U.S. patent application number 14/737053 was filed with the patent office on 2015-10-01 for therapeutic cd47 antibodies.
The applicant listed for this patent is Vasculox Inc.. Invention is credited to Hwai Wen CHANG, William A. FRAZIER, Gerhard FREY, Pamela T. MANNING.
Application Number | 20150274826 14/737053 |
Document ID | / |
Family ID | 50935081 |
Filed Date | 2015-10-01 |
United States Patent
Application |
20150274826 |
Kind Code |
A1 |
FRAZIER; William A. ; et
al. |
October 1, 2015 |
THERAPEUTIC CD47 ANTIBODIES
Abstract
Provided are monoclonal antibodies and antigen-binding fragments
thereof that bind to CD47 of multiple mammalian species, block the
binding of SIRPalpha and TSP1 to CD47, promote phagocytosis of
susceptible cancer cells, and reverse TSP1 inhibition of nitric
oxide signaling, as well as monoclonal antibodies and antigen
binding fragments thereof that compete with the former for binding
to CD47 and that exhibit similar biological activities. Also
provided are combinations of any of the foregoing. Such antibody
compounds are variously effective in 1) treating tissue ischemia
and ischemia-reperfusion injury (IRI) in the setting of organ
preservation and transplantation, pulmonary hypertension, sickle
cell disease, myocardial infarction, stroke, and other instances of
surgery and/or trauma in which IRI is a component of pathogenesis;
2) in treating autoimmune and inflammatory diseases; and 3) as
anti-cancer agents for treating susceptible cancer cells, promoting
their phagocytic uptake and clearance.
Inventors: |
FRAZIER; William A.; (St.
Louis, MO) ; MANNING; Pamela T.; (Chesterfield,
MO) ; FREY; Gerhard; (St. Louis, MO) ; CHANG;
Hwai Wen; (St. Louis, MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Vasculox Inc. |
St. Louis |
MO |
US |
|
|
Family ID: |
50935081 |
Appl. No.: |
14/737053 |
Filed: |
June 11, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14302348 |
Jun 11, 2014 |
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14737053 |
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PCT/US2013/074766 |
Dec 12, 2013 |
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14302348 |
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61833691 |
Jun 11, 2013 |
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61736301 |
Dec 12, 2012 |
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Current U.S.
Class: |
424/143.1 ;
530/387.3; 530/388.22 |
Current CPC
Class: |
A61K 2039/505 20130101;
A61P 25/00 20180101; C07K 2317/73 20130101; A61P 29/00 20180101;
A61P 35/00 20180101; A61P 37/02 20180101; A61P 9/10 20180101; C07K
2317/24 20130101; A61P 19/00 20180101; A61P 35/02 20180101; A61P
43/00 20180101; C07K 16/2896 20130101; A61P 19/02 20180101; A61P
37/06 20180101; A61P 17/06 20180101; C07K 2317/75 20130101; C07K
16/2803 20130101; C07K 2317/33 20130101; C07K 2317/71 20130101;
C07K 2317/92 20130101; A61P 5/14 20180101; C07K 2317/76 20130101;
A61P 1/04 20180101 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Claims
1. A monoclonal antibody, or antigen-binding fragment thereof,
that: (i) specifically binds human, rat, mouse, pig, cynomolgus
monkey, and dog CD47; (ii) blocks SIRPalpha and TSP1 binding to
CD47; (iii) promotes phagocytosis of cancer cells; and (iv)
reverses TSP1 inhibition of nitric oxide signaling.
2. The monoclonal antibody or antigen-binding fragment thereof of
claim 1, which is chimeric or humanized.
3. The monoclonal antibody, or antigen-binding fragment thereof, of
claim 2, which comprises three light chain complementarity
determining regions (LCDRs 1-3) and three heavy chain
complementarity determining regions (HCDRs 1-3), wherein:
TABLE-US-00032 LCDR 1 comprises the amino acid sequence (SEQ ID NO:
1) RSSQSLVHSNGNTYLH LCDR 2 comprises the amino acid sequence (SEQ
ID NO: 2) KVSYRFS; and LCDR 3 comprises the amino acid sequence
(SEQ ID NO: 3) SQNTHVPRT; HCDR1 comprises the amino acid sequence
(SEQ ID NO: 4) GYTFTNYYVF; HCDR 2 comprises the amino acid sequence
(SEQ ID NO: 5) DINPVNGDTNFNEKFKN; and HCDR 3 comprises the amino
acid sequence (SEQ ID NO: 6) GGYTMDY.
4. The monoclonal antibody, or antigen-binding fragment thereof of
claim 3, which comprises a light chain variable region (LCVR) and a
heavy chain variable region (HCVR), wherein said LCVR and said HCVR
comprise, respectively, amino acid sequences selected from among
the following combinations of LCVRs and HCVRs: SEQ ID NO:7 and SEQ
ID NO:57; SEQ ID NO:8 and SEQ ID NO:58; SEQ ID NO:9 and SEQ ID
NO:59; SEQ ID NO:10 and SEQ ID NO:60; SEQ ID NO:11 and SEQ ID
NO:61; SEQ ID NO:12 and SEQ ID NO:62; SEQ ID NO:13 and SEQ ID
NO:63; SEQ ID NO:14 and SEQ ID NO:64; SEQ ID NO:15 and SEQ ID
NO:65; SEQ ID NO:16 and SEQ ID NO:66; SEQ ID NO:17 and SEQ ID
NO:67; SEQ ID NO:18 and SEQ ID NO:68; SEQ ID NO:19 and SEQ ID
NO:69; SEQ ID NO:20 and SEQ ID NO:70; SEQ ID NO:21 and SEQ ID
NO:71; SEQ ID NO:22 and SEQ ID NO:72; SEQ ID NO:23 and SEQ ID
NO:73; SEQ ID NO:24 and SEQ ID NO:74; SEQ ID NO:25 and SEQ ID
NO:75; SEQ ID NO:26 and SEQ ID NO:76; SEQ ID NO:27 and SEQ ID
NO:77; SEQ ID NO:28 and SEQ ID NO:78; SEQ ID NO:29 and SEQ ID
NO:79; SEQ ID NO:30 and SEQ ID NO:80; and SEQ ID NO:31 and SEQ ID
NO:81, wherein each one of LCVR SEQ ID NOs:7-31 further comprises a
constant domain having the amino acid sequence shown in SEQ ID NO:
117, and wherein each one of HCVR SEQ ID NOs:57-81 comprises a
constant domain selected from among SEQ ID NO:118, SEQ ID NO:119,
SEQ ID NO:120, SEQ ID NO:121, and SEQ ID NO:124.
5. A monoclonal antibody, or antigen-binding fragment thereof, that
competes with said monoclonal antibody or antigen binding fragment
thereof of claim 4 for binding to human CD47.
6. A pharmaceutical composition, comprising said monoclonal
antibody or antigen-binding fragment thereof, or competing antibody
or antigen binding fragment thereof, of claim 1 and a
pharmaceutically or physiologically acceptable carrier, diluent, or
excipient.
7. A monoclonal antibody or antigen-binding fragment thereof, or
competing antibody or antigen binding fragment thereof, of claim 1
for use in human therapy.
8. The monoclonal antibody or antigen-binding fragment thereof, or
competing antibody or antigen binding fragment thereof of claim 1
for use in reducing, preventing, and/or treating
ischemia-reperfusion injury, or an autoimmune or inflammatory
disease in a human patient.
9. The monoclonal antibody or antigen-binding fragment thereof, or
competing antibody or antigen binding fragment thereof, of claim 8,
wherein said ischemia-reperfusion injury occurs in organ
transplantation, acute kidney injury, cardiopulmonary bypass
surgery, pulmonary hypertension, sickle cell disease, myocardial
infarction, stroke, surgical resections and reconstructive surgery,
reattachment of an appendage or other body part, skin grafting, or
trauma.
10. The monoclonal antibody, or antigen-binding fragment thereof,
or competing antibody or antigen binding fragment thereof, of claim
8, wherein said autoimmune or inflammatory disease is selected from
the group consisting of arthritis, multiple sclerosis, psoriasis,
Crohn's disease, inflammatory bowel disease, lupus, Grave's disease
and Hashimoto's thyroiditis, and ankylosing spondylitis.
11. The monoclonal antibody or antigen-binding fragment thereof, or
competing antibody or antigen binding fragment thereof of claim 1
for use in treating or reducing a susceptible cancer.
12. The monoclonal antibody or antigen binding fragment thereof, or
competing antibody or antigen binding fragment thereof, of claim
11, which promotes phagocytosis of cells of said susceptible
cancer.
13. The monoclonal antibody or antigen binding fragment thereof, or
competing antibody or antigen binding fragment thereof, of claim 11
wherein said susceptible cancer 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, 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, and a sarcoma.
14. The monoclonal antibody or antigen binding fragment thereof, or
competing antibody or antigen binding fragment thereof, of claim 13
wherein: said leukemia is selected from the group consisting of
systemic mastocytosis, acute lymphocytic (lymphoblastic) leukemia
(ALL), T cell--ALL, acute myeloid leukemia (AML), myelogenous
leukemia, chronic lymphocytic leukemia (CLL), multiple myeloma
(MM), chronic myeloid leukemia (CML), myeloproliferative
disorder/neoplasm, myelodysplastic syndrome, monocytic cell
leukemia, and plasma cell leukemia; wherein said lymphoma is
selected from the group consisting of 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, and
Waldenstrom's Macroglobulinemia; and wherein said sarcoma is
selected from the group consisting of osteosarcoma, Ewing sarcoma,
leiomyosarcoma, synovial sarcoma, alveolar soft part sarcoma,
angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and
chrondrosarcoma.
15. Use of said monoclonal antibody or antigen-binding fragment
thereof, or competing antibody or antigen binding fragment thereof,
of claim 1 to reduce, prevent, and/or treat ischemia-reperfusion
injury, or an autoimmune or inflammatory disease, in a human
patient.
16. Use of said monoclonal antibody or antigen-binding fragment
thereof, or competing antibody or antigen binding fragment thereof,
of claim 1 to reduce, prevent, and/or treat a susceptible
cancer.
17. Use of said monoclonal antibody or antigen-binding fragment
thereof, or competing antibody or antigen binding fragment thereof,
of claim 1 for the manufacture of a medicament to prevent, reduce,
and/or treat ischemia-reperfusion injury, or an autoimmune or
inflammatory disease, in a human patient.
18. The use of claim 17, wherein said ischemia-reperfusion injury
occurs in organ transplantation, acute kidney injury,
cardiopulmonary bypass surgery, pulmonary hypertension, sickle cell
disease, myocardial infarction, stroke, surgical resections and
reconstructive surgery, reattachment of an appendage or other body
part, skin grafting, and trauma.
19. The use of claim 17, wherein said autoimmune or inflammatory
disease is selected from among arthritis, multiple sclerosis,
psoriasis, Crohn's disease, inflammatory bowel disease, lupus,
Grave's disease and Hashimoto's thyroiditis, and ankylosing
spondylitis.
20. Use of said monoclonal antibody or antigen-binding fragment
thereof, or competing antibody or antigen binding fragment thereof,
of claim 1 for the manufacture of a medicament to treat or reduce a
susceptible cancer.
21. A monoclonal antibody, or antigen-binding fragment thereof, of
claim 1 that specifically binds to epitopes in the extracellular
IgV domain of CD47.
22. A monoclonal antibody, or antigen-binding fragment thereof, of
claim 5 that specifically binds to epitopes in the extracellular
IgV domain of CD47.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This Continuation-in-part application claims the benefit of
priority of Continuation-in-part application Ser. No. 14/302,348,
filed Jun. 11.sup.th 2014, PCT Application Serial No.
PCT/US2013/074766, filed Dec. 12, 2013, U.S. Provisional
Application Ser. No. 61/833,691, filed Jun. 11.sup.th 2013, and
U.S. Provisional Application Ser. No. 61/736,301, filed Dec.
12.sup.th 2012, the contents of each of which are herein
incorporated by reference in their entirety.
INCORPORATION OF SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled, "VLX0001-201CIP2 US_SequenceListing", created on
Jun. 11th 2015, which is 168,755 bytes in size. The information in
the electronic format of the Sequence Listing is incorporated
herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0003] The present disclosure relates to antibodies that bind CD47,
including that of humans and other mammalian species, and their use
in treating conditions and disorders, such as ischemia-reperfusion
injury (IRI) and cancers, mediated by this receptor.
[0004] CD47 is a cell surface receptor comprised of an
extracellular IgV set domain, a 5 membrane spanning transmembrane
domain, and a cytoplasmic tail that is alternatively spliced. Two
ligands bind CD47: thrombospondin-1 (TSP1), and signal inhibitory
receptor protein alpha (SIRPalpha). 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 pathway
in all vascular cells. The nitric oxide (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.
[0005] SIRPalpha is expressed on hematopoietic cells, including
macrophages and dendritic cells. When it engages CD47 on a
potential phagocytic target cell, phagocytosis is slowed or
prevented. The CD47-SIRPalpha interaction effectively sends a
"don't eat me" signal to the phagocyte. Thus, blocking the
SIRPalpha-CD47 interaction with a monoclonal antibody in this
therapeutic context can provide an effective anti-cancer therapy by
promoting, i.e., increasing, the uptake and clearance of cancer
cells by the host's immune system by increasing phagocytosis. This
mechanism is effective in leukemias, lymphomas, and many types of
solid tumors.
[0006] U.S. Pat. No. 8,236,313 contemplates antibodies that could
be useful in the field of ischemia and blood flow to reverse and/or
prevent tissue ischemia and related and associated tissue and cell
damage, including antibodies that block CD47.
[0007] U.S. Pat. No. 8,101,719 discloses humanized antibodies that
bind to CD47 for use in treating hematological disorders. Objects
of the invention include humanized anti-CD47 antibodies and small
antibody fragments exhibiting reduced antigenicity while retaining
their CD47 binding activity and apoptosis-inducing activity. Such
antibodies and small fragments are contemplated for use in treating
hematological disorders such as various types of leukemias,
malignant lymphoma, aplastic anemia, myelodysplastic syndromes, and
polycythemia vera.
[0008] PCT International Publication WO 2011/143624 discloses
chimeric and humanized anti-CD47 monoclonal antibodies for use as
reagents for the diagnosis and immunotherapy of diseases associated
with CD47 in humans, particularly in cancer therapy, for example to
increase phagocytosis of cancer cells expressing CD47. Preferred
antibodies are non-activating, i.e., block ligand binding, but do
not signal. Disclosed humanized B6H12 and 5F9 antibodies bound
soluble human CD47; B6H12 also bound human CD47 on the surface of
human CD47-transfected YB2/0 cells. Humanized B6H12 and 5F9
antibodies enabled phagocytosis of CFSE-labeled HL-60 cells by
mouse bone marrow- or peripheral blood-derived macrophages in
vitro, respectively. Humanized B6H12 utilized human VH-3-7 and
VK3-11 frameworks.
[0009] U.S. 2013/0142786 discloses non-activating anti-CD47
antibodies that increase the phagocytosis of CD47 expressing cells
and these humanized or chimeric anti-CD47 antibodies can be used
for therapeutic purposes, particularly in cancer therapy. Amino
acid sequences of murine and humanized mAb B6H12, 5F9, and 8B6
heavy and light chain variable regions are disclosed.
[0010] Han et al. (2000) J. Biol. Chem. 275(48):37984-37992
discloses the production of mouse anti-CD47 monoclonal antibodies
400 (IgG2b), 410 (IgG1), 420 (IgG2a), 430 (IgG2a), 440 (IgG1), 450
(IgG2a), 460 (IgG1), 470 (IgG2a), and 480 (IgG1) generated by
immunizing CD47-deficient mice with the extracellular domain of
human CD47. No amino acid sequences of these antibodies, or their
CDRs, are disclosed. Of these nine mAbs, three blocked macrophage
fusion: 430, 450, and 470. Han et al. discloses no data either
demonstrating or suggesting that CD47 plays a role in
ischemia-reperfusion injury, autoimmune or inflammatory diseases,
or cancer.
[0011] PCT International Publication WO 2013/119714 discloses
anti-CD47 antibodies that do not cause a significant level of
hemagglutination of human red blood cells.
[0012] There exists a need for antibodies to human CD47 that
selectively block the binding of TSP1 to CD47 to promote the
beneficial effects of nitric oxide-cGMP signaling and cAMP
signaling in the cardiovascular system in settings in which IRI
plays a role in pathogenesis. These situations/diseases include
organ transplantation, acute kidney injury, cardiopulmonary bypass
surgery, pulmonary hypertension, sickle cell disease, myocardial
infarction, stroke, surgical resections and reconstructive surgery,
reattachment of digits/body parts, skin grafting, and trauma. There
is also a need for antibodies that block the binding of SIRPalpha
to CD47, thus providing novel anti-cancer therapies.
[0013] Antibody compounds of the present disclosure meet these
needs. They bind to epitopes in the extracellular IgV domain of
CD47, variously inhibiting the binding of SIRPalpha and TSP1 to
CD47 and receptor activation. Antibodies that block TSP1 and
SIRPalpha binding should be therapeutically useful in preventing,
treating, or reducing many forms of IRI and treating cancers.
Antibodies that block SIRPalpha binding promote phagocytosis of
cancer cells. In view of these properties, SIRPalpha blocking
antibody compounds should be therapeutically useful in treating a
variety of cancers, including hematological cancers and solid
tumors.
SUMMARY OF THE INVENTION
[0014] Antibody compounds disclosed herein meet these needs by
exhibiting the following desirable therapeutic activities: [0015]
Binding to CD47 of multiple mammalian species; [0016] Blocking
SIRPalpha and TSP1 binding to CD47; [0017] Promoting phagocytosis
of cancer cells; and [0018] Reversing TSP1 inhibition of nitric
oxide signaling.
[0019] The present antibodies are useful in reducing, preventing,
and/or treating CD47-mediated diseases or conditions (e.g.,
ischemia reperfusion injury and cancers).
[0020] They bind to epitopes in the extracellular IgV domain of
CD47, inhibiting TSP1 and SIRPalpha binding to CD47, while inducing
little or no agonist activity and promoting tumor cell phagocytic
clearance. In view of these properties, antibody compounds of the
present disclosure should be therapeutically useful in treating
many forms of IRI and cancers.
[0021] In addition, the present antibody compounds can possess a
number of other desirable properties, including broad reactivity
with CD47 of a wide variety of mammalian species, including that of
human, mouse, rat, pig, cynomolgus monkey, and dog, making these
antibodies useful in both human and veterinary medicine. This
feature is further advantageous in that it facilitates preclinical
studies including, but not limited to, safety and efficacy studies,
in a variety of mammalian species, and therefore the development of
such antibodies as human and veterinary therapeutics.
[0022] Accordingly, the present disclosure provides:
[0023] [1] A monoclonal antibody, or antigen-binding fragment
thereof, that:
(i) specifically binds human, rat, mouse, pig, cynomolgus monkey,
and dog CD47; (ii) blocks SIRPalpha and TSP1 binding to CD47; (iii)
promotes phagocytosis of cancer cells; and (iv) reverses TSP1
inhibition of nitric oxide signaling.
[0024] [2] The monoclonal antibody or antigen-binding fragment
thereof of [1], which is chimeric or humanized.
[0025] [3] The monoclonal antibody, or antigen-binding fragment
thereof, of [1] or [2], which comprises three light chain
complementarity determining regions (LCDRs 1-3) and three heavy
chain complementarity determining regions (HCDRs 1-3), wherein:
[0026] LCDR 1 comprises the amino acid sequence RSSQSLVHSNGNTYLH
(SEQ ID NO:1);
[0027] LCDR 2 comprises the amino acid sequence KVSYRFS (SEQ ID
NO:2); and
[0028] LCDR 3 comprises the amino acid sequence SQNTHVPRT (SEQ ID
NO:3);
[0029] HCDR 1 comprises the amino acid sequence GYTFTNYYVF (SEQ ID
NO:4);
[0030] HCDR 2 comprises the amino acid sequence DINPVNGDTNFNEKFKN
(SEQ ID NO:5); and
[0031] HCDR 3 comprises the amino acid sequence GGYTMDY (SEQ ID
NO:6).
[0032] [4] The monoclonal antibody, or antigen-binding fragment
thereof, of any one of [1]-[3], which comprises a light chain
variable region (LCVR) and a heavy chain variable region (HCVR),
wherein said LCVR and said HCVR comprise, respectively, amino acid
sequences selected from among the following combinations of LCVRs
and HCVRs:
SEQ ID NO:7 and SEQ ID NO:57;
SEQ ID NO:8 and SEQ ID NO:58;
SEQ ID NO:9 and SEQ ID NO:59;
SEQ ID NO:10 and SEQ ID NO:60;
SEQ ID NO:11 and SEQ ID NO:61;
SEQ ID NO:12 and SEQ ID NO:62;
SEQ ID NO:13 and SEQ ID NO:63;
SEQ ID NO:14 and SEQ ID NO:64;
SEQ ID NO:15 and SEQ ID NO:65;
SEQ ID NO:16 and SEQ ID NO:66;
SEQ ID NO:17 and SEQ ID NO:67;
SEQ ID NO:18 and SEQ ID NO:68;
SEQ ID NO:19 and SEQ ID NO:69;
SEQ ID NO:20 and SEQ ID NO:70;
SEQ ID NO:21 and SEQ ID NO:71;
SEQ ID NO:22 and SEQ ID NO:72;
SEQ ID NO:23 and SEQ ID NO:73;
SEQ ID NO:24 and SEQ ID NO:74;
SEQ ID NO:25 and SEQ ID NO:75;
SEQ ID NO:26 and SEQ ID NO:76;
SEQ ID NO:27 and SEQ ID NO:77;
SEQ ID NO:28 and SEQ ID NO:78;
SEQ ID NO:29 and SEQ ID NO:79;
SEQ ID NO:30 and SEQ ID NO:80; and
SEQ ID NO:31 and SEQ ID NO:81,
[0033] wherein each one of LCVR SEQ ID NOs:7-31 further comprises a
constant domain having the amino acid sequence shown in SEQ ID
NO:117, and wherein each one of HCVR SEQ ID NOs:57-81 comprises a
constant domain selected from among SEQ ID NO:118, SEQ ID NO:119,
SEQ ID NO:120, SEQ ID NO:121, and SEQ ID NO:124.
[0034] [5] A monoclonal antibody, or antigen-binding fragment
thereof, that competes with said monoclonal antibody or
antigen-binding fragment thereof of any one of [1]-[4] for binding
to CD47, especially human CD47.
[0035] [6] A pharmaceutical composition, comprising said monoclonal
antibody or antigen-binding fragment thereof, or competing antibody
or antigen binding fragment thereof, of any one of [1]-[5], and a
pharmaceutically or physiologically acceptable carrier, diluent, or
excipient.
