natural
products and that some modification thereof--i.e.,
derivatization--may be desirable or necessary to render them ready
for conjugation.
[0233] A preferred DNA minor groove-binding alkylators is an analog
or a derivative of CC-1065 and the structurally related
duocarmycins, suitable examples of which are disclosed in Ng et
al., U.S. Pat. No. 7,087,600 B2 (2006); Ng et al., U.S. Pat. No.
6,989,452 B2 (2006); Ng et al., U.S. Pat. No. 7,129,261 B2 (2006);
Ng et al., WO 02/096910 A1 (2002); Boyd et al., US 2006/0024317 A1
(2006); Chen et al., US 2006/0004081 A1 (2006); Gangwar et al., US
2006/0247295 A1 (2006); Boyd et al., WO 2007/038658 A2 (2007);
Gangwar et al., WO 2007/051081 A1 (2007); Gangwar et al., WO
2007/059404 A2 (2007); Sufi et al., WO 2008/083312 A2 (2008); and
Chen et al., PCT Application No. PCT/US2008/054362, filed Feb. 20,
2008; the disclosures of which are incorporated herein by
reference. Such preferred partner molecules D can be represented by
formula (b):
##STR00002##
wherein ring system A is a substituted or unsubstituted aryl, a
substituted or unsubstituted heteroaryl, a substituted or
unsubstituted heterocycloalkyl group, such as phenyl or pyrrole; E
and G are independently H, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, a heteroatom, or a single
bond, or E and G are join to form a ring system selected from
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl and substituted or unsubstituted heterocycloalkyl; X is
O, S or NR.sup.23, where R.sup.23 is a H, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl, or
acyl;
R.sup.3 is (.dbd.O) or OH;
[0234] R.sup.4, R.sup.4', R.sup.5 and R.sup.5' are independently H,
substituted or unsubstituted alkyl, substituted or unsubstituted
aryl, substituted or unsubstituted heteroaryl, substituted or
unsubstituted heterocycloalkyl, halogen, NO.sub.2,
NR.sup.15R.sup.16, NC(O)R.sup.15, OC(.dbd.O)NR.sup.15R.sup.16,
OC(.dbd.O)OR.sup.15, C(.dbd.O)R.sup.15, SR.sup.15, OR.sup.15,
CR.sup.15.dbd.NR.sup.16, or O(CH.sub.2).sub.nN(CH.sub.3).sub.2,
where n is an integer from 1 to 20, or any adjacent pair of
R.sup.4, R.sup.4', R.sup.5 and R.sup.5', together with the carbon
atoms to which they are attached, are joined to form a substituted
or unsubstituted cycloalkyl or heterocycloalkyl ring system having
from 4 to 6 members; R.sup.15 and R.sup.16 are independently H,
substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
heterocycloalkyl or substituted or unsubstituted peptidyl, where
R.sup.15 and R.sup.16 together with the nitrogen atom to which they
are attached are optionally joined to form a substituted or
unsubstituted heterocycloalkyl ring system having from 4 to 6
members, optionally containing two or more heteroatoms; R.sup.6 is
a single bond which is either present or absent; R.sup.7 is
CH.sub.2--X.sup.1 or --CH.sub.2--, with the proviso that when
R.sup.6 is present, R.sup.6 and R.sup.7 are joined to form a
cyclopropyl ring; and X.sup.1 is a leaving group such as Cl, Br, F,
mesylate, or tosylate.
[0235] A partner molecule of formula (b) can be conjugated via one
of R.sup.3, R.sup.4, R.sup.4', R.sup.5, and R.sup.5', preferably
via R.sup.3 (when R.sup.3 is OH) or R.sup.4, more preferably via
R.sup.4. Also, R.sup.3 can carry a prodrugging moiety, as discussed
herein below.
[0236] A preferred partner molecule of formula (b) is represented
by formula (c):
##STR00003##
wherein R.sup.3, R.sup.4, R.sup.4', R.sup.5, R.sup.5', R.sup.6, and
R.sup.7 are as defined hereinabove; Z is O, NH, or N (lower alkyl);
and R.sup.1, R.sup.1', R.sup.2, and R.sup.2' are independently H,
substituted or unsubstituted lower alkyl, cyano, alkoxy, halogen,
C(.dbd.O)R.sup.8, or CO.sub.2R.sup.8, wherein R.sup.8 is
NR.sup.9R.sup.10 or OR.sup.9, wherein R.sup.9 and R.sup.10 are
independently H, substituted or unsubstituted alkyl, or substituted
or unsubstituted heteroalkyl.
[0237] A more preferred embodiment is shown in formula (d):
##STR00004##
wherein X.sup.1 is as previously defined and X.sup.2 is
##STR00005##
[0238] An example of a specific partner molecule D is represented
by formula (e):
##STR00006##
[0239] Where partner molecule D is a cytotoxin, it can be
prodrugged, that is, have attached to it a prodrug moiety whose
removal is required to activate it. Preferably, the prodrug group
(1) is removed by a reaction mechanism different from that for
cleaving the cytotoxin from the conjugate, (2) is not removed or is
only slowly removed while the conjugate is in circulation in the
blood plasma, but (3) is efficiently removed at the target tissue
or cell. Consequently, if the conjugate is adventitiously cleaved
before reaching the target tissue or cell, the cytotoxin is
released in its still-inactive prodrug form, eliminating or
reducing cytotoxicity towards non-target tissues or cells. That is,
the requirement for a second cleavage to activate the cytotoxin
provides a safety factor. In the instance of a cytotoxin according
to formulae (b), (c), (d) or (e), a preferred site for attachment
of a prodrug group is at the position labeled with a "4". To
increase the safety factor where cleavage of group C and removal of
the prodrug moiety are both mediated by enzymes, it is preferable
that different enzymes be involved.
[0240] Non-limiting examples of prodrug groups include esters,
carbamates, phosphates, and glycosides. To illustrate, the
4-position hydroxyl in the cytotoxins of formulae ((b)-(e) can be
prodrugged with the following prodrug moieties:
##STR00007##
[0241] Partner molecule D can also be a marker. The marker can be
any label that generates a detectable signal, such as a radiolabel,
a fluorescent label, or an enzyme that catalyzes a detectable
modification to a substrate. Markers (also called reporter groups
or detectable labels) are well known in the area of immunoassays,
biomedical research, and medical diagnosis and can be detected by
spectroscopic, photochemical, biochemical, immunochemical,
electrical, optical, or chemical means. The marker is preferably a
radioactive isotope, a fluorescent or chemiluminescent agent or
precursor thereof, a chromophore, an enzyme, or combinations
thereof. Examples of suitable enzymes are horseradish peroxidase,
alkaline phosphatase, .beta.-galactosidase, and glucose oxidase.
Fluorescent agents include fluorescein and its derivatives,
rhodamine and its derivatives, dansyl, umbelliferone, etc.
Chemiluminescent compounds include luciferin and
2,3-dihydrophthalazinediones such as luminol.
Linker --(X.sup.Z).sub.aC(X.sup.D).sub.b--
[0242] As noted above, the linker portion of a conjugate of this
invention comprises up to three elements: a cleavable group C and
optional spacers X.sup.Z and X.sup.D.
[0243] Cleavable group C is selected such that it is relatively
stable while the conjugate is in general circulation in the blood
plasma, but is readily cleaved once the conjugate reaches its site
of intended action. Preferably, the conjugate is internalized by
endocytosis by a target cell upon binding of antibody Z to an
antigen displayed on the surface of the target cell. Subsequently,
cleavage of group C occurs in a vesicular body of the target cell
(an early endosome, a late endosome, or, especially, a
lysosome).
[0244] In one embodiment, group C is a pH sensitive group. The pH
in blood plasma is slightly above neutral, while the pH inside a
lysosome is acidic, circa 5. Thus, a group C whose cleavage is acid
catalyzed will cleave at a rate several orders of magnitude faster
inside a lysosome than in the blood plasma rate. Examples of
suitable acid-sensitive groups include cis-aconityl amides and
hydrazones, as described in Shen et al., U.S. Pat. No. 4,631,190
(1986); Shen et al., U.S. Pat. No. 5,144,011 (1992); Shen et al.,
Biochem. Biophys. Res. Commun. 102, 1048-1054 (1981) and Yang et
al., Proc. Natl. Acad. Sci (USA), 85, 1189-1193 (1988); the
disclosures of which are incorporated herein by reference.
[0245] In another embodiment, group C is a disulfide. Disulfides
can be cleaved by a thiol-disulfide exchange mechanism, at a rate
dependent on the ambient thiol concentration. As the intracellular
concentration of glutathione and other thiols is higher than their
serum concentrations, the cleavage rate of a disulfide will be
higher intracellularly. Further, the rate of thiol-disulfide
exchange can be modulated by adjustment of the steric and
electronic characteristics of the disulfide (e.g., an alkyl-aryl
disulfide versus an alkyl-alkyl disulfide; substitution on the aryl
ring, etc.), enabling the design of disulfide linkages that have
enhanced serum stability or a particular cleavage rate. For
additional disclosures relating to disulfide cleavable groups in
conjugates, see, e.g., Thorpe et al., Cancer Res. 48, 6396-6403
(1988); Santi et al., US 2005/0287155 A1 (2005); Ng et al., U.S.
Pat. No. 6,989,452 B2 (2006); Ng et al., WO 2002/096910 A1 (2002);
Boyd et al., US 2006/0024317 A1 (2006); and Sufi et al., WO
2008/083312 A2 (2008); the disclosures of which are incorporated
herein by reference.
[0246] A preferred group C comprises a peptide bond that is cleaved
preferentially by a protease at the intended site of action, as
opposed to by a protease in the serum. Typically, group C comprises
from 1 to 20 amino acids, preferably from 1 to 6 amino acids, more
preferably from 1 to 3 amino acids. The amino acid(s) can be
natural and/or unnatural .alpha.-amino acids. Natural amino acids
are those encoded by the genetic code, as well as amino acids
derived therefrom, e.g., hydroxyproline, .gamma.-carboxyglutamate,
citrulline, and O-phosphoserine. The term amino acid also includes
amino acid analogs and mimetics. Analogs are compounds having the
same general H.sub.2N(R)CHCO.sub.2H structure of a natural amino
acid, except that the R group is not one found among the natural
amino acids. Examples of analogs include homoserine, norleucine,
methionine-sulfoxide, and methionine methyl sulfonium. An amino
acid mimetic is a compound that has a structure different from the
general chemical structure of an .alpha.-amino acid but functions
in a manner similar to one. The term "unnatural amino acid" is
intended to represent the "D" stereochemical form, the natural
amino acids being of the "L" form.
[0247] Preferably, group C contains an amino acid sequence that is
a cleavage recognition sequence for a protease. Many cleavage
recognition sequences are known in the art. See, e.g., Matayoshi et
al. Science 247: 954 (1990); Dunn et al. Meth. Enzymol. 241: 254
(1994); Seidah et al. Meth. Enzymol. 244: 175 (1994); Thornberry,
Meth. Enzymol. 244: 615 (1994); Weber et al. Meth. Enzymol. 244:
595 (1994); Smith et al. Meth. Enzymol. 244: 412 (1994); and
Bouvier et al. Meth. Enzymol. 248: 614 (1995); the disclosures of
which are incorporated herein by reference.
[0248] For conjugates that are not intended to be internalized by a
cell, a group C can be chosen such that it is cleaved by a protease
present in the extracellular matrix in the vicinity of the target
tissue, e.g., a protease released by nearby dying cells or a
tumor-associated protease. Exemplary extracellular tumor-associated
proteases are thimet oligopeptidase (TOP) and CD10.
[0249] For conjugates that are designed to be internalized by a
cell, group C preferably comprises an amino acid sequence selected
for cleavage by an endosomal or lysosomal protease, especially the
latter. Non-limiting examples of such proteases include cathepsins
B, C, D, H, L and S, especially cathepsin B. Cathepsin B
preferentially cleaves peptides at a sequence -AA.sup.2-AA.sup.1-
where AA.sup.1 is a basic or strongly hydrogen bonding amino acid
(such as lysine, arginine, or citrulline) and AA.sup.2 is a
hydrophobic amino acid (such as phenylalanine, valine, alanine,
leucine, or isoleucine), for example Val-Cit (where Cit denotes
citrulline) or Val-Lys. (Herein, amino acid sequences are written
in the N-to-C direction, as in
H.sub.2N-AA.sup.2-AA.sup.1-CO.sub.2H, unless the context clearly
indicates otherwise.) For additional information regarding
cathepsin-cleavable groups, see Dubowchik et al., Biorg. Med. Chem.
Lett. 8, 3341-3346 (1998); Dubowchik et al., Bioorg. Med. Chem.
Lett., 8 3347-3352 (1998); and Dubowchik et al., Bioconjugate Chem.
13, 855-869 (2002); the disclosures of which are incorporated by
reference.
[0250] In one embodiment, Group C is a peptide comprising the
two-amino acid sequence -AA.sup.2-AA.sup.1- wherein AA.sup.1 is
lysine, arginine, or citrulline and AA.sup.2 is phenylalanine,
valine, alanine, leucine or isoleucine. In another embodiment, C
consists of a sequence of one to five amino acids, selected from
the group consisting of Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys,
Pro-Val-Gly-Val-Val (SEQ ID NO:45), Ala-Asn-Val, Val-Leu-Lys,
Cit-Cit, Val-Lys, Lys, Cit, Ser, and Glu.
[0251] The preparation and design of cleavable groups C comprising
a single amino acid is further discussed in Chen et al., PCT
Application No. PCT/US2008/054362, filed Feb. 20, 2008, the
disclosure of which is incorporated herein by reference.
[0252] Group C can also be a photocleavable one, for example a
nitrobenzyl ether that is cleaved upon exposure to light.
[0253] Group C can be bonded directly to antibody Z or partner
molecule D; that is, spacers X.sup.Z and X.sup.D, as the case may
be, can be absent. For example, if group C is a disulfide, one of
the two sulfurs can be a cysteine residue or its surrogate on
antibody Z. Or, group C can be a hydrazone bonded to an aldehyde on
a carbohydrate side chain. Or, group C can be a peptide bond formed
with a lysine .epsilon.-amino group of antibody Z.
[0254] When present, spacer X.sup.Z provides spatial separation
between group C and antibody Z, lest the former sterically
interfere with antigen binding by latter or the latter sterically
interfere with cleavage of the former. Further, spacer X.sup.Z can
be used to confer increased solubility or decreased aggregation
properties to conjugates. A spacer X.sup.Z can comprise one or more
modular segments, which can be assembled in any number of
combinations. Examples of suitable segments for a spacer X.sup.Z
are:
##STR00008##
where the subscript r is 1 to 24, preferably 2 to 4. These segments
can be combined to make spacers X.sup.Z such as:
##STR00009##
[0255] Spacer X.sup.D, if present, provides spatial separation
between group C and partner molecule D, lest the latter interfere
sterically or electronically with cleavage of the former. Spacer
X.sup.D also can serve to introduce additional molecular mass and
chemical functionality into a conjugate. Generally, the additional
mass and functionality will affect the serum half-life and other
properties of the conjugate. Thus, through judicious selection of
spacer groups, the serum half-live of a conjugate can be modulated.
Spacer X.sup.D also can be assembled from modular segments, as
described above in the context of spacer X.sup.Z.
[0256] Either spacer X.sup.Z or X.sup.D, or both, can comprise a
self-immolating moiety. Briefly, a self-immolating moiety is a
moiety that (1) is bonded to group C and either antibody Z or
partner molecule D and (2) has a structure such that cleavage of
group C initiates a reaction sequence that results in the
self-immolating moiety to disbonding itself from antibody Z or
partner molecule D, as the case may be. In other words, a reaction
at a site distal from antibody Z or partner molecule D (cleavage of
group C) causes the X.sup.Z-Z or the X.sup.D-D bond to rupture as
well. The presence of a self-immolating moiety is desirable in the
case of spacer X.sup.D because, if, after cleavage of the
conjugate, spacer X.sup.D or a portion thereof remains attached to
partner molecule D, the biological activity of the latter may be
impaired. The use of a self-immolating moiety is especially
preferred where cleavable group C is a polypeptide.
