U.S. patent application number 15/871802 was filed with the patent office on 2018-05-24 for therapeutic cd47 antibodies.
The applicant listed for this patent is TIOMA THERAPEUTICS, INC.. Invention is credited to Juan C. ALMAGRO, Robert W. KARR, Pamela T. MANNING, Robyn PURO.
Application Number | 20180142019 15/871802 |
Document ID | / |
Family ID | 62019023 |
Filed Date | 2018-05-24 |
United States Patent
Application |
20180142019 |
Kind Code |
A1 |
MANNING; Pamela T. ; et
al. |
May 24, 2018 |
THERAPEUTIC CD47 ANTIBODIES
Abstract
Provided are anti-CD47 monoclonal antibodies (anti-CD47 mAbs)
with distinct functional profiles as described herein, methods to
generate anti-CD47 mAbs, and to methods of using these anti-CD47
mAbs as therapeutics for the prevention and treatment of solid and
hematological cancers, ischemia-reperfusion injury, cardiovascular
diseases, autoimmune diseases, inflammatory diseases or as
diagnostics for determining the level of CD47 in tissue
samples.
Inventors: |
MANNING; Pamela T.; (St.
Louis, MO) ; PURO; Robyn; (St. Louis, MO) ;
ALMAGRO; Juan C.; (St. Louis, MO) ; KARR; Robert
W.; (St. Louis, MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TIOMA THERAPEUTICS, INC. |
St. Louis |
MO |
US |
|
|
Family ID: |
62019023 |
Appl. No.: |
15/871802 |
Filed: |
January 15, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2017/057716 |
Oct 20, 2017 |
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15871802 |
|
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62475032 |
Mar 22, 2017 |
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62411319 |
Oct 21, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 5/38 20180101; C07K
2317/732 20130101; A61P 3/10 20180101; A61P 1/00 20180101; A61P
35/00 20180101; A61K 2039/505 20130101; A61P 21/04 20180101; A61P
17/06 20180101; C07K 16/2803 20130101; C07K 2317/24 20130101; C07K
2317/56 20130101; A61P 9/00 20180101; C07K 2317/565 20130101; C07K
2317/92 20130101; A61P 7/06 20180101; A61P 35/02 20180101; A61P
9/10 20180101; A61P 25/28 20180101; C07K 2317/33 20130101; A61P
19/02 20180101; C07K 2317/734 20130101; A61P 37/02 20180101; C07K
2317/76 20130101; C07K 2317/73 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61P 35/02 20060101 A61P035/02; A61P 35/00 20060101
A61P035/00; A61P 9/00 20060101 A61P009/00; A61P 19/02 20060101
A61P019/02; A61P 25/28 20060101 A61P025/28; A61P 17/06 20060101
A61P017/06; A61P 1/00 20060101 A61P001/00; A61P 37/02 20060101
A61P037/02; A61P 5/38 20060101 A61P005/38; A61P 21/04 20060101
A61P021/04; A61P 7/06 20060101 A61P007/06; A61P 3/10 20060101
A61P003/10; A61P 9/10 20060101 A61P009/10 |
Claims
1-101. (canceled)
102. A monoclonal antibody, or an antigen binding fragment thereof,
which: a. binds to human CD47; b. blocks SIRP (binding to human
CD47; c. increases phagocytosis of human tumor cells; d. induces
death of human tumor cells; e. exhibits pH-dependent binding to
human CD47 present on a cell; and wherein the monoclonal antibody,
or an antigen binding fragment thereof, further comprises one or
more characteristics selected from the group consisting of: i)
causes no detectable agglutination of human red blood cells, or
causes reduced agglutination of human red blood cells, and ii) has
reduced human red blood cell binding, or has minimal human red
blood cell binding.
103. The monoclonal antibody, or antigen binding fragment thereof,
of claim 102, wherein said monoclonal antibody, or an antigen
binding fragment thereof, possesses one or more among the following
characteristics: causes an increase in cell surface calreticulin
expression on human tumor cells; causes an increase in adenosine
triphosphate (ATP) release by human tumor cells; causes an increase
in high mobility group box 1 (HMGB1) release by human tumor cells;
causes an increase in annexin A1 release by human tumor cells;
causes an increase in Type I Interferon release by human tumor
cells; causes an increase in C-X-C Motif Chemokine Ligand 10
(CXCL10) release by human tumor cells; causes an increase in cell
surface protein disulfide-isomerase A3 (PDIA3) expression on human
tumor cells; causes an increase in cell surface heat shock protein
70 (HSP70) expression on human tumor cells; and causes an increase
in cell surface heat shock protein 90 (HSP90) expression on human
tumor cells.
104. The monoclonal antibody, or antigen binding fragment thereof,
of claim 102, wherein the antibody, or antigen binding fragment
thereof, is a chimeric or humanized antibody; or wherein the
monoclonal antibody, or antigen binding fragment thereof
cross-reacts with one or more species homologs of CD47.
105. The monoclonal antibody, or antigen binding fragment thereof,
of claim 102, wherein the antibody, or antigen binding fragment
thereof, causes no detectable agglutination of human red blood
cells and has minimal human red blood cell binding.
106. The monoclonal antibody, or antigen binding fragment thereof,
of claim 102, wherein the pH-dependent binding to human CD47 has a
greater affinity for human CD47 at an acidic pH compared to
physiological pH.
107. A method of treating a disease comprising an autoimmune
disease, an autoinflammatory disease, an inflammatory disease, a
cardiovascular disease, or a cancer in a subject, comprising
administration of the monoclonal antibody or antigen-binding
fragment thereof, of claim 102, wherein the subject comprises a
human or companion/pet animal, a working animal, a sport animal, a
zoo animal, or a valuable animal kept in captivity.
108. The method of claim 107, wherein the disease is selected from
the group consisting of a leukemia, a lymphoma, ovarian cancer,
breast cancer, endometrial cancer, colon cancer (colorectal
cancer), rectal cancer, bladder cancer, urothelial cancer, lung
cancer (non-small cell lung cancer, adenocarcinoma of the lung,
squamous cell carcinoma of the lung), bronchial cancer, bone
cancer, prostate cancer, pancreatic cancer, gastric cancer,
hepatocellular carcinoma, gall bladder cancer, bile duct cancer,
esophageal cancer, renal cell carcinoma, thyroid cancer, squamous
cell carcinoma of the head and neck (head and neck cancer),
testicular cancer, cancer of the endocrine gland, cancer of the
adrenal gland, cancer of the pituitary gland, cancer of the skin,
cancer of soft tissues, cancer of blood vessels, cancer of brain,
cancer of nerves, cancer of eyes, cancer of meninges, cancer of
oropharynx, cancer of hypopharynx, cancer of cervix, and cancer of
uterus, glioblastoma, meduloblastoma, astrocytoma, glioma,
meningioma, gastrinoma, neuroblastoma, melanoma, myelodysplastic
syndrome, a sarcoma, ischemia-reperfusion injury, heart failure,
arthritis, rheumatoid arthritis, multiple sclerosis, psoriasis,
psoriatic arthritis, Crohn's disease, inflammatory bowel disease,
ulcerative colitis, lupus, systemic lupus erythematous, juvenile
rheumatoid arthritis, juvenile idiopathic arthritis, Grave's
disease, Hashimoto's thyroiditis, Addison's disease, celiac
disease, dermatomyositis, multiple sclerosis, myasthenia gravis,
pernicious anemia, Sjogren syndrome, type I diabetes, vasculitis,
uveitis, atherosclerosis, and ankylosing spondylitis.
109. A monoclonal antibody, or an antigen binding fragment thereof,
which: a. binds to human CD47; b. blocks SIRP.alpha. binding to
human CD47; c. increases phagocytosis of human tumor cells; d.
induces death of human tumor cells; e. exhibits pH-dependent
binding to human CD47 present on a cell; f. exhibits reduced
binding to a normal cell; and wherein the monoclonal antibody, or
an antigen binding fragment thereof, further comprises one or more
characteristics selected from the group consisting of: i) causes no
detectable agglutination of human red blood cells, or causes
reduced agglutination of human red blood cells; and ii) has reduced
human red blood cell binding, or has minimal human red blood cell
binding.
110. The monoclonal antibody, or antigen binding fragment thereof,
of claim 109, wherein said monoclonal antibody, or an antigen
binding fragment thereof, further comprises one or more
characteristics selected from the group consisting of: causes an
increase in cell surface calreticulin expression on human tumor
cells; causes an increase in adenosine triphosphate (ATP) release
by human tumor cells; causes an increase in high mobility group box
1 (HMGB1) release by human tumor cells; causes an increase in
annexin A1 release by human tumor cells; causes an increase in Type
I Interferon release by human tumor cells; causes an increase in
C-X-C Motif Chemokine Ligand 10 (CXCL10) release by human tumor
cells; causes an increase in cell surface protein
disulfide-isomerase A3 (PDIA3) expression on human tumor cells;
causes an increase in cell surface heat shock protein 70 (HSP70)
expression on human tumor cells; and causes an increase in cell
surface heat shock protein 90 (HSP90) expression on human tumor
cells.
111. The monoclonal antibody, or antigen binding fragment thereof,
of claim 109, wherein the antibody, or antigen binding fragment
thereof, is a chimeric or humanized antibody; or wherein the
monoclonal antibody, or antigen binding fragment thereof
cross-reacts with one or more species homologs of CD47.
112. The monoclonal antibody, or antigen binding fragment thereof,
of claim 109, wherein the antibody, or antigen binding fragment
thereof, causes no detectable agglutination of human red blood
cells and has minimal human red blood cell binding.
113. The monoclonal antibody, or antigen binding fragment thereof,
of claim 109, wherein the pH-dependent binding to CD47 has a
greater affinity for CD47 at an acidic pH compared to physiological
pH.
114. A method of treating a disease comprising an autoimmune
disease, an autoinflammatory disease, an inflammatory disease, a
cardiovascular disease, or a cancer in a subject, comprising
administration of the monoclonal antibody or antigen-binding
fragment thereof, of claim 109, wherein subject comprises a human
or companion/pet animal, a working animal, a sport animal, a zoo
animal, or a valuable animal kept in captivity.
115. The method of claim 114, wherein the disease is selected from
the group consisting of a leukemia, a lymphoma, ovarian cancer,
breast cancer, endometrial cancer, colon cancer (colorectal
cancer), rectal cancer, bladder cancer, urothelial cancer, lung
cancer (non-small cell lung cancer, adenocarcinoma of the lung,
squamous cell carcinoma of the lung), bronchial cancer, bone
cancer, prostate cancer, pancreatic cancer, gastric cancer,
hepatocellular carcinoma, gall bladder cancer, bile duct cancer,
esophageal cancer, renal cell carcinoma, thyroid cancer, squamous
cell carcinoma of the head and neck (head and neck cancer),
testicular cancer, cancer of the endocrine gland, cancer of the
adrenal gland, cancer of the pituitary gland, cancer of the skin,
cancer of soft tissues, cancer of blood vessels, cancer of brain,
cancer of nerves, cancer of eyes, cancer of meninges, cancer of
oropharynx, cancer of hypopharynx, cancer of cervix, and cancer of
uterus, glioblastoma, meduloblastoma, astrocytoma, glioma,
meningioma, gastrinoma, neuroblastoma, melanoma, myelodysplastic
syndrome, a sarcoma, ischemia-reperfusion injury, heart failure,
arthritis, rheumatoid arthritis, multiple sclerosis, psoriasis,
psoriatic arthritis, Crohn's disease, inflammatory bowel disease,
ulcerative colitis, lupus, systemic lupus erythematous, juvenile
rheumatoid arthritis, juvenile idiopathic arthritis, Grave's
disease, Hashimoto's thyroiditis, Addison's disease, celiac
disease, dermatomyositis, multiple sclerosis, myasthenia gravis,
pernicious anemia, Sjogren syndrome, type I diabetes, vasculitis,
uveitis, atherosclerosis, and ankylosing spondylitis.
116. A monoclonal antibody, or an antigen binding fragment thereof,
which binds to human CD47, wherein said monoclonal antibody, or an
antigen binding fragment thereof, exhibits pH-dependent binding to
CD47 present on a cell.
117. The monoclonal antibody, or an antigen binding fragment
thereof, of claim 116, which: a. binds to human CD47; b. blocks
SIRP.alpha. binding to human CD47; c. increases phagocytosis of
human tumor cells; and d. induces death of human tumor cells.
118. The monoclonal antibody, or antigen binding fragment thereof,
of claim 116, wherein said monoclonal antibody, or an antigen
binding fragment thereof, further comprises one or more
characteristics selected from the group consisting of: causes an
increase in cell surface calreticulin expression on human tumor
cells; causes an increase in adenosine triphosphate (ATP) release
by human tumor cells; causes an increase in high mobility group box
1 (HMGB1) release by human tumor cells; causes an increase in
annexin A1 release by human tumor cells; causes an increase in Type
I Interferon release by human tumor cells; causes an increase in
C-X-C Motif Chemokine Ligand 10 (CXCL10) release by human tumor
cells; causes an increase in cell surface protein
disulfide-isomerase A3 (PDIA3) expression on human tumor cells;
causes an increase in cell surface heat shock protein 70 (HSP70)
expression on human tumor cells; and causes an increase in cell
surface heat shock protein 90 (HSP90) expression on human tumor
cells.
119. The monoclonal antibody, or antigen binding fragment thereof,
of claim 116, wherein the antibody, or antigen binding fragment
thereof, is a chimeric or humanized antibody; or wherein the
monoclonal antibody, or antigen binding fragment thereof
cross-reacts with one or more species homologs of CD47.
120. The monoclonal antibody, or antigen binding fragment thereof,
of claim 116, wherein the pH-dependent binding to CD47 has a
greater affinity for CD47 at an acidic pH compared to physiological
pH.
121. A method of treating a disease comprising an autoimmune
disease, an autoinflammatory disease, an inflammatory disease, a
cardiovascular disease, or a cancer in a subject, comprising
administration of the monoclonal antibody or antigen-binding
fragment thereof, of claim 116, wherein the subject comprises a
human or companion/pet animal, a working animal, a sport animal, a
zoo animal, or a valuable animal kept in captivity.
122. The method of claim 121, wherein the disease is selected from
the group consisting of a leukemia, a lymphoma, ovarian cancer,
breast cancer, endometrial cancer, colon cancer (colorectal
cancer), rectal cancer, bladder cancer, urothelial cancer, lung
cancer (non-small cell lung cancer, adenocarcinoma of the lung,
squamous cell carcinoma of the lung), bronchial cancer, bone
cancer, prostate cancer, pancreatic cancer, gastric cancer,
hepatocellular carcinoma, gall bladder cancer, bile duct cancer,
esophageal cancer, renal cell carcinoma, thyroid cancer, squamous
cell carcinoma of the head and neck (head and neck cancer),
testicular cancer, cancer of the endocrine gland, cancer of the
adrenal gland, cancer of the pituitary gland, cancer of the skin,
cancer of soft tissues, cancer of blood vessels, cancer of brain,
cancer of nerves, cancer of eyes, cancer of meninges, cancer of
oropharynx, cancer of hypopharynx, cancer of cervix, and cancer of
uterus, glioblastoma, meduloblastoma, astrocytoma, glioma,
meningioma, gastrinoma, neuroblastoma, melanoma, myelodysplastic
syndrome, a sarcoma, ischemia-reperfusion injury, heart failure,
arthritis, rheumatoid arthritis, multiple sclerosis, psoriasis,
psoriatic arthritis, Crohn's disease, inflammatory bowel disease,
ulcerative colitis, lupus, systemic lupus erythematous, juvenile
rheumatoid arthritis, juvenile idiopathic arthritis, Grave's
disease, Hashimoto's thyroiditis, Addison's disease, celiac
disease, dermatomyositis, multiple sclerosis, myasthenia gravis,
pernicious anemia, Sjogren syndrome, type I diabetes, vasculitis,
uveitis, atherosclerosis, and ankylosing spondylitis.
123. The monoclonal antibody, or antigen binding fragment thereof,
of claim 116, wherein the monoclonal antibody, or antigen binding
fragment thereof, further exhibits reduced binding to a normal
cell.
124. A monoclonal antibody, or an antigen binding fragment thereof,
which binds to human CD47, wherein said monoclonal antibody, or an
antigen binding fragment thereof, exhibits reduced binding to a
normal cell.
125. The monoclonal antibody, or an antigen binding fragment
thereof, of claim 124, which: a. binds to human CD47; b. blocks
SIRP.alpha. binding to human CD47; c. increases phagocytosis of
human tumor cells; and d. induces death of human tumor cells.
126. The monoclonal antibody, or antigen binding fragment thereof,
of claim 124, wherein the normal cell is selected from the group
consisting of an endothelial cell, a skeletal muscle cell, an
epithelial cell, a PBMC, a T cell, a red blood cell, a peripheral
blood mononuclear cell, a human aortic endothelial cell, a human
skeletal muscle cell, a human microvascular endothelial cell, a
human renal tubular epithelial cell, a human peripherial blood CD3+
cell, and a human peripheral blood mononuclear cell.
127. The monoclonal antibody, or antigen binding fragment thereof,
of claim 124, wherein said monoclonal antibody, or an antigen
binding fragment thereof, further comprises one or more
characteristics selected from the group consisting of: causes an
increase in cell surface calreticulin expression on human tumor
cells; causes an increase in adenosine triphosphate (ATP) release
by human tumor cells; causes an increase in high mobility group box
1 (HMGB1) release by human tumor cells; causes an increase in
annexin A1 release by human tumor cells; causes an increase in Type
I Interferon release by human tumor cells; causes an increase in
C-X-C Motif Chemokine Ligand 10 (CXCL10) release by human tumor
cells; causes an increase in cell surface protein
disulfide-isomerase A3 (PDIA3) expression on human tumor cells;
causes an increase in cell surface heat shock protein 70 (HSP70)
expression on human tumor cells; and causes an increase in cell
surface heat shock protein 90 (HSP90) expression on human tumor
cells.
128. The monoclonal antibody, or antigen binding fragment thereof,
of claim 124, wherein the antibody, or antigen binding fragment
thereof, is a chimeric or humanized antibody; or wherein the
monoclonal antibody, or antigen binding fragment thereof
cross-reacts with one or more species homologs of CD47.
129. A method of treating a disease comprising an autoimmune
disease, an autoinflammatory disease, an inflammatory disease, a
cardiovascular disease, or a cancer in a subject, comprising
administration of the monoclonal antibody or antigen-binding
fragment thereof, of claim 124, wherein the subject comprises a
human or companion/pet animal, a working animal, a sport animal, a
zoo animal, or a valuable animal kept in captivity.
130. The method of claim 129, wherein the disease is selected from
the group consisting of a leukemia, a lymphoma, ovarian cancer,
breast cancer, endometrial cancer, colon cancer (colorectal
cancer), rectal cancer, bladder cancer, urothelial cancer, lung
cancer (non-small cell lung cancer, adenocarcinoma of the lung,
squamous cell carcinoma of the lung), bronchial cancer, bone
cancer, prostate cancer, pancreatic cancer, gastric cancer,
hepatocellular carcinoma, gall bladder cancer, bile duct cancer,
esophageal cancer, renal cell carcinoma, thyroid cancer, squamous
cell carcinoma of the head and neck (head and neck cancer),
testicular cancer, cancer of the endocrine gland, cancer of the
adrenal gland, cancer of the pituitary gland, cancer of the skin,
cancer of soft tissues, cancer of blood vessels, cancer of brain,
cancer of nerves, cancer of eyes, cancer of meninges, cancer of
oropharynx, cancer of hypopharynx, cancer of cervix, and cancer of
uterus, glioblastoma, meduloblastoma, astrocytoma, glioma,
meningioma, gastrinoma, neuroblastoma, melanoma, myelodysplastic
syndrome, a sarcoma, ischemia-reperfusion injury, heart failure,
arthritis, rheumatoid arthritis, multiple sclerosis, psoriasis,
psoriatic arthritis, Crohn's disease, inflammatory bowel disease,
ulcerative colitis, lupus, systemic lupus erythematous, juvenile
rheumatoid arthritis, juvenile idiopathic arthritis, Grave's
disease, Hashimoto's thyroiditis, Addison's disease, celiac
disease, dermatomyositis, multiple sclerosis, myasthenia gravis,
pernicious anemia, Sjogren syndrome, type I diabetes, vasculitis,
uveitis, atherosclerosis, and ankylosing spondylitis.
131. The monoclonal antibody, or antigen binding fragment thereof,
of claim 124, wherein the monoclonal antibody, or antigen binding
fragment thereof, further exhibits pH-dependent binding to CD47
present on a cell.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of International
Application No. PCT/US2017/057716, filed Oct. 20, 2017, which
claims the benefit of U.S. Provisional Application No. 62/411,319,
filed Oct. 21, 2016, and U.S. Provisional Application No.
62/475,032, filed Mar. 22, 2017, the disclosures of which are
incorporated herein in their entirety.
FIELD OF THE DISCLOSURE
[0002] This disclosure is related generally to anti-CD47 monoclonal
antibodies (anti-CD47 mAbs) with distinct functional profiles as
described herein, methods to generate anti-CD47 mAbs, and methods
of using these anti-CD47 mAbs as therapeutics for the prevention
and treatment of solid and hematological cancers,
ischemia-reperfusion injury, cardiovascular diseases, autoimmune
diseases, or inflammatory diseases, or as diagnostics for
determining the level of CD47 in tissue samples.
BACKGROUND OF THE DISCLOSURE
[0003] CD47 is a cell surface receptor comprised of an
extracellular IgV set domain, a 5 transmembrane domain, and a
cytoplasmic tail that is alternatively spliced. Two ligands bind
CD47: signal inhibitory receptor protein .alpha. (SIRP.alpha.) and
thrombospondin-1 (TSP1). CD47 expression and/or activity has been
implicated in a number of diseases and disorders. Accordingly,
there exists a need for therapeutic compositions and methods for
treating diseases and conditions associated with CD47 in humans and
animals, including the prevention and treatment of solid and
hematological cancers, ischemia-reperfusion injury (IRI),
cardiovascular diseases, or an autoimmune or inflammatory
disease.
SUMMARY OF THE DISCLOSURE
[0004] The present disclosure describes anti-CD47 mAbs with
distinct functional profiles. These antibodies possess distinct
combinations of properties selected from the following: 1) exhibit
cross-reactivity with one or more species homologs of CD47; 2)
block the interaction between CD47 and its ligand SIRP.alpha.; 3)
increase phagocytosis of human tumor cells; 4) induce death of
susceptible human tumor cells; 5) do not induce cell death of human
tumor cells; 6) do not have reduced or minimal binding to human red
blood cells (hRBCs); 7) have reduced binding to hRBCs; 8) have
minimal binding to hRBCs; 9) cause reduced agglutination of hRBCs;
10) cause no detectable agglutination of hRBCs; 11) reverse TSP1
inhibition of the nitric oxide (NO) pathway; 12) do not reverse
TSP1 inhibition of the NO pathway; 13) cause loss of mitochondrial
membrane potential; 14) do not cause cause loss of mitochondrial
membrane potential; 15) cause an increase in cell surface
calreticulin expression on human tumor cells; 16) do not cause an
increase in cell surface calreticulin expression on human tumor
cells; 17) cause an increase in adenosine triphosphate (ATP)
release by human tumor cells; 18) do not cause an increase in
adenosine triphosphate (ATP) release by human tumor cells; 19)
cause an increase in high mobility group box 1 (HMGB1) release by
human tumor cells; 20) do not cause an increase in high mobility
group box 1 (HMGB1) release by human tumor cells; 21) cause an
increase in type I interferon release by human tumor cells; 22) do
not cause an increase in type I interferon release by human tumor
cells; 23) cause an increase in C-X-C Motif Chemokine Ligand 10
(CXCL10) release by human tumor cells; 24) do not cause an increase
in C-X-C Motif Chemokine Ligand 10 (CXCL10) release by human tumor
cells; 25) cause an increase in cell surface protein
disulfide-isomerase A3 (PDIA3) expression on human tumor cells; 26)
do not cause an increase in cell surface protein
disulfide-isomerase A3 (PDIA3) expression on human tumor cells; 27)
cause an increase in cell surface heat shock protein 70 (HSP70)
expression on human tumor cells; 28) do not cause an increase in
cell surface heat shock protein 70 (HSP70) expression on human
tumor cells; 29) cause an increase in cell surface heat shock
protein 90 (HSP90) expression on human tumor cells; 30) do not
cause an increase in cell surface heat shock protein 90 (HSP90)
expression on human tumor cells; 31) have reduced binding to normal
human cells, which includes, but is not limited to, endothelial
cells, skeletal muscle cells, epithelial cells, and peripheral
blood mononuclear cells (e.g., human aortic endothelial cells,
human skeletal muscle cells, human microvascular endothelial cells,
human renal tubular epithelial cells, human peripherial blood CD3+
cells, and human peripheral blood mononuclear cells); 32) do not
have reduced binding to normal human cells, which includes, but is
not limited to, endothelial cells, skeletal muscle cells,
epithelial cells, and peripheral blood mononuclear cells (e.g.,
human aortic endothelial cells, human skeletal muscle cells, human
microvascular endothelial cells, human renal tubular epithelial
cells, human peripherial blood CD3+ cells, and human peripheral
blood mononuclear cells); 33) have a greater affinity for human
CD47 at an acidic pH compared to physiological pH; 34) do not have
a greater affinity for human CD47 at an acidic pH compared to
physiological pH; and 35) cause an increase in annexin A1 release
by human tumor cells. The anti-CD47 mAbs of the disclosure are
useful in various therapeutic methods for treating diseases and
conditions associated with CD47 in humans and animals, including
the prevention and treatment of solid and hematological cancers,
autoimmune diseases, inflammatory diseases, IRI, and cardiovascular
diseases. The antibodies of the disclosure are also useful as
diagnostics to determine the level of CD47 expression in tissue
samples. Embodiments of the disclosure include isolated antibodies
and immunologically active binding fragments thereof;
pharmaceutical compositions comprising one or more of the anti-CD47
mAbs, preferably chimeric or humanized forms of said anti-CD47
mAbs; methods of therapeutic use of such anti-CD47 monoclonal
antibodies; and cell lines that produce these anti-CD47 mAbs.
[0005] The embodiments of the disclosure include the mAbs, or
antigen-binding fragments thereof, which are defined herein by
reference to specific structural characteristics, i.e., specified
amino acid sequences of either the CDRs or entire heavy chain or
light chain variable domains. All antibodies of the disclosure bind
to CD47.
[0006] The monoclonal antibodies, or antigen binding fragments
thereof, may comprise at least one, usually at least three, CDR
sequences as provided herein, usually in combination with framework
sequences from a human variable region or as an isolated CDR
peptide. In some embodiments, an antibody comprises at least one
light chain comprising the three light chain CDR sequences provided
herein situated in a variable region framework, which may be,
without limitation, a murine or human variable region framework,
and at least one heavy chain comprising the three heavy chain CDR
sequences provided herein situated in a variable region framework,
which may be, without limitation, a human or murine variable region
framework.
[0007] Some embodiments of the disclosure are anti-CD47 mAbs, or
antigen binding fragments thereof, comprising a heavy chain
variable domain comprising a variable heavy chain CDR1, variable
heavy chain CDR2, and a variable heavy chain CDR3, wherein said
variable heavy chain CDR1 comprises an amino acid sequence selected
from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, and SEQ ID
NO:3; said variable heavy chain CDR2 comprises an amino acid
sequence selected from the group consisting of: SEQ ID NO:4, SEQ ID
NO:5, and SEQ ID NO:6; and said variable heavy chain CDR3 comprises
an amino acid sequence selected from the group consisting of: SEQ
ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10.
[0008] The heavy chain variable (V.sub.H) domain may comprise any
one of the listed variable heavy chain CDR1 sequences (HCDR1) in
combination with any one of the variable heavy chain CDR2 sequences
(HCDR2) and any one of the variable heavy chain CDR3 sequences
(HCDR3). However, certain embodiments of HCDR1 and HCDR2 and HCDR3
are are provided that derive from a single common V.sub.H domain,
examples of which are described herein.
[0009] The antibody or antigen binding fragment thereof may
additionally comprise a light chain variable (V.sub.L) domain,
which is paired with the V.sub.H domain to form an antigen binding
domain. In some embodiments, light chain variable domains are those
comprising a variable light chain CDR1, variable light chain CDR2,
and a variable light chain CDR3, wherein said variable light chain
CDR1 comprises an amino acid sequence selected from the group
consisting of: SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID
NO:14; said variable light chain CDR2 optionally comprises an amino
acid sequence selected from the group consisting of: SEQ ID NO:15,
SEQ ID NO:16, and SEQ ID NO:17; and said variable light chain CDR3
optionally comprises an amino acid sequence selected from the group
consisting of: SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.
[0010] The light chain variable domain may comprise any one of the
listed variable light chain CDR1 sequences (LCDR1) in combination
with any one of the variable light chain CDR2 sequences (LCDR2) and
any one of the variable light chain CDR3 sequences (LCDR3).
However, certain embodiments of LCDR1 and LCDR2 and LCDR3 are
provided that derive from a single common V.sub.L domain, examples
of which are described herein.
[0011] Any given CD47 antibody or antigen binding fragment thereof
comprising a V.sub.H domain paired with a V.sub.L domain will
comprise a combination of 6 CDRs: variable heavy chain CDR1
(HCDR1), variable heavy chain CDR2 (HCDR2), variable heavy chain
CDR3 (HCDR3), variable light chain CDR1 (LCDR1), variable light
chain CDR2 (LCDR2), and variable light chain CDR3 (LCDR3). Although
all combinations of 6 CDRs selected from the CDR sequence groups
listed above are permissible, and within the scope of the
disclosure, certain combinations of 6 CDRs are provided.
[0012] In some embodiments, combinations of 6 CDRs include, but are
not limited to, the combinations of variable heavy chain CDR1
(HCDR1), variable heavy chain CDR2 (HCDR2), variable heavy chain
CDR3 (HCDR3), variable light chain CDR1 (LCDR1), variable light
chain CDR2 (LCDR2), and variable light chain CDR3 (LCDR3) selected
from the group consisting of: [0013] (i) HCDR1 comprising SEQ ID
NO:1, HCDR2 comprising SEQ ID NO:4, HCDR3 comprising SEQ ID NO:7,
LCDR1 comprising SEQ ID NO:11, LCDR2 comprising SEQ ID NO:15, LCDR3
comprising SEQ ID NO:18; [0014] (ii) HCDR1 comprising SEQ ID NO:1,
HCDR2 comprising SEQ ID NO:4, HCDR3 comprising SEQ ID NO:8, LCDR1
comprising SEQ ID NO:11, LCDR2 comprising SEQ ID NO:15, LCDR3
comprising SEQ ID NO:18; [0015] (iii) HCDR1 comprising SEQ ID NO:2,
HCDR2 comprising SEQ ID NO:5, HCDR3 comprising SEQ ID NO:9, LCDR1
comprising SEQ ID NO:12, LCDR2 comprising SEQ ID NO:16, LCDR3
comprising SEQ ID NO:19; [0016] (iv) HCDR1 comprising SEQ ID NO:2,
HCDR2 comprising SEQ ID NO:5, HCDR3 comprising SEQ ID NO:9, LCDR1
comprising SEQ ID NO:13, LCDR2 comprising SEQ ID NO:16, LCDR3
comprising SEQ ID NO:19; and [0017] (v) HCDR1 comprising SEQ ID
NO:3, HCDR2 comprising SEQ ID NO:6, HCDR3 comprising SEQ ID NO:10,
LCDR1 comprising SEQ ID NO:14, LCDR2 comprising SEQ ID NO: 17,
LCDR3 comprising SEQ ID NO:20.
[0018] In some embodiments, anti-CD47 mAbs include antibodies or
antigen binding fragments thereof, comprising a heavy chain
variable domain having an amino acid sequence selected from the
group consisting of: the amino acid sequences of SEQ ID NO:21, SEQ
ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26,
SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID
NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ
ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID
NO:40, and amino acid sequences exhibiting at least 90%, 95%, 97%,
98%, or 99% sequence identity to one of the recited sequences.
Alternatively, or in addition, anti-CD47 mAbs including antibodies
or antigen binding fragments thereof, may comprise a light chain
variable domain having an amino acid sequence selected from the
group consisting of: the amino acid sequences of SEQ ID NO:41, SEQ
ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46,
SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID
NO:51, and SEQ ID NO:52, and amino acid sequences exhibiting at
least 90%, 95%, 97%, 98%, or 99% sequence identity to one of the
recited sequences.
[0019] Although all possible pairing of V.sub.H domains and V.sub.L
domains selected from the V.sub.H and V.sub.L domain sequence
groups listed above are permissible, and within the scope of the
disclosure, some embodiments provide certain combinations of
V.sub.H and V.sub.L domains. Accordingly, in some embodiments,
anti-CD47 mAbs, or antigen binding fragments thereof, are those
comprising a combination of a heavy chain variable domain (V.sub.H)
and a light chain variable domain (V.sub.L), wherein the
combination is selected from the group consisting of: [0020] (i) a
heavy chain variable domain comprising the amino acid sequence of
SEQ ID NO:21 and a light chain variable domain comprising the amino
acid sequence SEQ ID NO:41; [0021] (ii) a heavy chain variable
domain comprising the amino acid sequence of SEQ ID NO:23 and a
light chain variable domain comprising the amino acid sequence SEQ
ID NO:43; [0022] (iii) a heavy chain variable domain comprising the
amino acid sequence of SEQ ID NO:34 and a light chain variable
domain comprising the amino acid sequence SEQ ID NO:49; [0023] (iv)
a heavy chain variable domain comprising the amino acid sequence of
SEQ ID NO:36 and a light chain variable domain comprising the amino
acid sequence SEQ ID NO:52; [0024] (v) a heavy chain variable
domain comprising the amino acid sequence of SEQ ID NO:38 and a
light chain variable domain comprising the amino acid sequence SEQ
ID NO:52; [0025] (vi) a heavy chain variable domain comprising the
amino acid sequence of SEQ ID NO:39 and a light chain variable
domain comprising the amino acid sequence SEQ ID NO:52; [0026]
(vii) a heavy chain variable domain comprising the amino acid
sequence of SEQ ID NO:24 and a light chain variable domain
comprising the amino acid sequence SEQ ID NO:43; [0027] (viii) a
heavy chain variable domain comprising the amino acid sequence of
SEQ ID NO:37 and a light chain variable domain comprising the amino
acid sequence SEQ ID NO:52; [0028] (ix) a heavy chain variable
domain comprising the amino acid sequence of SEQ ID NO:33 and a
light chain variable domain comprising the amino acid sequence SEQ
ID NO:48; [0029] (x) a heavy chain variable domain comprising the
amino acid sequence of SEQ ID NO:26 and a light chain variable
domain comprising the amino acid sequence SEQ ID NO:44; [0030] (xi)
a heavy chain variable domain comprising the amino acid sequence of
SEQ ID NO:27 and a light chain variable domain comprising the amino
acid sequence SEQ ID NO:44; and [0031] (xii) a heavy chain variable
domain comprising the amino acid sequence of SEQ ID NO:38 and a
light chain variable domain comprising the amino acid sequence SEQ
ID NO:51; [0032] (xiii) a heavy chain variable domain comprising
the amino acid sequence of SEQ ID NO:39 and a light chain variable
domain comprising the amino acid sequence SEQ ID NO:51; [0033]
(xiv) a heavy chain variable domain comprising the amino acid
sequence of SEQ ID NO:40 and a light chain variable domain
comprising the amino acid sequence SEQ ID NO:52; [0034] (xv) a
heavy chain variable domain comprising the amino acid sequence of
SEQ ID NO:36 and a light chain variable domain comprising the amino
acid sequence SEQ ID NO:51; [0035] (xvi) a heavy chain variable
domain comprising the amino acid sequence of SEQ ID NO:29 and a
light chain variable domain comprising the amino acid sequence SEQ
ID NO:47; [0036] (xvii) a heavy chain variable domain comprising
the amino acid sequence of SEQ ID NO:30 and a light chain variable
domain comprising the amino acid sequence SEQ ID NO:47; [0037]
(xviii) a heavy chain variable domain comprising the amino acid
sequence of SEQ ID NO:31 and a light chain variable domain
comprising the amino acid sequence SEQ ID NO:47; [0038] (xix) a
heavy chain variable domain comprising the amino acid sequence of
SEQ ID NO:32 and a light chain variable domain comprising the amino
acid sequence SEQ ID NO:47; [0039] (xx) a heavy chain variable
domain comprising the amino acid sequence of SEQ ID NO:33 and a
light chain variable domain comprising the amino acid sequence SEQ
ID NO:47; [0040] (xxi) a heavy chain variable domain comprising the
amino acid sequence of SEQ ID NO:29 and a light chain variable
domain comprising the amino acid sequence SEQ ID NO:48; [0041]
(xxii) a heavy chain variable domain comprising the amino acid
sequence of SEQ ID NO:30 and a light chain variable domain
comprising the amino acid sequence SEQ ID NO:48; [0042] (xxiii) a
heavy chain variable domain comprising the amino acid sequence of
SEQ ID NO:31 and a light chain variable domain comprising the amino
acid sequence SEQ ID NO:48; [0043] (xxiv) a heavy chain variable
domain comprising the amino acid sequence of SEQ ID NO:32 and a
light chain variable domain comprising the amino acid sequence SEQ
ID NO:48; [0044] (xxv) a heavy chain variable domain comprising the
amino acid sequence of SEQ ID NO:26 and a light chain variable
domain comprising the amino acid sequence SEQ ID NO:43; [0045]
(xxvi) a heavy chain variable domain comprising the amino acid
sequence of SEQ ID NO:27 and a light chain variable domain
comprising the amino acid sequence SEQ ID NO:43; [0046] (xxvii) a
heavy chain variable domain comprising the amino acid sequence of
SEQ ID NO:28 and a light chain variable domain comprising the amino
acid sequence SEQ ID NO:46; [0047] (xxviii) a heavy chain variable
domain comprising the amino acid sequence of SEQ ID NO:35 and a
light chain variable domain comprising the amino acid sequence SEQ
ID NO:50; [0048] (xxix) a heavy chain variable domain comprising
the amino acid sequence of SEQ ID NO:29 and a light chain variable
domain comprising the amino acid sequence SEQ ID NO:48; [0049]
(xxx) a heavy chain variable domain comprising the amino acid
sequence of SEQ ID NO:30 and a light chain variable domain
comprising the amino acid sequence SEQ ID NO:48; [0050] (xxxi) a
heavy chain variable domain comprising the amino acid sequence of
SEQ ID NO:31 and a light chain variable domain comprising the amino
acid sequence SEQ ID NO:48; [0051] (xxxii) a heavy chain variable
domain comprising the amino acid sequence of SEQ ID NO:32 and a
light chain variable domain comprising the amino acid sequence SEQ
ID NO:48; [0052] (xxxiii) a heavy chain variable domain comprising
the amino acid sequence of SEQ ID NO:37 and a light chain variable
domain comprising the amino acid sequence SEQ ID NO:51; and [0053]
(xxxiv) a heavy chain variable domain comprising the amino acid
sequence of SEQ ID NO:40 and a light chain variable domain
comprising the amino acid sequence SEQ ID NO:51.
[0054] In some embodiments, anti-CD47 antibodies or antigen binding
fragments thereof may also comprise a combination of a heavy chain
variable domain and a light chain variable domain wherein the heavy
chain variable domain comprises a V.sub.H sequence with at least
85% sequence identity, or at least 90% sequence identity, or at
least 95% sequence identity, or at least 97%, 98% or 99% sequence
identity, to the heavy chain amino acid sequences shown above in
(i) to (xxxiv) and/or the light chain variable domain comprises a
V.sub.L sequence with at least 85% sequence identity, or at least
90% sequence identity, or at least 95% sequence identity, or at
least 97%, 98% or 99% sequence identity, to the light chain amino
acid sequences shown above in (i) to (xxxiv). The specific V.sub.H
and V.sub.L pairings or combinations in parts (i) through (xxxiv)
may be preserved for anti-CD47 antibodies having V.sub.H and
V.sub.L domain sequences with a particular percentage sequence
identity to these reference sequences.
[0055] For all embodiments wherein the heavy chain and/or light
chain variable domains of the antibodies or antigen binding
fragments are defined by a particular percentage sequence identity
to a reference sequence, the V.sub.H and/or V.sub.L domains may
retain identical CDR sequences to those present in the reference
sequence such that the variation is present only within the
framework regions.
[0056] In another embodiment, CD47 antibodies, or antigen binding
fragments thereof, may comprise a combination of a heavy chain (HC)
and a light chain (LC), wherein the combination is selected from
the group consisting of: [0057] (i) a heavy chain comprising the
amino acid sequence of SEQ ID NO:78 and a light chain comprising
the amino acid sequence SEQ ID NO:67; [0058] (ii) a heavy chain
comprising the amino acid sequence of SEQ ID NO:79 and a light
chain comprising the amino acid sequence SEQ ID NO:69; [0059] (iii)
a heavy chain comprising the amino acid sequence of SEQ ID NO:80
and a light chain comprising the amino acid sequence SEQ ID NO:70;
[0060] (iv) a heavy chain comprising the amino acid sequence of SEQ
ID NO:81 and a light chain comprising the amino acid sequence SEQ
ID NO:71; [0061] (v) a heavy chain comprising the amino acid
sequence of SEQ ID NO:82 and a light chain comprising the amino
acid sequence SEQ ID NO:71; [0062] (vi) a heavy chain comprising
the amino acid sequence of SEQ ID NO:83 and a light chain
comprising the amino acid sequence SEQ ID NO:71; [0063] (vii) a
heavy chain comprising the amino acid sequence of SEQ ID NO:84 and
a light chain comprising the amino acid sequence SEQ ID NO:69;
[0064] (viii) a heavy chain comprising the amino acid sequence of
SEQ ID NO:85 and a light chain comprising the amino acid sequence
SEQ ID NO:71; [0065] (ix) a heavy chain comprising the amino acid
sequence of SEQ ID NO:86 and a light chain comprising the amino
acid sequence SEQ ID NO:72; [0066] (x) a heavy chain comprising the
amino acid sequence of SEQ ID NO:87 and a light chain comprising
the amino acid sequence SEQ ID NO:73; [0067] (xi) a heavy chain
comprising the amino acid sequence of SEQ ID NO:88 and a light
chain comprising the amino acid sequence SEQ ID NO:73; [0068] (xii)
a heavy chain comprising the amino acid sequence of SEQ ID NO:82
and a light chain comprising the amino acid sequence SEQ ID NO:74;
[0069] (xiii) a heavy chain comprising the amino acid sequence of
SEQ ID NO:83 and a light chain comprising the amino acid sequence
SEQ ID NO:74; [0070] (xiv) a heavy chain comprising the amino acid
sequence of SEQ ID NO:89 and a light chain comprising the amino
acid sequence SEQ ID NO:71; [0071] (xv) a heavy chain comprising
the amino acid sequence of SEQ ID NO:81 and a light chain
comprising the amino acid sequence SEQ ID NO:74; [0072] (xvi) a
heavy chain comprising the amino acid sequence of SEQ ID NO:90 and
a light chain comprising the amino acid sequence SEQ ID NO:75;
[0073] (xvii) a heavy chain comprising the amino acid sequence of
SEQ ID NO:91 and a light chain comprising the amino acid sequence
SEQ ID NO:75; [0074] (xviii) a heavy chain comprising the amino
acid sequence of SEQ ID NO:92 and a light chain comprising the
amino acid sequence SEQ ID NO:75; [0075] (xix) a heavy chain
comprising the amino acid sequence of SEQ ID NO:93 and a light
chain comprising the amino acid sequence SEQ ID NO:75; [0076] (xx)
a heavy chain comprising the amino acid sequence of SEQ ID NO:86
and a light chain comprising the amino acid sequence SEQ ID NO:75;
[0077] (xxi) a heavy chain comprising the amino acid sequence of
SEQ ID NO:94 and a light chain comprising the amino acid sequence
SEQ ID NO:72; [0078] (xxii) a heavy chain comprising the amino acid
sequence of SEQ ID NO:91 and a light chain comprising the amino
acid sequence SEQ ID NO:72; [0079] (xxiii) a heavy chain comprising
the amino acid sequence of SEQ ID NO:92 and a light chain
comprising the amino acid sequence SEQ ID NO:72; [0080] (xxiv) a
heavy chain comprising the amino acid sequence of SEQ ID NO:93 and
a light chain comprising the amino acid sequence SEQ ID NO:72;
[0081] (xxv) a heavy chain comprising the amino acid sequence of
SEQ ID NO:87 and a light chain comprising the amino acid sequence
SEQ ID NO:69; [0082] (xxvi) a heavy chain comprising the amino acid
sequence of SEQ ID NO:88 and a light chain comprising the amino
acid sequence SEQ ID NO:69; [0083] (xxvii) a heavy chain comprising
the amino acid sequence of SEQ ID NO:95 and a light chain
comprising the amino acid sequence SEQ ID NO:76; [0084] (xxviii) a
heavy chain comprising the amino acid sequence of SEQ ID NO:96 and
a light chain comprising the amino acid sequence SEQ ID NO:77;
[0085] (xxix) a heavy chain comprising the amino acid sequence of
SEQ ID NO:97 and a light chain comprising the amino acid sequence
SEQ ID NO:72; [0086] (xxx) a heavy chain comprising the amino acid
sequence of SEQ ID NO:98 and a light chain comprising the amino
acid sequence SEQ ID NO:72; [0087] (xxxi) a heavy chain comprising
the amino acid sequence of SEQ ID NO:99 and a light chain
comprising the amino acid sequence SEQ ID NO:72; [0088] (xxxii) a
heavy chain comprising the amino acid sequence of SEQ ID NO: 100
and a light chain comprising the amino acid sequence SEQ ID NO:72;
[0089] (xxxiii) a heavy chain comprising the amino acid sequence of
SEQ ID NO:85 and a light chain comprising the amino acid sequence
SEQ ID NO:74; [0090] (xxxiv) a heavy chain comprising the amino
acid sequence of SEQ ID NO:89 and a light chain comprising the
amino acid sequence SEQ ID NO:74; [0091] wherein the V.sub.H amino
acid sequence is at least 90%, 95%, 97%, 98% or 99% identical
thereto and the a V.sub.L amino acid sequence is at least 90%, 95%,
97%, 98% or 99% identical thereto.
[0092] In some embodiments, anti-CD47 antibodies as described
herein may also be characterized by combinations of properties
which are not exhibited by prior art anti-CD47 antibodies proposed
for human therapeutic use. Accordingly, in some embodiments,
anti-CD47 antibodies described herein are characterized by: [0093]
a. binds to human CD47; [0094] b. blocks SIRP.alpha. binding to
human CD47; [0095] c. increases phagocytosis of human tumor cells;
and [0096] d. induces death of susceptible human tumor cells.
[0097] In another embodiment described herein, the anti-CD47
antibodies are characterized by: [0098] a. binds to human CD47;
[0099] b. blocks SIRP.alpha. binding to human CD47; [0100] c.
increases phagocytosis of human tumor cells; [0101] d. induces
death of susceptible human tumor cells; and [0102] e. causes no
detectable agglutination of human red blood cells (hRBCs).