[0036] [7] A monoclonal antibody or antigen-binding fragment
thereof, or competing antibody or antigen binding fragment thereof,
of any one of [1]-[5] for use in human therapy or therapy of
companion/pet animals, working animals, sport animals, zoo animals,
or therapy of other valuable animals kept in captivity.
[0037] [8] The monoclonal antibody or antigen-binding fragment
thereof, or competing antibody or antigen binding fragment thereof,
of any one of [1]-[5] for use in treating ischemia-reperfusion
injury, or an autoimmune or inflammatory disease, in a human or
companion/pet animal, working animal, sport animal, zoo animal, or
other valuable animal kept in captivity.
[0038] [9] The monoclonal antibody or antigen-binding fragment
thereof, or competing antibody or antigen binding fragment thereof,
of [8], wherein said ischemia-reperfusion injury occurs in organ
transplantation, acute kidney injury, cardiopulmonary bypass
surgery, pulmonary hypertension, sickle cell disease, myocardial
infarction, stroke, surgical resections and reconstructive surgery,
reattachment of an appendage or other body part, skin grafting, or
trauma.
[0039] [10] The monoclonal antibody, or antigen-binding fragment
thereof, or competing antibody or antigen binding fragment thereof,
of [8], wherein said autoimmune or inflammatory disease is selected
from the group consisting of arthritis, multiple sclerosis,
psoriasis, Crohn's disease, inflammatory bowel disease, lupus,
Grave's disease and Hashimoto's thyroiditis, and ankylosing
spondylitis.
[0040] [11] The monoclonal antibody or antigen-binding fragment
thereof, or competing antibody or antigen binding fragment thereof,
of any one of [1]-[5] for use in treating a susceptible cancer.
[0041] [12] The monoclonal antibody or antigen binding fragment
thereof, or competing antibody or antigen binding fragment thereof,
of [11], which promotes phagocytosis of cells of said susceptible
cancer.
[0042] [13] The monoclonal antibody or antigen binding fragment
thereof, or competing antibody or antigen binding fragment thereof,
of [11] or [12], wherein said susceptible cancer 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, 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, melanoma, myelodysplastic
syndrome, and sarcomas including, but not limited to, osteosarcoma,
Ewing sarcoma, leiomyosarcoma, synovial sarcoma, alveolar soft part
sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma,
and chrondrosarcoma.
[0043] [14] The monoclonal antibody or antigen binding fragment
thereof, or competing antibody or antigen binding fragment thereof,
of any one of [11]-[13], wherein said leukemia is selected from the
group consisting of systemic mastocytosis, acute lymphocytic
(lymphoblastic) leukemia (ALL), T cell--ALL, acute myeloid leukemia
(AML), myelogenous leukemia, chronic lymphocytic leukemia (CLL),
multiple myeloma (MM), chronic myeloid leukemia (CML),
myeloproliferative disorder/neoplasm, myelodysplastic syndrome,
monocytic cell leukemia, and plasma cell leukemia; 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, and Waldenstrom's
Macroglobulinemia
[0044] [15] Use of said monoclonal antibody or antigen-binding
fragment thereof, or competing antibody or antigen binding fragment
thereof, of any one of [1]-[5] to treat ischemia-reperfusion
injury, or an autoimmune or inflammatory disease, in a human or
companion/pet animal, working animal, sport animal, zoo animal, or
other valuable animal kept in captivity.
[0045] [16] Use of said monoclonal antibody or antigen-binding
fragment thereof, or competing antibody or antigen binding fragment
thereof, of any one of [1]-[5] to treat a susceptible cancer.
[0046] [17] Use of said monoclonal antibody or antigen-binding
fragment thereof, or competing antibody or antigen binding fragment
thereof, of any one of [1]-[5] for the manufacture of a medicament
to treat ischemia-reperfusion injury, or an autoimmune or
inflammatory disease, in a human or companion/pet animal, working
animal, sport animal, zoo animal, or other valuable animal kept in
captivity.
[0047] [18] The use of [17], wherein said ischemia-reperfusion
injury occurs in organ transplantation, acute kidney injury,
cardiopulmonary bypass surgery, pulmonary hypertension, sickle cell
disease, myocardial infarction, stroke, surgical resections and
reconstructive surgery, reattachment of an appendage or other body
part, skin grafting, and trauma.
[0048] [19] The use of [17] or [18], wherein said autoimmune or
inflammatory disease is selected from among arthritis, multiple
sclerosis, psoriasis, Crohn's disease, inflammatory bowel disease,
lupus, Grave's disease and Hashimoto's thyroiditis, and ankylosing
spondylitis.
[0049] [20] Use of said monoclonal antibody or antigen-binding
fragment thereof, or competing antibody or antigen binding fragment
thereof, of any one of [1]-[5] for the manufacture of a medicament
to treat a susceptible cancer.
[0050] [21] A method of treating ischemia or ischemia-reperfusion
injury in a patient in need thereof, comprising administering to
said patient an effective amount of said monoclonal antibody or
antigen-binding fragment thereof, or competing antibody or antigen
binding fragment thereof, of any one of [1]-[5].
[0051] [22] The method of [21], wherein said patient is about to be
subjected to, or is experiencing, ischemia or ischemia-reperfusion
injury.
[0052] [23] The method of [21] or [22], wherein said patient is a
human.
[0053] [24] The method of [21] or [22], wherein said patient is a
companion/pet animal, working animal, sport animal, zoo animal, or
other valuable animal kept in captivity.
[0054] [25] The method of any one of [21]-[24], wherein said
ischemia occurs because said patient will undergo, or is
undergoing, a surgery selected from the group consisting of
integument surgery, soft tissue surgery, composite tissue surgery,
cosmetic surgery, surgical resections, reconstructive surgery, skin
graft surgery, and limb reattachment surgery.
[0055] [26] The method of [25], wherein said skin graft is an
autograft.
[0056] [27] The method of any one of [21]-[24], wherein said
iseheinia occurs because said patient will undergo, or is
undergoing, organ transplant surgery,
[0057] [28] The method of any one of [21]-[24], wherein said
ischemia-reperfusion injury occurs in organ transplantation, acute
kidney injury, cardiopulmonary bypass surgery, pulmonary
hypertension, sickle cell disease, myocardial infarction, stroke,
surgical resection, reconstructive surgery, reattachment of an
appendage or other body part, or skin grafting.
[0058] [29] The method of any one of [21]-[28], wherein said
monoclonal antibody or antigen-binding fragment thereof, or
competing monoclonal antibody or antigen binding fragment thereof,
is administered before, during, or after said subject undergoes
ischemia or surgery, or a combination of any of these time
periods.
[0059] [30] The method of any one of [21]-[29], further comprising
administering to said patient an effective amount of 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.
[0060] [31] The method of [30], wherein said nitric oxide donor or
precursor is selected from the group consisting of NO gas,
isosorbide dinitrate, nitrite, nitroprusside, nitroglycerin,
3-Morpholino-sydnonimine (SIN-1), S-nitroso-N-acetylpenicillamine
(SNAP), Diethylenetriamine/NO (DETA/NO), S-nitrosothiols,
Bidil.RTM., and arginine; and said agent that inhibits cyclic
nucleotide phosphodiesterases is selected from the group consisting
of sildenafil, tadalafil, vardenafil udenafil, and avanafil.
[0061] [32] A method of increasing tissue perfusion in a subject in
need thereof, comprising administering to said subject an effective
amount of a monoclonal antibody, or antigen-binding fragment
thereof, or competing antibody or antigen binding fragment thereof,
of any one of [1]-[5].
[0062] [33] The method of [32], wherein said subject has, or is at
risk of developing, at least one disease or condition selected from
the group consisting of ischemia-reperfusion injury, myocardial
infarction, myocardial ischemia, stroke, cerebral ischemia, sickle
cell anemia, and pulmonary hypertension.
[0063] [34] The method of [32], wherein said subject has, or is at
risk of developing, at least one disease or condition selected from
the group consisting of hypertension, atherosclerosis,
vasculopathy, ischemia secondary to diabetes, and peripheral
vascular disease.
[0064] [35] The method of [32], wherein the need for increased
tissue perfusion arises because said subject has had, is having, or
will have, a surgery selected from the group consisting of
integument surgery, soft tissue surgery, composite tissue surgery,
skin graft surgery, resection of a solid organ, and reattachment or
an appendage or other body part.
[0065] [36] The method of [35], wherein said skin graft is an
autograft.
[0066] [37] The method of [32], wherein the need for increased
tissue perfusion arises because said subject has had, is having, or
will have, organ transplant surgery.
[0067] [38] The method of any one of [32]-[37], further comprising
administering to said subject an effective amount of 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.
[0068] [39] The method of [38], wherein said nitric oxide donor or
precursor is selected from the group consisting of NO gas,
isosorbide dinitrate, nitrite, nitroprusside, nitroglycerin,
3-Morpholino-sydnonimine (SIN-1), S-nitroso-N-acetylpenicillamine
(SNAP), Diethylenetriamine/NO (DETA/NO), S-nitrosothiols,
Bidil.RTM., and arginine; and said agent that inhibits cyclic
nucleotide phosphodiesterases is selected from the group consisting
of sildenafil, tadalafil, vardenafil udenafil, and avanafil.
[0069] [40] A method of transplanting a donor organ from an organ
donor to an organ recipient, comprising any single step, any
combination of steps, or all steps selected from the group
consisting of steps i)-iii):
[0070] i) administering to said organ donor prior to, during, both
prior to and during, after, or any combination thereof, donation of
said donor organ an effective amount of said monoclonal antibody or
antigen-binding fragment thereof of any one of [1]-[5], and/or a
monoclonal antibody, or antigen-binding fragment thereof, that
competes with said monoclonal antibody or antigen-binding fragment
thereof of any one of [1]-[5] for binding to CD47;
[0071] ii) contacting said donor organ prior to, during, both prior
to and during, after, or any combination thereof, transplantation
to said organ recipient, and an effective amount of said monoclonal
antibody or antigen-binding fragment thereof of any one of [1]-[5],
and/or a monoclonal antibody, or antigen-binding fragment thereof,
that competes with said monoclonal antibody or antigen-binding
fragment thereof of any one of [1]-[5] for binding to CD47; and
[0072] iii) administering to said organ recipient prior to, during,
both prior to and during, after, or any combination thereof,
transplantation of said donor organ to said organ recipient, an
effective amount of said monoclonal antibody or antigen-binding
fragment thereof of any one of [1]-[5], and/or a monoclonal
antibody, or antigen-binding fragment thereof, that competes with
said monoclonal antibody or antigen-binding fragment thereof of any
one of [1]-[5] for binding to CD47.
[0073] [41] The method of [40], wherein said monoclonal antibody or
antigen-binding fragment thereof of any one of [1]-[5], or
monoclonal antibody, or antigen-binding fragment thereof, that
competes with said monoclonal antibody or antigen-binding fragment
thereof of any one of [1]-[5] for binding to CD47, reduces ischemia
reperfusion injury in said donor organ.
[0074] [42] The method of [40] or [41], further comprising
administering to said organ donor, said donor organ, said organ
recipient, or any combination thereof, an effective amount of a
nitric oxide donor, precursor, or both; a nitric oxide generating
topical agent; an agent that activates soluble guanylyl cyclase; or
an agent that inhibits cyclic nucleotide phosphodiesterases; or any
combination of any of the foregoing.
[0075] [43] The method of [42], wherein said nitric oxide donor or
precursor is selected from the group consisting of NO gas,
isosorbide dinitrate, nitrite, nitroprusside, nitroglycerin,
3-Morpholino-sydnonimine (SIN-1), S-nitroso-N-acetylpenicillamine
(SNAP), Diethylenetriamine/NO (DETA/NO), S-nitrosothiols,
Bidil.RTM., and arginine; and said agent that inhibits cyclic
nucleotide phosphodiesterases is selected from the group consisting
of sildenafil, tadalafil, vardenafil, udenafil, and avanafil.
[0076] [44] A method of treating an autoimmune or inflammatory
disease in a patient in need thereof, comprising administering to
said patient an effective amount of said monoclonal antibody, or
antigen-binding fragment thereof, or competing antibody or antigen
binding fragment thereof, of any one of [1]-[5].
[0077] [45] The method of [44], wherein said autoimmune or
inflammatory disease is selected from the group consisting of
arthritis, multiple sclerosis, psoriasis, Crohn's disease,
inflammatory bowel disease, lupus, Grave's disease and Hashimoto's
thyroiditis, and ankylosing spondylitis.
[0078] [46] The method of [44] or [45], wherein said patient is a
human.
[0079] [47] The method of [44] or [45], wherein said patient is a
companion/pet animal, working animal, sport animal, zoo animal, or
other valuable animal kept in captivity.
[0080] [48] The method of any one of [44]-[47], further comprising
administering to said patient an effective amount of 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.
[0081] [49] The method of [48], wherein said nitric oxide donor or
precursor is selected from the group consisting of NO gas,
isosorbide dinitrate, nitrite, nitroprusside, nitroglycerin,
3-Morpholino-sydnonimine (SIN-1), S-nitroso-N-acetylpenicillamine
(SNAP), Diethylenetriamine/NO (DETA/NO), S-nitrosothiols,
Bidil.RTM., and arginine; and said agent that inhibits cyclic
nucleotide phosphodiesterases is selected from the group consisting
of sildenafil, tadalafil, vardenafil, udenafil, and avanafil.
[0082] [50] A method of treating a susceptible cancer in a human or
companion/pet animal, working animal, sport animal, zoo animal, or
other valuable animal kept in captivity in need thereof, comprising
administering thereto an effective amount of a monoclonal antibody
or antigen binding fragment thereof, or competing antibody or
antigen binding fragment thereof, of any one of [1]-[5].
[0083] [51] The method of [50], wherein said susceptible cancer 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, 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, melanoma, myelodysplastic
syndrome, and sarcomas including, but not limited to, osteosarcoma,
Ewing sarcoma, leiomyosarcoma, synovial sarcoma, alveolar soft part
sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma,
and chrondrosarcoma.
[0084] [52] The method of [51], wherein said leukemia is selected
from the group consisting of systemic mastocytosis, acute
lymphocytic (lymphoblastic) leukemia (ALL), T cell--ALL, acute
myeloid leukemia (AML), myelogenous leukemia, chronic lymphocytic
leukemia (CLL), multiple myeloma (MM), chronic myeloid leukemia
(CML), myeloproliferative disorder/neoplasm, myelodysplastic
syndrome, monocytic cell leukemia, and plasma cell leukemia;
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, and
Waldenstrom's Macroglobulinemia. [53] The method of any one of
[50]-[52], wherein said monoclonal antibody or antigen-binding
fragment thereof, or competing antibody or antigen binding fragment
thereof, of any one of [1]-[45] increases phagocytosis of cells of
said susceptible cancer.
[0085] [54] The method of [53], wherein said monoclonal antibody or
antigen-binding fragment thereof, or competing antibody or antigen
binding fragment thereof, of any one of [1]-[5], increases
phagocytosis of cells of said susceptible cancer and inhibits
SIRPalpha binding to CD47.
[0086] [55] A humanized monoclonal antibody, or antigen-binding
fragment thereof, that specifically binds human CD47.
[0087] [56] The use or method of any one of the previously
described embodiments, further comprising administering to said
patient an anti-tumor therapeutic treatment selected from the group
consisting of surgery, radiation, an anti-tumor or anti-neoplastic
agent, and combinations of any of the foregoing.
[0088] [57] The use or method of [56], wherein said an anti-tumor
or anti-neoplastic agent is a small chemical molecule or a biologic
therapeutic.
[0089] [58] The use or method of [57], wherein said small chemical
molecule or biologic therapeutic is selected from the group
consisting of an alkylating agent; an antimetabolite; a natural
product; a miscellaneous agent used in cancer therapy; a hormone;
an antagonist; a monoclonal antibody or antigen-binding fragment
thereof; a cytokine; an antisense oligonucleotide; an siRNA; or a
miRNA.
[0090] [59] A method of enhancing the therapeutic effect of a
soluble guanylyl cyclase activator, comprising administering to a
patient in need thereof:
[0091] i) an effective amount of a soluble guanylyl cyclase
activator, and
[0092] ii) a monoclonal antibody or antigen-binding fragment
thereof, or competing antibody or antigen-binding fragment thereof,
of any one of [1]-[5] in an amount effective to enhance said
therapeutic effect of said soluble guanylyl cyclase activator.
[0093] [60] The method of [59], wherein said therapeutic effect
comprises treatment of ischemia-reperfusion injury, or an
autoimmune or inflammatory disease, in a human or companion/pet
animal, working animal, sport animal, zoo animal, or other valuable
animal kept in captivity.
[0094] [61] The method of [60], wherein said ischemia-reperfusion
injury occurs in organ transplantation, acute kidney injury,
cardiopulmonary bypass surgery, pulmonary hypertension, sickle cell
disease, myocardial infarction, stroke, surgical resections and
reconstructive surgery, reattachment of an appendage or other body
part, skin grafting, or trauma.
[0095] [62] The method of [60], wherein said autoimmune or
inflammatory disease is selected from the group consisting of
arthritis, multiple sclerosis, psoriasis, Crohn's disease,
inflammatory bowel disease, lupus, Grave's disease and Hashimoto's
thyroiditis, and ankylosing spondylitis.
[0096] [63] Use of said monoclonal antibody or antigen-binding
fragment thereof, or competing antibody or antigen-binding fragment
thereof, of any one of [1]-[5] for the manufacture of a medicament
to enhance the therapeutic effect of a soluble guanylyl cyclase
activator.
[0097] [64] The use of [63], wherein said therapeutic effect
comprises treatment of ischemia-reperfusion injury, or an
autoimmune or inflammatory disease, in a human or companion/pet
animal, working animal, sport animal, zoo animal, or other valuable
animal kept in captivity.
[0098] [65] The use of [64], wherein said ischemia-reperfusion
injury occurs in organ transplantation, acute kidney injury,
cardiopulmonary bypass surgery, pulmonary hypertension, sickle cell
disease, myocardial infarction, stroke, surgical resections and
reconstructive surgery, reattachment of an appendage or other body
part, skin grafting, and trauma.
[0099] [66] The use of [64], wherein said autoimmune or
inflammatory disease is selected from the group consisting of
arthritis, multiple sclerosis, psoriasis, Crohn's disease,
inflammatory bowel disease, lupus, Grave's disease and Hashimoto's
thyroiditis, and ankylosing spondylitis.
[0100] [67] A method of increasing the level of cGMP in vascular
cells, comprising administering to said cells:
[0101] i) an effective amount of a soluble guanylyl cyclase
activator, and
[0102] ii) a monoclonal antibody or antigen-binding fragment
thereof, or competing antibody or antigen-binding fragment thereof,
of any one of [1]-[5] in an amount effective to increase the level
of cGMP in said vascular cells.
[0103] [68] The monoclonal antibody or antigen-binding fragment
thereof, or competing antibody or antigen-binding fragment thereof,
of any one of [1]-[5], which is an IgG isotype selected from among
IgG1 isotype, IgG2 isotype, IgG3 isotype, and IgG4 isotypes
[0104] [69] A pharmaceutical composition, comprising said
monoclonal antibody or antigen-binding fragment thereof, or
competing antibody or antigen-binding fragment thereof, of [68],
and a pharmaceutically or physiologically acceptable carrier,
diluent, or excipient.
[0105] [70] The monoclonal antibody or antigen-binding fragment
thereof, or competing antibody or antigen-binding fragment thereof,
of [68] for use in human therapy or therapy of companion/pet
animals, working animals, sport animals, zoo animals, or therapy of
other valuable animals kept in captivity.
[0106] [71] The monoclonal antibody or antigen-binding fragment
thereof, or competing antibody or antigen-binding fragment thereof,
of [68] for use in treating ischemia-reperfusion injury, or an
autoimmune or inflammatory disease, in a human or companion/pet
animal, working animal, sport animal, zoo animal, or other valuable
animal kept in captivity.
[0107] [72] The monoclonal antibody or antigen-binding fragment
thereof, or competing antibody or antigen-binding fragment thereof,
of [68] for use in treating a susceptible cancer.
[0108] [73] Use of said monoclonal antibody or antigen-binding
fragment thereof, or competing antibody or antigen-binding fragment
thereof, of [68] to treat ischemia-reperfusion injury, or an
autoimmune or inflammatory disease, in a human or companion/pet
animal, working animal, sport animal, zoo animal, or other valuable
animal kept in captivity.
[0109] [74] Use of said monoclonal antibody or antigen-binding
fragment thereof, or competing antibody or antigen-binding fragment
thereof, of [68] to treat a susceptible cancer. [75] Use of said
monoclonal antibody or antigen-binding fragment thereof, or
competing antibody or antigen-binding fragment thereof, of [68] for
the manufacture of a medicament to treat ischemia-reperfusion
injury, or an autoimmune or inflammatory disease, in a human or
companion/pet animal, working animal, sport animal, zoo animal, or
other valuable animal kept in captivity.
[0110] [76] Use of said monoclonal antibody or antigen-binding
fragment thereof, or competing antibody or antigen-binding fragment
thereof, of [68] for the manufacture of a medicament to treat a
susceptible cancer.
[0111] Expressly encompassed herein is the use of the monoclonal
antibodies or antigen-binding fragments thereof of [68]-[76] in any
of the methods, uses, compositions, or any other embodiments
disclosed herein.
[0112] [77] The monoclonal antibody or antigen-binding fragment
thereof, or competing antibody or antigen-binding fragment thereof,
of any one of [1]-[5], wherein said monoclonal antibody or
antigen-binding fragment thereof, or competing antibody or
antigen-binding fragment thereof, is human IgG1 isotype, the human
IgG1 constant region is modified at amino acid Asn297 to prevent to
glycosylation; and/or at amino acid Leu 234 and/or Leu235 to alter
Fc receptor interactions; and/or to enhance FcRn binding; and/or to
alter antibody-dependent cellular cytotoxicity and/or
complement-dependent cytotoxicity; and/or to induce
heterodimerization, optionally further by introduction of a
disulfide bond;
[0113] i. when said monoclonal antibody or antigen-binding fragment
thereof, or competing antibody or antigen-binding fragment thereof,
is human IgG2 isotype, the human IgG2 constant region is modified
at amino acid Asn297 to prevent to glycosylation; and/or to enhance
FcRn binding; and/or to alter antibody-dependent cellular
cytotoxicity and/or complement-dependent cytotoxicity; and/or to
induce heterodimerization, optionally further by introduction of a
disulfide bond;
[0114] ii. when said monoclonal antibody or antigen-binding
fragment thereof, or competing antibody or antigen-binding fragment
thereof, is human IgG3 isotype, the human IgG3 constant region is
modified at amino acid Asn297 to prevent to glycosylation; and/or
at amino acid 435 to extend half-life; and/or to enhance FcRn
binding; and/or to alter antibody-dependent cellular cytotoxicity
and/or complement-dependent cytotoxicity; and/or to induce
heterodimerization, optionally further by introduction of a
disulfide bond;
[0115] iii. when said monoclonal antibody or antigen-binding
fragment thereof, or competing antibody or antigen-binding fragment
thereof, is human IgG4 isotype, the human IgG4 constant region is
modified within the hinge region to prevent or reduce strand
exchange; and/or at amino acid 235 to alter Fc receptor
interactions; and/or at amino acid Asn297 to prevent glycosylation;
and/or to enhance FcRn binding; and/or to alter antibody-dependent
cellular cytotoxicity; and/or complement-dependent cytotoxicity;
and/or to induce heterodimerization, optionally further by
introduction of a disulfide bond.