[0257] Exemplary self-immolating moieties (i)-(v) bonded to a
hydroxyl or amino group on a partner molecule D are shown
below:
##STR00010##
[0258] In each instance, the self-immolating moiety is the
structure between dotted lines a and b, with adjacent structural
features shown to provide context. Self-immolating moieties (i) and
(v) are bonded to a partner molecule D-NH.sub.2 (i.e., partner
molecule D is conjugated via an amino group), while self-immolating
moieties (ii), (iii), and (iv) are bonded to a partner molecule
D-OH (i.e., partner molecule D is conjugated via a hydroxyl group).
Cleavage of the amide bond at dotted line b (i.e., group C is a
peptide) releases the amide nitrogen as an amine nitrogen,
initiating a reaction sequence that results in the cleavage of the
bond at dotted line a and the consequent release of partner
molecule D-OH or D-NH.sub.2, as the case may be. For additional
disclosures regarding self-immolating moieties, see, Carl et al.,
J. Med. Chem., 24 (3), 479-480 (1981); Carl et al., WO 81/01145
(1981); Dubowchik et al., Pharmacology & Therapeutics, 83,
67-123 (1999); Firestone et al., U.S. Pat. No. 6,214,345 B1 (2001);
Toki et al., J. Org. Chem. 67, 1866-1872 (2002); Doronina et al.,
Nature Biotechnology 21 (7), 778-784 (2003) (erratum, p. 941); Boyd
et al., WO 2005/112919 (2005); Boyd et al., WO 2007/038658 (2007);
Sufi et al., WO 2008/083312 A2 (2008); Feng, U.S. Pat. No.
7,375,078 B2 (2008); and Senter et al., US 2003/0096743 A1 (2003);
the disclosures of which are incorporated by reference.
Examples of Conjugates
[0259] Examples of conjugates made with an antibody Z(SH).sub.m of
this invention (where m is 1, 2, 3, 4, or 5) are shown below.
Conjugates A-1 to A-6 and A-8 to A-15 are conjugates in which
cleavable group C comprises a peptide bond. Conjugates A-7 and A-16
are conjugates in which cleavable group C is a hydrazone.
Conjugates A-17 and A-18 are conjugates in which cleavable group C
is a disulfide. In conjugates A-1 to A-2, A-5 to A-9, A-11 to A-14,
and A-16, partner molecule D is a cytotoxin having a prodrug moiety
attached thereto. Conjugates A-10, A-11, A-14, and A-15 are
conjugates having a self-immolating moiety (two in the case of
conjugate A-10). Conjugates A-1 through A-8 and A-10 through A-18
illustrate the use of spacers having modular segments.
##STR00011## ##STR00012## ##STR00013## ##STR00014##
##STR00015##
[0260] Where present in the preceding formulae, Hal is Cl or Br and
R.sup.30 is the carboxyesterase-cleavable carbamate prodrug group
shown below:
##STR00016##
Preparation of Conjugates
[0261] Conjugates of this invention preferably are prepared by
first joining partner molecule D and linker
(X.sup.Z).sub.aC(X.sup.D).sub.b to form a moiety
D-(X.sup.Z).sub.aC(X.sup.D).sub.b--R.sup.31, where R.sup.31 is a
functional group suitable for reacting with a functional group on
antibody Z, to form the conjugate. Examples of suitable groups
R.sup.21 include:
##STR00017##
where R.sup.32 is Cl, Br, F, mesylate, or tosylate and R.sup.33 is
Cl, Br, I, F, OH, --O--N-succinimidyl, --O-(4-nitrophenyl),
--O-pentafluorophenyl, or --O-tetrafluorophenyl. The preparation of
suitable moieties D-(X.sup.Z).sub.aC(X.sup.D).sub.b--R.sup.31 is
disclosed in Ng et al., U.S. Pat. No. 7,087,600 B2 (2006); Ng et
al., U.S. Pat. No. 6,989,452 B2 (2006); Ng et al., U.S. Pat. No.
7,129,261 B2 (2006); Ng et al., WO 02/096910 A1 (2002); Boyd et
al., US 2006/0024317 A1 (2006); Chen et al., US 2006/0004081 A1
(2006); Gangwar et al., US 2006/0247295 A1 (2006); Boyd et al., WO
2007/038658 A2 (2007); Gangwar et al., WO 2007/051081 A1 (2007);
Gangwar et al., WO 2007/059404 A2 (2007); Sufi et al., WO
2008/083312 A2 (2008); and Chen et al., PCT Application No.
PCT/US2008/054362, filed Feb. 20, 2008; the disclosures of which
are incorporated herein by reference.
[0262] In a preferred embodiment (formula M), R.sup.31 is a
maleimide group and the functional group on antibody Z is a thiol
group as illustrated following, using conjugate A-2 where Hal is Cl
and antibody Z(SH).sub.m:
##STR00018##
[0263] The following is an illustrative procedure, based on
introduction of free thiol groups into an antibody by reaction of
its lysine .epsilon.-amino groups with 2-iminothiolane, followed by
reaction with a drug-linker moiety
D-(X.sup.Z).sub.aC(X.sup.D).sub.b--R.sup.31, where R.sup.31 is
maleimide. Initially the antibody is buffer exchanged into 0.1 M
phosphate buffer (pH 8.0) containing 50 mM NaCl and 2 mM DTPA and
concentrated to 5-10 mg/mL. Thiolation is achieved through addition
of 2-iminothiolane to the antibody. The amount of 2-iminothiolane
to be added can be determined by a preliminary experiment and
varies from antibody to antibody. In the preliminary experiment, a
titration of increasing amounts of 2-iminothiolane is added to the
antibody, and following incubation with the antibody for 1 h at
room temperature, the antibody is desalted into 50 mM pH 6.0 HEPES
buffer using a Sephadex G-25 column and the number of thiol groups
introduced determined rapidly by reaction with dithiodipyridine
(DTDP). Reaction of thiol groups with DTDP results in liberation of
thiopyridine, which can be monitored spectroscopically at 324 nm.
Samples at a protein concentration of 0.5-1.0 mg/mL are typically
used. The absorbance at 280 nm can be used to accurately determine
the concentration of protein in the samples, and then an aliquot of
each sample (0.9 mL) is incubated with 0.1 mL DTDP (5 mM stock
solution in ethanol) for 10 min at room temperature. Blank samples
of buffer alone plus DTDP are also incubated alongside. After 10
min, absorbance at 324 nm is measured and the number of thiol
groups is quantitated using an extinction coefficient for
thiopyridine of 19,800 M.sup.-1.
[0264] Typically a thiolation level of three thiol groups per
antibody is desired in this procedure. For example, with some
antibodies this can be achieved by adding a 15-fold molar excess of
2-iminothiolane followed by incubation at room temperature for 1 h.
The antibody is then incubated with 2-iminothiolane at the desired
molar ratio and then desalted into conjugation buffer (50 mM pH 6.0
HEPES buffer containing 5 mM glycine and 2 mM DTPA). The thiolated
material is maintained on ice while the number of thiols introduced
is quantitated as described above.
[0265] After verification of the number of thiols introduced, the
drug-linker moiety D-(X.sup.Z).sub.aC(X.sup.D).sub.b--R.sup.31 is
added at a 3-fold molar excess per thiol. The conjugation reaction
is allowed to proceed in conjugation buffer also containing a final
concentration of 5% dimethylsulfoxide (DMSO), or similar
alternative solvent. Commonly, the drug-linker stock solution is
dissolved in 100% DMSO. The stock solution is added directly to the
thiolated antibody, which has enough DMSO added to bring the final
concentration to 10%, or pre-diluted in conjugation buffer
containing a final concentration of 10% DMSO, followed by addition
to an equal volume of thiolated antibody.
[0266] The conjugation reaction mixture is incubated at room
temperature for 2 h with stirring. Following incubation, the
conjugation reaction mixture is centrifuged and filtered through a
0.2 .mu.m filter. Purification of the conjugate can be achieved
through chromatography using a number of methods. In one method,
the conjugate is purified using size-exclusion chromatography on a
Sephacryl 5200 column pre-equilibrated with 50 mM pH 7.2 HEPES
buffer containing 5 mM glycine and 50 mM NaCl. Chromatography is
carried out at a linear flow rate of 28 cm/h. Fractions containing
conjugate are collected, pooled and concentrated. In an alternative
method, purification can be achieved through ion-exchange
chromatography. Conditions vary from antibody to antibody and
should to be optimized in each case. For example, antibody-drug
conjugate reaction mix is applied to an SP-Sepharose column
pre-equilibrated in 50 mM pH 5.5 HEPES containing 5 mM glycine. The
antibody conjugate is eluted using a gradient of 0-1 M NaCl in
equilibration buffer at pH 5.5. Relevant fractions containing the
conjugate are pooled and dialyzed against formulation buffer (50 mM
pH 7.2 HEPES buffer containing 5 mM glycine and 100 mM NaCl).
[0267] Those skilled in the art will understand that the
above-described conditions and methodology are exemplary and
non-limiting and that other approaches for conjugating antibodies
are known in the art and usable in the present invention.
ADEPT
[0268] In another embodiment, an antibody according to this
invention is conjugated to an enzyme for use in antibody-directed
enzyme prodrug therapy (ADEPT). In ADEPT, an enzyme is guided to a
tumor site by the antibody to which it is conjugated. There, the
enzyme acts on a subsequently administered prodrug to release
locally the corresponding active drug. See, e.g., Melton et al., J.
Natl Cancer Inst. 88(3/4), 153-165 (1996). Exemplary enzymes that
can be conjugated for use in ADEPT include carboxypeptidase A and
G2, alkaline phosphatase, .beta.-glucuronidase, .beta.-lactamase,
.beta.-glucosidase, penicillin amidase, aminopeptidase, cytosine
deaminase, and nitroreductase.
[0269] Because ADEPT does not require the release of the enzyme
from the antibody, the presence of a cleavable group between the
antibody and the enzyme is not mandatory. Thus, an ADEPT conjugate
can be represented by formula (f)
Z-X-D (f)
where Z is an antibody of this invention; D is an enzyme, and X is
a linker connecting Z and D.
Immunoconjugates
[0270] Immunoconjugates that include one or more cytotoxins are
referred to as "immunotoxins".
[0271] Cytotoxins can be conjugated to antibodies of the invention
using linker technology available in the art. Examples of linker
types that have been used to conjugate a cytotoxin to an antibody
include, but are not limited to, hydrazones, thioethers, esters,
disulfides and peptide-containing linkers. A linker can be chosen
that is, for example, susceptible to cleavage by low pH within the
lysosomal compartment or susceptible to cleavage by proteases, such
as proteases preferentially expressed in tumor tissue such as
cathepsins (e.g., cathepsins B, C, D).
Bispecific Molecules
[0272] In another aspect, the present invention features bispecific
molecules comprising an anti-CADM1 antibody, or a fragment thereof,
of the invention. An antibody of the invention, or antigen-binding
portions thereof, can be derivatized or linked to another
functional molecule, e.g., another peptide or protein (e.g.,
another antibody or ligand for a receptor) to generate a bispecific
molecule that binds to at least two different binding sites or
target molecules. The antibody of the invention may in fact be
derivatized or linked to more than one other functional molecule to
generate multispecific molecules that bind to more than two
different binding sites and/or target molecules; such multispecific
molecules are also intended to be encompassed by the term
"bispecific molecule" as used herein. To create a bispecific
molecule of the invention, an antibody of the invention can be
functionally linked (e.g., by chemical coupling, genetic fusion,
noncovalent association or otherwise) to one or more other binding
molecules, such as another antibody, antibody fragment, peptide or
binding mimetic, such that a bispecific molecule results.
[0273] Accordingly, the present invention includes bispecific
molecules comprising at least one first binding specificity for
CADM1 and a second binding specificity for a second target epitope.
In a particular embodiment of the invention, the second target
epitope is an Fc receptor, e.g., human Fc.gamma.RI (CD64) or a
human Fc.alpha. receptor (CD89). Therefore, the invention includes
bispecific molecules capable of binding both to Fc.gamma.R or
Fc.alpha.R expressing effector cells (e.g., monocytes, macrophages
or polymorphonuclear cells (PMNs)), and to target cells expressing
CADM1. These bispecific molecules target CADM1 expressing cells to
effector cell and trigger Fc receptor-mediated effector cell
activities, such as phagocytosis of CADM1 expressing cells,
antibody dependent cell-mediated cytotoxicity (ADCC), cytokine
release, or generation of superoxide anion.
[0274] In an embodiment of the invention in which the bispecific
molecule is multispecific, the molecule can further include a third
binding specificity, in addition to an anti-Fc binding specificity
and an anti-CADM1 binding specificity. In one embodiment, the third
binding specificity is an anti-enhancement factor (EF) portion,
e.g., a molecule which binds to a surface protein involved in
cytotoxic activity and thereby increases the immune response
against the target cell. The "anti-enhancement factor portion" can
be an antibody, functional antibody fragment or a ligand that binds
to a given molecule, e.g., an antigen or a receptor, and thereby
results in an enhancement of the effect of the binding determinants
for the Fc receptor or target cell antigen. The "anti-enhancement
factor portion" can bind an Fc receptor or a target cell antigen.
Alternatively, the anti-enhancement factor portion can bind to an
entity that is different from the entity to which the first and
second binding specificities bind. For example, the
anti-enhancement factor portion can bind a cytotoxic T-cell (e.g.
via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immune cell
that results in an increased immune response against the target
cell).
[0275] In one embodiment, the bispecific molecules of the invention
comprise as a binding specificity at least one antibody, or an
antibody fragment thereof, including, e.g., an Fab, Fab',
F(ab').sub.2, Fv, Fd, dAb or a single chain Fv. The antibody may
also be a light chain or heavy chain dimer, or any minimal fragment
thereof such as a Fv or a single chain construct as described in
U.S. Pat. No. 4,946,778 to Ladner et al., the contents of which is
expressly incorporated by reference.
[0276] In one embodiment, the binding specificity for an Fc.gamma.
receptor is provided by a monoclonal antibody, the binding of which
is not blocked by human immunoglobulin G (IgG). As used herein, the
term "IgG receptor" refers to any of the eight .gamma.-chain genes
located on chromosome 1. These genes encode a total of twelve
transmembrane or soluble receptor isoforms which are grouped into
three Fc.gamma. receptor classes: Fc.gamma.RI (CD64), Fc.gamma.RII
(CD32), and Fc.gamma.RIII (CD16). In one preferred embodiment, the
Fc.gamma. receptor a human high affinity Fc.gamma.RI. The human
Fc.gamma.RI is a 72 kDa molecule, which shows high affinity for
monomeric IgG (10.sup.8-10.sup.9 M.sup.-1).
[0277] The production and characterization of certain preferred
anti-Fc.gamma. monoclonal antibodies are described in PCT
Publication WO 88/00052 and in U.S. Pat. No. 4,954,617 to Fanger et
al., the teachings of which are fully incorporated by reference
herein. These antibodies bind to an epitope of Fc.gamma.RI,
Fc.gamma.RII or Fc.gamma.RIII at a site which is distinct from the
Fc.gamma. binding site of the receptor and, thus, their binding is
not blocked substantially by physiological levels of IgG. Specific
anti-Fc.gamma.RI antibodies useful in this invention are mAb 22,
mAb 32, mAb 44, mAb 62 and mAb 197. The hybridoma producing mAb 32
is available from the American Type Culture Collection, ATCC
Accession No. HB9469. In other embodiments, the anti-Fc.gamma.
receptor antibody is a humanized form of monoclonal antibody 22
(H22). The production and characterization of the H22 antibody is
described in Graziano, R. F. et al. (1995) J. Immunol 155 (10):
4996-5002 and PCT Publication WO 94/10332 to Tempest et al. The H22
antibody producing cell line was deposited at the American Type
Culture Collection under the designation HA022CL1 and has the
accession no. CRL 11177.
[0278] In still other preferred embodiments, the binding
specificity for an Fc receptor is provided by an antibody that
binds to a human IgA receptor, e.g., an Fc-alpha receptor
(Fc.alpha.RI (CD89)), the binding of which is preferably not
blocked by human immunoglobulin A (IgA). The term "IgA receptor" is
intended to include the gene product of one .alpha.-gene
(Fc.alpha.RI) located on chromosome 19. This gene is known to
encode several alternatively spliced transmembrane isoforms of 55
to 110 kDa. Fc.alpha.RI (CD89) is constitutively expressed on
monocytes/macrophages, eosinophilic and neutrophilic granulocytes,
but not on non-effector cell populations. Fc.alpha.RI has medium
affinity (.apprxeq.5.times.10.sup.7 M.sup.-1) for both IgA1 and
IgA2, which is increased upon exposure to cytokines such as G-CSF
or GM-CSF (Morton, H. C. et al. (1996) Critical Reviews in
Immunology 16:423-440). Four Fc.alpha.RI-specific monoclonal
antibodies, identified as A3, A59, A62 and A77, which bind
Fc.alpha.RI outside the IgA ligand binding domain, have been
described (Monteiro, R. C. et al. (1992) J. Immunol. 148:1764).