[0103] In yet another embodiment described herein, the anti-CD47
antibodies are characterized by: [0104] a. binds to human CD47;
[0105] b. blocks SIRP.alpha. binding to human CD47; [0106] c.
increases phagocytosis of human tumor cells; [0107] d. induces
death of susceptible human tumor cells; and [0108] e. causes
reduced agglutination of human red blood cells (hRBCs).
[0109] In another embodiment described herein, the anti-CD47
antibodies are characterized by: [0110] a. binds to human CD47;
[0111] b. blocks SIRP.alpha. binding to human CD47; [0112] c.
increases phagocytosis of human tumor cells; [0113] d. induces
death of susceptible human tumor cells; and [0114] e. has reduced
hRBC binding.
[0115] In another embodiment described herein, the anti-CD47
antibodies are characterized by: [0116] a. binds to human CD47,
[0117] b. blocks SIRP.alpha. binding to human CD47, [0118] c.
increases phagocytosis of human tumor cells, [0119] d. causes no
detectable agglutination of human red blood cells (hRBCs); and
[0120] e. has minimal binding to hRBCs.
[0121] In another embodiment described herein, the anti-CD47
antibodies are characterized by: [0122] a. binds to human CD47;
[0123] b. blocks SIRP.alpha. binding to human CD47; [0124] c.
increases phagocytosis of human tumor cells; [0125] d. causes no
detectable agglutination of human red blood cells (hRBCs); and
[0126] e. has reduced hRBC binding.
[0127] Additional embodiments of the anti-CD47 antibodies described
herein, are also characterized by combinations of properties which
are not exhibited by prior art anti-CD47 antibodies proposed for
human therapeutic use. Accordingly, anti-CD47 antibodies as
described herein may be further characterized by one or more among
the following characteristics: [0128] a. causes an increase in cell
surface calreticulin expression on human tumor cells; [0129] b.
causes an increase in adenosine triphosphate (ATP) release by human
tumor cells; [0130] c. causes an increase in high mobility group
box 1 (HMGB1) release by human tumor cells; [0131] d. causes an
increase in annexin A1 release by human tumor cells; [0132] e.
causes an increase in Type I Interferon release by human tumor
cells; [0133] f. causes an increase in C-X-C Motif Chemokine Ligand
10 (CXCL10) release by human tumor cells; [0134] g. causes an
increase in cell surface protein disulfide-isomerase A3 (PDIA3)
expression on human tumor cells; [0135] h. causes an increase in
cell surface heat shock protein 70 (HSP70) expression on human
tumor cells; and [0136] i. causes an increase in cell surface heat
shock protein 90 (HSP90) expression on human tumor cells.
[0137] In another embodiment described herein, the monoclonal
antibody, or antigen binding fragment thereof binds to human,
non-human primate, mouse, rabbit, and rat CD47.
[0138] In yet another embodiment described herein, the monoclonal
antibody, or antigen binding fragment thereof specifically also
binds to non-human primate CD47, wherein non-human primate may
include, but is not limited to, cynomolgus monkey, green monkey,
rhesus monkey, and squirrel monkey.
[0139] In yet another embodiment described herein, the monoclonal
antibody, or antigen binding fragment thereof, has reduced binding
to normal human cells, which includes, but is not limited to,
endothelial cells, skeletal muscle cells, epithelial cells, and
peripheral blood mononuclear cells (e.g., human aortic endothelial
cells, human skeletal muscle cells, human microvascular endothelial
cells, human renal tubular epithelial cells, human peripherial
blood CD3+ cells, and human peripheral blood mononuclear
cells).
[0140] In yet another embodiment described herein, the monoclonal
antibody, or antigen binding fragment thereof, has a greater have a
greater affinity for human CD47 at an acidic pH compared to
physiological pH.
[0141] In some embodiments, the monoclonal antibody, or antigen
binding fragment thereof, may additionally possess one or more of
the following characteristics: 1) exhibit cross-reactivity with one
or more species homologs of CD47; 2) block the interaction between
CD47 and its ligand SIRP.alpha.; 3) increase phagocytosis of human
tumor cells; 4) induce death of susceptible human tumor cells; 5)
do not induce cell death of human tumor cells; 6) do not have
reduced or minimal binding to human red blood cells (hRBCs); 7)
have reduced binding to hRBCs; 8) have minimal binding to hRBCs; 9)
cause reduced agglutination of hRBCs; 10) cause no detectable
agglutination of hRBCs; 11) reverse TSP1 inhibition of the nitric
oxide (NO) pathway; 12) do not reverse TSP1 inhibition of the NO
pathway; 13) cause loss of mitochondrial membrane potential; 14) do
not cause cause loss of mitochondrial membrane potential; 15) cause
an increase in cell surface calreticulin expression on human tumor
cells; 16) do not cause an increase in cell surface calreticulin
expression on human tumor cells; 17) cause an increase in adenosine
triphosphate (ATP) release by human tumor cells; 18) do not cause
an increase in adenosine triphosphate (ATP) release by human tumor
cells; 19) cause an increase in high mobility group box 1 (HMGB1)
release by human tumor cells; 20) do not cause an increase in high
mobility group box 1 (HMGB1) release by human tumor cells; 21)
cause an increase in type I interferon release by human tumor
cells; 22) do not cause an increase in type I interferon release by
human tumor cells; 23) cause an increase in C-X-C Motif Chemokine
Ligand 10 (CXCL10) release by human tumor cells; 24) do not cause
an increase in C-X-C Motif Chemokine Ligand 10 (CXCL10) release by
human tumor cells; 25) cause an increase in cell surface protein
disulfide-isomerase A3 (PDIA3) expression on human tumor cells; 26)
do not cause an increase in cell surface protein
disulfide-isomerase A3 (PDIA3) expression on human tumor cells; 27)
cause an increase in cell surface heat shock protein 70 (HSP70)
expression on human tumor cells; 28) do not cause an increase in
cell surface heat shock protein 70 (HSP70) expression on human
tumor cells; 29) cause an increase in cell surface heat shock
protein 90 (HSP90) expression on human tumor cells; 30) do not
cause an increase in cell surface heat shock protein 90 (HSP90)
expression on human tumor cells; 31) have reduced binding to normal
human cells, which includes, but is not limited to, endothelial
cells, skeletal muscle cells, epithelial cells, and peripheral
blood mononuclear cells (e.g., human aortic endothelial cells,
human skeletal muscle cells, human microvascular endothelial cells,
human renal tubular epithelial cells, human peripherial blood CD3+
cells, and human peripheral blood mononuclear cells); 32) do not
have reduced binding to normal human cells, which includes, but is
not limited to, endothelial cells, skeletal muscle cells,
epithelial cells, and peripheral blood mononuclear cells (e.g.,
human aortic endothelial cells, human skeletal muscle cells, human
microvascular endothelial cells, human renal tubular epithelial
cells, human peripherial blood CD3+ cells, and human peripheral
blood mononuclear cells); 33) have a greater affinity for human
CD47 at an acidic pH compared to physiological pH; 34) do not have
a greater affinity for human CD47 at an acidic pH compared to
physiological pH; and 35) cause an increase in annexin A1 release
by human tumor cells.
[0142] Various forms of the anti-CD47 mAbs disclosed are
contemplated herein. For example, the anti-CD47 mAbs can be
full-length humanized antibodies with human frameworks and constant
regions of the isotypes, IgA, IgD, IgE, IgG, and IgM, more
particularly, IgG1, IgG2, IgG3, IgG4, and in some cases with
various mutations to alter Fc receptor function or prevent Fab arm
exchange or an antibody fragment, e.g., a F(ab')2 fragment, a F(ab)
fragment, a single chain Fv fragment (scFv), etc., as disclosed
herein.
[0143] In some embodiments, pharmaceutical or veterinary
compositions are provided that comprise one or more of the
anti-CD47 mAbs or fragments disclosed herein, optionally chimeric
or humanized forms, and a pharmaceutically acceptable carrier,
diluent, or excipient.
[0144] Prior to the present disclosure, there was a need to
identify anti-CD47 mAbs that possess the functional profiles as
described herein. The anti-CD47 mAbs of the present disclosure
exhibit distinct combinations of properties, particularly
combinations of properties that render the mAbs particularly
advantageous or suitable for use in human therapy, particularly in
the prevention and/or treatment of solid and hematological cancers,
ischemia-reperfusion injury, autoimmune and/or inflammatory
diseases.
[0145] In some embodiments, the disclosure provides a monoclonal
antibody, or an antigen binding fragment thereof, which: binds to
human CD47; blocks SIRP.alpha. binding to human CD47; increases
phagocytosis of human tumor cells; and induces death of human tumor
cells; wherein said monoclonal antibody, or an antigen binding
fragment thereof, exhibits pH-dependent binding to CD47 present on
a cell. In other embodiments, the disclosure provides a monoclonal
antibody, or an antigen binding fragment thereof, which: binds to
human CD47; blocks SIRP.alpha. binding to human CD47; increases
phagocytosis of human tumor cells; wherein said monoclonal
antibody, or an antigen binding fragment thereof, exhibits
pH-dependent binding to CD47 present on a cell. In other
embodiments, the disclosure provides a monoclonal antibody, or an
antigen binding fragment thereof, which: binds to human CD47;
blocks SIRP.alpha. binding to human CD47; increases phagocytosis of
human tumor cells; and induces death of human tumor cells; wherein
said monoclonal antibody, or an antigen binding fragment thereof,
exhibits reduced binding to normal cells. In one embodiment, these
cells may be an endothelial cell, a skeletal muscle cell, an
epithelial cell, a PBMC or a RBC (e.g., human aortic endothelial
cells, human skeletal muscle cells, human microvascular endothelial
cells, human renal tubular epithelial cells, human peripherial
blood CD3+ cells, human peripheral blood mononuclear cells or human
RBC). In other embodiments, the disclosure provides a monoclonal
antibody, or an antigen binding fragment thereof, which: binds to
human CD47; blocks SIRP.alpha. binding to human CD47; increases
phagocytosis of human tumor cells; wherein said monoclonal
antibody, or an antigen binding fragment thereof, exhibits reduced
binding to normal cells. In one embodiment, these cells may be an
endothelial cell, a skeletal muscle cell, an epithelial cell, a
PBMC or a RBC (e.g., human aortic endothelial cells, human skeletal
muscle cells, human microvascular endothelial cells, human renal
tubular epithelial cells, human peripherial blood CD3+ cells, human
peripheral blood mononuclear cells or human RBC). In another
embodiment, the monoclonal antibody, or an antigen binding fragment
thereof, exhibits both pH dependent binding and reduced binding to
a cell.
[0146] Further scope of the applicability of the present disclosure
will become apparent from the detailed description provided below.
However, it should be understood that the detailed description and
specific examples, while indicating some embodiments of the
disclosure, are given by way of illustration only since various
changes and modifications within the spirit and scope of the
disclosure will become apparent to those skilled in the art from
this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0147] The above and other aspects, features, and advantages of the
present disclosure will be better understood from the following
detailed descriptions taken in conjunction with the accompanying
drawing(s), all of which are given by way of illustration only, and
are not limitative of the present disclosure.
[0148] FIG. 1A. Binding of VLX4 Humanized mAbs to Human OV10 Cells
Expressing Human CD47. Binding of VLX4 humanized mAbs (VLX4hum_01
IgG1, VLX4hum_02 IgG1, VLX4hum_01 IgG4PE, and VLX4hum_02 IgG4PE) to
human CD47 was determined using a OV10 cell line expressing human
CD47 (OV10 hCD47) cell-based ELISA. OV10 hCD47 cells were plated
into 96 well plates and were confluent at the time of assay.
Various concentrations of mAbs were added to the cells for 1 hr.
Cells were washed and then incubated with HRP-labelled secondary
antibody for 1 hr followed by addition of peroxidase substrate.
[0149] FIG. 1B. Binding of VLX4 Humanized mAbs to Human OV10 Cells
Expressing Human CD47. Binding of VLX4 humanized mAbs (VLX4hum_06
IgG4PE, VLX4hum_07 IgG4PE, VLX4hum_12 IgG4PE, and VLX4hum_13
IgG4PE) to human CD47 was determined using an OV10 CD47 cell-based
ELISA. OV10 hCD47 cells were plated into 96 well plates and were
confluent at the time of assay. Various concentrations of VLX4
representative mAbs were added to the cells for 1 hr. Cells were
washed and then incubated with HRP-labelled secondary antibody for
1 hr followed by addition of peroxidase substrate.
[0150] FIG. 2A. Binding of VLX4 Humanized mAbs to Human RBCs
(hRBCs). Binding of VLX4 humanized mAbs (VLX4hum_01 IgG, VLX4hum_02
IgG, VLX4hum_01 IgG4PE, and VLX4hum_02 IgG4PE) to human CD47 was
determined using freshly isolated hRBCs. hRBCs were incubated for
60 minutes at 37.degree. C. with various concentrations of VLX4
mAbs, washed and incubated for 1 hr with FITC-labelled donkey
anti-human antibody. Cells were washed and antibody binding
measured using flow cytometry.
[0151] FIG. 2B. Binding of VLX4 Humanized mAbs to Human RBCs.
Binding of VLX4 humanized mAbs (VLX4hum_07 IgG4PE, VLX4hum_12
IgG4PE, and VLX4hum_13 IgG4PE) to human CD47 was determined using
freshly isolated hRBCs. hRBCs were incubated for 60 minutes at
37.degree. C. with various concentrations of VLX4 mAbs, washed and
incubated for 1 hr with FITC-labelled donkey anti-human antibody.
Cells were washed and antibody binding measured using flow
cytometry.
[0152] FIG. 3A. Binding of VLX8 Humanized mAbs to Human OV10 hCD47
Cells. Binding of VLX8 IgG4PE chimera (xi) or humanized mAbs
(VLX8hum_01 IgG4PE, VLX8hum_04 IgG4PE, VLX8hum_07 IgG4PE, and
VLX8hum_09 IgG4PE) to human CD47 was determined using an OV10 hCD47
cell-based ELISA. OV10 hCD47 cells were plated into 96 well plates
and were confluent at the time of assay. Various concentrations of
VLX8 representative mAbs were added to the cells for 1 hr. Cells
were washed and then incubated with HRP-labelled secondary antibody
for 1 hr followed by addition of peroxidase substrate.
[0153] FIG. 3B. Binding of VLX8 Humanized mAbs to Human OV10 hCD47
Cells. Binding of VLX8 chimera or humanized mAbs (VLX8hum_06 IgG2,
VLX8hum_07 IgG2, VLX8hum_08 IgG2, and VLX8hum_09 IgG2) to human
CD47 was determined using an OV10 hCD47 cell-based ELISA. OV10
hCD47 cells were plated into 96 well plates and were confluent at
the time of assay. Various concentrations of VLX8 representative
mAbs were added to the cells for 1 hr. Cells were washed and then
incubated with HRP-labelled secondary antibody for 1 hr followed by
addition of peroxidase substrate.
[0154] FIG. 4A. Binding of VLX8 Humanized mAbs to Human RBCs.
Binding of VLX8 IgG4PE xi or humanized mAbs (VLX8hum_01 IgG4PE,
VLX8hum_03 IgG4PE, VLX8hum_07 IgG4PE, and VLX8hum_10 IgG4PE) to
human CD47 was determined using freshly isolated human RBCs. RBCs
were incubated for 1 hr at 37.degree. C. with various
concentrations of VLX8 mAbs, washed and incubated for 1 hr with
FITC-labelled donkey anti-human antibody. Cells were washed and
antibody binding measured using flow cytometry.
[0155] FIG. 4B. Binding of VLX8 Humanized mAbs to Human RBCs.
Binding of VLX8 IgG4PE xi or humanized mAbs (VLX8hum_06 IgG2,
VLX8hum_07 IgG2, VLX8hum_08 IgG2 and VLX8hum_09 IgG2) to human CD47
was determined using freshly isolated human RBCs. RBCs were
incubated for 1 hr at 37.degree. C. with various concentrations of
VLX8 mAbs, washed and incubated for 1 hr with FITC-labelled donkey
anti-human antibody. Cells were washed and antibody binding
measured using flow cytometry.
[0156] FIG. 5A. Binding of VLX9 Humanized mAbs to Human OV10 hCD47
Cells. Binding of VLX9 IgG2 xi or humanized mAbs (VLX9hum_01 IgG2,
VLX9hum_02 IgG2, VLX9hum_03 IgG2, VLX9hum_04 IgG2 and VLX9hum_05
IgG2) to human CD47 was determined using an OV10 human CD47
cell-based ELISA. OV10 hCD47 cells were plated into 96 well plates
and were confluent at the time of assay. Various concentrations of
mAbs were added to the cells for 1 hr. Cells were washed and then
incubated with HRP-labelled secondary antibody for 1 hr followed by
addition of peroxidase substrate.
[0157] FIG. 5B. Binding of VLX9 Humanized mAbs to Human OV10 hCD47
Cells. Binding of VLX9 IgG2 xi or humanized mAbs (VLX9hum_06 IgG2,
VLX9hum_07 IgG2, VLX9hum_08 IgG2, VLX9hum_09 IgG2 and VLX9hum_10
IgG2) to human CD47 was determined using a OV10 hCD47 cell-based
ELISA. OV10 hCD47 cells were plated into 96 well plates and were
confluent at the time of assay. Various concentrations of mAbs were
added to the cells for 1 hr. Cells were washed and then incubated
with HRP-labelled secondary antibody for 1 hr followed by addition
of peroxidase substrate.
[0158] FIG. 6A. Specific Binding of VLX Humanized mAbs to CD47.
Binding of VLX humanized mAb VLX4hum_07 IgG4PE to wildtype and CD47
knockout Jurkat cells was determined by flow cytometry. Various
concentrations of mAbs were added to 1.times.10.sup.4 cells for 1
hr. The cells were washed and then incubated with FITC-labelled
secondary antibody for 1 hr. Cells were washed and antibody binding
measured using flow cytometry.
[0159] FIG. 6B. Specific Binding of VLX Humanized mAbs to CD47.
Binding of VLX humanized mAb VLX9hum_04 IgG2 to wildtype and CD47
knockout Jurkat cells was determined by flow cytometry. Various
concentrations of mAbs were added to 1.times.10.sup.4 cells for 1
hr. The cells were washed and then incubated with FITC-labelled
secondary antibody for 1 hr. Cells were washed and antibody binding
measured using flow cytometry.
[0160] FIG. 7. Binding of VLX9 Humanized mAbs to Human RBCs.
Binding of VLX9 IgG2 xi or humanized VLX9 mAbs to human CD47
(VLX9hum_01 IgG2, VLX9hum_02 IgG2 and VLX9hum_07 IgG2) was
determined using freshly isolated human hRBCs. RBCs were incubated
for 60 minutes at 37.degree. C. with various concentrations of VLX9
mAbs, washed and incubated for 1 hr with FITC-labelled donkey
anti-human antibody. Cells were washed and antibody binding
measured using flow cytometry.
[0161] FIG. 8A. Binding of VLX Humanized mAbs to Human Aortic
Endothelial Cells (HAEC). Binding of VLX humanized mAbs (VLX4hum_07
IgG4PE, VLX8hum_10 IgG4PE, VLX8hum_11 IgG4PE, VLX4hum_01 IgG4PE,
VLX9hum_06 IgG2, VLX9hum_08 IgG2, VLX9hum_09 IgG2, VLX9hum_03 IgG2
and VLX9hum_04 IgG2) to HAEC was determined by flow cytometry. HAEC
were removed from the flask using acutase. Various concentrations
of mAbs were added to 1.times.10.sup.4 cells for 1 hr. The cells
were washed and then incubated with FITC-labelled secondary
antibody for 1 hr followed by measurement of FITC label by flow
cytometry.
[0162] FIG. 8B. Binding of VLX Humanized mAbs to Skeletal Human
Muscle Cells (SkMC). Binding of VLX humanized mAbs (VLX4hum_07
IgG4PE, VLX8hum_10 IgG4PE, VLX8hum_11 IgG4PE, VLX4hum_01 IgG4PE,
VLX9hum_06 IgG2, VLX9hum_08 IgG2, VLX9hum_09 IgG2, VLX9hum_03 IgG2
and VLX9hum_04 IgG2) to SkMc was determined by flow cytometry. SkMC
were removed from the flask using acutase. Various concentrations
of mAbs were added to 1.times.10.sup.4 cells for 1 hr. The cells
were washed and then incubated with FITC-labelled secondary
antibody for 1 hr followed by measurement of FITC label by flow
cytometry.
[0163] FIG. 8C. Binding of VLX Humanized mAbs to Human Lung
Microvascular Endothelial Cells (HMVEC-L). Binding of VLX humanized
mAbs (VLX4hum_07 IgG4PE, VLX8hum_10 IgG4PE, VLX8hum_11 IgG4PE,
VLX4hum_01 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2, VLX9hum_09
IgG2, VLX9hum_03 IgG2 and VLX9hum_04 IgG2) to HMVEC-L was
determined by flow cytometry. HMVEC-L were removed from the flask
using acutase. Various concentrations of mAbs were added to
1.times.10.sup.4 cells for 1 hr. The cells were washed and then
incubated with FITC-labelled secondary antibody for 1 hr followed
by measurement of FITC label by flow cytometry.
[0164] FIG. 8D. Binding of VLX Humanized mAbs to Human Renal
Tubular Epithelial Cells (RTEC). Binding of VLX humanized mAbs
(VLX4hum_07 IgG4PE, VLX8hum_10 IgG4PE, VLX8hum_11 IgG4PE,
VLX4hum_01 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2, VLX9hum_09
IgG2, VLX9hum_03 IgG2 and VLX9hum_04 IgG2) to RTEC by flow
cytometry. RTEC were removed from the flask using acutase. Various
concentrations of mAbs were added to 1.times.10.sup.4 cells for 1
hr. The cells were washed and then incubated with FITC-labelled
secondary antibody for 1 hr followed by measurement of FITC label
by flow cytometry.
[0165] FIG. 8E. Binding of VLX Humanized mAbs to Human Peripheral
Blood CD3.sup.+ Cells. Binding of VLX humanized mAbs (VLX4hum_07
IgG4PE, VLX8hum_10 IgG4PE, VLX8hum_11 IgG4PE, VLX4hum_01 IgG4PE,
VLX9hum_06 IgG2, VLX9hum_08 IgG2, VLX9hum_09 IgG2, VLX9hum_03 IgG2
and VLX9hum_04 IgG2) to CD3.sup.+ cells was determined by flow
cytometry. PBMC were plated into 96 well plates. Various
concentrations of mAbs were added to the cells for 1 hr. Cells were
washed and then incubated with FITC-labelled secondary antibody and
(APC)-labelled anti-CD3 antibody for 1 hr followed by measurement
of FITC-labelled APC-positive cells by flow cytometry.
[0166] FIG. 8F. Binding of VLX Humanized mAbs to Human Peripheral
Blood Mononuclear Cells (PBMC). Binding of VLX humanized mAbs
(VLX4hum_07 IgG4PE, VLX8hum_10 IgG4PE, VLX8hum_11 IgG4PE,
VLX4hum_01 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2, VLX9hum_09
IgG2, VLX9hum_03 IgG2 and VLX9hum_04 IgG2) to PBMC was determined
by flow cytometry. PBMCs were plated into 96 well plates. Various
concentrations of mAbs were added to the cells for 1 hr. Cells were
washed and then incubated with FITC-labelled secondary antibody for
1 hr followed by measurement of FITC label by flow cytometry.
[0167] FIG. 9A. pH Dependent and pH Independent Binding of
Humanized mAb to His-CD47. Binding of VLX9hum_09 IgG2 to human CD47
was determined using a solid-phase CD47 ELISA assay. His-CD47 was
adsorbed to microtiter wells, washed and various concentrations of
humanized mAbs were added to the wells for 1 hr at pH 6 or 8. The
wells were washed and then incubated with HRP-labelled secondary
antibody for 1 hour followed by addition of peroxidase
substrate.
[0168] FIG. 9B. pH Dependent and pH Independent Binding of
Humanized mAb to His-CD47. Binding of VLX9hum_04 IgG2 to human CD47
was determined using a solid-phase CD47 ELISA assay. His-CD47 was
adsorbed to microtiter wells, washed and various concentrations of
humanized mAbs were added to the wells for 1 hr at pH 6 or 8. The
wells were washed and then incubated with HRP-labelled secondary
antibody for 1 hour followed by addition of peroxidase
substrate.
[0169] FIG. 9C. pH Dependent and pH Independent Binding of
Humanized mAb to His-CD47. Binding of VLX4hum_07 IgG4PE to human
CD47 was determined using a solid-phase CD47 ELISA assay. His-CD47
was adsorbed to microtiter wells, washed and various concentrations
of humanized mAbs were added to the wells for 1 hr at pH 6 or 8.
The wells were washed and then incubated with HRP-labelled
secondary antibody for 1 hour followed by addition of peroxidase
substrate.
[0170] FIG. 9D. pH Dependent and pH Independent Binding of
Humanized mAb to His-CD47. Binding of VLX8hum_10 IgG4PE to human
CD47 was determined using a solid-phase CD47 ELISA assay. His-CD47
was adsorbed to microtiter wells, washed and various concentrations
of humanized mAbs were added to the wells for 1 hr at pH 6 or 8.
The wells were washed and then incubated with HRP-labelled
secondary antibody for 1 hour followed by addition of peroxidase
substrate.
[0171] FIG. 10. VLX4, VLX8, and VLX9 Humanized mAbs Block
SIRP.alpha. binding to CD47 on Human Jurkat Cells.
1.5.times.10.sup.6 Jurkat cells were incubated with 5 .mu.g/ml of
VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE,
VLX4hum_07 IgG4PE, VLX8hum_10 IgG4PE, VLX4hum_11 IgG4PE, VLX9hum_03
IgG2, VLX9hum_06 IgG2, and VLX9hum_08 IgG2) or a control antibody
or no antibody in RPMI containing 10% FBS for 30 min at 37.degree.
C. An equal volume of media containing fluorescently labelled
SIRP.alpha.-Fc fusion protein was added and incubated for an
additional 30 min at 37.degree. C. Cells were washed and binding
was assessed using flow cytometry.
[0172] FIG. 11. VLX4 CD47 Chimeric mAbs Increase Phagocytosis of
Human Jurkat Cells by Human Macrophages. Human macrophages were
plated at a concentration of 1.times.10.sup.4 cells per well in a
96 well plate and allowed to adhere for 24 hrs. 5.times.10.sup.4
CFSE-labelled human Jurkat cells and 1 .mu.g/ml of the VLX4
chimeric mAbs (VLX4 IgG1 xi, VLX4 IgG1 N297Q xi, VLX4 IgG4PE xi,
VLX4 IgG4 S228P xi) were added to the macrophage cultures and
incubated at 37.degree. C. for 2 hrs. Non-phagocytosed Jurkat cells
were removed and macrophage cultures were washed extensively.
Macrophages were trypsinized and stained for CD14. Flow cytometry
was used to determine the percentage of CD14.sup.+/CFSE.sup.+ cells
in the total CD14.sup.+ population.
[0173] FIG. 12A. VLX4 Humanized mAbs Increase Phagocytosis of Human
Jurkat Cells by Human Macrophages. Human macrophages were plated at
a concentration of 1.times.10.sup.4 cells per well in a 96 well
plate and allowed to adhere for 24 hrs. 5.times.10.sup.4
CFSE-labelled human Jurkat cells and 1 .mu.g/ml of antibody
(VLX4hum_01 IgG1 and VLX4hum_01 IgG4PE) were added to the
macrophage cultures and incubated at 37.degree. C. for 2 hrs.
Non-phagocytosed Jurkat cells were removed and macrophage cultures
were washed extensively. Macrophages were trypsinized and stained
for CD14. Flow cytometry was used to determine the percentage of
CD14.sup.+/CFSE.sup.+ cells in the total CD14.sup.+ population.
[0174] FIG. 12B. VLX4 Humanized mAbs Increase Phagocytosis of Human
Jurkat Cells by Human Macrophages. Human macrophages were plated at
a concentration of 1.times.10.sup.4 cells per well in a 96 well
plate and allowed to adhere for 24 hrs. 5.times.10.sup.4
CFSE-labelled human Jurkat cells and 1 .mu.g/ml of antibody (VLX4
IgG4PE xi, VLX4hum_06 IgG4PE, VLX4hum_07 IgG4PE, VLX4hum_012 IgG4PE
and VLX4hum_13 IgG4PE) were added to the macrophage cultures and
incubated at 37.degree. C. for 2 hrs. Non-phagocytosed Jurkat cells
were removed and macrophage cultures were washed extensively.
Macrophages were trypsinized and stained for CD14. Flow cytometry
was used to determine the percentage of CD14.sup.+/CFSE.sup.+ cells
in the total CD14.sup.+ population.
[0175] FIG. 13A. VLX8 CD47 Chimeric mAbs Increase Phagocytosis of
Human Jurkat Cells by Human Macrophages. Human macrophages were
plated at a concentration of 1.times.10.sup.4 cells per well in a
96 well plate and allowed to adhere for 24 hrs. 5.times.10.sup.4
CFSE-labelled human Jurkat cells and 1 .mu.g/ml of the VLX8
chimeric mAbs (VLX8 IgG1 N297Q xi and VLX8 Ig4PE xi) were added to
the macrophage cultures and incubated at 37.degree. C. for 2 hrs.
Non-phagocytosed Jurkat cells were removed and macrophage cultures
were washed extensively. Macrophages were trypsinized and stained
for CD14. Flow cytometry was used to determine the percentage of
CD14.sup.+/CFSE.sup.+ cells in the total CD14.sup.+ population.
[0176] FIG. 13B. VLX8 Humanized mAbs Increase Phagocytosis of Human
Jurkat Cells by Human Macrophages. Human macrophages were plated at
a concentration of 1.times.10.sup.4 cells per well in a 96 well
plate and allowed to adhere for 24 hrs. 5.times.10.sup.4
CFSE-labelled human Jurkat cells and 1 .mu.g/ml of antibody (VLX8
IgG4PE xi, VLX8hum_01 IgG4PE, VLX8hum_03 IgG4PE, VLX8hum_07 IgG4PE,
VLX8hum_08 IgG4PE and VLX8hum_09 IgG4PE) were added to the
macrophage cultures and incubated at 37.degree. C. for 2 hrs.
Non-phagocytosed Jurkat cells were removed and macrophage cultures
were washed extensively. Macrophages were trypsinized and stained
for CD14. Flow cytometry was used to determine the percentage of
CD14.sup.+/CFSE.sup.+ cells in the total CD14.sup.+ population.
[0177] FIG. 14A. VLX9 CD47 Chimeric mAbs Increase Phagocytosis of
Human Jurkat Cells by Human Macrophages. Human macrophages were
plated at a concentration of 1.times.10.sup.4 cells per well in a
96 well plate and allowed to adhere for 24 hours. 5.times.10.sup.4
CFSE-labelled human Jurkat cells and 1 .mu.g/ml of the VLX9
chimeric mAbs (VLX9 IgG1 N297 xi, VLX9 IgG2 xi and VLX9 IgG4PE xi)
were added to the macrophage cultures and incubated at 37.degree.
C. for two hours. Non-phagocytosed Jurkat cells were removed and
macrophage cultures were washed extensively. Macrophages were
trypsinized and stained for CD14. Flow cytometry was used to
determine the percentage of CD14+/CFSE+ cells in the total CD14+
population.
[0178] FIG. 14B. VLX9 Humanized mAbs Increase Phagocytosis of Human
Jurkat Cells by Human Macrophages. Human macrophages were plated at
a concentration of 1.times.10.sup.4 cells per well in a 96 well
plate and allowed to adhere for 24 hours. 5.times.10.sup.4
CFSE-labelled human Jurkat cells and 1 .mu.g/ml of antibody (VLX9
IgG2 xi, VLX9hum_01 IgG2, VLX9hum_02 IgG2, VLX9hum_03 IgG2,
VLX9hum_04 IgG2, VLX9hum_05 IgG2, VLX9hum_06 IgG2, VLX9hum_07 IgG2,
VLX9hum_08 IgG2, VLX9hum_09 IgG2 and VLX9hum_10 IgG2) were added to
the macrophage cultures and incubated at 37.degree. C. for two
hours. Non-phagocytosed Jurkat cells were removed and macrophage
cultures were washed extensively. Macrophages were trypsinized and
stained for CD14. Flow cytometry was used to determine the
percentage of CD14+/CFSE+ cells in the total CD14+ population.
[0179] FIG. 15A. Induction of Cell Death in Human Jurkat Cells by
Soluble VLX4 Humanized mAbs. Jurkat cells (1.times.10.sup.4) were
incubated with 1 .mu.g/ml VLX4 humanized mAbs (VLX4hum_01 IgG,
VLX4hum_01 IgG4PE, VLX4hum_02 IgG1, VLX4hum_02 IgG4PE) in RPMI
media for 24 hours at 37.degree. C. Cells were then stained with
annexin V and the signal was detected by flow cytometry. The data
are shown as % of cells that are annexin V positive (annexin
V+).
[0180] FIG. 15B. Induction of Cell Death in Human Jurkat Cells by
Soluble VLX4 Humanized mAbs. Jurkat cells (1.times.10.sup.4) were
incubated with 1 .mu.g/ml VLX4 humanized mAbs (VLX4hum_01 IgG,
VLX4hum_01 IgG4PE, VLX4hum_02 IgG1, VLX4hum_02 IgG4PE) in RPMI
media for 24 hours at 37.degree. C. Cells were then stained with
annexin V and 7-AAD and analyzed by flow cytometry. The data are
shown as % of the cells that are annexin V positive/7-AAD negative
(annexin V.sup.+/7-AAD.sup.-).
[0181] FIG. 15C. Induction of Cell Death in Human Jurkat Cells by
Soluble VLX4 Humanized mAbs. Jurkat cells (1.times.10.sup.4) were
incubated with 1 .mu.g/ml VLX4 humanized mAbs (VLX4hum_01 IgG,
VLX4hum_01 IgG4PE, VLX4hum_02 IgG1, VLX4hum_02 IgG4PE) in RPMI
media for 24 hours at 37.degree. C. Cells were then stained with
annexin V and 7-AAD and analyzed by flow cytometry. The data are
shown as % of cells that are annexin V positive/7-AAD positive
(annexin V.sup.+/7-AAD.sup.+).
[0182] FIG. 15D. Induction of Cell Death in Human Jurkat Cells by
Soluble VLX4 Humanized mAbs. Jurkat cells (1.times.10.sup.4) were
incubated with 1 .mu.g/ml VLX4 humanized mAbs (VLX4hum_06 IgG4PE,
VLX4hum_07 IgG4PE, VLX4hum_08 IgG4PE, VLX4hum_11 IgG4PE, VLX4hum_12
IgG4PE, VLX4hum_13 IgG4PE) in RPMI media for 24 hours at 37.degree.
C. Cells were then stained with annexin V and 7-AAD and analyzed by
flow cytometry. The data are shown as the % of cells that are
annexin V positive (annexin V+).
[0183] FIG. 15E. Induction of Cell Death in Human Jurkat Cells by
Soluble VLX4 Humanized mAbs. Jurkat cells (1.times.10.sup.4) were
incubated with 1 .mu.g/ml VLX4 humanized mAbs (VLX4hum_06 IgG4PE,
VLX4hum_07 IgG4PE, VLX4hum_08 IgG4PE, VLX4hum_11 IgG4PE, VLX4hum_12
IgG4PE, VLX4hum_13 IgG4PE) in RPMI media for 24 hours at 37.degree.
C. Cells were then stained with annexin V and 7-AAD by flow
cytometry. The data are shown as the % of cells that are annexin V
positive/7-AAD negative (annexin V.sup.+/7-AAD.sup.-).
[0184] FIG. 15F. Induction of Cell Death in Human Jurkat Cells by
Soluble VLX4 Humanized mAbs. Jurkat cells (1.times.10.sup.4) were
incubated with 1 .mu.g/ml VLX4 humanized mAbs (VLX4hum_06 IgG4PE,
VLX4hum_07 IgG4PE, VLX4hum_08 IgG4PE, VLX4hum_11 IgG4PE, VLX4hum_12
IgG4PE, VLX4hum_13 IgG4PE) in RPMI media for 24 hours at 37.degree.
C. Cells were then stained with annexin V and and 7-AAD and
analyzed by flow cytometry. The data are shown as the % of cells
that are annexin V positive/7-AAD positive
(annexin.sup.+/7-AAD.sup.+).
[0185] FIG. 16A. Induction of Cell Death in Human Jurkat Cells by
Soluble VLX8 CD47 Chimeric mAbs. Jurkat cells (1.times.10.sup.4)
were incubated with 1 .mu.g/ml VLX8 chimeric mAbs (VLX8 IgG1 N297Q
xi and VLX8 IgG4PE xi) in RPMI media for 24 hrs at 37.degree. C.
Cells were then stained with annexin V and analyzed by flow
cytometry. The data are presented as % of cells that are annexin V
positive (annexin V+).
[0186] FIG. 16B. Induction of Cell Death in Human Jurkat Cells by
Soluble VLX8 Chimeric mAbs. Jurkat cells (1.times.10.sup.4) were
incubated with 1 .mu.g/ml VLX8 chimeric mAbs (VLX8 IgG1 N297Q xi
and VLX8 IgG4PE xi) in RPMI media for 24 hrs at 37.degree. C. Cells
were then stained with annexin V and 7-AAD and analyzed by flow
cytometry. The data are presented as the % of cells that are
annexin V positive/7-AAD negative (annexin
V.sup.+/7-AAD.sup.-).
[0187] FIG. 16C. Induction of Cell Death in Human Jurkat Cells by
Soluble VLX8 Chimeric mAbs. Jurkat cells (1.times.10.sup.4) were
incubated with 1 .mu.g/ml VLX8 chimeric mAbs (VLX8 IgG1 N297Q xi
and VLX8 IgG4PE (xi) in RPMI media for 24 hrs at 37.degree. C.
Cells were then stained with annexin V and 7-AAD and analyzed by
flow cytometry. The data are presented as the % of cells that are
annexin V positive/7-AAD positive (annexin
V.sup.+/7-AAD.sup.+).
[0188] FIG. 16D. Induction of Cell Death in Human Jurkat Cells by
Soluble VLX8 Humanized mAbs. Jurkat cells (1.times.10.sup.4) were
incubated with 1 .mu.g/ml VLX8 humanized mAbs (VLX8hum_02 IgG4PE,
VLX8hum_04 IgG4PE, VLX8hum_07 IgG4PE and VLX8hum_08 IgG4PE) and
chimeric mAb VLX8 IgG4PE in RPMI media for 24 hrs at 37.degree. C.
Cells were then stained with annexin V and analyzed by flow
cytometry. The data are presented as the % of cells that are
annexin V positive (annexin V+).
[0189] FIG. 16E. Induction of Cell Death in Human Jurkat Cells by
Soluble VLX8 Humanized mAbs. Jurkat cells (1.times.10.sup.4) were
incubated with 1 .mu.g/ml VLX8 humanized mAbs (VLX8hum_02 IgG4PE,
VLX8hum_04 IgG4PE, VLX8hum_07 IgG4PE and VLX8hum_08 IgG4PE) and
chimeric mAb VLX8 IgG4PE in RPMI media for 24 hrs at 37.degree. C.
Cells were then stained with annexin V and 7-AAD and analyzed by
flow cytometry. The data are shown as the % of cells that are
annexin V positive/7-AAD negative (annexin
V.sup.+/7-AAD.sup.-).
[0190] FIG. 16F. Induction of Cell Death in Human Jurkat Cells by
Soluble VLX8 Humanized mAbs. Jurkat cells (1.times.10.sup.4) were
incubated with 1 .mu.g/ml VLX8 humanized mAbs (VLX8hum_02 IgG4PE,
VLX8hum_04 IgG4PE, VLX8hum_07 IgG4PE and VLX8hum_08 IgG4PE) and
chimeric mAb VLX8 IgG4PE in RPMI media for 24 hrs at 37.degree. C.
Cells were then stained with annexin V and 7-AAD and analyzed by
flow cytometry. The data are shown as the % of cells that are
annexin V positive/7-AAD positive (annexin
V.sup.+/7-AAD.sup.+).
[0191] FIG. 17A. Induction of Cell Death of Human Jurkat Cells by
Soluble VLX9 Chimeric mAbs. 1.times.10.sup.4 Jurkat cells were
incubated with 1 .mu.g/ml of the VLX9 CD47 chimeric mAbs (VLX9 IgG1
N297Q xi, VLX9 IgG2 xi and VLX9 IgG4PE xi) in RPMI media for 24
hours 37.degree. C. Cells were then stained with annexin V and the
signal analyzed by flow cytometry. The data are shown as % of cells
that are annexin V positive (annexin V+).
[0192] FIG. 17B. Induction of Cell Death of Human Jurkat Cells by
Soluble VLX9 Chimeric mAbs. 1.times.10.sup.4 Jurkat cells were
incubated with 1 .mu.g/ml of the VLX9 CD47 chimeric mAbs (VLX9 IgG1
N297Q xi, VLX9 IgG2 xi and VLX9 IgG4PE xi) in RPMI media for 24
hours 37.degree. C. Cells were then stained with annexin V and
7-AAD and analyzed by flow cytometry. The data are shown as % of
cells that are annexin V positive/7-AAD negative (annexin
V.sup.+/7-AAD.sup.-).
[0193] FIG. 17C. Induction of Cell Death of Human Jurkat Cells by
Soluble VLX9 Chimeric mAbs. 1.times.10.sup.4 Jurkat cells were
incubated with 1 .mu.g/ml of the VLX9 CD47 chimeric mAbs (VLX9 IgG1
N297Q xi, VLX9 IgG2 xi and VLX9 IgG4PE xi) in RPMI media for 24
hours 37.degree. C. Cells were then stained with annexin V and
7-AAD and analyzed by flow cytometry. The data are shown as % of
cells that are annexin V positive/7-AAD positive (annexin
V.sup.+/7-AAD.sup.+).
[0194] FIG. 17D. Induction of Cell Death in Human Jurkat Cells by
Soluble VLX9 Humanized mAbs. Jurkat cells (1.times.10.sup.4) were
incubated with 1 .mu.g/ml VLX9 humanized mAbs (VLX9hum_01 to 10
IgG2) and chimeric mAb VLX9 IgG2 xi in RPMI media for 24 hours at
37.degree. C. Cells were then stained with annexin V and the signal
was detected by flow cytometry. VLX9 IgG2 (xi) is a murine/human
chimera. The data are shown as % of cells that are annexin V
positive (annexin V.sup.+).
[0195] FIG. 17E. Induction of Cell Death in Human Jurkat Cells by
Soluble VLX9 Humanized mAbs. Jurkat cells (1.times.10.sup.4) were
incubated with 1 .mu.g/ml VLX9 humanized mAbs (VLX9hum_01 to 10
IgG2) and chimeric mAb VLX9 IgG2 xi in RPMI media for 24 hours at
37.degree. C. Cells were then stained with annexin V and 7-AAD and
analyzed by flow cytometry. VLX9 IgG2 (xi) is a murine/human
chimera. The data are shown as % of cells that are annexin V
positive/7-AAD negative (annexin V.sup.+/7-AAD.sup.-).
[0196] FIG. 17F. Induction of Cell Death in Human Jurkat Cells by
Soluble VLX9 Humanized mAbs. Jurkat cells (1.times.10.sup.4) were
incubated with 1 .mu.g/ml VLX9 humanized mAbs (VLX9hum_01 to 10
IgG2) and chimeric mAb VLX9 IgG2 xi in RPMI media for 24 hours at
37.degree. C. Cells were then stained with annexin V and 7-AAD and
analyzed by flow cytometry. VLX9 IgG2 (xi) is a murine/human
chimera. The data are shown as the % of cells that are annexin V
positive/7-AAD positive (annexin V.sup.+/7-AAD.sup.+).
[0197] FIG. 18. Induction of Mitochondrial Depolarization in Human
Raji Cells by Soluble VLX4, VLX8 and VLX9 Humanized mAbs.
1.times.10.sup.4 Raji cells were incubated with 10 .mu.g/ml of
VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE,
VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_03 IgG2, VLX9hum_06
IgG2 and VLX9hum_08 IgG2), a negative IgG control antibody or 1
.mu.M of mitoxantrone as a positive control in RPMI media at
37.degree. C. for 24 hours. Cells were washed and the change in
JC-1 dye fluorescence was assessed using flow cytometry. The data
are expressed as % of cells with mitochondrial depolarization.
[0198] FIG. 19. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Cause an
Increase in Cell Surface Calreticulin Expression on Human Raji
Cells. 1.times.10.sup.4 Raji cells were incubated with 10 .mu.g/ml
of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE,
VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08
IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1
.mu.M of mitoxantrone as a positive control in RPMI media at
37.degree. C. for 24 hours. Cells were washed and calreticulin
expression was assessed using flow cytometry. The data are
expressed as % of cells that are calreticulin positive.
[0199] FIG. 20. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Cause an
Increase in Cell Surface Protein Disulfide-Isomerase A3 (PDIA3)
Expression on Human Raji Cells. 1.times.10.sup.4 Raji cells were
incubated with 10 .mu.g/ml of VLX4, VLX8 and VLX9 CD47 humanized
mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_l1 IgG4PE,
VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative
IgG control antibody or 1 .mu.M of mitoxantrone as a positive
control in RPMI media at 37.degree. C. for 24 hours. Cells were
washed and PDIA3 expression was assessed using flow cytometry. The
data are expressed as % of cells that are PDIA3 positive.
[0200] FIG. 21. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase
Cell Surface HSP70 Expression on Human Raji Cells. 1.times.10.sup.4
Raji cells were incubated with 10 .mu.g/ml of VLX4, VLX8 and VLX9
CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE,
VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03
IgG2), a negative IgG control antibody or 1 .mu.M of mitoxantrone
as a positive control in RPMI media at 37.degree. C. for 24 hours.
Cells were washed and HSP70 expression was assessed using flow
cytometry. The data are expressed as % of cells that are HSP70
positive.
[0201] FIG. 22. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase
Cell Surface HSP90 Expression on Human Raji Cells. 1.times.10.sup.4
Raji cells were incubated with 10 .mu.g/ml of VLX4, VLX8 and VLX9
CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE,
VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03
IgG2), a negative IgG control antibody or 1 .mu.M of mitoxantrone
as a positive control in RPMI media at 37.degree. C. for 24 hours.
Cells were washed and HSP90 expression was assessed using flow
cytometry. The data are expressed as % of cells that are HSP90
positive.
[0202] FIG. 23. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase
Release of Adenosine Triphosphate (ATP) by Human Raji Cells.
1.times.10.sup.4 Raji cells were incubated with .mu.g/ml of VLX4,
VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07
IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and
VLX9hum_03 IgG2), a negative IgG control antibody or 1 .mu.M of
mitoxantrone as a positive control in RPMI media at 37.degree. C.
for 24 hours. Cell-free supernatant was collected and analyzed
using an ATP determination kit. The data are expressed as pM ATP in
the supernatant.
[0203] FIG. 24. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Cause an
Increase in Release of High Mobility Group Box 1 (HMGB1) by Human
Raji Cells. 1.times.10.sup.4 Raji cells were incubated with 10
.mu.g/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01
IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_03 IgG2,
VLX9hum_06 IgG2 and VLX9hum_08 IgG2), a negative IgG control
antibody or 1 .mu.M of mitoxantrone as a positive control in RPMI
media at 37.degree. C. for 24 hours. Cell-free supernatant was
collected and analyzed using an HMGB1 immunoassay. The data are
expressed as ng/ml of HMGB1 in the supernatant.