[0116] [78] The monoclonal antibody or antigen-binding fragment
thereof, or competing antibody or antigen-binding fragment thereof,
of [77], wherein:
[0117] i. when said monoclonal antibody or antigen-binding fragment
thereof, or competing antibody or antigen-binding fragment thereof,
is human IgG1 isotype, the human IgG1 constant region is modified
at amino acid Asn297 to prevent to glycosylation by modification of
Asn297.fwdarw.Ala (N297A) or Asn297.fwdarw.Gln(N297Q); and/or at
amino acid Leu 234 by modification of Leu234.fwdarw.Ala (L234A)
and/or Leu235 by modification of Leu235.fwdarw.Glu (L235E) or
Leu235.fwdarw.Ala (L235A) or at both amino acid 234 and 235 by
modification of Leu234.fwdarw.Ala and Leu235.fwdarw.Ala to alter Fc
receptor interactions; and/or to enhance FcRn binding by
modification of Met252.fwdarw.Tyr, Ser254.fwdarw.Thr,
Thr256.fwdarw.Glu, Met428.fwdarw.Leu, or Asn434.fwdarw.Ser; and/or
to alter antibody-dependent cellular cytotoxicity and/or
complement-dependent cytotoxicity; and/or to induce
heterodimerization by modification of Thr366.fwdarw.Trp, and
optionally further by introduction of a disulfide bond by
modification of Ser354.fwdarw.Cys and Tyr349.fwdarw.Cys on opposite
CH3 domains;
[0118] ii. when said monoclonal antibody or antigen-binding
fragment thereof, or competing antibody or antigen-binding fragment
thereof, is human IgG2 isotype, the human IgG2 constant region is
modified at amino acid Asn297 to prevent to glycosylation by
modification of Asn297.fwdarw.Ala or Asn297.fwdarw.Gln; and/or to
enhance FcRn binding by modification of Met252.fwdarw.Tyr,
Ser254.fwdarw.Thr, Thr256.fwdarw.Glu, Met428.fwdarw.Leu, or
Asn434.fwdarw.Ser; and/or to alter antibody-dependent cellular
cytotoxicity and/or complement-dependent cytotoxicity; and/or to
induce heterodimerization by modification of Thr366.fwdarw.Trp, and
optionally further by introduction of a disulfide bond by
modification of Ser354.fwdarw.Cys and Tyr349.fwdarw.Cys on opposite
CH3 domains;
[0119] iii. when said monoclonal antibody or antigen-binding
fragment thereof, or competing antibody or antigen-binding fragment
thereof, is human IgG3 isotype, the human IgG3 constant region is
modified at amino acid Asn297 to prevent to glycosylation by
modification of Asn297.fwdarw.Ala or Asn297.fwdarw.Gln; and/or at
amino acid 435 to extend half-life by modification of
Arg435.fwdarw.His; and/or to enhance FcRn binding by modification
of Met252.fwdarw.Tyr, Ser254.fwdarw.Thr, Thr256.fwdarw.Glu,
Met428.fwdarw.Leu, or Asn434.fwdarw.Ser; and/or to alter
antibody-dependent cellular cytotoxicity and/or
complement-dependent cytotoxicity; and/or to induce
heterodimerization by modification of Thr366.fwdarw.Trp, and
optionally further by introduction of a disulfide bond by
modification of Ser354.fwdarw.Cys and Tyr349.fwdarw.Cys on opposite
CH3 domains;
[0120] iv. when said monoclonal antibody or antigen-binding
fragment thereof, or competing antibody or antigen-binding fragment
thereof, is human IgG4 isotype, the human IgG4 constant region is
modified within the hinge region to prevent or reduce strand
exchange by modification of Ser228.fwdarw.Pro; and/or at amino acid
235 to alter Fc receptor interactions by modification of
Leu235.fwdarw.Glu, or by modification within the hinge and at amino
acid 235 by modifying Ser228.fwdarw.Pro and Leu235.fwdarw.Glu;
and/or at amino acid Asn297 to prevent glycosylation by
modification of Asn297.fwdarw.Ala; and/or to enhance FcRn binding
by modification of Met252.fwdarw.Tyr, Ser254.fwdarw.Thr,
Thr256.fwdarw.Glu, Met428.fwdarw.Leu, or Asn434.fwdarw.Ser; and/or
to alter antibody-dependent cellular cytotoxicity and/or
complement-dependent cytotoxicity; and/or to induce
heterodimerization by modification of Thr366.fwdarw.Trp, and
optionally further by introduction of a disulfide bond by
modification of Ser354.fwdarw.Cys and Tyr349.fwdarw.Cys on opposite
CH3 domains.
[0121] Expressly encompassed herein is the use of the monoclonal
antibodies or antigen binding fragments thereof of [77]-[78] in any
of the methods, uses, compositions, or any other embodiments
disclosed herein.
[0122] Further scope of the applicability of the present antibody
compounds and methods will become apparent from the detailed
description provided below. However, it should be understood that
the detailed description and specific examples, while indicating
particular 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
[0123] 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, in which:
[0124] FIGS. 1A, 1B, 1C, and 1D show cross species binding curves
to human, mouse, rat, and porcine RBCs, respectively, generated
using various concentrations of purified antibodies from clones
(Cl) Cl 1, Cl 1.1 (hum01 IgG1 N297Q), Cl 13, and Cl 13.1 (hum13
IgG1 N297Q) as described in Example 3. Clones Cl 1 and Cl 13 are as
described in Table 3. Clones Cl 1.1 and Cl 13.1 are Fc mutants of
clones Cl 1 and Cl 13, respectively, modified to reduce Fc effector
function. Each has an Asn297.fwdarw.Gln (N297Q) mutation in the Fc
domain (Sazinsky et al. (2008) PNAS 105(51):20167-20172). All of
these clones exhibit concentration-dependent binding to all of the
species of RBCs tested. RBCs are incubated for 60 minutes on ice
with various concentrations of purified antibodies from clones Cl
1, Cl 1.1, Cl 13, and Cl 13.1. Cells are then washed with cold PBS
containing EDTA, incubated for an additional hour on ice with FITC
labeled donkey anti-human antibody, washed, and antibody binding is
analyzed using a BD FACS Aria Cell Sorter (Becton Dickinson) or a
C6 Accuri Flow Cytometer (Becton Dickinson).
[0125] FIG. 2 shows CD47 mAb-mediated phagocytosis of Jurkat cells.
Target (Jurkat) and effector (human macrophages) are combined at a
target effector ratio of 4:1, 10 .mu.g/ml of the CD47 mAbs added
and incubated for 2 hours at 37 C. Percent phagocytosis is
determined by flow cytometry as the percentage of
CFSE.sup.+/CD14.sup.+ cells from the total CD14.sup.+ population.
All of the clones, Clone 1, Clone 1.1, Clone 1-IgG4 (Clone 1 IgG4
S228P, Clone 1.3), Clone 13, Clone 13.1, and Clone 13-IgG4 (Clone
13 IgG4 S228P, Clone 13.3), except the negative control mAb 2D3,
increase phagocytosis of the Jurkat cells by the macrophages
irrespective of their affinity to activate Fc receptors.
[0126] FIG. 3 shows reversal of TSP1 inhibition of NO-stimulated
cGMP production by humanized antibodies of the present disclosure.
As described in Example 5, Jurkat JE6.1 cells are incubated
overnight in serum-free medium and then incubated with humanized
antibodies of the present disclosure or the control chimeric mAb,
and with or without TSP1, followed by treatment with or without a
NO donor. Cells are lysed 5 minutes later and cGMP is measured.
None of the present humanized antibody clones tested, or the
control chimeric mAb, has an effect on basal cGMP levels. The
control chimeric antibody reverses the TSP1 inhibition, as do
humanized Clones 1, 9, 11, and 24 disclosed herein (Cl 1; Cl 9; Cl
11; Cl 24, respectively). PBS: phosphate buffered saline; TSP or
TSP: thrombospondin-1; DEA: diethylamine NONOate; chim: chimeric
antibody >VxP037-01LC (SEQ ID NO:7))/>VxP037-01HC (SEQ ID
NO:57).
[0127] FIG. 4 shows reversal of TSP1 inhibition of NO-stimulated
cGMP production by humanized antibodies of the present disclosure.
As described in Example 5, Jurkat JE6.1 cells are incubated
overnight in serum-free medium and then incubated with purified
humanized Clone 1 and 13 antibodies, or PBS as the control, and
with or without TSP1, followed by treatment with or without a NO
donor. Cells are lysed 5 minutes later and cGMP is measured. The
humanized antibody clones or PBS have no effect on basal cGMP
levels. The humanized Clones 1 and 13 reverse the TSP1 inhibition,
while PBS has no effect. PBS: phosphate buffered saline; TSP or
TSP1: thrombospondin-1; DEA: diethylamine NONOate.
[0128] FIG. 5 shows that treatment of a donor kidney with Clone 1
(Cl 1) at the time of organ harvest is effective in reducing IRI
and kidney damage in vivo in a rat kidney transplantation model as
assessed by measuring serum creatinine. A syngeneic rat renal
transplantation model of IRI with bilaterally nephrectomized
recipients is used to evaluate the effect of the anti-CD47
monoclonal antibody Clone 1 on graft function following
transplantation. Male Lewis rats weighing 275-300 g are used as
both donor and recipient animals. Donor kidneys are flushed with 50
.mu.g of purified Clone 1 or vehicle (phosphate buffered saline, pH
7.2), stored at 4.degree. C. in University of Wisconsin
preservation (WU) solution for 6 hours, and then transplanted. Two
days following transplantation, kidney function is assessed by
measuring serum creatinine. Treatment of donor kidneys with Clone 1
results in improved kidney function compared to controls as
measured by a reduction in serum creatinine.
[0129] FIG. 6 shows the survival in a DCD (donation after cardiac
death) rat kidney transplant model. Male Lewis rats weighing
275-300 g underwent 60 minutes of warm ischemia, prior to flushing
the donor kidneys with 50 .mu.g of purified Clone 1.1 or an IgG
control mAb. Kidneys are then stored at 4.degree. C. in University
of Wisconsin preservation solution (UW) for 6 hours prior to
transplantation. In this experiment, survival is monitored over a 7
day time period. All animals that received the IgG control
mAb-treated kidney died within 4 days. In contrast, survival is
significantly prolonged in the animals that received the Clone1.1
treated kidney, with 30% of the animals surviving for the 7 day
duration of the experiment. This shows that with extended periods
of warm ischemia, treatment of donor kidneys with Clone 1.1 reduces
IRI and increases survival of the recipient.
[0130] FIG. 7 shows that purified, humanized antibody Clone 13 (Cl
13) reduces tumor burden in vivo in a syngeneic mouse Acute
Promyelocytic Leukemia (APL) model. Murine APL cells (B6APL1) are
injected intravenously into C57BL/6 mice randomized into three
groups (5-10 mice per group): Group 1: no APL; Group 2: APL with no
treatment; Group 3: APL treated with anti-CD47mAb Cl 13. Antibody
treatment is initiated on the day of tumor inoculation (day 0), and
given in single doses of 10 .mu.g/dose (0.4 mg/kg) by
intraperitoneal injection on days 0, 3, and 6. Circulating APL
cells (representing the tumor burden) are evaluated at day 25
following tumor inoculation by flow cytometry
(CD34.sup.+/CD117.sup.+ cells). Mice treated with Cl 13 have
reduced tumor burden compared to untreated mice at 25 days after
tumor inoculation, demonstrating anti-tumor activity of this
humanized clone.
[0131] FIG. 8 shows that treatment with the CD47 mAb Clone 1.1
significantly reduced tumor growth of the human HepG2
hepatocellular carcinoma model. Male NSG mice are obtained from The
Jackson Laboratory (Bar Harbor, Me.) and housed in cages in
temperature and light-controlled environments with access to water
and food ad libitum. For the heterotopic xenograft model,
HepG2-luc2 cells (Perkin Elmer, Waltham, Mass. #134280) are
suspended in DMEM containing 25% (v/v), and 1,000,000 cells
implanted subcutaneously into the dorsal subcutaneous space of 4-
to 8-wk-old NSG mice. After 2 weeks of growth, antibody treatment
is begun with twice-weekly intraperitoneal injections of 15 mg/kg
of either anti-CD47 antibody Clone 1.1 or an IgG control for 6
weeks. Tumor volumes are calculated twice weekly using
(length.times.width)/0.6. After 6 weeks of treatment, animals are
euthanized and tumors were resected, weighed, and fixed in 10%
formalin.
[0132] FIG. 9 shows binding of Clone 1.1 (IgG1 N297Q) and clone 1.3
(IgG4 S228P) to cynomolgus monkey RBCs by FACS (described
previously). Both mAbs exhibited similar binding affinities to
cynomolgus RBCs.
[0133] FIG. 10 shows that Clones 1.1, 1.2 (IgG4 with mutations of
residue S228 to P and of residue L235 to E to reduce Fc effector
function, and 1.3 (IgG4 with mutation of residue S228 to P)
increase phagocytosis of Jurkat cells by human macrophages while
control IgG or the mouse 2D3 antibody that binds to CD47, but does
not block the CD47/SIRPalpha interaction, do not.
[0134] FIG. 11 shows Clones 1.1, 1.2, 1.3, 13.1, 13.2 and 13.3 all
block the interaction of CD47 (expressed on the Jurkat cells) with
SIPRalpha while a control antibody that does not bind to CD47 did
not block the CD47/SIRPalpha interaction. Absorbance was read at
450 nm.
DETAILED DESCRIPTION OF THE INVENTION
[0135] The following detailed description is provided to aid those
skilled in the art in practicing the various embodiments of the
present disclosure described herein, including all the methods,
uses, compositions, etc., described herein. Even so, the following
detailed description should not be construed to unduly limit the
present disclosure, as modifications and variations in the
embodiments herein discussed may be made by those of ordinary skill
in the art without departing from the spirit or scope of the
present discoveries.
[0136] Any feature, or combination of features, described herein
is(are) included within the scope of the present disclosure,
provided that the features included in any such combination are not
mutually inconsistent as will be apparent from the context, this
specification, and the knowledge of one of ordinary skill in the
art. Additional advantages and aspects of the present disclosure
are apparent in the following detailed description and claims.
[0137] The contents of all publications, patent applications,
patents, and other references mentioned herein are incorporated by
reference herein in their entirety. In case of conflict, the
present specification, including explanations of terms, will
control.
[0138] Antibody compounds of the present disclosure can bind to
epitopes in the extracellular IgV domain of CD47, inhibiting TSP1
and SIRPalpha binding to CD47 and receptor activation, while
inducing little or no agonist activity, and promoting tumor cell
phagocytic clearance. In view of these properties, antibody
compounds of the present disclosure should be therapeutically
useful in treating many forms of IRI and cancers.
[0139] The present antibody compounds can also possess a number of
other desirable properties, including broad reactivity with CD47 of
a wide variety of mammalian species, including that of human,
mouse, rat, pig, cynomolgus monkey, and/or dog, i.e., any
individual one of these mammalian species, or various combinations
thereof, making these antibodies useful in both human and
veterinary medicine. This broad reactivity is further advantageous
in that it facilitates preclinical studies including, but not
limited to, safety and efficacy studies, in a variety of mammalian
species, and therefore the development of such antibodies as human
and veterinary therapeutics.
[0140] Thus, antibody compounds of the present disclosure exhibit
the following desirable therapeutic activities: [0141] Binding to
CD47 of multiple mammalian species; [0142] Blocking SIRPalpha and
TSP1 binding to CD47; [0143] Promoting phagocytosis of cancer
cells; and [0144] Reversing TSP1 inhibition of nitric oxide
signaling and are therefore useful in treating ischemia reperfusion
injury and cancers.
DEFINITIONS
[0145] The following definitions are provided to aid the reader in
understanding the various aspects of the present disclosure. Unless
defined otherwise, all technical and scientific terms used herein
have the same meaning as commonly understood by those of ordinary
skill in the art to which this disclosure pertains.
[0146] A full-length antibody as it exists naturally is an
immunoglobulin molecule comprising two heavy (H) chains and two
light (L) chains interconnected by disulfide bonds. The amino
terminal portion of each chain 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 primarily responsible for effector
function.
[0147] The CDRs are interspersed with regions that are more
conserved, termed framework regions ("FR"). 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. While the light chain CDRs and heavy chain CDRs
disclosed herein are numbered 1, 2, and 3, respectively, it is not
necessary that they be employed in the corresponding antibody
compound light and heavy chain variable regions in that numerical
order, i.e., they can be present in any numerical order in a light
or heavy chain variable region, respectively.
[0148] Light chains are classified as kappa or lambda, and are
characterized by a particular constant region as known in the art.
Heavy chains are classified as gamma, mu, alpha, delta, or epsilon,
and define the isotype of an antibody as IgG, IgM, IgA, IgD, or
IgE, respectively. IgG antibodies can be further divided into
subclasses, e.g., IgG, IgG2, IgG3, IgG4. Each heavy chain type is
characterized by a particular constant region with a sequence well
known in the art.
[0149] The monoclonal antibodies and other antibody compounds
useful in the methods and compositions described herein can be any
of these isotypes. Furthermore, any of these isotypes can comprise
amino acid modifications as follows.
[0150] In some embodiments, the antibody constant region is of
human IgG1 isotype.
[0151] In some embodiments, the human IgG1 constant region is
modified at amino acid Asn297 (Kabat Numbering) to prevent to
glycosylation of the antibody. For example, this modification can
be Asn297.fwdarw.Ala (N297A) or Asn297.fwdarw.Gln (N297Q) (Sazinsky
et al. (2008) PNAS 105(51):20167-20172).
[0152] In some embodiments, the constant region of the antibody is
modified at amino acid Leu234 (Kabat Numbering) to alter Fc
receptor interactions. For example, this modification can be
Leu234.fwdarw.Ala (L234A).
[0153] In some embodiments, the constant region of the antibody is
modified at amino acid Leu235 (Kabat Numbering) to alter Fc
receptor interactions. For example, this modification can be
Leu235.fwdarw.Glu (L235E) or Leu235.fwdarw.Ala (L235A).
[0154] In some embodiments, the constant region of the antibody is
altered at both amino acid 234 and 235. For example, these
modifications can be Leu234.fwdarw.Ala and Leu235.fwdarw.Ala
(L234A/L235A) (EU index of Kabat et al. (1991) Sequences of
Proteins of Immunological Interest).
[0155] In some embodiments, the constant region of the antibody is
of human IgG2 isotype.
[0156] In some embodiments, the human IgG2 constant region is
modified at amino acid Asn297 (Kabat Numbering) to prevent to
glycosylation of the antibody. For example, this modification can
be Asn297.fwdarw.Ala (N297A) or Asn297.fwdarw.Gln(N297Q).
[0157] In some embodiments, the constant region of the antibody is
of human IgG3 isotype.
[0158] In some embodiments, the human IgG3 constant region is
modified at amino acid Asn297 (Kabat Numbering) to prevent to
glycosylation of the antibody. For example, this modification can
be Asn297.fwdarw.Ala (N297A) or Asn297.fwdarw.Gln(N297Q).
[0159] In some embodiments, the human IgG3 constant region is
modified at amino acid 435 to extend the half-life. For example,
this modification can be Arg435.fwdarw.His (R435H) (EU index of
Kabat et al. (1991) Sequences of Proteins of Immunological
Interest).
[0160] In some embodiments, the constant region of the antibody is
of human IgG4 isotype.
[0161] In some embodiments, the human IgG4 constant region is
modified within the hinge region to prevent or reduce strand
exchange. For example, this modification can be Ser228.fwdarw.Pro
(S228P) (Angal et al. (1993) Molecular Immunology
30(1):105-108).
[0162] In other embodiments, the human IgG4 constant region is
modified at amino acid 235 to alter Fc receptor interactions. For
example, this can be Leu235.fwdarw.Glu (L235E).
[0163] In some embodiments, the human IgG4 constant region is
modified within the hinge and at amino acid 228 and in the Fc at
amino acid 235. For example, this can be Ser228.fwdarw.Pro and
Leu235.fwdarw.Glu (S228P/L235E).
[0164] In some embodiments, the human IgG4 constant region is
modified at amino acid Asn297 (Kabat Numbering) to prevent to
glycosylation of the antibody. For example, this can be
Asn297.fwdarw.Ala (N297A). (EU index of Kabat et al. (1991)
Sequences of Proteins of Immunological Interest).
[0165] In some embodiments, the human IgG constant region is
modified to enhance FcRn binding. Examples of Fc mutations that
enhance binding to FcRn are Met252.fwdarw.Tyr, Ser254.fwdarw.Thr,
Thr256.fwdarw.Glu (M252Y, S254T, and T256E, respectively) (Kabat
numbering, Dall'Acqua et al. (2006) J. Biol. Chem. 281(33)
23514-23524), or Met428.fwdarw.Leu and Asn434.fwdarw.Ser (M428L,
N434S) (Zalevsky et al. (2010) Nature Biotech. 28(2):157-159). (EU
index of Kabat et al. (1991) Sequences of Proteins of Immunological
Interest).
[0166] In some embodiments, the human IgG constant region is
modified to alter antibody-dependent cellular cytotoxicity (ADCC)
and/or complement-dependent cytotoxicity (CDC), e.g., the amino
acid modifications described in Natsume et al. (2008) Cancer Res.
68(10):3863-72; Idusogie et al. (2001) J. Immunol. 166(4):2571-5;
Moore et al. (2010) mAbs 2(2):181-189; Lazar et al. (2006) PNAS
103(11):4005-4010; Shields et al. (2001) J. Biol. Chem.
276(9):6591-6604; Stavenhagen et al. (2007) Cancer Res.
67(18):8882-8890; Stavenhagen et al. (2008) Advan. Enzyme Regul.
48:152-164; Alegre et al. (1992) J. Immunol. 148:3461-3468;
reviewed in Kaneko and Niwa (2011) Biodrugs 25(1):1-11.