[0279] Fc.alpha.RI and Fc.gamma.RI are preferred trigger receptors
for use in the bispecific molecules of the invention because they
are (1) expressed primarily on immune effector cells, e.g.,
monocytes, PMNs, macrophages and dendritic cells; (2) expressed at
high levels (e.g., 5,000-100,000 per cell); (3) mediators of
cytotoxic activities (e.g., ADCC, phagocytosis); and (4) mediate
enhanced antigen presentation of antigens, including self-antigens,
targeted to them.
[0280] While human monoclonal antibodies are preferred, other
antibodies which can be employed in the bispecific molecules of the
invention are murine, chimeric and humanized monoclonal
antibodies.
[0281] The bispecific molecules of the present invention can be
prepared by conjugating the constituent binding specificities,
e.g., the anti-FcR and anti-CADM1 binding specificities, using
methods known in the art. For example, each binding specificity of
the bispecific molecule can be generated separately and then
conjugated to one another. When the binding specificities are
proteins or peptides, a variety of coupling or cross-linking agents
can be used for covalent conjugation. Examples of cross-linking
agents include protein A, carbodiimide,
N-succinimidyl-5-acetyl-thioacetate (SATA),
5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide
(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and
sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohaxane-1-carboxylate
(sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med.
160:1686; Liu, M A et al. (1985) Proc. Natl. Acad. Sci. USA
82:8648). Other methods include those described in Paulus (1985)
Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science
229:81-83, and Glennie et al. (1987) J. Immunol. 139: 2367-2375).
Preferred conjugating agents are SATA and sulfo-SMCC, both
available from Pierce Chemical Co. (Rockford, Ill.).
[0282] When the binding specificities are antibodies, they can be
conjugated via sulfhydryl bonding of the C-terminus hinge regions
of the two heavy chains. In a particularly preferred embodiment,
the hinge region is modified to contain an odd number of sulfhydryl
residues, preferably one, prior to conjugation.
[0283] Alternatively, both binding specificities can be encoded in
the same vector and expressed and assembled in the same host cell.
This method is particularly useful where the bispecific molecule is
a mAb.times.mAb, mAb.times.Fab, Fab.times.F(ab').sub.2 or ligand x
Fab fusion protein. A bispecific molecule of the invention can be a
single chain molecule comprising one single chain antibody and a
binding determinant, or a single chain bispecific molecule
comprising two binding determinants. Bispecific molecules may
comprise at least two single chain molecules. Methods for preparing
bispecific molecules are described for example in U.S. Pat. Nos.
5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786;
5,013,653; 5,258,498; and 5,482,858, all of which are expressly
incorporated herein by reference.
[0284] Binding of the bispecific molecules to their specific
targets can be confirmed by, for example, enzyme-linked
immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis,
bioassay (e.g., growth inhibition), or Western Blot assay. Each of
these assays generally detects the presence of protein-antibody
complexes of particular interest by employing a labeled reagent
(e.g., an antibody) specific for the complex of interest. For
example, the FcR-antibody complexes can be detected using e.g., an
enzyme-linked antibody or antibody fragment which recognizes and
specifically binds to the antibody-FcR complexes. Alternatively,
the complexes can be detected using any of a variety of other
immunoassays. For example, the antibody can be radioactively
labeled and used in a radioimmunoassay (RIA) (see, for example,
Weintraub, B., Principles of Radioimmunoassays, Seventh Training
Course on Radioligand Assay Techniques, The Endocrine Society,
March, 1986, which is incorporated by reference herein). The
radioactive isotope can be detected by such means as the use of a
.gamma. counter or a scintillation counter or by
autoradiography.
Antibody Fragments and Antibody Mimetics
[0285] The instant invention is not limited to traditional
antibodies and may be practiced through the use of antibody
fragments and antibody mimetics. As detailed below, a wide variety
of antibody fragment and antibody mimetic technologies have now
been developed and are widely known in the art. While a number of
these technologies, such as domain antibodies, Nanobodies, and
UniBodies make use of fragments of, or other modifications to,
traditional antibody structures, there are also alternative
technologies, such as Affibodies, DARPins, Anticalins, Avimers, and
Versabodies that employ binding structures that, while they mimic
traditional antibody binding, are generated from and function via
distinct mechanisms.
[0286] Domain Antibodies (dAbs) are the smallest functional binding
units of antibodies, corresponding to the variable regions of
either the heavy (VH) or light (VL) chains of human antibodies.
Domain Antibodies have a molecular weight of approximately 13 kDa.
Domantis has developed a series of large and highly functional
libraries of fully human VH and VL dAbs (more than ten billion
different sequences in each library), and uses these libraries to
select dAbs that are specific to therapeutic targets. In contrast
to many conventional antibodies, Domain Antibodies are well
expressed in bacterial, yeast, and mammalian cell systems. Further
details of domain antibodies and methods of production thereof may
be obtained by reference to U.S. Pat. Nos. 6,291,158; 6,582,915;
6,593,081; 6,172,197; 6,696,245; US Serial No. 2004/0110941;
European patent application No. 1433846 and European Patents
0368684 & 0616640; WO05/035572, WO04/101790, WO04/081026,
WO04/058821, WO04/003019 and WO03/002609, each of which is herein
incorporated by reference in its entirety.
[0287] Nanobodies are antibody-derived therapeutic proteins that
contain the unique structural and functional properties of
naturally-occurring heavy-chain antibodies. These heavy-chain
antibodies contain a single variable domain (VHH) and two constant
domains (CH2 and CH3). Importantly, the cloned and isolated VHH
domain is a perfectly stable polypeptide harboring the full
antigen-binding capacity of the original heavy-chain antibody.
Nanobodies have a high homology with the VH domains of human
antibodies and can be further humanized without any loss of
activity. Importantly, Nanobodies have a low immunogenic potential,
which has been confirmed in primate studies with Nanobody lead
compounds.
[0288] Nanobodies combine the advantages of conventional antibodies
with important features of small molecule drugs. Like conventional
antibodies, Nanobodies show high target specificity, high affinity
for their target and low inherent toxicity. However, like small
molecule drugs they can inhibit enzymes and readily access receptor
clefts. Furthermore, Nanobodies are extremely stable, can be
administered by means other than injection (see e.g. WO 04/041867,
which is herein incorporated by reference in its entirety) and are
easy to manufacture. Other advantages of Nanobodies include
recognizing uncommon or hidden epitopes as a result of their small
size, binding into cavities or active sites of protein targets with
high affinity and selectivity due to their unique 3-dimensional,
drug format flexibility, tailoring of half-life and ease and speed
of drug discovery.
[0289] Nanobodies are encoded by single genes and are efficiently
produced in almost all prokaryotic and eukaryotic hosts e.g. E.
coli (see e.g. U.S. Pat. No. 6,765,087, which is herein
incorporated by reference in its entirety), molds (for example
Aspergillus or Trichoderma) and yeast (for example Saccharomyces,
Kluyveromyces, Hansenula or Pichia) (see e.g. U.S. Pat. No.
6,838,254, which is herein incorporated by reference in its
entirety). The production process is scalable and multi-kilogram
quantities of Nanobodies have been produced. Because Nanobodies
exhibit a superior stability compared with conventional antibodies,
they can be formulated as a long shelf-life, ready-to-use
solution.
[0290] The Nanoclone method (see e.g. WO 06/079372, which is herein
incorporated by reference in its entirety) is a proprietary method
for generating Nanobodies against a desired target, based on
automated high-throughout selection of B-cells and could be used in
the context of the instant invention.
[0291] UniBodies are another antibody fragment technology; however
this one is based upon the removal of the hinge region of IgG4
antibodies. The deletion of the hinge region results in a molecule
that is essentially half the size of traditional IgG4 antibodies
and has a univalent binding region rather than the bivalent binding
region of IgG4 antibodies. It is also well known that IgG4
antibodies are inert and thus do not interact with the immune
system, which may be advantageous for the treatment of diseases
where an immune response is not desired, and this advantage is
passed onto UniBodies. For example, UniBodies may function to
inhibit or silence, but not kill, the cells to which they are
bound. Additionally, UniBody binding to cancer cells do not
stimulate them to proliferate. Furthermore, because UniBodies are
about half the size of traditional IgG4 antibodies, they may show
better distribution over larger solid tumors with potentially
advantageous efficacy. UniBodies are cleared from the body at a
similar rate to whole IgG4 antibodies and are able to bind with a
similar affinity for their antigens as whole antibodies. Further
details of UniBodies may be obtained by reference to patent
WO2007/059782, which is herein incorporated by reference in its
entirety.
[0292] Affibody molecules represent a new class of affinity
proteins based on a 58-amino acid residue protein domain, derived
from one of the IgG-binding domains of staphylococcal protein A.
This three helix bundle domain has been used as a scaffold for the
construction of combinatorial phagemid libraries, from which
Affibody variants that target the desired molecules can be selected
using phage display technology (Nord K, Gunneriusson E, Ringdahl J,
Stahl S, Uhlen M, Nygren P A, Binding proteins selected from
combinatorial libraries of an .alpha.-helical bacterial receptor
domain, Nat Biotechnol 1997; 15:772-7. Ronmark J, Gronlund H, Uhlen
M, Nygren P A, Human immunoglobulin A (IgA)-specific ligands from
combinatorial engineering of protein A, Eur J Biochem 2002;
269:2647-55). The simple, robust structure of Affibody molecules in
combination with their low molecular weight (6 kDa), make them
suitable for a wide variety of applications, for instance, as
detection reagents (Ronmark J, Hansson M, Nguyen T, et al,
Construction and characterization of affibody-Fc chimeras produced
in Escherichia coli, J Immunol Methods 2002; 261:199-211) and to
inhibit receptor interactions (Sandstorm K, Xu Z, Forsberg G,
Nygren P A, Inhibition of the CD28-CD80 co-stimulation signal by a
CD28-binding Affibody ligand developed by combinatorial protein
engineering, Protein Eng 2003; 16:691-7). Further details of
Affibodies and methods of production thereof may be obtained by
reference to U.S. Pat. No. 5,831,012 which is herein incorporated
by reference in its entirety.
[0293] Labeled Affibodies may also be useful in imaging
applications for determining abundance of Isoforms.
[0294] DARPins (Designed Ankyrin Repeat Proteins) are one example
of an antibody mimetic DRP (Designed Repeat Protein) technology
that has been developed to exploit the binding abilities of
non-antibody polypeptides. Repeat proteins such as ankyrin or
leucine-rich repeat proteins, are ubiquitous binding molecules,
which occur, unlike antibodies, intra- and extracellularly. Their
unique modular architecture features repeating structural units
(repeats), which stack together to form elongated repeat domains
displaying variable and modular target-binding surfaces. Based on
this modularity, combinatorial libraries of polypeptides with
highly diversified binding specificities can be generated. This
strategy includes the consensus design of self-compatible repeats
displaying variable surface residues and their random assembly into
repeat domains.
[0295] DARPins can be produced in bacterial expression systems at
very high yields and they belong to the most stable proteins known.
Highly specific, high-affinity DARPins to a broad range of target
proteins, including human receptors, cytokines, kinases, human
proteases, viruses and membrane proteins, have been selected.
DARPins having affinities in the single-digit nanomolar to
picomolar range can be obtained.
[0296] DARPins have been used in a wide range of applications,
including ELISA, sandwich ELISA, flow cytometric analysis (FACS),
immunohistochemistry (IHC), chip applications, affinity
purification or Western blotting. DARPins also proved to be highly
active in the intracellular compartment for example as
intracellular marker proteins fused to green fluorescent protein
(GFP). DARPins were further used to inhibit viral entry with IC50
in the pM range. DARPins are not only ideal to block
protein-protein interactions, but also to inhibit enzymes.
Proteases, kinases and transporters have been successfully
inhibited, most often an allosteric inhibition mode. Very fast and
specific enrichments on the tumor and very favorable tumor to blood
ratios make DARPins well suited for in vivo diagnostics or
therapeutic approaches.
[0297] Additional information regarding DARPins and other DRP
technologies can be found in US Patent Application Publication No.
2004/0132028, and International Patent Application Publication No.
WO 02/20565, both of which are hereby incorporated by reference in
their entirety.
[0298] Anticalins are an additional antibody mimetic technology,
however in this case the binding specificity is derived from
lipocalins, a family of low molecular weight proteins that are
naturally and abundantly expressed in human tissues and body
fluids. Lipocalins have evolved to perform a range of functions in
vivo associated with the physiological transport and storage of
chemically sensitive or insoluble compounds. Lipocalins have a
robust intrinsic structure comprising a highly conserved
.beta.-barrel which supports four loops at one terminus of the
protein. These loops form the entrance to a binding pocket and
conformational differences in this part of the molecule account for
the variation in binding specificity between individual
lipocalins.
[0299] While the overall structure of hypervariable loops supported
by a conserved .beta.-sheet framework is reminiscent of
immunoglobulins, lipocalins differ considerably from antibodies in
terms of size, being composed of a single polypeptide chain of
160-180 amino acids which is marginally larger than a single
immunoglobulin domain.
[0300] Lipocalins are cloned and their loops are subjected to
engineering in order to create Anticalins. Libraries of
structurally diverse Anticalins have been generated and Anticalin
display allows the selection and screening of binding function,
followed by the expression and production of soluble protein for
further analysis in prokaryotic or eukaryotic systems. Studies have
successfully demonstrated that Anticalins can be developed that are
specific for virtually any human target protein can be isolated and
binding affinities in the nanomolar or higher range can be
obtained.
[0301] Anticalins can also be formatted as dual targeting proteins,
so-called Duocalins. A Duocalin binds two separate therapeutic
targets in one easily produced monomeric protein using standard
manufacturing processes while retaining target specificity and
affinity regardless of the structural orientation of its two
binding domains.
[0302] Modulation of multiple targets through a single molecule is
particularly advantageous in diseases known to involve more than a
single causative factor. Moreover, bi- or multivalent binding
formats such as Duocalins have significant potential in targeting
cell surface molecules in disease, mediating agonistic effects on
signal transduction pathways or inducing enhanced internalization
effects via binding and clustering of cell surface receptors.
Furthermore, the high intrinsic stability of Duocalins is
comparable to monomeric Anticalins, offering flexible formulation
and delivery potential for Duocalins.
[0303] Additional information regarding Anticalins can be found in
U.S. Pat. No. 7,250,297 and International Patent Application
Publication No. WO 99/16873, both of which are hereby incorporated
by reference in their entirety.
[0304] Another antibody mimetic technology useful in the context of
the instant invention are Avimers. Avimers are evolved from a large
family of human extracellular receptor domains by in vitro exon
shuffling and phage display, generating multidomain proteins with
binding and inhibitory properties. Linking multiple independent
binding domains has been shown to create avidity and results in
improved affinity and specificity compared with conventional
single-epitope binding proteins. Other potential advantages include
simple and efficient production of multitarget-specific molecules
in Escherichia coli, improved thermostability and resistance to
proteases. Avimers with sub-nanomolar affinities have been obtained
against a variety of targets.
[0305] Additional information regarding Avimers can be found in US
Patent Application Publication Nos. 2006/0286603, 2006/0234299,
2006/0223114, 2006/0177831, 2006/0008844, 2005/0221384,
2005/0164301, 2005/0089932, 2005/0053973, 2005/0048512,
2004/0175756, all of which are hereby incorporated by reference in
their entirety.
[0306] Versabodies are another antibody mimetic technology that
could be used in the context of the instant invention. Versabodies
are small proteins of 3-5 kDa with >15% cysteines, which form a
high disulfide density scaffold, replacing the hydrophobic core
that typical proteins have. The replacement of a large number of
hydrophobic amino acids, comprising the hydrophobic core, with a
small number of disulfides results in a protein that is smaller,
more hydrophilic (less aggregation and non-specific binding), more
resistant to proteases and heat, and has a lower density of T-cell
epitopes, because the residues that contribute most to MHC
presentation are hydrophobic. All four of these properties are
well-known to affect immunogenicity, and together they are expected
to cause a large decrease in immunogenicity.