[0204] FIG. 25. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase
CXCL10 Release by Human Raji Cells. 1.times.10.sup.4 Raji cells
were incubated with 10 .mu.g/ml of VLX4, VLX8 and VLX9 CD47
humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11
IgG4PE, VLX9hum_03 IgG2, VLX9hum_06 IgG2 and VLX9hum_08 IgG2), a
negative IgG control antibody or 1 .mu.M of mitoxantrone as a
positive control in RPMI media at 37.degree. C. for 24 hours.
Cell-free supernatant was collected and analyzed using an CXCL10
immunoassay. The data are expressed as pg/ml of CXCL10 in the
supernatant.
[0205] FIG. 26. Induction Mitochondrial Depolarization in Human
Jurkat Cells by Soluble VLX4, VLX8 and VLX9 Humanized mAbs.
1.times.10.sup.4 Jurkat cells were incubated with 10 .mu.g/ml of
VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE,
VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08
IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1
.mu.M of mitoxantrone as a positive control in RPMI media at
37.degree. C. for 24 hours. Cells were washed and the change in
JC-1 dye fluorescence was assessed using flow cytometry. The data
are expressed as % of cells with mitochondrial depolarization.
[0206] FIG. 27. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase
Cell Surface Calreticulin Expression on Human Jurkat Cells.
1.times.10.sup.4 Jurkat cells were incubated with 10 .mu.g/ml of
VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE,
VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08
IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1
.mu.M of mitoxantrone as a positive control in RPMI media at
37.degree. C. for 24 hours. Cells were washed and calreticulin
expression was assessed using flow cytometry. The data are
expressed as % of cells that are calreticulin positive.
[0207] FIG. 28. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase
Cell Surface PDIA3 Expression on Human Jurkat Cells.
1.times.10.sup.4 Jurkat cells were incubated with 10 .mu.g/ml of
VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE,
VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08
IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1
.mu.M of mitoxantrone as a positive control in RPMI media at
37.degree. C. for 24 hours. Cells were washed and PDIA3 expression
was assessed using flow cytometry. The data are expressed as % of
cells that are PDIA3 positive.
[0208] FIG. 29. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase
Cell Surface HSP70 Expression on Human Jurkat Cells.
1.times.10.sup.4 Jurkat cells were incubated with 10 .mu.g/ml of
VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE,
VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08
IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1
.mu.M of mitoxantrone as a positive control in RPMI media at
37.degree. C. for 24 hours. Cells were washed and HSP70 expression
was assessed using flow cytometry. The data are expressed as % of
cells that are HSP70 positive.
[0209] FIG. 30. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase
Cell Surface HSP90 Expression on Human Jurkat Cells.
1.times.10.sup.4 Jurkat cells were incubated with 10 .mu.g/ml of
VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE,
VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08
IgG2 and VLX9hum_03 IgG2), a negative IgG control antibody or 1
.mu.M of mitoxantrone as a positive control in RPMI media at
37.degree. C. for 24 hours. Cells were washed and HSP90 expression
was assessed using flow cytometry. The data are expressed as % of
cells that are HSP90 positive.
[0210] FIG. 31. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase
ATP Release by Human Jurkat Cells. 1.times.10.sup.4 Jurkat cells
were incubated with 10 .mu.g/ml of VLX4, VLX8 and VLX9 CD47
humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11
IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a
negative IgG control antibody or 1 .mu.M of mitoxantrone as a
positive control in RPMI media at 37.degree. C. for 24 hours.
Cell-free supernatant was collected and analyzed using an ATP
determination kit. The data are expressed as pM ATP in the
supernatant.
[0211] FIG. 32. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase
HMGB1 Release by Human Jurkat Cells. 1.times.10.sup.4 Jurkat cells
were incubated with 10 .mu.g/ml of VLX4, VLX8 and VLX9 CD47
humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11
IgG4PE, VLX9hum_03 IgG2, VLX9hum_06 IgG2 and VLX9hum_08 IgG2), a
negative IgG control antibody or 1 .mu.M of mitoxantrone as a
positive control in RPMI media at 37.degree. C. for 24 hours.
Cell-free supernatant was collected and analyzed using an HMGB1
immunoassay. The data are expressed as ng/ml of HMGB1 in the
supernatant.
[0212] FIG. 33A. Agglutination of hRBCs by VLX4 Humanized mAbs.
Hemagglutination was assessed following incubation of hRBCs with
various concentrations of humanized VLX4 mAbs (VLX4hum_01 IgG1 and
VLX4hum_01 IgG4PE). Blood was diluted (1:50) and washed 3 times
with PBS/EDTA/BSA. hRBCs were added to U-bottomed 96 well plates
with equal volumes of the antibodies (75 .mu.l) and incubated for 3
hrs at 37.degree. C. and overnight at 4.degree. C.
[0213] FIG. 33B. Agglutination of hRBCs by VLX8 Chimeric and
Humanized mAbs. Hemagglutination was assessed following incubation
of hRBCs with various concentrations of humanized VLX8 mAbs
(VLX8hum_01 IgG4PE, VLX8hum_02 IgG4PE VLX8hum_03 IgG4PE, VLX8hum_08
IgG4PE, VLX8hum_09 IgG4PE, VLX8hum_10 IgG4PE and VLX8hum_11 IgG4PE)
and the chimeric mAb VLX8 IgG4PE xi. Blood was diluted (1:50) and
washed 3 times with PBS/EDTA/BSA. hRBCs were added to U-bottomed 96
well plates with equal volumes of the antibodies (75 .mu.l) and
incubated for 3 hrs at 37.degree. C. and overnight at 4.degree.
C.
[0214] FIG. 34A. Agglutination of Human RBCs by VLX9 Humanized
mAbs. Hemagglutination was assessed following incubation of human
RBCs with various concentrations of VLX9 IgG2 chimera (xi) and
humanized VLX9 mAbs (VLX9hum_01 to 05 IgG2). Blood was diluted
(1:50) and washed 3 times with PBS/EDTA/BSA. RBCs were added to
U-bottomed 96 well plates with equal volumes of the antibodies (75
.mu.l) and incubated for 3 hrs at 37.degree. C. and overnight at
4.degree. C.
[0215] FIG. 34B. Agglutination of Human RBCs by VLX9 Humanized
mAbs. Hemagglutination was assessed following incubation of human
RBCs with various concentrations of VLX9 IgG2 chimera (xi) and
humanized VLX9 mAbs (VLX9hum_06 to _10 IgG2). Blood was diluted
(1:50) and washed 3 times with PBS/EDTA/BSA. RBCs were added to
U-bottomed 96 well plates with equal volumes of the antibodies (75
.mu.l) and incubated for 3 hrs at 37.degree. C. and overnight at
4.degree. C.
[0216] FIG. 35. VLX4 Humanized mAb Reduces Tumor Growth in Raji
Xenograft Model. Female NSG mice were inoculated subcutaneously in
the right flank with 0.1 mL of a 30% RPMI/70% Matrigel.TM. mixture
containing a suspension of 5.times.10.sup.6 Raji tumor cells. Five
days following inoculation, tumor volumes were measured and mice
with palpable tumor volumes of 31-74 mm.sup.3 were randomized into
8-10/group. VLX4hum_07 IgG4PE or PBS (control) administration was
initiated at this time. Mice were treated with 5 mg/kg of antibody
5.times./week for 4 weeks by intraperitoneal injection. Tumor
volumes and body weights were recorded twice weekly.
[0217] FIG. 36. VLX8 Humanized mAb Reduces Tumor Growth in Raji
Xenograft Model. Female NSG mice were inoculated subcutaneously in
the right flank with 0.1 mL of a 30% RPMI/70% Matrigel.TM. mixture
containing a suspension of 5.times.10.sup.6 Raji tumor cells. Five
days following inoculation, tumor volumes were measured and mice
with palpable tumor volumes of 31-74 mm.sup.3 were randomized into
8-10/group. VLX8hum_10 IgG4PE or PBS (control) administration was
initiated at this time. Mice were treated with 5 mg/kg of antibody
5.times./week for 4 weeks by intraperitoneal injection. Tumor
volumes and body weights were recorded twice weekly.
[0218] FIG. 37. VLX9 Humanized mAb Reduces Tumor Growth in Raji
Xenograft Model. Female NSG mice were inoculated subcutaneously in
the right flank with 0.1 mL of a 30% RPMI/70% Matrigel.TM. mixture
containing a suspension of 5.times.10.sup.6 Raji tumor cells. Five
days following inoculation, tumor volumes were measured and mice
with palpable tumor volumes of 31-74 mm.sup.3 were randomized into
8-10/group. VLX9hum_08 IgG2 or PBS (control) administration was
initiated at this time. Mice were treated with 5 mg/kg of antibody
5.times./week for 4 weeks by intraperitoneal injection. Tumor
volumes and body weights were recorded twice weekly.
[0219] FIG. 38A. Hemoglobin Levels in Blood Following
Administration of a Humanized VLX9 mAb to Cynomolgus Monkeys by
Intravenous Infusion. VLX9hum_08 IgG2 or vehicle were administered
as a one hour intravenous infusion a dose of 5 mg/kg on day 1 and a
dose of 15 mg/kg on day 18. Hemoglobin levels were monitored
throughout the study and normalized to control values.
[0220] FIG. 38B. RBC Levels in Blood Following Administration of
Humanized VLX9 mAbs to Cynomolgus Monkeys by Intravenous Infusion.
VLX9hum_08 IgG2 or vehicle was administered as a one hour
intraveneous infusion a dose of 5 mg/kg on day 1 and a dose of 15
mg/kg on day 18. RBC levels were monitored throughout the study and
normalized to control values.
DETAILED DESCRIPTION OF THE DISCLOSURE
Definitions
[0221] Unless otherwise defined, scientific and technical terms
used in connection with the present disclosure shall have the
meanings that are commonly understood by those of ordinary skill in
the art. Further, unless otherwise required by context, singular
terms shall include pluralities and plural terms shall include the
singular. Generally, nomenclatures utilized in connection with, and
techniques of, cell and tissue culture, molecular biology, and
protein and oligo or polynucleotide chemistry and hybridization
described herein are those well-known and commonly used in the
art.
[0222] As used herein, the term "CD47," "integrin-associated
protein (IAP)," "ovarian cancer antigen OA3," "Rh-related antigen,"
and "MERG" are synonymous and may be used interchangeably.
[0223] The term "anti-CD47 antibody" refer to an antibody of the
disclosure which is intended for use as a therapeutic or diagnostic
agent, and therefore will typically possess the binding affinity
required to be useful as a therapeutic and/or diagnostic agent.
[0224] As used herein, the term "antibody" refers to immunoglobulin
molecules and immunologically active portions of immunoglobulin
(Ig) molecules, i.e., molecules that contain an antigen binding
site that specifically binds (immunoreacts with) an antigen. By
"specifically binds" or "immunoreacts with" or "directed against"
is meant that the antibody reacts with one or more antigenic
determinants of the desired antigen and does not react with other
polypeptides or binds at a much lower affinity (Kd>10.sup.-6).
Antibodies include but are not limited to, polyclonal, monoclonal,
chimeric, Fab fragments, Fab' fragments, F(ab')2 fragments, single
chain Fv fragments, and one-armed antibodies.
[0225] As used herein, the term "monoclonal antibody" (mAb) as
applied to the present antibody compounds refers to an antibody
that is derived from a single copy or clone including, for example,
any eukaryotic, prokaryotic, or phage clone, and not the method by
which it is produced. mAbs of the present disclosure preferably
exist in a homogeneous or substantially homogeneous population.
Complete mAbs contain 2 heavy chains and 2 light chains.
[0226] An "antibody fragment" refers to a molecule other than an
intact antibody that comprises a portion of an intact antibody that
binds the antigen to which the intact antibody binds. Examples of
antibody fragments include but are not limited to Fv, Fab, Fab',
Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain
antibody molecules (e.g., scFv); and multispecific antibodies
formed from antibody fragments.
[0227] As disclosed herein, "antibody compounds" refers to mAbs and
antigen-binding fragments thereof. Additional antibody compounds
exhibiting similar functional properties according to the present
disclosure can be generated by conventional methods. For example,
mice can be immunized with human CD47 or fragments thereof, the
resulting antibodies can be recovered and purified, and
determination of whether they possess binding and functional
properties similar to or the same as the antibody compounds
disclosed herein can be assessed by the methods described in
Examples 3-17 below. Antigen-binding fragments can also be prepared
by conventional methods. Methods for producing and purifying
antibodies and antigen-binding fragments are well known in the art
and can be found, for example, in Harlow and Lane (1988)
Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., chapters 5-8 and 15.
[0228] The monoclonal antibodies encompass antibodies in which a
portion of the heavy and/or light chain is identical with, or
homologous to, corresponding sequences in murine antibodies, in
particular the murine CDRs, while the remainder of the chain(s) is
(are) identical with, or homologous to, corresponding sequences in
human antibodies. Other embodiments of the disclosure include
antigen-binding fragments of these monoclonal antibodies that
exhibit binding and biological properties similar or identical to
the monoclonal antibodies. The antibodies of the present disclosure
can comprise kappa or lambda light chain constant regions, and
heavy chain IgA, IgD, IgE, IgG, or IgM constant regions, including
those of IgG subclasses IgG1, IgG2, IgG3, and IgG4 and in some
cases with various mutations to alter Fc receptor function.
[0229] The monoclonal antibodies containing the presently disclosed
murine CDRs can be prepared by any of the various methods known to
those skilled in the art, including recombinant DNA methods.
[0230] Reviews of current methods for antibody engineering and
improvement can be found, for example, in P. Chames, Ed., (2012)
Antibody Engineering: Methods and Protocols, Second Edition
(Methods in Molecular Biology, Book 907), Humana Press, ISBN-10:
1617799734; C. R. Wood, Ed., (2011) Antibody Drug Discovery
(Molecular Medicine and Medicinal Chemistry, Book 4), Imperial
College Press; R. Kontermann and S. Dubel, Eds., (2010) Antibody
Engineering Volumes 1 and 2 (Springer Protocols), Second Edition;
and W. Strohl and L. Strohl (2012) Therapeutic antibody
engineering: Current and future advances driving the strongest
growth area in the pharmaceutical industry, Woodhead
Publishing.
[0231] Methods for producing and purifying antibodies and
antigen-binding fragments are well known in the art and can be
found, for example, in Harlow and Lane (1988) Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., chapters 5-8 and 15.
[0232] A full-length antibody as it exists naturally is a "Y"
shaped immunoglobulin (Ig) molecule comprising four polypeptide
chains: two identical heavy (H) chains and two identical light (L)
chains, interconnected by disulfide bonds. The amino terminal
portion of each chain, termed the fragment antigen binding region
(FAB), includes a variable region of about 100-110 or more amino
acids primarily responsible for antigen recognition via the
complementarity determining regions (CDRs) contained therein. The
carboxy-terminal portion of each chain defines a constant region
(the "Fc" region) primarily responsible for effector function.
[0233] The CDRs are interspersed with regions that are more
conserved, termed frameworks ("FRs"). Amino acid sequences of many
FRs are well known in the art. Each light chain variable region
(LCVR) and heavy chain variable region (HCVR) is composed of 3 CDRs
and 4 FRs, arranged from amino-terminus to carboxy-terminus in the
following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDRs
of the light chain are referred to as "LCDR1, LCDR2, and LCDR3" and
the 3 CDRs of the heavy chain are referred to as "HCDR1, HCDR2, and
HCDR3." The CDRs contain most of the residues which form specific
interactions with the antigen. The numbering and positioning of CDR
amino acid residues within the LCVR and HCVR regions are in
accordance with the well-known Kabat numbering convention Kabat et
al. (1991) Sequences of Proteins of Immunological Interest, Fifth
Edition. NIH Publication No. 91-3242.
[0234] As described herein, the "antigen-binding site" can also be
defined as the "hypervariable regions," "HVRs," or "HVs," and refer
to the structurally hypervariable regions of antibody variable
domains as defined by Chothia and Lesk (Chothia and Lesk, Mol.
Biol. 196:901-917, 1987). There are six HVRs, three in VH (H1, H2,
H3) and three in VL (L1, L2, L3). The CDRs used herein are as
defined by Kabat except in H-CDR1, which is extended to include
H1.
[0235] There are five types of mammalian immunoglobulin (Ig) heavy
chains, denoted by the Greek letters .alpha. (alpha), .delta.
(delta), .epsilon. (epsilon), .gamma. (gamma), and .mu. (mu), which
define the class or isotype of an antibody as IgA, IgD, IgE, IgG,
or IgM, respectively. IgG antibodies can be further divided into
subclasses, for example, IgG1, IgG2, IgG3, and IgG4.
[0236] Each heavy chain type is characterized by a particular
constant region with a sequence well known in the art. The constant
region is identical in all antibodies of the same isotype, but
differs in antibodies of different isotypes. Heavy chains .gamma.,
.mu., and .delta. have a constant region composed of three tandem
immunoglobulin (Ig) domains, and a hinge region for added
flexibility. Heavy chains t and E have a constant region composed
of four Ig domains.
[0237] The hinge region is a flexible amino acid stretch that links
the Fc and Fab portions of an antibody. This regions contains
cysteine residues that can form disulfide bonds, connecting two
heavy chains together.
[0238] The variable region of the heavy chain differs in antibodies
produced by different B cells, but is the same for all antibodies
produced by a single B cell or B cell clone. The variable region of
each heavy chain is approximately 110 amino acids long and is
composed of a single Ig domain.
[0239] In mammals, light chains are classified as kappa (.kappa.)
or lambda (.lamda.), and are characterized by a particular constant
region as known in the art. A light chain has two successive
domains: one variable domain at the amino-terminal end, and one
constant domain at the carboxy-terminal end. Each antibody contains
two light chains that are always identical; only one type of light
chain, .kappa. or .lamda., is present per antibody in mammals.
[0240] The Fc region, composed of two heavy chains that contribute
three or four constant domains depending on the class of the
antibody, plays a role in modulating immune cell activity. By
binding to specific proteins, the Fc region ensures that each
antibody generates an appropriate immune response for a given
antigen. The Fc region also binds to various cell receptors, such
as Fc receptors, and other immune molecules, such as complement
proteins. By doing this, it mediates different physiological
effects, including opsonization, cell lysis, and degranulation of
mast cells, basophils and eosinophils.
[0241] As used herein, the term "epitope" refers to a specific
arrangement of amino acids located on a peptide or protein to which
an antibody or antibody fragment binds. Epitopes often consist of a
chemically active surface grouping of molecules such as amino acids
or sugar side chains, and have specific three dimensional
structural characteristics as well as specific charge
characteristics. Epitopes can be linear, i.e., involving binding to
a single sequence of amino acids, or conformational, i.e.,
involving binding to two or more sequences of amino acids in
various regions of the antigen that may not necessarily be
contiguous in the linear sequence.
[0242] As used herein, the terms "specifically binds," "bind
specifically," "specific binding," and the like as applied to the
present antibody compounds refer to the ability of a specific
binding agent (such as an antibody) to bind to a target molecular
species in preference to binding to other molecular species with
which the specific binding agent and target molecular species are
admixed. A specific binding agent is said specifically to recognize
a target molecular species when it can bind specifically to that
target.
[0243] As used herein, the term "binding affinity" refers to the
strength of binding of one molecule to another at a site on the
molecule. If a particular molecule will bind to or specifically
associate with another particular molecule, these two molecules are
said to exhibit binding affinity for each other. Binding affinity
is related to the association constant and dissociation constant
for a pair of molecules as measured in a 1:1 interaction.
Affinities as used herein to describe interactions between
molecules of the described methods which can be used to compare the
relative strength with which one molecule (e.g., an antibody or
other specific binding partner) will bind two other molecules
(e.g., two versions or variants of a peptide) in a univalent
interaction. The concepts of binding affinity, association
constant, and dissociation constant are well known.
[0244] As used herein, the term "apparent binding affinity" refers
to the apparent strength of binding of one molecule to another at a
site on the molecule. If a particular molecule will bind to or
specifically associate with another particular molecule, these two
molecules are said to exhibit binding affinity for each other.
Apparent binding affinity is related to the association constant
and dissociation constant for a pair of molecules, and relates to a
non 1:1 or multivalent association between the pair of molecules.
Apparent affinities as used herein to describe interactions between
molecules of the described methods are observed in empirical
studies, which can be used to compare the relative strength with
which one molecule (e.g., an antibody or other specific binding
partner) will bind two other molecules (e.g., two versions or
variants of a peptide). The concept of binding affinity may be
described as apparent Kd, apparent binding constant, EC.sub.50 or
other measurements of binding.
[0245] As used herein, the term "sequence identity" means the
percentage of identical nucleotide or amino acid residues at
corresponding positions in two or more sequences when the sequences
are aligned to maximize sequence matching, i.e., taking into
account gaps and insertions. Identity can be readily calculated by
known methods, including but not limited to those described in:
Computational Molecular Biology, Lesk, A. M., ed., Oxford
University Press, New York, 1988; Biocomputing: Informatics and
Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;
Computer Analysis of Sequence Data, Part I, Griffin, A. M., and
Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence
Analysis in Molecular Biology, von Heinje, G., Academic Press,
1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J.,
eds., M Stockton Press, New York, 1991; and Carillo, H., and
Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods to
determine identity are designed to give the largest match between
the sequences tested. Moreover, methods to determine identity are
codified in publicly available computer programs.
[0246] Optimal alignment of sequences for comparison can be
conducted, for example, by the local homology algorithm of Smith
& Waterman, by the homology alignment algorithms, by the search
for similarity method or, by computerized implementations of these
algorithms (GAP, BESTFIT, PASTA, and TFASTA in the GCG Wisconsin
Package, available from Accelrys, Inc., San Diego, Calif., United
States of America), or by visual inspection. See generally,
Altschul, S. F. et al., J. Mol. Biol. 215: 403-410 (1990) and
Altschul et al. Nucl. Acids Res. 25: 3389-3402 (1997).
[0247] One example of an algorithm that is suitable for determining
percent sequence identity and sequence similarity is the BLAST
algorithm, which is described in (Altschul, S. et al., NCBI NLM NIH
Bethesda, Md. 20894; and Altschul, S. et al., J. Mol. Biol. 215:
403-410 (1990). Software for performing BLAST analyses is publicly
available through the National Center for Biotechnology
Information. This algorithm involves first identifying high scoring
sequence pairs (HSPs) by identifying short words of length W in the
query sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighborhood word score
threshold.
[0248] These initial neighborhood word hits act as seeds for
initiating searches to find longer HSPs containing them. The word
hits are then extended in both directions along each sequence for
as far as the cumulative alignment score can be increased.
Cumulative scores are calculated using, for nucleotide sequences,
the parameters M (reward score for a pair of matching residues;
always; 0) and N (penalty score for mismatching residues; always;
0). For amino acid sequences, a scoring matrix is used to calculate
the cumulative score. Extension of the word hits in each direction
are halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value, the cumulative score
goes to zero or below due to the accumulation of one or more
negative-scoring residue alignments, or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a word length (W) of 11, an
expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison
of both strands. For amino acid sequences, the BLASTP program uses
as defaults a word length (W) of 3, an expectation (E) of 10, and
the BLOSUM62 scoring matrix.
[0249] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences. One measure of similarity
provided by the BLAST algorithm is the smallest sum probability
(P(N)), which provides an indication of the probability by which a
match between two nucleotide or amino acid sequences would occur by
chance. For example, a test nucleic acid sequence is considered
similar to a reference sequence if the smallest sum probability in
a comparison of the test nucleic acid sequence to the reference
nucleic acid sequence is in one embodiment less than about 0.1, in
another embodiment less than about 0.01, and in still another
embodiment less than about 0.001.
[0250] As used herein, the terms "humanized," "humanization," and
the like, refer to grafting of the murine monoclonal antibody CDRs
disclosed herein to human FRs and constant regions. Also
encompassed by these terms are possible further modifications to
the murine CDRs, and human FRs, by the methods disclosed in, for
example, Kashmiri et al. (2005) Methods 36(1):25-34 and Hou et al.
(2008) J. Biochem. 144(1):115-120, respectively, to improve various
antibody properties, as discussed below.
[0251] As used herein, the term "humanized antibodies" refers to
mAbs and antigen binding fragments thereof, including antibody
compounds, that have binding and functional properties similar to
those disclosed herein, and that have FRs and constant regions that
are substantially human or fully human surrounding CDRs derived
from a non-human antibody.
[0252] As used herein, the term "FR" or "framework sequence" refers
to any one of FRs 1 to 4. Humanized antibodies and antigen binding
fragments encompassed by the present disclosure include molecules
wherein any one or more of FRs 1 to 4 is substantially or fully
human, i.e., wherein any of the possible combinations of individual
substantially or fully human FRs 1 to 4, is present. For example,
this includes molecules in which FR1 and FR2, FR1 and FR3, FR1,
FR2, and FR3, etc., are substantially or fully human. Substantially
human frameworks are those that have at least 80% sequence identity
to a known human germline framework sequence. Preferably, the
substantially human frameworks have at least 85%, at least 86%, at
least 87%, at least 88%, at least 89%, at least 90%, at least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at least 97%, at least 98%, or at least 99% sequence identity,
to a framework sequence disclosed herein, or to a known human
germline framework sequence.
[0253] Fully human frameworks are those that are identical to a
known human germline framework sequence. Human FR germline
sequences can be obtained from the international ImMunoGeneTics
(IMGT) database and from The Immunoglobulin FactsBook by
Marie-Paule Lefranc and Gerard Lefranc, Academic Press, 2001, the
contents of which are herein incorporated by reference in their
entirety.
[0254] The Immunoglobulin Facts Book is a compendium of the human
germline immunoglobulin genes that are used to create the human
antibody repertoire, and includes entries for 203 genes and 459
alleles, with a total of 837 displayed sequences. The individual
entries comprise all the human immunoglobulin constant genes, and
germline variable, diversity, and joining genes that have at least
one functional or open reading frame allele, and which are
localized in the three major loci. For example, germline light
chain FRs can be selected from the group consisting of: IGKV3D-20,
IGKV2-30, IGKV2-29, IGKV2-28, IGKV1-27, IGKV3-20, IGKV1-17,
IGKV1-16, 1-6, IGKV1-5, IGKV1-12, IGKV1D-16, IGKV2D-28, IGKV2D-29,
IGKV3-11, IGKV1-9, IGKV1-39, IGKV1D-39, IGKV1D-33, and IGKJ1-5; and
germline heavy chain FRs can be selected from the group consisting
of: IGHV1-2, IGHV1-18, IGHV1-46, IGHV1-69, IGHV2-5, IGHV2-26,
IGHV2-70, IGHV1-3, IGHV1-8, IGHV3-9, IGHV3-11, IGHV3-15, IGHV3-20,
IGHV3-66, IGHV3-72, IGHV3-74, IGHV4-31, IGHV3-21, IGHV3-23,
IGHV3-30, IGHV3-48, IGHV4-39, IGHV4-59, IGHV5-51, and IGHJ1-6.
[0255] Substantially human FRs are those that have at least 80%
sequence identity to a known human germline FR sequence.
Preferably, the substantially human frameworks have at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, or at least 99% sequence identity, to a framework sequences
disclosed herein, or to a known human germline framework
sequence.
[0256] CDRs encompassed by the present disclosure include not only
those specifically disclosed herein, but also CDR sequences having
sequence identities of at least 80%, at least 85%, at least 90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least
99% sequence identity to a CDR sequence disclosed herein.
Alternatively, CDRs encompassed by the present disclosure include
not only those specifically disclosed herein, but also CDR
sequences having 1, 2, 3, 4, or 5 amino acid changes at
corresponding positions compared to CDR sequences disclosed herein.
Such sequence identical, or amino acid modified, CDRs preferably
bind to the antigen recognized by the intact antibody.
[0257] Humanized antibodies in addition to those disclosed herein
exhibiting similar functional properties according to the present
disclosure can be generated using several different methods,
including those disclosed by Almagro et al. (Frontiers in
Biosciences. Humanization of antibodies. (2008) Jan. 1;
13:1619-33).
[0258] In one approach, the parent antibody compound CDRs are
grafted into a human framework that has a high sequence identity
with the parent antibody compound framework. The sequence identity
of the new framework will generally be at least 80%, at least 85%,
at least 86%, at least 87%, at least 88%, at least 89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least
99% sequence identical to the sequence of the corresponding
framework in the parent antibody compound. In the case of
frameworks having fewer than 100 amino acid residues, one, two,
three, four, five, six, seven, eight, nine, or ten amino acid
residues can be changed. This grafting may result in a reduction in
binding affinity compared to that of the parent antibody. If this
is the case, the framework can be back-mutated to the parent
framework at certain positions based on specific criteria disclosed
by Queen et al. (1991) Proc. Natl. Acad. Sci. USA 88:2869.
Additional references describing methods useful to generate
humanized variants based on homology and back mutations include as
described in Olimpieri et al. (Bioinformatics 2015 Feb. 1;
31(3):434-435) and U.S. Pat. Nos. 4,816,397, 5,225,539, and
5,693,761; and the method of Winter and co-workers (Jones et al.
(1986) Nature 321:522-525; Riechmann et al. (1988) Nature
332:323-327; and Verhoeyen et al. (1988) Science
239:1534-1536).
[0259] Humanization began with chimerization, a method developed
during the first half of the 1980's (Morrison, S. L., M. J.
Johnson, L. A. Herzenberg & V. T. Oi: Chimeric human antibody
molecules: mouse antigen-binding domains with human constant region
domains. Proc. Natl. Acad. Sci. USA 81, 6851-5 (1984)), consisting
of combining the variable (V) domains of murine antibodies with
human constant (C) domains to generate molecules with .about.70% of
human content.
[0260] Several different methods can be used to generate humanized
antibodies, which are described herein. In one approach, the parent
antibody compound CDRs are grafted into a human FR that has a high
sequence identity with the parent antibody compound framework. The
sequence identity of the new FR will generally be at least 60%, at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, or at least 99% identical to the sequence of the corresponding
FR in the parent antibody compound. In the case of FRs having fewer
than 100 amino acid residues, one, two, three, four, five, or more
amino acid residues can be changed. This grafting may result in a
reduction in binding affinity compared to that of the parent
antibody. If this is the case, the FR can be back-mutated to the
parent framework at certain positions based on specific criteria
disclosed by Queen et al. (1991) Proc. Natl. Acad. Sci. USA
88:2869. Additional references describing methods useful to
generate humanized variants based on homology and back mutations
include as described in Olimpieri et al. (Bioinformatics. 2015 Feb.
1; 31(3):434-435) and U.S. Pat. Nos. 4,816,397, 5,225,539, and
5,693,761; and the method of Winter and co-workers (Jones et al.
(1986) Nature 321:522-525; Riechmann et al. (1988) Nature
332:323-327; and Verhoeyen et al. (1988) Science
239:1534-1536).
[0261] The identification of residues to consider for back-mutation
can be carried out as described below. When an amino acid falls
under the following category, the framework amino acid of the human
germ-line sequence that is being used (the "acceptor FR") is
replaced by a framework amino acid from a framework of the parent
antibody compound (the "donor FR"):
[0262] (a) the amino acid in the human FR of the acceptor framework
is unusual for human frameworks at that position, whereas the
corresponding amino acid in the donor immunoglobulin is typical for
human frameworks at that position;
[0263] (b) the position of the amino acid is immediately adjacent
to one of the CDRs; or
[0264] (c) any side chain atom of a framework amino acid is within
about 5-6 angstroms (center-to-center) of any atom of a CDR amino
acid in a three dimensional immunoglobulin model.
[0265] When each of the amino acids in the human FR of the acceptor
framework and a corresponding amino acid in the donor framework is
generally unusual for human frameworks at that position, such amino
acid can be replaced by an amino acid typical for human frameworks
at that position. This back-mutation criterion enables one to
recover the activity of the parent antibody compound.
[0266] Another approach to generating humanized antibodies
exhibiting similar functional properties to the antibody compounds
disclosed herein involves randomly mutating amino acids within the
grafted CDRs without changing the framework, and screening the
resultant molecules for binding affinity and other functional
properties that are as good as, or better than, those of the parent
antibody compounds. Single mutations can also be introduced at each
amino acid position within each CDR, followed by assessing the
effects of such mutations on binding affinity and other functional
properties. Single mutations producing improved properties can be
combined to assess their effects in combination with one
another.
[0267] Further, a combination of both of the foregoing approaches
is possible. After CDR grafting, one can back-mutate specific FRs
in addition to introducing amino acid changes in the CDRs. This
methodology is described in Wu et al. (1999, J. Mol. Biol. 294:
151-162).
[0268] Applying the teachings of the present disclosure, a person
skilled in the art can use common techniques, e.g., site-directed
mutagenesis, to substitute amino acids within the presently
disclosed CDR and FR sequences and thereby generate further
variable region amino acid sequences derived from the present
sequences. Up to all naturally occurring amino acids can be
introduced at a specific substitution site. The methods disclosed
herein can then be used to screen these additional variable region
amino acid sequences to identify sequences having the indicated in
vivo functions. In this way, further sequences suitable for
preparing humanized antibodies and antigen-binding portions thereof
in accordance with the present disclosure can be identified.
Preferably, amino acid substitution within the frameworks is
restricted to one, two, three, four, or five positions within any
one or more of the four light chain and/or heavy chain FRs
disclosed herein. Preferably, amino acid substitution within the
CDRs is restricted to one, two, three, four, or five positions
within any one or more of the three light chain and/or heavy chain
CDRs. Combinations of the various changes within these FRs and CDRs
described above are also possible.
[0269] That the functional properties of the antibody compounds
generated by introducing the amino acid modifications discussed
above conform to those exhibited by the specific molecules
disclosed herein can be confirmed by the methods in Examples
disclosed herein.
[0270] As described above, to circumvent the problem of eliciting
human anti-murine antibody (HAMA) response in patients, murine
antibodies have been genetically manipulated to progressively
replace their murine content with the amino acid residues present
in their human counterparts by grafting their complementarity
determining regions (CDRs) onto the variable light (VL) and
variable heavy (VH) frameworks of human immunoglobulin molecules,
while retaining those murine framework residues deemed essential
for the integrity of the antigen-combining site. However, the
xenogeneic CDRs of the humanized antibodies may evoke
anti-idiotypic (anti-Id) response in patients.
[0271] To minimize the anti-Id response, a procedure to humanize
xenogeneic antibodies by grafting onto the human frameworks only
the CDR residues most crucial in the antibody-ligand interaction,
called "SDR grafting", has been developed, wherein only the crucial
specificity determining residues (SDRs) of CDRS are grafted onto
the human frameworks.
[0272] This procedure, described in Kashmiri et al. (2005, Methods
36(1):25-34), involves identification of SDRs through the help of a
database of the three-dimensional structures of the
antigen-antibody complexes of known structures, or by mutational
analysis of the antibody-combining site. An alternative approach to
humanization involving retention of more CDR residues is based on
grafting of the `abbreviated` CDRs, the stretches of CDR residues
that include all the SDRs. Kashmiri et al. also discloses a
procedure to assess the reactivity of humanized antibodies to sera
from patients who had been administered the murine antibody.
[0273] Another strategy for constructing human antibody variants
with improved immunogenic properties is disclosed in Hou et al.
(2008, J. Biochem. 144(1):115-120). These authors developed a
humanized antibody from 4C8, a murine anti-human CD34 monoclonal
antibody, by CDR grafting using a molecular model of 4C8 built by
computer-assisted homology modelling. Using this molecular model,
the authors identified FR residues of potential importance in
antigen binding. A humanized version of 4C8 was generated by
transferring these key murine FR residues onto a human antibody
framework that was selected based on homology to the murine
antibody FR, together with the murine CDR residues. The resulting
humanized antibody was shown to possess antigen-binding affinity
and specificity similar to that of the original murine antibody,
suggesting that it might be an alternative to murine anti-CD34
antibodies routinely used clinically.
[0274] Embodiments of the present disclosure encompass antibodies
created to avoid recognition by the human immune system containing
CDRs disclosed herein in any combinatorial form such that
contemplated mAbs can contain the set of CDRs from a single murine
mAb disclosed herein, or light and heavy chains containing sets of
CDRs comprising individual CDRs derived from two or three of the
disclosed murine mAbs. Such mAbs can be created by standard
techniques of molecular biology and screened for desired activities
using assays described herein. In this way, the disclosure provides
a "mix and match" approach to create novel mAbs comprising a
mixture of CDRs from the disclosed murine mAbs to achieve new, or
improved, therapeutic activities.
[0275] Monoclonal antibodies or antigen-binding fragments thereof
encompassed by the present disclosure that "compete" with the
molecules disclosed herein are those that bind human CD47 at
site(s) that are identical to, or overlapping with, the site(s) at
which the present molecules bind. Competing monoclonal antibodies
or antigen-binding fragments thereof can be identified, for
example, via an antibody competition assay. For example, a sample
of purified or partially purified human CD47 extracellular domain
can be bound to a solid support. Then, an antibody compound, or
antigen binding fragment thereof, of the present disclosure and a
monoclonal antibody or antigen-binding fragment thereof suspected
of being able to compete with such disclosure antibody compound are
added. One of the two molecules is labelled. If the labelled
compound and the unlabelled compound bind to separate and discrete
sites on CD47, the labelled compound will bind to the same level
whether or not the suspected competing compound is present.
However, if the sites of interaction are identical or overlapping,
the unlabelled compound will compete, and the amount of labelled
compound bound to the antigen will be lowered. If the unlabelled
compound is present in excess, very little, if any, labelled
compound will bind. For purposes of the present disclosure,
competing monoclonal antibodies or antigen-binding fragments
thereof are those that decrease the binding of the present antibody
compounds to CD47 by about 50%, about 60%, about 70%, about 80%,
about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,
about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,
about 97%, about 98%, or about 99%. Details of procedures for
carrying out such competition assays are well known in the art and
can be found, for example, in Harlow and Lane (1988) Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. Such assays can be made quantitative by using purified
antibodies. A standard curve is established by titrating one
antibody against itself, i.e., the same antibody is used for both
the label and the competitor. The capacity of an unlabelled
competing monoclonal antibody or antigen-binding fragment thereof
to inhibit the binding of the labelled molecule to the plate is
titrated. The results are plotted, and the concentrations necessary
to achieve the desired degree of binding inhibition are
compared.
[0276] Whether mAbs or antigen-binding fragments thereof that
compete with antibody compounds of the present disclosure in such
competition assays possess the same or similar functional
properties of the present antibody compounds can be determined via
these methods in conjunction with the methods described in Examples
below. In various embodiments, competing antibodies for use in the
therapeutic methods encompassed herein possess biological
activities as described herein in the range of from about 50% to
about 100% or about 125%, or more, compared to that of the antibody
compounds disclosed herein. In some embodiments, competing
antibodies possess about 50%, about 60%, about 70%, about 80%,
about 85%, about 90%, about 91%, about 92%, about 93%, about 94%,
about 95%, about 96%, about 97%, about 98%, about 99%, or identical
biological activity compared to that of the antibody compounds
disclosed herein as determined by the methods disclosed in the
Examples presented below.
[0277] The mAbs or antigen-binding fragments thereof, or competing
antibodies useful in the compositions and methods can be any of the
isotypes described herein. Furthermore, any of these isotypes can
comprise further amino acid modifications as follows.
[0278] The monoclonal antibody or antigen-binding fragment thereof,
or competing antibody described herein can be of the human IgG1
isotype.
[0279] The human IgG1 constant region of the monoclonal antibody,
antigen-binding fragment thereof, or competing antibody described
herein can be modified to alter antibody half-life. Antibody
half-life is regulated in large part by Fc-dependent interactions
with the neonatal Fc receptor (Roopenian and Alikesh, 2007). The
human IgG1 constant region of the monoclonal antibody,
antigen-binding fragment thereof, or competing antibody can be
modified to increase half-life include, but are not limited to
amino acid modifications N434A, T307A/E380A/N434A (Petkova et al.,
2006, Yeung et al., 2009); M252Y/S254T/T256E (Dall'Acqua et al.,
2006); T250Q/M428L (Hinton et al., 2006); and M428L/N434S (Zalevsky
et al., 2010).
[0280] As opposed to increasing half-life, there are some
circumstances where decreased half-life would be desired, such as
to reduce the possibility of adverse events associated with high
Antibody-Dependent Cellular Cytotoxicity (ADCC) and
Complement-Dependent Cytotoxicity (CDC) antibodies (Presta 2008).
The human IgG1 constant region of the monoclonal antibody,
antigen-binding fragment thereof, or competing antibody described
herein can be modified to decrease half-life and/or decrease
endogenous IgG include, but are not limited to amino acid
modifications I253A (Petkova et al., 2006); P257I/N434H,
D376V/N434H (Datta-Mannan et al., 2007); and
M252Y/S254T/T256E/H433K/N434F (Vaccaro et al., 2005).
[0281] The human IgG1 constant region of the monoclonal antibody,
antigen-binding fragment thereof, or competing antibody described
herein can be modified to increase or decrease antibody effector
functions. These antibody effector functions include, but are not
limited to, Antibody-Dependent Cellular Cytotoxicity (ADCC),
Complement-Dependent Cytotoxicity (CDC), Antibody-Dependent
Cellular Phagocytosis (ADCP), C1q binding, and altered binding to
Fc receptors.
[0282] The human IgG1 constant region of the monoclonal antibody,
antigen-binding fragment thereof, or competing antibody described
herein can be modified to increase antibody effector function
include, but are not limited to amino acid modifications
S298A/E333A/K334 (Shields et al., 2001); S239D/I332E and
S239D/A330L/I332E (Lazar et al., 2006); F234L/R292P/Y300L,
F234L/R292P/Y300L/P393L, and F243L/R292P/Y300L/V305I/P396L
(Stevenhagen et al., 2007); G236A, G236A/S239D/I332E, and
G236A/S239D/A330L/I332E (Richards et al., 2008); K326A/E333A,
K326A/E333S and K326W/E333S (Idusogie et al., 2001); S267E and
S267E/L328F (Smith et al., 2012); H268F/S324T, S267E/H268F,
S267E/S234T, and S267E/H268F/S324T (Moore et al., 2010);
S298G/T299A (Sazinsky et al., 2008); E382V/M428I (Jung et al.,
2010).
[0283] The human IgG1 constant region of the monoclonal antibody,
antigen-binding fragment thereof, or competing antibody described
herein can be modified to decrease antibody effector function
include, but are not limited to amino acid modifications N297A and
N297Q (Bolt et al., 1993, Walker et al., 1989); L234A/L235A (Xu et
al., 2000);
K214T/E233P/L234V/L235A/G236-deleted/A327G/P331A/D356E/L358M
(Ghevaert et al., 2008); C226S/C229S/E233P/L234V/L235A (McEarchern
et al., 2007); S267E/L328F (Chu et al., 2008).
[0284] The human IgG1 constant region of the monoclonal antibody,
antigen-binding fragment thereof, or competing antibody described
herein can be modified to decrease antibody effector function
include, but are not limited to amino acid modifications
V234A/G237A (Cole et al., 1999); E233D, G237D, P238D, H268Q, H268D,
P271G, V309L, A330S, A330R, P331S, H268Q/A330S/V309L/P331S,
H268D/A330S/V309L/P331S, H268Q/A330R/V309L/P331S,
H268D/A330R/V309L/P331S, E233D/A330R, E233D/A330S,
E233D/P271G/A330R, E233D/P271G/A330S, G237D/H268D/P271G,
G237D/H268Q/P271G, G237D/P271G/A330R, G237D/P271G/A330S,
E233D/H268D/P271G/A330R, E233D/H268Q/P271G/A330R,
E233D/H268D/P271G/A330S, E233D/H268Q/P271G/A330S, G237D/H268D/P271
G/A330R, G237D/H268Q/P271G/A330R, G237D/H268D/P271G/A330S,
G237D/H268Q/P271G/A330S, E233D/G237D/H268D/P271G/A330R,
E233D/G237D/H268Q/P271G/A330R, E233D/G237D/H268D/P271G/A330S,
E233D/G237D/H268Q/P271G/A330S, P238D/E233D/A330R,
P238D/E233D/A330S, P238D/E233D/P271G/A330R,
P238D/E233D/P271G/A330S, P238D/G237D/H268D/P271 G,
P238D/G237D/H268Q/P271G, P238D/G237D/P271G/A330R,
P238D/G237D/P271G/A330S, P238D/E233D/H268D/P271G/A330R,
P238D/E233D/H268Q/P271G/A330R, P238D/E233D/H268D/P271G/A330S,
P238D/E233D/H268Q/P271G/A330S, P238D/G237D/H268D/P271G/A330R,
P238D/G237D/H268Q/P271G/A330R, P238D/G237D/H268D/P271G/A330S,
P238D/G237D/H268Q/P271G/A330S, P238D/E233D/G237D/H268D/P271G/A330R,
P238D/E233D/G237D/H268Q/P271 G/A330R,
P238D/E233D/G237D/H268D/P271G/A330S,
P238D/E233D/G237D/H268Q/P271G/A330S (An et al., 2009, Mimoto,
2013).
[0285] The monoclonal antibody or antigen-binding fragment thereof,
or competing antibody described herein can be of the human IgG2
isotype.
[0286] The human IgG2 constant region of the monoclonal antibody,
antigen-binding fragment thereof, or competing antibody described
herein can be modified to increase or decrease antibody effector
functions. These antibody effector functions include, but are not
limited to, Antibody-Dependent Cellular Cytotoxicity (ADCC),
Complement-Dependent Cytotoxicity (CDC), Antibody-Dependent
Cellular Phagocytosis (ADCP), and C1q binding, and altered binding
to Fc receptors.
[0287] The human IgG2 constant region of the monoclonal antibody,
antigen-binding fragment thereof, or competing antibody described
herein can be modified to increase antibody effector function
include, but are not limited to the amino acid modification
K326A/E333S (Idusogie et al., 2001).
[0288] The human IgG2 constant region of the monoclonal antibody,
antigen-binding fragment thereof, or competing antibody described
herein can be modified to decrease antibody effector function
include, but are not limited to amino acid modifications
V234A/G237A (Cole et al., 1999); V234A, G237A, P238S, H268A, E233D,
G237D, P238D, H268Q, H268D, P271G, V309L, A330S, A330R, P331S,
P238S/H268A, V234A/G237A/P238S/H268A/V309L/A330S/P331S,
H268Q/A330S/V309L/P331S, H268D/A330S/V309L/P331S,
H268Q/A330R/V309L/P331S, H268D/A330R/V309L/P331S, E233D/A330R,
E233D/A330S, E233D/P271G/A330R, E233D/P271G/A330S,
G237D/H268D/P271G, G237D/H268Q/P271G, G237D/P271G/A330R,
G237D/P271G/A330S, E233D/H268D/P271G/A330R,
E233D/H268Q/P271G/A330R, E233D/H268D/P271G/A330S,
E233D/H268Q/P271G/A330S, G237D/H268D/P271G/A330R,
G237D/H268Q/P271G/A330R, G237D/H268D/P271G/A330S,
G237D/H268Q/P271G/A330S, E233D/G237D/H268D/P271G/A330R,
E233D/G237D/H268Q/P271G/A330R, E233D/G237D/H268D/P271G/A330S,
E233D/G237D/H268Q/P271G/A330S, P238D/E233D/A330R,
P238D/E233D/A330S, P238D/E233D/P271G/A330R,
P238D/E233D/P271G/A330S, P238D/G237D/H268D/P271 G,
P238D/G237D/H268Q/P271G, P238D/G237D/P271G/A330R,
P238D/G237D/P271G/A330S, P238D/E233D/H268D/P271G/A330R,
P238D/E233D/H268Q/P271G/A330R, P238D/E233D/H268D/P271G/A330S,
P238D/E233D/H268Q/P271G/A330S, P238D/G237D/H268D/P271G/A330R,
P238D/G237D/H268Q/P271G/A330R, P238D/G237D/H268D/P271G/A330S,
P238D/G237D/H268Q/P271G/A330S, P238D/E233D/G237D/H268D/P271G/A330R,
P238D/E233D/G237D/H268Q/P271 G/A330R,
P238D/E233D/G237D/H268D/P271G/A330S,
P238D/E233D/G237D/H268Q/P271G/A330S (An et al., 2009, Mimoto,
2013).