[0167] In some embodiments, the human IgG constant region is
modified to induce heterodimerization. For example, having an amino
acid modification within the CH3 domain at Thr366, which when
replaced with a more bulky amino acid, such as Trp (T366W), is able
to preferentially pair with a second CH3 domain having amino acid
modifications to less bulky amino acids at positions Thr366,
Leu368, and Tyr407, e.g., Ser, Ala, and Val, respectively
(T366S/L368A/Y407V). Heterodimerization via CH3 modifications can
be further stabilized by the introduction of a disulfide bond, for
example by changing Ser354 to Cys (S354C) and Tyr349 to Cys (Y349C)
on opposite CH3 domains (reviewed in Carter (2001) Journal of
Immunological Methods 248:7-15).
[0168] 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, and
can be chimeric or humanized. Complete mAbs contain two heavy
chains and two light chains.
[0169] "Antigen binding fragments" of such monoclonal antibodies
may be desirable for certain applications due to their small size
and consequent superior tissue distribution, and include, for
example, Fab fragments, Fab' fragments, F(ab').sub.2 fragments, Fd
fragments, single chain Fv fragments (ScFv), and one-armed
antibodies comprising a light chain and a heavy chain. Preferred
antigen binding fragments are those that bind to the antigen
recognized by the intact antibody. Fc fragments can also be
obtained. Monoclonal antibodies and antigen-binding fragments
thereof of the present disclosure can be produced, for example, by
recombinant technologies, phage display technologies, synthetic
technologies, e.g., CDR-grafting, or combinations of such
technologies, or other technologies known in the art, including
proteolytic digestion of intact antibodies.
[0170] "Antibody compounds" refers to mAbs, antigen-binding
fragments thereof such as Fabs, etc., and competing antibodies,
disclosed herein that specifically bind CD47 of various species,
including human, rat, mouse, pig, cynomolgus monkey, and dog CD47,
and that exhibit the properties disclosed herein. Thus, the term
"mAb" as used herein with respect to antibodies encompassed by the
present disclosure includes Fabs and competing antibodies.
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
disclosed in Examples 3, 4, and 5, 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,
ISBN 0-87969-314-2.
[0171] The phrase "humanized antibodies" refers to monoclonal
antibodies and antigen binding fragments thereof, including the
antibody compounds disclosed herein, that have binding and
functional properties according to the disclosure similar to those
disclosed herein, and that have framework and constant regions that
are substantially human or fully human surrounding CDRs derived
from a non-human antibody. "Framework region" or "framework
sequence" refers to any one of framework regions 1 to 4. Humanized
antibodies and antigen binding fragments encompassed by the present
disclosure include molecules wherein any one or more of framework
regions 1 to 4 is substantially or fully human, i.e., wherein any
of the possible combinations of individual substantially or fully
human framework regions 1 to 4, is present. For example, this
includes molecules in which framework region 1 and framework region
2, framework region 1 and framework region 3, framework region 1,
2, and 3, 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.
[0172] 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 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 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, 5, or 6
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.
[0173] As used herein, the phrase "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.
[0174] 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)).
[0175] 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; & 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.
[0176] 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.
[0177] 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.
[0178] Fully human frameworks are those that are identical to a
known human germline framework sequence. Human framework germline
sequences can be obtained from ImMunoGeneTics (IMGT) via their
website or from The Immunoglobulin FactsBook by Marie-Paule Lefranc
and Gerard Lefranc, Academic Press, 2001, ISBN 012441351. For
example, germline light chain frameworks can be selected from the
group consisting of: A11, A17, A18, A19, A20, A27, A30, LI, L1I,
L12, L2, L5, L15, L6, L8, O12, O2, and 08, and germline heavy chain
framework regions can be selected from the group consisting of:
VH2-5, VH2-26, VH2-70, VH3-20, VH3-72, VHI-46, VH3-9, VH3-66,
VH3-74, VH4-31, VHI-18, VHI-69, VI-13-7, VH3-11, VH3-15, VH3-21,
VH3-23, VH3-30, VH3-48, VH4-39, VH4-59, and VH5-5I.
[0179] Humanized antibodies in addition to those disclosed herein
exhibiting similar functional properties according to the present
disclosure can be generated using several different methods. 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 in humanizing mouse antibodies include U.S. Pat. Nos.
4,816,397; 5,225,539; and 5,693,761; computer programs ABMOD and
ENCAD as described in Levitt (1983) J. Mol. Biol. 168:595-620; 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.
[0180] The identification of residues to consider for back-mutation
can be carried out as follows: 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 framework") is replaced
by a framework amino acid from a framework of the parent antibody
compound (the "donor framework"): (a) the amino acid in the human
framework region 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; (b) the position of the amino acid is immediately
adjacent to one of the CDRs; or (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.
[0181] When each of the amino acids in the human framework region
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.
[0182] Another approach to generating human engineered 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.
[0183] Single mutations producing improved properties can be
combined to assess their effects in combination with one
another.
[0184] Further, a combination of both of the foregoing approaches
is possible. After CDR grafting, one can back-mutate specific
framework regions 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.
[0185] The method described in Example 1 below can also be
employed.
[0186] 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 framework 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, including conservative
amino acid substitutions as are well known to those of ordinary
skill in the art. The methods disclosed herein can then be used to
screen these additional variable region amino acid sequences to
identify sequences having the indicated in vitro and/or in vivo
functions. In this way, further sequences suitable for preparing
human engineered antibodies and antigen-binding portions thereof in
accordance with the present disclosure can be identified. In some
embodiments, amino acid substitution within the frameworks can
include one, two, three, four, five, six, seven, eight, nine, or
ten positions within any one or more of the 4 light chain and/or
heavy chain framework regions disclosed herein. In some
embodiments, amino acid substitution within the CDRs is restricted
to one, two, three, four, or five positions within any one or more
of the 3 light chain and/or heavy chain CDRs. Combinations of the
various changes within these framework regions and CDRs described
above are also possible.
[0187] That the functional properties of the antibody compounds
generated by introducing the amino acid modifications discussed
above conform to, and are comparable to, those exhibited by the
specific molecules disclosed herein can be confirmed by the methods
disclosed in the Examples below.
[0188] 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.
[0189] "Binding affinity" is a term that 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, but it is not critical to the methods herein
that these constants be measured or determined. Rather, affinities
as used herein to describe interactions between molecules of the
described methods are generally apparent affinities (unless
otherwise specified) 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
concepts of binding affinity, association constant, and
dissociation constant are well known.
[0190] 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.
[0191] 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 labeled. If the labeled compound
and the unlabeled compound bind to separate and discrete sites on
CD47, the labeled 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 unlabeled
compound will compete, and the amount of labeled compound bound to
the antigen will be lowered. If the unlabeled compound is present
in excess, very little, if any, labeled 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., pages 567-569, ISBN
0-87969-314-2. 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 unlabeled
competing monoclonal antibody or antigen-binding fragment thereof
to inhibit the binding of the labeled molecule to the plate is
titrated. The results are plotted, and the concentrations necessary
to achieve the desired degree of binding inhibition are
compared.
[0192] Whether monoclonal antibodies 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 3-5, 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.
[0193] The 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, symptom, etc.,
of a disease or pathological condition after it has begun to
develop.
[0194] Acute events and chronic conditions can be treated. In an
acute event, an antibody or antigen binding fragment thereof is
administered at the onset of a symptom, disorder, condition,
disease, or procedure, and is discontinued when the acute event
ends, or in the case of organ transplantation to the organ, at the
time of organ harvest and/or to the transplant recipient at the
time of organ transplantation. In contrast, a chronic symptom,
disorder, condition, or disease is treated over a more protracted
time frame.
[0195] The 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.
[0196] 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. In some embodiments, an effective dose
will generally be from about 0.01 mg/kg to about 50 mg/kg, or about
0.05 mg/kg to about 10 mg/kg of the compositions of the present
disclosure in the individual to which it is administered.
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, or from
about 0.1 mg/kg to about 50 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/m.sup.2; etc.
[0197] 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 a biomarker, such as serum
biomarkers of injury of the treated organ, including but not
limited to liver, kidney, lung, intestine, pancreas and heart,
changes in pulmonary artery pressures, cell surface CD47 expression
in tumor or non-tumor tissues, tumor regression, circulating tumor
cells or tumor stem cells, etc. 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.
[0198] The antibody compounds of the present disclosure can be used
as medicaments in human and veterinary medicine, administered by a
variety of routes. 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.
[0199] Antibody compounds can be administered by any number of
routes including, but not limited to, oral, intravenous,
intramuscular, intra-arterial, intramedullary, intraperitoneal,
intrathecal, intraventricular, transdermal, transcutaneous,
topical, subcutaneous, intranasal, enteral, sublingual,
intravaginal, intravesiciular or rectal routes. 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.
[0200] Direct delivery of the compositions can generally be
accomplished by injection, subcutaneously, intraperitoneally,
intravenously, or intramuscularly, or delivered to the interstitial
space of a tissue. The compositions can also be administered into a
lesion such as a tumor. Dosage treatment may be a single dose
schedule or a multiple dose schedule.
[0201] In some embodiments, such compositions are formulated for
parenteral administration by, for example, intravenous,
intramuscular, subcutaneous, etc., administration by infusion,
injection, implantation, etc., as is well known in the art.
Examples include bolus injection or continuous infusion.
Intratumoral administration, for example by injection, is also
contemplated.
[0202] Such pharmaceutical compositions can be prepared by methods
well known in the art. See, e.g., Remington: The Science and
Practice of Pharmacy, 21.sup.st Edition (2005), Lippincott Williams
& Wilkins, Philadelphia, Pa., and comprise one or more antibody
compounds disclosed herein, and a pharmaceutically or veterinary
acceptable, e.g., physiologically acceptable, carrier, diluent, or
excipient.
Combination Therapies
[0203] The therapeutic methods encompassed herein include the use
of the antibodies disclosed herein alone, and/or in combinations
with one another, and/or with antigen-binding fragments thereof,
and/or with competing antibodies exhibiting appropriate
biological/therapeutic activity, as well, i.e., all possible
combinations of these antibody compounds.
[0204] In addition, the present therapeutic methods also encompass
the use of these antibodies, antigen-binding fragments thereof,
competing antibodies, etc., and combinations thereof further in
combination with: (1) any one or more of the nitric oxide donor,
precursor, or nitric oxide generating topical agents, and/or agents
that activate soluble guanylyl cyclase, and/or agents that inhibit
cyclic nucleotide phosphodiesterases disclosed herein, or (2) any
one or more anti-tumor therapeutic treatments selected from
surgery, radiation, anti-tumor or anti-neoplastic agents, and
combinations of any of these, 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.
Combinations of Antibody Compounds
[0205] It should be noted that in all of the therapeutic methods
disclosed and claimed herein, the monoclonal antibodies or antigen
binding fragments thereof, and monoclonal antibodies or antigen
binding fragments thereof that compete with these monoclonal
antibodies or antigen binding fragments thereof of the present
disclosure that bind to CD47, can be used alone, or in any
appropriate combinations with one another, to achieve the greatest
treatment efficacy.
Further Therapeutic Combinations to Treat IRI-Related
Indications
[0206] In addition to administering the combinations of antibody
compounds as described immediately above, 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 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.
[0207] 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.
[0208] 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.
[0209] The agent that inhibits cyclic nucleotide phosphodiesterases
can be selected from sildenafil, tadalafil, vardenafil, udenafil,
and avanafil.
Further Therapeutic Combinations to Treat Cancer Indications
[0210] In addition to the foregoing, the methods of the present
disclosure, for example those related to treatment of cancer
indications, can further comprise 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, etc., conventionally used, or currently
being developed, to treat cancer. This includes antibodies and
antigen-binding fragments other than those disclosed herein,
cytokines, antisense oligonucleotides, siRNAs, miRNAs, etc.
[0211] 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.
[0212] 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; and monoclonal
antibodies.
[0213] 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.
These agents that are involved in the immune response include IL-10
(Interleukin-10, human cytokin synthesis inhibitory factor, CSIF)
and Galectins.
[0214] 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 SIRPalpha, are
administered to a patient, increasing 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 Costimulator 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 (IL2R.alpha., Cluster of
Differentiation 25, Interleukin-2 Receptor .alpha. chain, IL-2
Receptor .alpha. 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
1 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, PDCDiLG2, 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, ENTPD 1), and CD73 (Ecto-5'-nucleotidase,
5'-nucleotidase, 5'-NT, Cluster of Differentiation 73), including
antibodies and small molecules, and agonists of 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 small molecules and antibodies, are also specifically
contemplated herein.
[0215] YERVOY.RTM. (ipilimumab; Bristol-Meyers Squibb) is an
example of an approved anti-CTLA-4 antibody.
[0216] KEYTRUDA.RTM. (pembrolizumab; Merck) and OPDIVO.RTM.
(nivolumab; Bristol-Meyers Squibb Company) are examples of approved
anti-PD-1 antibodies.
[0217] Another useful class of compounds for the combination
therapies contemplated herein includes modulators of SIRPalpha/CD47
binding such as antibodies to SIRPalpha, as well as soluble protein
fragments of this ligand, or CD47 itself, acting as "decoy"
molecules inhibiting binding of, or interfering with binding of,
SIRPalpha to CD47.
[0218] The present disclosure encompasses therapeutic methods
comprising not only the administration of any of the individual
monoclonal antibodies, antigen binding fragments thereof, or
competing antibodies disclosed herein with any one or more of the
molecules discussed immediately above, but also combinations of the
disclosed monoclonal antibodies, antigen-binding fragments thereof,
and competing antibodies in combinations with any one or more of
the molecules discussed immediately above, i.e., all possible
permutations and combinations of the presently disclosed
molecules.
[0219] The term "tumor" refers to all neoplastic cell growth and
proliferation, whether malignant or benign, and all pre-cancerous
and cancerous cells and tissues. The terms "cancer", "cancerous",
and "tumor" are not mutually exclusive as used herein.
[0220] 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, carcinomas, lymphomas, blastomas, sarcomas,
and leukemias.
[0221] The term "susceptible cancer" as used herein refers to a
cancer, cells of which express CD47 and that are responsive to
treatment with an antibody or antigen binding fragment thereof, or
competing antibody or antigen binding fragment thereof, of the
present disclosure. Exemplary susceptible cancers include, but are
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), multiple myeloma (MM), chronic myeloid leukemia
(CML), myeloproliferative disorder/neoplasm, myelodysplastic
syndrome, monocytic cell leukemia, and plasma cell leukemia;
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, and
Waldenstrom's Macroglobulinemia, ovarian cancer, breast cancer,
endometrial cancer, colon cancer (colorectal cancer), rectal
cancer, bladder 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, melanoma, myelodysplastic syndrome, and sarcomas
including, but not limited to, osteosarcoma, Ewing sarcoma,
leiomyosarcoma, synovial sarcoma, alveolar soft part sarcoma,
angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and
chrondrosarcoma.
[0222] "Phagocytosis" of cancer cells refers to the engulfment and
digestion of such cells by macrophages, and the eventual digestion
or degradation of these cancer cells and their release
extracellularly, or intracellularly, to undergo further processing.
Anti-CD47 monoclonal antibodies that block SIRPalpha binding to
CD47, the "don't eat me" signal which is highly expressed on cancer
cells as compared with normal cells, induce macrophage phagocytosis
of cancer cells. SIRPalpha binding to CD47 on cancer cells would
otherwise allow these cells to escape macrophage phagocytosis.
[0223] The terms "promote", "promoting", and the like are used
herein synonymously with "increase", "increasing", etc.
[0224] "Ischemia" refers to a vascular phenomenon in which a
decrease in the blood supply to a bodily organ, tissue, or part is
caused, for instance, by constriction or obstruction of one or more
blood vessels. Ischemia sometimes results from vasoconstriction or
thrombosis or embolism. Ischemia can lead to direct ischemic
injury, tissue damage due to cell death caused by reduced oxygen
supply. Ischemia can occur acutely, as during surgery, or from
trauma to tissue incurred in accidents, injuries and war settings,
or following harvest of organs intended for subsequent
transplantation, for example. It can also occur sub-acutely, as
found in atherosclerotic peripheral vascular disease, where
progressive narrowing of blood vessels leads to inadequate blood
flow to tissues and organs.
[0225] When a tissue is subjected to ischemia, a sequence of
chemical events is initiated that may ultimately lead to cellular
dysfunction and necrosis. If ischemia is ended by the restoration
of blood flow, a second series of injurious events ensue, producing
additional injury. Thus, whenever there is a transient decrease or
interruption of blood flow in a subject, the resultant injury
involves two components--the direct injury occurring during the
ischemic interval, and the indirect or reperfusion injury that
follows.
[0226] "Ischemic stroke" can be caused by several different kinds
of diseases. The most common problem is narrowing of the arteries
in the neck or head. This is most often caused by atherosclerosis,
or gradual cholesterol deposition. If the arteries become too
narrow, blood cells may collect in them and form blood clots
(thrombi). These blood clots can block the artery where they are
formed (thrombosis), or can dislodge and become trapped in arteries
closer to the brain (embolism). Cerebral stroke can occur when
atherosclerotic plaque separates away partially from the vessel
wall and occludes the flow of blood through the blood vessel.
[0227] "Reperfusion" refers to restoration of blood flow to tissue
that is ischemic, due to decrease in blood flow. Reperfusion is a
procedure for treating infarction or other ischemia, by enabling
viable ischemic tissue to recover, thus limiting further necrosis.
However, reperfusion can itself further damage the ischemic tissue,
causing reperfusion injury.
[0228] In addition to the immediate injury that occurs during
deprivation of blood flow, "ischemic/reperfusion injury" involves
tissue injury that occurs after blood flow is restored. Current
understanding is that much of this injury is caused by chemical
products, free radicals, and active biological agents released by
the ischemic tissues.
[0229] "Nitric oxide 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.
[0230] "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 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.
[0231] "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.
[0232] Examples of "an agent that inhibits cyclic nucleotide
phosphodiesterases" include sildenafil, tadalafil, vardenafil,
udenafil, and avanafil.
[0233] The singular terms "a", "an", and "the" include plural
referents unless context clearly indicates otherwise. Similarly,
the word "or" is intended to include "and" unless the context
clearly indicates otherwise. Hence, comprising A or B means
including A, or B, or A and B.
[0234] The term "about" as used herein is a flexible word with a
meaning similar to "approximately" or "nearly". The term "about"
indicates that exactitude is not claimed, but rather a contemplated
variation. Thus, as used herein, the term "about" means within 1 or
2 standard deviations from the specifically recited value, or .+-.a
range of up to 20%, up to 15%, up to 10%, up to 9%, up to 8%, up to
7%, up to 6%, up to 5%, up to 4%, up to 3%, up to 2%, or up to 1%
compared to the specifically recited value.
[0235] The term "comprising" as used in a claim herein is
open-ended, and means that the claim must have all the features
specifically recited therein, but that there is no bar on
additional features that are not recited being present as well. The
term "comprising" leaves the claim open for the inclusion of
unspecified ingredients even in major amounts. The term "consisting
essentially of" in a claim means that the invention necessarily
includes the listed ingredients, and is open to unlisted
ingredients that do not materially affect the basic and novel
properties of the invention. A "consisting essentially of" claim
occupies a middle ground between closed claims that are written in
a closed "consisting of" format and fully open claims that are
drafted in a "comprising` format". These terms can be used
interchangeably herein if, and when, this may become necessary.
[0236] Furthermore, the use of the term "including", as well as
other related forms, such as "includes" and "included", is not
limiting.
CD47 and Ischemia-Reperfusion Injury (IRI)
[0237] 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. Thus, blocking the TSP1-CD47 pathway,
as with the antibody compounds disclosed herein, will provide more
robust functioning of these endogenous protective pathways.
[0238] The humanized anti-CD47 antibodies, antigen binding
fragments thereof, and competing antibodies and antigen binding
fragments thereof, of the present disclosure can be used in the
methods disclosed in U.S. Pat. No. 8,236,313, the contents of which
are herein incorporated by reference in their entirety.
CD47 and Cancer
[0239] CD47 has been identified as a novel therapeutic target in
hematologic cancers (Majeti et al. (2009) Cell 138(2):286-99), as
well as in solid tumors such as colon, prostate, breast, and brain
cancers (Willingham et al. (2012) Proc Natl Acad Sci USA
109(17):6662-7). Many human cancers up-regulate cell surface
expression of CD47, and those expressing the highest levels of CD47
are 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
SIRPalpha, an inhibitory receptor that prevents phagocytosis of
CD47-bearing cells (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. Anti-CD47 mAbs (CD47mAbs) that
block the CD47/SIRPalpha interaction enhance phagocytosis of cancer
cells in vitro and contribute to control of tumor burden in
published human to mouse xenograft tumor models.
[0240] Antibodies that block CD47 and prevent its binding to
SIRPalpha ("blocking mAbs") have shown efficacy in human tumor in
mouse (xenograft) tumor models. Such blocking CD47mAbs exhibiting
this property promote (increase) the phagocytosis of cancer cells
by macrophages, which can reduce tumor burden (Majeti et al. (2009)
Cell 138(2):286-99) and may ultimately lead to generation of an
adaptive immune response to the tumor (Tseng et al. (2013) Proc
Natl Acad Sci USA. 110(27):11103-8).
Therapeutic Indications
IRI-Related and Autoimmune/Inflammatory Conditions
[0241] 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 and inflammatory 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, to 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; sickle cell disease (crisis); myocardial
infarction; stroke; surgically-induced ischemia; acute kidney
disease/kidney failure; any other condition in which IRI occurs and
contributes to the pathogenesis of disease; and
autoimmune/inflammatory diseases, including arthritis, multiple
sclerosis, psoriasis, Crohn's disease, inflammatory bowel disease,
lupus, Grave's disease and Hashimoto's thyroiditis, and ankylosing
spondylitis.
[0242] 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.
Susceptible Cancers
[0243] Presently disclosed mAbs and antigen binding fragments
thereof effective as cancer therapeutics 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), multiple
myeloma (MM), chronic myeloid leukemia (CML), myeloproliferative
disorder/neoplasm, myelodysplastic syndrome, monocytic cell
leukemia, and plasma cell leukemia; 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, and Waldenstrom's Macroglobulinemia,
ovarian cancer, breast cancer, endometrial cancer, colon cancer
(colorectal cancer), rectal cancer, bladder 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, melanoma, myelodysplastic
syndrome, and sarcomas including, but not limited to, osteosarcoma,
Ewing sarcoma, leiomyosarcoma, synovial sarcoma, alveolar soft part
sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma,
and chrondrosarcoma.
[0244] In certain cases, it may be advantageous to administer the
mAb directly to the cancer by injection into the tumor.
[0245] Since CD47 expression is up-regulated on many cancers, it
may also be desirable to use one or more of the disclosed mAbs as
imaging and diagnostic agents when labeled with radioactive or
other tracers known to those skilled in the art of in vivo imaging
of cancers/tumors.
[0246] The following examples describe various aspects of the
present disclosure, but should not be considered as limiting the
disclosure only to these particularly disclosed embodiments. The
materials and methods employed in these examples are for
illustrative purposes, and are not intended to limit the practice
of the present disclosure thereto. Any materials and methods
similar or equivalent to those described herein as would be
apparent to one of ordinary skill in the art can be used in the
practice or testing of the present compounds and methods.