[0307] The inspiration for Versabodies comes from the natural
injectable biopharmaceuticals produced by leeches, snakes, spiders,
scorpions, snails, and anemones, which are known to exhibit
unexpectedly low immunogenicity. Starting with selected natural
protein families, by design and by screening the size,
hydrophobicity, proteolytic antigen processing, and epitope density
are minimized to levels far below the average for natural
injectable proteins.
[0308] Given the structure of Versabodies, these antibody mimetics
offer a versatile format that includes multi-valency,
multi-specificity, a diversity of half-life mechanisms, tissue
targeting modules and the absence of the antibody Fc region.
Furthermore, Versabodies are manufactured in E. coli at high
yields, and because of their hydrophilicity and small size,
Versabodies are highly soluble and can be formulated to high
concentrations. Versabodies are exceptionally heat stable (they can
be boiled) and offer extended shelf-life.
[0309] Additional information regarding Versabodies can be found in
US Patent Application Publication No. 2007/0191272 which is hereby
incorporated by reference in its entirety.
[0310] The detailed description of antibody fragment and antibody
mimetic technologies provided above is not intended to be a
comprehensive list of all technologies that could be used in the
context of the instant specification. For example, and also not by
way of limitation, a variety of additional technologies including
alternative polypeptide-based technologies, such as fusions of
complimentary determining regions as outlined in Qui et al., Nature
Biotechnology, 25(8) 921-929 (2007), which is hereby incorporated
by reference in its entirety, as well as nucleic acid-based
technologies, such as the RNA aptamer technologies described in
U.S. Pat. Nos. 5,789,157, 5,864,026, 5,712,375, 5,763,566,
6,013,443, 6,376,474, 6,613,526, 6,114,120, 6,261,774, and
6,387,620, all of which are hereby incorporated by reference, could
be used in the context of the instant invention.
Pharmaceutical Compositions
[0311] In another aspect, the present invention provides a
composition, e.g., a pharmaceutical composition, containing one or
a combination of monoclonal antibodies, or antigen-binding
portion(s) thereof, of the present invention, formulated together
with a pharmaceutically acceptable carrier. Such compositions may
include one or a combination of (e.g., two or more different)
antibodies, or immunoconjugates or bispecific molecules of the
invention. For example, a pharmaceutical composition of the
invention can comprise a combination of antibodies (or
immunoconjugates or bispecifics) that bind to different epitopes on
the target antigen or that have complementary activities.
[0312] Pharmaceutical compositions of the invention also can be
administered in combination therapy, i.e., combined with other
agents. For example, the combination therapy can include an
anti-CADM1 antibody of the present invention combined with at least
one other anti-inflammatory or immunosuppressant agent. Examples of
therapeutic agents that can be used in combination therapy are
described in greater detail below in the section on uses of the
antibodies of the invention.
[0313] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like that are physiologically compatible.
Preferably, the carrier is suitable for intravenous, intramuscular,
subcutaneous, parenteral, spinal or epidermal administration (e.g.,
by injection or infusion). Depending on the route of
administration, the active compound, i.e., antibody,
immunoconjugate, or bispecific molecule, may be coated in a
material to protect the compound from the action of acids and other
natural conditions that may inactivate the compound.
[0314] The pharmaceutical compounds of the invention may include
one or more pharmaceutically acceptable salts. A "pharmaceutically
acceptable salt" refers to a salt that retains the desired
biological activity of the parent compound and does not impart any
undesired toxicological effects (see e.g., Berge, S. M., et al.
(1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid
addition salts and base addition salts. Acid addition salts include
those derived from nontoxic inorganic acids, such as hydrochloric,
nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous
and the like, as well as from nontoxic organic acids such as
aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic
acids, hydroxy alkanoic acids, aromatic acids, aliphatic and
aromatic sulfonic acids and the like. Base addition salts include
those derived from alkaline earth metals, such as sodium,
potassium, magnesium, calcium and the like, as well as from
nontoxic organic amines, such as N,N'-dibenzylethylenediamine,
N-methylglucamine, chloroprocaine, choline, diethanolamine,
ethylenediamine, procaine and the like.
[0315] A pharmaceutical composition of the invention also may
include a pharmaceutically acceptable anti-oxidant. Examples of
pharmaceutically acceptable antioxidants include: (1) water soluble
antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium
bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)
oil-soluble antioxidants, such as ascorbyl palmitate, butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin,
propyl gallate, alpha-tocopherol, and the like; and (3) metal
chelating agents, such as citric acid, ethylenediamine tetraacetic
acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the
like.
[0316] Examples of suitable aqueous and nonaqueous carriers that
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0317] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of presence of microorganisms may be ensured
both by sterilization procedures, supra, and by the inclusion of
various antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents which delay absorption
such as aluminum monostearate and gelatin.
[0318] Pharmaceutically acceptable carriers include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersion. The use
of such media and agents for pharmaceutically active substances is
known in the art. Except insofar as any conventional media or agent
is incompatible with the active compound, use thereof in the
pharmaceutical compositions of the invention is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0319] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
liposome, or other ordered structure suitable to high drug
concentration. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent that
delays absorption, for example, monostearate salts and gelatin.
[0320] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by sterilization
microfiltration. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle that
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and
freeze-drying (lyophilization) that yield a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0321] The amount of active ingredient which can be combined with a
carrier material to produce a single dosage form will vary
depending upon the subject being treated, and the particular mode
of administration. The amount of active ingredient which can be
combined with a carrier material to produce a single dosage form
will generally be that amount of the composition which produces a
therapeutic effect. Generally, out of one hundred percent, this
amount will range from about 0.01 percent to about ninety-nine
percent of active ingredient, preferably from about 0.1 percent to
about 70 percent, most preferably from about 1 percent to about 30
percent of active ingredient in combination with a pharmaceutically
acceptable carrier.
[0322] Dosage regimens are adjusted to provide the optimum desired
response (e.g., a therapeutic response). For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
subjects to be treated; each unit contains a predetermined quantity
of active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the invention are
dictated by and directly dependent on (a) the unique
characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active compound for the treatment
of sensitivity in individuals.
[0323] For administration of the antibody, the dosage ranges from
about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the
host body weight. For example dosages can be 0.3 mg/kg body weight,
1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10
mg/kg body weight or within the range of 1-10 mg/kg. An exemplary
treatment regime entails administration once per week, once every
two weeks, once every three weeks, once every four weeks, once a
month, once every 3 months or once every three to 6 months.
Preferred dosage regimens for an anti-CADM1 antibody of the
invention include 1 mg/kg body weight or 3 mg/kg body weight via
intravenous administration, with the antibody being given using one
of the following dosing schedules: (i) every four weeks for six
dosages, then every three months; (ii) every three weeks; (iii) 3
mg/kg body weight once followed by 1 mg/kg body weight every three
weeks.
[0324] In some methods, two or more monoclonal antibodies with
different binding specificities are administered simultaneously, in
which case the dosage of each antibody administered falls within
the ranges indicated. Antibody is usually administered on multiple
occasions. Intervals between single dosages can be, for example,
weekly, monthly, every three months or yearly. Intervals can also
be irregular as indicated by measuring blood levels of antibody to
the target antigen in the patient. In some methods, dosage is
adjusted to achieve a plasma antibody concentration of about 1-1000
.mu.g/ml and in some methods about 25-300 .mu.g/ml.
[0325] Alternatively, antibody can be administered as a sustained
release formulation, in which case less frequent administration is
required. Dosage and frequency vary depending on the half-life of
the antibody in the patient. In general, human antibodies show the
longest half life, followed by humanized antibodies, chimeric
antibodies, and nonhuman antibodies. The dosage and frequency of
administration can vary depending on whether the treatment is
prophylactic or therapeutic. In prophylactic applications, a
relatively low dosage is administered at relatively infrequent
intervals over a long period of time. Some patients continue to
receive treatment for the rest of their lives. In therapeutic
applications, a relatively high dosage at relatively short
intervals is sometimes required until progression of the disease is
reduced or terminated, and preferably until the patient shows
partial or complete amelioration of symptoms of disease.
Thereafter, the patient can be administered a prophylactic
regime.
[0326] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of the present invention may be varied
so as to obtain an amount of the active ingredient which is
effective to achieve the desired therapeutic response for a
particular patient, composition, and mode of administration,
without being toxic to the patient. The selected dosage level will
depend upon a variety of pharmacokinetic factors including the
activity of the particular compositions of the present invention
employed, or the ester, salt or amide thereof, the route of
administration, the time of administration, the rate of excretion
of the particular compound being employed, the duration of the
treatment, other drugs, compounds and/or materials used in
combination with the particular compositions employed, the age,
sex, weight, condition, general health and prior medical history of
the patient being treated, and like factors well known in the
medical arts.
[0327] A "therapeutically effective dosage" of an anti-CADM1
antibody of the invention preferably results in a decrease in
severity of disease symptoms, an increase in frequency and duration
of disease symptom-free periods, or a prevention of impairment or
disability due to the disease affliction. For example, for the
treatment of CADM1.sup.+ tumors, a "therapeutically effective
dosage" preferably inhibits cell growth or tumor growth by at least
about 20%, more preferably by at least about 40%, even more
preferably by at least about 60%, and still more preferably by at
least about 80% relative to untreated subjects. The ability of a
compound to inhibit tumor growth can be evaluated in an animal
model system predictive of efficacy in human tumors. Alternatively,
this property of a composition can be evaluated by examining the
ability of the compound to inhibit cell growth, such inhibition can
be measured in vitro by assays known to the skilled practitioner. A
therapeutically effective amount of a therapeutic compound can
decrease tumor size, or otherwise ameliorate symptoms in a subject.
One of ordinary skill in the art would be able to determine such
amounts based on such factors as the subject's size, the severity
of the subject's symptoms, and the particular composition or route
of administration selected.
[0328] A composition of the present invention can be administered
via one or more routes of administration using one or more of a
variety of methods known in the art. As will be appreciated by the
skilled artisan, the route and/or mode of administration will vary
depending upon the desired results. Preferred routes of
administration for antibodies of the invention include intravenous,
intramuscular, intradermal, intraperitoneal, subcutaneous, spinal
or other parenteral routes of administration, for example by
injection or infusion. The phrase "parenteral administration" as
used herein means modes of administration other than enteral and
topical administration, usually by injection, and includes, without
limitation, intravenous, intramuscular, intraarterial, intrathecal,
intracapsular, intraorbital, intracardiac, intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular,
intraarticular, subcapsular, subarachnoid, intraspinal, epidural
and intrasternal injection and infusion.
[0329] Alternatively, an antibody of the invention can be
administered via a non-parenteral route, such as a topical,
epidermal or mucosal route of administration, for example,
intranasally, orally, vaginally, rectally, sublingually or
topically.
[0330] The active compounds can be prepared with carriers that will
protect the compound against rapid release, such as a controlled
release formulation, including implants, transdermal patches, and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Many methods for the preparation of such
formulations are patented or generally known to those skilled in
the art. See, e.g., Sustained and Controlled Release Drug Delivery
Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York,
1978.
[0331] Therapeutic compositions can be administered with medical
devices known in the art. For example, in a preferred embodiment, a
therapeutic composition of the invention can be administered with a
needleless hypodermic injection device, such as the devices
disclosed in U.S. Pat. No. 5,399,163; 5,383,851; 5,312,335;
5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of
well-known implants and modules useful in the present invention
include: U.S. Pat. No. 4,487,603, which discloses an implantable
micro-infusion pump for dispensing medication at a controlled rate;
U.S. Pat. No. 4,486,194, which discloses a therapeutic device for
administering medicants through the skin; U.S. Pat. No. 4,447,233,
which discloses a medication infusion pump for delivering
medication at a precise infusion rate; U.S. Pat. No. 4,447,224,
which discloses a variable flow implantable infusion apparatus for
continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses
an osmotic drug delivery system having multi-chamber compartments;
and U.S. Pat. No. 4,475,196, which discloses an osmotic drug
delivery system. These patents are incorporated herein by
reference. Many other such implants, delivery systems, and modules
are known to those skilled in the art.
[0332] In certain embodiments, the human monoclonal antibodies of
the invention can be formulated to ensure proper distribution in
vivo. For example, the blood-brain barrier (BBB) excludes many
highly hydrophilic compounds. To ensure that the therapeutic
compounds of the invention cross the BBB (if desired), they can be
formulated, for example, in liposomes. For methods of manufacturing
liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and
5,399,331. The liposomes may comprise one or more moieties which
are selectively transported into specific cells or organs, thus
enhance targeted drug delivery (see, e.g., V. V. Ranade (1989) J.
Clin. Pharmacol. 29:685). Exemplary targeting moieties include
folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et
al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res.
Commun. 153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS
Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother.
39:180); surfactant protein A receptor (Briscoe et al. (1995) Am.
J. Physiol. 1233:134); p 120 (Schreier et al. (1994) J. Biol. Chem.
269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett.
346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods
4:273.
Uses and Methods
[0333] The antibodies, particularly the human antibodies, antibody
compositions and methods of the present invention have numerous in
vitro and in vivo diagnostic and therapeutic utilities involving
the diagnosis and treatment of CADM1 mediated disorders. For
example, these molecules can be administered to cells in culture,
in vitro or ex vivo, or to human subjects, e.g., in vivo, to treat,
prevent and to diagnose a variety of disorders. As used herein, the
term "subject" is intended to include human and non-human animals.
Non-human animals include all vertebrates, e.g., mammals and
non-mammals, such as non-human primates, sheep, dogs, cats, cows,
horses, chickens, amphibians, and reptiles. Preferred subjects
include human patients having disorders mediated by CADM1 activity.
The methods are particularly suitable for treating human patients
having a disorder associated with aberrant CADM1 expression. When
antibodies to CADM1 are administered together with another agent,
the two can be administered in either order or simultaneously.
[0334] Given the specific binding of the antibodies of the
invention for CADM1, the antibodies of the invention can be used to
specifically detect CADM1 expression on the surface of cells and,
moreover, can be used to purify CADM1 via immunoaffinity
purification.
[0335] Furthermore, given the expression of CADM1 on various tumor
cells, the human antibodies, antibody compositions and methods of
the present invention can be used to treat a subject with a
tumorigenic disorder, e.g., a disorder characterized by the
presence of tumor cells expressing CADM1 including, for example,
small cell lung cancer, adult T-cell leukemia, neuroendocrine
cancers including those of lung, adrenal, pituitary, GI-tract,
kidney, liver (including hepatocellular carcinomas), pancreas
(including insulinomas and glucagonomas), glioblastomas, and
carcinoid tumors including those of pancreas, lung, GI tract,
liver, or kidney.
[0336] In one embodiment, the antibodies (e.g., human monoclonal
antibodies, multispecific and bispecific molecules and
compositions) of the invention can be used to detect levels of
CADM1, or levels of cells which contain CADM1 on their membrane
surface, which levels can then be linked to certain disease
symptoms. Alternatively, the antibodies can be used to inhibit or
block CADM1 function which, in turn, can be linked to the
prevention or amelioration of certain disease symptoms, thereby
implicating CADM1 as a mediator of the disease. This can be
achieved by contacting a sample and a control sample with the
anti-CADM1 antibody under conditions that allow for the formation
of a complex between the antibody and CADM1. Any complexes formed
between the antibody and CADM1 are detected and compared in the
sample and the control.
[0337] In another embodiment, the antibodies (e.g., human
antibodies, multispecific and bispecific molecules and
compositions) of the invention can be initially tested for binding
activity associated with therapeutic or diagnostic use in vitro.
For example, compositions of the invention can be tested using the
flow cytometric assays described in the Examples below.
[0338] The antibodies (e.g., human antibodies, multispecific and
bispecific molecules, immunoconjugates and compositions) of the
invention have additional utility in therapy and diagnosis of CADM1
related diseases. For example, the human monoclonal antibodies, the
multispecific or bispecific molecules and the immunoconjugates can
be used to elicit in vivo or in vitro one or more of the following
biological activities: to inhibit the growth of and/or kill a cell
expressing CADM1; to mediate phagocytosis or ADCC of a cell
expressing CADM1 in the presence of human effector cells, or to
block CADM1 ligand binding to CADM1.