[0289] The Fc region of a human IgG2 of the monoclonal antibody,
antigen-binding fragment thereof, or competing antibody described
herein can be modified to alter isoform and/or agonistic activity,
include, but are not limited to amino acid modifications C127S (CH1
domain), C232S, C233S, C232S/C233S, C236S, and C239S (White et al.,
2015, Lightle et al., 2010).
[0290] The monoclonal antibody or antigen-binding fragment thereof,
or competing antibody described herein can be of the human IgG3
isotype.
[0291] The human IgG3 constant region of the monoclonal antibody,
or antigen binding fragment thereof, wherein said human IgG3
constant region of the monoclonal antibody, or antigen-binding
fragment thereof can be modified at one or more amino acid(s) to
increase antibody half-life, Antibody-Dependent Cellular
Cytotoxicity (ADCC), Complement-Dependent Cytotoxicity (CDC), or
apoptosis activity.
[0292] The human IgG3 constant region of the monoclonal antibody,
or antigen-binding fragment thereof, wherein said human IgG3
constant region of the monoclonal antibody, or antigen-binding
fragment thereof can be modified at amino acid R435H to increase
antibody half-life.
[0293] The monoclonal antibody or antigen-binding fragment thereof,
or competing antibody described herein can be of the human IgG4
isotype.
[0294] The human IgG4 constant region of the monoclonal antibody,
antigen-binding fragment thereof, or competing antibody described
herein can be modified to decrease antibody effector functions.
These antibody effector functions include, but are not limited to,
Antibody-Dependent Cellular Cytotoxicity (ADCC) and
Antibody-Dependent Cellular Phagocytosis (ADCP).
[0295] The human IgG4 constant region of the monoclonal antibody,
antigen-binding fragment thereof, or competing antibody described
herein can be modified to prevent Fab arm exchange and/or decrease
antibody effector function include, but are not limited to amino
acid modifications F234A/L235A (Alegre et al., 1994); S228P, L235E
and S228P/L235E (Reddy et al., 2000).
[0296] As used herein, the term "tumor" refers to all neoplastic
cell growth and proliferation, whether malignant or benign, and all
pre-cancerous and cancerous cells and tissues.
[0297] The terms "cancer," "cancerous," and "tumor" are not
mutually exclusive as used herein.
[0298] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by aberrant cell growth/proliferation. Examples of cancers include,
but are not limited to, carcinoma, lymphoma (i.e., Hodgkin's and
non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More
particular examples of such cancers include squamous cell cancer,
small-cell lung cancer, non-small cell lung cancer, adenocarcinoma
of the lung, squamous carcinoma of the lung, cancer of the
peritoneum, hepatocellular cancer, gastrointestinal cancer,
pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver
cancer, bladder cancer, hepatoma, breast cancer, colon cancer,
colorectal cancer, endometrial or uterine carcinoma, salivary gland
carcinoma, kidney cancer, liver cancer, prostate cancer, vulval
cancer, thyroid cancer, hepatic carcinoma, leukemia and other
lymphoproliferative disorders, and various types of head and neck
cancer.
[0299] The term "susceptible cancer" as used herein refers to a
cancer, cells of which express CD47, and are responsive to
treatment with an anti-CD47 antibody or antigen binding fragment
thereof, or competing antibody or antigen binding fragment thereof,
of the present disclosure.
[0300] "Nitric oxide (NO) donor, precursor, or nitric oxide
generating topical agent" refers to a compound or agent that either
delivers NO, or that can be converted to NO through enzymatic or
non-enzymatic processes. Examples include, but are not limited to,
NO gas, isosorbide dinitrite, nitrite, nitroprusside,
nitroglycerin, 3-Morpholinosydnonimine (SIN-1),
S-nitroso-N-acetyl-penicillamine (SNAP), Diethylenetriamine/NO
(DETA/NO), S-nitrosothiols, Bidil.RTM., and arginine.
[0301] "Soluble guanylyl cyclase (sGC)" is the receptor for nitric
oxide in vascular smooth muscle. In the cardiovascular system,
nitric oxide is endogenously generated by endothelial nitric oxide
synthase from L-arginine, and activates soluble guanylyl cyclase in
adjacent vascular smooth muscle cells to increase cGMP levels,
inducing vascular relaxation. Nitric oxide binds to the normally
reduced heme moiety of soluble guanylyl cyclase, and increases the
formation of cGMP from GTP, leading to a decrease in intracellular
calcium, vasodilation, and anti-inflammatory effects. Oxidation of
the heme iron on sGC decreases responsiveness of the enzyme to
nitric oxide, and promotes vasoconstriction. The nitric
oxide-sGC-cGMP pathway therefore plays an important role in
cardiovascular diseases. Nitrogen-containing compounds such as
sodium azide, sodium nitrite, hydroxylamine, nitroglycerin, and
sodium nitroprusside have been shown to stimulate sGC, causing an
increase in cGMP, and vascular relaxation. In contrast to
stimulators of sGC, which bind to reduced sGC, activators of sGC
activate the oxidized or heme-deficient sGC enzyme that is not
responsive to nitric oxide, i.e., they stimulate sGC independent of
redox state. While stimulators of of sGC can enhance the
sensitivity of reduced sGC to nitric oxide, activators of sGC can
increase sGC enzyme activity even when the enzyme is oxidized and
is therefore less, or unresponsive, to nitric oxide. Thus, sGC
activators are non-nitric oxide based. Note the reviews of Nossaman
et al. (2012) Critical Care Research and Practice, Volume 2012,
article 290805, and Derbyshire and Marletta (2012) Ann. Rev.
Biochem. 81:533-559.
[0302] "An agent that activates soluble guanylyl cyclase" refers,
for example, to organic nitrates (Artz et al. (2002) J. Biol. Chem.
277:18253-18256); protoporphyrin IX (Ignarro et al. (1982) Proc.
Natl. Acad. Sci. USA 79:2870-2873); YC-1 (Ko et al. (1994) Blood
84:4226-4233); BAY 41-2272 and BAY 41-8543 (Stasch et al. (2001
Nature 410 (6825): 212-5), CMF-1571, and A-350619 (reviewed in
Evgenov et al. (2006) Nat. Rev. Drug. Discov. 5:755-768); BAY
58-2667 (Cinaciguat; Frey et al. (2008) Journal of Clinical
Pharmacology 48 (12): 1400-10); BAY 63-2521 (Riociguat; Mittendorf
et al. (2009) Chemmedchem 4 (5): 853-65). Additional soluble
guanylyl cyclase activators are disclosed in Stasch et al. (2011)
Circulation 123:2263-2273; Derbyshire and Marletta (2012) Ann. Rev.
Biochem. 81:533-559, and Nossaman et al. (2012) Critical Care
Research and Practice, Volume 2012, Article ID 290805, pages
1-12.
[0303] cGMP can also be increased by inhibiting degradation using
phosphodiesterase inhibitors. Examples of "an agent that inhibits
cyclic nucleotide phosphodiesterases" include, tadalafil,
vardenafil, udenafil, and sildenafil avanafil.
[0304] As used herein, term "treating" or "treat" or "treatment"
means slowing, interrupting, arresting, controlling, stopping,
reducing, or reversing the progression or severity of a sign,
symptom, disorder, condition, or disease, but does not necessarily
involve a total elimination of all disease-related signs, symptoms,
conditions, or disorders. The term "treating" and the like refer to
a therapeutic intervention that ameliorates a sign or symptom of a
disease or pathological condition after it has begun to
develop.
[0305] As used herein, term "effective amount" refers to the amount
or dose of an antibody compound of the present disclosure which,
upon single or multiple dose administration to a patient or organ,
provides the desired treatment or prevention.
[0306] The precise effective amount for any particular subject will
depend upon their size and health, the nature and extent of their
condition, and the therapeutics or combination of therapeutics
selected for administration. The effective amount for a given
patient is determined by routine experimentation and is within the
judgment of a clinician. Therapeutically effective amounts of the
present antibody compounds can also comprise an amount in the range
of from about 0.1 mg/kg to about 150 mg/kg, from about 0.1 mg/kg to
about 100 mg/kg, from about 0.1 mg/kg to about 50 mg/kg, or from
about 0.05 mg/kg to about 10 mg/kg per single dose administered to
a harvested organ or to a patient. Known antibody-based
pharmaceuticals provide guidance in this respect. For example,
Herceptin.TM. is administered by intravenous infusion of a 21 mg/ml
solution, with an initial loading dose of 4 mg/kg body weight and a
weekly maintenance dose of 2 mg/kg body weight; Rituxan.TM. is
administered weekly at 375 mg/m2; for example.
[0307] A therapeutically effective amount for any individual
patient can be determined by the health care provider by monitoring
the effect of the antibody compounds on tumor regression,
circulating tumor cells, tumor stem cells or anti-tumor responses.
Analysis of the data obtained by these methods permits modification
of the treatment regimen during therapy so that optimal amounts of
antibody compounds of the present disclosure, whether employed
alone or in combination with one another, or in combination with
another therapeutic agent, or both, are administered, and so that
the duration of treatment can be determined as well. In this way,
the dosing/treatment regimen can be modified over the course of
therapy so that the lowest amounts of antibody compounds used alone
or in combination that exhibit satisfactory efficacy are
administered, and so that administration of such compounds is
continued only so long as is necessary to successfully treat the
patient. Known antibody-based pharmaceuticals provide guidance
relating to frequency of administration e.g., whether a
pharmaceutical should be delivered daily, weekly, monthly, etc.
Frequency and dosage may also depend on the severity of
symptoms.
[0308] In some embodiments antibody compounds of the present
disclosure can be used as medicaments in human and veterinary
medicine, administered by a variety of routes including, but not
limited to, oral, intravenous, intramuscular, intra-arterial,
intramedullary, intraperitoneal, intrathecal, intraventricular,
transdermal, transcutaneous, topical, subcutaneous, intratumoral,
intranasal, enteral, sublingual, intravaginal, intravesiciular or
rectal routes. The compositions can also be administered directly
into a lesion such as a tumor. Dosage treatment may be a single
dose schedule or a multiple dose schedule. Hypo sprays may also be
used to administer the pharmaceutical compositions. Typically, the
therapeutic compositions can be prepared as injectables, either as
liquid solutions or suspensions. Solid forms suitable for solution
in, or suspension in, liquid vehicles prior to injection can also
be prepared. Veterinary applications include the treatment of
companion/pet animals, such as cats and dogs; working animals, such
as guide or service dogs, and horses; sport animals, such as horses
and dogs; zoo animals, such as primates, cats such as lions and
tigers, bears, etc.; and other valuable animals kept in
captivity.
[0309] Such pharmaceutical compositions can be prepared by methods
well known in the art. See, e.g., Remington: The Science and
Practice of Pharmacy, 21st Edition (2005), Lippincott Williams
& Wilkins, Philadelphia, Pa., and comprise one or more antibody
compounds disclosed herein, and a pharmaceutically or veterinarily
acceptable, for example, physiologically acceptable, carrier,
diluent, or excipient.
[0310] The present disclosure describes anti-CD47 mAbs with
distinct functional profiles. These antibodies possess distinct
combinations of properties selected from the following: These
antibodies possess distinct combinations of properties selected
from the following: 1) exhibit cross-reactivity with one or more
species homologs of CD47; 2) block the interaction between CD47 and
its ligand SIRP.alpha.; 3) increase phagocytosis of human tumor
cells; 4) induce death of susceptible human tumor cells; 5) do not
induce cell death of human tumor cells; 6) do not have reduced or
minimal binding to human red blood cells (hRBCs); 7) have reduced
binding to hRBCs; 8) have minimal binding to hRBCs; 9) cause
reduced agglutination of hRBCs; 10) cause no detectable
agglutination of hRBCs; 11) reverse TSP1 inhibition of the nitric
oxide (NO) pathway; 12) do not reverse TSP1 inhibition of the NO
pathway; 13) cause loss of mitochondrial membrane potential; 14) do
not cause cause loss of mitochondrial membrane potential; 15) cause
an increase in cell surface calreticulin expression on human tumor
cells; 16) do not cause an increase in cell surface calreticulin
expression on human tumor cells; 17) cause an increase in adenosine
triphosphate (ATP) release by human tumor cells; 18) do not cause
an increase in adenosine triphosphate (ATP) release by human tumor
cells; 19) cause an increase in high mobility group box 1 (HMGB1)
release by human tumor cells; 20) do not cause an increase in high
mobility group box 1 (HMGB1) release by human tumor cells; 21)
cause an increase in type I interferon release by human tumor
cells; 22) do not cause an increase in type I interferon release by
human tumor cells; 23) cause an increase in C-X-C Motif Chemokine
Ligand 10 (CXCL10) release by human tumor cells; 24) do not cause
an increase in C-X-C Motif Chemokine Ligand 10 (CXCL10) release by
human tumor cells; 25) cause an increase in cell surface protein
disulfide-isomerase A3 (PDIA3) expression on human tumor cells; 26)
do not cause an increase in cell surface protein
disulfide-isomerase A3 (PDIA3) expression on human tumor cells; 27)
cause an increase in cell surface heat shock protein 70 (HSP70)
expression on human tumor cells; 28) do not cause an increase in
cell surface heat shock protein 70 (HSP70) expression on human
tumor cells; 29) cause an increase in cell surface heat shock
protein 90 (HSP90) expression on human tumor cells; 30) do not
cause an increase in cell surface heat shock protein 90 (HSP90)
expression on human tumor cells; 31) have reduced binding to normal
human cells, which includes, but is not limited to, endothelial
cells, skeletal muscle cells, epithelial cells, and peripheral
blood mononuclear cells (e.g., human aortic endothelial cells,
human skeletal muscle cells, human microvascular endothelial cells,
human renal tubular epithelial cells, human peripherial blood CD3+
cells, and human peripheral blood mononuclear cells); 32) do not
have reduced binding to normal human cells, which includes, but is
not limited to, endothelial cells, skeletal muscle cells,
epithelial cells, and peripheral blood mononuclear cells (e.g.,
human aortic endothelial cells, human skeletal muscle cells, human
microvascular endothelial cells, human renal tubular epithelial
cells, human peripherial blood CD3+ cells, and human peripheral
blood mononuclear cells); 33) have a greater affinity for human
CD47 at an acidic pH compared to physiological pH; 34) do not have
a greater affinity for human CD47 at an acidic pH compared to
physiological pH; and 35) cause an increase in annexin A1 release
by human tumor cells.
[0311] The anti-CD47 antibodies and antigen binding fragments
thereof of the present disclosure possess combinations of
properties that are distinct from the anti-CD47 antibodies of the
prior art. These properties and characteristics will now be
described in further detail. As used herein, the term "binds to
human CD47" refers to binding with an apparent Kd greater than 50
nM, for example, in a solid phase ELISA assay or cell based
assay.
[0312] As used herein, the terms "apparent binding affinity and
apparent Kd" are determined by non-linear fit (Prism GraphPad
software) of the binding data at the various antibody
concentrations.
Binding to CD47 of Different Species
[0313] The anti-CD47 antibodies, and antigen binding fragments
thereof, of the present disclosure bind human CD47. In certain
embodiments, the anti-CD47 antibodies exhibit cross-reactivity with
one or more species homologs of CD47, for example CD47 homologs of
non-human primate origin. In certain embodiments, the anti-CD47
antibodies and antigen binding fragments thereof of the present
disclosure bind to human CD47 and to CD47 of non-human primate,
mouse, rat, and/or rabbit origin. The cross-reactivity with other
species homologs can be particularly advantageous in the
development and testing of therapeutic antibodies. For example,
pre-clinical toxicology testing of therapeutic antibodies is
frequently carried out in non-human primate species including, but
not limited to, cynomolgus monkey, green monkey, rhesus monkey and
squirrel monkey. Cross-reactivity with these species homologs can
therefore be particularly advantageous for the development of
antibodies as clinical candidates.
[0314] As used herein, the term "cross-reacts with one or more
species homologs of CD47" refers to binding with an apparent Kd
greater than 50 nM.
Blocking the Interaction Between CD47 and SIRP.alpha. and Promoting
Phagocytosis
[0315] CD47, also known as integrin associated protein (IAP), is a
50 kDa cell surface receptor that is comprised of an extracellular
N-terminal IgV domain, a five membrane-spanning transmembrane
domain, and a short C-terminal intracellular tail that is
alternatively spliced.
[0316] Two ligands bind to CD47: Signal Regulatory Protein alpha
(SIRP.alpha.) and Thrombospondin-1 (TSP1). TSP1 is present in
plasma and synthesized by many cells, including platelets.
SIRP.alpha. is expressed on hematopoietic cells, which include
macrophages and dendritic cells.
[0317] When SIRP.alpha. on a phagocyte engages CD47 on a target
cell, this interaction prevents phagocytosis of the target cell.
The interaction of CD47 and SIRP.alpha. effectively sends a "don't
eat me" signal to the phagocyte (Oldenborg et al. Science 288:
2051-2054, 2000). Blocking the interaction of SIRP.alpha. and CD47
with an anti-CD47 mAb in a therapeutic context can provide an
effective anti-cancer treatment by promoting the uptake and
clearance of cancer cells by the host's immune system. Thus, an
important functional characteristic of some anti-CD47 mAbs is the
ability to block the interaction of CD47 and SIRP.alpha., resulting
in phagocytosis of CD47 expressing tumor cells by phagocytes
including macrophages. Several anti-CD47 mAbs have been shown to
block the interaction of CD47 and SIRP.alpha., including B6H12
(Seiffert et al. Blood 94:3633-3643,1999; Latour et al. J. Immunol.
167: 2547-2554, 2001; Subramanian et al. Blood 107: 2548-2556,
2006; Liu et al. J Biol. Chem. 277: 10028-10036, 2002; Rebres et
al. J. Cellular Physiol. 205: 182-193, 2005), BRIC126
(Vernon-Wilson et al. Eur J Immunol. 30: 2130-2137, 2000;
Subramanian et al. Blood 107: 2548-2556, 2006), CC2C6 (Seiffert et
al. Blood 94:3633-3643,1999), 1F7 (Rebres et al. J. Cellular
Physiol. 205: 182-193, 2005), 5F9 (Liu et al. PLoS One. 2015 Sep.
21; 10(9): e0137345) and CC-90002 (Narla et al. Proc Am Assoc
Cancer Res 58: 1200, 2017; abst 469)4. B6H12 and BRIC126 have also
been shown to cause phagocytosis of human tumor cells by human and
mouse macrophages (Willingham et al. Proc Natl Acad Sci USA
109(17):6662-6667, 2012; Chao et al. Cell 142:699-713, 2012; EP 2
242 512 B1). Other existing anti-CD47 mAbs, such as 2D3, does not
block the interaction of CD47 and SIRP.alpha. (Seiffert et al.
Blood 94:3633-3643,1999; Latour et al. J. Immunol. 167: 2547-2554,
2001; Rebres et al. J. Cellular Physiol. 205: 182-193, 2005), and
does not cause phagocytosis of tumor cells (Willingham et al. Proc
Natl Acad Sci USA 109(17):6662-6667, 2012; Chao et al. Cell
142:699-713, 2012; EP 2 242 512 B1).
[0318] As used herein, the term "blocks SIRP.alpha. binding to
human CD47" refers to a greater than 50% reduction of
SIRP.alpha.-Fc binding to CD47 on cells by an anti-CD47 mAb
compared to either untreated cells or cells treated with a negative
antibody.
[0319] The anti-CD47 mAbs of the disclosure described herein, block
the interaction of CD47 and SIRP.alpha. and increase phagocytosis
of human tumor cells.
[0320] "Phagocytosis" of cancer cells refers to the engulfment and
digestion of such cells by phagocytes including, but not limited
to, macrophages and dendritic cells, and the eventual digestion or
degradation of these cancer cells and the release of digested or
degraded cellular components extracellularly, or intracellularly to
undergo further processing. Anti-CD47 monoclonal antibodies that
block SIRP.alpha. binding to CD47 increase phagocytosis of cancer
cells. SIRP.alpha. binding to CD47 on cancer cells would otherwise
allow these cells to escape phagocytosis. The cancer cell may be
viable or living cancer cells.
[0321] As used herein, the term "increases phagocytosis of human
tumor cells" refers to a greater than 2-fold increase in
phagocytosis of human tumor cells by human macrophages in the
presence of an anti-CD47 mAb compared to either untreated cells or
cells treated with a negative control antibody.
Inducing Death of Tumor Cells
[0322] Some soluble anti-CD47 mAbs initiate a cell death program on
binding to CD47 on tumor cells, resulting in collapse of
mitochrondrial membrane potential, loss of ATP generating capacity,
increased cell surface expression of phosphatidylserine (detected
by increased staining for annexin V) and cell death without the
participation of caspases or fragmentation of DNA. Such soluble
anti-CD47 mAbs have the potential to treat a variety of solid and
hematological cancers. Several soluble anti-CD47 mAbs which have
been shown to induce tumor cell death, including MABL-1, MABL-2 and
fragments thereof (U.S. Pat. No. 8,101,719; Uno et al. Oncol Rep.
17: 1189-94, 2007; Kikuchi et al. Biochem Biophys Res. Commun. 315:
912-8, 2004), Ad22 (Pettersen et al. J. Immuno. 166: 4931-4942,
2001; Lamy et al. J. Biol. Chem. 278: 23915-23921, 2003), and 1F7
(Manna et al. J. Immunol. 170: 3544-3553, 2003; Manna et al. Cancer
Research, 64: 1026-1036, 2004). Some of the anti-CD47 mAbs of the
disclosure described herein induce cell death of human tumor
cells.
[0323] Induction of cell death refers to the ability of certain of
the soluble anti-CD47 antibodies, murine antibodies, chimeric
antibodies, humanized antibodies, or antigen-binding fragments
thereof (and competing antibodies and antigen-binding fragments
thereof) disclosed herein to kill cancer cells via a cell
autonomous mechanism without participation of complement or other
cells including, but not limited to, T cells, neutrophils, natural
killer cells, macrophages, or dendritic cells.
[0324] The terms "inducing cell death" or "kills" and the like, are
used interchangeably herein.
[0325] As used herein, the term "induces death of human tumor
cells" refers to increased binding of annexin V (in the presence of
calcium) and increased 7-aminoactinomycin D (7-AAD) or propidium
iodide uptake in response to treatment with an anti-CD47 mAb. These
features may be quantitated in three cell populations: annexin V
positive (annexin V.sup.+), annexin V positive/7-AAD negative
(annexin V.sup.+/7-AAD.sup.-) and annexin V positive/7-AAD positive
(annexin V.sup.+/7-AAD.sup.+) by flow cytometry. Induction of cell
death may be defined by a greater than 2-fold increase in each of
the above cell populations in human tumor cells caused by soluble
anti-CD47 mAb compared to the background obtained with the negative
control antibody, (humanized, isotype-matched antibody) or
untreated cells.
[0326] Another indicator of cell death is loss of mitochondrial
function and membrane potential by the tumor cells as assayed by
one of several available measures (potentiometric fluorescent dyes
such as DiO-C6 or JC1 or formazan-based assays such as MTT or
WST-1).
[0327] As used herein, the term "causes loss of mitochondrial
membrane potential" refers to a statistically significant
(p<0.05) decrease in mitochondrial membrane potential by a
soluble anti-CD47 mAb compared to the background obtained with a
negative control, humanized isotype-matched antibody or no
treatment.
[0328] Among the present humanized or chimeric mAbs, those that
induce cell death of human tumor cells cause increased annexin V
binding similar to the findings reported for anti-CD47 mAbs Ad22
(Pettersen et al. J. Immunol. 166: 4931-4942, 2001; Lamy et al. J.
Biol. Chem. 278: 23915-23921, 2003); 1F7 (Manna and Frazier J.
Immunol. 170:3544-3553, 2003; Manna and Frazier Cancer Res.
64:1026-1036, 2004); and MABL-1 and 2 (U.S. Pat. No. 7,531,643 B2;
U.S. Pat. No. 7,696,325 B2; U.S. Pat. No. 8,101,719 B2).
[0329] Cell viability assays are described in NCI/NIH guidance
manual that describes numerous types of cell based assays that can
be used to assess induction of cell death caused by CD47
antibodies: "Cell Viability Assays", Terry L Riss, PhD, Richard A
Moravec, BS, Andrew L Niles, MS, Helene A Benink, PhD, Tracy J
Worzella, MS, and Lisa Minor, PhD. Contributor Information,
published May 1, 2013.
Binding to hRBCs
[0330] CD47 is expressed on human erythrocytes (hRBCs) (Brown. J
Cell Biol. 111: 2785-2794, 1990; Avent. Biochem J., (1988) 251:
499-505; Knapp. Blood, (1989) Vol. 74, No. 4, 1448-1450; Oliveira
et al. Biochimica et Biophysica Acta 1818: 481-490, 2012; Petrova
P. et al. Cancer Res 2015; 75(15 Suppl): Abstract no. 4271). It has
been shown that anti-CD47 mAbs bind to RBCs, including B6H12 (Brown
et al. J. Cell Biol., 1990, Oliveira et al. Biochimica et
Biophysica Acta 1818: 481-490, 2012, Petrova P. et al. Cancer Res
2015; 75(15 Suppl): Abstract no. 4271), BRIC125 (Avent. Biochem J.,
(1988) 251: 499-505), BRIC126 (Avent. Biochem J., (1988) 251:
499-505; Petrova P. et al. Cancer Res 2015; 75(15 Suppl): Abstract
no. 4271), 5F9 (Uger R. et al. Cancer Res 2014; 74(19 Suppl):
Abstract no. 5011, Liu et al. PLoS One. 2015 Sep. 21; 10(9):
e0137345; Sikic B. et al. J Clin Oncol 2016; 34 (suppl; abstract
3019)), anti-CD47 antibodies disclosed in US Patent Publication
2014/0161799, WO Publication 2014/093678, US Patent Publication
2014/0363442, and CC2C6 (Petrova P. et al. Cancer Res 2015; 75(15
Suppl): Abstract no. 4271, Uger R. et al. Cancer Res 2014; 74(19
Suppl): Abstract no. 5011). It has also been shown that a
SIRP.alpha.-Fc fusion protein, which binds to human CD47, has
reduced binding to human RBCs compared to other human cells (Uger
R. et al. Cancer Res 2014; 74(19 Suppl: Abstract no. 5011; Petrova
et al. Clin Cancer Res 23: 1068-1079, 2017). Binding to RBCs can be
reduced by generation of bi-specific antibodies with only one CD47
binding arm (Masternak et al. Cancer Res 2015; 75(15 Suppl):
Abstract no. 2482). Because some anti-CD47 mAbs have been shown to
result in reduction of RBCs when administered to cynomolgus monkeys
(Mounho-Zamora B. et al. The Toxicologist, Supplement to
Toxicological Sciences, 2015; 144 (1): Abstract 596: 127, Liu et
al. PLoS One. 2015 Sep. 21; 10(9): e0137345; Pietsch et al. Cancer
Res 2015; 75(15 Suppl): Abstract nr 2470), it is highly desirable
to identify anti-CD47 mAbs that have reduced or minimal binding to
CD47-expressing RBCs.
[0331] As used herein, the terms "red blood cell(s)" and
"erythrocyte(s)" are synonymous and used interchangeably
herein.
[0332] As used herein, the term "reduced binding to hRBCs" refers
to an apparent Kd of an anti-CD47 mAb binding to a hRBC which is
8-fold or greater than the apparent Kd on a human tumor cell,
wherein the tumor cell is an OV10 hCD47 cell (human OV10 ovarian
cancer cell line expressing human CD47).
[0333] As used herein, the term "minimal binding" or "MB" refers to
no measurable binding to hRBCs at an anti-CD47 mAb concentration up
to 5,000 pM.
[0334] Prior to the disclosure described herein, no monospecific
anti-CD47 mAbs have been reported that have minimal binding to
human RBCs expressing CD47.
[0335] Some of the anti-CD47 mAbs, disclosed herein, have reduced
or minimal binding to human RBCs.
Binding to Human Endothelial Cells and Other Normal Human Cells
[0336] In addition to expression/overexpression on most
hematological malignancies and solid tumors (Willingham et al.,
Proc Natl Acad Sci 2012), CD47 is also expressed, by many but not
all, normal cell types, including, but not limited to RBCs (see
previous section), lymphocytes and mononuclear cells, endothelial
cells, and brain, liver, muscle cells and/or tissues (Brown et al.,
J Cell Biol 1990; Reinhold et al., J Cell Sci. 1995; Matozaki et
al., Cell 2009; Stefanidakis et al., Blood 2008; Xiao et al.,
Cancer Letters 2015). Because of this expression, it is expected
that some anti-CD47 mAbs would bind to these normal cell
types/tissues in addition to the cancer cells which are the
therapeutic target. It is therefore desirable to identify anti-CD47
mAbs that either have reduced or minimal binding to some of these
normal cells to both reduce potential non-desired effects on these
normal cells and also allow more available antibody for binding to
the tumor cells. Anti-CD47 mAbs with such reduced or minimal
binding to normal cells have not been described.
[0337] As used herein, the terms "reduced binding to normal human
cells which includes, but is not limited to, endothelial cells,
skeletal muscle cells, epithelial cells, and peripheral blood
mononuclear cells (e.g., human aortic endothelial cells, human
skeletal muscle cells, human microvascular endothelial cells, human
renal tubular epithelial cells, human peripherial blood CD3+ cells,
and human peripheral blood mononuclear cells) refers to the
apparent Kd of an anti-CD47 mAb binding to these cells which is
8-fold or greater than the apparent Kd of the anti-CD47 mAb binding
to a human tumor cell, wherein the tumor cell is OV10 hCD47.
[0338] As used herein, the term "minimal binding" or "MB" refers to
no measurable binding of an antibody or other molecule as described
herein to normal human cells which includes, but is not limited to,
endothelial cells, skeletal muscle cells, epithelial cells, and
peripheral blood mononuclear cells (e.g., human aortic endothelial
cells, human skeletal muscle cells, human microvascular endothelial
cells, human renal tubular epithelial cells, human peripherial
blood CD3+ cells, and human peripheral blood mononuclear cells) at
an anti-CD47 mAb concentration up to 5,000 pM.
Agglutination of RBCs
[0339] Red blood cell (RBC) agglutination or hemagglutination is a
homotypic interaction that occurs when RBCs aggregate or clump
together following incubation with various agents, including
antibodies to RBC antigens and cell surface proteins such as CD47.
Many anti-CD47 antibodies have been reported to cause
hemagglutination of washed human RBCs in vitro, in a concentration
dependent manner, including B6H12, BRIC126, MABL-1, MABL-2, CC2C6,
and 5F9 (Uger R. et al. Cancer Res 2014; 74(19 Suppl): Abstract no.
5011, U.S. Pat. No. 9,045,541, Uno et al. Oncol Rep. 17: 1189-94,
2007; Kikuchi et al. Biochem Biophys Res. Commun. 315: 912-8, 2004;
Sikic B. et al. J Clin Oncol 2016; 34 (suppl; abstract 3019)). This
functional effect requires binding to RBCs by an intact, bivalent
antibody and can be reduced or eliminated by generating antibody
fragments, either a F(ab') or svFv (Uno et al. Oncol Rep. 17:
1189-94, 2007; Kikuchi et al. Biochem Biophys Res. Commun. 315:
912-8, 2004) or bi-specific antibodies with only one CD47 binding
arm (Masternak et al. Cancer Res 2015; 75(15 Suppl): Abstract no.
2482). Other functional properties of these fragments, including
cell killing, were shown to be either reduced or retained in these
fragments (Uno et al. Oncol Rep. 17: 1189-94, 2007; Kikuchi et al.
Biochem Biophys Res. Commun. 315: 912-8, 2004). The mouse antibody
2D3 is an example of an anti-CD47 antibody that binds to CD47 on
red blood cells but does not cause hemagglutination (U.S. Pat. No.
9,045,541, Petrova et al. Cancer Res 2015; 75(15 Suppl): Abstract
no. 4271).
[0340] Hemagglutination has been shown to be reduced/eliminated by
reducing the binding selectively to human RBCs, but not other
cells, using a SIRP.alpha.-Fc fusion protein (Uger R. et al. Blood
2013; 122(21): 3935). In addition, mouse anti-CD47 mAb 2A1 and
humanized versions of 2A1 have been reported to block
CD47/SIRP.alpha. but do not exhibit hemagglutination activity in a
washed RBC assay (U.S. Pat. No. 9,045,541; Narla et al. Proc Am
Assoc Cancer Res 58: 1200, 2017; abst 4694). A small number of a
panel of mouse anti-human CD47 antibodies (8 of 23) were reported
to not cause hemagglutination of human RBCs (Pietsch E et al. Blood
Cancer Journal (2017) 7, e536; doi:10.1038/bcj.2017.7). Therefore,
prior to the disclosure described herein, there was a need to
identify CD47 mAbs that block SIRP.alpha./CD47 binding, have
reduced or minimal binding to RBCs and/or cause no detectable
hemagglutination. The term "agglutination" refers to cellular
clumping, while the term "hemagglutination" refers to clumping of a
specific subset of cells, i.e., RBCs. Thus, hemagglutination is a
type of agglutination.
[0341] As used herein, the term "reduced hemagglutination" refers
to detectable agglutination activity of hRBCs at anti-CD47 mAb
concentrations greater than or equal to 1.85 .mu.g/ml, and no
measurable activity at concentrations less than 1.85 .mu.g/ml in a
washed RBC assay, as visualized by discrete punctate dot compared
to a diffuse pattern that represents hemagglutination.
[0342] As used herein, the term "no detectable hemagglutination"
refers to no visible or detectable agglutination activity of hRBCs
at anti-CD47 mAb concentrations greater or equal to 0.3 pg/ml to a
concentration less than or equal to 10 .mu.g/ml in a washed RBC
assay, as visualized by discrete punctate dot compared to a diffuse
pattern that represents hemagglutination.
[0343] Some of the anti-CD47 antibodies described herein, cause
reduced or no detectable hemagglutination of human RBCs.
Modulation of the NO Pathway
[0344] As noted above, TSP1 is also a ligand for CD47. The
TSP1/CD47 pathway opposes the beneficial effects of the NO pathway
in many cell types, including, but not limited to, vascular cells.
The NO pathway consists of any of three enzymes (nitric oxide
synthases, NOS I, NOS II and NOS III) that generate bioactive gas
NO using arginine as a substrate. NO can act within the cell in
which it is produced, or in neighboring cells, to activate the
enzyme soluble guanylyl cyclase that produces the messenger
molecule cyclic GMP (cGMP). The proper functioning of the NO/cGMP
pathway is essential for protecting the cardiovascular system
against stresses including, but not limited to, those resulting
from wounding, inflammation, hypertension, metabolic syndrome,
ischemia, and IRI. In the context of these cellular stresses the
inhibition of the NO/cGMP pathway by the TSP1/CD47 system
exacerbates the effects of stress. This is a particular problem in
the cardiovascular system where both cGMP and cAMP play important
protective roles. There are many cases in which ischemia and
reperfusion injury cause or contribute to disease, trauma, and poor
outcomes of surgical procedures.
[0345] As disclosed herein, one of more of the chimeric or
humanized anti-CD47 antibodies will reverse TSP1 inhibition of cGMP
production. Reversal will be complete (>80%) or intermediate
(20%-80%). This reversal of TSP1 inhibition of cGMP production will
demonstrate that the anti-CD47 mAbs have the ability to increase NO
signaling and suggest utility in protecting the cardiovascular
system against stresses including, but not limited to, those
resulting from wounding, inflammation, hypertension, metabolic
syndrome, ischemia, and ischemia-reperfusion injury (IRI).
Additional assay systems, for example smooth muscle cell
contraction, will also be expected to show that some of the
chimeric or humanized antibodies reverse the inhibitory actions of
TSP1 on downstream effects resulting from the activation of NO
signaling.
[0346] As disclosed herein, "complete reversal of NO pathway
inhibition" refers to greater than 80% reversal of TSP1 inhibition
of NO signaling by an anti-CD47 mAb compared to a negative control,
humanized isotype-matched antibody or no treatment.
[0347] As disclosed herein, "intermediate reversal of NO pathway
inhibition" refers to 20-80% reversal of TSP1 inhibition of NO
signaling by an anti-CD47 mAb compared to a negative control,
humanized isotype-matched antibody or no treatment.
[0348] As disclosed herein, "no reversal of NO pathway inhibition"
refers to less than 20% reversal of TSP1 inhibition of NO signaling
by an anti-CD47 mAb compared to a negative control, humanized
isotype-matched antibody or no treatment.
Immunogenic Cell Death
[0349] The concept of immunogenic cell death (ICD) has emerged in
recent years. This form of cell death, unlike non-immunogenic cell
death, stimulates an immune response against antigens from cancer
cells. ICD is induced by specific chemotherapy drugs, including
anthracyclines (doxorubicin, daurorubicin and mitoxantrone) and
oxaliplatin, but not by cisplatin and other chemotherapy drugs. ICD
is also induced by bortezomib, cardiac glycosides, photodynamic
therapy and radiation Galluzi et al. Nat Rev Immunol 17: 97-111,
2016). The distinctive characteristics of ICD of tumor cells are
the release from or exposure on tumor cell surfaces of specific
ligands: 1) the pre-apoptotic cell surface exposure of
calreticulin, 2) the secretion of adenosine triphosphate (ATP), 3)
release of high mobility group box 1 (HMGB1), 4) annexin A1
release, 5) type I interferon release and 6) C-X-C motif chemokine
ligand 10 (CXCL10) release. These ligands are endogenous
damage-associated molecular patterns (DAMPs), which include the
cell death-associated molecules (CDAMs) (Kroemer et al. Annu Rev
Immunol 31: 51-72, 2013). Importantly, each of these ligands
induced during ICD binds to specific receptors, referred to as
pattern recognition receptors (PRRs), that contribute to an
anti-tumor immune response. ATP binds the purinergic receptors PY2,
G-protein coupled, 2 (P2RY2) and PX2, ligand-gated ion channel, 7
(P2RX7) on dendritic cells causing dendritic cell recruitment and
activation, respectively. Annexin A1 binds to formyl peptide
receptor 1 (FPR1) on dendritic cells causing dendritic cell homing.
Calreticulin expressed on the surface of tumor cells binds to LRP1
(CD91) on dendritic cells promoting antigen uptake by dendritic
cells. HMGB1 binds to toll-like receptor 4 (TLR4) on dendritic
cells to cause dendritic cell maturation. As a component of ICD,
tumor cells release type I interferon leading to signaling via the
type I interferon receptor and the release of the CXCL10 which
favors the recruitment of effector CXCR3+ T cells Together, the
actions of these ligands on their receptors facilitate recruitment
of DCs into the tumor, the engulfment of tumor antigens by DCs and
optimal antigen presentation to T cells. Kroemer et al. have
proposed that a precise combination of the CDAMs mentioned above
elicited by ICD can overcome the mechanisms that normally prevent
the activation of anti-tumor immune responses (Kroemer et al. Annu
Rev Immunol 31: 51-72, 2013; Galluzi et al. Nat Rev Immunol 17:
97-111, 2016). When mouse tumor cells treated in vitro with
ICD-inducing modalities are administered in vivo to syngeneic mice,
they provide effective vaccination that leads to an anti-tumor
adaptive immune response, including memory. This vaccination effect
cannot be tested in xenograft tumor models because the mice used in
these studies lack a complete immune system. The available data
indicate that ICD effects induced by chemotherapy or radiation will
promote an adaptive anti-tumor immune response in cancer patients.
The components of ICD are described in more detail below.
[0350] In 2005, it was reported that tumor cells which were dying
in response to anthracycline chemotherapy in vitro caused an
effective anti-tumor immune response when administered in vivo in
the absence of adjuvant (Casares et al. J Exp Med 202: 16911701,
2005). This immune response protected mice from subsequent
re-challenge with viable cells of the same tumor and caused
regression of established tumors. Anthracyclines (doxorubicin,
daunorubicin and idarubicin) and mitomycin C induced tumor cell
apoptosis with caspase activation, but only apoptosis induced by
anthracyclines resulted in immunogenic cell death. Caspase
inhibition did not inhibit cell death induced by doxorubicin but
did suppress the immunogenicity of tumor cells dying in response to
doxorubicin. The central roles of dendritic cells and CD8+ T cells
in the immune response elicited by doxorubicin-treated apoptotic
tumor cells were established by the demonstration that depletion of
these cells abolished the immune response in vivo.
[0351] Calreticulin is one of the most abundant proteins in the
endoplasmic reticulum (ER). Calreticulin was shown to rapidly
translocate preapoptotically from the ER lumen to the surface of
cancer cells in response to multiple ICD inducers, including
anthracyclines (Obeid et al. Nat Med 13: 54-61, 2007; Kroemer et
al. Annu Rev Immunol 31: 51-72, 2013). Blockade or knockdown of
calretiulin suppressed the phagocytosis of anthracycline-treated
tumor cells by dendritic cells and abolished their immunogenicity
in mice. The exposure of calreticulin caused by anthracyclines or
oxaliplatin is activated by an ER stress response that involves the
phosphorylation of the eukaryotic translation initiation factor
eIF2.alpha. by the PKR-like ER kinase. Calretiulin, which has a
prominent function as an "eat-me" signal (Gardai et al. Cell 123:
321-334, 2005) binds to LRP1 (CD91) on dendritic cells and
macrophages resulting in phagocytosis of the calreticulin
expressing cell, unless the calreticulin-expressing cell expresses
a don't eat me signal, such as CD47. Calreticulin also signals
through CD91 on antigen presenting cells to cause the release of
proinflammatory cytokines and to program Th17 cell responses. In
summary, calreticulin expressed as part of immunogenic cell death
stimulates antigen presenting cells to engulf dying cells, process
their antigens and prime an immune response.
[0352] In addition to calreticulin, protein disulfide-isomerase A3
(PDIA3), also called Erp57, was shown to translocate from the ER to
the surface of tumor cells following treatment with mitoxantrone,
oxaliplatin and irradiation with UVC light (Panaretakis et al. Cell
Death Differ 15: 1499-1509, 2008; Panaretakis et al. EMBL J 28:
578-590, 2009). A human ovarian cancer cell line, primary ovarian
cancer cells and a human prostate cancer cell line expressed
cell-surface calreticulin, HSP70 and HSP90 following treatment with
the anthracyclines doxorubicin and idarubicin (Fucikova et al.
Cancer Res 71: 4821-4833, 2011). HSP70 and HSP90 bind to the PRR
LRP1 on antigen presenting cells; the PRR to which PDIA3 binds has
not been identified (Galluzi et al. Nat Rev Immunol 17: 97-111,
2016).
[0353] TLR4 was shown to be required for cross-presentation of
dying tumor cells and to control tumor antigen processing and
presentation. Among proteins that were known to bind to and
stimulate TLR4, HMGB1 was uniquely released by mouse tumor cells in
which ICD was induced by irradiation or doxorubicin (Apetoh et al.
Nat Med 13: 1050-1059, 2007). The highly efficient induction of an
in vivo anti-tumor immune by doxorubicin treatment of mouse tumor
cells required the presence of HMGB1 and TLR4, as demonstrated by
abrogation of the immune response by inhibition of HMGB1 and
knock-out TLR4. These preclinical findings are clinically relevant.
Patients with breast cancer who carry a TLR4 loss-of-function
allele relapse more quickly after radiotherapy and chemotherapy
than those carrying the normal TLR4 allele.
[0354] Ghiringhelli et al. showed that mouse tumor cells treated
with oxaliplatin, doxorubicin and mitoxanthrone in vitro released
ATP and that the ATP binds to the purinergic receptors PY2,
G-protein coupled, 2 (P2RY2) and PX2, ligand-gated ion channel, 7
(P2RX7) on dendritic cells (Ghiringhelli et al. Nat Med 15:
1170-1178, 2009). Binding of ATP to P2RX7 on DCs triggers the
NOD-like receptor family, pyrin domain containing-3 protein
(NLRP3)-dependent caspase-1 activation complex (inflammasome),
allowing for the secretion of interleukin-10 (IL-13), which is
essential for the priming of interferon-gamma-producing CD8+ T
cells by dying tumor cells. Therefore, the ATP-elicited production
of IL-10 by DCs appears to be one of the critical factors for the
immune system to perceive cell death induced by certain
chemotherapy drugs as immunogenic. This paper also reports that
HMGB1, a TLR4 agonist, also contributes to the stimulation of the
NLRP3 inflammasome in DCs and the secretion of IL-13. These
preclinical results have been shown to have clinical relevance; in
a breast cancer cohort, the presence of the P2RX7 loss-of-function
allele had a significant negative prognostic impact of metastatic
disease-free survival. ATP binding to P2RY2 causes the recruitment
of myeloid cells into the tumor microenvironment (Vacchelli et al.
Oncoimmunology 5: e1118600, 2016)
[0355] Michaud et al. demonstrated that autophagy is required for
the immunogenicity of chemotherapy-induced cell death (Michaud et
al. Science 334: 1573-1577, 2011). Release of ATP from dying tumor
cells required autophagy and autophagy-competent, but not
autophagy-deficient, mouse tumors attracted dendritic cells and T
lymphocytes into the tumor microenvironment in response to
chemotherapy that induces ICD.
[0356] Ma et al. addressed the question of how chemotherapy-induced
cell death leads to efficient antigen presentation to T cells (Ma
et al. Immunity 38: 729-741, 2013). They found that at specific
kind of tumor infiltrating lymphocyte,
CD11c.sup.+CD11b.sup.+Ly6C.sup.hi cells, are particularly important
for the induction of anticancer immune responses by
anthracyclines.
[0357] ATP released by dying cancer cells recruited myeloid cells
into tumors and stimulated the local differentiation of
CD11c.sup.+CD11b.sup.+Ly6C.sup.hi cells. These cells were shown to
be particularly efficient in capturing and presenting tumor cell
antigens and, after adoptive transfer into naive mice, conferring
protection to challenge with living tumor cells of the same cell
line.
[0358] It has been shown that anthracyclines stimulate the rapid
production of type I interferons by tumor cells after activation of
TLR3 (Sistugu et al. Nat Med 20: 1301-1309, 2014). Type I
interferons (IFN) bind to IFN-.alpha. and IFN-.beta. receptors on
cancer cells and trigger autocrine and paracrine signaling pathways
that result in release of CXCL10. Tumors lacking Tlr3 or Ifnar
failed to respond to chemotherapy unless type I IFN or CXCL10,
respectively, was supplied. These preclinical findings have
clinical relevance. A type I IFN-related gene expression signature
predicted clinical responses to anthracycline-based chemotherapy in
independent cohorts of breast cancer patients.