Example 1
Production of CD47 Antibodies
[0247] 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 target chimeric, non-human antibody
VxP037-01LC/VxP037-01HC (SEQ ID NO:7/SEQ ID NO:57) are determined
following commonly accepted rules disclosed, for example, 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 CDR
fragments are synthesized and combined with pools of frameworks to
generate full length variable domains. The humanized variable
domains are then combined with a secretion signal and human kappa
and human IgG1 constant domains, and cloned into a mammalian
expression system (e.g., OptiCHO System, Life Technologies,
Carlsbad, Calif.) to generate a library of humanized IgG, IgG2, and
IgG4 variants. An aliquot of the library is sequenced to ensure
high diversity and integrity of the reading frames of the
individual clones. Aliquots of the humanized variant library are
then re-arrayed as single clones into 96 well plates, mini-prepped
(e.g., 96 well Miniprep Kit, Qiagen Hilden, Germany), and
transfected into CHO cells (Lipofectamine transfection protocol as
recommended by Life Technologies, Carlsbad, Calif.). Transfected
CHO cells are grown in DMEM medium with 10% FBS (both from Life
Technologies, Carlsbad, Calif.) at 37.degree. C. under 5% CO.sub.2.
The humanized variants are expressed as full length IgG1 molecules,
and secreted into the medium.
[0248] The cell culture supernatant containing the humanized IgG
variants is then screened for binding to the target antigen, CD47.
In parallel, the concentration of each variant is determined in
order to calculate specific activity for each clone. The specific
activity of each clone is compared to the specific activity of
chimeric clone VxP037-01 LC-Pro/VxPO37-01HC-Pro (SEQ ID NO:107/SEQ
ID NO:109) expressed on the same plate, and normalized. Top hits
from each plate are re-arrayed and re-screened for confirmation.
The final candidates are selected by specific activity, functional
activity, expression level, and sequence diversity, as well as
other criteria, as described below.
[0249] A non-glycosylated version (IgG1-N297Q) was created by site
directed mutagenesis of heavy chain position 297 to change the
asparagine to glutamine (pVxP037-01-HC-IgG1 N297Q-Pro; SEQ ID
NO:111). IgG2, IgG4-S228P and IgG4-2SS8P-L235E isotypes were
constructed by cloning the heavy chain variable domain in frame
with the human IgG2 and IgG4-S228P constant domains
(pVxK7b-037-hum01-HC-IgG2-Pro, SEQ ID NO:112 or
pVxK7b-037-hum01-HC_IgG4 S228P-Pro, SEQ ID NO:113,
pVxK7b-037-hum01-HC-IgG4 S228P L235E-Pro, SEQ ID NO: 122).
Example 2
CD47 Antibody Sequences
[0250] The amino acid sequences of the light chain and heavy chain
variable regions, the complete light and heavy chains, and the
respective encoding nucleotide sequences of the foregoing, of the
present human engineered antibodies are listed below in the section
entitled "Amino Acid and Nucleic Acid Sequences."
[0251] Also included in this list are complete light chain
sequences (SEQ ID NO:107/SEQ ID NO:108), complete heavy chain amino
acid and respectively encoding nucleotide sequences of humanized
IgG1 (SEQ ID NO:109/SEQ ID NO:110), complete heavy chain amino acid
and respectively encoding nucleotide sequences of humanized IgG1
with a N.fwdarw.Q mutation at amino acid position 297 (SEQ ID
NO:111/SEQ ID NO:114), IgG2 (SEQ ID NO:112/SEQ ID NO:115), and IgG4
(SEQ ID NO:113/SEQ ID NO: 116) antibodies.
[0252] SEQ ID NO:117 shows the amino acid sequence of framework
4+the light chain constant domain amino acid sequence of chimeric
complete light chain amino acid sequence SEQ ID NO:107.
[0253] SEQ ID NOs: 118, 119, 120, 121, and 124 show the amino acid
sequences of framework 4+the heavy chain constant domain amino acid
sequences of complete heavy chain amino acid sequences SEQ ID
NOs:109, 111, 112, 113, and 122 respectively.
[0254] All the light chain variable regions SEQ ID NOs: 7-31 can
further comprise SEQ ID NO:117, and all the heavy chain amino acid
sequences SEQ ID NOs:57-81 can further comprise any of SEQ ID
NOs:118, 119, 120, 121, and 124 thereby describing complete
antibody sequences encompassed by this disclosure.
[0255] The light chain and heavy chain CDR amino acid sequences are
shown in Tables 1 and 2, respectively.
TABLE-US-00001 TABLE 1 Light Chain CDRs. CDR1 CDR2 CDR3
RSSQSLVHSNGNTYLH KVSYRFS SQNTHVPRT (SEQ ID NO: 1) (SEQ ID NO: 2)
(SEQ ID NO: 3)
TABLE-US-00002 TABLE 2 Heavy Chain CDRs. CDR1 CDR2 CDR3 GYTFTNYYVF
DINPVNGDTNFNEKFKN GGYTMDY (SEQ ID NO: 4) (SEQ ID NO: 5) (SEQ ID NO:
6)
Example 3
Binding of Antibodies to CD47 of Different Species
[0256] Cross species reactivity of humanized antibodies of the
present disclosure is determined using freshly isolated red blood
cells (RBCs), which display CD47 on their surface, from human,
mouse, rat, pig, cynomolgus monkey, and dog according to the
methods disclosed in Kamel et al. (2010) Blood. Transfus.
8(4):260-266.
[0257] Supernatants containing secreted antibodies are collected
from CHO cells transiently transfected with plasmids encoding
antibody clones and used as collected, or antibodies are further
purified from the supernatants using standard methods. Transfected
CHO cells are grown in F-12 medium containing 10% heat inactivated
fetal bovine serum (BioWest; S01520). Antibody concentration in the
supernatants is determined utilizing a quantitative ELISA. ELISA
plates are coated with a donkey anti-human FC antibody (Sigma;
Catalog #12136) at 10 .mu.g/ml overnight at 4.degree. C. (Promega;
Catalog #W4031). Plates are washed with PBS, and then blocked with
casein blocking solution (ThermoScientific; Catalog #37532) for 60
minutes at room temperature. Plates are again washed with PBS,
tissue culture supernatants are added, and the plates are incubated
for 60 minutes at room temperature. Plates are then washed three
times with PBS and incubated with peroxidase-conjugated goat
anti-human IgG (Jackson Immunoresearch Labs; Catalog #109-035-003)
for 60 minutes at room temperature. Plates are washed three times
with PBS, and the peroxidase substrate
3,3'-5,5'-tetramethylbenzidine is added (Sigma; Catalog #T4444).
Reactions are terminated by the addition of HCl to 0.7N, and
absorbance at 450 nM is determined using a Tecan model Infinite
M200 plate reader.
[0258] RBCs are incubated for 60 minutes on ice with tissue culture
supernatants containing the secreted humanized antibodies at a
concentration of 10 ng/ml in a solution of phosphate buffered
saline, pH 7.2, 2.5 mM EDTA (PBS+E), or with various concentrations
of purified antibodies. Cells are then washed with cold PBS+E, and
incubated for an additional hour on ice with FITC labeled donkey
anti-human antibody (Jackson Immuno Research Labs, West Grove, Pa.;
Catalogue #709-096-149) in PBS+E. Cells are then washed with PBS+E,
and antibody binding is analyzed using a BD FACSAria Cell Sorter
(Becton Dickinson) or a C6 Accuri Flow Cytometer (Becton
Dickinson). Antibody binding is quantitated by comparison of mean
fluorescence values relative to that of chimeric antibody
>VxP037-01LC (SEQ ID NO:7))/>VxPO37-01HC (SEQ ID NO:57). The
mean fluorescence value for each antibody is divided by the mean
fluorescence value for the chimeric antibody.
[0259] The results obtained from the supernatants are shown in
Table 3, where "Chimera" represents chimeric antibody
>VxP037-01LC (SEQ ID NO:7))/>VxPO37-01HC (SEQ ID NO:57;
complete sequences VxPO37-01LC-Pro/VxP037-01HC-Pro (SEQ ID
NO:107/SEQ ID NO:109)), Clone 1 represents >pVxK7b-037-hum01-LC
(SEQ ID NO:8)/>pVxK7b-037-hum01-HC (SEQ ID NO:58), Clone 2
represents >pVxK7b-037-hum02-LC (SEQ ID
NO:9)/>pVxK7b-037-hum02-HC (SEQ ID NO:59), and so on similarly
for remaining clones 3-24. Each antibody also contains a light
chain constant domain (SEQ ID:117) and a heavy chain constant
domain selected from among SEQ ID NO:118, SEQ ID NO:119, SEQ ID
NO:120, and SEQ ID NO:121.
TABLE-US-00003 TABLE 3 Binding of Humanized Antibodies to CD47 on
the Surface of Red Blood Cells of Different Mammalian Species.
Clone No. Human Mouse Rat Pig Dog Chimera 1.0 1.0 1.0 1.0 1.0 1 1.1
1.7 2.7 1.3 1.0 2 1.0 1.2 2.6 1.2 1.0 3 0.7 0.9 1.7 0.9 0.9 4 0.6
0.6 1.0 0.6 0.6 5 1.0 1.0 2.2 1.2 1.1 6 0.9 1.2 2.1 1.1 1.1 7 0.5
0.4 0.8 0.9 0.8 8 0.7 0.7 1.2 0.8 0.8 9 1.2 1.4 3.7 1.6 1.0 10 1.1
1.2 2.9 1.5 1.1 11 0.8 0.7 1.2 1.2 0.8 12 0.8 0.6 1.3 1.4 0.9 13
1.2 1.3 3.1 1.4 1.0 14 1.1 1.5 3.2 1.4 1.3 15 1.0 1.3 2.4 1.2 1.1
16 0.9 1.0 2.1 1.1 1.1 17 0.8 0.9 2.1 1.3 1.3 18 1.0 1.3 2.2 1.2
1.5 19 0.7 1.0 2.6 1.3 1.2 20 1.3 1.5 1.9 1.7 1.1 21 1.2 1.2 2.8
1.4 1.1 22 1.1 1.2 2.8 1.4 1.0 23 1.2 1.4 3.3 1.7 1.1 24 0.8 0.7
1.2 1.1 1.0
[0260] FIG. 1 shows cross species binding curves to human, mouse,
rat, and porcine RBCs (panels A, B, C, and D, respectively,
generated using various concentrations of purified antibodies from
clones Cl 1, Cl 1.1, Cl 13, and Cl 13.1. Clones Cl 1 and Cl 13 are
as described above in Table 3. Clones Cl 1.1 and Cl 13.1 are Fc
mutants of clones Cl 1 and Cl 13, respectively, modified to reduce
effector function. Each has an Asn297.fwdarw.Gln (N297Q) mutation
in the Fc domain (Sazinsky et al. (2008) PNAS 105(51):20167-20172).
All of these clones exhibit concentration-dependent binding to all
of the species of RBCs tested. These clones also bind to cynomolgus
monkey RBCs as shown in FIG. 9 by the concentration-dependent
binding of Clones 1.1 (IgG 1 N297Q mutation) and 1.3 (IgG4 S228P
mutation).
[0261] Table 4 shows the apparent affinities of these clones to
human RBCs determined by non-linear fits (Prism GraphPad software)
of the median fluorescence intensities at various antibody
concentrations. Clones 1, 1.1, 13, and 13.1 all have apparent Kd
values in the low nanomolar range.
TABLE-US-00004 TABLE 4 Binding Affinity of Humanized Antibodies to
CD47 on Human RBCs. Clone 1 Clone 1.1 Clone 13 Clone 13.1 Kd
(ng/ml) 226.2 307.1 86.4 182.9 Kd (apparent) 1.51 2.04 0.58 1.21
nM
[0262] Binding activities of humanized clones 1, 3, 5, 8, 13, 14,
17, 20 and 23, either IgG1 N297Q (Clone 1.1, Clone 13.1, etc.) or
an IgG4 S228P L235E (Clones 1.2, etc.), to human and mouse CD47
were determined using cell-based ELISA assays with either human
OV10 or mouse 4T1 cells expressing cell surface CD47. OV10 cells
are grown in IMDM medium containing 10% heat inactivated fetal
bovine serum (BioWest; S01520) and 4T1 cells are grown in RMPI
medium containing 10% heat inactivated fetal bovine serum (BioWest;
S01520). One day before assay, 3.times.10.sup.4 cells are plated in
96 well cell bind plates (Corning #3300, VWR #66025-626) so that
they are 95-100% confluent at the time of assay. Cells are washed
and various concentrations of purified antibodies added in either
IMDM or RPMI at 37.degree. C. for 1 hour in 95% O.sub.2/5%
CO.sub.2. Cells are then washed with media and incubated for an
additional hour at 37.degree. C. with HRP labeled secondary
anti-human antibody (Promega) diluted 1/2500 in media. Cells are
washed three times with PBS, and the peroxidase substrate
3,3',5,5'-tetramethylbenzidine is added (Sigma; Catalog #T4444).
Reactions are terminated by the addition of HCl to 0.7N, and
absorbance at 450 nM is determined using a Tecan model Infinite
M200 plate reader. The apparent binding affinities of these clones
to human and mouse cells is determined by non-linear fits (Prism
GraphPad software). As shown in Table 5, all of the humanized
clones bind to both human and mouse tumor cells with apparent
affinities in the picomolar range. Agglutination of human RBCs is
assessed following incubation of human RBCs with various
concentrations of humanized blocking only clones. Blood is diluted
(1:50) and washed 3 times with PBS/EDTA/BSA. RBCs are 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. As shown in Table 3, while all clones bind similarly
to human RBCs, they exhibit different agglutination activities.
Clones 3.1, 3.2, 13.1, 13.2, 20.1, and 20.2 cause agglutination of
human RBCs, whereas Clones 1.1 (IgG1 N297Q), 1.2 (IgG4 S228P
L235E), 5.1, 5.2, 8.1, 8.2, 14.1, 14.2, 17.1, 17.2, 23.1 and 23.2
do not.
TABLE-US-00005 TABLE 5 Humanized Blocking-Only mAb. CD47 Binding
Affinity and Red Blood Cell Hemagglutination. Human OV10_ Mouse
ELISA 4T1_ Hemag- Kd ELISA glutination [pM] Kd [pM] Human RBC Clone
1.1 147.7 131.6 No hum01 IgG4 S228P L235E Clone 1.2 150.5 175.5 No
hum13 IgG1 N297Q Clone 13.1 88.6 86.7 Yes hum 13 IgG4 S228P L235E
Clone 13.2 107.8 177.8 Yes hum03 IgG1 N297Q Clone 3.1 115.3 131.7
Yes hum03 IgG4 S228P L235E Clone 3.2 129.3 202 Yes hum05 IgG1 N297Q
Clone 5.1 124.7 112.4 No hum05 IgG4 S228P L235E Clone 5.2 107.5
171.8 No hum08 IgG1 N297Q Clone 8.1 114.9 205 No hum08 IgG4 S228P
L235E Clone 8.2 121.4 94.9 No hum14 IgG1 N297Q Clone 14.1 96.2 94.9
No hum14 IgG4 S228P L235E Clone 14.2 89.9 101.8 No hum17 IgG1 N297Q
Clone 17.1 85.5 197.9 No hum17 IgG4 S228P L235E Clone 17.2 120.9
147 No hum20 IgG1 N297Q Clone 20.1 120.9 236.9 Yes hum20 IgG4 S228P
L235E Clone 20.2 113.5 354 Yes hum23 IgG1 N297Q Clone 23.1 99.6
92.2 No hum23 IgG4 S228P L235E Clone 23.2 194.6 192.2 No
[0263] These data demonstrate that all of the humanized CD47 mAb
clones disclosed herein bind to CD47 of a variety of different
mammalian species in addition to human CD47, confirming the useful
cross-species reactivity of these antibodies and that certain of
these antibodies do not cause agglutination of human RBCs
Example 4
Antibodies to CD47 Enhance Phagocytosis
[0264] To assess the effect of humanized CD47 mAbs on phagocytosis
of tumor cells by marcrophages in vitro the following method is
employed using flow cytometry, essentially as described by
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.
[0265] Human derived macrophages are derived from leukapheresis of
healthy human peripheral blood incubated in human AB serum (Sigma
Aldrich) for 24 hours in culture. After 24 hours, all non-adherent
cells are removed and the remaining adherent macrophages are
incubated in RPMI medium (10% fetal bovine serum (FBS; Hyclone) and
antibiotics) for two weeks. In additional experiments, human
macrophages are derived from human peripheral blood and incubated
in AIM-V media (Life Technologies) for 7-10 days. For the in vitro
phagocytosis assay, macrophages are re-plated at a concentration of
5.times.10.sup.4 cells per well in 1 ml of RPMI media in a 24 well
plate and allowed to adhere for 24 hours. Once the effector
macrophages have adhered to the culture dish, the target cancer
cells (Jurkat) are labeled with 1 .mu.M 5(6)-Carboxyfluorescein
diacetate N-succinimidyl ester (CFSE; Sigma Aldrich) and added to
the macrophage cultures at a concentration of 2.times.10.sup.5
cells in 1 ml of RPMI media (4:1 target to effector ratio).
CD47mAbs (1-10 .mu.g/ml) are added immediately upon mixture of
target and effector cells and allowed to incubate at 37.degree. C.
for 2-3 hours. After 2-3 hours, all non-phagocytosed cells are
removed and the remaining cells are washed three times with
phosphate buffered saline (PBS; Sigma Aldrich). Cells are
trypsinized, collected into microcentrifuge tubes and incubated in
100 ng of allophycocyanin (APC) labeled CD14 antibodies (BD
biosciences) for 30 minutes. Cells are washed once and analyzed by
flow cytometry (Accuri C6; BD biosciences) for the percentage of
CD14 positive cells that are also CFSE positive indicating complete
phagocytosis.
[0266] As shown in FIG. 2, both Clone 1 and Clone 13 humanized IgG1
mAbs increase phagocytosis of Jurkat cells by human macrophages
grown in serum. Clones 1.1 and Clone 13.1 are identical to Clones 1
and 13 except for a mutation of residue N297 to Q that reduces the
affinity of the IgG1 molecule to the Fc receptor. The IgG4 versions
of Clone 1 and Clone 13 with a mutation of residue S228 to P to
reduce chain strand exchange (also designated as Clones 1.3 and
13.3) also increase phagocytosis of Jurkat cells. The IgG4 isotype
also has reduced affinity for activating Fc receptors. As shown in
FIG. 10, the IgG4 version of Clone 1 with a mutation of residue
S228 to P and a further mutation of residue L235 to E to reduce Fc
effector function (designated as Clone 1.2) also increases
phagocytosis of Jurkat cells.
[0267] Therefore, all isotypes/mutants of Clone 1 and 13 enhance
phagocytosis via blocking the CD47/SIRPalpha interaction.
Example 5
Antibodies to CD47 Regulate Nitric Oxide Signaling
[0268] The purpose of this experiment is to demonstrate that
humanized antibody clones 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.
[0269] The method employed to measure cGMP is 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) are used as these cells retain the
NO-cGMP signaling pathway when grown in culture and exhibit a
robust and reproducible inhibitory response to TSP1 ligation of
CD47. Cells are 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 .mu.g mL
streptomycin (Sigma; Catalogue #P4222) at densities less than
1.times.10.sup.6 cells/mL. For the cGMP assay, cells are 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.
[0270] 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, are then 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), is added to half the wells at a final
concentration of 1 .mu.M. Five minutes later, the cells are lysed
with buffer supplied in the cGMP kit, and aliquots of each well are
assayed for cGMP content.
[0271] As shown in FIGS. 3 and 4, none of the present humanized
antibody clones tested, or the chimeric control mAb, has an effect
on basal cGMP levels. As expected, the chimeric antibody
VxP037-01LC-Pro/VxP037-01HC-Pro (SEQ ID NO:107/SEQ ID NO: 109))
reverses the TSP1 inhibition.
[0272] Humanized Clones 1, 9, 11, 13, and 24 of the present
disclosure also significantly reverse TSP1 inhibition,
demonstrating that they have the ability to increase NO signaling
(FIGS. 3 and 4), suggesting their 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).
Example 6
Reduction of Ischemia-Reperfusion Injury In Vivo
[0273] The purpose of this experiment is to demonstrate that a
humanized antibody clone disclosed herein, i.e., Clone 1, that is
shown to regulate nitric oxide signaling in vitro in Example 5, is
effective in reducing IRI and kidney damage in vivo in a rat kidney
transplant model under standard conditions, i.e., with no warm
ischemic time but with cold ischemic time. IRI significantly
contributes to delayed graft function and inflammation leading to
graft loss, and is exacerbated by the thrombospondin-1/CD47 system
through inhibition of nitric oxide signaling.
[0274] A syngeneic rat renal transplantation model of IRI with
bilaterally nephrectomized recipients is used to evaluate the
effect of the anti-CD47 monoclonal antibody Clone 1 on graft
function following transplantation as described in Schumacher et
al. (2003) Microsurg. 23:389-394 and Karatzas et al. (2007)
Microsug. 27:668-672.
[0275] Male Lewis rats weighing 275-300 g are obtained from Charles
River Laboratories (Wilmington, Mass.). Donor kidneys are flushed
with 50 .mu.g of purified Clone 1 or vehicle (phosphate buffered
saline, pH 7.2), and stored at 4.degree. C. in University of
Wisconsin preservation solution (UW) for 6 hours prior to
transplantation. Two days following transplantation, kidney
function is assessed by measuring serum creatinine by standard
methodology.
[0276] As shown in FIG. 5, CD47mAb Clone 1 perfusion of donor
kidneys results in improved kidney function compared to controls as
measured by a reduction in serum creatinine.
[0277] An additional experiment is shown that also demonstrates the
ability of CD47mAbs of the present disclosure to improve kidney
function of extended criteria organs that have also undergone a 60
minute period of warm ischemic time in addition to a 6 hour cold
ischemic time. Male Lewis rats weighing 275-300 g underwent 60
minutes of warm ischemia, prior to flushing the donor kidneys with
50 .mu.g of purified Clone 1.1 or an IgG control mAb. Kidneys are
stored at 4.degree. C. in University of Wisconsin preservation
solution (UW) for 6 hours prior to transplantation. In this
experiment, survival is monitored over a 7 day time period.
[0278] As shown in FIG. 6, all animals that received the IgG
control mAb-treated kidney die within 4 days. In contrast, survival
is significantly increased in the animals that received the
Clone1.1 treated kidney, with 30% of the animals surviving for the
7 day duration of the experiment.
[0279] Together, these experiments show that with both standard and
extended criteria donor kidneys, Clone 1 and Clone 1.1 reduce IRI
and increase kidney function and survival outcomes,
respectively.