[0339] In a particular embodiment, the antibodies (e.g., human
antibodies, multispecific and bispecific molecules and
compositions) are used in vivo to treat, prevent or diagnose a
variety of CADM1-related diseases. Examples of CADM1-related
diseases include, among others, human cancer tissues representing
small cell lung cancer, neuroendocrine pancreatic cancer, liver
cancer, lung carcinoids, and gastro-intestinal carcinoids.
[0340] Suitable routes of administering the antibody compositions
(e.g., human monoclonal antibodies, multispecific and bispecific
molecules and immunoconjugates) of the invention in vivo and in
vitro are well known in the art and can be selected by those of
ordinary skill. For example, the antibody compositions can be
administered by injection (e.g., intravenous or subcutaneous).
Suitable dosages of the molecules used will depend on the age and
weight of the subject and the concentration and/or formulation of
the antibody composition.
[0341] As previously described, human anti-CADM1 antibodies of the
invention can be co-administered with one or other more therapeutic
agents, e.g., a cytotoxic agent, a radiotoxic agent or an
immunosuppressive agent. The antibody can be linked to the agent
(as an immunocomplex) or can be administered separate from the
agent. In the latter case (separate administration), the antibody
can be administered before, after or concurrently with the agent or
can be co-administered with other known therapies, e.g., an
anti-cancer therapy, e.g., radiation. Such therapeutic agents
include, among others, anti-neoplastic agents such as doxorubicin
(adriamycin), cisplatin bleomycin sulfate, carmustine,
chlorambucil, and cyclophosphamide hydroxyurea which, by
themselves, are only effective at levels which are toxic or
subtoxic to a patient. Cisplatin is intravenously administered as a
100 mg/kg dose once every four weeks and adriamycin is
intravenously administered as a 60-75 mg/ml dose once every 21
days. Co-administration of the human anti-CADM1 antibodies, or
antigen binding fragments thereof, of the present invention with
chemotherapeutic agents provides two anti-cancer agents which
operate via different mechanisms which yield a cytotoxic effect to
human tumor cells. Such co-administration can solve problems due to
development of resistance to drugs or a change in the antigenicity
of the tumor cells which would render them unreactive with the
antibody.
[0342] Target-specific effector cells, e.g., effector cells linked
to compositions (e.g., human antibodies, multispecific and
bispecific molecules) of the invention can also be used as
therapeutic agents. Effector cells for targeting can be human
leukocytes such as macrophages, neutrophils or monocytes. Other
cells include eosinophils, natural killer cells and other IgG- or
IgA-receptor bearing cells. If desired, effector cells can be
obtained from the subject to be treated. The target-specific
effector cells can be administered as a suspension of cells in a
physiologically acceptable solution. The number of cells
administered can be in the order of 10.sup.8-10.sup.9 but will vary
depending on the therapeutic purpose. In general, the amount will
be sufficient to obtain localization at the target cell, e.g., a
tumor cell expressing CADM1, and to affect cell killing by, e.g.,
phagocytosis. Routes of administration can also vary.
[0343] Therapy with target-specific effector cells can be performed
in conjunction with other techniques for removal of targeted cells.
For example, anti-tumor therapy using the compositions (e.g., human
antibodies, multispecific and bispecific molecules) of the
invention and/or effector cells armed with these compositions can
be used in conjunction with chemotherapy. Additionally, combination
immunotherapy may be used to direct two distinct cytotoxic effector
populations toward tumor cell rejection. For example, anti-CADM1
antibodies linked to anti-Fc-gamma RI or anti-CD3 may be used in
conjunction with IgG- or IgA-receptor specific binding agents.
[0344] Bispecific and multispecific molecules of the invention can
also be used to modulate Fc.gamma.R or Fc.gamma.R levels on
effector cells, such as by capping and elimination of receptors on
the cell surface. Mixtures of anti-Fc receptors can also be used
for this purpose.
[0345] The compositions (e.g., human, humanized, or chimeric
antibodies, multispecific and bispecific molecules and
immunoconjugates) of the invention which have complement binding
sites, such as portions from IgG1, -2, or -3 or IgM which bind
complement, can also be used in the presence of complement. In one
embodiment, ex vivo treatment of a population of cells comprising
target cells with a binding agent of the invention and appropriate
effector cells can be supplemented by the addition of complement or
serum containing complement. Phagocytosis of target cells coated
with a binding agent of the invention can be improved by binding of
complement proteins. In another embodiment target cells coated with
the compositions (e.g., human antibodies, multispecific and
bispecific molecules) of the invention can also be lysed by
complement. In yet another embodiment, the compositions of the
invention do not activate complement.
[0346] The compositions (e.g., human, humanized, or chimeric
antibodies, multispecific and bispecific molecules and
immunoconjugates) of the invention can also be administered
together with complement. In certain embodiments, the instant
disclosure provides compositions comprising human antibodies,
multispecific or bispecific molecules and serum or complement.
These compositions can be advantageous when the complement is
located in close proximity to the human antibodies, multispecific
or bispecific molecules. Alternatively, the human antibodies,
multispecific or bispecific molecules of the invention and the
complement or serum can be administered separately.
[0347] Also within the scope of the present invention are kits
comprising the antibody compositions of the invention (e.g., human
antibodies, bispecific or multispecific molecules, or
immunoconjugates) and instructions for use. The kit can further
contain one or more additional reagents, such as an
immunosuppressive reagent, a cytotoxic agent or a radiotoxic agent,
or one or more additional human antibodies of the invention (e.g.,
a human antibody having a complementary activity which binds to an
epitope in the CADM1 antigen distinct from the first human
antibody).
[0348] Accordingly, patients treated with antibody compositions of
the invention can be additionally administered (prior to,
simultaneously with, or following administration of a human
antibody of the invention) with another therapeutic agent, such as
a cytotoxic or radiotoxic agent, which enhances or augments the
therapeutic effect of the human antibodies.
[0349] In other embodiments, the subject can be additionally
treated with an agent that modulates, e.g., enhances or inhibits,
the expression or activity of Fc.gamma. or Fc.gamma. receptors by,
for example, treating the subject with a cytokine. Preferred
cytokines for administration during treatment with the
multispecific molecule include of granulocyte colony-stimulating
factor (G-CSF), granulocyte-macrophage colony-stimulating factor
(GM-CSF), interferon-.gamma. (IFN-.gamma.), and tumor necrosis
factor (TNF).
[0350] The compositions (e.g., human antibodies, multispecific and
bispecific molecules) of the invention can also be used to target
cells expressing Fc.gamma.R or CADM1, for example for labeling such
cells. For such use, the binding agent can be linked to a molecule
that can be detected. Thus, the invention provides methods for
localizing ex vivo or in vitro cells expressing Fc receptors, such
as Fc.gamma.R, or CADM1. The detectable label can be, e.g., a
radioisotope, a fluorescent compound, an enzyme, or an enzyme
co-factor.
[0351] In a particular embodiment, the invention provides methods
for detecting the presence of CADM1 antigen in a sample, or
measuring the amount of CADM1 antigen, comprising contacting the
sample, and a control sample, with a human monoclonal antibody, or
an antigen binding portion thereof, which specifically binds to
CADM1, under conditions that allow for formation of a complex
between the antibody or portion thereof and CADM1. The formation of
a complex is then detected, wherein a difference complex formation
between the sample compared to the control sample is indicative the
presence of CADM1 antigen in the sample.
[0352] In other embodiments, the invention provides methods for
treating a CADM1 mediated disorder in a subject, e.g., human
cancers, including small cell lung cancer, neuroendocrine cancers
including those of lung, adrenal, pituitary, GI-tract, kidney,
liver, pancreas (including insulinomas and glucagonomas), and
carcinoid tumors including those of pancreas, lung, GI tract, liver
or kidney.
[0353] In yet another embodiment, immunoconjugates of the invention
can be used to target compounds (e.g., therapeutic agents, labels,
cytotoxins, radiotoxins immunosuppressants, etc.) to cells which
have CADM1 cell surface receptors by linking such compounds to the
antibody. For example, an anti-CADM1 antibody can be conjugated to
any of the toxin compounds described in U.S. Pat. Nos. 6,281,354
and 6,548,530, US patent publication Nos. 20030050331, 20030064984,
20030073852, and 20040087497, or published in WO 03/022806. Thus,
the invention also provides methods for localizing ex vivo or in
vivo cells expressing CADM1 (e.g., with a detectable label, such as
a radioisotope, a fluorescent compound, an enzyme, or an enzyme
co-factor). Alternatively, the immunoconjugates can be used to kill
cells which have CADM1 cell surface receptors by targeting
cytotoxins or radiotoxins to CADM1.
[0354] The present invention is further illustrated by the
following examples which should not be construed as further
limiting. The contents of all figures and all references, patents
and published patent applications cited throughout this application
are expressly incorporated herein by reference.
EXAMPLES
Example 1
Generation of Human Monoclonal Antibodies Against CADM1 Antigen
[0355] A recombinant fusion protein composed of the extracellular
domain of the CADM1 (CADM1 ECD) linked to a non-CADM1 polypeptide
(his protein) (SEQ ID NO:44) was generated by standard recombinant
methods and used as antigen for immunization (see below).
Transgenic HuMAb Mouse.RTM. and KM Mouse.RTM. Strains
[0356] Fully human monoclonal antibodies to CADM1 were prepared
using HCo7 and HCo27 strains of the transgenic HuMAb Mouse.RTM. and
the KM strain of transgenic transchromosomic mice, each of which
express human antibody genes. In each of these mouse strains, the
endogenous mouse kappa light chain gene has been homozygously
disrupted as described in Chen et al. (1993) EMBO J. 12:811-820 and
the endogenous mouse heavy chain gene has been homozygously
disrupted as described in Example 1 of PCT Publication WO 01/09187.
Each of these mouse strains carries a human kappa light chain
transgene, KCo5, as described in Fishwild et al. (1996) Nature
Biotechnology 14:845-851. The HCo7 strain carries the HCo7 human
heavy chain transgene as described in U.S. Pat. Nos. 5,545,806;
5,625,825; and 5,545,807. The HCo27 strain carries the HCo27 human
heavy chain transgene as described in PCT Publication WO 01/09187.
The KM Mouse.RTM. strain contains the SC20 transchromosome as
described in PCT Publication WO 02/43478.
HuMab and KM Immunizations
[0357] To generate fully human monoclonal antibodies to CADM1,
HuMab mice of the HCo7, HCo27 and KM Mouse strains were immunized
with purified recombinant CADM1-ECD-his protein. General
immunization schemes for these mice are described in Lonberg, N. et
al (1994) Nature 368(6474): 856-859; Fishwild, D. et al. (1996)
Nature Biotechnology 14: 845-851 and PCT Publication WO 98/24884.
The mice were 6-16 weeks of age upon the first infusion of antigen.
A purified recombinant preparation (5-50 .mu.g) of CADM1-ECD-his
protein was used to immunize the HuMab and KM Mouse.RTM..
[0358] Transgenic mice were immunized with the antigen in Ribi
adjuvant either intraperitonealy (IP), subcutaneously (Sc) or via
footpad (FP) in 3-21 days intervals (up to a total of 9
immunizations). The immune response was monitored by retroorbital
bleeds. The plasma was screened by ELISA (as described below), and
mice with sufficient titers of anti-CADM1 human immunoglobulin were
used for fusions. Mice were boosted intravenously with antigen 3
and 2 days before sacrifice and removal of the spleen. Typically,
10-20 fusions for each antigen were performed. Several dozen mice
were immunized for each antigen.
Selection of a HuMab Mouse.RTM. or KM Mouse.RTM. Animal Producing
Anti-CADM1 Antibodies
[0359] To select a HuMab Mouse.RTM. or KM Mouse.RTM. animal
producing antibodies that bound CADM1, sera from immunized mice was
tested by ELISA as described by Fishwild, D. et al. (1996)(supra).
Briefly, microtiter plates were coated with purified recombinant
CADM1 at 1-2 .mu.g/ml in PBS, 50 .mu.l/wells incubated 4.degree. C.
overnight then blocked with 200 .mu.l/well of 5% chicken serum in
PBS/Tween (0.05%). Dilutions of plasma from CADM1-immunized mice
were added to each well and incubated for 1-2 hours at ambient
temperature. The plates were washed with PBS/Tween and then
incubated with a goat-anti-human IgG Fc polyclonal antibody
conjugated with horseradish peroxidase (HRP) for 1 hour at room
temperature. After washing, the plates were developed with ABTS
substrate (Moss Inc, product: ABTS-1000) and analyzed by
spectrophotometer at OD 415-495. Mice that developed the highest
titers of anti-CADM1 antibodies were used for fusions. Fusions were
performed as described below and hybridoma supernatants were tested
for anti-CADM1 activity by ELISA and FACS.
Generation of Hybridomas Producing Human Monoclonal Antibodies to
CADM1
[0360] The mouse splenocytes, isolated from a HuMab Mouse.RTM.
and/or a KM Mouse.RTM., were fused with a mouse myeloma cell line
using electric field based electrofusion using a Cyto Pulse large
chamber cull fusion electroporator (Cyto Pulse Sciences, Inc., Glen
Burnie, Md.). Briefly, single cell suspensions of splenic
lymphocytes from immunized mice were fused to equal number of Sp2/0
nonsecreting mouse myeloma cells (ATCC, CRL 1581). Cells were
plated at a density of approximately 2.times.10.sup.4/well in flat
bottom microtiter plates, which were then incubated in selective
medium containing 10% fetal bovine serum, 10% P388D1 (ATCC, CRL
TIB-63) conditioned medium, 3-5% origen (IGEN) in DMEM (Mediatech,
CRL 10013, with high glucose, L-glutamine and sodium pyruvate) plus
5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 mg/ml gentamycin and
1.times.HAT (Sigma, CRL P-7185). After 1-2 weeks, cells were
cultured in medium in which the HAT was replaced with HT.
Approximately 10-14 days after cell plating, supernatants from
individual wells were screened first for whether they contained
human g,k antibodies. The supernatants which were scored positive
for human g,k were then subsequently screened-by ELISA and FACS
(described above) for human anti-CADM1 monoclonal IgG antibodies.
The antibody secreting hybridomas were transferred to 24 well
plates, screened again and, if still positive for human anti-CADM1
IgG monoclonal antibodies, were subcloned at least twice by
limiting dilution. The stable subclones were then cultured in vitro
to generate small amounts of antibody in tissue culture medium for
further characterization.
[0361] Hybridoma clones PTA021_A1, PTA021_A2, and PTA021_A3
generated from a KM Mouse.RTM., were selected for further
analysis.
Example 2
Structural Characterization of Human Monoclonal Antibodies
PTA021_A1, PTA021_A2, or PTA021_A3
[0362] The cDNA sequences encoding the heavy and light chain
variable regions of the PTA021_A1, PTA021_A2, and PTA021_A3
monoclonal antibodies were obtained from the PTA021_A1, PTA021_A2,
and PTA021_A3 hybridomas, respectively, using standard PCR
techniques and were sequenced using standard DNA sequencing
techniques.
[0363] The antibody sequences may be mutagenized to revert back to
germline residues at one or more residues. For example, the
PTA021_A1 heavy chain variable region can be mutagenized to reflect
the germline sequence at specific sites (e.g., residue 30) to
remove glycosylation sites (e.g., a N30Q mutation).
[0364] The nucleotide and amino acid sequences of the heavy chain
variable region of PTA021_A1 are shown in FIG. 1A and in SEQ ID
NO:19 and 25, respectively.
[0365] The nucleotide and amino acid sequences of the light chain
variable region of PTA021_A1 are shown in FIG. 1B and in SEQ ID
NO:28 and 22, respectively.
[0366] Comparison of the PTA021_A1 heavy chain immunoglobulin
sequence to the known human germline immunoglobulin heavy chain
sequences demonstrated that the PTA021_A1 heavy chain utilizes a
V.sub.H segment from human germline V.sub.H 2-05, a D segment from
the human germline 6-6, and a JH segment from human germline JH 5b.
The alignment of the PTA021_A1 V.sub.H sequence to the germline
V.sub.H 2-05 sequence is shown in FIG. 4. Further analysis of the
PTA021_A1 V.sub.H sequence using the Kabat system of CDR region
determination led to the delineation of the heavy chain CDR1, CDR2
and CD3 regions as shown in FIGS. 1A and 4, and in SEQ ID NOs:1, 4
and 7, respectively.