[0359] Another receptor on dendritic cells that is involved in
chemotherapy-induced anti-cancer immune response was recently
identified: formyl peptide receptor-1, which binds annexin A1
(Vacchelli et al. Science 350: 972-978, 2015). Vacchelli et al.
designed a screen to identify candidate genetic defects that
negatively affect responses to chemotherapy. They identified a
loss-of-function allele of the gene encoding formyl peptide
receptor 1 (FPR1) that was associated with poor metastatis-free
survival and overall survival in breast and colorectal cancer
patients receiving adjuvant chemotherapy. The therapeutic effects
of anthracyclines were abrogated in tumor-bearing Fpr1-/- mice due
to impaired antitumor immunity. FPR1-deficient DCs did not approach
dying tumor cells and, therefore, could not elicit antitumor T cell
immunity. Two anthracyclines, doxorubicin and mitoxantrone,
stimulated the secretion of annexin A1, one of four known ligands
of FPR1. FPR1 and annexin A1 promoted stable interactions between
dying cancer cells and human or mouse leukocytes.
[0360] In addition to anthracyclines and oxaliplatin, other drugs
have been shown to induce immunogenic cell death. Cardiac
glycosides, including clinically used digoxin and digitoxin, were
also shown to be efficient inducers of immunogenic cell death of
tumor cells (Menger et al. Sci Transl Med 4: 143ra99, 2012). Other
chemotherapy agents and cancer drugs that have been reported to
induce DAMP expression or release are bleomycin, bortezomib,
cyclophosphamide, paclitaxel, vorinistat and cisplatin (Garg et al.
Front Immunol 588: 1-24, 2015; Menger et al. Sci Transl Med 4:
143ra99, 2012; Martins et al. Oncogene 30: 1147-1158, 2011).
Importantly, these results have clinical relevance. Administration
of digoxin during chemotherapy had a significant positive impact on
the overall survival of patients with breast, colorectal, head and
neck, and hepatocellular cancers, but failed to improve overall
survival of lung and prostate cancer patients.
[0361] The anti-CD20 monoclonal antibody rituximab has improved
outcomes in multiple B-cell malignancies. The success of rituximab,
referred to as a type I anti-CD20 mAb, led to the development of
type II anti-CD20 mAbs, including obinutuzumab and tositumomab.
Cheadle et al., investigated the induction of immunogenic cell
death by anti-CD20 mAbs (Cheadle et al. Brit J Haematol 162:
842-862, 2013). They found that the cell death induced by
obinutuzumab and tositumomab is a form of immunogenic cell death
characterized by the release of HMGB1, HSP90 and ATP. A type I
anti-CD20 mAb did not cause release of HMGB1, HSP90 and ATP.
Incubation of supernatants from a human tumor cell line treated
with obinutuzumab caused maturation of human dendritic cells,
consistent with the previously described effects of HMGB1 and ATP
on dendritic cells. In contrast to the results reported by Cheadle
et al., Zhao et al. reported that both type I and II anti-CD20 mAbs
increased HMGB1 release from human diffuse large B cell lymphoma
cell lines, but did not cause ATP release or cell surface
expression of calreticulin (Zhao et al. Oncotarget 6: 27817-27831,
2015).
[0362] The release from or exposure on tumor cell surfaces of the
DAMPs calreticulin, ATP, HMGB1, annexin A1, type I interferon
release, CXCL10, PDIA3, HSP70 and/or HSP90 in response to anti-CD47
mAbs has not been reported. As disclosed herein, anti-CD47 mAbs
cause release from or exposure on tumor cell surfaces of one or
more of the DAMPs listed above (characteristics of ICD), an
unexpected result. These DAMPS are expected to promote a
therapeutically beneficial adaptive anti-tumor immune response.
[0363] As disclosed herein, "causes an increase in cell surface
calreticulin expression on human tumor cells" refers to a
statistically significant increase (p<0.05) in calreticulin
expression by a soluble anti-CD47 mAb compared to the background
obtained with a negative control, humanized isotype-matched
antibody or no treatment.
[0364] As disclosed herein, the term "the release of" is synonymous
with secretion and is defined as the extracellular appearance of
ATP, HMGB1, annexin A1, type I interferon and CXCL10.
[0365] As disclosed herein, "cause an increase in the release of
adenosine triphosphate by human tumor cells" refers to a
statistically significant increase (p<0.05) in ATP in the
supernatant caused by a soluble anti-CD47 mAb compared to the
background obtained with a negative control, humanized
isotype-matched antibody or no treatment.
[0366] As disclosed herein, "cause an increase in the release of
high mobility group box 1 by human tumor cells" refers to a
statistically significant increase (p<0.05) in HMGB1 in the
supernatant caused by a soluble anti-CD47 mAb compared to the
background obtained with a negative control, humanized
isotype-matched antibody or no treatment.
[0367] As disclosed herein, "causes an increase in the release of
type I interferon by human tumor cells" refers to a statistically
significant increase (p<0.05) in type I interferon in the
supernatant or type I interferon mRNA caused by a soluble anti-CD47
mAb compared to the background obtained with a negative control,
humanized isotype-matched antibody or no treatment.
[0368] As disclosed herein, "causes an increase in the release of
C-X-C Motif Chemokine Ligand 10 (CXCL10) by human tumor cells"
refers to a statistically significant increase (p<0.05) in
CXCL10 in the supernatant or CXCL10 mRNA caused by a soluble
anti-CD47 mAb compared to the background obtained with a negative
control, humanized isotype-matched antibody or no treatment.
[0369] As disclosed herein, "causes an increase in cell surface
PDIA3 expression on human tumor cells" refers to a statistically
significant increase (p<0.05) in PDIA3 expression by a soluble
anti-CD47 mAb compared to the background obtained with a negative
control, humanized isotype-matched antibody or no treatment.
[0370] As disclosed herein, "causes an increase in cell surface
HSP70 expression on human tumor cells" refers to a statistically
significant increase (p<0.05) in HSP70 expression by a soluble
anti-CD47 mAb compared to the background obtained with a negative
control, humanized isotype-matched antibody or no treatment.
[0371] As disclosed herein, "causes an increase in cell surface
HSP90 expression on human tumor cells" refers to statistically
significant increase (p<0.05) in HSP90 expression by a soluble
anti-CD47 mAb compared to the background obtained with a negative
control, humanized isotype-matched antibody or no treatment.
pH Dependence of Anti-CD47 mAb Binding
[0372] Most antibody binding, particularly in the blood compartment
and to normal cells occurs at physiological pH (approximately 7.4).
Therefore, the binding affinity of therapeutic mAbs is normally
assessed in vitro at physiological pH. However, the tumor
microenvironment (TME) is more acidic in nature, with pH values
below 7.4. This appears to be due to a number of differences
including hypoxia, anaerobic glycolysis leading to the production
of lactic acid and hydrolysis of ATP (Tannock and Rotin, Cancer Res
1989; Song et al., Cancer Drug Discovery and Development 2006; Chen
and Pagel, Advan Radiol 2015). The acidic pH may provide an
advantage to the tumor by promoting invasiveness, metastatic
behavior, chronic autophagy, resistance to chemotherapies and
reduced efficacy of immune cells in the tumor microenvironment
(Estrella et al. Cancer Res 2013; Wojtkowiak et al., Cancer Res
2012; Song et al., Cancer Drug Discovery and Development 2006;
Barar, BioImpacts, 2012). The identification of anti-CD47
antibodies with the property of increased binding affinity at
acidic pH would confer a therapeutic advantage with higher binding
to CD47 on tumor cells within the acidic TME compared to cells at
physiological pH. Antibodies with pH-dependent properties have been
generated with the goal of recycling antibodies. However, in
contrast to exhibiting the properties of enhanced binding at acidic
pH, these bind with high affinity to their target antigen at
physiological pH, but release their target at acidic pH (Bonvin et
al., mAbs 2015; Igawa and Hattori, Biochem Biophys Acta 2014).
[0373] As disclosed herein, "has a greater affinity for CD47 at an
acidic pH compared to physiological pH" refers to an apparent Kd
that is increased 5-fold or more at acidic pH (<7.4) compared to
physiological pH (7.4).
Combinations of Functional Properties
[0374] In some embodiments, the anti-CD47 antibodies described
herein, are also characterized by combinations of properties which
are not exhibited by prior art anti-CD47 antibodies proposed for
human therapeutic use. Accordingly, in some embodiments, anti-CD47
antibodies described herein may be characterized by: [0375] a.
binds to human CD47; [0376] b. blocks SIRP.alpha. binding to human
CD47; [0377] c. increases phagocytosis of human tumor cells; and
[0378] d. induces death of susceptible human tumor cells.
[0379] In another embodiment described herein, the anti-CD47
antibodies are characterized by: [0380] a. binds to human CD47;
[0381] b. blocks SIRP.alpha. binding to human CD47; [0382] c.
increases phagocytosis of human tumor cells; [0383] d. induces
death of susceptible human tumor cells; and [0384] e. causes no
detectable agglutination of human red blood cells (hRBCs).
[0385] In yet another embodiment described herein, the anti-CD47
antibodies are characterized by: [0386] a. binds to human CD47;
[0387] b. blocks SIRP.alpha. binding to human CD47; [0388] c.
increases phagocytosis of human tumor cells; [0389] d. induces
death of susceptible human tumor cells; and [0390] e. causes
reduced agglutination of human red blood cells (hRBCs).
[0391] In another embodiment described herein, the anti-CD47
antibodies are characterized by: [0392] a. binds to human CD47;
[0393] b. blocks SIRP.alpha. binding to human CD47; [0394] c.
increases phagocytosis of human tumor cells; [0395] d. induces
death of susceptible human tumor cells; and [0396] e. has reduced
hRBC binding.
[0397] In another embodiment described herein, the anti-CD47
antibodies are characterized by: [0398] a. binds to human CD47;
[0399] b. blocks SIRP.alpha. binding to human CD47; [0400] c.
increases phagocytosis of human tumor cells; [0401] d. causes no
detectable agglutination of human red blood cells (hRBCs); and
[0402] e. has minimal binding to hRBCs.
[0403] In another embodiment described herein, the anti-CD47
antibodies are characterized by: [0404] a. binds to human CD47;
[0405] b. blocks SIRP.alpha. binding to human CD47; [0406] c.
increases phagocytosis of human tumor cells; [0407] d. causes no
detectable agglutination of human red blood cells (hRBCs); and
[0408] e. has reduced hRBC binding.
[0409] In another embodiment described herein, the monoclonal
antibody, or antigen binding fragment thereof binds to human,
non-human primate, mouse, rabbit, and rat CD47.
[0410] In yet another embodiment described herein, the monoclonal
antibody, or antigen binding fragment thereof specifically also
binds to non-human primate CD47, wherein non-human primate may
include, but is not limited to, cynomolgus monkey, green monkey,
rhesus monkey and squirrel monkey.
[0411] In another embodiment, the anti-CD47 monoclonal antibody, or
antigen binding fragment thereof, may additionally possess one or
more of the following characteristics: 1) exhibit cross-reactivity
with one or more species homologs of CD47; 2) block the interaction
between CD47 and its ligand SIRP.alpha.; 3) increase phagocytosis
of human tumor cells; 4) induce death of susceptible human tumor
cells; 5) do not induce cell death of human tumor cells; 6) do not
have reduced or minimal binding to human red blood cells (hRBCs);
7) have reduced binding to hRBCs; 8) have minimal binding to hRBCs;
9) cause reduced agglutination of hRBCs; 10) cause no detectable
agglutination of hRBCs; 11) reverse TSP1 inhibition of the nitric
oxide (NO) pathway; 12) do not reverse TSP1 inhibition of the NO
pathway; 13) cause loss of mitochondrial membrane potential; 14) do
not cause cause loss of mitochondrial membrane potential; 15) cause
an increase in cell surface calreticulin expression on human tumor
cells; 16) do not cause an increase in cell surface calreticulin
expression on human tumor cells; 17) cause an increase in adenosine
triphosphate (ATP) release by human tumor cells; 18) do not cause
an increase in adenosine triphosphate (ATP) release by human tumor
cells; 19) cause an increase in high mobility group box 1 (HMGB1)
release by human tumor cells; 20) do not cause an increase in high
mobility group box 1 (HMGB1) release by human tumor cells; 21)
cause an increase in type I interferon release by human tumor
cells; 22) do not cause an increase in type I interferon release by
human tumor cells; 23) cause an increase in C-X-C Motif Chemokine
Ligand 10 (CXCL10) release by human tumor cells; 24) do not cause
an increase in C-X-C Motif Chemokine Ligand 10 (CXCL10) release by
human tumor cells; 25) cause an increase in cell surface protein
disulfide-isomerase A3 (PDIA3) expression on human tumor cells; 26)
do not cause an increase in cell surface protein
disulfide-isomerase A3 (PDIA3) expression on human tumor cells; 27)
cause an increase in cell surface heat shock protein 70 (HSP70)
expression on human tumor cells; 28) do not cause an increase in
cell surface heat shock protein 70 (HSP70) expression on human
tumor cells; 29) cause an increase in cell surface heat shock
protein 90 (HSP90) expression on human tumor cells; 30) do not
cause an increase in cell surface heat shock protein 90 (HSP90)
expression on human tumor cells; 31) have reduced binding to normal
human cells, which includes, but is not limited to, endothelial
cells, skeletal muscle cells, epithelial cells, and peripheral
blood mononuclear cells (e.g., human aortic endothelial cells,
human skeletal muscle cells, human microvascular endothelial cells,
human renal tubular epithelial cells, human peripherial blood CD3+
cells, and human peripheral blood mononuclear cells); 32) do not
have reduced binding to normal human cells, which includes, but is
not limited to, endothelial cells, skeletal muscle cells,
epithelial cells, and peripheral blood mononuclear cells (e.g.,
human aortic endothelial cells, human skeletal muscle cells, human
microvascular endothelial cells, human renal tubular epithelial
cells, human peripherial blood CD3+ cells, and human peripheral
blood mononuclear cells); 33) have a greater affinity for human
CD47 at an acidic pH compared to physiological pH; 34) do not have
a greater affinity for human CD47 at an acidic pH compared to
physiological pH; and 35) cause an increase in annexin A1 release
by human tumor cells.
[0412] In some embodiments, a monoclonal antibody, or an antigen
binding fragment thereof, is provided, which: binds to human CD47;
blocks SIRP.alpha. binding to human CD47; increases phagocytosis of
human tumor cells; and induces death of human tumor cells; wherein
said monoclonal antibody, or an antigen binding fragment thereof,
exhibits pH-dependent binding to CD47 present on a cell. In other
embodiments, the disclosure provides a monoclonal antibody, or an
antigen binding fragment thereof, which: binds to human CD47;
blocks SIRP.alpha. binding to human CD47; increases phagocytosis of
human tumor cells; and induces death of human tumor cells; wherein
said monoclonal antibody, or an antigen binding fragment thereof,
exhibits reduced binding to normal cells. In some embodiments, a
cell to which such an antibody may bind may be of any cell type as
described herein. In other embodiments, a monoclonal antibody as
described herein, or an antigen binding fragment thereof, may
exhibit any combination of characteristics provided in the present
disclosure. For example, a monoclonal antibody may beneficially
exhibit both pH dependent binding and reduced binding to a cell.
These cells may be an endothelial cell, a skeletal muscle cell, an
epithelial cell, a PBMC or a RBC (e.g., human aortic endothelial
cells, human skeletal muscle cells, human microvascular endothelial
cells, human renal tubular epithelial cells, human peripherial
blood CD3+ cells, human peripheral blood mononuclear cells or human
RBC). Such characteristics may be exhibited individually or in any
combination as described herein. As used herein, pH dependent
binding of an antibody of the disclosure may refer to altered
binding of the antibody at a particular pH, for example an antibody
that exhibits increased binding affinity at acidic pH.
CD47 Antibodies
[0413] Many human cancers up-regulate cell surface expression of
CD47 and those expressing the highest levels of CD47 appear to be
the most aggressive and the most lethal for patients. Increased
CD47 expression is thought to protect cancer cells from phagocytic
clearance by sending a "don't eat me" signal to macrophages via
SIRP.alpha., an inhibitory receptor that prevents phagocytosis of
CD47-bearing cells (Oldenborg et al. Science 288: 2051-2054, 2000;
Jaiswal et al. (2009) Cell 138(2):271-851; Chao et al. (2010)
Science Translational Medicine 2(63):63ra94). Thus, the increase of
CD47 expression by many cancers provides them with a cloak of
"selfness" that slows their phagocytic clearance by macrophages and
dendritic cells.
[0414] Antibodies that block CD47 and prevent its binding to
SIRP.alpha. have shown efficacy in human tumor in murine
(xenograft) tumor models. Such blocking anti-CD47 mAbs exhibiting
this property increase the phagocytosis of cancer cells by
macrophages, which can reduce tumor burden (Majeti et al. (2009)
Cell 138 (2): 286-99; U.S. Pat. No. 9,045,541; Willingham et al.
(2012) Proc Natl Acad. Sci. USA 109(17):6662-6667; Xiao et al.
(2015) Cancer Letters 360:302-309; Chao et al. (2012) Cell
142:699-713; Kim et al. (2012) Leukemia 26:2538-2545).
[0415] Anti-CD47 mAbs have also been shown to promote an adaptive
immune response to tumors in vivo (Tseng et al. (2013) PNAS 110
(27):11103-11108; Soto-Pantoja et al. (2014) Cancer Res. 74 (23):
6771-6783; Liu et al. (2015) Nat. Med. 21 (10): 1209-1215; Xu et
al. (2017) Immunity 47: 363-373).
[0416] However, there are mechanisms by which anti-CD47 mAbs can
attack transformed cells that have not yet been exploited in the
treatment of cancer. Multiple groups have shown that particular
anti-human CD47 mAbs induce cell death of human tumor cells.
Anti-CD47 mAb Ad22 induces cell death of multiple human tumor cells
lines (Pettersen et al. J. Immunol. 166: 4931-4942, 2001; Lamy et
al. J. Biol. Chem. 278: 23915-23921, 2003). AD22 was shown to
indice rapid mitochondrial dysfunction and rapid cell death with
early phosphatidylserine exposure and a drop in mitochondrial
membrane potential (Lamy et al. J. Biol. Chem. 278: 23915-23921,
2003). Anti-CD47 mAb MABL-2 and fragments thereof induce cell death
of human leukemia cell lines, but not normal cells in vitro and had
an anti-tumor effect in in vivo xenograft models. (Uno et al.
(2007) Oncol. Rep. 17 (5): 1189-94). Anti-human CD47 mAb 1F7
induces cell death of human T cell leukemias (Manna and Frazier
(2003) J. Immunol. 170: 3544-53) and several breast cancers (Manna
and Frazier (2004) Cancer Research 64 (3):1026-36). 1F7 kills
CD47-bearing tumor cells without the action of complement or cell
mediated killing by NK cells, T cells, or macrophages. Instead,
anti-CD47 mAb 1F7 acts via a non-apoptotic mechanism that involves
a direct CD47-dependent attack on mitochondria, discharging their
membrane potential and destroying the ATP-generating capacity of
the cell leading to rapid cell death. It is noteworthy that
anti-CD47 mAb 1F7 does not kill resting leukocytes, which also
express CD47, but only those cells that are "activated" by
transformation. Thus, normal circulating cells, many of which
express CD47, are spared while cancer cells are selectively killed
by the tumor-toxic CD47 mAb (Manna and Frazier (2003) J. Immunol.
170: 3544-53). This mechanism can be thought of as a proactive,
selective and direct attack on tumor cells in contrast to the
passive mechanism of causing phagocytosis by simply blocking
CD47/SIRP.alpha. binding. Importantly, mAb 1F7 also blocks binding
of SIRP.alpha. to CD47 (Rebres et al., J. Cellular Physiol. 205:
182-193, 2005) and thus it can act via two mechanisms: (1) direct
tumor toxicity, and (2) causing phagocytosis of cancer cells. A
single mAb that can accomplish both functions may be superior to
one that only blocks CD47/SIRP.alpha. binding.
[0417] An additional mechanism by which anti-CD47 mAbs can be
exploited in the treatment of cancer is through the promotion of an
anti-tumor immune response. The discovery that anti-CD47 mAbs cause
tumor cells to release DAMPs that cause maturation, activation and
homing of DCs and attraction of T cells connects anti-CD47 mAb
treatment to the development of the therapeutically desirable
anti-tumor immune response. Anti-CD47 mAbs have not been previously
shown to cause tumor cell release of ATP, HMGB1, annexin A1, type I
interferons and CXCL10 and tumor cell expression of calreticulin,
PDIA3, HSP70 and HSP90.
[0418] Following periods of tissue ischemia, the initiation of
blood flow causes damage referred to as "ischemia-reperfusion
injury" or IRI. IRI contributes to poor outcomes in many surgical
procedures where IRI occurs due to the necessity to stop blood flow
for a period of time, in many forms/causes of trauma in which blood
flow is interrupted and later restored by therapeutic intervention
and in procedures required for organ transplantation,
cardio/pulmonary bypass procedures, reattachment of severed body
parts, reconstructive and cosmetic surgeries and other situations
involving stopping and restarting blood flow. Ischemia itself
causes many physiological changes that, by themselves would
eventually lead to cell and tissue necrosis and death. Reperfusion
poses its own set of damaging events including generation of
reactive oxygen species, thrombosis, inflammation and cytokine
mediated damage. The pathways that are limited by the TSP1-CD47
system are precisely those that would be of most benefit in
combating the damage of IRI, including the NO pathway. Thus,
blocking the TSP1-CD47 pathway, as with the antibodies disclosed
herein, will provide more robust functioning of these endogenous
protective pathways. Anti-CD47 mAbs have been shown to reduce organ
damage in rodent models of renal warm ishchemia (Rogers et al. J Am
Soc Nephrol. 23: 1538-1550, 2012), liver ischemia-reperfusion
injury (Isenberg et al. Surgery. 144: 752-761, 2008), renal
transplantation (Lin et al. Transplantation. 98: 394-401, 2014;
Rogers et al. Kidney Interantional. 90: 334-347, 2016)) and liver
transplantation, including steatotic livers (Xiao et al. Liver
Transpl. 21: 468-477, 2015; Xiao et al. Transplantation. 100:
1480-1489, 2016). In addition, anti-CD47 mAb caused significant
reductions of right ventricular systolic pressure and right
ventricular hypertrophy in the monocrotaline model of pulmonary
arterial hypertension (Bauer et al. Cardiovasc Res. 93: 682-693,
2012). Studies in skin flap models have shown that modulation of
CD47, including with anti-CD47 mAbs, inhibits TSP1-mediated CD47
signaling. This results in inceased activity of the NO pathway,
resulting in reduced IRI (Maxhimer et al. Plast Reconstr Surg. 124:
1880-1889, 2009; Isenberg et al. Arterioscler Throm Vasc Biol. 27:
2582-2588, 2007; Isenberg et al. Curr Drug Targets. 9: 833-841,
2008; Isenberg et al. Ann Surg. 247: 180-190, 2008) Anti-CD47 mAbs
have also been shown to be efficacious in models of other
cardiovascular diseases. In the mouse transverse aortic
constriction model of pressure overload left ventricular heart
failure, anti-CD47 mAb mitigated cardiac myocyte hypertrophy,
decreased left ventricular fibrosis, prevented an increase in left
ventricular weight, decreased ventricular stiffness, and normalized
changes in the pressure volume loop profile (Sharifi-Sanjani et al.
J Am Heart Assoc., 2014). An anti-CD47 mAb ameliorated
atherosclerosis in multiple mouse models (Kojima et al. Nature.,
2016).
Cancer Indications
[0419] Presently disclosed are anti-CD47 mAbs and antigen binding
fragments thereof effective as cancer therapeutics which can be
administered to patients, preferably parenterally, with susceptible
hematologic cancers and solid tumors including, but not limited to,
leukemias, including systemic mastocytosis, acute lymphocytic
(lymphoblastic) leukemia (ALL), T cell--ALL, acute myeloid leukemia
(AML), myelogenous leukemia, chronic lymphocytic leukemia (CLL),
chronic myeloid leukemia (CML), myeloproliferative
disorder/neoplasm, monocytic cell leukemia, and plasma cell
leukemia; multiple myeloma (MM); Waldenstrom's Macroglobulinemia;
lymphomas, including histiocytic lymphoma and T cell lymphoma, B
cell lymphomas, including Hodgkin's lymphoma and non-Hodgkin's
lymphoma, such as low grade/follicular non-Hodgkin's lymphoma
(NHL), cell lymphoma (FCC), mantle cell lymphoma (MCL), diffuse
large cell lymphoma (DLCL), small lymphocytic (SL) NHL,
intermediate grade/follicular NHL, intermediate grade diffuse NHL,
high grade immunoblastic NHL, high grade lymphoblastic NHL, high
grade small non-cleaved cell NHL, bulky disease NHL; solid tumors,
including ovarian cancer, breast cancer, endometrial cancer, colon
cancer (colorectal cancer), rectal cancer, bladder cancer,
urothelial cancer, lung cancer (non-small cell lung cancer,
adenocarcinoma of the lung, squamous cell carcinoma of the lung),
bronchial cancer, bone cancer, prostate cancer, pancreatic cancer,
gastric cancer, hepatocellular carcinoma (liver cancer, hepatoma),
gall bladder cancer, bile duct cancer, esophageal cancer, renal
cell carcinoma, thyroid cancer, squamous cell carcinoma of the head
and neck (head and neck cancer), testicular cancer, cancer of the
endocrine gland, cancer of the adrenal gland, cancer of the
pituitary gland, cancer of the skin, cancer of soft tissues, cancer
of blood vessels, cancer of brain, cancer of nerves, cancer of
eyes, cancer of meninges, cancer of oropharynx, cancer of
hypopharynx, cancer of cervix, and cancer of uterus, glioblastoma,
meduloblastoma, astrocytoma, glioma, meningioma, gastrinoma,
neuroblastoma, myelodysplastic syndrome, and sarcomas including,
but not limited to, osteosarcoma, Ewing's sarcoma, leiomyosarcoma,
synovial sarcoma, alveolar soft part sarcoma, angiosarcoma,
liposarcoma, fibrosarcoma, rhabdomyosarcoma, chrondrosarcoma, and
melanoma.
Treatment of Cancer
[0420] As is well known to those of ordinary skill in the art,
combination therapies are often employed in cancer treatment as
single-agent therapies or procedures may not be sufficient to treat
or cure the disease or condition. Conventional cancer treatments
often involve surgery, radiation treatment, the administration of a
combination of cytotoxic drugs to achieve additive or synergistic
effects, and combinations of any or all of these approaches.
Especially useful chemotherapeutic and biologic therapy
combinations employ drugs that work via different mechanisms of
action, increasing cancer cell control or killing, increasing the
ability of the immune system to control cancer cell growth,
reducing the likelihood of drug resistance during therapy, and
minimizing possible overlapping toxicities by permitting the use of
reduced doses of individual drugs.
[0421] Classes of conventional anti-tumor/anti-neoplastic agents
useful in the combination therapies encompassed by the present
methods are disclosed, for example, in Goodman & Gilman's The
Pharmacological Basis of Therapeutics, Twelfth Edition (2010) L. L.
Brunton, B. A. Chabner, and B. C. Knollmann Eds., Section VIII,
"Chemotherapy of Neoplastic Diseases", Chapters 60-63, pp.
1665-1770, McGraw-Hill, NY, and include, for example, alkylating
agents, antimetabolites, natural products, a variety of
miscellaneous agents, hormones and antagonists, targeted drugs,
monoclonal antibodies and other protein therapeutics.
[0422] In addition to the foregoing, the methods of the present
disclosure are related to treatment of cancer indications and
further comprises treating the patient via surgery, radiation,
and/or administering to a patient in need thereof an effective
amount of a chemical small molecule or biologic drug including, but
not limited to, a peptide, polypeptide, protein, nucleic acid
therapeutic, conventionally used or currently being developed, to
treat tumorous conditions. This includes antibodies and
antigen-binding fragments, other than those disclosed herein,
cytokines, antisense oligonucleotides, siRNAs, and miRNAs.
[0423] The therapeutic methods disclosed and claimed herein include
the use of the antibodies disclosed herein alone, and/or in
combinations with one another, and/or with antigen-binding
fragments thereof of the present disclosure that bind to CD47,
and/or with competing antibodies exhibiting appropriate
biological/therapeutic activity, as well, for example, all possible
combinations of these antibody compounds to achieve the greatest
treatment efficacy.
[0424] In addition, the present therapeutic methods also encompass
the use of these antibodies, antigen-binding fragments thereof,
competing antibodies and combinations thereof further in
combination with: (1) any one or more anti-tumor therapeutic
treatments selected from surgery, radiation, anti-tumor,
anti-neoplastic agents, and combinations of any of these, or (2)
any one or more of anti-tumor biological agents, or (3) equivalents
of any of the foregoing of (1) or (2) as would be apparent to one
of ordinary skill in the art, in appropriate combination(s) to
achieve the desired therapeutic treatment effect for the particular
indication.
[0425] Antibody and small molecule drugs that increase the immune
response to cancer by modulating co-stimulatory or inhibitory
interactions that influence the T cell response to tumor antigens,
including inhibitors of immune checkpoints and modulators of
co-stimulatory molecules, are also of particular interest in the
context of the combination therapeutic methods encompassed herein
and include, but are not limited to, other anti-CD47 antibodies.
Administration of therapeutic agents that bind to the CD47 protein,
for example, antibodies or small molecules that bind to CD47 and
prevent interaction between CD47 and SIRP.alpha., are administered
to a patient, causing the clearance of cancer cells via
phagocytosis. The therapeutic agent that binds to the CD47 protein
is combined with a therapeutic agent such as an antibody, a
chemical small molecule or biologic drug disclosed herein, directed
against one or more additional cellular targets of CD70 (Cluster of
Differentiation 70), CD200 (OX-2 membrane glycoprotein, Cluster of
Differentiation 200), CD154 (Cluster of Differentiation 154, CD40L,
CD40 ligand, Cluster of Differentiation 40 ligand), CD223
(Lymphocyte-activation gene 3, LAG3, Cluster of Differentiation
223), KIR (Killer-cell immunoglobulin-like receptors), GITR
(TNFRSF18, glucocorticoid-induced TNFR-related protein,
activation-inducible TNFR family receptor, AITR, Tumor necrosis
factor receptor superfamily member 18), CD28 (Cluster of
Differentiation 28), CD40 (Cluster of Differentiation 40, Bp50,
CDW40, TNFRSF5, Tumor necrosis factor receptor superfamily member
5, p50), CD86 (B7-2, Cluster of Differentiation 86), CD160 (Cluster
of Differentiation 160, BY55, NK1, NK28), CD258 (LIGHT, Cluster of
Differentiation 258, Tumor necrosis factor ligand superfamily
member 14, TNFSF14, HVEML, HVEM ligand, herpesvirus entry mediator
ligand, LTg), CD270 (HVEM, Tumor necrosis factor receptor
superfamily member 14, herpesvirus entry mediator, Cluster of
Differentiation 270, LIGHTR, HVEA), CD275 (ICOSL, ICOS ligand,
Inducible T-cell co-stimulator ligand, Cluster of Differentiation
275), CD276 (B7-H3, B7 homolog 3, Cluster of Differentiation 276),
OX40L (OX40 Ligand), B7-H4 (B7 homolog 4, VTCN1, V-set
domain-containing T-cell activation inhibitor 1), GITRL
(Glucocorticoid-induced tumor necrosis factor receptor-ligand,
glucocorticoid-induced TNFR-ligand), 4-1BBL (4-1BB ligand), CD3
(Cluster of Differentiation 3, T3D), CD25 (IL2Ru, Cluster of
Differentiation 25, Interleukin-2 Receptor a chain, IL-2 Receptor a
chain), CD48 (Cluster of Differentiation 48, B-lymphocyte
activation marker, BLAST-1, signaling lymphocytic activation
molecule 2, SLAMF2), CD66a (Ceacam-1, Carcinoembryonic
antigen-related cell adhesion molecule 1, biliary glycoprotein,
BGP, BGP1, BGPI, Cluster of Differentiation 66a), CD80 (B7-1,
Cluster of Differentiation 80), CD94 (Cluster of Differentiation
94), NKG2A (Natural killer group 2A, killer cell lectin-like
receptor subfamily D member 1, KLRD1), CD96 (Cluster of
Differentiation 96, TActILE, T cell activation increased late
expression), CD112 (PVRL2, nectin, Poliovirus receptor-related 2,
herpesvirus entry mediator B, HVEB, nectin-2, Cluster of
Differentiation 112), CD115 (CSF1R, Colony stimulating factor 1
receptor, macrophage colony-stimulating factor receptor, M-CSFR,
Cluster of Differentiation 115), CD205 (DEC-205, LY75, Lymphocyte
antigen 75, Cluster of Differentiation 205), CD226 (DNAM1, Cluster
of Differentiation 226, DNAX Accessory Molecule-1, PTA1, platelet
and T cell activation antigen 1), CD244 (Cluster of Differentiation
244, Natural killer cell receptor 2B4), CD262 (DR5, TrailR2,
TRAIL-R2, Tumor necrosis factor receptor superfamily member 10b,
TNFRSF10B, Cluster of Differentiation 262, KILLER, TRICK2, TRICKB,
ZTNFR9, TRICK2A, TRICK2B), CD284 (Toll-like Receptor-4, TLR4,
Cluster of Differentiation 284), CD288 (Toll-like Receptor-8, TLR8,
Cluster of Differentiation 288), TNFSF15 (Tumor necrosis factor
superfamily member 15, Vascular endothelial growth inhibitor, VEGI,
TL1A), TDO2 (Tryptophan 2,3-dioxygenase, TPH2, TRPO), IGF-1R (Type
I Insulin-like Growth Factor), GD2 (Disialoganglioside 2), TMIGD2
(Transmembrane and immunoglobulin domain-containing protein 2),
RGMB (RGM domain family, member B), VISTA (V-domain
immunoglobulin-containing suppressor of T-cell activation, B7-H5,
B7 homolog 5), BTNL2 (Butyrophilin-like protein 2), Btn
(Butyrophilin family), TIGIT (T cell Immunoreceptor with Ig and
ITIM domains, Vstm3, WUCAM), Siglecs (Sialic acid binding Ig-like
lectins), Neurophilin, VEGFR (Vascular endothelial growth factor
receptor), ILT family (LIRs, immunoglobulin-like transcript family,
leukocyte immunoglobulin-like receptors), NKG families (Natural
killer group families, C-type lectin transmembrane receptors), MICA
(MHC class I polypeptide-related sequence A), TGF.beta.
(Transforming growth factor .beta.), STING pathway (Stimulator of
interferon gene pathway), Arginase (Arginine amidinase, canavanase,
L-arginase, arginine transamidinase), EGFRvIII (Epidermal growth
factor receptor variant III), and HHLA2 (B7-H7, B7y, HERV-H
LTR-associating protein 2, B7 homolog 7), inhibitors of PD-1
(Programmed cell death protein 1, PD-1, CD279, Cluster of
Differentiation 279), PD-L1 (B7-H1, B7 homolog 1, Programmed
death-ligand 1, CD274, cluster of Differentiation 274), PD-L2
(B7-DC, Programmed cell death 1 ligand 2, PDCD1LG2, CD273, Cluster
of Differentiation 273), CTLA-4 (Cytotoxic T-lymphocyte-associated
protein 4, CD152, Cluster of Differentiation 152), BTLA (B- and
T-lymphocyte attenuator, CD272, Cluster of Differentiation 272),
Indoleamine 2,3-dioxygenase (IDO, IDO1), TIM3 (HAVCR2, Hepatitis A
virus cellular receptor 2, T cell immunoglobulin mucin-3, KIM-3,
Kidney injury molecule 3, TIMD-3, T cell immunoglobulin
mucin-domain 3), A2A adenosine receptor (ADO receptor), CD39
(ectonucleoside triphosphate diphosphohydrolase-1, Cluster of
Differentiation 39, ENTPDi), and CD73 (Ecto-5'-nucleotidase,
5'-nucleotidase, 5'-NT, Cluster of Differentiation 73), CD27
(Cluster of Differentiation 27), ICOS (CD278, Cluster of
Differentiation 278, Inducible T-cell Co-stimulator), CD137 (4-1BB,
Cluster of Differentiation 137, tumor necrosis factor receptor
superfamily member 9, TNFRSF9), OX40 (CD134, Cluster of
Differentiation 134), and TNFSF25 (Tumor necrosis factor receptor
superfamily member 25), including antibodies, small molecules, and
agonists, are also specifically contemplated herein. Additional
agents include IL-10 (Interleukin-10, human cytokine synthesis
inhibitory factor, CSIF) and Galectins.
[0426] YERVOY.RTM. (ipilimumab; Bristol-Meyers Squibb) is an
example of an approved anti-CTLA-4 antibody.
[0427] KEYTRUDA.RTM. (pembrolizumab; Merck) and OPDIVO.RTM.
(nivolumab; Bristol-Meyers Squibb Company) are examples of approved
anti-PD-1 antibodies.
[0428] TECENTRIQ.RTM. (atezolizumab; Roche) is an example of an
approved anti-PD-L1 antibody.
Ischemia-Reperfusion Injury (IRI)-Related, Autoimmune,
Autoinflammatory, Inflammatory, Cardiovascular Conditions and
Diseases
[0429] Administration of a CD47 mAb or antigen binding fragment
thereof disclosed herein can be used to treat a number of diseases
and conditions in which IRI is a contributing feature, and to treat
various autoimmune, autoinflammatory, inflammatory and
cardiovascular diseases. These include: organ transplantation in
which a mAb or antigen binding fragment thereof of the present
disclosure is administered to the donor prior to organ harvest, to
the harvested donor organ in the organ preservation solution, to
the recipient patient, or to any combination thereof; skin
grafting; surgical resections or tissue reconstruction in which
such mAb or fragment is administered either locally by injection to
the affected tissue or parenterally to the patient; reattachment of
body parts; treatment of traumatic injury; pulmonary hypertension;
pulmonary arterial hypertension; sickle cell disease (crisis);
myocardial infarction; cerebrovascular disease; stroke;
surgically-induced ischemia; acute kidney disease/kidney failure;
any other condition in which IRI occurs and contributes to the
pathogenesis of disease; autoimmune and inflammatory diseases,
including arthritis, rheumatoid arthritis, multiple sclerosis,
psoriasis, psoriatic arthritis, Crohn's disease, inflammatory bowel
disease, ulcerative colitis, lupus, systemic lupus erythematous,
juvenile rheumatoid arthritis, juvenile idiopathic arthritis,
Grave's disease, Hashimoto's thyroiditis, Addison's disease, celiac
disease, dermatomyositis, multiple sclerosis, myasthenia gravis,
pernicious anemia, Sjogren syndrome, type I diabetes, vasculitis,
uveitis, and ankylosing spondylitis; autoinflammatory diseases,
including familial Mediterranean fever, neonatal onset multisystem
inflammatory disease, tumor necrosis factor (TNF)
receptor-associated periodic syndrome, deficiency of the
interleukin-1 receptor antagonist, Behget's disease; cardiovascular
diseases, including coronary heart disease, coronary artery
disease, atherosclerosis, myocardial infarction, heart failure, and
left ventricular heart failure.
[0430] Anti-CD47 mAbs and antigen binding fragments thereof of the
present disclosure can also be used to increase tissue perfusion in
a subject in need of such treatment. Such subjects can be
identified by diagnostic procedures indicating a need for increased
tissue perfusion. In addition, the need for increased tissue
perfusion may arise because the subject has had, is having, or will
have, a surgery selected from integument surgery, soft tissue
surgery, composite tissue surgery, skin graft surgery, resection of
a solid organ, organ transplant surgery, or reattachment or an
appendage or other body part.
Treatment of Ischemia-Reperfusion Injury (IRI)-Related
Indications
[0431] The methods of the present disclosure, for example those
related to treatment of IRI-related indications, can further
comprise administering to a patient in need thereof an effective
amount of therapeutic agent that binds to the CD47 protein and a
nitric oxide donor, precursor, or both; a nitric oxide generating
topical agent; an agent that activates soluble guanylyl cyclase; an
agent that inhibits cyclic nucleotide phosphodiesterases; or any
combination of any of the foregoing.
[0432] In these methods, the nitric oxide donor or precursor can be
selected from NO gas, isosorbide dinitrate, nitrite, nitroprusside,
nitroglycerin, 3-Morpholinosydnonimine (SIN-1),
S-nitroso-N-acetylpenicillamine (SNAP), Diethylenetriamine/NO
(DETA/NO), S-nitrosothiols, Bidil.RTM., and arginine.
[0433] The agent that activates soluble guanylyl cyclase can be a
non-NO (nitric oxide)-based chemical activator of soluble guanylyl
cyclase that increases cGMP levels in vascular cells. Such agents
bind soluble guanylyl cyclase in a region other than the NO binding
motif, and activate the enzyme regardless of local NO or reactive
oxygen stress (ROS). Non-limiting examples of chemical activators
of soluble guanylyl cyclase include organic nitrates (Artz et al.
(2002) J. Biol. Chem. 277:18253-18256); protoporphyrin IX (Ignarro
et al. (1982) Proc. Natl. Acad. Sci. USA 79:2870-2873); YC-1 (Ko et
al. (1994) Blood 84:4226-4233); BAY 41-2272 and BAY 41-8543 (Stasch
et al. (2001 Nature 410 (6825): 212-5), CMF-1571, and A-350619
(reviewed in Evgenov et al. (2006) Nat. Rev. Drug. Discov.
5:755-768); BAY 58-2667 (Cinaciguat; Frey et al. (2008) Journal of
Clinical Pharmacology 48 (12): 1400-10); BAY 63-2521 (Riociguat;
Mittendorf et al. (2009) Chemmedchem 4 (5): 853-65). Additional
soluble guanylyl cyclase activators are disclosed in Stasch et al.
(2011) Circulation 123:2263-2273; Derbyshire and Marletta (2012)
Ann. Rev. Biochem. 81:533-559, and Nossaman et al. (2012) Critical
Care Research and Practice, Volume 2012, Article ID 290805, pages
1-12.
[0434] The agent that inhibits cyclic nucleotide phosphodiesterases
can be selected from, tadalafil, vardenafil, udenafil, sildenafil
and avanafil.
Treatment of Autoimmune, Autoinflammatory, Inflammatory, and
Cardiovascular Diseases
[0435] A therapeutic agent that binds to the CD47 protein for the
treatment of an autoimmune, autoinflammatory, inflammatory disease
and/or cardiovascular disease can be combined with one or more
therapeutic agent(s) such as an antibody, a chemical small
molecule, or biologic or a medical or surgical procedure which
include, but are not limited to the following. For the treatment of
autoimmune, autoinflammatory and inflammatory diseases, the
combined therapeutic agents are: hydroxychloroquine, leflunomide,
methotrexate, minocycline, sulfasalazine, abatacept, rituximab,
tocilizumab, anti-TNF inhibitors or blockers (adalimumab,
etanercept, infliximab, certolizumab pegol, golimumab),
non-steroidal anti-inflammatory drugs, glucocorticoids,
corticosteroids, intravenous immunoglobulin, anakinra, canakinumab,
rilonacept, cyclophosphamide, mycophenolate mofetil, azathioprine,
6-mercaptopurine, belimumab, beta interferons, glatiramer acetate,
dimethyl fumarate, fingolimod, teriflunomide, natalizumab,
5-aminosalicylic acid, mesalamine, cyclosporine, tacrolimus,
pimecrolimus, vedolizumab, ustekinumab, secukinumab, ixekizumab,
apremilast, budesonide and tofacitinib. For the treatment of
atherosclerosis, the combined therapeutic agents or procedures are:
medical procedures and/or surgery, including percutaneous coronary
intervention (coronary angioplasty and stenting), coronary artery
bypass grafting, and carotid endarterectomy; therapeutic agents,
including angiotensin-converting enzyme (ACE) inhibitors (including
ramipril, quinapril, captopril, and enalapril), calcium channel
blockers (including amiodipine, nifedipine, verapamil, felodipine
and diltiazem), angiotensin-receptor blockers (including eposartan,
olmesarten, azilsartan, valsartan, telmisartan, losartan,
candesartan, and irbesartan), the combination of ezetimibe and
simvastatin, PCSK9 inhibitors (including alirocumab and
evolocumab), anacetrapib, and HMG-CoA inhibitors (including
atorvastatin, pravastatin, simvastatin, rosuvastatin, pitavastatin,
lovastatin and fluvastatin). For the treatment of heart failure,
the combined therapeutic agents are: ACE inhibitors, angiotensin
receptor blockers, angiotensin receptor neprilsyn inhibitors
(including the combination of sacubitril and valsartan), diuretics,
digoxin, inotropes, beta blockers and aldosterone antagonists. For
the treatment of pumonary hypertension the combined therapeutic
agents are: sildenafil, tadalafil, ambrisentan, bosentan,
macitentan, riociguat, treprostinil, epoprostenol, iloprost, and
selexipag.
[0436] As disclosed herein, the anti-CD47 mAb is administered
before, at the same time or after the combined therapeutic agents
or medical or surgical procedures.
[0437] Another useful class of compounds for the combination
therapies contemplated herein includes modulators of
SIRP.alpha./CD47 binding such as antibodies to SIRP.alpha., as well
as soluble protein fragments of this ligand, or CD47 itself,
inhibiting binding of, or interfering with binding of, SIRP.alpha.
to CD47. It should be noted that the therapeutic methods
encompassed herein include the use of the antibodies disclosed
herein alone, in combination with one another, and/or with
antigen-binding fragments thereof as well, for example, all
possible combinations of these antibody compounds.
[0438] The examples illustrate various embodiments of the present
disclosure, but should not be considered as limiting the disclosure
to only these particularly disclosed embodiments.
Diagnostics for CD47 Expression
[0439] Diagnostics (including complementary and companion) have
been an area of focus in the field of oncology. A number of
diagnostic assays have been developed for targeted therapeutics
such as Herceptin (Genentech), Tarceva (OSI
Pharmaceuticals/Genentech), Iressa (Astra Zeneca), and Erbitux
(Imclone/Bristol Myers Squibb). The anti-CD47 mAbs antibodies of
the disclosure are particularly well-suited to use in diagnostic
applications. Accordingly, the disclosure provides a method to
measure CD47 expression in tumor and/or immune cells, using an
anti-CD47 mAb of the disclosure.
[0440] The anti-CD47 mAbs of the disclosure may be used in a
diagnostic assay and/or in vitro method to measure CD47 expression
in tumor and/or immune cells present in a patient's tumor sample.