Example 7
Acute Promyelocytic Leukemia (APL) Anti-Tumor Activity In Vivo
[0280] The purpose of this experiment is to demonstrate that a
humanized antibody clone disclosed herein, i.e., Clone 13, reduces
tumor burden in vivo in a mouse leukemia model.
[0281] The anti-tumor activity of the anti-CD47mAb Clone13 (Cl 13;
clone number as described above in Examples 2 and 3) is determined
in a syngeneic murine model of Acute Promyelocytic Leukemia (APL)
as described in Ramirez et al. (2009) Blood 113:6206-6214.
[0282] Murine APL cells (B6APL1) are injected intravenously into
C57BL/6 mice that are randomized into three groups (5-10 mice per
group): Group 1: no APL; Group 2: APL with no treatment; Group 3:
APL with anti-CD47mAb Cl 13treatment. Antibody treatment is
initiated on the day of tumor inoculation (day 0), and given in
single doses of 10 .mu.g/dose (0.4 mg/kg) in phosphate buffered
saline, pH 7.2, by intraperitoneal injection on days 0, 3, and
6.
[0283] Tumor burden is evaluated at day 25 following tumor cell
inoculation. Blood samples from each mouse are analyzed for white
blood cell count using an automated hemocytometer, and circulating
APL cells (representing the tumor burden) are quantified by flow
cytometry (CD34.sup.+/CD117.sup.+ cells).
[0284] As shown in FIG. 7, mice treated with Cl 13 have reduced
tumor burden compared to untreated mice at 25 days after tumor
inoculation, thus demonstrating anti-tumor activity of this
humanized clone.
Example 8
HepG2 Anti-Tumor Activity In Vivo
[0285] The purpose of this experiment is to demonstrate that a
humanized antibody clone disclosed herein, i.e., Clone 1.1, reduces
tumor burden in vivo in a mouse xenograft model of human
hepatocellular carcinoma (HCC).
[0286] Male NSG mice are obtained from The Jackson Laboratory (Bar
Harbor, Me.) and housed in cages in temperature and
light-controlled environments with access to water and food ad
libitum. For the heterotopic xenograft model, HepG2-luc2 cells
(Perkin Elmer, Waltham, Mass. #134280) are suspended in DMEM
containing 25% (v/v), and 1,000,000 cells implanted subcutaneously
into the dorsal subcutaneous space of 4- to 8-wk-old NSG mice.
After 2 weeks of growth, antibody treatment is begun with
twice-weekly intraperitoneal injections of 15 mg/kg of either
anti-CD47 antibody Clone 1.1 or an IgG control for 6 weeks. Tumor
volumes are calculated twice weekly using (length.times.width)/0.6.
After 6 weeks of treatment, animals are euthanized and tumors were
resected, weighed, and fixed in 10% formalin.
[0287] As shown in FIG. 8, treatment with the CD47 mAb Clone 1.1
significantly reduced tumor growth of the HepG2 tumors (p<0.01),
demonstrating anti-tumor efficacy on solid tumors.
[0288] Embodiments of the disclosure being thus described, it will
be obvious that the same may be varied in many ways. Such
variations are not to be regarded as a departure from the spirit
and scope of the disclosure, and all such modifications as would be
obvious to one skilled in the art are intended to be included
within the scope of the following claims.
Example 9
Antibodies to CD47 Block CD47/SIRPalpha Binding
[0289] To assess the effect of humanized CD47 mAbs on binding of
CD47 to SIRPalpha in vitro the following method is employed using
binding of CD47 expressing Jurkat cells to SIPRalpha bound to
plates.
[0290] Polystyrene 96 well tissue culture plates were coated for 60
minutes at 37 degrees with 2 .mu.g/ml SIRP-Fc fusion protein (R and
D Systems, cat #4546-SA). Unbound SIRP-Fc fusion was removed and
nonspecific protein binding sites were blocked with casein for 60
minutes at 37 degrees (ThermoScientific cat #37528). Blocking
solution was removed and plates were washed with PBS. Jurkat cells
in RPMI growth medium containing 10% FBS were added (100,000
cells/well), with or without CD47 antibodies at 1 ug/ml. Cells were
incubated at 37 degrees for 60 minutes. Cells were aspirated and
wells gently washed twice with PBS. Growth medium containing WST-1
reagent was added and plates incubated at 37 degrees for 2 hrs
(Cayman Scientific cat #10008883). Absorbance was read at 450 nm.
As shown in FIG. 11, Clones 1.1, 1.2, 1.3, 13.1, 13.2 and 13.3 all
block the interaction of CD47 (expressed on the Jurkat cells) with
SIPRalpha while a control antibody that does not bind to CD47 does
not block the CD47/SIRPalpha interaction.
Amino Acid and Nucleic Acid Sequences
Light Chain Variable Region Amino Acid Sequences
Murine Sequence
TABLE-US-00006 [0291]>VxP037-01LC: Underlined amino acid
sequences represent CDRs (SEQ ID NO: 7)
DVVMTQTPLSLSVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPK
LLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTHVP RTFGQG
Humanized Light Chain Sequences
TABLE-US-00007 [0292]>pVxK7b-037-hum01-LC (SEQ ID NO: 8)
DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYQQKPGKAPK
LLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVP RTFGQG
>pVxK7b-037-hum02-LC (SEQ ID NO: 9)
DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYQQKPGQAPR
LLIYKVSYRFSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCSQNTHVP RTFGQG
>pVxK7b-037-hum03-LC (SEQ ID NO: 10)
DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYQQKPGKAPK
LLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVP RTFGQG
>pVxK7b-037-hum04-LC (SEQ ID NO: 11)
DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTYLHWYLQKPGQSPQ
LLIYKVSYRFSGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCSQNTHVP RTFGQG
>pVxK7b-037-hum05-LC (SEQ ID NO: 12)
DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQ
LLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVP RTFGQG
>pVxK7b-037-hum06-LC (SEQ ID NO: 13)
DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTYLHWYLQKPGQSPQ
LLIYKVSYRFSGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCSQNTHVP RTFGQG
>pVxK7b-037-hum07-LC (SEQ ID NO: 14)
DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQ
LLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVP RTFGQG
>pVxK7b-037-hum08-LC (SEQ ID NO: 15)
DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYQQKPGKAPK
LLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVP RTFGQG
>pVxK7b-037-hum09-LC (SEQ ID NO: 16)
DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYQQKPGKAPK
LLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVP RTFGQG
>pVxK7b-037-hum10-LC (SEQ ID NO: 17)
DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQ
LLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVP RTFGQG
>pVxK7b-037-hum11-LC (SEQ ID NO: 18)
EIVLTQSPATLSVSPGERATLSCRSSQSLVHSNGNTYLHWYQQKPGQAPR
LLIYKVSYRFSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCSQNTHVP RTFGQG
>pVxK7b-037-hum12-LC (SEQ ID NO: 19)
DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYQQKPGKAPK
LLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVP RTFGQG
>pVxK7b-037-hum13-LC (SEQ ID NO: 20)
DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYQQKPGKAPK
LLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVP RTFGQG
>pVxK7b-037-hum14-LC (SEQ ID NO: 21)
DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYQQKPGKAPK
LLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVP RTFGQG
>pVxK7b-037-hum15-LC (SEQ ID NO: 22)
AIQLTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTYLHWYQQKPGQAPR
LLIYKVSYRFSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCSQNTHVP RTFGQG
>pVxK7b-037-hum16-LC (SEQ ID NO: 23)
AIQLTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTYLHWYQQKPGQAPR
LLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVP RTFGQG
>pVxK7b-037-hum17-LC (SEQ ID NO: 24)
AIQLTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTYLHWYQQKPGQAPR
LLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVP RTFGQG
>pVxK7b-037-hum18-LC (SEQ ID NO: 25)
EIVLTQSPATLSVSPGERATLSCRSSQSLVHSNGNTYLHWYQQKPGQAPR
LLIYKVSYRFSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCSQNTHVP RTFGQG
>pVxK7b-037-hum19-LC (SEQ ID NO: 26)
DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYQQKPGKAPK
LLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVP RTFGQG
>pVxK7b-037-hum20-LC (SEQ ID NO: 27)
DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQ
LLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVP RTFGQG
>pVxK7b-037-hum21-LC (SEQ ID NO: 28)
AIQLTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTYLHWYQQKPGQAPR
LLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVP RTFGQG
>pVxK7b-037-hum22-LC (SEQ ID NO: 29)
EIVLTQSPATLSVSPGERATLSCRSSQSLVHSNGNTYLHWYQQKPGQAPR
LLIYKVSYRFSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCSQNTHVP RTFGQG
>pVxK7b-037-hum23-LC (SEQ ID NO: 30)
DIVMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYQQKPGKAPK
LLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVP RTFGQG
>pVxK7b-037-hum24-LC (SEQ ID NO: 31)
AIQLTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTYLHWYQQKPGQAPR
LLIYKVSYRFSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCSQNTHVP RTFGQG
Murine Light Chain Variable Region Nucleic Acid Sequence
TABLE-US-00008 [0293]>VxP037-01LC (SEQ ID NO: 32)
GATGTTGTTATGACCCAAACTCCACTCTCCCTGTCTGTCAGTCTTGGAGA
TCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATG
GAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAG
CTCCTGATCTACAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTT
CAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGG
AGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAATACACATGTTCCT
CGGACGTTCGGCCAAGGAG
Humanized Light Chain Variable Region Nucleic Acid Sequences
TABLE-US-00009 [0294]>pVxK7b-037-hum01-LC (SEQ ID NO: 33)
GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGA
GCCGGCCTCCATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATG
GAAACACCTATTTACATTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAG
CTCCTGATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTT
CAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGG
AGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCT
CGGACGTTCGGCCAAGGG >pVxK7b-037-hum02-LC (SEQ ID NO: 34)
GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACA
GCCGGCCTCCATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATG
GAAACACCTATTTACATTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGG
CTCCTCATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCATCAAGGTT
CAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGC
AGCCTGATGATTTTGCAACTTATTACTGTTCTCAAAATACACATGTTCCT
CGGACGTTCGGCCAAGGG >pVxK7b-037-hum03-LC (SEQ ID NO: 35)
GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACA
GCCGGCCTCCATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATG
GAAACACCTATTTACATTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAG
CTCCTGATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTT
CAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGG
AGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCT
CGGACGTTCGGCCAAGGG >pVxK7b-037-hum04-LC (SEQ ID NO: 36)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCACTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATG
GAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAG
CTCCTGATCTATAAAGTTTCCTACCGATTTTCTGGGATCCCAGCCAGGTT
CAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGC
AGTCTGAAGATTTTGCAGTTTATTACTGTTCTCAAAATACACATGTTCCT
CGGACGTTCGGCCAAGGG >pVxK7b-037-hum05-LC (SEQ ID NO: 37)
GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGA
GCCGGCCTCCATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATG
GAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAG
CTCCTGATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTT
CAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGG
AGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCT
CGGACGTTCGGCCAAGGG >pVxK7b-037-hum06-LC (SEQ ID NO: 38)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCACTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATG
GAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAG
CTCCTGATCTATAAAGTTTCCTACCGATTTTCTGGGATCCCAGCCAGGTT
CAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGC
AGTCTGAAGATTTTGCAGTTTATTACTGTTCTCAAAATACACATGTTCCT
CGGACGTTCGGCCAAGGG >pVxK7b-037-hum07-LC (SEQ ID NO: 39)
GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGA
GCCGGCCTCCATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATG
GAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAG
CTCCTGATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTT
CAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGG
AGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCT
CGGACGTTCGGCCAAGGG >pVxK7b-037-hum08-LC (SEQ ID NO: 40)
GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGA
GCCGGCCTCCATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATG
GAAACACCTATTTACATTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAG
CTCCTGATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTT
CAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGG
AGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCT
CGGACGTTCGGCCAAGGG >pVxK7b-037-hum09-LC (SEQ ID NO: 41)
GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACA
GCCGGCCTCCATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATG
GAAACACCTATTTACATTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAG
CTCCTGATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTT
CAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGG
AGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCT
CGGACGTTCGGCCAAGGG >pVxK7b-037-hum10-LC (SEQ ID NO: 42)
GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGA
GCCGGCCTCCATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATG
GAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAG
CTCCTGATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTT
CAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGG
AGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCT
CGGACGTTCGGCCAAGGG >pVxK7b-037-hum11-LC (SEQ ID NO: 43)
GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGA
AAGAGCCACCCTCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATG
GAAACACCTATTTACATTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGG
CTCCTCATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCCTCGAGGTT
CAGTGGCAGTGGATCTGGGACAGATTTCACCTTTACCATCAGTAGCCTGG
AAGCTGAAGATGCTGCAACATATTACTGTTCTCAAAATACACATGTTCCT
CGGACGTTCGGCCAAGGG >pVxK7b-037-hum12-LC (SEQ ID NO: 44)
GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGA
GCCGGCCTCCATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATG
GAAACACCTATTTACATTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAG
CTCCTGATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTT
CAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGG
AGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCT
CGGACGTTCGGCCAAGGG >pVxK7b-037-hum13-LC (SEQ ID NO: 45)
GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGA
GCCGGCCTCCATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATG
GAAACACCTATTTACATTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAG
CTCCTGATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTT
CAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGG
AGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCT
CGGACGTTCGGCCAAGGG >pVxK7b-037-hum14-LC (SEQ ID NO: 46)
GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACA
GCCGGCCTCCATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATG
GAAACACCTATTTACATTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAG
CTCCTGATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTT
CAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGG
AGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCT
CGGACGTTCGGCCAAGGG >pVxK7b-037-hum15-LC (SEQ ID NO: 47)
GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCACTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATG
GAAACACCTATTTACATTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGG
CTCCTCATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCATCAAGGTT
CAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGC
AGCCTGATGATTTTGCAACTTATTACTGTTCTCAAAATACACATGTTCCT
CGGACGTTCGGCCAAGGG >pVxK7b-037-hum16-LC (SEQ ID NO: 48)
GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCACTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATG
GAAACACCTATTTACATTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGG
CTCCTCATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTT
CAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGG
AGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCT
CGGACGTTCGGCCAAGGG >pVxK7b-037-hum17-LC (SEQ ID NO: 49)
GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCACTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATG
GAAACACCTATTTACATTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGG
CTCCTCATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTT
CAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGG
AGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCT
CGGACGTTCGGCCAAGGG >pVxK7b-037-hum18-LC (SEQ ID NO: 50)
GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGA
AAGAGCCACCCTCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATG
GAAACACCTATTTACATTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGG
CTCCTCATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCCTCGAGGTT
CAGTGGCAGTGGATCTGGGACAGATTTCACCTTTACCATCAGTAGCCTGG
AAGCTGAAGATGCTGCAACATATTACTGTTCTCAAAATACACATGTTCCT
CGGACGTTCGGCCAAGGG >pVxK7b-037-hum19-LC (SEQ ID NO: 51)
GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACA
GCCGGCCTCCATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATG
GAAACACCTATTTACATTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAG
CTCCTGATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTT
CAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGG
AGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCT
CGGACGTTCGGCCAAGGG >pVxK7b-037-hum20-LC (SEQ ID NO: 52)
GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGA
GCCGGCCTCCATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATG
GAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAG
CTCCTGATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTT
CAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGG
AGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCT
CGGACGTTCGGCCAAGGG >pVxK7b-037-hum21-LC (SEQ ID NO: 53)
GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCACTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATG
GAAACACCTATTTACATTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGG
CTCCTCATCTATAAAGTTTCCTACCGATTT
TCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCAC
ACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGTT
CTCAAAATACACATGTTCCTCGGACGTTCGGCCAAGGG >pVxK7b-037-hum22-LC (SEQ
ID NO: 54) GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGA
AAGAGCCACCCTCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATG
GAAACACCTATTTACATTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGG
CTCCTCATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCCTCGAGGTT
CAGTGGCAGTGGATCTGGGACAGATTTCACCTTTACCATCAGTAGCCTGG
AAGCTGAAGATGCTGCAACATATTACTGTTCTCAAAATACACATGTTCCT
CGGACGTTCGGCCAAGGG >pVxK7b-037-hum23-LC (SEQ ID NO: 55)
GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGA
GCCGGCCTCCATCTCCTGCAGATCTAGTCAGAGCCTTGTACACAGTAATG
GAAACACCTATTTACATTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAG
CTCCTGATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTT
CAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGG
AGGCTGAGGATGTTGGAGTTTATTACTGTTCTCAAAATACACATGTTCCT
CGGACGTTCGGCCAAGGG >pVxK7b-037-hum24-LC (SEQ ID NO: 56)
GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCACTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATG
GAAACACCTATTTACATTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGG
CTCCTCATCTATAAAGTTTCCTACCGATTTTCTGGGGTCCCATCAAGGTT
CAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGC
AGCCTGATGATTTTGCAACTTATTACTGTTCTCAAAATACACATGTTCCT
CGGACGTTCGGCCAAGGG
Heavy Chain Variable Region Amino Acid Sequences
Murine Heavy Chain Variable Region Amino Acid Sequence: Underlined
Amino Acid Sequences Indicate CDRs
TABLE-US-00010 [0295]>VxP037-01HC (SEQ ID NO: 57)
EVQLQQFGAELVKPGASMKLSCKASGYTFTNYYVFWVKQRPGQGLEWIGD
INPVNGDTNFNEKFKNKATLTVDKSSTTTYLQLSSLTSEDSAVYYCTRGG YTMDYWGQG
Humanized Heavy Chain Variable Region Amino Acid Sequences
TABLE-US-00011 [0296]>pVxK7b-037-hum01-HC (SEQ ID NO: 58)
QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGD
INPVNGDTNFNEKFKNRVTISADKSISTAYLQWSSLKASDTAMYYCARGG YTMDYWGQG
>pVxK7b-037-hum02-HC (SEQ ID NO: 59)
QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGD
INPVNGDTNFNEKFKNRVTISADKSISTAYLQWSSLKASDTAMYYCARGG YTMDYWGQG
>pVxK7b-037-hum03-HC (SEQ ID NO: 60)
EVQLVQSGAEVKKPGESLRISCKGSGYTFTNYYVFWVRQARGQRLEWIGD
INPVNGDTNFNEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARGG YTMDYWGQG
>pVxK7b-037-hum04-HC (SEQ ID NO: 61)
QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQAPGKGLEWVSD
INPVNGDTNFNEKFKNRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGG YTMDYWGQG
>pVxK7b-037-hum05-HC (SEQ ID NO: 62)
QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQAPGKGLEWVSD
INPVNGDTNFNEKFKNRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGG YTMDYWGQG
>pVxK7b-037-hum06-HC (SEQ ID NO: 63)
QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGD
INPVNGDTNFNEKFKNRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGG YTMDYWGQG
>pVxK7b-037-hum07-HC (SEQ ID NO: 64)
QVQLQESGPGLVKPGATVKISCKVSGYTFTNYYVFWVRQARGQRLEWIGD
INPVNGDTNFNEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARGG YTMDYWGQG
>pVxK7b-037-hum08-HC (SEQ ID NO: 65)
QITLKESGPTLVKPTQTLTLTCTFSGYTFTNYYVFWIRQSPSRGLEWLGD
INPVNGDTNFNEKFKNRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGG YTMDYWGQG
>pVxK7b-037-hum09-HC (SEQ ID NO: 66)
QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGD
INPVNGDTNFNEKFKNRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGG YTMDYWGQG
>pVxK7b-037-hum10-HC (SEQ ID NO: 67)
QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGD
INPVNGDTNFNEKFKNRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGG YTMDYWGQG
>pVxK7b-037-hum11-HC (SEQ ID NO: 68)
QVQLQESGPGLVKPGATVKISCKVSGYTFTNYYVFWVRQARGQRLEWIGD
INPVNGDTNFNEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARGG YTMDYWGQG
>pVxK7b-037-hum12-HC (SEQ ID NO: 69)
QVQLQESGPGLVKPGATVKISCKVSGYTFTNYYVFWVRQARGQRLEWIGD
INPVNGDTNFNEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARGG YTMDYWGQG
>pVxK7b-037-hum13-HC (SEQ ID NO: 70)
EVQLVQSGAEVKKPGESLRISCKGSGYTFTNYYVFWIRQSPSRGLEWLGD
INPVNGDTNFNEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARGG YTMDYWGQG
>pVxK7b-037-hum14-HC (SEQ ID NO: 71)
QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGD
INPVNGDTNFNEKFKNRVTISADKSISTAYLQWSSLKASDTAMYYCARGG YTMDYWGQG
>pVxK7b-037-hum15-HC (SEQ ID NO: 72)
QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGD
INPVNGDTNFNEKFKNRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGG YTMDYWGQG
>pVxK7b-037-hum16-HC (SEQ ID NO: 73)
QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGD
INPVNGDTNFNEKFKNRVTISADKSISTAYLQWSSLKASDTAMYYCARGG YTMDYWGQG
>pVxK7b-037-hum17-HC (SEQ ID NO: 74)
EVQLVQSGAEVKKPGATVKISCKVSGYTFTNYYVFWIRQPPGKGLEWIGD
INPVNGDTNFNEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARGG YTMDYWGQG
>pVxK7b-037-hum18-HC (SEQ ID NO: 75)
EVQLVQSGAEVKKPGESLRISCKGSGYTFTNYYVFWIRQSPSRGLEWLGD
INPVNGDTNFNEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARGG YTMDYWGQG
>pVxK7b-037-hum19-HC (SEQ ID NO: 76)
EVQLVQSGAEVKKPGESLRISCKGSGYTFTNYYVFWIRQSPSRGLEWLGD
INPVNGDTNFNEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARGG YTMDYWGQG
>pVxK7b-037-hum20-HC (SEQ ID NO: 77)
QITLKESGPTLVKPTQTLTLTCTFSGYTFTNYYVFWVRQAPGQGLEWMGD
INPVNGDTNFNEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARGG YTMDYWGQG
>pVxK7b-037-hum21-HC (SEQ ID NO: 78)
QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGD
INPVNGDTNFNEKFKNRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGG YTMDYWGQG
>pVxK7b-037-hum22-HC (SEQ ID NO: 79)
QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGD
INPVNGDTNFNEKFKNRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGG YTMDYWGQG
>pVxK7b-037-hum23-HC (SEQ ID NO: 80)
QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGD
INPVNGDTNFNEKFKNRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGG YTMDYWGQG
>pVxK7b-037-hum24-HC (SEQ ID NO: 81)
QVQLQESGPGLVKPGATVKISCKVSGYTFTNYYVFWVRQARGQRLEWIGD
INPVNGDTNFNEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARGG YTMDYWGQG
Murine Heavy Chain Variable Region Nucleic Acid Sequence
TABLE-US-00012 [0297]>VxP037-01HC (SEQ ID NO: 82)
GAGGTCCAGCTGCAGCAGTTTGGGGCTGAACTGGTGAAGCCTGGGGCTTC
AATGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAACTACTATG
TATTCTGGGTGAAACAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAC
ATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAA
GGCCACACTGACTGTAGACAAGTCCTCCACCACAACATACTTGCAACTCA
GCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGAGGGGGT
TATACTATGGACTACTGGGGTCAAGGA
Humanized Heavy Chain Variable Region Nucleic Acid Sequences
TABLE-US-00013 [0298]>pVxK7b-037-hum01-HC (SEQ ID NO: 83)
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATG
TATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGAC
ATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAG
AGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGA
GCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGAGGGGGT
TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum02-HC (SEQ ID NO: 84)
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATG
TATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGAC
ATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAG
AGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGA
GCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGAGGGGGT
TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum03-HC (SEQ ID NO: 85)
GAAGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTC
TCTGAGGATCTCCTGTAAGGGTTCTGGCTACACCTTCACCAACTACTATG
TATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGAC
ATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAG
AGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGA
GCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGGT
TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum04-HC (SEQ ID NO: 86)
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATG
TATTCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCAGTGAC
ATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAG
AGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGA
GCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGGGGGT
TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum05-HC (SEQ ID NO: 87)
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATG
TATTCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCAGTGAC
ATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAG
AGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGA
GCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGGGGGT
TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum06-HC (SEQ ID NO: 88)
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATG
TATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGAC
ATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAG
ACTCACCATCTCCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGA
CCAACATGGACCCTGTGGACACAGCCACGTATTACTGTGCAAGAGGGGGT
TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum07-HC (SEQ ID NO: 89)
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTGGGGCTAC
AGTGAAAATCTCCTGCAAGGTTTCTGGCTACACCTTCACCAACTACTATG
TATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGAC
ATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAG
AGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGA
GCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGGT
TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum08-HC (SEQ ID NO: 90)
CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACAGAC
CCTCACGCTGACCTGCACCTTCTCTGGCTACACCTTCACCAACTACTATG
TATTCTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGTGAC
ATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAG
ATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGA
ACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGT
TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum09-HC (SEQ ID NO: 91)
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATG
TATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGAC
ATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAG
ACTCACCATCTCCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGA
CCAACATGGACCCTGTGGACACAGCCACGTATTACTGTGCAAGAGGGGGT
TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum10-HC (SEQ ID NO: 92)
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATG
TATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGAC
ATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAG
ACTCACCATCTCCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGA
CCAACATGGACCCTGTGGACACAGCCACGTATTACTGTGCAAGAGGGGGT
TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum11-HC (SEQ ID NO: 93)
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTGGGGCTAC
AGTGAAAATCTCCTGCAAGGTTTCTGGCTACACCTTCACCAACTACTATG
TATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGAC
ATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAG
AGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGA
GCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGGT
TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum12-HC (SEQ ID NO: 94)
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTGGGGCTAC
AGTGAAAATCTCCTGCAAGGTTTCTGGCTACACCTTCACCAACTACTATG
TATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGAC
ATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAG
AGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGA
GCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGGT
TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum13-HC (SEQ ID NO: 95)
GAAGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTC
TCTGAGGATCTCCTGTAAGGGTTCTGGCTACACCTTCACCAACTACTATG
TATTCTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGTGAC
ATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAG
AGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGA
GCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGGT
TATACTATGGACTACTGGGGCCAGGA >pVxK7b-037-hum14-HC (SEQ ID NO: 96)
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATG
TATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGAC
ATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAG
AGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGA
GCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGAGGGGGT
TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum15-HC (SEQ ID NO: 97)
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATG
TATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGAC
ATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAG
ACTCACCATCTCCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGA
CCAACATGGACCCTGTGGACACAGCCACGTATTACTGTGCAAGAGGGGGT
TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum16-HC (SEQ ID NO: 98)
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATG
TATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGAC
ATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAG
AGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGA
GCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGAGGGGGT
TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum17-HC (SEQ ID NO: 99)
GAGGTCCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCTAC
AGTGAAAATCTCCTGCAAGGTTTCTGGCTACACCTTCACCAACTACTATG
TATTCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGTGAC
ATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAG
AGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGA
GCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGGT
TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum18-HC (SEQ ID NO:
100) GAAGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTC
TCTGAGGATCTCCTGTAAGGGTTCTGGCTACACCTTCACCAACTACTATG
TATTCTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGTGAC
ATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAG
AGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGA
GCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGGT
TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum19-HC (SEQ ID NO:
101) GAAGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTC
TCTGAGGATCTCCTGTAAGGGTTCTGGCTACACCTTCACCAACTACTATG
TATTCTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGTGAC
ATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAG
AGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGA
GCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGGT
TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum20-HC (SEQ ID NO:
102) CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACAGAC
CCTCACGCTGACCTGCACCTTCTCTGGCTACACCTTCACCAACTACTATG
TATTCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGTGAC
ATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAG
AGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGA
GCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGGT
TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum21-HC (SEQ ID NO:
103) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATG
TATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGAC
ATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAG
ACTCACCATCTCCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGA
CCAACATGGACCCTGTGGACACAGCCACGTATTACTGTGCAAGAGGGGGT
TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum22-HC (SEQ ID NO:
104) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATG
TATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGAC
ATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAG
ACTCACCATCTCCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGA
CCAACATGGACCCTGTGGACACAGCCACGTATTACTGTGCAAGAGGGGGT
TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum23-HC (SEQ ID NO:
105) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATG
TATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGAC
ATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAG
ACTCACCATCTCCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGA
CCAACATGGACCCTGTGGACACAGCCACGTATTACTGTGCAAGAGGGGGT
TATACTATGGACTACTGGGGCCAGGGA >pVxK7b-037-hum24-HC (SEQ ID NO:
106) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTGGGGCTAC
AGTGAAAATCTCCTGCAAGGTTTCTGGCTACACCTTCACCAACTACTATG
TATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGAC
ATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAG
AGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGA
GCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGGGGT
TATACTATGGACTACTGGGGCCAGGGA
Chimeric Complete Light Chain Amino Acid Sequence
[0299] >VxPO37-01-LC-Pro, below, represents a full length
chimeric light chain variable domain (SEQ ID NO:7)+a constant
domain amino acid sequence. The underlined amino acid
sequence=framework 4+the constant domain. All full length humanized
light chain sequences can contain a light chain variable region
sequence selected from SEQ ID NOs:7-31 in combination with
framework 4+the same constant domain as VxPO37-01-LC-Pro. However,
while present, this constant domain is not shown for all the
complete humanized light chain amino acid sequences.