[0367] Comparison of the PTA021_A1 light chain immunoglobulin
sequence to the known human germline immunoglobulin light chain
sequences demonstrated that the PTA021_A1 light chain utilizes a
V.sub.K segment from human germline V.sub.K L15 and a JK segment
from human germline JK 4. The alignment of the PTA021_A1 V.sub.K
sequence to the germline V.sub.K L15 sequence is shown in FIG. 7.
Further analysis of the PTA021_A1 V.sub.K sequence using the Kabat
system of CDR region determination led to the delineation of the
light chain CDR1, CDR2 and CD3 regions as shown in FIGS. 1B and 7,
and in SEQ ID NOs:10, 13 and 16, respectively.
[0368] The nucleotide and amino acid sequences of the heavy chain
variable region of PTA021_A2 are shown in FIG. 2A and in SEQ ID
NO:26 and 20, respectively.
[0369] The nucleotide and amino acid sequences of the light chain
variable region of PTA021_A2 are shown in FIG. 2B and in SEQ ID
NO:29 and 23, respectively.
[0370] Comparison of the PTA021_A2 heavy chain immunoglobulin
sequence to the known human germline immunoglobulin heavy chain
sequences demonstrated that the PTA021_A2 heavy chain utilizes a
V.sub.H segment from human germline V.sub.H 2-05, a D segment from
the human germline 6-6, and a JH segment from human germline JH 5b.
The alignment of the PTA021_A2 V.sub.H sequence to the germline
V.sub.H 2-05 sequence is shown in FIG. 5. Further analysis of the
PTA021_A2 V.sub.H sequence using the Kabat system of CDR region
determination led to the delineation of the heavy chain CDR1, CDR2
and CD3 regions as shown in FIGS. 2A and 5, and in SEQ ID NOs:2, 5
and 8, respectively.
[0371] Comparison of the PTA021_A2 light chain immunoglobulin
sequence to the known human germline immunoglobulin light chain
sequences demonstrated that the PTA021_A2 light chain utilizes a
V.sub.K segment from human germline V.sub.K L15 and a JK segment
from human germline JK 4. The alignment of the PTA021_A2 V.sub.L
sequence to the germline V.sub.K L15 sequence is shown in FIG. 8.
Further analysis of the PTA021_A2 V.sub.K sequence using the Kabat
system of CDR region determination led to the delineation of the
light chain CDR1, CDR2 and CD3 regions as shown in FIGS. 2B and 8,
and in SEQ ID NOs:11, 14 and 17, respectively.
[0372] The nucleotide and amino acid sequences of the heavy chain
variable region of PTA021_A3 are shown in FIG. 3A and in SEQ ID
NO:27 and 21, respectively.
[0373] The nucleotide and amino acid sequences of the light chain
variable region of PTA021_A3 are shown in FIG. 3B and in SEQ ID
NO:30 and 24, respectively.
[0374] Comparison of the PTA021_A3 heavy chain immunoglobulin
sequence to the known human germline immunoglobulin heavy chain
sequences demonstrated that the PTA021_A3 heavy chain utilizes a
V.sub.H segment from human germline V.sub.H 2-05, a D segment from
the human germline 6-6, and a JH segment from human germline JH
JH5b. The alignment of the PTA021_A3 V.sub.H sequence to the
germline V.sub.H 2-05 sequence is shown in FIG. 6. Further analysis
of the PTA021_A3 V.sub.H sequence using the Kabat system of CDR
region determination led to the delineation of the heavy chain
CDR1, CDR2 and CD3 regions as shown in FIGS. 3A and 6, and in SEQ
ID NOs: 3, 6 and 9, respectively.
[0375] Comparison of the PTA021_A3 light chain immunoglobulin
sequence to the known human germline immunoglobulin light chain
sequences demonstrated that the PTA021_A3 light chain utilizes a
V.sub.K segment from human germline V.sub.K L15 and a JK segment
from human germline JK 4. The alignment of the PTA021_A3 V.sub.L
sequence to the germline V.sub.K L15 sequence is shown in FIG. 9.
Further analysis of the PTA021_A3 V.sub.L sequence using the Kabat
system of CDR region determination led to the delineation of the
light chain CDR1, CDR2 and CD3 regions as shown in FIGS. 3B and 9,
and in SEQ ID NOs:12, 15, and 18, respectively.
Example 3
Characterization of Binding Properties of CADM1 Monoclonal
Antibodies Flow Cytometry Studies
[0376] In this example, the binding of mAbs PTA021_A1, PTA021_A2,
PTA021_A3 and the non-fucosylated version of PTA021_A3 (NF) to cell
surface CADM1 was examined by flow cytometry.
[0377] To test the ability of the antibodies to bind to cell
surface CADM1 protein, the antibodies were incubated with different
CADM1-expressing cells: NCI_H69 (ATCC Designation HTB-119.TM.), a
human small cell lung cancer cell line; DMS79 (ATCC Designation
CRL-2049.TM.), a human small cell lung cancer line; SKOV3 (ATCC
Designation HTB-77.TM.), a human ovarian cancer cell line; A549
(ATCC Designation CCL-185.TM.), a human non-small cell lung cancer
cell line; SkMel28 (ATCC Designation HTB-72.TM.), a human melanoma
cell line and 786-O (ATCC Designation CRL-1932.TM.), a human renal
cell carcinoma cell line. For the flow cytometry studies, the
PTA021_A1, PTA021_A2, PTA021_A3 and PTA021_A3 (NF) monoclonal
antibodies were diluted with cold 1.times.PBS+0.1% BSA to 40
.mu.g/ml. For the binding reaction, 50 .mu.l of diluted antibody
solution was added to a 50 .mu.l cell suspension containing
4.times.10.sup.5 cells and the mixture was incubated on ice for
30-60 minutes. The cells were then washed three times with
1.times.PBS+0.1% BSA. A 1:50 dilution of R-phycoerythrin-labeled
goat anti-human IgG F.gamma. F(ab)2 fragment (Jackson
ImmunoResearch Labs, Cat. #109-116-098) was added and the mixture
was incubated on ice for 1 hour, followed by washing twice with
cold 1.times.PBS+0.1% BSA. After the final wash, 200 .mu.l of cold
1.times.PBS+0.1% BSA was added to each solution and analysis of
antibody binding was carried out by FACS.
[0378] FIG. 10 and Table 1 below show the results of the flow
cytometry analysis and the EC.sub.50 for binding to the NCI-H69
cell line. The results demonstrate that all three monoclonal
antibodies bind effectively to cell-surface human CADM1.
TABLE-US-00001 TABLE 1 Binding of Anti-CADM1 Antibodies to Cells
Expressing Human CADM1 Antibody NCI-H69 Cells EC.sub.50 (nM)
PTA021_A1 0.588 PTA021_A2 0.667 PTA021_A3 0.6742
[0379] FIGS. 11A and 11B show the results of the flow cytometry
analysis in NCI-H69 and DMS79 small cell lung cancer cell
lines.
[0380] FIG. 12 shows the results of flow cytometric analysis on
antibody PTA021_A3 in the SKOV3 ovarian cancer cell line.
[0381] FIG. 13 shows the results of flow cytometric analysis on
antibody PTA021_A3 in the A549 non-small cell lung cancer cell
line.
[0382] FIG. 14 shows the results of flow cytometric analysis on
antibody PTA021_A3 in 786-O renal cell carcinoma and SkMel28
melanoma cell lines.
[0383] The results of FIGS. 11 to 14 demonstrate that PTA021_A3
binds effectively to cell surface human CADM1 in different
CADM1-expressing cells.
[0384] FIG. 15 shows the results of flow cytometric analysis on the
non-fucosylated version of PTA021_A3 (NF) in NCI_H69 and DMS79
small cell lung cancer cell lines. These results demonstrate that
non-fucosylation does not affect the binding of PTA021_A3 to cell
surface human CADM1.
Example 4
Antibody-Dependent Cellular Cytotoxicity Mediated by Anti-CADM1
mAbs
[0385] To determine the ability of the anti-CADM1 mAbs to kill
CADM1-expressing cells in the presence of effector cells via
antibody dependent cellular cytotoxicity (ADCC), two cell lines,
NCI_H69 and DMS79, were used as the target cells.
[0386] Human effector cells were prepared from whole blood as
follows. Human peripheral blood mononuclear cells were purified
from heparinized whole blood by standard Ficoll-paque separation.
The cells were resuspended in RPMI1640 media containing 10% FBS
(heat-inactivated) and 200 U/ml of human IL-2 and incubated
overnight at 37.degree. C. The following day, the cells were
collected and washed four times in culture media and resuspended at
1.times.10.sup.7 cells/ml. Target cells were prepared by incubating
with BATDA reagent (Perkin Elmer, Wellesley, Mass.) at 2.5 .mu.l
BATDA per 1.times.10.sup.6 target cells/mL for 20 minutes at
37.degree. C. The target cells were washed four times, spun down
and brought to a final volume of 1.times.10.sup.5 cells/ml.
[0387] The target cells were tested for antibody specific ADCC to
the human anti-CADM1 monoclonal antibodies using the Delfia
fluorescence emission analysis as follows. NCI-H69 or DMS79 cells
(100 .mu.l of labeled target cells, 10.sup.4 cells/well) were
incubated with 50 .mu.l of effector cells (10.sup.6 cells/well) and
50 .mu.l of antibody (10 ug/ml final concentration). A target to
effector ratio of 1:25 was used throughout the experiments. In all
studies, a human IgG1 isotype control was used as a negative
control. Cells were spun down at 2000 rpm and incubated for one
hour incubation at 37.degree. C. The supernatants were then
collected, submitted to centrifugation and 20 .mu.l of supernatant
was transferred to a flat bottom plate, to which 180 .mu.l of Eu
solution (Perkin Elmer, Wellesley, Mass.) was added and read in a
RubyStar reader (BMG Labtech). The % lysis was calculated as
follows: (sample release-spontaneous release*100)/(maximum
release-spontaneous release), where the spontaneous release is the
fluorescence from wells which contain target cells plus effector
cells and maximum release is the fluorescence from wells containing
target cells and have been treated with 2% Triton-X.
[0388] The results are summarized below in Table 2 and FIGS. 16A
and 16B, which demonstrate that PTA021_A1, PTA021_A2, and PTA021_A3
are capable of specifically inducing ADCC on CADM1 expressing
cancer cell lines.
TABLE-US-00002 TABLE 2 Antibody Dependent Cellular Cytotoxicity
Mediated by PTA021_A1, PTA021_A2 and PTA021_A3 Antibodies %
Specific Lysis: Antibody % Specific Lysis: NCI-H69 DMS79 PTA021_A1
24.6 +/- 4.5 37.76 +/- 7.8 PTA021_A2 15.9 +/- 1.9 34.66 +/- 3.1
PTA021_A3 29.8 +/- 1.6 61.0 +/- 1.5 Isotype Control 3.3 +/- 4.6
2.96 +/- 1.9
Example 5
CADM1 Antibody Internalization
[0389] The monoclonal antibodies PTA021_A1, PTA021_A2, and
PTA021_A3 were shown to be internalized by NCI-H69 and DMS79 cells
upon binding to the cells using a Hum-Zap assay. The Hum-ZAP assay
showed internalization of the anti-CADM1 monoclonal antibodies
through binding of an anti-human IgG secondary antibody conjugated
to the toxin saporin. (Advanced Targeting System, San Diego,
Calif., IT-22-100). First, PTA021_A1, PTA021_A2, and PTA021_A3 were
bound to the surface of the NCI-H69 or DMS79 cells. Then, the
Hum-ZAP antibodies were bound to the primary antibodies. Next, the
primary antibody/Hum-ZAP complex was internalized by the cells. The
entrance of Saporin into the cells resulted in protein synthesis
inhibition and eventual cell death.
[0390] The Hum-ZAP assay was conducted as follows. Each of the
cells was seeded at a density of 3.times.10.sup.3 cells per well.
The anti-CADM1 monoclonal antibodies or an isotype control human
IgG were serially diluted then added to the cells. The Hum-ZAP was
then added at a concentration of 2 .mu.g/ml and the plates allowed
to incubate for 96 hours. Cell viability in the plates was detected
by CellTiter-Glo.RTM. Luminescent Cell Viability Assay kit
(Promega, G7571) and the plates were read at 490 nM by a
Luminomitor (Tuner BioSystems, Sunnyvale, Calif.). The data was
analyzed by Prism (Graphpad). Cell death was proportional to the
concentration of PTA021_A1, PTA021_A2, and PTA021_A3 monoclonal
antibodies. FIGS. 17A and 17B show that the anti-CADM1 monoclonal
antibodies were efficiently internalized by DMS79 and NCI-H69 cells
respectively as compared to the hIgG1 isotype control antibody.
Example 6
CADM1 is Co-Localized with LAMP1
[0391] The monoclonal antibody PTA021_A3 was shown to co-localize
with LAMP1 and thus to be internalized. PTA021_A3 was bound to
NCI-H69 cells, washed and incubated at 37.degree. C. for 45
minutes. The PTA021_A3 antibody was tracked via a FITC labeled
anti-human antibody.
[0392] Some cells were permeabilized and stained with anti-human
LAMP1, detected with TRITC labeled secondary.
[0393] The results showed that PTA021_A3 and LAMP1 are co-localized
in the endosomes.
Example 7
Anti-CADM1 Antibodies Bind to Cancer Tissues
[0394] Anti-CADM1 monoclonal antibodies PTA021_A1, PTA021_A2, and
PTA021_A3 were shown to bind human cancer tissues including tissues
representing small cell lung cancer, neuroendocrine pancreatic
cancer, liver cancer, lung carcinoids, and gastro-intestinal
carcinoids. Biopsies from cancer patients were obtained and the
antibodies used for immunohistochemistry staining (Cytomyx, Mass.).
5 .mu.m tissues cores were used. After drying for 30 minutes on
slides, the tissue sections were fixed with acetone at room
temperature for 5 minutes. Slides were rinsed in PBS and then
blocked with serum-free protein and peroxidase blocker (Dako S2001,
CO) and subsequently incubated with primary antibody complex at 5
.mu.g/ml for 45 minutes at room temperature. Next, the slides were
washed and incubated for 30 minutes with FITC-conjugated secondary
antibody (Jackson Immunoresearch Lab, 109-097-003) and washed again
with PBS and incubated with polymer HRP conjugates (Dako, Colo.,
K4063) for 20 minutes. Chromogen (Dako K3464) was used as a
substrate, resulting in brown staining. Slides were mounted in
Faramount Aqueous Mounting Media (Dako, S3025). PTA021_A1,
PTA021_A2 and PTA021_A3 were shown to bind specifically to tumor
cells of the types listed above. When stained with these monoclonal
antibodies, other organs exhibit negative or non-specific staining,
which include uterus, lung, liver, kidney, colon, cervix, breast,
bone marrow, cerebellum, cerebrum, esophagus, heart, prostate,
placenta, pituitary, ovary, mesothelia, tonsil, skin, small
intestine, skeletal muscle, stomach, spleen, thymus, and thyroid.
The data demonstrate that anti-CADM1 HuMabs PTA021_A1, PTA021_A2,
and PTA021_A3 recognize CADM1 expressed on tumors, including those
tumors of small cell lung cancer, non-small-cell lung cancer
(including squamous carcinomas and adenocarcinomas), neuroendocrine
cancers (including those of the pancreas, lung and
gastro-intestinal tract), liver cancer, lung carcinoids, breast
cancer, colon cancer, prostate cancer, ovarian cancer, kidney
cancer, and gastro-intestinal carcinoids.
[0395] Immunohistochemistry on antibody PTA021_A3 in small cell
lung cancer tissue samples showed 75% prevalence of high level
(+++) membrane staining of all tumor cells. Immunohistochemistry on
antibody PTA021_A3 in liver cancer tissue samples showed positive
staining in 75%-95% of samples with 45% of tumors showing high
level (+++) membrane staining of all tumor cells. Further studies
on PTA021_A3 were carried out on smaller sample sets of non-small
cell lung cancer, breast cancer, prostate cancer, colorectal
cancer, ovarian cancer and renal cell carcinoma. Prevalence of
positive staining in non-small cell lung carcinoma, breast
carcinoma and renal clear cell carcinoma was .about.66%; in
prostate carcinoma and ovarian carcinoma prevalence was 100% and
colorectal adenocarcinoma showed positive staining in 17% of
samples. Neuroendocrine tumors of the lung, pancreas and
gastro-intestinal tract were also tested with lung carcinoids and
neuroendocrine pancreatic samples showing 75% prevalence and
gastro-intestinal tract neuroendocrine tumors showing positive
staining in 65% of samples.