In particular, the anti-CD47 mAbs of the disclosure may bind CD47
on approximately 1% or more of tumor and/or immune cells present in
a patient's sample as compared to a reference level. In another
embodiment, the anti-CD47 mAbs may bind CD47 on approximately 5% or
more of tumor and/or immune cells in a patient's sample as compared
to a reference level, for example, or binding at least 10%, or at
least 20%, or at least 30%, or at least about 40%, or at least
about 50%, or at least about 60%, or at least about 70%, or at
least about 80%, or at least about 90%, or between 10-100% as
compared to a reference level. In yet another embodiment, the
anti-CD47 mAbs may bind CD47 on tumor and/or immune cells in a
patient's sample to at least about a 2-fold increase as compared to
a reference level, or at least about 3-fold, or at least about a
4-fold, or at least about a 5-fold or at least about a 8-fold
increase, or between 2-fold and 8-fold, or about 10-fold or greater
as compared to a reference level. As described herein, the
measurement of CD47 expression in a patient's sample provides
biological and/or clinical information that enables decision making
about the development and use of a potential drug therapy, notably
the use of anti-CD47 antibodies for treating solid and
hematological cancers, autoimmune disease, inflammatory disease,
atherosclerosis, heart failure, in which the CD47 receptor plays a
role.
[0441] In one embodiment, the in vitro method comprises, obtaining
a patient sample, contacting the patient sample with a monoclonal
antibody, or antigen-binding fragment thereof, which specifically
binds to an epitope within the sequence of SEQ ID NO:66, and
assaying for binding of the antibody to the patient sample, wherein
binding of the antibody to the patient sample is diagnostic of CD47
expression in a patient sample.
[0442] Accordingly, a diagnostic assay in accordance with the
disclosure may comprise contacting tumor and/or immune cells in a
patient's sample with an anti-CD47 mAb, or an antigen binding
fragment thereof, and assaying for binding of the anti-CD47 mAb to
a patient's tumor sample, wherein binding of the anti-CD47 mAb to
the patient sample is diagnostic of CD47 expression. Preferably,
the patient's sample is a sample containing tumor cells. In this
case, binding of the anti-CD47 mAb of the disclosure, or antigen
binding fragment thereof, to the tumor cells may be assessed for
CD47 expression. The levels of CD47 expression by tumor cells
and/or immune cells of a patient's tumor sample may be predictive
of clinical outcome in a patient.
[0443] Increased binding of anti-CD47 mAbs binding to cells in a
patient's sample is associated with increased CD47 expression. In
one embodiment, the anti-CD47 mAbs of the disclosure may bind to
approximately 5% or more of tumor cells in a patient's sample and
this may indicate that the patient would benefit from rapid
intervention to a solid and hematological cancer. A diagnostic
assay of this sort may be used to determine suitable therapeutic
regimes for solid and hematological cancers with relatively high
binding of anti-CD47 mAbs of the disclosure, i.e., increased CD47
expression.
[0444] It will be appreciated that the diagnostic assay disclosed
herein has a number of advantages. The most important of these
advantages is that the diagnostic assay of the disclosure may allow
the user a greater deal of confidence in the CD47 expression in
tumor and/or immune cells. The increased sensitivity of the
diagnostic assay of the disclosure allows detection of CD47 in a
patient's sample at lower levels than has previously been the
case.
[0445] The anti-CD47 mAbs of the disclosure may be used as a
diagnostic assay in relation to many forms of cancer. Particular
forms of cancer that may advantageously be investigated for CD47
expression include susceptible hematologic cancers and solid tumors
including, but not limited to, leukemias, lymphomas, and solid
tumors.
[0446] The diagnostic assays of the disclosure may utilize any
suitable means for detecting binding of an anti-CD47 mAb to measure
CD47 expression. Suitable methods may be selected with reference to
the nature of any reporter moiety used to label the anti-CD47 mAbs
of the disclosure. Suitable techniques include, but are by no means
limited to, flow cytometry, and enzyme linked immunosorbent assays
(ELISA) and assays utilizing nanoparticles.
EXAMPLES
Example 1
Amino Acid Sequences
Light Chain CDRs
TABLE-US-00001 [0447] LCDR1 LCDR2 LCDR3 Vx4-LCDR1 Vx4-LCDR2
Vx4-LCDR3 RSRQSIVHTNGNTYLG KVSNRFS FQGSHVPYT (SEQ ID NO: 11) (SEQ
ID NO: 15) (SEQ ID NO: 18) Vx8-LCDR1 Vx8-LCDR2 Vx8-LCDR3
RASQDISNYLN YTSRLYS QQGNTLPWT (SEQ ID NO: 12) (SEQ ID NO: 16) (SEQ
ID NO: 19) Vx8-LCDR1 RASQSISNYLN (SEQ ID NO: 13) Vx9-LCDR1
Vx9-LCDR2 Vx9-LCDR3 RSSQNIVQSNGNTYLE KVFHRFS FQGSHVPWT (SEQ ID NO:
14) (SEQ ID NO: 17) (SEQ ID NO: 20)
Heavy Chain CDRs
TABLE-US-00002 [0448] HCDR1 HCDR2 HCDR3 Vx4-HCDR1 Vx4-HCDR2
Vx4-HCDR3 GYTFTNYVIH YIYPYNDGILYNEKFKG GGYYVPDY (SEQ ID NO: 1) (SEQ
ID NO: 4) (SEQ ID NO: 7) Vx4-HCDR3 GGYYVYDY (SEQ ID NO: 8)
Vx8-HCDR1 Vx8-HCDR2 Vx8-HCDR3 GYSFTNYYIH YIDPLNGDTTYNQKFKG GGKRAMDY
(SEQ ID NO: 2) (SEQ ID NO: 5) (SEQ ID NO: 9) Vx9-HCDR1 Vx9-HCDR2
Vx9-HCDR3 GYTFTNYWIH YTDPRTDYTEYNQKFKD GGRVGLGY (SEQ ID NO: 3) (SEQ
ID NO: 6) (SEQ ID NO: 10)
Murine Light Chain Variable Domains
TABLE-US-00003 [0449]>Vx4murL01 (SEQ ID NO: 41)
DVLMTQTPLSLPVNLGDQASISCRSRQSIVHTNGNTYLGWFLQKPGQSPK
LLIYKVSNRFSGVPDRFSGSGSGTDFTLTISRVEAEDLGVYYCFQGSHVP YTFGGGTKLEIK.
>Vx4murL02 (SEQ ID NO: 42)
DVLMTQTPLSLPVNLGDQASISCRSRQSIVHTNGNTYLGWFLQKPGQSPK
LLIYKVSNRFSGVPDRFSGSGSGTDFTLTISRVEAEDLGVYYCFQGSHVP YTFGQGTKVEIK.
>Vx8murL03 (SEQ ID NO: 46)
DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYY
TSRLYSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGG GTKLEIK.
>Vx9murL04 (SEQ ID NO: 50)
DVFMTQTPLSLPVSLGDQASISCRSSQNIVQSNGNTYLEWYLQKPGQSPK
LLIYKVFHRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVP WTFGGGTKVEIK
Murine Heavy Chain Variable Domains
TABLE-US-00004 [0450]>Vx4murH01 (SEQ ID NO: 21)
EVQLQQSGPELVKPGASVKMSCKASGYTFTNYVIHWVKRRPGQGLEWIGY
IYPYNDGILYNEKFKGKATVTSDKSSSTAYMDLSSLTSEDSAVYYCTRGG
YYVPDYWGQGTTLTVSS. >Vx4mur-H02 (SEQ ID NO: 22)
EVQLQQSGPELVKPGASVKMSCKASGYTFTNYVIHWVKRRPGQGLEWIGY
IYPYNDGILYNEKFKGKATVTSDKSSSTAYMDLSSLTSEDSAVYYCTRGG
YYVPDYWGQGTLVTVSS. >Vx8murH03 (SEQ ID NO: 28)
EVQLQQSGPELMKPGASVKISCKASGYSFTNYYIHWVNQSHGKSLEWIGY
IDPLNGDTTYNQKFKGKATLTVDKSSSTAYMRLSSLTSADSAVYYCARGG
KRAMDYWGQGTSVTVSS. >Vx9murH04 (SEQ ID NO: 35)
QVQLQQFGAELAKPGASVQMSCKASGYTFTNYWIHWVKQRPGQGLEWIGY
TDPRTDYTEYNQKFKDKATLAADRSSSTAYMRLSSLTSEDSAVYYCAGGG
RVGLGYWGHGSSVTVSS
Human Light Chain Variable Domains
TABLE-US-00005 [0451]>Vx4humL01 (SEQ ID NO: 43)
DIVMTQSPLSLPVTPGEPASISCRSRQSIVHTNGNTYLGWYLQKPGQSP
RLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEADDVGIYYCFQGSH VPYTFGQGTKLEIK
>Vx4humL02 (SEQ ID NO: 44)
DVVMTQSPLSLPVTLGQPASISCRSRQSIVHTNGNTYLGWFQQRPGQSP
RRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSH VPYTFGQGTKLEIK
>Vx4humL03 (SEQ ID NO: 45)
DIVMTQSPDSLAVSLGERATINCRSRQSIVHTNGNTYLGWYQQKPGQPP
KLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCFQGSH VPYTFGQGTKLEIK
>Vx8humL04 (SEQ ID NO: 47)
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIY
YTSRLYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPWTF GQGTKVEIK.
>Vx8humL05 (SEQ ID NO: 48)
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKLLIY
YTSRLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTF GQGTKVEIK.
>Vx8humL06 (SEQ ID NO: 49)
DIVMTQSPLSLPVTPGEPASISCRASQDISNYLNWYLQKPGQSPRLLIY
YTSRLYSGVPDRFSGSGSGTDFTLKISRVEADDVGIYYCQQGNTLPWTF GQGTKLEIK
>Vx9humL07 (SEQ ID NO: 51)
DVVMTQSPLSLPVTLGQPASISCRSSQNIVQSNGNTYLEWFQQRPGQSP
RRLIYKVFHRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSH VPYTFGQGTKLEIK.
>Vx9humL08 (SEQ ID NO: 52)
DIVMTQSPDSLAVSLGERATINCRSSQNIVQSNGNTYLEWYQQKPGQPPK
LLIYKVFHRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCFQGSHVP
YTFGQGTKLEIK.
Human Heavy Chain Variable Domains
TABLE-US-00006 [0452]>Vx4humH01 (SEQ ID NO: 23)
QVQLVQSGAEVKKPGASVQVSCKASGYTFTNYVIHWLRQAPGQGLEWMGY
IYPYNDGILYNEKFKGRVTMTSDTSISTAYMELSSLRSDDTAVYYCARGG
YYVPDYWGQATLVTVSS. >Vx4humH02 (SEQ ID NO: 24)
QVQLVQSGAEVKKPGASVQVSCKASGYTFTNYVIHWLRQAPGQGLEWMGY
IYPYNDGILYNEKFKGRVTMTSDTSISTAYMELSSLRSDDTAVYYCARGG
YYVYDYWGQATLVTVSS. >Vx4humH03 (SEQ ID NO: 25)
EVQLVQSGAEVKKPGATVKISCKVSGYTFTNYVIHWVQQAPGKGLEWMGY
IYPYNDGILYNEKFKGRVTITADTSTDTAYMELSSLRSEDTAVYYCATGG
YYVPDYWGQGTTVTVSS >Vx4humH04 (SEQ ID NO: 26)
EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYVIHWVRQMPGKGLEWMGY
IYPYNDGILYNEKFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARGG
YYVPDYWGQGTTVTVSS >Vx4humH05 (SEQ ID NO: 27)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYVIHWVRQAPGQGLEWMGY
IYPYNDGILYNEKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGG
YYVPDYWGQGTTVTVSS >Vx8humH06 (SEQ ID NO: 29)
QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGY
IDPLNGDTTYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGG
KRAMDYWGQGTLVTVSS. >Vx8humH07 (SEQ ID NO: 30)
QVQLVQSGAEVKKPGSSVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGY
IDPLNGDTTYNQKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARGG
KRAMDYWGQGTLVTVSS. >Vx8humH08 (SEQ ID NO: 31)
EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIHWVRQMPGKGLEWMGY
IDPLNGDTTYNQKFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARGG
KRAMDYWGQGTLVTVSS. >Vx8humH09 (SEQ ID NO: 32)
QVQLVQSGAEVKKPGSSVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGY
IDPLNGDTTYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGG
KRAMDYWGQGTLVTVSS. >Vx8humH10 (SEQ ID NO: 33)
EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIHWVRQMPGKGLEWMGY
IDPLNGDTTYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGG
KRAMDYWGRGTLVTVSS. >Vx8humH11 (SEQ ID NO: 34)
QVQLVQSGAEVKKPGASVQVSCKASGYSFTNYYIHWLRQAPGQGLEWMGY
IDPLNGDTTYNQKFKGRVTMTSDTSISTAYMELSSLRSDDTAVYYCARGG
KRAMDYWGQATLVTVSS >Vx9humH12 (SEQ ID NO: 36)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIHWVRQAPGQGLEWMGY
TDPRTDYTEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGG
RVGLGYWGQGTLVTVSS. >Vx9humH13 (SEQ ID NO: 37)
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWIHWVRQAPGQGLEWMGY
TDPRTDYTEYNQKFKDRVTITADESTSTAYMELSSLRSEDTAVYYCARGG
RVGLGYWGQGTLVTVSS. >Vx9humH14 (SEQ ID NO: 38)
[1]EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYWIHWVRQMPGKGLEW
MGYTDPRTDYTEYNQKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCA
RGGRVGLGYWGQGTLVTVSS. >Vx9humH15 (SEQ ID NO: 39)
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWIHWVRQAPGQGLEWMGY
TDPRTDYTEYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGG
RVGLGYWGQGTLVTVSS. >Vx9humH16 (SEQ ID NO: 40)
EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYWIHWVRQMPGKGLEWMGY
TDPRTDYTEYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGG
RVGLGYWGQGTLVTVSS.
Human IgG-Fc
TABLE-US-00007 [0453]>Human Fc IgG1 (SEQ ID NO: 53)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Human Fc IgG1-N297Q (SEQ ID NO:
54) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Human Fc-IgG2 (SEQ ID NO: 56)
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVER
KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP
EVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKC
KVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG
FYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK. >Human Fc-IgG3 (SEQ ID NO: 57)
ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVEL
KTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSC
DTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED
PEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYK
CKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVK
GFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQG
NIFSCSVMHEALHNRFTQKSLSLSPGK >Human Fc-IgG4 (SEQ ID NO: 58)
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES
KYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED
PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG
NVFSCSVMHEALHNHYTQKSLSLSLG. >Human Fc-IgG4 S228P (SEQ ID NO: 59)
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES
KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED
PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG
NVFSCSVMHEALHNHYTQKSLSLSLG. >Human Fc-IgG4PE (SEQ ID NO: 60)
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES
KYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED
PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG
NVFSCSVMHEALHNHYTQKSLSLSLGK >Human Fc-IgG4PE' (SEQ ID NO: 101)
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES
KYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED
PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG
NVFSCSVMHEALHNHYTQKSLSLSLG >Human kappa LC (SEQ ID NO: 61)
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG
NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC. >Rat
Fc-IgG2c (SEQ ID NO: 62)
ARTTAPSVYPLVPGCSGTSGSLVTLGCLVKGYFPEPVTVKWNSGALSSGV
HTFPAVLQSGLYTLSSSVTVPSSTWSSQTVTCSVAHPATKSNLIKRIEPR
RPKPRPPTDICSCDDNLGRPSVFIFPPKPKDILMITLTPKVTCVVVDVSE
EEPDVQFSWFVDNVRVFTAQTQPHEEQLNGTFRVVSTLHIQHQDWMSGKE
FKCKVNNKDLPSPIEKTISKPRGKARTPQVYTIPPPREQMSKNKVSLTCM
VTSFYPASISVEWERNGELEQDYKNTLPVLDSDESYFLYSKLSVDTDSWM
RGDIYTCSVVHEALHNHHTQKNLSRSPGK. >Rat kappa LC (SEQ ID NO: 63)
RADAAPTVSIFPPSMEQLTSGGATVVCFVNNFYPRDISVKWKIDGSEQRD
GVLDSVTDQDSKDSTYSMSSTLSLTKVEYERHNLYTCEVVHKTSSSPVVK SFNRNEC.
Rabbit IgG-Fc
TABLE-US-00008 [0454]>Rabbit IgG (SEQ ID NO: 64)
GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGV
RTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTC
SKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQ
FTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVH
NKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYP
SDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFT
CSVMHEALHNHYTQKSISRSPGK. >Rabbit kappa LC (SEQ ID NO: 65)
RDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTG
IENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFN RGDC. >CD47
(SEQ ID NO: 66) MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQN
TTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKM
DKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPI
FAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPG
EYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYI
LAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVE.
Chimera and Human Light Chains
TABLE-US-00009 [0455]>Vx4murL01 Full length (SEQ ID NO: 67)
DVLMTQTPLSLPVNLGDQASISCRSRQSIVHTNGNTYLGWFLQKPGQSPK
LLIYKVSNRFSGVPDRFSGSGSGTDFTLTISRVEAEDLGVYYCFQGSHVP
YTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE
VTHQGLSSPVTKSFNRGEC. >Vx4murL01 Full length (SEQ ID NO: 68)
DVLMTQTPLSLPVNLGDQASISCRSRQSIVHTNGNTYLGWFLQKPGQSPK
LLIYKVSNRFSGVPDRFSGSGSGTDFTLTISRVEAEDLGVYYCFQGSHVP
YTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE
VTHQGLSSPVTKSFNRGEC. >Vx4humL01 Full length LC (SEQ ID NO: 69)
DIVMTQSPLSLPVTPGEPASISCRSRQSIVHTNGNTYLGWYLQKPGQSPR
LLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEADDVGIYYCFQGSHVP
YTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE
VTHQGLSSPVTKSFNRGEC. >Vx8humL03 Full length LC (SEQ ID NO: 70)
DIVMTQSPLSLPVTPGEPASISCRASQDISNYLNWYLQKPGQSPRLLIYY
TSRLYSGVPDRFSGSGSGTDFTLKISRVEADDVGIYYCQQGNTLPWTFGQ
GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC.
>Vx9humL02 Full length LC (SEQ ID NO: 71)
DIVMTQSPDSLAVSLGERATINCRSSQNIVQSNGNTYLEWYQQKPGQPPK
LLIYKVFHRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCFQGSHVP
YTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE
VTHQGLSSPVTKSFNRGEC. >Vx8humL02 Full length LC (SEQ ID NO: 72)
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKLLIYY
TSRLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC.
>Vx4humL02 Full length LC (SEQ ID NO: 73)
DVVMTQSPLSLPVTLGQPASISCRSRQSIVHTNGNTYLGWFQQRPGQSPR
RLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVP
YTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE
VTHQGLSSPVTKSFNRGEC. >Vx9humL07 Full length LC (SEQ ID NO: 74)
DVVMTQSPLSLPVTLGQPASISCRSSQNIVQSNGNTYLEWFQQRPGQSPR
RLIYKVFHRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVP
YTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE
VTHQGLSSPVTKSFNRGEC. >Vx8humL01 Full length LC (SEQ ID NO: 75)
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYY
TSRLYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPWTFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC.
>Vx8murL03 Full length LC (SEQ ID NO: 76)
DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYY
TSRLYSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGG
GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC.
>Vx9mur_L04 Full length LC (SEQ ID NO: 77)
DVFMTQTPLSLPVSLGDQASISCRSSQNIVQSNGNTYLEWYLQKPGQSPK
LLIYKVFHRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVP
WTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE
VTHQGLSSPVTKSFNRGEC.
Chimera and Human Heavy Chains
TABLE-US-00010 [0456]>Vx4murH01 Full length HC (SEQ ID NO: 78)
EVQLQQSGPELVKPGASVKMSCKASGYTFTNYVIHWVKRRPGQGLEWIGY
IYPYNDGILYNEKFKGKATVTSDKSSSTAYMDLSSLTSEDSAVYYCTRGG
YYVPDYWGQGTTLTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC
NVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP
SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx4humH01 Full
length HC (SEQ ID NO: 79)
QVQLVQSGAEVKKPGASVQVSCKASGYTFTNYVIHWLRQAPGQGLEWMGY
IYPYNDGILYNEKFKGRVTMTSDTSISTAYMELSSLRSDDTAVYYCARGG
YYVPDYWGQATLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC
NVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP
SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx8humH11 Full
length HC (SEQ ID NO: 80)
QVQLVQSGAEVKKPGASVQVSCKASGYSFTNYYIHWLRQAPGQGLEWMGY
IDPLNGDTTYNQKFKGRVTMTSDTSISTAYMELSSLRSDDTAVYYCARGG
KRAMDYWGQATLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC
NVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP
SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx9humH12 Full
length HC (SEQ ID NO: 81)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIHWVRQAPGQGLEWMGY
TDPRTDYTEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGG
RVGLGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTC
NVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS
VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Vx9humH14 Full
length HC (SEQ ID NO: 82)
EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYWIHWVRQMPGKGLEWMGY
TDPRTDYTEYNQKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARGG
RVGLGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTC
NVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS
VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Vx9humH15 Full
length HC (SEQ ID NO: 83)
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWIHWVRQAPGQGLEWMGY
TDPRTDYTEYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGG
RVGLGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTC
NVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS
VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Vx4humH02 Full
length HC (SEQ ID NO: 84)
QVQLVQSGAEVKKPGASVQVSCKASGYTFTNYVIHWLRQAPGQGLEWMGY
IYPYNDGILYNEKFKGRVTMTSDTSISTAYMELSSLRSDDTAVYYCARGG
YYVYDYWGQATLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC
NVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP
SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx9humH13 Full
length HC (SEQ ID NO: 85)
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWIHWVRQAPGQGLEWMGY
TDPRTDYTEYNQKFKDRVTITADESTSTAYMELSSLRSEDTAVYYCARGG
RVGLGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTC
NVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS
VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Vx8humH10 Full
length HC (SEQ ID NO: 86)
EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIHWVRQMPGKGLEWMGY
IDPLNGDTTYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGG
KRAMDYWGRGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC
NVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP
SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx4humH04 Full
length HC (SEQ ID NO: 87)
EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYVIHWVRQMPGKGLEWMGY
IYPYNDGILYNEKFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARGG
YYVPDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC
NVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP
SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx4humH05 Full
length HC (SEQ ID NO: 88)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYVIHWVRQAPGQGLEWMGY
IYPYNDGILYNEKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGG
YYVPDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC
NVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP
SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx9humH16 Full
length HC (SEQ ID NO: 89)
EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYWIHWVRQMPGKGLEWMGY
TDPRTDYTEYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGG
RVGLGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTC
NVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS
VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Vx8humH06 Full
length HC (SEQ ID NO: 90)
[2]QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQGLEW
MGYIDPLNGDTTYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCA
RGGKRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT
YTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDT
LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT
LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx8humH07 Full
length HC (SEQ ID NO: 91)
QVQLVQSGAEVKKPGSSVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGY
IDPLNGDTTYNQKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARGG
KRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC
NVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP
SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx8humH08 Full
length HC (SEQ ID NO: 92)
EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIHWVRQMPGKGLEWMGY
IDPLNGDTTYNQKFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARGG
KRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC
NVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP
SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx8humH09 Full
length HC (SEQ ID NO: 93)
QVQLVQSGAEVKKPGSSVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGY
IDPLNGDTTYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGG
KRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC
NVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP
SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx8humH06 Full
length HC (SEQ ID NO: 94)
QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGY
IDPLNGDTTYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGG
KRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC
NVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP
SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx8mur-H03 Full
length HC (SEQ ID NO: 95)
EVQLQQSGPELMKPGASVKISCKASGYSFTNYYIHWVNQSHGKSLEWIGY
IDPLNGDTTYNQKFKGKATLTVDKSSSTAYMRLSSLTSADSAVYYCARGG
KRAMDYWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC
NVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP
SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx9mur-H04 Full
length HC (SEQ ID NO: 96)
QVQLQQFGAELAKPGASVQMSCKASGYTFTNYWIHWVKQRPGQGLEWIGY
TDPRTDYTEYNQKFKDKATLAADRSSSTAYMRLSSLTSEDSAVYYCAGGG
RVGLGYWGHGSSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTC
NVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS
VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Vx8humH06 Full
length HC (SEQ ID NO: 97)
QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGY
IDPLNGDTTYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGG
KRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTC
NVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS
VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Vx8humH07 Full
length HC (SEQ ID NO: 98)
QVQLVQSGAEVKKPGSSVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGY
IDPLNGDTTYNQKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARGG
KRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTC
NVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS
VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Vx8humH08 Full
length HC (SEQ ID NO: 99)
EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIHWVRQMPGKGLEWMGY
IDPLNGDTTYNQKFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARGG
KRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTC
NVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS
VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Vx8humH09 Full
length HC (SEQ ID NO: 100)
QVQLVQSGAEVKKPGSSVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGY
IDPLNGDTTYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGG
KRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTC
NVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS
VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.
Example 2
Production of CD47 Antibodies
[0457] Chimeric antibodies disclosed herein comprise a mouse heavy
chain variable domain and a light chain variable domain combined
with a human kappa or human Fc IgG1, IgG1-N297Q, IgG2, IgG4, IgG4
S228P, and IgG4 PE constant domains, respectively. These were
designed to incorporate a secretion signal and cloned into a
mammalian expression system, and transfected into CHO cells to
generate chimeric (murine-human) antibodies. The chimeric variants
were expressed as full length IgG molecules, secreted into the
medium, and purified using protein A.
[0458] Multiple methods for humanizing antibodies are well-known to
those of ordinary skill in the art. One such method, as used
herein, has previously been described (Making and Using Antibodies
a Practical Handbook, Second Edition, Ed. Matthew R. Kase, Chapter
15: Humanization of Antibodies, Juan Carlos Almagro et al., CRC
Press 2013). As such, the humanized antibodies disclosed herein
comprise frameworks derived from the human genome. The collection
covers the diversity found in the human germ line sequences,
yielding functionally expressed antibodies in vivo. The
complementarity determining regions (CDRs) in the light and heavy
chain variable regions of the murine and chimeric (murine-human)
are described herein and were determined by following commonly
accepted rules disclosed in "Protein Sequence and Structure
Analysis of Antibody Variable Domains," In: Antibody Engineering
Lab Manual, eds. S. Duebel and R. Kontermann, Springer-Verlag,
Heidelberg (2001)). The human light chain variable domains were
then designed. The humanized variable domains were then combined
with a secretion signal and human kappa and human Fc IgG1,
IgG1-N297Q, IgG2, IgG3, IgG4 S228P and IgG4 PE constant domains,
cloned into a mammalian expression system, and transfected into CHO
cells to generate humanized mAbs. The humanized variants were
expressed as full length IgG molecules, secreted into the medium
and purified using protein A.
[0459] A non-glycosylated version (IgG1-N297Q) was created by site
directed mutagenesis of heavy chain position 297 to change the
asparagine to glutamine (Human Fc IgG1-N297Q, SEQ ID NO:54). An
IgG4 variant was created by site-directed mutagenesis at position
228 to change the serine to proline thereby preventing in vivo Fab
arm exchange. An IgG4 double mutant was created by site-directed
mutagenesis at positions 228 (serine to proline) and 235 (leucine
to glutamate) to prevent Fab arm exchange and to further reduce Fc
effector function. IgG2, IgG3, IgG4 S228P, and IgG4PE isotypes were
constructed by cloning the heavy chain variable domain in frame
with the human IgG2, IgG3, IgG4 S228P, and IgG4PE constant domains
(Human Fc-IgG2, SEQ ID NO:56 Human Fc-IgG3, SEQ ID NO:57; Human
Fc-IgG4 S228P, SEQ ID NO:59; and Human Fc-IgG4PE, SEQ ID
NO:60).
Example 3
Binding of CD47 Monoclonal Antibodies (mAbs)
[0460] The binding of chimeric (murine-human) and humanized
antibodies of the present disclosure was determined by ELISA using
OV10 cells transfected with human CD47 (OV10 hCD47) or using
freshly isolated human red blood cells (hRBCs), which display CD47
on their surface (Kamel et al. (2010) Blood. Transfus.
8(4):260-266).
[0461] Binding activities of VLX4, VLX8, and VLX9 chimeric (xi) and
humanized mAbs were determined using a cell-based ELISA assay with
human OV10 hCD47 cells expressing cell surface human CD47. OV10
hCD47 cells were grown in IMDM medium containing 10% heat
inactivated fetal bovine serum (BioWest; S01520). One day before
assay, 3.times.10.sup.4 cells were plated in 96 well cell bind
plates (Corning #3300, VWR #66025-626) and were 95-100% confluent
at the time of assay. Cells were washed, various concentrations of
purified antibodies added in IMDM and incubated at 37.degree. C.
for 1 hr in 95% O.sub.2/5% CO.sub.2. Cells were then washed with
media and incubated for an additional hour at 37.degree. C. with
HRP labelled secondary anti-human antibody (Promega) diluted 1/2500
in media. Cells were washed three times with PBS, and the
peroxidase substrate 3,3', 5,5'-tetramethylbenzidine was added
(Sigma; Catalog #T4444). Reactions were terminated by the addition
of HCl to 0.7N, and absorbance at 450 nM determined using a Tecan
model Infinite M200 plate reader. The apparent binding affinities
of these clones to human OV10 hCD47 cells was determined by
non-linear fit (Prism GraphPad software).
[0462] Binding activities of chimeric and humanized VLX4, VLX8, and
VLX9 mAbs to human CD47 on hRBCs were also determined using flow
cytometry. Blood was obtained from normal volunteers and RBCs were
washed 3 times with phosphate buffered saline, pH 7.2 containing
2.5 mM EDTA (PBS+E). hRBCs were incubated for 60 min at 37.degree.
C. with various concentrations of the chimeric or humanized
antibodies in a PBS+E. Cells were then washed with cold PBS+E and
incubated for an additional hour on ice with FITC labelled donkey
anti-human antibody (Jackson Immuno Research Labs, West Grove, Pa.;
Catalogue #709-096-149) in PBS+E. Cells were washed with PBS+E,
antibody binding was analyzed using a C6 Accuri Flow Cytometer
(Becton Dickinson) and apparent binding affinities determined by
non-linear fit (Prism GraphPad software) of the median fluorescence
intensities at the various antibody concentrations.
[0463] All of the VLX4 chimeric (murine-human) mAbs bound to human
OV10 hCD47 tumor cells with apparent affinities in the picomolar
(pM) range (Table 1).
[0464] Similarly, the humanized VLX4 mAbs bound to human OV10 hCD47
tumor cells in a concentration-dependent manner (FIG. 1A and FIG.
1B) with apparent binding affinities ranging from the picomolar to
low nanomolar range (Table 2).
[0465] All of the chimeric VLX4 mAbs bound to human RBCs with
apparent Kd values in the picomolar range and these were similar to
the K.sub.d values obtained for OV10 hCD47 tumor cells by ELISA
(Table 1).
[0466] The humanized VLX4 mAbs VLX4hum_01 IgG1 N297Q, VLX4hum_02
IgG1 N297Q, VLX4hum_01 IgG4PE, VLX4hum_02 IgG4PE, VLX4hum_12
IgG4PE, and VLX4hum_13 IgG4PE bound to human RBCs with Kd values
similar to those obtained for OV10 hCD47 tumor cells whereas
VLX4hum_06 IgG4PE and VLX4hum_07 IgG4 PE exhibited reduced binding
to hRBCs (FIG. 2A, FIG. 2B, and Table 2). This differential binding
of the humanized mAbs to tumor cells and RBCs was unexpected as the
VLX4 IgG4PE chimeric mAb bound with similar apparent Kd values to
both tumor and RBC CD47 (Table 1).
[0467] As shown in Table 1, all the VLX8 chimeric mAbs bound to
human OV10 hCD47 tumor cells in a concentration-dependent manner
with apparent affinities in the picomolar (pM) range.
[0468] Similarly, the humanized VLX8 mAbs bound to human OV10 hCD47
tumor cells in a concentration-dependent manner (FIG. 3A and FIG.
3B) with apparent affinities in the picomolar range (Table 2).
[0469] All the VLX8 chimeric mAbs bound to hRBCs with apparent
K.sub.d values in the picomolar range and these were similar to the
apparent K.sub.d values obtained for OV10 hCD47 tumor cells by
ELISA (Table 1).
[0470] The VLX8 humanized mAbs VLX8hum_01 IgG4PE, VLX8hum_02
IgG4PE, VLX8hum_03 IgG4PE, VLX8hum_04 IgG4PE, VLX8hum_05 IgG4 PE,
and VLX8hum_06 IgG4PE, VLX8hum_07 IgG4PE, VLX8hum_08 IgG4 PE,
VLX8hum_09 IgG4 PE, VLX8hum_11 IgG4 PE, VLX8hum_06 IgG2, VLX8hum_07
IgG2, VLX8hum_08 and VLX8hum_09 IgG2 IgG2 bound to human RBCs with
Kd values similar to the values obtained for OV10 hCD47 tumor cells
whereas VLX8hum_10 IgG4PE exhibited reduced to hRBCs (FIG. 4A, FIG.
4B, and Table 2). This differential binding of the humanized mAbs
to tumor cells and RBCs was unexpected as the VLX8 IgG4PE chimeric
mAb bound with similar apparent Kd values to both tumor and RBC
CD47 (Table 1).
[0471] Table 1 shows the apparent binding affinities of VLX9
chimeric mAbs to human OV10 hCD47 cells and to human RBCs. All of
the chimeric mAbs bound to OV10 hCD47 tumor cells with apparent
binding constants in the picomolar range. Similarly, the humanized
VLX9 mAbs bound to human OV10 hCD47 tumor cells in a
concentration-dependent manner (FIG. 5A and FIG. 5B) with apparent
affinities in the picomolar to nanomolar range (Table 2).
[0472] All the VLX9 chimeric mAbs bound to hRBCs with apparent Kd
values in the picomolar range and these were similar to the
apparent K.sub.d values obtained for OV10 hCD47 tumor cells by
ELISA (Table 1). In contrast to the chimeric mAbs, the VLX9
humanized mAbs VLX9hum_01 IgG2, VLX9hum_02 IgG2 and VLX9hum_07 IgG2
exhibited reduced binding to human RBCs (FIG. 7, Table 2). By
contrast, the humanized mAbs VLX9hum_03 IgG2, VLX9hum_04 IgG2,
VLX9hum_05 IgG2, VLX9hum_06 IgG2, VLX9hum_08 IgG2, VLX9hum_09 IgG2
and VLX9hum_10 IgG2 exhibited no measureable binding to RBCs up to
5,000 pM (Table 2). This differential binding of the humanized mAbs
to tumor cells and RBCs was unexpected as the VLX9 IgG2 chimeric
mAbs all bound with similar apparent Kd values to both tumor and
RBC CD47 (Table 1).
[0473] Specific binding of CD47 humanized mAbs was demonstrated
using Jurkat wildtype and Jurkat CD47 knockout (KO) cells. Jurkat
wildtype and Jurkat CD47 KO cells were grown in RPMI medium
containing 10% heat inactivated fetal bovine serum (BioWest;
S01520). The cells were washed and 1.times.10.sup.4 cells were
resuspended media and incubated with various antibody
concentrations for one hour at 370 in 5% CO.sub.2. Cells were then
washed twice with 1.times.PBS and then resuspended 1:1000 in
secondary antibody (goat anti-human IgG (H+L) FITC-labelled,
Jackson Labs, 109-095-003) for one hour at 37.degree. in 5%
CO.sub.2. Cells were then washed twice with 1.times.PBS and
resuspended in 1.times.PBS. Median fluorescence intensity was
determined by flow cytometry and the apparent binding affinities
determined by non-linear fit (Prism GraphPad software).
[0474] As shown in FIG. 6, VLX4hum_07 IgG4PE (FIG. 6A) and
VLX9hum_09 IgG2 (FIG. 6B) bound to Jurkat cells expressing CD47,
whereas no binding is observed to Jurkat CD47KO cells.
TABLE-US-00011 TABLE 1 Binding of VLX4, VLX8, and VLX9 Chimeric
(xi) mAbs to OV10 hCD47 Cells and Human Red Blood Cells (hRBCs). Kd
(pM) OV10 hCD47 Kd (pM) HA Cell-based ELISA hRBC hRBC VLX4 IgG1
(xi) 315 104 Yes VLX4 IgG1 N297Q (xi) 258 92 Yes VLX4 IgG2 (xi) 431
184 Yes VLX4 IgG4 S228P (xi) 214 99 No VLX4 IgG4 PE(xi) 225 303 No
VLX8 IgG1 N297Q (xi) 42 91 Yes VLX8 IgG4 PE (xi) 56 77 Yes VLX9
IgG1 (xi) 280 381 Yes VLX9 IgG1 N297Q (xi) 275 190 Yes VLX9 IgG2
(xi) 880 742 Yes VLX9 IgG4 PE (xi) 293 126 Yes
TABLE-US-00012 TABLE 2 Binding of VLX4, VLX8, and VLX9 Humanized
mAbs to Human OV10 hCD47 and Human Red Blood Cells (hRBCs). Kd (pM)
OV10 hCD47 Kd (pM) HA Cell-based ELISA hRBC hRBC VLX4hum_01 IgG1 73
23 Yes VLX4hum_02 IgG1 80 70 Yes VLX4hum_01 IgG4 PE 82 80 No
VLX4hum_02 IgG4 PE 95 75 R*** VLX4hum_06 IgG4 PE 196 >33,000**
Yes VLX4hum_07 IgG4 PE 209 >33,000** Yes VLX4hum_12 IgG4 PE 56
263 Yes VLX4hum_13 IgG4 PE 62 340 Yes VLX8hum_01 IgG4 PE 54 209 No
VLX8hum_02 IgG4 PE 50 221 No VLX8hum_03 IgG4 PE 67 183 No
VLX8hum_04 IgG4 PE 49 119 No VLX8hum_05 IgG4 PE 68 264 No
VLX8hum_06 IgG4 PE 61 274 Yes VLX8hum_07 IgG4 PE 24 241 Yes
VLX8hum_08 IgG4 PE 97 217 Yes VLX8hum_09 IgG4 PE 82 336 Yes
VLX8hum_10 IgG4 PE 183 >33,000** Yes VLX8hum_11 IgG4 PE 90 87 No
VLX8hum_06 IgG2 403 246 Yes VLX8hum_07 IgG2 460 671 Yes VLX8hum_08
IgG2 464 820 Yes VLX8hum_09 IgG2 680 1739 Yes VLX9hum_01 IgG2 162
1653** No VLX9hum_02 IgG2 227 4103** No VLX9hum_03 IgG2 606 *MB No
VLX9hum_04 IgG2 823 *MB No VLX9hum_05 IgG2 6372 *MB No VLX9hum_06
IgG2 547 *MB No VLX9hum_07 IgG2 341 >66,000** ***R VLX9hum_08
IgG2 688 *MB No VLX9hum_09 IgG2 8340 *MB No VLX9hum_10 IgG2 12232
*MB No *MB--Minimal biniding; no measurable binding detected at mAb
concentration up to 5,000 pM. **Reduced RBC binding. ***R--Reduced
hemagglutination.
[0475] Cross-species binding of humanized VLX4, VLX8, and VLX9 mAbs
was determined using flow cytometry. Mouse, rat, rabbit or
cynomolgus monkey RBCs were incubated for 60 min on at 37.degree.
C. with various concentrations of the humanized antibodies in a
solution of phosphate buffered saline, pH 7.2, 2.5 mM EDTA (PBS+E).
Cells were then washed with cold PBS+E, and incubated for an
additional hr on ice with FITC labelled donkey anti-human antibody
(Jackson Immuno Research Labs, West Grove, Pa.; Catalogue
#709-096-149) in PBS+E. Cells were washed with PBS+E, and antibody
binding analyzed using a C6 Accuri Flow Cytometer (Becton
Dickinson).
[0476] Table 3 shows the apparent binding affinities of the
humanized VLX4 and VLX8 mAbs to RBCs from mouse, rat, and
cynomolgus monkey determined by non-linear fit (Prism GraphPad
software) of the median fluorescence intensities at various
antibody concentrations. This data demonstrates that humanized VLX4
and VLX8 mAbs bind to mouse, rat, rabbit (data not shown) and
cynomolgus monkey RBCs with apparent Kd values in the picomolar to
nanomolar range.
TABLE-US-00013 TABLE 3 Binding of VLX4 and VLX8 Humanized mAbs to
Mouse, Rat and Cynomolgus Monkey RBCs. Kd (pM) Kd (pM) Kd (pM)
Cynomolgus Mouse RBC Rat RBC Monkey RBC VLX4hum_01 IgG4 PE 13001
30781 56 VLX4hum_07 IgG4 PE 15192 14274 13522 VLX8hum_11 IgG4 PE
9123 8174 55
Example 4
Binding of Humanized Anti-CD47 mAbs Determined by Surface Plasmon
Resonance
[0477] Binding of soluble anti-CD47 mAbs to recombinant human
His-CD47 was measured in vitro by surface plasmon resonance on a
Biacore 2000. An Anti-Human IgG (GE Lifesciences) was amine coupled
to a CM5 chip on flow cells 1 and 2. The humanized mAbs VLX4hum_07
IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_08 IgG2 or VLX9hum_03 IgG2
diluted in HBS-EP.sup.+ running buffer (pH 7.2) were captured onto
flow cell 2. Multi-cycle kinetics were determined using 0 to 1000
nM His-tagged human CD47 (Acro Biosystems) diluted in HBS-EP.sup.+
running buffer (pH 7.2) with contact time of 180 seconds and
dissociation time of 300 seconds. A 1:1 binding model was employed
for kinetic analysis of binding curves. The on-rate, off-rate and
Dissociation constants for VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE,
VLX9hum_08 IgG2 and VLX9hum_03 IgG2 are shown in Table 4.
TABLE-US-00014 TABLE 4 Binding of VLX4, VLX8 and VLX9 Humanized
mAbs to Human Recombinant His-CD47 by Surface Plasmon Resonance at
pH 7.2. k.sub.a k.sub.d K.sub.D (nM) VLX4hum_07 IgG4PE 1.7e.sup.5
8.7e.sup.-4 5.1 VLX8hum_11 IgG4PE 6.8e.sup.5 7.9e.sup.-4 1.2
VLX9_08 IgG2 7.6e.sup.4 6.5e.sup.-4 8.6 VLX9_03 IgG2 6.5e.sup.4
7.3e.sup.-4 11.1
Example 5
Differential Binding of Anti-CD47 mAbs
[0478] Some soluble CD47 antibodies described herein have been
shown to differentially bind to normal cells. This additional
property of selective binding is expected to have advantages
compared to mAbs that bind with equal affinity to normal and tumor
cells. Anti-CD47 mAbs with such reduced binding have not been
described.
[0479] Binding by soluble anti-CD47 mAbs is measured in vitro.
Binding activities of VLX4, VLX8, and VLX9 humanized mAbs were
determined using a flow cytometry based binding assay with human
aortic endothelial cells (HAEC), skeletal muscle cells (SkMC),
human lung microvascular endothelial cells (HMVEC-L), renal tubular
epithelial cells (RTEC), CD3.sup.+ cells or peripheral blood
mononuclear cells (PBMC). HAEC, SkMC, HMVEC-L and RTEC cells were
purchased from Lonza and cultured according to the manufacturer's
recommendations. Adherent cells were removed from the culture flask
with accutase, resuspended in the recommended media and
1.times.10.sup.4 cells were incubated with various antibody
concentrations for one hour at 37.degree., 5% CO.sub.2. For
non-adherent cells, 1.times.10.sup.4 cells were resuspended in the
recommended media and incubated with various antibody
concentrations for one hour at 37.degree., 5% CO.sub.2. Cells were
then washed twice with 1.times.PBS and then resuspended 1:1000 in
secondary antibody (goat anti-human IgG (H+L)--FITC, Jackson Labs,
109-095-003) for one hour at 37.degree. C., 5% CO.sub.2.
[0480] PBMC were isolated by ficoll gradient and were incubated
with an FcR blocking reagent (Miltenyi Biotec) for 10 min at
4.degree. C. per manufacturer's recommendation immediately
preceeding the addition of various concentrations of antibodies
diluted in PBS. CD3 cells were detected using an allophycocyanin
(APC)-labelled anti-CD3 antibody (BD BioSciences) which was added
at the same time as the FITC-labelled goat anti-human IgG (H+L)
antibody. Cells were washed twice with 1.times.PBS and antibody
binding was assessed by flow cytometry analysis.
[0481] As shown in FIG. 8A, VLX4 and VLX8 humanized mAbs bound to
HAEC cells whereas VLX9 humanized mAbs had reduced or minimal
binding to HAEC cells as compared to tumor cells (Table 5). VLX9
humanized mAbs also showed reduced binding to SkMC cells (FIG. 8B),
reduced or minimal binding to HMVEC-L cells (FIG. 8C), reduced
binding to RPTEC cells (FIG. 8D) as compared to binding to tumor
cells (Table 5). Reduced binding of VLX9 humanized mAbs was also
observed to CD3.sup.+ cells (FIG. 8E) and PBMC (FIG. 8F) as
compared to tumor cells (Table 5). This selective binding imparts
an additional desirable antibody property and potential therapeutic
benefit in the treatment of cancer.
TABLE-US-00015 TABLE 5 VLX4, VLX8 and VLX9 Humanized mAbs Binding
to Normal Cells. Kd (pM) OV10 hCD47 Cell-based Kd (pM) Kd (pM) Kd
(pM) Kd (pM) Kd (pM) Kd (pM) Kd (pM) ELISA hRBC HAEC HMVEC-L SKMC
RPTEC CD3.sup.+ PBMC VLX4hum_01 IgG4 PE 82 80 118 72 5 26 220 269
VLX4hum_07 IgG4 PE 209 >33,000** 747 792 630 784 440 499
VLX8hum_10 IgG4 PE 183 >33,000** 1104 2113** 461 491 91 106
VLX8hum_11 IgG4 PE 90 87 34 20 7 26 144 156 VLX9hum_03 IgG2 606 MB*
MB* >200,000** >200,000** >200,000** 10863** 10232**
VLX9hum_04 IgG2 823 MB* MB* MB* >200,000** >200,000** 7426**
7619** VLX9hum_06 IgG2 547 MB* >200,000** 71619** 23483** 4847**
19354** 17904** VLX9hum_08 IgG2 688 MB* >200,000** >200,000**
34783** >200,000** 28287** 24486** VLX9hum_09 IgG2 8340 MB* MB*
MB* MB* >200,000** 56146** 48348** *MB--Minimal binding, no
measureable binding detected at mAb concentration up to 5,000 pM.
**Reduced binding.
Example 6
pH Dependent and Independent Binding of Humanized Anti-CD47
mAbs
[0482] Some soluble anti-CD47 mAbs described herein have been shown
to bind tumor cells at acidic pH with greater affinity compared to
physiologic pH. This additional property is expected to have
advantages compared to mAbs that bind at similar affinities to CD47
at both acidic and physiologic pH, in part due to the acidic nature
of the tumor microenvironment (Tannock and Rotin, Cancer Res 1989;
Song et al. Cancer Drug Discovery and Development 2006; Chen and
Pagel, Advan Radiol 2015).
[0483] Binding by soluble anti-CD47 mAbs to immobilized recombinant
human CD47 and to human CD47 expressed on cells was measured in
vitro. For the in vitro binding to recombinant CD47, His-CD47
(AcroBiosystems) was adsorbed to high-binding microtiter plates
overnight at 4.degree. C. The wells were washed and varying
concentrations of anti-CD47 mAbs were added to the wells in buffers
with a of either pH 6 or pH 8 for 1 hour. The wells were washed and
then incubated with HRP-labelled secondary antibody for 1 hour at
pH 6 or pH 8 followed by addition of peroxidase substrate. The
apparent affinities were calculated using non-linear fit model
(Graphpad Prism).