TABLE-US-00014 >VxP037-01-LC-Pro (SEQ ID NO: 107)
DVVMTQTPLSLSVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPK
LLIYKVSYRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTHVP
RTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE
VTHQGLSSPVTKSFNRGEC
Chimeric Complete Light Chain Nucleic Acid Sequence
[0300] The underlined nucleic acid sequence encodes the underlined
protein sequence in >VxPO37-01-LC-Pro, above.
TABLE-US-00015 >VxP037-01-LC-DNA (SEQ ID NO: 108)
GATGTTGTTATGACCCAAACTCCACTCTCCCTGTCTGTCAGTCTTGGAGA
TCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATG
GAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAG
CTCCTGATCTACAAAGTTTCCTACCGATTTTCTGGGGTCCCAGACAGGTT
CAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGG
AGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAATACACATGTTCCT
CGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGC
ACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAA
CTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAA
GTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAG
TGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCC
TGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAA
GTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGG AGAGTGTTGA
Chimeric and Humanized Complete Heavy Chain Amino Acid
Sequences
[0301] >VxP037-01-HC-Pro, below, represents a full length
chimeric heavy chain variable domain (SEQ ID NO:57)+a constant
domain amino acid sequence. The underlined amino acid
sequence=framework 4+the constant IgG1 domain. All full length
humanized heavy chain sequences can contain a heavy chain variable
region sequence selected from SEQ ID NOs:57-81 in combination with
framework 4+the same constant domain as VxP037-01-HC-Pro. However,
while present, this constant domain is not shown for all the
complete humanized heavy chain amino acid sequences.
Chimeric Complete Heavy Chain Amino Acid Sequence
TABLE-US-00016 [0302]>VxP037-01-HC-Pro (SEQ ID NO: 109)
EVQLQQFGAELVKPGASMKLSCKASGYTFTNYYVFWVKQRPGQGLEWIGD
INPVNGDTNFNEKFKNKATLTVDKSSTTTYLQLSSLTSEDSAVYYCTRGG
YTMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Chimeric Complete Heavy Chain Nucleic Acid Sequence
[0303] The underlined nucleic acid sequence encodes the underlined
protein sequence in >VxP037-01-HC-Pro, above.
TABLE-US-00017 >VxP037-01-HC-DNA (SEQ ID NO: 110)
GAGGTCCAGCTGCAGCAGTTTGGGGCTGAACTGGTGAAGCCTGGGGCTTC
AATGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAACTACTATG
TATTCTGGGTGAAACAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAC
ATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAA
GGCCACACTGACTGTAGACAAGTCCTCCACCACAACATACTTGCAACTCA
GCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGAGGGGGT
TATACTATGGACTACTGGGGCCAGGGAACGCTGGTCACCGTCAGCTCAGC
CTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCA
CCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCC
GAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCA
CACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCG
TGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAAC
GTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAA
ATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCC
TGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC
ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCA
CGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGC
ATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGT
GTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGA
GTACAAGTGCAAGGTCAGCAACAAAGCCCTCCCAGCCCCCATCGAGAAAA
CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTG
CCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCT
GGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATG
GGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGAC
GGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCA
GCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACC
ACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
Complete IgG1 N2970, IgG2, IgG4 S228P, and IgG4 S228P L235E Heavy
Chain Amino Acid Sequences
[0304] >VxP037-01-HC-IgG1 N297Q-Pro, below, represents a full
length heavy chain variable domain (SEQ ID NO:57)+a constant domain
amino acid sequence. The underlined amino acid sequence=framework
4+the constant IgG1 constant domain containing a N-Q mutation at
amino acid position 297. All full length humanized heavy chain
sequences can contain a heavy chain variable region sequence
selected from SEQ ID NOs:57-81 in combination with framework 4+the
same constant domain as >VxP037-01-HC-IgG1 N297Q-Pro. However,
while present, this constant domain is not shown for all the
complete humanized heavy chain amino acid sequences.
TABLE-US-00018 >VxP037-01-HC-IgG1-N297Q-Pro (SEQ ID NO: 111)
EVQLQQFGAELVKPGASMKLSCKASGYTFTNYYVFWVKQRPGQGLEWIGD
INPVNGDTNFNEKFKNKATLTVDKSSTTTYLQLSSLTSEDSAVYYCTRGG
YTMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSR
[0305] >pVxK7b-037-hum01-HC-IgG2-Pro, below, represents a full
length heavy chain variable domain (SEQ ID NO:58)+a constant domain
amino acid sequence. The underlined amino acid sequence=framework
4+the constant IgG2 domain. All full length humanized heavy chain
sequences can contain a heavy chain variable region sequence
selected from SEQ ID NOs:57-81 in combination with framework 4+the
same constant domain as >pVxK7b-037-hum01-HC-IgG2-Pro. However,
while present, this constant domain is not shown for all the
complete humanized heavy chain amino acid sequences.
TABLE-US-00019 >pVxK7b-037-hum01-HC-IgG2-Pro (SEQ ID NO: 112)
QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGD
INPVNGDTNFNEKFKNRVTISADKSISTAYLQWSSLKASDTAMYYCARGG
YTMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCN
VDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSV
LTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSR
EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYNTTPPMLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0306] >pVxK7b-037-hum01-HC-IgG4 S228P-Pro, below, represents a
full length heavy chain variable domain (SEQ ID NO:58)+a constant
domain amino acid sequence. The underlined amino acid
sequence=framework 4+the constant IgG4 S228P domain. All full
length humanized heavy chain sequences can contain a heavy chain
variable region sequence selected from SEQ ID NOs:57-81 in
combination with framework 4+the same constant domain as
>pVxK7b-037-hum01-HC-IgG4 S228P-Pro. However, while present,
this constant domain is not shown for all the complete humanized
heavy chain amino acid sequences.
TABLE-US-00020 >pVxK7b-037-hum01-HC-IgG4 S228P-Pro (SEQ ID NO:
113) QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGD
INPVNGDTNFNEKFKNRVTISADKSISTAYLQWSSLKASDTAMYYCARGG
YTMDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN
VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS
QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
[0307] >pVxK7b-037-hum01-HC-IgG4 S228P L235E-Pro, below,
represents a full length heavy chain variable domain (SEQ ID
NO:58)+a constant domain amino acid sequence. The underlined amino
acid sequence=framework 4+the constant IgG4 S228P L235E domain. All
full length humanized heavy chain sequences can contain a heavy
chain variable region sequence selected from SEQ ID NOs:57-81 in
combination with framework 4+the same constant domain as
>pVxK7b-037-hum01-HC-IgG4 S228P-L235E-Pro. However, while
present, this constant domain is not shown for all the complete
humanized heavy chain amino acid sequences.
TABLE-US-00021 >pVxK7b-037-hum01-HC-IgG4 S228P L235E-Pro (SEQ ID
NO: 122) QVQLQESGPGLVKPSQTLSLTCTVSGYTFTNYYVFWVRQARGQRLEWIGD
INPVNGDTNFNEKFKNRVTISADKSISTAYLQWSSLKASDTAMYYCARGG
YTMDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN
VDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS
QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
Complete IgG1 N2970, IgG2, IgG4 S228P, and IgG4 IgG4 S228P L235E
Heavy Chain Nucleic Acid Sequences
[0308] The underlined nucleic acid sequence encodes the underlined
protein sequence in >VxP037-01-HC-IgG1 N297Q-Pro, above.
TABLE-US-00022 >VxP037-01-HC-IgG1 N297Q-DNA (SEQ ID NO: 114)
GAGGTCCAGCTGCAGCAGTTTGGGGCTGAACTGGTGAAGCCTGGGGCTTC
AATGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAACTACTATG
TATTCTGGGTGAAACAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAC
ATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAA
GGCCACACTGACTGTAGACAAGTCCTCCACCACAACATACTTGCAACTCA
GCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGAGGGGGT
TATACTATGGACTACTGGGGCCAGGGAACGCTGGTCACCGTCAGCTCAGC
CTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCA
CCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCC
GAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCA
CACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCG
TGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAAC
GTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAA
ATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCC
TGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC
ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCA
CGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGC
ATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACCAGAGCACGTACCGT
GTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGA
GTACAAGTGCAAGGTCAGCAACAAAGCCCTCCCAGCCCCCATCGAGAAAA
CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTG
CCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCT
GGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATG
GGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGAC
GGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCA
GCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACC
ACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
[0309] The underlined nucleic acid sequence encodes the underlined
protein sequence in >VxP037-hum 01-HC-IgG2-Pro, above.
TABLE-US-00023 >pVxK7b-037-hum01-HC-IgG2-DNA (SEQ ID NO: 115)
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATG
TATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGAC
ATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAG
AGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGA
GCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGAGGGGGT
TATACTATGGACTACTGGGGCCAGGGAACGCTGGTCACCGTCAGCTCAGC
CTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTGCTCCAGGAGCA
CCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCC
GAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCA
CACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCG
TGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAAC
GTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAA
ATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGT
CAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG
ACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGA
GGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGA
CAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTC
CTCACCGTCGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAA
GGTGTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAA
CCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGG
GAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT
CTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGA
ACAACTACAACACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTC
CTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGT
CTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGA
AGAGCCTCTCCCTGTCTCCGGGTAAA
[0310] The underlined nucleic acid sequence encodes the underlined
protein sequence in >VxP037-hum 01-HC-IgG4 S228P-Pro, above.
TABLE-US-00024 >pVxK7b-037-hum01-HC-IgG4 S228P-DNA (SEQ ID NO:
116) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATG
TATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGAC
ATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAG
AGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGA
GCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGAGGGGGT
TATACTATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGTCCTCCGC
TTCCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCA
CCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCC
GAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCA
CACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCG
TGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAAC
GTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAA
ATATGGTCCCCCATGCCCACCGTGCCCAGCACCTGAGTTCCTGGGGGGAC
CATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCC
CGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCC
CGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCA
AGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGC
GTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTG
CAAGGTGTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCA
AAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCC
CAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG
CTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG
AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTC
TTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAA
TGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACAC
AGAAGAGCCTCTCCCTGTCTCTGGGTAAA
[0311] The underlined nucleic acid sequence encodes the underlined
protein sequence in >VxP037-hum 01-HC-IgG4 S228P L235E-Pro,
above.