Example 8
Effect of Fucosylated and Non-Fucosylated Anti-CADM1 Antibodies on
Liver Cancer Tumor Growth in a Mouse Xenograft Model
[0396] The effect of PTA021_A3 and the non-fucosylated version of
PTA021_A3(NF) on the growth of liver cancer derived HepG2 cells in
a mouse xenograft model was examined. In this xenograft model, SCID
mice (CB17, from Charles River Laboratories, Hollister, Calif.)
were implanted with 2.5.times.10.sup.6 HepG2 cells/mouse and the
HepG2 cells were allowed to grow for ca. 30 days. The mice were
then randomized and treated intraperitoneally (i.p.) as follows in
Table 3:
TABLE-US-00003 TABLE 3 Immunization Protocol for HEPG2 Tumor
Xenograft Model Dose (Ab mg/kg) Dose Day Vehicle Day 0 Isotype IgG1
10 Day 0, 4, 7, 11 Isotype nf IgG1 10 Day 0, 4, 7, 11 CADM1
PTA021_A3 10, 3 Day 0, 4, 7, 11 parental IgG1 CADM1 PTA021_A3 nf
10, 3 Day 0, 4, 7, 11 IgG1 Nexavar Sorafenib 20 (Q1Dx3), 10 p.o.
Q1Dx8, Day 0-7
Nexavar Sorafenib, a chemical-based multiple kinase inhibitor used
to treat renal cell carcinoma and liver cancer, was used as a
control for targeted therapy.
[0397] As seen in FIG. 18, treatment with the PTA021_A3 and
PTA021_A3 NF antibodies significantly reduced tumor growth rate,
with the non-fucosylated PTA021_A3 NF antibody being the more
potent.
Example 9
Anti-CADM1 Antibody-Drug Conjugate Inhibits HepG2 Cell Growth in a
Mouse Xenograft Model
[0398] The effect of a PTA021_A3 conjugate according to formula M
(hereinafter referred to as "PTA021_A3-Formula A conjugate" or
"PTA021_A3-Formula M") on the growth of hepatocellular carcinoma
derived HepG2 cells in a mouse xenograft model was examined. In
this xenograft model, SCID mice (CB 17, from Charles River
Laboratories, Hollister, Calif.) were implanted with
2.5.times.10.sup.6 HepG2 cells/mouse and the HepG2 cells were
allowed to grow for ca. 30 days. The mice were then randomized and
treated intraperitoneally (i.p.) with PTA021_A3-Formula M conjugate
(0.1 umole/kg, or 0.03 umol/kg). DT, and anti diptheria toxin
antibody, was used as a non binding isotype control. Nexavar
Sorafenib, a chemical-based multiple kinase inhibitor used to treat
renal cell carcinoma and liver cancer, was used as a control for
targeted therapy.
[0399] As seen in FIG. 19, treatment with the PTA021_A3-Formula M
conjugate significantly inhibited tumor growth rate in a dose
dependent fashion.
Example 10
Anti-CADM1 Antibody-Drug Conjugate Inhibits DMS79 Cell Growth in a
Mouse Xenograft Model
[0400] The effect of PTA021_A3-Formula M on the growth of small
cell lung cancer derived DMS79 cells in a mouse xenograft model was
examined. In this xenograft model, SCID mice (CB 17, from Charles
River Laboratories, Hollister, Calif.) were implanted with
5.times.10.sup.6 DMS79 cells/mouse and the DMS79 cells were allowed
to grow until the mean tumor volume was ca. 200 mm.sup.3. The mice
were then randomized and treated intraperitoneally (i.p.) with
PTA021_A3-Formula M conjugate in two studies.
[0401] As seen in FIG. 20A, treatment with the PTA021_A3-Formula M
conjugate at 0.3 .mu.mole/kg caused complete tumor regression in
all mice throughout the study (day 88). As seen in FIG. 20B,
treatment with the PTA021_A3-Formula M conjugate caused tumor
regression at the 0.1 umol/kg dose through the study which
completed on day 60. The low dose of 0.03 umol/kg caused
significant delay in tumor growth.
Example 11
Antibody-Dependent Cellular Cytotoxicity Mediated by PTA021_A3 and
Non-Fucosylated PTA021_A3
[0402] A fluorescence cytotoxicity assay was used to determine the
ability of the non fucosylated PTA021_A3 anti-CADM1 mAb to kill
CADM1-expressing cells in the presence of effector cells via
antibody dependent cellular cytotoxicity (ADCC with HepG2, 786-O
and DMS79 cells).
[0403] Human effector cells were prepared from whole blood as
follows. Human peripheral blood mononuclear cells were purified
from heparinized whole blood by standard Ficoll-paque separation.
The cells were resuspended in RPMI1640 media containing 10% FBS
(heat-inactivated) and 200 U/ml of human IL-2 and incubated
overnight at 37.degree. C. The following day, the cells were
collected and washed four times in culture media and resuspended at
2.times.10.sup.7 cells/ml. Target CHO-mesothelin cells were
prepared by incubating with BATDA reagent (Perkin Elmer, Wellesley,
Mass.) at 2.5 BATDA per 1.times.10.sup.6 target cells/mL for 20
minutes at 37.degree. C. The target cells were washed four times,
spun down and brought to a final volume of 1.times.10.sup.5
cells/ml.
[0404] The HepG2, 786-O and DMS79 cells were tested for antibody
specific ADCC to the human anti-CADM1 PTA021_A3 monoclonal antibody
and the non-fucosylated preparation of PTA021_A3 using the Delfia
fluorescence emission analysis as follows. HepG2, 786-O or DMS79
(100 .mu.l of labeled target cells, 10.sup.4 cells/well) were
incubated with 50 .mu.l of effector cells (10.sup.6 cells/well) and
50 .mu.l of antibody (10 ug/ml final concentration). A target to
effector ratio of 1:100 was used throughout the experiments. In all
studies, a human IgG1 isotype control was used as a negative
control. Cells were spun down at 2000 rpm and incubated for one
hour at 37.degree. C. The supernatants were then collected,
submitted to centrifugation and 20 .mu.l of supernatant was
transferred to a flat bottom plate, to which 180 .mu.l of Eu
solution (Perkin Elmer, Wellesley, Mass.) was added and read in a
RubyStar reader (BMG Labtech). The % lysis was calculated as
follows: (sample release-spontaneous release*100)/(maximum
release-spontaneous release), where the spontaneous release is the
fluorescence from wells which contain target cells plus effector
cells and maximum release is the fluorescence from wells containing
target cells and have been treated with 2% Triton-X.
[0405] FIGS. 21, 22 and 23 show both PTA021_A3 and non-fucosylated
PTA021_A3 are capable of eliciting ADCC on HepG2, 786-O and DMS79
cells and that the non-fucosylated PTA021_A3 is the more potent
antibody. The same effect can be observed on other cell lines that
express CADM1 such as the SCLC line NCI_H69.
Example 12
Anti-CADM1 Antibody/Cisplatin Inhibits DMS79 Cell Growth in a Mouse
Xenograft Model
[0406] The effect of PTA021_A3 alone and in combination with
cisplatin on the growth of SCLC derived DMS79 cells in a mouse
xenograft model was examined. In this xenograft model, SCID mice
(CB17, from Charles River Laboratories, Hollister, Calif.) were
implanted with 5.times.10.sup.6 DMS79 cells/mouse and the DMS79
cells were allowed to grow until the tumors reached an average of
200 mm3. The mice were then randomized and treated
intraperitoneally (i.p.) as shown in the tumor volume plot.
[0407] As seen in FIG. 24, treatment with PTA021_A3 alone delays
tumor growth, and in combination with cisplatin shows significant
and synergistic anti-tumor activity.
Example 13
Assessment of Toxicity of PTA021_A3NF, PTA021_A3, and
PTA021_A3-Formula M
[0408] The toxicity profile of anti CADM1 antibodies, including
nonfucosylated antibodies and antibody-drug conjugates, are
assessed in cynomolgus macaques using e.g., PTA021_A3 and its
derivatives. IHC assay results have shown that cynomolgus monkeys
and humans show similar patterns of expression of CADM1 when
subjected to IHC using PTA021_A3. Additionally, the CADM1 protein
shows high identity in cynos and humans. Multiple IV doses between
e.g. 0.1 mg/kg and 100 mg/kg are used to determine a maximum
tolerated dose for use to identify doses appropriate for clinical
trials in humans.
[0409] An exploratory toxicology study was conducted on PTA021_A3
and the non-fucosylated version of PTA021_A3(NF) in cynomolgus
macaques. 8 four-year old non-naive (naive to biologics but had
been previously treated in a small-molecule pharmacokinetic study)
cynomolgus monkeys were used in the study assessed in two groups
with two males and two females in each group. The animals in Group
1 were treated with the non-fucosylated PTA021_A3(NF) antibody in a
single intravenous (IV) dose at 1 mg/kg. The animals in Group 2
were treated with the PTA021_A3 antibody in a single IV dose at 1
mg/kg.
[0410] Clinical observations and mortality/morbidity were assessed
twice daily am and pm. Body weight, hematology and blood chemistry
were analysed on days--11, 0, 1, 3, 8, 15 and 22. Hematology
assessments included RBC, HGB, HCT, PLT, MCH, MCV, Reticulocytes,
WBC and Differential Count. Blood chemistry assessments included
AST, ALT, ALP, CRE, BUN, GLU, CHO, TP, ALB, TBIL, LDH, TG, Ca, P,
A/G, K, Na and Cl.
[0411] There were no significant overt observations (e.g. feeding
behaviour) during the study. There were no observable effects on
neuroendocrine tissues (e.g. hormonal changes which would lead to
behavioural changes and signs of distress). There was also no
evidence of pain or tremor noted. FIGS. 25 to 32 show that all of
the analyses conducted were within normal expected values.
[0412] FIGS. 25A and 25B show the Body Weight data for male monkeys
and female monkeys respectively.
[0413] FIGS. 26A and 26B show the Glucose data for male monkeys and
female monkeys respectively.
[0414] FIGS. 27A and 27B show the Alanine Transaminase data for
male monkeys and female monkeys respectively.
[0415] FIGS. 28A and 28B show the Aspartate Transaminase data for
male monkeys and female monkeys respectively.
[0416] FIGS. 29A and 29B show the Alkaline Phosphatase data for
male monkeys and female monkeys respectively.
[0417] FIGS. 30A and 30B show the Lactate Dehydrogenase data for
male monkeys and female monkeys respectively.
[0418] FIGS. 31A and 31B show the RBC data for male monkeys and
female monkeys respectively.
[0419] FIGS. 32A and 32B show the WBC data for male monkeys and
female monkeys respectively.
Example 14
Assessment of Efficacy of PTA021_A3NF, PTA021_A3, and
PTA021_A3-Formula M
[0420] CADM1 has been shown to be expressed in many cancer types by
IHC using the PTA021_A3 HuMAb. Cell lines representing cancer tumor
models are used to demonstrate efficacy of naked and toxin
conjugated anti CADM1 antibodies to treat various cancers. For
example, PTA021_A3 and derivatives thereof are used by themselves
or in combination with standards of care in efficacy models of
renal cancer (786-O cells), melanoma (SkMel28 cells) and small cell
lung cancer (DMS79 cells) where CADM1 is expressed. PTA021_A3 and
other anti-CADM1 antibodies may be used with xenograft animal
models using CADM1-expressing cell lines representing cancers such
as neuroendocrine cancers, colon cancer, breast cancer, ovarian
cancer, and prostate cancer.
[0421] All references cited in this specification, including
without limitation all papers, publications, patents, patent
applications, presentations, texts, reports, manuscripts,
brochures, books, internet postings, journal articles, periodicals,
product fact sheets, and the like, one hereby incorporated by
reference into this specification in their entireties. The
discussion of the references herein is intended to merely summarize
the assertions made by their authors and no admission is made that
any reference constitutes prior art and Applicants' reserve the
right to challenge the accuracy and pertinence of the cited
references.
[0422] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to those of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the dependant claims.
TABLE-US-00004 SUMMARY OF SEQUENCE LISTING SEQ ID NO: SEQUENCE 1
V.sub.H CDR1 amino acid PTA021_A1 2 V.sub.H CDR1 amino acid
PTA021_A2 3 V.sub.H CDR1 amino acid PTA021_A3 4 V.sub.H CDR2 amino
acid PTA021_A1 5 V.sub.H CDR2 amino acid PTA021_A2 6 V.sub.H CDR2
amino acid PTA021_A3 7 V.sub.H CDR3 amino acid PTA021_A1 8 V.sub.H
CDR3 amino acid PTA021_A2 9 V.sub.H CDR3 amino acid PTA021_A3 10
V.sub.K CDR1 amino acid PTA021_A1 11 V.sub.K CDR1 amino acid
PTA021_A2 12 V.sub.K CDR1 amino acid PTA021_A3 13 V.sub.K CDR2
amino acid PTA021_A1 14 V.sub.K CDR2 amino acid PTA021_A2 15
V.sub.K CDR2 amino acid PTA021_A3 16 V.sub.K CDR3 amino acid
PTA021_A1 17 V.sub.K CDR3 amino acid PTA021_A2 18 V.sub.K CDR3
amino acid PTA021_A3 19 V.sub.H amino acid PTA021_A1 20 V.sub.H
amino acid PTA021_A2 21 V.sub.H amino acid PTA021_A3 22 VK amino
acid PTA021_A1 23 VK amino acid PTA021_A2 24 VK amino acid
PTA021_A3 25 V.sub.H n.t. PTA021_A1 26 V.sub.H n.t. PTA021_A2 27
V.sub.H n.t. PTA021_A3 28 V.sub.K n.t. PTA021_A1 29 V.sub.K n.t.