[0484] For analysis of pH dependent binding by surface plasmon
resonance using a Biacore 2000, an Anti-Human IgG (GE Lifesciences)
was amine coupled to a CM5 chip on flow cells 1 and 2. An Fc-tagged
human CD47 (Acro Biosystems) was diluted in PBS-EP.sup.+ running
buffer (pH 7.5, 6.5 or 6.0) and captured onto flow cell 2.
Multi-cycle kinetics were determined using 0 to 100 nM VLX8hum_11
Fab or VLX9hum_08 Fab diluted in PBS-EP.sup.+ running buffer (pH
7.5, 6.5 or 6.0) with contact time of 180 seconds and dissociation
time of 300 seconds. A 1:1 binding model was employed for kinetic
analysis of binding curves.
[0485] For the in vitro binding to cells expressing CD47, Jurkat
cells were grown in RPMI medium containing 10% heat inactivated
fetal bovine serum (BioWest; S01520). The cells were washed and
1.times.10.sup.4 cells were resuspended in PBS supplementated with
2% FBS at either pH 7.4 or 6.5 and incubated with various antibody
concentrations for 1 hour at 37.degree. C. Cells were then washed
twice and resuspended with 1:1000 of secondary antibody (goat
anti-human IgG (H+L) labelled with Alexa488, JacksonImmunoresearch)
for 1 hour at 37.degree. C. at pH 6 or pH 8. Cells were then washed
twice and the median fluorescence intensity was determined by flow
cytometry. The apparent binding affinities were determined by
non-linear fit (Prism GraphPad software).
[0486] As shown in FIG. 9A and FIG. 9B, the soluble VLX9 humanized
mAbs (VLX9hum_09 IgG2 and VLX9hum_04 IgG2) bound to His-CD47 with
greater affinity at the more acidic pH 6.0 than at pH 8.0. Neither
VLX4hum_07 IgG4PE (FIG. 9C) nor VLX8hum_10 IgG4PE (FIG. 9D)
displayed pH dependent binding. In addition, the murine VLX9
antibody and VLX9 chimeric antibodies containing human Fc from
isoytpes IgG1, IgG2 and IgG4PE did not display pH dependence (Table
6) whereas VLX9hum_04 as either an IgG, IgG2 or an IgG4PE
demonstrated greater binding to His-CD47 at acidic pH (Table 7).
The apparent binding affinities for additional humanized mAbs to
recombinant human CD47 are shown in Table 8. All humanized VLX9
mAbs exhibited pH dependent binding whereas the VLX4 and VLX8
humanized mAbs did not. To determine the effect of pH on on-rates,
off-rates and dissociation constants, Biacore analysis was
performed for humanized mAbs VLX8hum_11 Fab fragment and VLX9hum_08
Fab at pH 6, pH 6.5 and pH 7.5. The VLX9hum_08 Fab exhibited pH
dependent binding that increased with decreasing pH wheras the
VLX8hum_11 Fab did not. The on-rate, off-rate and dissociation
constants for VLX8hum_11 Fab and VLX9hum_08 Fab are shown in Table
9. Table 10 illustrates the pH dependent binding exhibited by
VLX9hum_04 IgG2 to CD47 expressed on Jurkat cells. No pH dependent
binding was exhibited by VLX4hum_07 IgG4PE. This pH dependence of
the VLX9 humanized mAbs imparts an additional desirable antibody
property and therapeutic benefit in the treatment of cancer.
TABLE-US-00016 TABLE 6 Murine VLX9 and mouse-human chimeric VLX9
Binding to CD47 is not pH Dependent KD (pM) KD (pM) pH 6 pH 8 VLX9
IgG (murine) 91 76 VLX9 IgG1-N297Q (xi) 99 135 VLX9 IgG2 (xi) 130
137 VLX9 IgG4PE (xi) 133 160
TABLE-US-00017 TABLE 7 VLX9hum_04 Humanized mAbs Bind to CD47 in a
pH Dependent Manner and Binding is not Isotype Specific KD (pM) KD
(pM) pH 6 pH 8 VLX9hum_04 Ig1-N297Q 215 >33,000 VLX9hum_04 IgG2
470 >33,000 VLX9hum_04 IgG4PE 256 >33,000
TABLE-US-00018 TABLE 8 pH Dependent and Independent Binding of
VLX4, VLX8 and VLX9 Humanized mAbs. K.sub.D (pM) K.sub.D (pM) pH 6
pH 8 VLX9hum_03 IgG2 48 >33,000 VLX9hum_04 IgG2 43 >33,000
VLX9hum_06 IgG2 61 >33,000 VLX9hum_08 IgG2 65 >33,000
VLX9hum_09 IgG2 138 >33,000 VLX4hum_07 IgG4PE 63 92 VLX4hum_01
IgG4PE 47 75 VLX8hum_10 IgG4PE 52 79 VLX8hum_11 IgG4PE 64 92
TABLE-US-00019 TABLE 9 pH Independent and Dependent Binding of
VLX8hum_11 Fab and VLX9hum_08 Fab to Recombinant Human CD47 k.sub.a
k.sub.d K.sub.D (nM) VLX8hum_11 Fab 1.35e.sup.6 2.29e.sup.-3 1.7 nM
(pH 7.5) VLX8hum_11 Fab 2.14e.sup.6 2.78e.sup.-3 1.3 nM (pH 6.5)
VLX8hum_11 Fab 1.64e.sup.6 2.63e.sup.-3 1.6 nM (pH 6.0) VLX9hum_03
Fab 1.43e.sup.5 1.13e.sup.-2 79 nM (pH 7.5) VLX9hum_08 Fab
1.74e.sup.5 9.74e.sup.-4 5.6 nM (pH 6.5) VLX9hum_08 Fab 1.95e.sup.5
9.94e.sup.-4 5.1 nM (pH 6.0)
TABLE-US-00020 TABLE 10 pH Dependent and Independent Binding of
VLX4 and VLX9 Humanized mAbs to Jurkat Cells KD (pM) KD (pM) pH 6.5
pH 7.4 VLX4hum_07 IgG4PE 69 23 VLX9hum_04 IgG2 231 1526
Example 7
CD47 Antibodies Block CD47/SIRP.alpha. Binding
[0487] To assess the effect of humanized CD47 mAbs on binding of
CD47 to SIRP.alpha. in vitro the following method is employed using
the binding of fluorescently-labelled SIRP.alpha.-Fc fusion protein
to CD47 expressing Jurkat cells.
[0488] SIRP.alpha.-Fc fusion protein (R&D Systems, cat
#4546-SA) was labelled using an Alexa Fluor.RTM. antibody labelling
kit (Invitrogen Cat No. A20186) according to the manufacturers
specifications. 1.5.times.10.sup.6 Jurkat cells were incubated with
humanized mAbs (5 .mu.g/ml), a human control antibody in RPMI
containing 10% media or media alone for 30 min at 37.degree. C. An
equal volume of fluorescently labelled SIRP.alpha.-Fc fusion
protein was added and incubated for an additional 30 min at
37.degree. C. Cells were washed once with PBS and the amount of
labelled SIRP.alpha.-Fc bound to the Jurkat cells analyzed by flow
cytometry.
[0489] As shown in FIG. 10, the humanized VLX4, VLX8 and VLX9 mAbs
(VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_10 IgG4PE,
VLX8hum_11 IgG4PE, VLX9hum_03 IgG2, VLX9hum_06 IgG2 and VLX9hum_08
IgG2) blocked the interaction of CD47 expressed on the Jurkat cells
with soluble SIPR.alpha., while the human control antibody (which
does not bind to CD47) or media alone, did not block the
CD47/SIRP.alpha. interaction.
Example 8
CD47 Antibodies Increase Phagocytosis
[0490] To assess the effect of chimeric (murine-human) and
humanized VLX4, VLX8, and VLX9 CD47 mAbs on phagocytosis of tumor
cells by macrophages in vitro the following method is employed
using flow cytometry (Willingham et al. (2012) Proc Natl Acad Sci
USA 109(17):6662-7 and Tseng et al. (2013) Proc Natl Acad Sci USA
110(27): 11103-8).
[0491] Human derived macrophages were derived from leukapheresis of
healthy human peripheral blood and incubated in AIM-V media (Life
Technologies) for 7-10 days. For the in vitro phagocytosis assay,
macrophages were re-plated at a concentration of 1.times.10.sup.4
cells per well in 100 ul of AIM-V media in a 96-well plate and
allowed to adhere for 24 hrs. Once the effector macrophages adhered
to the culture dish, the target human cancer cells (Jurkat) were
labelled with 1 .mu.M 5(6)-Carboxyfluorescein diacetate
N-succinimidyl ester (CFSE; Sigma Aldrich) and added to the
macrophage cultures at a concentration of 5.times.10.sup.4 cells in
1 ml of AIM-V media (5:1 target to effector ratio). VLX4, VLX8, and
VLX9 CD47 mAbs (1 .mu.g/ml) were added immediately upon mixture of
target and effector cells and allowed to incubate at 37.degree. C.
for 2-3 hours. After 2-3 hrs, all non-phagocytosed cells were
removed and the remaining cells washed three times with phosphate
buffered saline (PBS; Sigma Aldrich). Cells were then trypsinized,
collected into microcentrifuge tubes, and incubated in 100 ng of
allophycocyanin (APC) labelled CD14 antibodies (BD Biosciences) for
30 minutes, washed once, and analyzed by flow cytometry (Accuri C6;
BD Biosciences) for the percentage of CD14.sup.+ cells that were
also CFSE.sup.+ indicating complete phagocytosis.
[0492] As shown in FIG. 11, the VLX4 chimeric mAbs VLX4 IgG1 xi,
VLX4 IgG1 N297Q xi, VLX4 IgG4PE xi, and VLX4 IgG4 S228P xi
increased phagocytosis of Jurkat cells by human macrophages by
blocking the CD47/SIRP.alpha. interaction. This enhanced
phagocytosis is independent of Fc function.
[0493] Similarly, as shown in FIG. 12A and FIG. 12B, humanized mAbs
VLX4hum_01 IgG1, VLX4hum_01 IgG4PE, VLX4hum_06 IgG4PE, VLX4hum_07
IgG4PE, VLX4hum_12 IgG4PE, and VLX4hum_13 IgG4PE increased
phagocytosis of Jurkat cells by human macrophages by blocking the
CD47/SIRP.alpha. interaction. This enhanced phagocytosis is
independent of Fc function.
[0494] As shown in FIG. 13A, the VLX8 chimeric mAbs VLX8 IgG1 N297Q
xi and VLX8 IgG4PE xi increase phagocytosis of Jurkat cells by
human macrophages by blocking the CD47/SIRP.alpha. interaction.
This enhanced phagocytosis is independent of Fc function.
[0495] Similarly, as shown in FIG. 13B, humanized mAbs VLX8hum_01
IgG4PE, VLX8hum_03 IgG4PE, VLX8hum_07 IgG4PE, VLX8hum_08 IgG4PE,
and VLX8hum_09 IgG4PE and chimeric mAb VLX8 IgG4PE xi increased
phagocytosis of Jurkat cells by human macrophage by blocking the
CD47/SIRP.alpha. interaction.
[0496] As shown in FIG. 14A, the VLX9 IgG1 N297Q xi, VLX9 IgG2 xi
and VLX9 IgG4PE xi chimeric mAbs all increased phagocytosis of
Jurkat cells by human macrophages by blocking the CD47/SIRP.alpha.
interaction. This enhanced phagocytosis is independent of Fc
effector function. Similarly as shown in FIG. 14B, all of the
humanized VLX9 IgG2 mAbs (VLX9hum_01 to _10 IgG2) increased
phagocytosis of Jurkat cells.
Example 9
Induction of Cell Death by Soluble CD47 Antibodies
[0497] Some soluble CD47 antibodies have been shown to induce
selective cell death of tumor cells. This additional property of
selective toxicity to cancer cells is expected to have advantages
compared to mAbs that only block SIRP.alpha. binding to CD47.
[0498] Induction of cell death by soluble anti-CD47 mAbs is
measured in vitro (Manna et al. (2003) J. Immunol. 107 (7):
3544-53). For the in vitro cell death assay, 1.times.10.sup.5
transformed human T cells (Jurkat cells) were incubated with
soluble humanized VLX4, VLX8, and VLX9 CD47 mAbs (1 .mu.g/ml) for
24 hrs at 37.degree. C. As cell death occurs, mitochondrial
membrane potential is decreased, the inner leaflet of the cell
membrane is inverted, exposing phosphatidylserines (PS), and
propidium iodide (PI) or 7-aminoactinomycin D (7-AAD) is able to
incorporate into nuclear DNA. In order to detect these cellular
changes, cells were then stained with fluorescently labelled
annexin V and PI or 7-aminoactinomycin D (7-AAD) (BD Biosciences)
and the signal detected using an Accuri C6 flow cytometer (BD
Biosciences). The increase in PS exposure is determined by
measuring the percent increase in annexin V signal and the percent
of dead cells by measuring the percent increase in PI or 7-AAD
signal. Annexin V positive (annexin V.sup.+) or annexin V
positive/7-AAD negative (annexin V.sup.+/7-AAD.sup.-) cells are
observed in early stages of cell death and annexin V positive/7-AAD
positive (annexin V.sup.+/7-AAD.sup.+) cells are dead cells.
Importantly for therapeutic purposes, these mAbs induce cell death
of tumor cells directly and do not require complement or the
intervention of other cells (e.g., NK cells, T cells, or
macrophages) to kill. Thus, the mechanism is independent of both
other cells and of Fc effector function. Therefore, therapeutic
antibodies developed from these mAbs can be engineered to reduce Fc
effector functions such as ADCC and CDC and thereby limit the
potential for side effects common to humanized mAbs with intact Fc
effector functions.
[0499] As shown in FIG. 15A-F, the soluble VLX4 humanized mAbs
induced increased PS exposure and cell death of Jurkat cells as
measured by increased % of the cells that are annexin V.sup.+ (FIG.
15A and FIG. 15D), annexin V.sup.+/7-AAD.sup.- (FIG. 15B and FIG.
15E), or annexin V.sup.+/7-AAD.sup.+ (FIG. 15C and FIG. 15F). The
humanized mAbs VLX4hum_01 IgG1, VLX4hum_01 IgG4PE, VLX4hum_02 IgG1,
VLX4hum_02 IgG4PE, VLX4hum_06 IgG4 PE, VLX4hum_07 IgG4PE,
VLX4hum_12 IgG4PE, and VLX4hum_13 IgG4PE caused increased PS
exposure and cell death. In contrast, the humanized mAbs VLX4hum_08
IgG4PE and VLX4hum_11 IgG4PE did not cause increased PS exposure
and cell death of Jurkat cells. Induction of cell death and the
promotion of phagocytosis of susceptible cancer cells imparts an
additional desirable antibody property and potential therapeutic
benefit in the treatment of cancer.
[0500] As shown in FIGS. 16A-F, the soluble VLX8 chimeric and
humanized mAbs induced increased PS exposure and cell death of
Jurkat cells as measured by the % of the cells that are annexin
V.sup.+ (FIGS. 16A, D), annexin V.sup.+/7-AAD.sup.- (FIGS. 16B, E),
or annexin V.sup.+/7-AAD.sup.+ (FIGS. 16C, F). The chimeric mAbs,
VLX8 IgG1 N297Q xi and VLX8 IgG4PE xi, and the humanized mAbs,
VLX8hum_07 IgG4PE and VLX8hum_08 IgG4PE, induced increased PS
exposure and cell death of Jurkat cells. In contrast, the humanized
mAbs VLX8hum_02 IgG4PE and VLX8hum_04 IgG4PE did not cause
increased PS exposure and cell death of Jurkat cells. Induction of
cell death and the promotion of phagocytosis of susceptible cancer
cells imparts an additional desirable antibody property and
potential therapeutic benefit in the treatment of cancer.
[0501] As shown in FIG. 17A-FIG. 17F, the soluble VLX9 chimeric and
humanized antibodies induced increased PS exposure and cell death
of Jurkat cells as measured by % of the cells that are annexin
V.sup.+ (FIG. 17A and FIG. 17D), annexin V.sup.+/7-AAD.sup.- (FIG.
17B and FIG. 17E), or annexin V.sup.+/7-AAD.sup.+ (FIG. 17C and
FIG. 17F). The chimeric VLX9 IgG2xi mAb and the humanized mAbs
VLX9hum_06 IgG2, VLX9hum_07 IgG2, VLX9hum_08 IgG2, and VLX9hum_09
IgG2 induced increased PS exposure and cell death of Jurkat cells.
In contrast, the humanized mAbs VLX9hum_01 IgG2, VLX9hum_02 IgG2,
VLX9hum_03 IgG2, VLX9hum_04 IgG2, VLX9hum_05 IgG2 and VLX9hum_010
IgG2 did not cause increased PS exposure and cell death of Jurkat
cells. Induction of cell death and the promotion of phagocytosis of
susceptible cancer cells imparts an additional desirable antibody
property and potential therapeutic benefit in the treatment of
cancer. Importantly, chimeric and humanized mAbs that cause cell
death of tumor cells do not cause cell death of normal cells.
Example 10
Damage-Associated Molecular Pattern (DAMP) Expression and Release,
Mitochondrial Depolarization and Cell Death Caused by Humanized
Anti-CD47 mAb
[0502] Humanized Anti-CD47 mAbs Cause Loss of Mitochondrial
Membrane Potential
[0503] These experiments demonstrate that humanized anti-CD47 mAbs
of the present disclosure exhibit the ability to induce the loss of
mitochondrial membrane potential in tumor cell as described
previously (Manna and Frazier, 2014 Journal of Immunology
170(7):3544-3553).
[0504] Loss of mitochondrial membrane potential in the tumor cell
was determined using JC-1 dye (Thermo; Catalogue #M34152). Human
Raji lymphoma cells (ATCC, Manassas, Va.; Catalog # CCL-86) or
other cells types that express sufficient levels of CD47 will be
used. Cells were grown in RPMI-1640 medium containing 10% (v/v)
heat inactivated fetal bovine serum (BioWest; Catalogue # S01520),
100 units/mL penicillin, 100 lag mL streptomycin (Sigma; Catalogue
# P4222) at densities less than 1.times.10.sup.6 cells/mL. For this
assay, Raji cells were plated in 96 well tissue culture plates at a
density of 1.times.10.sup.5 cells/ml RPMI-1640 medium containing
10% (v/v) heat inactivated fetal bovine serum (BioWest; Catalog #
S01520), 100 units/mL penicillin, 100 .mu.g/mL streptomycin (Sigma;
#P4222).
[0505] The humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07
IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06 IgG2 VLX9hum_08 IgG2 and
VLX9hum_03 IgG2) as disclosed herein, purified from transient
transfections in CHO cells as described above, as well as the
control chimeric antibody, were added at a final concentration of
10 .mu.g/ml. As a positive control for loss of mitochondrial
membrane potential, cells were treated with 1 .mu.M of
chemotherapeutic anthracycline mitoxantrone. The cells were
incubated at 37.degree. C. for 24 hours, after which the cells were
harvested, washed twice with PBS, and incubated for 30 minutes with
JC-1 dye as described above, diluted 1:2000 in PBS. After 30
minutes the cells were washed twice with PBS, resuspended in 100
.mu.l of PBS, and analyzed for the percent of cells that shift
their fluorescence emission from red to green by flow cytometry
(Accuri C6, Becton Dickinson, Franklin Lakes, N.J.). Results are
presented as means.+-.SEM and analyzed for statistical significance
using ANOVA in GraphPad Prism 6.
[0506] Some of the chimeric or humanized antibodies induce the loss
of mitochondrial membrane potential in the tumor cell. As shown in
FIG. 18, the percent of cells with mitochondrial membrane
depolarization in all anti-CD47 mAb treated cultures was
significantly increased (p<0.05) compared to an isotype control.
This increase in the amount of mitochondrial membrane
depolarization demonstrates that anti-CD47 chimeric or humanized
antibodies induce mitochondrial depolarization that leads to cell
death in human tumor cells.
Humanized Anti-CD47 mAbs Cause Increase in Cell Surface
Calreticulin Expression
[0507] These experiments demonstrate that humanized anti-CD47 mAbs
of the present disclosure exhibit the ability to expose the
endoplasmic reticulum resident chaperone calreticulin on the
surface of the tumor cell as, for example, described previously
using chemotherapeutic anthracyclines such as doxorubicin and
mitoxantrone, as disclosed by Obeid et al. (2007) Nat. Med.
13(1):54-61.
[0508] Cell surface exposure of calreticulin was determined using a
rabbit monoclonal antibody against calreticulin conjugated to Alexa
Fluor 647 (Abcam; Catalogue #ab196159). Human Raji lymphoma cells
(ATCC, Manassas, Va.; Catalog # CCL-86) or other cells types that
express sufficient levels of CD47 will be used. Cells were grown in
RPMI-1640 medium containing 10% (v/v) heat inactivated fetal bovine
serum (BioWest; Catalogue # S01520), 100 units/mL penicillin, 100
lag mL streptomycin (Sigma; Catalogue # P4222) at densities less
than 1.times.10.sup.6 cells/mL. For this assay, cells were plated
in 96 well tissue culture plates at a density of 1.times.10.sup.5
cells/ml RPMI-1640 medium containing 10% (v/v) heat inactivated
fetal bovine serum (BioWest; Catalog # S01520), 100 units/mL
penicillin, 100 .mu.g/mL streptomycin (Sigma; #P4222).
[0509] The humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07
IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2
VLX9hum_03 IgG2) as disclosed herein, purified from transient
transfections in CHO cells as described above, as well as the
control chimeric antibody, were added at a final concentration of
10 .mu.g/ml. As a positive control for calreticulin exposure, cells
were treated with 1 .mu.M of chemotherapeutic anthracycline
mitoxantrone. The cells were incubated at 37.degree. C. for 24
hours, after which the cells were harvested, washed twice with PBS,
and incubated for 30 minutes with anti-calreticulin antibody as
described above, diluted 1:200 in PBS. After 30 minutes the cells
were washed twice with PBS, resuspended in 100 .mu.l of PBS, and
analyzed for the mean fluorescence intensity of the
anti-calreticulin antibody signal as well as the percent of cells
that stain positive for cell surface calreticulin by flow cytometry
(Accuri C6, Becton Dickinson, Franklin Lakes, N.J.). Results are
presented as means.+-.SEM and analyzed for statistical significance
using ANOVA in GraphPad Prism 6.
[0510] As shown in FIG. 19, the humanized antibodies induced the
preapoptotic exposure of calreticulin on the tumor cell surface.
The percent of calreticulin positive cells in all anti-CD47 mAb
treated cultures was significantly increased (p<0.05) compared
to an isotype control. This increase in the exposure of
calreticulin on the cell surface demonstrates that some of the
humanized antibodies induce DAMPs from tumor cells that can lead to
phagocytosis of tumor cells and processing of tumor antigen by
innate immune cells.
Humanized Anti-CD47 mAbs Cause Increased Protein
Disulfide-Isomerase 3 (PDIA3) Expression
[0511] These experiments demonstrate that humanized anti-CD47 mAbs
of the present disclosure exhibit the ability to expose the
endoplasmic reticulum resident chaperone PDIA3 on the surface of
the tumor cell as, for example, described previously using
chemotherapeutic anthracyclines such as doxorubicin and
mitoxantrone, as disclosed by Panaretakis et al. (2008) Cell Death
& Differentiation 15:1499-1509.
[0512] Cell surface exposure of PDIA3 was determined using a mouse
monoclonal antibody against PDIA3 conjugated to FITC (Abcam;
Catalogue #ab183396). Human Raji lymphoma cells (ATCC, Manassas,
Va.; Catalog # CCL-86) or other cells types that express sufficient
levels of CD47 will be used. Cells were grown in RPMI-1640 medium
containing 10% (v/v) heat inactivated fetal bovine serum (BioWest;
Catalogue # S01520), 100 units/mL penicillin, 100 .mu.g mL
streptomycin (Sigma; Catalogue # P4222) at densities less than
1.times.10.sup.6 cells/mL. For this assay, cells were plated in 96
well tissue culture plates at a density of 1.times.10.sup.5
cells/ml RPMI-1640 medium containing 10% (v/v) heat inactivated
fetal bovine serum (BioWest; Catalog # S01520), 100 units/mL
penicillin, 100 .mu.g/mL streptomycin (Sigma; #P4222).
[0513] The humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07
IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and
VLX9hum_03 IgG2) as disclosed herein, purified from transient
transfections in CHO cells as described above, as well as the
control chimeric antibody, were added at a final concentration of
10 .mu.g/ml. As a positive control for PDIA3 exposure, cells were
treated with 1 .mu.M of chemotherapeutic anthracycline
mitoxantrone. The Raji cells were incubated at 37.degree. C. for 24
hours, after which the cells were harvested, washed twice with PBS,
and incubated for 30 minutes with anti-PDIA3 antibody as described
above, diluted 1:200 in PBS. After 30 minutes the cells were washed
twice with PBS, resuspended in 100 .mu.l of PBS, and analyzed for
the mean fluorescence intensity of the anti-PDIA3 antibody signal
as well as the percent of cells that stain positive for cell
surface calreticulin by flow cytometry (Accuri C6, Becton
Dickinson, Franklin Lakes, N.J.). Results are presented as
means.+-.SEM and analyzed for statistical significance using ANOVA
in GraphPad Prism 6.
[0514] Some of the chimeric or humanized antibodies induce the
preapoptotic exposure of PDIA3 on the tumor cell surface. As shown
in FIG. 20, the percent of PDIA3 positive cells in all the soluble
anti-CD47 mAb treated cultures was significantly increased
(p<0.05) compared to the background obtained with a negative
control, humanized isotype-matched antibody. This increase in the
exposure of PDIA3 on the cell surface demonstrates that some of the
chimeric or humanized antibodies induce DAMPs from tumor cells that
can lead to phagocytosis of tumor cells and processing of tumor
antigen by innate immune cells.
Humanized Anti-CD47 mAbs Cause Increased Cell Surface HSP70
Expression
[0515] These experiments demonstrate that humanized anti-CD47 mAbs
of the present disclosure exhibit the ability to expose the
endoplasmic reticulum resident chaperone HSP70 on the surface of
the tumor cell as, for example, described previously using
chemotherapeutic anthracyclines such as doxorubicin and
mitoxantrone, as disclosed by Fucikova et al. (2011) Cancer
Research 71(14):4821-4833.
[0516] Cell surface exposure of HSP70 was determined using a mouse
monoclonal antibody against HSP70 conjugated to Phycoerythrin
(Abcam; Catalogue #ab65174). Human Raji lymphoma cells (ATCC,
Manassas, Va.; Catalog # CCL-86) or other cells types that express
sufficient levels of CD47 were used. Cells were grown in RPMI-1640
medium containing 10% (v/v) heat inactivated fetal bovine serum
(BioWest; Catalogue # S01520), 100 units/mL penicillin, 100 .mu.g
mL streptomycin (Sigma; Catalogue # P4222) at densities less than
1.times.10.sup.6 cells/mL. For this assay, cells were plated in 96
well tissue culture plates at a density of 1.times.10.sup.5
cells/ml RPMI-1640 medium containing 10% (v/v) heat inactivated
fetal bovine serum (BioWest; Catalog # S01520), 100 units/mL
penicillin, 100 .mu.g/mL streptomycin (Sigma; #P4222).
[0517] The humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07
IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and
VLX9hum_03 IgG2) as disclosed herein, purified from transient
transfections in CHO cells as described above, as well as the
control chimeric antibody, were added at a final concentration of
10 .mu.g/ml. As a positive control for HSP70 exposure, Raji cells
were treated with 1 .mu.M of chemotherapeutic anthracycline
mitoxantrone. The cells were incubated at 37.degree. C. for 24
hours, after which the cells were harvested, washed twice with PBS,
and incubated for 30 minutes with anti-HSP70 antibody as described
above, diluted 1:200 in PBS. After 30 minutes the cells were washed
twice with PBS, resuspended in 100 .mu.l of PBS, and analyzed for
the mean fluorescence intensity of the anti-HSP70 antibody signal
as well as the percent of cells that stain positive for cell
surface calreticulin by flow cytometry (Accuri C6, Becton
Dickinson, Franklin Lakes, N.J.). Results are presented as
means.+-.SEM and analyzed for statistical significance using ANOVA
in GraphPad Prism 6.
[0518] Some of the chimeric or humanized antibodies induce the
preapoptotic exposure of HSP70 on the tumor cell surface. As shown
in FIG. 21, the percent of HSP70 positive cells in all anti-CD47
mAb treated cultures was significantly increased (p<0.05)
compared to those seen in isotype control treated cultures. This
increase in the exposure of HSP70 on the cell surface demonstrates
that some of the chimeric or humanized antibodies induce DAMPs from
tumor cells and can lead to phagocytosis of tumor cells and
processing of tumor antigen by innate immune cells.
Humanized Anti-CD47 mAbs Cause Increased Cell Surface HSP90
Expression
[0519] These experiments demonstrate that humanized anti-CD47 mAbs
of the present disclosure expose the endoplasmic reticulum resident
chaperone HSP70 on the surface of the tumor cell as, for example,
described previously using chemotherapeutic anthracyclines such as
doxorubicin and mitoxantrone, as disclosed by Fucikova et al.
(2011) Cancer Research 71(14):4821-4833.
[0520] Cell surface exposure of HSP90 was determined using a mouse
monoclonal antibody against HSP70 conjugated to Phycoerythrin
(Abcam; Catalogue #ab65174). Human Raji lymphoma cells (ATCC,
Manassas, Va.; Catalog # CCL-86) or other cells types that express
sufficient levels of CD47 were used. Cells are grown in RPMI-1640
medium containing 10% (v/v) heat inactivated fetal bovine serum
(BioWest; Catalogue # S01520), 100 units/mL penicillin, 100 .mu.g
mL streptomycin (Sigma; Catalogue # P4222) at densities less than
1.times.10.sup.6 cells/mL. For this assay, cells were plated in 96
well tissue culture plates at a density of 1.times.10.sup.5
cells/ml RPMI-1640 medium containing 10% (v/v) heat inactivated
fetal bovine serum (BioWest; Catalog # S01520), 100 units/mL
penicillin, 100 .mu.g/mL streptomycin (Sigma; #P4222).
[0521] The humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07
IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and
VLX9hum_03 IgG2) as disclosed herein, purified from transient
transfections in CHO cells as described above, as well as the
control chimeric antibody, were added at a final concentration of
10 .mu.g/ml. As a positive control for HSP90 exposure, cells were
treated with 1 .mu.M of chemotherapeutic anthracycline
mitoxantrone. The Raji cells were incubated at 37.degree. C. for 24
hours, after which the cells were harvested, washed twice with PBS,
and incubated for 30 minutes with anti-HSP70 antibody as described
above, diluted 1:200 in PBS. After 30 minutes the cells were washed
twice with PBS, resuspended in 100 .mu.l of PBS, and analyzed for
the mean fluorescence intensity of the anti-HSP70 antibody signal
as well as the percent of cells that stain positive for cell
surface calreticulin by flow cytometry (Accuri C6, Becton
Dickinson, Franklin Lakes, N.J.). Results are presented as
means.+-.SEM and analyzed for statistical significance using ANOVA
in GraphPad Prism 6.
[0522] Some of the chimeric or humanized antibodies induce the
preapoptotic exposure of HSP90 on the tumor cell surface. As shown
in FIG. 22, the percent of HSP90 positive cells in soluble
anti-CD47 mAb-treated cultures was significantly increased
(p<0.05) compared to the background obtained with a negative
control, humanized isotype-matched antibody, except for VLXhum_06
IgG2 and VLX4hum_01 IgG4PE (ns, not significant). This increase in
the exposure of HSP90 on the cell surface demonstrates that some of
the chimeric or humanized antibodies induce DAMPs from tumor cells
and can lead to phagocytosis of tumor cells and processing of tumor
antigen by innate immune cells.
Humanized Anti-CD47 mAbs Cause Increased ATP Release
[0523] These experiments demonstrate that humanized anti-CD47 mAbs
of the present disclosure induce increased release of adenosine
triphosphate (ATP) from the tumor cell as described previously
using anthracycline chemotherapy drugs (Martins et al., 2014 Cell
Death and Differentiation 21:79-91).
[0524] Release of ATP from the tumor cell is determined by
quantitative bioluminescence assay as described by the manufacturer
(Molecular Probes; Catalogue #A22066). Human Raji lymphoma cells
(ATCC, Manassas, Va.; Catalog # CCL-86) or other cells types that
express sufficient levels of CD47 were used. Cells were grown in
RPMI-1640 medium containing 10% (v/v) heat inactivated fetal bovine
serum (BioWest; Catalogue # S01520), 100 units/mL penicillin, 100
.mu.g mL streptomycin (Sigma; Catalogue # P4222) at densities less
than 1.times.10.sup.6 cells/mL. For this assay, cells were plated
in 96 well tissue culture plates at a density of 1.times.10.sup.5
cells/ml RPMI-1640 medium containing 10% (v/v) heat inactivated
fetal bovine serum (BioWest; Catalog # S01520), 100 units/mL
penicillin, 100 .mu.g/mL streptomycin (Sigma; #P4222).
[0525] The humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07
IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and
VLX9hum_03) as disclosed herein, purified from transient
transfections in CHO cells as described above, as well as the
control chimeric antibody, were added at a final concentration of
10 .mu.g/ml. As a positive control for ATP release, cells were
treated with 1 .mu.M of chemotherapeutic anthracycline
mitoxantrone. The cells were incubated at 37.degree. C. for 24
hours, after which the cell-free supernatant was collected and
stored at -80.degree. C. After all samples have been collected, 10
.mu.l of each sample was tested by the ATP determination kit as
described above. Final concentrations were determined by comparing
experimental values to a standard curve and displayed as the
concentration of ATP (.mu.M) released by tumor cells in response to
antibody treatment. Results are presented as means.+-.SEM and
analyzed for statistical significance using ANOVA in GraphPad Prism
6.
[0526] The humanized antibodies increased the release of ATP from
the tumor cells. As shown in FIG. 23, the amount of released ATP in
all anti-CD47 mAb treated cultures was significantly increased
(p<0.05) compared to an isotype control. This increase in the
release of ATP demonstrates that some of the chimeric or humanized
antibodies induce the release of ATP from tumor cells and can lead
to dendritic cell migration through its cognate purinergic
receptors.
Humanized Anti-CD47 mAbs Cause HMGB1 Release
[0527] These experiments demonstrate that humanized anti-CD47 mAbs
of the present disclosure increase the release of the non-histone
chromatin protein high-mobility group box 1 (HMGB1) from the tumor
cell as described previously using chemotherapy agents, such as
oxaliplatin (Tesniere et al., 2010 Oncogene, 29:482-491) and
mitoxantrone (Michaud et al., 2011 Science 334:1573-1577).
[0528] Release of HMGB1 protein from the tumor cell was determined
by enzyme immunoassay as described by the manufacturer (IBL
International; Hamburg, Germany, Catalogue #ST51011). Human Raji
lymphoma cells (ATCC, Manassas, Va.; Catalog # CCL-86) or other
cells types that express sufficient levels of CD47 were used. Cells
will be grown in RPMI-1640 medium containing 10% (v/v) heat
inactivated fetal bovine serum (BioWest; Catalogue # S01520), 100
units/mL penicillin, 100 .mu.g mL streptomycin (Sigma; Catalogue
#P4222) at densities less than 1.times.10.sup.6 cells/mL. For this
assay, cells were plated in 96 well tissue culture plates at a
density of 1.times.10.sup.5 cells/ml RPMI-1640 medium containing
10% (v/v) heat inactivated fetal bovine serum (BioWest; Catalog #
S01520), 100 units/mL penicillin, 100 .mu.g/mL streptomycin (Sigma;
#P4222).
[0529] The humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07
IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and
VLX9hum_03 IgG2) as disclosed herein, purified from transient
transfections in CHO cells as described above, as well as the
control chimeric antibody, will then be added at a final
concentration of 10 .mu.g/ml.
[0530] As a positive control for HMGB1 release, Raji cells were
treated with 1 .mu.M of chemotherapeutic anthracycline
mitoxantrone. The cells were incubated at 37.degree. C. for 24
hours, after which the cell-free supernatant was collected and
stored at -80.degree. C. After all samples have been collected, 10
.mu.l of each sample was tested by HMGB1 ELISA as described above.
Final concentrations were determined by comparing experimental
values to a standard curve and reported as the concentration of
HMGB1 (ng/ml) released by tumor cells in response to antibody
treatment. Results are presented as means.+-.SEM and analyzed for
statistical significance using ANOVA in GraphPad Prism 6.
[0531] As shown in FIG. 24, the humanized antibodies increased the
release of HMGB1 protein from the tumor cells. The amount of
released HMGB1 protein in all anti-CD47 mAb treated cultures was
significantly increased (p<0.05) compared to an isotype control,
except for VLX9hum_06 IgG2 (ns, not significant). This increase in
the release of HMGB1 demonstrates that some of the chimeric or
humanized antibodies induce release of DAMPs from tumor cells and
can lead to dendritic cell activation.
Humanized Anti-CD47 mAbs Cause CXCL10 Release
[0532] These experiments demonstrate that humanized anti-CD47 mAbs
of the present disclosure increase the production and release of
the chemokine CXCL10 from the human tumor cells as described
previously using anthracycline chemotherapy drugs (Sistigu et al.,
2014 Nat. Med. 20(11):1301-1309).
[0533] Release of the CXCL10 from the tumor cell was determined by
enzyme immunoassay as described by the manufacturer (R&D
Systems; Catalogue #DIP100). Human Raji lymphoma cells (ATCC,
Manassas, Va.; Catalog # CCL-86) or other cells types that express
sufficient levels of CD47 will be used. Cells were grown in
RPMI-1640 medium containing 5% (v/v) heat inactivated fetal bovine
serum (BioWest; Catalogue # S01520), 100 units/mL penicillin, 100
.mu.g mL streptomycin (Sigma; Catalogue # P4222) at densities less
than 1.times.10.sup.6 cells/mL. For this assay, cells were plated
in 96 well tissue culture plates at a density of 1.times.10.sup.5
cells/ml RPMI-1640 medium containing 5% (v/v) heat inactivated
fetal bovine serum (BioWest; Catalog # S01520), 100 units/mL
penicillin, 100 .mu.g/mL streptomycin (Sigma; #P4222).
[0534] The humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07
IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and
VLX9hum_03 IgG2) as disclosed herein, purified from transient
transfections in CHO cells as described above, as well as the
control chimeric antibody, were added at a final concentration of
10 .mu.g/ml. As a positive control for CXCL10 release, Raji cells
were treated with 1 .mu.M of the chemotherapeutic anthracycline
mitoxantrone. The cells were incubated at 37.degree. C. for 24
hours, after which the cell-free supernatant was collected and
stored at -80.degree. C. After all samples have been collected, 10
.mu.l of each sample was tested by the CXCL10 ELISA as described
above. Final concentrations were determined by comparing
experimental values to a standard curve and displayed as the
concentration of CXCL10 (pg/ml) released by tumor cells in response
to antibody treatment.
[0535] Some of the chimeric or humanized antibodies induce release
of CXCL10 by human tumor cells. As shown in FIG. 25, the amount of
released CXCL10 in all anti-CD47 mAb treated cultures significantly
increased (p<0.05) compared to an isotype control. This increase
in the release of CXCL10 demonstrates that some of the chimeric or
humanized antibodies induce the release of CXCL10 from tumor cells
and suggest a role in the recruitment of immune cells to the
tumor.
Example 11
Damage-Associated Molecular Pattern (DAMP) Expression and Release,
Mitochondrial Depolarization and Cell Death Caused by Humanized
Anti-CD47 mAbs
[0536] These studies were conducted as described in Example 10,
except that the human Jurkat T ALL cell line (ATCC, Manassas, Va.;
Catalog # TIB-152) was used.
Humanized Anti-CD47 mAbs Cause Loss of Mitochondrial Membrane
Potential
[0537] As shown in FIG. 26, the humanized mAbs (VLX4hum_01 IgG4PE,
VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08
IgG2 and VLX9hum_03 IgG2) caused a significant increase in the
percent of cells with mitochondrial membrane depolarization
(p<0.05) compared to an isotype control. This increase in the
amount of mitochondrial membrane depolarization demonstrates that
some of the chimeric or humanized antibodies induce cell death in
human tumor cells.
Humanized Anti-CD47 mAbs Cause Increase in Cell Surface
Calreticulin Expression
[0538] As shown in FIG. 27, the humanized antibodies (VLX4hum_01
IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06 IgG2,
VLX9hum_08 IgG2 and VLX9hum_03 IgG2) induced the preapoptotic
exposure of calreticulin on the tumor cell surface. The percent of
calreticulin positive cells in all anti-CD47 mAb treated cultures
were significantly increased (p<0.05) compared to an isotype
control, except VLX9hum_03 IgG2 (ns). This increase in the exposure
of calreticulin on the cell surface demonstrated that some of the
humanized antibodies induce DAMPs from tumor cells and can lead to
phagocytosis of tumor cells and processing of tumor antigen by
innate immune cells.
Humanized Anti-CD47 mAbs Cause Increase in Cell Surface PDIA3
Expression
[0539] As shown in FIG. 28, the percent of PDIA3 positive cells in
soluble anti-CD47 mAb (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE,
VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03
IgG2) treated cultures were significantly increased (p<0.05)
compared to the background obtained with a negative control,
humanized isotype-matched antibody. This increase in the exposure
of PDIA3 on the cell surface demonstrates that some of the chimeric
or humanized antibodies induce DAMPs from tumor cells and can lead
to phagocytosis of tumor cells and processing of tumor antigen by
innate immune cells.
Humanized Anti-CD47 mAbs Cause Increase in Cell Surface HSP70
Expression
[0540] As shown in FIG. 29, the percent of HSP70 positive cells in
anti-CD47 mAb (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11
IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2)
treated cultures were significantly increased (p<0.05) compared
to those seen in isotype control treated cultures. Although each of
the anti-CD47 mAbs caused a statistically significant increase in
HSP70 expression, mitoxantrone did not. This increase in the
exposure of HSP70 on the cell surface demonstrates that some of the
chimeric or humanized antibodies induce DAMPs from tumor cells and
can lead to phagocytosis of tumor cells and processing of tumor
antigen by innate immune cells.
Humanized Anti-CD47 mAbs Cause Increase in Cell Surface HSP90
Expression
[0541] As shown in FIG. 30, the percent of HSP90 positive cells in
soluble anti-CD47 mAb (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE,
VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03
IgG2) treated cultures were significantly increased (p<0.05)
compared to the background obtained with a negative control,
humanized isotype-matched antibody. This increase in the exposure
of HSP90 on the cell surface demonstrates that some of the chimeric
or humanized antibodies induce DAMPs from tumor cells and can lead
to phagocytosis of tumor cells and processing of tumor antigen by
innate immune cells.
Humanized Anti-CD47 mAbs Cause Increase in ATP Release
[0542] As shown in FIG. 31, the amount of released ATP in humanized
anti-CD47 mAb (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11
IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2)
treated cultures was significantly increased (p<0.05) compared
to an isotype control. Although each of the anti-CD47 mAbs caused a
statistically significant increase in HSP70 expression,
mitoxantrone did not (ns). This increase in the release of ATP will
demonstrates that some of the chimeric or humanized antibodies
induce the release of ATP from tumor cells and can lead to
dendritic cell migration through its cognate purinergic
receptors.
Humanized Anti-CD47 mAbs Cause Increase in HMGB1 Release
[0543] As shown in FIG. 32, the amount of released HMGB1 protein in
anti-CD47 mAb (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11
IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2)
treated cultures was significantly increased (p<0.05) compared
to an isotype control, except for VLX4hum_01 IgG4PE (ns). This
increase in the release of HMGB1 demonstrates that some of the
chimeric or humanized antibodies induce DAMPs from tumor cells and
can lead to dendritic cell activation.
Example 12
Hemagglutination of Human Red Blood Cells (hRBCs)
[0544] Many CD47 antibodies, including B6H12, BRIC126, MABL1,
MABL2, CC2C6, 5F9, have been shown to cause hemagglutination (HA)
of washed RBCs in vitro or in vivo (Petrova P. et al. Cancer Res
2015; 75(15 Suppl): Abstract nr 4271; U.S. Pat. No. 9,045,541; Uno
et al. Oncol Rep. 17: 1189-94, 2007; Kikuchi et al. Biochem Biophys
Res. Commun. 315: 912-8, 2004; Sikic B. et al. J Clin Oncol 2016;
34 (suppl; abstract 3019)). Hemagglutination of hRBCs was assessed
following incubation of hRBCs with various concentrations of
chimeric and humanized VLX4, VLX8, and VLX9 mAbs in vitro
essentially as described by Kikuchi et al. Biochem Biophys Res.
Commun (2004) 315:912-918. Blood was obtained from healthy donors,
diluted (1:50) in PBS/1 mM EDTA/BSA and washed 3 times with
PBS/EDTA/BSA. hRBCs were added to U-bottomed 96 well plates with
equal volumes of the antibodies (75 .mu.l of each) and incubated
for 3 hrs at 37.degree. C. and overnight at 4.degree. C. A tight
RBC pellet is observed with antibodies that do not cause
hemagglutination, and a diffuse, hazy pattern is observed with
antibodies that cause hemagglutination.
[0545] As shown in FIG. 33A and Tables 1 and 2, The VLX4hum_01 IgG1
caused visible hemagglutination of hRBCs, whereas the humanized
VLX4hum_01 IgG4PE mAb did not (mAb concentrations 50 .mu.g/ml to
0.3 ng/ml). The lack of detectable hemagglutination by VLX4hum_01
IgG4 PE imparts an additional desirable antibody property and
potential therapeutic benefit in the treatment of cancer.
[0546] As shown in FIG. 33B and Tables 1 and 2, the chimeric
antibody VLX8 IgG4PE (xi) and the humanized antibodies VLX8hum_08
IgG4PE, VLX8hum_09 IgG4PE, and VLX8hum_10 IgG4PE caused visible
hemagglutination of hRBCs, whereas the VLX8 humanized Abs
VLX8hum_01 IgG4PE, VLX8hum_02 IgG4 PE, VLX8hum_03 IgG4 PE and
VLX8hum_11 IgG4PE did not (mAb concentrations 50 .mu.g/ml to 0.3
ng/ml).
[0547] The lack of detectable hemagglutination by humanized
antibodies VLX4hum_01 IgG4PE, VLX8hum_01 IgG4PE, VLX8hum_02 IgG4
PE, VLX8hum_03 IgG4 PE and VLX8hum_11 IgG4 PE imparts an additional
desirable antibody property and a potential therapeutic benefit in
the treatment of cancer.
[0548] As shown in FIG. 34A and FIG. 34B, the chimeric antibody
VLX9 IgG2 xi caused visible hemagglutination of hRBCs, whereas all
of the humanized VLX9 mAbs except for VLX9hum_07 IgG2, did not
cause detectable hemagglutination (at concentrations from 50 ug/ml
to 0.3 .mu.g/ml). However, the amount of detectable
hemagglutination caused by VLX9hum_07 was reduced compared to the
VLX9 IgG2 chimeric mAb. Again, the reduced or lack of detectable
hemagglutination by the VLX9 humanized mAbs imparts an additional
desirable antibody property and a potential therapeutic benefit in
the treatment of cancer.
Example 13
Anti-Tumor Activity In Vivo
[0549] The purpose of this experiment was to demonstrate that VLX4,
VLX8 and VLX9 humanized antibodies, exemplified by VLX4_07 IgG4PE,
VLX8_10 IgG4PE and VLX9hum_08 IgG2, reduce tumor burden in vivo in
a mouse xenograft model of lymphoma.
[0550] Raji human Burkitt's lymphoma cells (ATCC #CCL-86, Manassas,
Va.) were maintained in RPMI-1640 (Lonza; Walkersville, Md.)
supplemented with 10% Fetal Bovine Serum (FBS; Omega Scientific;
Tarzana, Calif.) within a 5% CO.sub.2 atmosphere. Cultures were
expanded in tissue culture flasks.
[0551] Female NSG (NOD-Cg-Prkdc.sup.scidI12rg.sup.tm1Wjl/SzJ) were
obtained from Jackson Laboratory (Bar Harbor, Me.) at 5-6 weeks of
age. Mice were acclimated prior to handling and housed in
microisolator cages (Lab Products, Seaford, Del.) under specific
pathogen-free conditions. Mice were fed Teklad Global Diet.RTM.
2920x irradiated laboratory animal diet (Envigo, Formerly Harlan;
Indianapolis, Ind.) and provided autoclaved water ad libitum. All
procedures were carried out under Institutional Animal Care and Use
guidelines.
[0552] Female NSG mice were inoculated subcutaneously in the right
flank with 0.1 mL of a 30% RPMI/70% Matrigel.TM. (BD Biosciences;
Bedford, Mass.) mixture containing a suspension of 5.times.10.sup.6
Raji tumor cells. Five days following inoculation, digital calipers
were used to measure width and length diameters of the tumor. Tumor
volumes were calculated utilizing the formula: tumor volume
(mm.sup.3)=(a.times.b.sup.2/2) where `b` is the smallest diameter
and `a` is the largest diameter. Mice with palpable tumor volumes
of 31-74 mm.sup.3 were randomized into 8-10/group and VLX9hum_08 or
PBS (control) administration was initiated at this time. Mice were
treated with 5 mg/kg of antibody 5.times./week for 4 weeks by
intraperitoneal injection. Tumor volumes and body weights were
recorded twice weekly.
[0553] As shown in FIG. 35, treatment with the humanized VLX4hum_07
IgG4PE significantly reduced tumor growth of the Raji tumors
(p<0.05, two-way ANOVA), demonstrating anti-tumor efficacy in
vivo.
[0554] As shown in FIG. 36, treatment with the humanized anti-CD47
mAb, VLX8hum_10 IgG4PE significantly reduced (p<0.0001, two-way
ANOVA) tumor growth of the Raji tumors, demonstrating anti-tumor
efficacy in vivo.
[0555] As shown in FIG. 37, treatment with the humanized anti-CD47
mAb, VLX9hum_08 IgG2 significantly reduced (p<0.05, two-way
ANOVA) tumor growth of the Raji tumors, demonstrating anti-tumor
efficacy in vivo.
Example 14
Effect on Circulating Red Blood Cell Parameters
[0556] The purpose of this experiment is to demonstrate that VLX9
humanized antibodies that do not bind to human RBC in vitro (Table
2), exemplified by hum1017_08 IgG2, do not cause a reduction in
either hemoglobin (Hg) or circulating RBCs following administration
to cynomolgus monkeys.
[0557] Female Chinese cynomolgus monkeys (Charles River
Laboratories, Houston, Tex.) 2.5-3 kg were used in accordance with
the Institutional Animal Care and Use guidelines. VLX9hum_08 IgG2
or vehicle (PBS) was administered as a 1 hour intravenous infusion
on day 1 at a dose of 5 mg/kg and on day 18 at a dose of 15 mg/kg
(3 animals/group). Hematological parameters were measured
throughout the study on days -7, -3 (not shown), pre-dose, 3, 8,
12, 18 (pre-dose), 20, 25, 29, 35 and 41 and compared/normalized to
the means values of control animals. The pre-treatment RBC and Hg
values on day 0 in the VLX9hum_08 IgG2 group were lower than the
control group. Following treatment with either dose of VLX9hum_08
IgG2, there were minimal changes (<10%) in Hg (FIG. 38A) or RBC
counts (FIG. 38B) compared to the control group demonstrating that
VLX9hum_08 IgG2 causes minimal reductions in RBC hematological
parameters when administered to cynomolgus monkeys.
Example 15
Antibodies to CD47 Regulate Nitric Oxide Signaling
[0558] TSP1 binding to CD47 activates the heterotrimeric G protein
Gi, which leads to suppression of intracellular cyclic AMP (cAMP)
levels. In addition, the TSP1/CD47 pathway opposes the beneficial
effects of the nitric oxide (NO) pathway in all vascular cells. The
NO pathway consists of any of three nitric oxide synthase enzymes
(NOS I, NOS II and NOS III) that generate bioactive gas NO using
arginine as a substrate. NO can act within the cell in which it is
produced or in neighboring cells, to activate the enzyme soluble
guanylyl cyclase that produces the messenger molecule cyclic GMP
(cGMP). The proper functioning of the NO/cGMP pathway is essential
for protecting the cardiovascular system against stresses
including, but not limited to, those resulting from wounding,
inflammation, hypertension, metabolic syndrome, ischemia, and
ischemia-reperfusion injury (IRI). In the context of these cellular
stresses, the inhibition of the NO/cGMP pathway by the TSP1/CD47
system exacerbates the effects of stress. This is a particular
problem in the cardiovascular system where both cGMP and cAMP play
important protective roles. There are many cases in which ischemia
and reperfusion injury cause or contribute to disease, trauma, and
poor outcomes of surgical procedures.
[0559] The purpose of these experiment will be to demonstrate that
humanized anti-CD47 mAbs of the present disclosure exhibit the
ability to reverse TSP1-mediated inhibition of NO-stimulated cGMP
synthesis as, for example, described previously using mouse
monoclonal antibodies to CD47 as disclosed by Isenberg et al.
(2006) J. Biol. Chem. 281:26069-80, or alternatively other
downstream markers of or effects resulting from NO signaling, for
example smooth muscle cell relaxation or platelet aggregation as
described previously by Miller et al. (2010) Br J. Pharmacol. 159:
1542-1547.
[0560] The method employed that will be to measure cGMP as
described by the manufacturer (CatchPoint Cyclic-GMP Fluorescent
Assay Kit, Molecular Devices, Sunnyvale, Calif.). Jurkat JE6.1
cells (ATCC, Manassas, Va.; Catalog # TIB-152) or other cells types
that retain the NO/cGMP signaling pathway when grown in culture and
exhibit a robust and reproducible inhibitory response to TSP1
ligation of CD47 will be used. Cells will be grown in Iscove's
modified Dulbeccco's medium containing 5% (v/v) heat inactivated
fetal bovine serum (BioWest; Catalogue # S01520), 100 units/mL
penicillin, 100 ag mL streptomycin (Sigma; Catalogue # P4222) at
densities less than 1.times.106 cells/mL. For the cGMP assay, cells
will be plated in 96 well tissue culture plates at a density of
1.times.10.sup.5 cells/ml in Iscoves modified Dulbecco's medium
containing 5% (v/v) heat inactivated fetal bovine serum (BioWest;
Catalog # S01520), 100 units/mL penicillin, 100 .mu.g/mL
streptomycin (Sigma; #P4222) for 24 hours and then transferred to
serum free medium overnight.
[0561] The humanized antibodies as disclosed herein, purified from
transient transfections in CHO cells as described above in Example
3, as well as the control chimeric antibody, will then be added at
a final concentration of 20 ng/ml, followed 15 minutes later by 0
or 1 .mu.g/ml human TSP1 (Athens Research and Technology, Athens,
Ga., Catalogue #16-20-201319). After an additional 15 minutes, the
NO donor, diethylamine (DEA) NONOate (Cayman Chemical, Ann Arbor,
Mich., Catalog #82100), will be added to half the wells at a final
concentration of 1 .mu.M. Five minutes later, the cells will be
lysed with buffer supplied in the cGMP kit, and aliquots of each
well assayed for cGMP content.
[0562] It is anticipated that some of the chimeric or humanized
antibodies will reverse TSP1 inhibition of cGMP. Reversal will be
complete (>80%) or intermediate (20%-80%). This reversal of TSP1
inhibition of cGMP will demonstrate that they have the ability to
increase NO signaling and suggest utility in protecting the
cardiovascular system against stresses including, but not limited
to, those resulting from wounding, inflammation, hypertension,
metabolic syndrome, ischemia, and ischemia-reperfusion injury
(IRI). Additional assay systems, for example smooth muscle cell
contraction, will also be expected to show that some of the
chimeric or humanized antibody clones reverse the inhibitory
actions of TSP on downstream effects resulting from the activation
of NO signaling.
Sequence CWU 1
1
101110PRTHomo sapiens 1Gly Tyr Thr Phe Thr Asn Tyr Val Ile His 1 5
10 210PRTHomo sapiens 2Gly Tyr Ser Phe Thr Asn Tyr Tyr Ile His 1 5
10 310PRTHomo sapiens 3Gly Tyr Thr Phe Thr Asn Tyr Trp Ile His 1 5
10 417PRTHomo sapiens 4Tyr Ile Tyr Pro Tyr Asn Asp Gly Ile Leu Tyr
Asn Glu Lys Phe Lys 1 5 10 15 Gly 517PRTHomo sapiens 5Tyr Ile Asp
Pro Leu Asn Gly Asp Thr Thr Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly
617PRTHomo sapiens 6Tyr Thr Asp Pro Arg Thr Asp Tyr Thr Glu Tyr Asn
Gln Lys Phe Lys 1 5 10 15 Asp 78PRTHomo sapiens 7Gly Gly Tyr Tyr
Val Pro Asp Tyr 1 5 88PRTHomo sapiens 8Gly Gly Tyr Tyr Val Tyr Asp
Tyr 1 5 98PRTHomo sapiens 9Gly Gly Lys Arg Ala Met Asp Tyr 1 5
108PRTHomo sapiens 10Gly Gly Arg Val Gly Leu Gly Tyr 1 5
1116PRTHomo sapiens 11Arg Ser Arg Gln Ser Ile Val His Thr Asn Gly
Asn Thr Tyr Leu Gly 1 5 10 15 1211PRTHomo sapiens 12Arg Ala Ser Gln
Asp Ile Ser Asn Tyr Leu Asn 1 5 10 1311PRTHomo sapiens 13Arg Ala
Ser Gln Ser Ile Ser Asn Tyr Leu Asn 1 5 10 1416PRTHomo sapiens
14Arg Ser Ser Gln Asn Ile Val Gln Ser Asn Gly Asn Thr Tyr Leu Glu 1
5 10 15 157PRTHomo sapiens 15Lys Val Ser Asn Arg Phe Ser 1 5
167PRTHomo sapiens 16Tyr Thr Ser Arg Leu Tyr Ser 1 5 177PRTHomo
sapiens 17Lys Val Phe His Arg Phe Ser 1 5 189PRTHomo sapiens 18Phe
Gln Gly Ser His Val Pro Tyr Thr 1 5 199PRTHomo sapiens 19Gln Gln
Gly Asn Thr Leu Pro Trp Thr 1 5 209PRTHomo sapiens 20Phe Gln Gly
Ser His Val Pro Trp Thr 1 5 21117PRTHomo sapiens 21Glu Val Gln Leu
Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val
Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30
Val Ile His Trp Val Lys Arg Arg Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Ile Leu Tyr Asn Glu Lys
Phe 50 55 60 Lys Gly Lys Ala Thr Val Thr Ser Asp Lys Ser Ser Ser
Thr Ala Tyr 65 70 75 80 Met Asp Leu Ser Ser Leu Thr Ser Glu Asp Ser
Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Gly Tyr Tyr Val Pro Asp
Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115
22117PRTHomo sapiens 22Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Val Ile His Trp Val Lys Arg
Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Pro
Tyr Asn Asp Gly Ile Leu Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys
Ala Thr Val Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met
Asp Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90
95 Thr Arg Gly Gly Tyr Tyr Val Pro Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110 Val Thr Val Ser Ser 115 23117PRTHomo sapiens 23Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15
Ser Val Gln Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20
25 30 Val Ile His Trp Leu Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45 Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Ile Leu Tyr Asn
Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Ser Asp Thr Ser
Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Asp
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Tyr Val
Pro Asp Tyr Trp Gly Gln Ala Thr Leu 100 105 110 Val Thr Val Ser Ser
115 24117PRTHomo sapiens 24Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Gln Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Val Ile His Trp Leu Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Tyr
Pro Tyr Asn Asp Gly Ile Leu Tyr Asn Glu Lys Phe 50 55 60 Lys Gly
Arg Val Thr Met Thr Ser Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Gly Gly Tyr Tyr Val Tyr Asp Tyr Trp Gly Gln Ala Thr
Leu 100 105 110 Val Thr Val Ser Ser 115 25117PRTHomo sapiens 25Glu
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10
15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30 Val Ile His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu
Trp Met 35 40 45 Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Ile Leu Tyr
Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Thr
Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Gly Gly Tyr Tyr
Val Pro Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser
Ser 115 26117PRTHomo sapiens 26Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys
Gly Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Val Ile His Trp Val
Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile
Tyr Pro Tyr Asn Asp Gly Ile Leu Tyr Asn Glu Lys Phe 50 55 60 Lys
Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70
75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr
Cys 85 90 95 Ala Arg Gly Gly Tyr Tyr Val Pro Asp Tyr Trp Gly Gln
Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 27117PRTHomo
sapiens 27Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Asn Tyr 20 25 30 Val Ile His Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Tyr Pro Tyr Asn Asp
Gly Ile Leu Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met
Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg
Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Gly Gly Tyr Tyr Val Pro Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110
Val Thr Val Ser Ser 115 28117PRTHomo sapiens 28Glu Val Gln Leu Gln
Gln Ser Gly Pro Glu Leu Met Lys Pro Gly Ala 1 5 10 15 Ser Val Lys
Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30 Tyr
Ile His Trp Val Asn Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40
45 Gly Tyr Ile Asp Pro Leu Asn Gly Asp Thr Thr Tyr Asn Gln Lys Phe
50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr
Ala Tyr 65 70 75 80 Met Arg Leu Ser Ser Leu Thr Ser Ala Asp Ser Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Lys Arg Ala Met Asp Tyr
Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115
29117PRTHomo sapiens 29Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Ser Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asp Pro
Leu Asn Gly Asp Thr Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg
Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Gly Lys Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110 Val Thr Val Ser Ser 115 30117PRTHomo sapiens 30Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr 20
25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45 Gly Tyr Ile Asp Pro Leu Asn Gly Asp Thr Thr Tyr Asn
Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser
Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Lys Arg Ala
Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser
115 31117PRTHomo sapiens 31Glu Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly
Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Arg
Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asp
Pro Leu Asn Gly Asp Thr Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Gly
Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80
Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85
90 95 Ala Arg Gly Gly Lys Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr
Leu 100 105 110 Val Thr Val Ser Ser 115 32117PRTHomo sapiens 32Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10
15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr
20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45 Gly Tyr Ile Asp Pro Leu Asn Gly Asp Thr Thr Tyr
Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu
Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Lys Arg
Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser
Ser 115 33117PRTHomo sapiens 33Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys
Gly Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val
Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile
Asp Pro Leu Asn Gly Asp Thr Thr Tyr Ser Pro Ser Phe 50 55 60 Gln
Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70
75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr
Cys 85 90 95 Ala Arg Gly Gly Lys Arg Ala Met Asp Tyr Trp Gly Arg
Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 34117PRTHomo
sapiens 34Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15 Ser Val Gln Val Ser Cys Lys Ala Ser Gly Tyr Ser
Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Leu Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asp Pro Leu Asn Gly
Asp Thr Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Met
Thr Ser Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser
Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Gly Gly Lys Arg Ala Met Asp Tyr Trp Gly Gln Ala Thr Leu 100 105 110
Val Thr Val Ser Ser 115 35117PRTHomo sapiens 35Gln Val Gln Leu Gln
Gln Phe Gly Ala Glu Leu Ala Lys Pro Gly Ala 1 5 10 15 Ser Val Gln
Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp
Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40
45 Gly Tyr Thr Asp Pro Arg Thr Asp Tyr Thr Glu Tyr Asn Gln Lys Phe
50 55 60 Lys Asp Lys Ala Thr Leu Ala Ala Asp Arg Ser Ser Ser Thr
Ala Tyr 65 70 75 80 Met Arg Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala
Val Tyr Tyr Cys 85 90 95 Ala Gly Gly Gly Arg Val Gly Leu Gly Tyr
Trp Gly His Gly Ser Ser 100 105 110 Val Thr Val Ser Ser 115
36117PRTHomo sapiens 36Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Ile His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Thr Asp Pro
Arg Thr Asp Tyr Thr Glu Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg
Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Gly Arg Val Gly Leu Gly Tyr Trp Gly Gln Gly Thr Leu
100 105 110 Val Thr Val Ser Ser 115 37117PRTHomo sapiens 37Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20
25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45 Gly Tyr Thr Asp Pro Arg Thr Asp Tyr Thr Glu Tyr Asn
Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Thr Ala Asp Glu Ser
Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Arg Val Gly
Leu Gly Tyr Trp Gly Gln Gly Thr Leu 100
105 110 Val Thr Val Ser Ser 115 38117PRTHomo sapiens 38Glu Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser
Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25
30 Trp Ile His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
35 40 45 Gly Tyr Thr Asp Pro Arg Thr Asp Tyr Thr Glu Tyr Asn Gln
Lys Phe 50 55 60 Lys Asp Gln Val Thr Ile Ser Ala Asp Lys Ser Ile
Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp
Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Arg Val Gly Leu
Gly Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
39117PRTHomo sapiens 39Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Ile His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Thr Asp Pro
Arg Thr Asp Tyr Thr Glu Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg
Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Gly Arg Val Gly Leu Gly Tyr Trp Gly Gln Gly Thr Leu
100 105 110 Val Thr Val Ser Ser 115 40117PRTHomo sapiens 40Glu Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15
Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asn Tyr 20
25 30 Trp Ile His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp
Met 35 40 45 Gly Tyr Thr Asp Pro Arg Thr Asp Tyr Thr Glu Tyr Ser
Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser
Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser
Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Arg Val Gly
Leu Gly Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser
115 41112PRTHomo sapiens 41Asp Val Leu Met Thr Gln Thr Pro Leu Ser
Leu Pro Val Asn Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg
Ser Arg Gln Ser Ile Val His Thr 20 25 30 Asn Gly Asn Thr Tyr Leu
Gly Trp Phe Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu
Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85
90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys 100 105 110 42112PRTHomo sapiens 42Asp Val Leu Met Thr Gln Thr
Pro Leu Ser Leu Pro Val Asn Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile
Ser Cys Arg Ser Arg Gln Ser Ile Val His Thr 20 25 30 Asn Gly Asn
Thr Tyr Leu Gly Trp Phe Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro
Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55
60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe
Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys 100 105 110 43112PRTHomo sapiens 43Asp Ile Val Met
Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro
Ala Ser Ile Ser Cys Arg Ser Arg Gln Ser Ile Val His Thr 20 25 30
Asn Gly Asn Thr Tyr Leu Gly Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Asp Asp Val Gly Ile Tyr
Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys 100 105 110 44112PRTHomo sapiens 44Asp
Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10
15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Arg Gln Ser Ile Val His Thr
20 25 30 Asn Gly Asn Thr Tyr Leu Gly Trp Phe Gln Gln Arg Pro Gly
Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Phe
Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val
Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 45112PRTHomo
sapiens 45Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser
Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ser Arg Gln Ser
Ile Val His Thr 20 25 30 Asn Gly Asn Thr Tyr Leu Gly Trp Tyr Gln
Gln Lys Pro Gly Gln Pro 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val
Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Gln
Ala Glu Asp Val Ala Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His
Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110
46107PRTHomo sapiens 46Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu
Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala
Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys
Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg
Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu
Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90
95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 47107PRTHomo
sapiens 47Asp 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 Asp
Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Tyr Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala
Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 48107PRTHomo sapiens
48Asp 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 Ser Ile Ser Asn
Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Tyr 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 Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105 49107PRTHomo sapiens 49Asp Ile Val
Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu
Pro Ala Ser Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25
30 Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile
35 40 45 Tyr Tyr Thr Ser Arg Leu Tyr Ser Gly Val Pro Asp Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser
Arg Val Glu Ala 65 70 75 80 Asp Asp Val Gly Ile Tyr Tyr Cys Gln Gln
Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys 100 105 50112PRTHomo sapiens 50Asp Val Phe Met Thr Gln
Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser
Ile Ser Cys Arg Ser Ser Gln Asn Ile Val Gln Ser 20 25 30 Asn Gly
Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45
Pro Lys Leu Leu Ile Tyr Lys Val Phe His Arg Phe Ser Gly Val Pro 50
55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys
Phe Gln Gly 85 90 95 Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys 100 105 110 51112PRTHomo sapiens 51Asp Val Val
Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln
Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val Gln Ser 20 25
30 Asn Gly Asn Thr Tyr Leu Glu Trp Phe Gln Gln Arg Pro Gly Gln Ser
35 40 45 Pro Arg Arg Leu Ile Tyr Lys Val Phe His Arg Phe Ser Gly
Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 52112PRTHomo sapiens
52Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1
5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ser Ser Gln Asn Ile Val Gln
Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Lys Pro
Gly Gln Pro 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Phe His Arg
Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Gln Ala Glu Asp
Val Ala Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110
53330PRTHomo sapiens 53Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90
95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215
220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys 325 330 54330PRTHomo sapiens 54 Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10
15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145
150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu 165 170 175 Glu Gln Tyr Gln Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230
235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 325 330 55326PRTHomo sapiens 55 Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser
Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe
Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val
Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser
His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155
160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln
Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Gly Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr
Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr
Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280
285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys 325 56326PRTHomo
sapiens 56Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys
Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr
Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn
Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val
Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110
Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115
120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp 130 135 140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val
Leu Thr Val Val His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 Ala Pro Ile Glu
Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235
240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
245 250 255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly
Lys 325 57377PRTHomo sapiens 57Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70
75 80 Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys 85 90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His
Thr Cys Pro 100 105 110 Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro
Pro Pro Cys Pro Arg 115 120 125 Cys Pro Glu Pro Lys Ser Cys Asp Thr
Pro Pro Pro Cys Pro Arg Cys 130 135 140 Pro Glu Pro Lys Ser Cys Asp
Thr Pro Pro Pro Cys Pro Arg Cys Pro 145 150 155 160 Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175 Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180 185 190
Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr 195
200 205 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu 210 215 220 Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr
Val Leu His 225 230 235 240 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys 245 250 255 Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Thr Lys Gly Gln 260 265 270 Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 275 280 285 Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300 Ser Asp
Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 305 310 315
320 Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu
325 330 335 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Ile 340 345 350 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
Arg Phe Thr Gln 355 360 365 Lys Ser Leu Ser Leu Ser Pro Gly Lys 370
375 58326PRTHomo sapiens 58Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85
90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala
Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val
Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210
215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr
Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys
Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu
Ser Leu Ser Leu Gly 325 59326PRTHomo sapiens 59Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser
Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40
45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys
Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln
Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170
175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295
300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
305 310 315 320 Leu Ser Leu Ser Leu Gly 325 60327PRTHomo sapiens
60Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1
5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Glu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135
140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260
265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325
61107PRTHomo sapiens 61Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90
95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105
62329PRTRattus norvegicus 62Ala Arg Thr Thr Ala Pro Ser Val Tyr Pro
Leu Val Pro Gly Cys Ser 1 5 10 15 Gly Thr Ser Gly Ser Leu Val Thr
Leu Gly Cys Leu Val Lys Gly Tyr 20 25 30 Phe Pro Glu Pro Val Thr
Val Lys Trp Asn Ser Gly Ala Leu Ser Ser 35 40 45 Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Gly Leu Tyr Thr Leu 50 55 60 Ser Ser
Ser Val Thr Val Pro Ser Ser Thr Trp Ser Ser
Gln Thr Val 65 70 75 80 Thr Cys Ser Val Ala His Pro Ala Thr Lys Ser
Asn Leu Ile Lys Arg 85 90 95 Ile Glu Pro Arg Arg Pro Lys Pro Arg
Pro Pro Thr Asp Ile Cys Ser 100 105 110 Cys Asp Asp Asn Leu Gly Arg
Pro Ser Val Phe Ile Phe Pro Pro Lys 115 120 125 Pro Lys Asp Ile Leu
Met Ile Thr Leu Thr Pro Lys Val Thr Cys Val 130 135 140 Val Val Asp
Val Ser Glu Glu Glu Pro Asp Val Gln Phe Ser Trp Phe 145 150 155 160
Val Asp Asn Val Arg Val Phe Thr Ala Gln Thr Gln Pro His Glu Glu 165
170 175 Gln Leu Asn Gly Thr Phe Arg Val Val Ser Thr Leu His Ile Gln
His 180 185 190 Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val
Asn Asn Lys 195 200 205 Asp Leu Pro Ser Pro Ile Glu Lys Thr Ile Ser
Lys Pro Arg Gly Lys 210 215 220 Ala Arg Thr Pro Gln Val Tyr Thr Ile
Pro Pro Pro Arg Glu Gln Met 225 230 235 240 Ser Lys Asn Lys Val Ser
Leu Thr Cys Met Val Thr Ser Phe Tyr Pro 245 250 255 Ala Ser Ile Ser
Val Glu Trp Glu Arg Asn Gly Glu Leu Glu Gln Asp 260 265 270 Tyr Lys
Asn Thr Leu Pro Val Leu Asp Ser Asp Glu Ser Tyr Phe Leu 275 280 285
Tyr Ser Lys Leu Ser Val Asp Thr Asp Ser Trp Met Arg Gly Asp Ile 290
295 300 Tyr Thr Cys Ser Val Val His Glu Ala Leu His Asn His His Thr
Gln 305 310 315 320 Lys Asn Leu Ser Arg Ser Pro Gly Lys 325
63107PRTRattus norvegicus 63Arg Ala Asp Ala Ala Pro Thr Val Ser Ile
Phe Pro Pro Ser Met Glu 1 5 10 15 Gln Leu Thr Ser Gly Gly Ala Thr
Val Val Cys Phe Val Asn Asn Phe 20 25 30 Tyr Pro Arg Asp Ile Ser
Val Lys Trp Lys Ile Asp Gly Ser Glu Gln 35 40 45 Arg Asp Gly Val
Leu Asp Ser Val Thr Asp Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr
Ser Met Ser Ser Thr Leu Ser Leu Thr Lys Val Glu Tyr Glu 65 70 75 80
Arg His Asn Leu Tyr Thr Cys Glu Val Val His Lys Thr Ser Ser Ser 85
90 95 Pro Val Val Lys Ser Phe Asn Arg Asn Glu Cys 100 105
64323PRTOryctolagus cuniculus 64Gly Gln Pro Lys Ala Pro Ser Val Phe
Pro Leu Ala Pro Cys Cys Gly 1 5 10 15 Asp Thr Pro Ser Ser Thr Val
Thr Leu Gly Cys Leu Val Lys Gly Tyr 20 25 30 Leu Pro Glu Pro Val
Thr Val Thr Trp Asn Ser Gly Thr Leu Thr Asn 35 40 45 Gly Val Arg
Thr Phe Pro Ser Val Arg Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu
Ser Ser Val Val Ser Val Thr Ser Ser Ser Gln Pro Val Thr Cys 65 70
75 80 Asn Val Ala His Pro Ala Thr Asn Thr Lys Val Asp Lys Thr Val
Ala 85 90 95 Pro Ser Thr Cys Ser Lys Pro Thr Cys Pro Pro Pro Glu
Leu Leu Gly 100 105 110 Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 115 120 125 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser Gln 130 135 140 Asp Asp Pro Glu Val Gln Phe
Thr Trp Tyr Ile Asn Asn Glu Gln Val 145 150 155 160 Arg Thr Ala Arg
Pro Pro Leu Arg Glu Gln Gln Phe Asn Ser Thr Ile 165 170 175 Arg Val
Val Ser Thr Leu Pro Ile Ala His Gln Asp Trp Leu Arg Gly 180 185 190
Lys Glu Phe Lys Cys Lys Val His Asn Lys Ala Leu Pro Ala Pro Ile 195
200 205 Glu Lys Thr Ile Ser Lys Ala Arg Gly Gln Pro Leu Glu Pro Lys
Val 210 215 220 Tyr Thr Met Gly Pro Pro Arg Glu Glu Leu Ser Ser Arg
Ser Val Ser 225 230 235 240 Leu Thr Cys Met Ile Asn Gly Phe Tyr Pro
Ser Asp Ile Ser Val Glu 245 250 255 Trp Glu Lys Asn Gly Lys Ala Glu
Asp Asn Tyr Lys Thr Thr Pro Ala 260 265 270 Val Leu Asp Ser Asp Gly
Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val 275 280 285 Pro Thr Ser Glu
Trp Gln Arg Gly Asp Val Phe Thr Cys Ser Val Met 290 295 300 His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Ile Ser Arg Ser 305 310 315
320 Pro Gly Lys 65104PRTOryctolagus cuniculus 65Arg Asp Pro Val Ala
Pro Thr Val Leu Ile Phe Pro Pro Ala Ala Asp 1 5 10 15 Gln Val Ala
Thr Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys Tyr 20 25 30 Phe
Pro Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln Thr 35 40
45 Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys Thr
50 55 60 Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr
Asn Ser 65 70 75 80 His Lys Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr
Thr Ser Val Val 85 90 95 Gln Ser Phe Asn Arg Gly Asp Cys 100
66293PRTOryctolagus cuniculus 66Met Trp Pro Leu Val Ala Ala Leu Leu
Leu Gly Ser Ala Cys Cys Gly 1 5 10 15 Ser Ala Gln Leu Leu Phe Asn
Lys Thr Lys Ser Val Glu Phe Thr Phe 20 25 30 Cys Asn Asp Thr Val
Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala 35 40 45 Gln Asn Thr
Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp 50 55 60 Ile
Tyr Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp 65 70
75 80 Phe Ser Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp
Ala 85 90 95 Ser Leu Lys Met Asp Lys Ser Asp Ala Val Ser His Thr
Gly Asn Tyr 100 105 110 Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly
Glu Thr Ile Ile Glu 115 120 125 Leu Lys Tyr Arg Val Val Ser Trp Phe
Ser Pro Asn Glu Asn Ile Leu 130 135 140 Ile Val Ile Phe Pro Ile Phe
Ala Ile Leu Leu Phe Trp Gly Gln Phe 145 150 155 160 Gly Ile Lys Thr
Leu Lys Tyr Arg Ser Gly Gly Met Asp Glu Lys Thr 165 170 175 Ile Ala
Leu Leu Val Ala Gly Leu Val Ile Thr Val Ile Val Ile Val 180 185 190
Gly Ala Ile Leu Phe Val Pro Gly Glu Tyr Ser Leu Lys Asn Ala Thr 195
200 205 Gly Leu Gly Leu Ile Val Thr Ser Thr Gly Ile Leu Ile Leu Leu
His 210 215 220 Tyr Tyr Val Phe Ser Thr Ala Ile Gly Leu Thr Ser Phe
Val Ile Ala 225 230 235 240 Ile Leu Val Ile Gln Val Ile Ala Tyr Ile
Leu Ala Val Val Gly Leu 245 250 255 Ser Leu Cys Ile Ala Ala Cys Ile
Pro Met His Gly Pro Leu Leu Ile 260 265 270 Ser Gly Leu Ser Ile Leu
Ala Leu Ala Gln Leu Leu Gly Leu Val Tyr 275 280 285 Met Lys Phe Val
Glu 290 67219PRTHomo sapiens 67Asp Val Leu Met Thr Gln Thr Pro Leu
Ser Leu Pro Val Asn Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys
Arg Ser Arg Gln Ser Ile Val His Thr 20 25 30 Asn Gly Asn Thr Tyr
Leu Gly Trp Phe Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu
Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70
75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln
Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu
Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195
200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
68219PRTHomo sapiens 68Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu
Pro Val Asn Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser
Arg Gln Ser Ile Val His Thr 20 25 30 Asn Gly Asn Thr Tyr Leu Gly
Trp Phe Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile
Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser
Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90
95 Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 69219PRTHomo
sapiens 69Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr
Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Arg Gln Ser
Ile Val His Thr 20 25 30 Asn Gly Asn Thr Tyr Leu Gly Trp Tyr Leu
Gln Lys Pro Gly Gln Ser 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val
Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu
Ala Asp Asp Val Gly Ile Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His
Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115
120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 210 215 70214PRTHomo sapiens 70Asp Ile
Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20
25 30 Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu
Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Tyr Ser Gly Val Pro Asp Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
Ser Arg Val Glu Ala 65 70 75 80 Asp Asp Val Gly Ile Tyr Tyr Cys Gln
Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150
155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
71219PRTHomo sapiens 71Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu
Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ser
Ser Gln Asn Ile Val Gln Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu
Trp Tyr Gln Gln Lys Pro Gly Gln Pro 35 40 45 Pro Lys Leu Leu Ile
Tyr Lys Val Phe His Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser
Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Phe Gln Gly 85 90
95 Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 72214PRTHomo
sapiens 72Asp 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 Ser
Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Tyr 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 Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe
Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155
160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
73219PRTHomo sapiens 73Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu
Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser
Arg Gln Ser Ile Val His Thr 20 25 30 Asn Gly Asn Thr Tyr Leu Gly
Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile
Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser
Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90
95 Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 74219PRTHomo
sapiens 74Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr
Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn
Ile Val Gln Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Phe Gln
Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Lys Val
Phe His Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu
Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His
Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115
120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 210 215 75214PRTHomo sapiens 75Asp 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 Asp Ile Ser Asn Tyr 20
25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Tyr Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile
Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln
Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150
155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
76214PRTMus musculus 76Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu
Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala
Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys
Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg
Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu
Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90
95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe
Asn Arg Gly Glu Cys 210 77219PRTMus musculus 77Asp Val Phe Met Thr
Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala
Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val Gln Ser 20 25 30 Asn
Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40
45 Pro Lys Leu Leu Ile Tyr Lys Val Phe His Arg Phe Ser Gly Val Pro
50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr
Cys Phe Gln Gly 85 90 95 Ser His Val Pro Trp Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170
175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
215 78444PRTMus musculus 78Glu Val Gln Leu Gln Gln Ser Gly Pro Glu
Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Val Ile His Trp Val Lys
Arg Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr
Pro Tyr Asn Asp Gly Ile Leu Tyr Asn Glu Lys Phe 50 55 60 Lys Gly
Lys Ala Thr Val Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80
Met Asp Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85
90 95 Thr Arg Gly Gly Tyr Tyr Val Pro Asp Tyr Trp Gly Gln Gly Thr
Thr 100 105 110 Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr
Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly
Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205
Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210
215 220 Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu
Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu
Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser
Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330
335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu
Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 79444PRTHomo sapiens
79Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1
5 10 15 Ser Val Gln Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn
Tyr 20 25 30 Val Ile His Trp Leu Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Ile Leu
Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Ser Asp
Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg
Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr
Tyr Val Pro Asp Tyr Trp Gly Gln Ala Thr Leu 100 105 110 Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala
Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135
140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn
Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val
Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro
Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260
265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser
Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385
390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp
Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
Gly Lys 435 440 80444PRTHomo sapiens 80Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Gln Val Ser
Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30 Tyr Ile His
Trp Leu Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly
Tyr Ile Asp Pro Leu Asn Gly Asp Thr Thr Tyr Asn Gln Lys Phe 50 55
60 Lys Gly Arg Val Thr Met Thr Ser Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Gly Gly Lys Arg Ala Met Asp Tyr Trp Gly
Gln Ala Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser
Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185
190 Leu Gly Thr
Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr
Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215
220 Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe
225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp
Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn
Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340
345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr
Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Leu Gly Lys 435 440 81443PRTHomo sapiens 81Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10
15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45 Gly Tyr Thr Asp Pro Arg Thr Asp Tyr Thr Glu Tyr
Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr
Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Arg Val
Gly Leu Gly Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro
Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145
150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser Ser Asn 180 185 190 Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val
Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Thr Val Glu
Arg Lys Cys Cys Val Glu Cys Pro 210 215 220 Pro Cys Pro Ala Pro Pro
Val Ala Gly Pro Ser Val Phe Leu Phe Pro 225 230 235 240 Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 245 250 255 Cys
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn 260 265
270 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu
Thr Val 290 295 300 Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser 305 310 315 320 Asn Lys Gly Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Glu 340 345 350 Glu Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360 365 Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 370 375 380 Asn
Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe 385 390
395 400 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly 405 410 415 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr 420 425 430 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 82443PRTHomo sapiens 82Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys
Gly Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Ile His Trp Val
Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Tyr Thr
Asp Pro Arg Thr Asp Tyr Thr Glu Tyr Asn Gln Lys Phe 50 55 60 Lys
Asp Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70
75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr
Cys 85 90 95 Ala Arg Gly Gly Arg Val Gly Leu Gly Tyr Trp Gly Gln
Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn 180 185 190
Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195
200 205 Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys
Pro 210 215 220 Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe
Leu Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr 245 250 255 Cys Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Gln Phe Asn 260 265 270 Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg 275 280 285 Glu Glu Gln Phe
Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val 290 295 300 Val His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 305 310 315
320 Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys
325 330 335 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Glu 340 345 350 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe 355 360 365 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu 370 375 380 Asn Asn Tyr Lys Thr Thr Pro Pro
Met Leu Asp Ser Asp Gly Ser Phe 385 390 395 400 Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 405 410 415 Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 420 425 430 Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 83443PRTHomo
sapiens 83Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Asn Tyr 20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Thr Asp Pro Arg Thr Asp
Tyr Thr Glu Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile
Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Gly Gly Arg Val Gly Leu Gly Tyr Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115
120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser Ser Asn 180 185 190 Phe Gly Thr Gln Thr Tyr
Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp
Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro 210 215 220 Pro Cys
Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro 225 230 235
240 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
245 250 255 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln
Phe Asn 260 265 270 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Val
Val Ser Val Leu Thr Val 290 295 300 Val His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser 305 310 315 320 Asn Lys Gly Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 340 345 350 Glu
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360
365 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
370 375 380 Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly
Ser Phe 385 390 395 400 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly 405 410 415 Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr 420 425 430 Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 435 440 84444PRTHomo sapiens 84Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Gln
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Val
Ile His Trp Leu Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45 Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Ile Leu Tyr Asn Glu Lys Phe
50 55 60 Lys Gly Arg Val Thr Met Thr Ser Asp Thr Ser Ile Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Tyr Val Tyr Asp Tyr
Trp Gly Gln Ala Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser
Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170
175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro
Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly
Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly
Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg
Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295
300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile
Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe
Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420
425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440
85443PRTHomo sapiens 85Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Ile His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Thr Asp Pro
Arg Thr Asp Tyr Thr Glu Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg
Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Gly Arg Val Gly Leu Gly Tyr Trp Gly Gln Gly Thr Leu
100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala
Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn 180 185 190 Phe Gly Thr
Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr
Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro 210 215
220 Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro
225 230 235 240 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr 245 250 255 Cys Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Gln Phe Asn 260
265 270 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val
Leu Thr Val 290 295 300 Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser 305 310 315 320 Asn Lys Gly Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 340 345 350 Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360 365 Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 370 375 380
Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe 385
390 395 400 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly 405 410 415 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr 420 425 430 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 435 440 86444PRTHomo sapiens 86Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys
Lys Gly Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp
Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Tyr
Ile Asp Pro Leu Asn Gly Asp Thr Thr Tyr Ser Pro Ser Phe 50 55 60
Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65
70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr
Tyr Cys 85 90 95 Ala Arg Gly Gly Lys Arg Ala Met Asp Tyr Trp Gly
Arg Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser
Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185
190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro
Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser
Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val
Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu
Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310
315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr
Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440
87444PRTHomo sapiens 87Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser
Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Val Ile His Trp Val Arg Gln
Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Tyr Pro
Tyr Asn Asp Gly Ile Leu Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Gln
Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu
Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90
95 Ala Arg Gly Gly Tyr Tyr Val Pro Asp Tyr Trp Gly Gln Gly Thr Thr
100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala
Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr
Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr
Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215
220 Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe
225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp
Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn
Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340
345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr
Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Leu Gly Lys 435 440 88444PRTHomo sapiens 88Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10
15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30 Val Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45 Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Ile Leu Tyr
Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Thr Asp Thr
Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser
Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Tyr
Val Pro Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro
Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145
150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val
Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu
Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu
Phe Glu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr
Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265
270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile
Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390
395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
Lys 435 440 89443PRTHomo sapiens 89Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys
Lys Gly Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Ile His Trp
Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Tyr
Thr Asp Pro Arg Thr Asp Tyr Thr Glu Tyr Ser Pro Ser Phe 50 55 60
Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65
70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr
Tyr Cys 85 90 95 Ala Arg Gly Gly Arg Val Gly Leu Gly Tyr Trp Gly
Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser
Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn 180 185
190 Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
195 200 205 Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu
Cys Pro 210 215 220 Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val
Phe Leu Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr 245 250 255 Cys Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Gln Phe Asn 260 265 270 Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 275 280 285 Glu Glu Gln
Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val 290 295 300 Val
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 305 310
315 320 Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr
Lys 325 330 335 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu 340 345 350 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe 355 360 365 Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu 370 375 380 Asn Asn Tyr Lys Thr Thr Pro
Pro Met Leu Asp Ser Asp Gly Ser Phe 385 390 395 400 Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 405 410 415 Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 420 425 430
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 90444PRTHomo
sapiens 90Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser
Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asp Pro Leu Asn Gly
Asp Thr Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Gly Gly Lys Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115
120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr
Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp
Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys
Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235
240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val
Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 91444PRTHomo
sapiens 91Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser
Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asp Pro Leu Asn Gly
Asp Thr Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile
Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Gly Gly Lys Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115
120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr
Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp
Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys
Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235
240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val
Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360
365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys
Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Leu Gly Lys 435 440 92444PRTHomo sapiens 92Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu
Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30
Tyr Ile His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35
40 45 Gly Tyr Ile Asp Pro Leu Asn Gly Asp Thr Thr Tyr Asn Gln Lys
Phe 50 55 60 Lys Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser
Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr
Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Lys Arg Ala Met Asp
Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165
170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys
Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr
Gly Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Phe Glu Gly
Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val
Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285
Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290
295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe
Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410
415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440
93444PRTHomo sapiens 93Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Ser Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asp Pro
Leu Asn Gly Asp Thr Thr Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg
Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Gly Lys Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala
Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr
Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr
Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215
220 Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe
225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp
Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn
Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340
345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr
Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Leu Gly Lys 435 440 94444PRTHomo sapiens 94Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10
15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr
20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45 Gly Tyr Ile Asp Pro Leu Asn Gly Asp Thr Thr Tyr
Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr
Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Lys Arg
Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro
Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145
150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val
Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu
Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu
Phe Glu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr
Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265
270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile
Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390
395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
Lys 435 440 95444PRTMus musculus 95Glu Val Gln Leu Gln Gln Ser Gly
Pro Glu Leu Met Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys
Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp
Val Asn Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Tyr
Ile Asp Pro Leu Asn Gly Asp Thr Thr Tyr Asn Gln Lys Phe 50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65
70 75 80 Met Arg Leu Ser Ser Leu Thr Ser Ala Asp Ser Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Gly Gly Lys Arg Ala Met Asp Tyr Trp Gly
Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser
Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185
190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro
Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser
Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val
Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu
Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310
315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr
Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405
410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435
440 96443PRTMus musculus 96Gln Val Gln Leu Gln Gln Phe Gly Ala Glu
Leu Ala Lys Pro Gly Ala 1 5 10 15 Ser Val Gln Met Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Ile His Trp Val Lys
Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Thr Asp
Pro Arg Thr Asp Tyr Thr Glu Tyr Asn Gln Lys Phe 50 55 60 Lys Asp
Lys Ala Thr Leu Ala Ala Asp Arg Ser Ser Ser Thr Ala Tyr 65 70 75 80
Met Arg Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85
90 95 Ala Gly Gly Gly Arg Val Gly Leu Gly Tyr Trp Gly His Gly Ser
Ser 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr
Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn 180 185 190 Phe Gly
Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205
Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro 210
215 220 Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe
Pro 225 230 235 240 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr 245 250 255 Cys Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Gln Phe Asn 260 265 270 Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser
Thr Phe Arg Val Val Ser Val Leu Thr Val 290 295 300 Val His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 305 310 315 320 Asn
Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 325 330
335 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
340 345 350 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe 355 360 365 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu 370 375 380 Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu
Asp Ser Asp Gly Ser Phe 385 390 395 400 Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly 405 410 415 Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr 420 425 430 Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys 435 440 97443PRTHomo sapiens 97Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10
15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr
20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45 Gly Tyr Ile Asp Pro Leu Asn Gly Asp Thr Thr Tyr
Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr
Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Lys Arg
Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro
Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145
150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser Ser Asn 180 185 190 Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val
Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Thr Val Glu
Arg Lys Cys Cys Val Glu Cys Pro 210 215 220 Pro Cys Pro Ala Pro Pro
Val Ala Gly Pro Ser Val Phe Leu Phe Pro 225 230 235 240 Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 245 250 255 Cys
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn 260 265
270 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu
Thr Val 290 295 300 Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser 305 310 315 320 Asn Lys Gly Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Glu 340 345 350 Glu Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360 365 Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 370 375 380 Asn
Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe 385 390
395 400 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly 405 410 415 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr 420 425 430 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 98443PRTHomo sapiens 98Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile
Asp Pro Leu Asn Gly Asp Thr Thr Tyr Asn Gln Lys Phe 50 55 60 Lys
Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70
75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Gly Gly Lys Arg Ala Met Asp Tyr Trp Gly Gln
Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn 180 185 190
Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195
200 205 Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys
Pro 210 215 220 Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe
Leu Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr 245 250 255 Cys Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Gln Phe Asn 260 265 270 Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg 275 280 285 Glu Glu Gln Phe
Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val 290 295 300 Val His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 305 310 315
320 Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys
325 330 335 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Glu 340 345 350 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe 355 360 365 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu 370 375 380 Asn Asn Tyr Lys Thr Thr Pro Pro
Met Leu Asp Ser Asp Gly Ser Phe 385 390 395 400 Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 405 410 415 Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 420 425 430 Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 99443PRTHomo
sapiens 99Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser
Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Met Pro Gly
Lys Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asp Pro Leu Asn Gly
Asp Thr Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Gln Val Thr Ile
Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser
Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg
Gly Gly Lys Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115
120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser Ser Asn 180 185 190 Phe Gly Thr Gln Thr Tyr
Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp
Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro 210 215 220 Pro Cys
Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro 225 230 235
240 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
245 250 255 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln
Phe Asn 260 265 270 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Val
Val Ser Val Leu Thr Val 290 295 300 Val His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser 305 310 315 320 Asn Lys Gly Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 340 345 350 Glu
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360
365 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
370 375 380 Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly
Ser Phe 385 390 395 400 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly 405 410 415 Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr 420 425 430 Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 435 440 100443PRTHomo sapiens 100Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Tyr Ile Asp Pro Leu Asn Gly Asp Thr Thr Tyr Ala Gln Lys
Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser
Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Lys Arg Ala Met Asp
Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165
170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
Asn 180 185 190 Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys
Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys
Cys Val Glu Cys Pro 210 215 220 Pro Cys Pro Ala Pro Pro Val Ala Gly
Pro Ser Val Phe Leu Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr 245 250 255 Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn 260 265 270 Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 275 280 285
Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val 290
295 300 Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser 305 310 315 320 Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Thr Lys 325 330 335 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu 340 345 350 Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe 355 360 365 Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 370 375 380 Asn Asn Tyr Lys
Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe 385 390 395 400 Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 405 410
415 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
420 425 430 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
101326PRTHomo sapiens 101Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro
Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Glu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155
160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280
285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly 325
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