PTA021_A2 30 V.sub.K n.t. PTA021_A3 31 V.sub.H 2-05 germline amino
acid 32 V.sub.H 2-05 germline amino acid 33 V.sub.H 2-05 germline
amino acid 34 V.sub.K L15 germline amino acid 35 V.sub.K L15
germline amino acid 36 V.sub.K L15 germline amino acid 37 V.sub.H
JH5b germline amino acid 38 V.sub.H JH5b germline amino acid 39
V.sub.H JH5b germline amino acid 40 JK4 germline amino acid 41 JK4
germline amino acid 42 JK4 germline amino acid 43 CADM1 construct
44 CADM1 ECD-6HIS 45 Conjugate peptide
Sequence CWU 1
1
4617PRTHomo sapiens 1Thr Ser Gly Val Gly Val Gly 1 5 27PRTHomo
sapiens 2Thr Ser Gly Val Gly Val Gly 1 5 37PRTHomo sapiens 3Thr Ser
Gly Val Gly Val Gly 1 5 416PRTHomo sapiens 4Leu Ile Tyr Trp Asp Asp
Asp Lys Arg Tyr Ser Pro Ser Leu Lys Ser 1 5 10 15 516PRTHomo
sapiens 5Leu Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Ser Pro Ser Leu
Lys Ser 1 5 10 15 616PRTHomo sapiens 6Leu Ile Tyr Trp Asp Asp Asp
Lys Arg Tyr Ser Pro Ser Leu Lys Ser 1 5 10 15 715PRTHomo sapiens
7Arg Arg Val Glu Trp Val Ala Leu Ala Gly Asn Trp Phe Asp Pro 1 5 10
15 815PRTHomo sapiens 8Arg Arg Val Glu Trp Phe Ala Leu Ala Gly Asn
Trp Phe Asp Pro 1 5 10 15 915PRTHomo sapiens 9Arg Arg Val Glu Trp
Val Thr Leu Ala Gly Asn Trp Phe Asp Pro 1 5 10 15 1011PRTHomo
sapiens 10Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala 1 5 10
1111PRTHomo sapiens 11Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala 1
5 10 1211PRTHomo sapiens 12Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu
Ala 1 5 10 137PRTHomo sapiens 13Gly Ala Ser Ser Leu Gln Ser 1 5
147PRTHomo sapiens 14Ala Ala Ser Ser Leu Gln Ser 1 5 157PRTHomo
sapiens 15Ala Ala Ser Ser Leu Gln Ser 1 5 169PRTHomo sapiens 16Gln
Gln Tyr Asn Ser Tyr Pro Leu Thr 1 5 179PRTHomo sapiens 17Gln Gln
Tyr Asn Ser Tyr Pro Leu Thr 1 5 189PRTHomo sapiens 18Gln Gln Tyr
Asn Ser Tyr Pro Leu Thr 1 5 19125PRTHomo sapiens 19Gln Ile Thr Leu
Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu
Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Asn Thr Ser 20 25 30
Gly Val Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35
40 45 Trp Leu Ala Leu Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Ser Pro
Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys
Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp
Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala His Arg Arg Val Glu Trp Val
Ala Leu Ala Gly Asn Trp Phe 100 105 110 Asp Pro Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser 115 120 125 20125PRTHomo sapiens 20Gln Ile
Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1 5 10 15
Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20
25 30 Gly Val Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu
Glu 35 40 45 Trp Leu Ala Leu Ile Tyr Trp Asp Asp Asp Lys Arg Tyr
Ser Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Thr
Ser Lys Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro
Val Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Ala His Arg Arg Val Glu
Trp Phe Ala Leu Ala Gly Asn Trp Phe 100 105 110 Asp Pro Trp Gly Gln
Gly Ser Leu Val Thr Val Ser Ser 115 120 125 21125PRTHomo sapiens
21Gln Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1
5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr
Ser 20 25 30 Gly Val Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys
Ala Leu Glu 35 40 45 Trp Leu Ala Leu Ile Tyr Trp Asp Asp Asp Lys
Arg Tyr Ser Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Thr Lys
Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met
Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala His Arg Arg
Val Glu Trp Val Thr Leu Ala Gly Asn Trp Phe 100 105 110 Asp Pro Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 22107PRTHomo
sapiens 22Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly
Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys
Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Gly Ala Ser Ser Leu Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Asn Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95 Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 23107PRTHomo sapiens
23Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser
Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys
Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys 100 105 24107PRTHomo sapiens 24Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25
30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile
35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Asn Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Tyr Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys 100 105 25375DNAHomo sapiensCDS(1)..(375) 25cag atc acc
ttg aag gag tct ggt cct acg ctg gtg aaa ccc aca cag 48Gln Ile Thr
Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1 5 10 15 acc
ctc acg ctg acc tgc acc ttc tct ggg ttc tca ctc aat act agt 96Thr
Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Asn Thr Ser 20 25
30 gga gtg ggt gtg ggc tgg atc cgt cag ccc cca gga aag gcc ctg gag
144Gly Val Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu
35 40 45 tgg ctt gca ctc att tat tgg gac gat gat aag cgc tac agc
cca tct 192Trp Leu Ala Leu Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Ser
Pro Ser 50 55 60 ctg aag agc agg ctc acc atc acc aag gac acc tcc
aaa aac cag gtg 240Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Thr Ser
Lys Asn Gln Val 65 70 75 80 gtc ctt aca atg acc aac atg gac cct gtg
gac aca gcc aca tat tac 288Val Leu Thr Met Thr Asn Met Asp Pro Val
Asp Thr Ala Thr Tyr Tyr 85 90 95 tgt gca cac agg aga gtt gaa tgg
gtc gcc ctg gca ggg aac tgg ttc 336Cys Ala His Arg Arg Val Glu Trp
Val Ala Leu Ala Gly Asn Trp Phe 100 105 110 gac ccc tgg ggc cag gga
acc ctg gtc acc gtc tcc tca 375Asp Pro Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 115 120 125 26375DNAHomo sapiensCDS(1)..(375) 26cag
atc acc ttg aag gag tct ggt cct acg ctg gtg aaa ccc aca cag 48Gln
Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1 5 10
15 acc ctc acg ctg acc tgc acc ttc tct ggg ttc tca ctc agc act agt
96Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser
20 25 30 gga gtg ggt gtg ggc tgg atc cgt cag ccc cca gga aag gcc
ctg gag 144Gly Val Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala
Leu Glu 35 40 45 tgg ctt gca ctc att tat tgg gat gat gat aag cgc
tac agc cca tct 192Trp Leu Ala Leu Ile Tyr Trp Asp Asp Asp Lys Arg
Tyr Ser Pro Ser 50 55 60 ctg aag agc agg ctc acc atc acc aag gac
acc tcc aaa aac cag gtg 240Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp
Thr Ser Lys Asn Gln Val 65 70 75 80 gtc ctt aca atg acc aac atg gac
cct gtg gac aca gcc ata tat tac 288Val Leu Thr Met Thr Asn Met Asp
Pro Val Asp Thr Ala Ile Tyr Tyr 85 90 95 tgt gcg cac agg aga gtt
gag tgg ttc gcc ctg gca ggg aac tgg ttc 336Cys Ala His Arg Arg Val
Glu Trp Phe Ala Leu Ala Gly Asn Trp Phe 100 105 110 gac ccc tgg ggc
cag gga tcc ctg gtc acc gtc tcc tca 375Asp Pro Trp Gly Gln Gly Ser
Leu Val Thr Val Ser Ser 115 120 125 27375DNAHomo
sapiensCDS(1)..(375) 27cag atc acc ttg aag gag tct ggt cct acg ctg
gtg aaa ccc aca cag 48Gln Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu
Val Lys Pro Thr Gln 1 5 10 15 acc ctc acg ctg acc tgc acc ttc tct
ggg ttc tca ctc agt act agt 96Thr Leu Thr Leu Thr Cys Thr Phe Ser
Gly Phe Ser Leu Ser Thr Ser 20 25 30 gga gtg ggt gtg ggc tgg atc
cgt cag ccc cca gga aag gcc ctg gag 144Gly Val Gly Val Gly Trp Ile
Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 tgg ctt gca ctc att
tat tgg gac gat gat aag cgc tac agc cca tct 192Trp Leu Ala Leu Ile
Tyr Trp Asp Asp Asp Lys Arg Tyr Ser Pro Ser 50 55 60 ctg aag agc
agg ctc acc atc acc aag gac acc tcc aaa aac cag gtg 240Leu Lys Ser
Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 gtc
ctt aca atg acc aac atg gac cct gtg gac aca gcc aca tat tac 288Val
Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90
95 tgt gca cac agg aga gtt gag tgg gtc acc ctg gca ggg aac tgg ttc
336Cys Ala His Arg Arg Val Glu Trp Val Thr Leu Ala Gly Asn Trp Phe
100 105 110 gac ccc tgg ggc cag gga acc ctg gtc acc gtc tcc tca
375Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125
28321DNAHomo sapiensCDS(1)..(321) 28gac atc cag atg acc cag tct cca
tcc tca ctg tct gca tct gta gga 48Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 gac aga gtc acc atc act
tgt cgg gcg agt cag ggt att agc agc tgg 96Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 tta gcc tgg tat
cag cag aaa cca gag aaa gcc cct aag tcc ctg atc 144Leu Ala Trp Tyr
Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 tat ggt
gca tcc agt ttg caa agt ggg gtc cca tca agg ttc agc ggc 192Tyr Gly
Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
agt gga tct ggg aca gat ttc act ctc acc atc agc aac ctg cag cct
240Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Leu Gln Pro
65 70 75 80 gaa gat ttt gca act tat tac tgc caa cag tat aat agt tac
cct ctc 288Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr
Pro Leu 85 90 95 act ttc ggc gga ggg acc aag gtg gag atc aaa 321Thr
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 29321DNAHomo
sapiensCDS(1)..(321) 29gac atc cag atg acc cag tct cca tcc tca ctg
tct gca tct gta gga 48Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 gac aga gtc acc atc act tgt cgg gcg
agt cag ggt att agc agc tgg 96Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Ser Trp 20 25 30 tta gcc tgg tat cag cag aaa
cca gag aaa gcc cct aag tcc ctg atc 144Leu Ala Trp Tyr Gln Gln Lys
Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 tat gct gca tcc agt
ttg caa agt ggg gtc cca tca agg ttc agc ggc 192Tyr Ala Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 agt gga tct
ggg aca gat ttc act ctc acc atc agc agc ctg cag cct 240Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 gaa
gat ttt gca act tat tac tgc caa cag tat aat agt tac cct ctc 288Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90
95 act ttc ggc gga ggg acc aag gtg gag atc aaa 321Thr Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys 100 105 30321DNAHomo
sapiensCDS(1)..(321) 30gac atc cag atg acc cag tct cca tcc tca ctg
tct gca tct gta gga 48Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 gac aga gtc acc atc act tgt cgg gcg
agt cag ggt att agc agc tgg 96Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Ser Trp 20 25 30 tta gcc tgg tat cag cag aaa
cca gag aaa gcc cct aag tcc ctg atc 144Leu Ala Trp Tyr Gln Gln Lys
Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 tat gct gca tcc agt
ttg caa agt ggg gtc cca tca agg ttc agc ggc 192Tyr Ala Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 agt gga tct
ggg aca gat ttc act ctc acc atc agc aac ctg cag cct 240Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Leu Gln Pro 65 70 75 80 gaa
gat ttt gca act tat tac tgc caa cag tat aat agt tac cct ctc 288Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90
95 act ttc ggc gga ggg acc aag gtg gag atc aaa 321Thr Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys 100 105 31100PRTHomo sapiens 31Gln Ile
Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1 5 10 15
Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20
25 30 Gly Val Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu
Glu 35 40 45 Trp Leu Ala Leu Ile Tyr Trp Asn Asp Asp Lys Arg Tyr
Ser Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Thr
Ser Lys Asn Gln Val 65 70 75 80 Val Leu Thr
Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys
Ala His Arg 100 32100PRTHomo sapiens 32Gln Ile Thr Leu Lys Glu Ser
Gly Pro Thr Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr
Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Val Gly
Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp
Leu Ala Leu Ile Tyr Trp Asn Asp Asp Lys Arg Tyr Ser Pro Ser 50 55
60 Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val
65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr
Tyr Tyr 85 90 95 Cys Ala His Arg 100 33100PRTHomo sapiens 33Gln Ile
Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1 5 10 15
Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20
25 30 Gly Val Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu
Glu 35 40 45 Trp Leu Ala Leu Ile Tyr Trp Asn Asp Asp Lys Arg Tyr
Ser Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Thr
Ser Lys Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro
Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala His Arg 100
3495PRTHomo sapiens 34Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro 85 90 95
3595PRTHomo sapiens 35Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro 85 90 95
3695PRTHomo sapiens 36Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro 85 90 95
3716PRTHomo sapiens 37Asn Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 1 5 10 15 3816PRTHomo sapiens 38Asn Trp Phe Asp
Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 15
3916PRTHomo sapiens 39Asn Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 1 5 10 15 4012PRTHomo sapiens 40Leu Thr Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys 1 5 10 4112PRTHomo sapiens 41Leu
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 1 5 10 4212PRTHomo
sapiens 42Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 1 5 10
43387PRTHomo sapiens 43Met Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu
Ala Gly Arg Ala Leu 1 5 10 15 Ala Gln Asn Leu Phe Thr Lys Asp Val
Thr Val Ile Glu Gly Glu Val 20 25 30 Ala Thr Ile Ser Cys Gln Val
Asn Lys Ser Asp Asp Ser Val Ile Gln 35 40 45 Leu Leu Asn Pro Asn
Arg Gln Thr Ile Tyr Phe Arg Asp Phe Arg Pro 50 55 60 Leu Lys Asp
Ser Arg Phe Gln Leu Leu Asn Phe Ser Ser Ser Glu Leu 65 70 75 80 Lys
Val Ser Leu Thr Asn Val Ser Ile Ser Asp Glu Gly Arg Tyr Phe 85 90
95 Cys Gln Leu Tyr Thr Asp Pro Pro Gln Glu Ser Tyr Thr Thr Ile Thr
100 105 110 Val Leu Val Pro Pro Arg Asn Leu Met Ile Asp Ile Gln Lys
Asp Thr 115 120 125 Ala Val Glu Gly Glu Glu Ile Glu Val Asn Cys Thr
Ala Met Ala Ser 130 135 140 Lys Pro Ala Thr Thr Ile Arg Trp Phe Lys
Gly Asn Thr Glu Leu Lys 145 150 155 160 Gly Lys Ser Glu Val Glu Glu
Trp Ser Asp Met Tyr Thr Val Thr Ser 165 170 175 Gln Leu Met Leu Lys
Val His Lys Glu Asp Asp Gly Val Pro Val Ile 180 185 190 Cys Gln Val
Glu His Pro Ala Val Thr Gly Asn Leu Gln Thr Gln Arg 195 200 205 Tyr
Leu Glu Val Gln Tyr Lys Pro Gln Val His Ile Gln Met Thr Tyr 210 215
220 Pro Leu Gln Gly Leu Thr Arg Glu Gly Asp Ala Leu Glu Leu Thr Cys
225 230 235 240 Glu Ala Ile Gly Lys Pro Gln Pro Val Met Val Thr Trp
Val Arg Val 245 250 255 Asp Asp Glu Met Pro Gln His Ala Val Leu Ser
Gly Pro Asn Leu Phe 260 265 270 Ile Asn Asn Leu Asn Lys Thr Asp Asn
Gly Thr Tyr Arg Cys Glu Ala 275 280 285 Ser Asn Ile Val Gly Lys Ala
His Ser Asp Tyr Met Leu Tyr Val Tyr 290 295 300 Asp Ser Arg Ala Gly
Glu Glu Gly Ser Ile Arg Ala Val Asp His Ala 305 310 315 320 Val Ile
Gly Gly Val Val Ala Val Val Val Phe Ala Met Leu Cys Leu 325 330 335
Leu Ile Ile Leu Gly Arg Tyr Phe Ala Arg His Lys Gly Thr Tyr Phe 340
345 350 Thr His Glu Ala Lys Gly Ala Asp Asp Ala Ala Asp Ala Asp Thr
Ala 355 360 365 Ile Ile Asn Ala Glu Gly Gly Gln Asn Asn Ser Glu Glu
Lys Lys Glu 370 375 380 Tyr Phe Ile 385 44326PRTHomo sapiens 44Met
Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu Ala Gly Arg Ala Leu 1 5 10
15 Thr Gln Asn Leu Phe Thr Lys Asp Val Thr Val Ile Glu Gly Glu Val
20 25 30 Ala Thr Ile Ser Cys Gln Val Asn Lys Ser Asp Asp Ser Val
Ile Gln 35 40 45 Leu Leu Asn Pro Asn Arg Gln Thr Ile Tyr Phe Arg
Asp Phe Arg Pro 50 55 60 Leu Lys Asp Ser Arg Phe Gln Leu Leu Asn
Phe Ser Ser Ser Glu Leu 65 70 75 80 Lys Val Ser Leu Thr Asn Val Ser
Ile Ser Asp Glu Gly Arg Tyr Phe 85 90 95 Cys Gln Leu Tyr Thr Asp
Pro Pro Gln Glu Ser Tyr Thr Thr Ile Thr 100 105 110 Val Leu Val Pro
Pro Arg Asn Leu Met Ile Asp Ile Gln Lys Asp Thr 115 120 125 Ala Val
Glu Gly Glu Glu Ile Glu Val Asn Cys Thr Ala Met Ala Ser 130 135 140
Lys Pro Ala Thr Thr Ile Arg Trp Phe Lys Gly Asn Thr Glu Leu Lys 145
150 155 160 Gly Lys Ser Glu Val Glu Glu Trp Ser Asp Met Tyr Thr Val
Thr Ser 165 170 175 Gln Leu Met Leu Lys Val His Lys Glu Asp Asp Gly
Val Pro Val Ile 180 185 190 Cys Gln Val Glu His Pro Ala Val Thr Gly
Asn Leu Gln Thr Gln Arg 195 200 205 Tyr Leu Glu Val Gln Tyr Lys Pro
Gln Val His Ile Gln Met Thr Tyr 210 215 220 Pro Leu Gln Gly Leu Thr
Arg Glu Gly Asp Ala Leu Glu Leu Thr Cys 225 230 235 240 Glu Ala Ile
Gly Lys Pro Gln Pro Val Met Val Thr Trp Val Arg Val 245 250 255 Asp
Asp Glu Met Pro Gln His Ala Val Leu Ser Gly Pro Asn Leu Phe 260 265
270 Ile Asn Asn Leu Asn Lys Thr Asp Asn Gly Thr Tyr Arg Cys Glu Ala
275 280 285 Ser Asn Ile Val Gly Lys Ala His Ser Asp Tyr Met Leu Tyr
Val Tyr 290 295 300 Asp Ser Arg Ala Gly Glu Glu Gly Ser Ile Arg Ala
Val Asp Ala Ser 305 310 315 320 His His His His His His 325
455PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide conjugate 45Pro Val Gly Val Val 1 5
4615PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 46Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser 1 5 10 15
* * * * *
References