TABLE-US-00025 > pVxK7b-037-hum01-HC-IgG4 S228P L235E-DNA (SEQ
ID NO: 123) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGAC
CCTGTCCCTCACCTGCACTGTCTCTGGCTACACCTTCACCAACTACTATG
TATTCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGAC
ATTAATCCTGTCAATGGTGATACTAACTTCAATGAGAAATTCAAGAACAG
AGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGA
GCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGAGGGGGT
TATACTATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGTCCTCCGC
TTCCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCA
CCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCC
GAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCA
CACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCG
TGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAAC
GTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAA
ATATGGTCCCCCATGCCCACCGTGCCCAGCACCTGAGTTCGAGGGGGGAC
CATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCC
CGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCC
CGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCA
AGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGC
GTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTG
CAAGGTGTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCA
AAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCC
CAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG
CTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG
AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTC
TTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAA
TGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACAC
AGAAGAGCCTCTCCCTGTCTCTGGGTAAA
Framework 4+Light Chain Constant Domain Amino Acid Sequence
TABLE-US-00026 [0312] (SEQ ID NO: 117)
TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL
SSPVTKSFNRGEC
Framework 4+Heavy Chain Constant IgG1 Domain
TABLE-US-00027 [0313] (SEQ ID NO: 118)
TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH
QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK
NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Framework 4+Heavy Chain Constant IgG1 N297Q Domain
TABLE-US-00028 [0314] (SEQ ID NO: 119)
TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLH
QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
Framework 4+Heavy Chain Constant IgG2 Domain
TABLE-US-00029 [0315] (SEQ ID NO: 120)
WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV
SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKP
SNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH
QDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTK
NQVSLTCLVKGFYPSDIAVEWESNGQPENNYNTTPPMLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Framework 4+Heavy Chain Constant IgG4 S228P Domain
TABLE-US-00030 [0316] (SEQ ID NO: 121)
TTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK
VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVV
VDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDW
LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD
KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
Framework 4+Heavy Chain Constant IgG4 S228P L235E Domain
TABLE-US-00031 [0317] (SEQ ID NO: 124)
TTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK
VDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVV
VDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDW
LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD
KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
Sequence CWU 1
1
124116PRTArtificial Sequencelight chain complementarity determining
region 1Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu
His 1 5 10 15 27PRTArtificial Sequencelight chain complementarity
determining region 2Lys Val Ser Tyr Arg Phe Ser 1 5 39PRTArtificial
Sequencelight chain complementarity determining region 3Ser Gln Asn
Thr His Val Pro Arg Thr 1 5 410PRTArtificial Sequenceheavy chain
complementarity determining region 4Gly Tyr Thr Phe Thr Asn Tyr Tyr
Val Phe 1 5 10 517PRTArtificial Sequenceheavy chain complementarity
determining region 5Asp Ile Asn Pro Val Asn Gly Asp Thr Asn Phe Asn
Glu Lys Phe Lys 1 5 10 15 Asn 67PRTArtificial Sequenceheavy chain
complementarity determining region 6Gly Gly Tyr Thr Met Asp Tyr 1 5
7106PRTArtificial Sequencelight chain variable region 7Asp Val Val
Met Thr Gln Thr Pro Leu Ser Leu Ser Val Ser Leu Gly 1 5 10 15 Asp
Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25
30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Tyr 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 Phe Cys Ser Gln Asn 85 90 95 Thr His Val Pro Arg Thr Phe Gly
Gln Gly 100 105 8106PRTArtificial Sequencelight chain variable
region 8Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro
Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu
Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln
Lys Pro Gly Lys Ala 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser
Tyr 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 Ser Gln Asn 85 90 95 Thr His Val
Pro Arg Thr Phe Gly Gln Gly 100 105 9106PRTArtificial Sequencelight
chain variable region 9Asp 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 Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His
Trp Tyr Gln Gln Lys Pro Gly Gln Ala 35 40 45 Pro Arg Leu Leu Ile
Tyr Lys Val Ser Tyr Arg Phe Ser Gly Val Pro 50 55 60 Ser Arg Phe
Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile 65 70 75 80 Ser
Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Ser Gln Asn 85 90
95 Thr His Val Pro Arg Thr Phe Gly Gln Gly 100 105
10106PRTArtificial Sequencelight chain variable region 10Asp 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 Ser Leu Val His Ser 20
25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys
Ala 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Tyr 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 Ser Gln Asn 85 90 95 Thr His Val Pro Arg Thr Phe
Gly Gln Gly 100 105 11106PRTArtificial Sequencelight chain variable
region 11Asp 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 Ser Ser Gln Ser
Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu
Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val
Ser Tyr Arg Phe Ser Gly Ile Pro 50 55 60 Ala Arg Phe Ser Gly Ser
Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Gln
Ser Glu Asp Phe Ala Val Tyr Tyr Cys Ser Gln Asn 85 90 95 Thr His
Val Pro Arg Thr Phe Gly Gln Gly 100 105 12106PRTArtificial
Sequencelight chain variable region 12Asp Ile Val Met Thr Gln Thr
Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile
Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn
Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro
Gln Leu Leu Ile Tyr Lys Val Ser Tyr 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 Ser
Gln Asn 85 90 95 Thr His Val Pro Arg Thr Phe Gly Gln Gly 100 105
13106PRTArtificial Sequencelight chain variable region 13Asp 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 Ser Ser Gln Ser Leu Val His Ser 20
25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln
Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Tyr Arg Phe Ser
Gly Ile Pro 50 55 60 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu
Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Gln Ser Glu Asp Phe Ala
Val Tyr Tyr Cys Ser Gln Asn 85 90 95 Thr His Val Pro Arg Thr Phe
Gly Gln Gly 100 105 14106PRTArtificial Sequencelight chain variable
region 14Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr
Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser
Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu
Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val
Ser Tyr 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 Ser Gln Asn 85 90 95 Thr His
Val Pro Arg Thr Phe Gly Gln Gly 100 105 15106PRTArtificial
Sequencelight chain variable region 15Asp Ile Val Met Thr Gln Thr
Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile
Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn
Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala 35 40 45 Pro
Lys Leu Leu Ile Tyr Lys Val Ser Tyr 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 Ser
Gln Asn 85 90 95 Thr His Val Pro Arg Thr Phe Gly Gln Gly 100 105
16106PRTArtificial Sequencelight chain variable region 16Asp 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 Ser Leu Val His Ser 20
25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys
Ala 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Tyr 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 Ser Gln Asn 85 90 95 Thr His Val Pro Arg Thr Phe
Gly Gln Gly 100 105 17106PRTArtificial Sequencelight chain variable
region 17Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr
Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser
Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu
Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val
Ser Tyr 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 Ser Gln Asn 85 90 95 Thr His
Val Pro Arg Thr Phe Gly Gln Gly 100 105 18106PRTArtificial
Sequencelight chain variable region 18Glu Ile Val Leu Thr Gln Ser
Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu
Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn
Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala 35 40 45 Pro
Arg Leu Leu Ile Tyr Lys Val Ser Tyr Arg Phe Ser Gly Val Pro 50 55
60 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile
65 70 75 80 Ser Ser Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Ser
Gln Asn 85 90 95 Thr His Val Pro Arg Thr Phe Gly Gln Gly 100 105
19106PRTArtificial Sequencelight chain variable region 19Asp Ile
Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20
25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys
Ala 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Tyr 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 Ser Gln Asn 85 90 95 Thr His Val Pro Arg Thr Phe
Gly Gln Gly 100 105 20106PRTArtificial Sequencelight chain variable
region 20Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr
Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser
Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln
Gln Lys Pro Gly Lys Ala 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val
Ser Tyr 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 Ser Gln Asn 85 90 95 Thr His
Val Pro Arg Thr Phe Gly Gln Gly 100 105 21106PRTArtificial
Sequencelight chain variable region 21Asp 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 Ser Leu Val His Ser 20 25 30 Asn Gly Asn
Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala 35 40 45 Pro
Lys Leu Leu Ile Tyr Lys Val Ser Tyr 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 Ser
Gln Asn 85 90 95 Thr His Val Pro Arg Thr Phe Gly Gln Gly 100 105
22106PRTArtificial Sequencelight chain variable region 22Ala Ile
Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Leu Val His Ser 20
25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln
Ala 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Tyr Arg Phe Ser
Gly Val Pro 50 55 60 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu
Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Gln Pro Asp Asp Phe Ala
Thr Tyr Tyr Cys Ser Gln Asn 85 90 95 Thr His Val Pro Arg Thr Phe
Gly Gln Gly 100 105 23106PRTArtificial Sequencelight chain variable
region 23Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser
Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln
Gln Lys Pro Gly Gln Ala 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val
Ser Tyr 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 Ser Gln Asn 85 90 95 Thr His
Val Pro Arg Thr Phe Gly Gln Gly 100 105 24106PRTArtificial
Sequencelight chain variable region 24Ala Ile Gln Leu Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile
Thr Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn
Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala 35 40 45 Pro
Arg Leu Leu Ile Tyr Lys Val Ser Tyr 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 Ser
Gln Asn 85 90 95 Thr His Val Pro Arg Thr Phe Gly Gln Gly 100 105
25106PRTArtificial Sequencelight chain variable region 25Glu Ile
Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20
25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln
Ala 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Tyr Arg Phe Ser
Gly Val Pro 50 55 60 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Phe Thr Ile 65 70 75 80 Ser Ser Leu Glu Ala Glu Asp Ala Ala
Thr Tyr Tyr Cys Ser Gln Asn 85 90 95 Thr His Val Pro Arg Thr Phe
Gly Gln Gly 100 105 26106PRTArtificial Sequencelight chain variable
region 26Asp 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 Ser Leu Val His Ser
20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly
Lys Ala 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Tyr 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 Ser Gln Asn 85 90 95 Thr His Val Pro Arg Thr
Phe Gly Gln Gly 100 105 27106PRTArtificial Sequencelight chain
variable region 27Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro
Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser
Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp
Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr
Lys Val Ser Tyr 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 Ser Gln Asn 85 90 95
Thr His Val Pro Arg Thr Phe Gly Gln Gly 100 105 28106PRTArtificial
Sequencelight chain variable region 28Ala Ile Gln Leu Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile
Thr Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn
Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala 35 40 45 Pro
Arg Leu Leu Ile Tyr Lys Val Ser Tyr 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 Ser
Gln Asn 85 90 95 Thr His Val Pro Arg Thr Phe Gly Gln Gly 100 105
29106PRTArtificial Sequencelight chain variable region 29Glu Ile
Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20
25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln
Ala 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Tyr Arg Phe Ser
Gly Val Pro 50 55 60 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Phe Thr Ile 65 70 75 80 Ser Ser Leu Glu Ala Glu Asp Ala Ala
Thr Tyr Tyr Cys Ser Gln Asn 85 90 95 Thr His Val Pro Arg Thr Phe
Gly Gln Gly 100 105 30106PRTArtificial Sequencelight chain variable
region 30Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr
Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser
Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln
Gln Lys Pro Gly Lys Ala 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val
Ser Tyr 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 Ser Gln Asn 85 90 95 Thr His
Val Pro Arg Thr Phe Gly Gln Gly 100 105 31106PRTArtificial
Sequencelight chain variable region 31Ala Ile Gln Leu Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile
Thr Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn
Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala 35 40 45 Pro
Arg Leu Leu Ile Tyr Lys Val Ser Tyr Arg Phe Ser Gly Val Pro 50 55
60 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile
65 70 75 80 Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Ser
Gln Asn 85 90 95 Thr His Val Pro Arg Thr Phe Gly Gln Gly 100 105
32319DNAArtificial Sequencelight chain variable region 32gatgttgtta
tgacccaaac tccactctcc ctgtctgtca gtcttggaga tcaagcctcc 60atctcttgca
gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg
120tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc
ctaccgattt 180tctggggtcc cagacaggtt cagtggcagt ggatcaggga
cagatttcac actcaagatc 240agcagagtgg aggctgagga tctgggagtt
tatttctgct ctcaaaatac acatgttcct 300cggacgttcg gccaaggag
31933318DNAArtificial Sequencelight chain variable region
33gatattgtga tgacccagac tccactctcc ctgcccgtca cccctggaga gccggcctcc
60atctcctgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg
120tatcagcaga aaccagggaa agctcctaag ctcctgatct ataaagtttc
ctaccgattt 180tctggggtcc cagacaggtt cagtggcagt gggtcaggca
ctgatttcac actgaaaatc 240agcagggtgg aggctgagga tgttggagtt
tattactgtt ctcaaaatac acatgttcct 300cggacgttcg gccaaggg
31834318DNAArtificial Sequencelight chain variable region
34gatgttgtga tgactcagtc tccactctcc ctgcccgtca cccttggaca gccggcctcc
60atctcctgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg
120taccagcaga aacctggcca ggctcccagg ctcctcatct ataaagtttc
ctaccgattt 180tctggggtcc catcaaggtt cagcggcagt ggatctggga
cagaattcac tctcaccatc 240agcagcctgc agcctgatga ttttgcaact
tattactgtt ctcaaaatac acatgttcct 300cggacgttcg gccaaggg
31835318DNAArtificial Sequencelight chain variable region
35gatgttgtga tgactcagtc tccactctcc ctgcccgtca cccttggaca gccggcctcc
60atctcctgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg
120tatcagcaga aaccagggaa agctcctaag ctcctgatct ataaagtttc
ctaccgattt 180tctggggtcc cagacaggtt cagtggcagt gggtcaggca
ctgatttcac actgaaaatc 240agcagggtgg aggctgagga tgttggagtt
tattactgtt ctcaaaatac acatgttcct 300cggacgttcg gccaaggg
31836318DNAArtificial Sequencelight chain variable region
36gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc
60atcacttgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg
120tacctgcaga agccagggca gtctccacag ctcctgatct ataaagtttc
ctaccgattt 180tctgggatcc cagccaggtt cagtggcagt gggtctggga
cagagttcac tctcaccatc 240agcagcctgc agtctgaaga ttttgcagtt
tattactgtt ctcaaaatac acatgttcct 300cggacgttcg gccaaggg
31837318DNAArtificial Sequencelight chain variable region
37gatattgtga tgacccagac tccactctcc ctgcccgtca cccctggaga gccggcctcc
60atctcctgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg
120tacctgcaga agccagggca gtctccacag ctcctgatct ataaagtttc
ctaccgattt 180tctggggtcc cagacaggtt cagtggcagt gggtcaggca
ctgatttcac actgaaaatc 240agcagggtgg aggctgagga tgttggagtt
tattactgtt ctcaaaatac acatgttcct 300cggacgttcg gccaaggg
31838318DNAArtificial Sequencelight chain variable region
38gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc
60atcacttgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg
120tacctgcaga agccagggca gtctccacag ctcctgatct ataaagtttc
ctaccgattt 180tctgggatcc cagccaggtt cagtggcagt gggtctggga
cagagttcac tctcaccatc 240agcagcctgc agtctgaaga ttttgcagtt
tattactgtt ctcaaaatac acatgttcct 300cggacgttcg gccaaggg
31839318DNAArtificial Sequencelight chain variable region
39gatattgtga tgacccagac tccactctcc ctgcccgtca cccctggaga gccggcctcc
60atctcctgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg
120tacctgcaga agccagggca gtctccacag ctcctgatct ataaagtttc
ctaccgattt 180tctggggtcc cagacaggtt cagtggcagt gggtcaggca
ctgatttcac actgaaaatc 240agcagggtgg aggctgagga tgttggagtt
tattactgtt ctcaaaatac acatgttcct 300cggacgttcg gccaaggg
31840318DNAArtificial Sequencelight chain variable region
40gatattgtga tgacccagac tccactctcc ctgcccgtca cccctggaga gccggcctcc
60atctcctgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg
120tatcagcaga aaccagggaa agctcctaag ctcctgatct ataaagtttc
ctaccgattt 180tctggggtcc cagacaggtt cagtggcagt gggtcaggca
ctgatttcac actgaaaatc 240agcagggtgg aggctgagga tgttggagtt
tattactgtt ctcaaaatac acatgttcct 300cggacgttcg gccaaggg
31841318DNAArtificial Sequencelight chain variable region
41gatgttgtga tgactcagtc tccactctcc ctgcccgtca cccttggaca gccggcctcc
60atctcctgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg
120tatcagcaga aaccagggaa agctcctaag ctcctgatct ataaagtttc
ctaccgattt 180tctggggtcc cagacaggtt cagtggcagt gggtcaggca
ctgatttcac actgaaaatc 240agcagggtgg aggctgagga tgttggagtt
tattactgtt ctcaaaatac acatgttcct 300cggacgttcg gccaaggg
31842318DNAArtificial Sequencelight chain variable region
42gatattgtga tgacccagac tccactctcc ctgcccgtca cccctggaga gccggcctcc
60atctcctgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg
120tacctgcaga agccagggca gtctccacag ctcctgatct ataaagtttc
ctaccgattt 180tctggggtcc cagacaggtt cagtggcagt gggtcaggca
ctgatttcac actgaaaatc 240agcagggtgg aggctgagga tgttggagtt
tattactgtt ctcaaaatac acatgttcct 300cggacgttcg gccaaggg
31843318DNAArtificial Sequencelight chain variable region
43gaaattgtgt tgacacagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc
60ctctcctgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg
120taccagcaga aacctggcca ggctcccagg ctcctcatct ataaagtttc
ctaccgattt 180tctggggtcc cctcgaggtt cagtggcagt ggatctggga
cagatttcac ctttaccatc 240agtagcctgg aagctgaaga tgctgcaaca
tattactgtt ctcaaaatac acatgttcct 300cggacgttcg gccaaggg
31844318DNAArtificial Sequencelight chain variable region
44gatattgtga tgacccagac tccactctcc ctgcccgtca cccctggaga gccggcctcc
60atctcctgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg
120tatcagcaga aaccagggaa agctcctaag ctcctgatct ataaagtttc
ctaccgattt 180tctggggtcc cagacaggtt cagtggcagt gggtcaggca
ctgatttcac actgaaaatc 240agcagggtgg aggctgagga tgttggagtt
tattactgtt ctcaaaatac acatgttcct 300cggacgttcg gccaaggg
31845318DNAArtificial Sequencelight chain variable region
45gatattgtga tgacccagac tccactctcc ctgcccgtca cccctggaga gccggcctcc
60atctcctgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg
120tatcagcaga aaccagggaa agctcctaag ctcctgatct ataaagtttc
ctaccgattt 180tctggggtcc cagacaggtt cagtggcagt gggtcaggca
ctgatttcac actgaaaatc 240agcagggtgg aggctgagga tgttggagtt
tattactgtt ctcaaaatac acatgttcct 300cggacgttcg gccaaggg
31846318DNAArtificial Sequencelight chain variable region
46gatgttgtga tgactcagtc tccactctcc ctgcccgtca cccttggaca gccggcctcc
60atctcctgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg
120tatcagcaga aaccagggaa agctcctaag ctcctgatct ataaagtttc
ctaccgattt 180tctggggtcc cagacaggtt cagtggcagt gggtcaggca
ctgatttcac actgaaaatc 240agcagggtgg aggctgagga tgttggagtt
tattactgtt ctcaaaatac acatgttcct 300cggacgttcg gccaaggg
31847318DNAArtificial Sequencelight chain variable region
47gccatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc
60atcacttgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg
120taccagcaga aacctggcca ggctcccagg ctcctcatct ataaagtttc
ctaccgattt 180tctggggtcc catcaaggtt cagcggcagt ggatctggga
cagaattcac tctcaccatc 240agcagcctgc agcctgatga ttttgcaact
tattactgtt ctcaaaatac acatgttcct 300cggacgttcg gccaaggg
31848318DNAArtificial Sequencelight chain variable region
48gccatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc
60atcacttgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg
120taccagcaga aacctggcca ggctcccagg ctcctcatct ataaagtttc
ctaccgattt 180tctggggtcc cagacaggtt cagtggcagt gggtcaggca
ctgatttcac actgaaaatc 240agcagggtgg aggctgagga tgttggagtt
tattactgtt ctcaaaatac acatgttcct 300cggacgttcg gccaaggg
31849318DNAArtificial Sequencelight chain variable region
49gccatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc
60atcacttgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg
120taccagcaga aacctggcca ggctcccagg ctcctcatct ataaagtttc
ctaccgattt 180tctggggtcc cagacaggtt cagtggcagt gggtcaggca
ctgatttcac actgaaaatc 240agcagggtgg aggctgagga tgttggagtt
tattactgtt ctcaaaatac acatgttcct 300cggacgttcg gccaaggg
31850318DNAArtificial Sequencelight chain variable region
50gaaattgtgt tgacacagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc
60ctctcctgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg
120taccagcaga aacctggcca ggctcccagg ctcctcatct ataaagtttc
ctaccgattt 180tctggggtcc cctcgaggtt cagtggcagt ggatctggga
cagatttcac ctttaccatc 240agtagcctgg aagctgaaga tgctgcaaca
tattactgtt ctcaaaatac acatgttcct 300cggacgttcg gccaaggg
31851318DNAArtificial Sequencelight chain variable region
51gatgttgtga tgactcagtc tccactctcc ctgcccgtca cccttggaca gccggcctcc
60atctcctgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg
120tatcagcaga aaccagggaa agctcctaag ctcctgatct ataaagtttc
ctaccgattt 180tctggggtcc cagacaggtt cagtggcagt gggtcaggca
ctgatttcac actgaaaatc 240agcagggtgg aggctgagga tgttggagtt
tattactgtt ctcaaaatac acatgttcct 300cggacgttcg gccaaggg
31852318DNAArtificial Sequencelight chain variable region
52gatattgtga tgacccagac tccactctcc ctgcccgtca cccctggaga gccggcctcc
60atctcctgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg
120tacctgcaga agccagggca gtctccacag ctcctgatct ataaagtttc
ctaccgattt 180tctggggtcc cagacaggtt cagtggcagt gggtcaggca
ctgatttcac actgaaaatc 240agcagggtgg aggctgagga tgttggagtt
tattactgtt ctcaaaatac acatgttcct 300cggacgttcg gccaaggg
31853318DNAArtificial Sequencelight chain variable region
53gccatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc
60atcacttgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg
120taccagcaga aacctggcca ggctcccagg ctcctcatct ataaagtttc
ctaccgattt 180tctggggtcc cagacaggtt cagtggcagt gggtcaggca
ctgatttcac actgaaaatc 240agcagggtgg aggctgagga tgttggagtt
tattactgtt ctcaaaatac acatgttcct 300cggacgttcg gccaaggg
31854318DNAArtificial Sequencelight chain variable region
54gaaattgtgt tgacacagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc
60ctctcctgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg
120taccagcaga aacctggcca ggctcccagg ctcctcatct ataaagtttc
ctaccgattt 180tctggggtcc cctcgaggtt cagtggcagt ggatctggga
cagatttcac ctttaccatc 240agtagcctgg aagctgaaga tgctgcaaca
tattactgtt ctcaaaatac acatgttcct 300cggacgttcg gccaaggg
31855318DNAArtificial Sequencelight chain variable region
55gatattgtga tgacccagac tccactctcc ctgcccgtca cccctggaga gccggcctcc
60atctcctgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg
120tatcagcaga aaccagggaa agctcctaag ctcctgatct ataaagtttc
ctaccgattt 180tctggggtcc cagacaggtt cagtggcagt gggtcaggca
ctgatttcac actgaaaatc 240agcagggtgg aggctgagga tgttggagtt
tattactgtt ctcaaaatac acatgttcct 300cggacgttcg gccaaggg
31856318DNAArtificial Sequencelight chain variable region
56gccatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc
60atcacttgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg
120taccagcaga aacctggcca ggctcccagg ctcctcatct ataaagtttc
ctaccgattt 180tctggggtcc catcaaggtt cagcggcagt ggatctggga
cagaattcac tctcaccatc 240agcagcctgc agcctgatga ttttgcaact
tattactgtt ctcaaaatac acatgttcct 300cggacgttcg gccaaggg
31857109PRTArtificial Sequenceheavy chain variable region 57Glu Val
Gln Leu Gln Gln Phe Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15
Ser Met Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20
25
30 Tyr Val Phe Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45 Gly Asp Ile Asn Pro Val Asn Gly Asp Thr Asn Phe Asn Glu
Lys Phe 50 55 60 Lys Asn Lys Ala Thr Leu Thr Val Asp Lys Ser Ser
Thr Thr Thr Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp
Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Gly Tyr Thr Met Asp
Tyr Trp Gly Gln Gly 100 105 58109PRTArtificial Sequenceheavy chain
variable region 58Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val
Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly
Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Val Phe Trp Val Arg Gln Ala
Arg Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Asp Ile Asn Pro Val
Asn Gly Asp Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Asn Arg 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 Thr Met Asp Tyr Trp Gly Gln Gly 100 105
59109PRTArtificial Sequenceheavy chain variable region 59Gln Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Thr Phe Thr Asn Tyr 20
25 30 Tyr Val Phe Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp
Ile 35 40 45 Gly Asp Ile Asn Pro Val Asn Gly Asp Thr Asn Phe Asn
Glu Lys Phe 50 55 60 Lys Asn Arg 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 Thr Met
Asp Tyr Trp Gly Gln Gly 100 105 60109PRTArtificial Sequenceheavy
chain variable region 60Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Arg Ile Ser Cys Lys Gly Ser
Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Val Phe Trp Val Arg Gln
Ala Arg Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Asp Ile Asn Pro
Val Asn Gly Asp Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Asn Arg
Val Thr Ile Thr Ala Asp Lys 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 Tyr Thr Met Asp Tyr Trp Gly Gln Gly 100 105
61109PRTArtificial Sequenceheavy chain variable region 61Gln Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Thr Phe Thr Asn Tyr 20
25 30 Tyr Val Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ser Asp Ile Asn Pro Val Asn Gly Asp Thr Asn Phe Asn
Glu Lys Phe 50 55 60 Lys Asn Arg Val Thr Ile Ser Val Asp Thr Ser
Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Thr Met
Asp Tyr Trp Gly Gln Gly 100 105 62109PRTArtificial Sequenceheavy
chain variable region 62Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser
Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Val Phe Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Asp Ile Asn Pro
Val Asn Gly Asp Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Asn Arg
Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Gly Tyr Thr Met Asp Tyr Trp Gly Gln Gly 100 105
63109PRTArtificial Sequenceheavy chain variable region 63Gln Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Thr Phe Thr Asn Tyr 20
25 30 Tyr Val Phe Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp
Ile 35 40 45 Gly Asp Ile Asn Pro Val Asn Gly Asp Thr Asn Phe Asn
Glu Lys Phe 50 55 60 Lys Asn Arg Leu Thr Ile Ser Lys Asp Thr Ser
Lys Asn Gln Val Val 65 70 75 80 Leu Thr Met Thr Asn Met Asp Pro Val
Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Thr Met
Asp Tyr Trp Gly Gln Gly 100 105 64109PRTArtificial Sequenceheavy
chain variable region 64Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val 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 Tyr Val Phe Trp Val Arg Gln
Ala Arg Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Asp Ile Asn Pro
Val Asn Gly Asp Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Asn Arg
Val Thr Ile Thr Ala Asp Lys 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 Tyr Thr Met Asp Tyr Trp Gly Gln Gly 100 105
65109PRTArtificial Sequenceheavy chain variable region 65Gln Ile
Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1 5 10 15
Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Tyr Thr Phe Thr Asn Tyr 20
25 30 Tyr Val Phe Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu Trp
Leu 35 40 45 Gly Asp Ile Asn Pro Val Asn Gly Asp Thr Asn Phe Asn
Glu Lys Phe 50 55 60 Lys Asn Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Thr Met
Asp Tyr Trp Gly Gln Gly 100 105 66109PRTArtificial Sequenceheavy
chain variable region 66Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser
Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Val Phe Trp Val Arg Gln
Ala Arg Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Asp Ile Asn Pro
Val Asn Gly Asp Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Asn Arg
Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val 65 70 75 80 Leu
Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys 85 90
95 Ala Arg Gly Gly Tyr Thr Met Asp Tyr Trp Gly Gln Gly 100 105
67109PRTArtificial Sequenceheavy chain variable region 67Gln Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Thr Phe Thr Asn Tyr 20
25 30 Tyr Val Phe Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp
Ile 35 40 45 Gly Asp Ile Asn Pro Val Asn Gly Asp Thr Asn Phe Asn
Glu Lys Phe 50 55 60 Lys Asn Arg Leu Thr Ile Ser Lys Asp Thr Ser
Lys Asn Gln Val Val 65 70 75 80 Leu Thr Met Thr Asn Met Asp Pro Val
Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Thr Met
Asp Tyr Trp Gly Gln Gly 100 105 68109PRTArtificial Sequenceheavy
chain variable region 68Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val 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 Tyr Val Phe Trp Val Arg Gln
Ala Arg Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Asp Ile Asn Pro
Val Asn Gly Asp Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Asn Arg
Val Thr Ile Thr Ala Asp Lys 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 Tyr Thr Met Asp Tyr Trp Gly Gln Gly 100 105
69109PRTArtificial Sequenceheavy chain variable region 69Gln Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val 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 Tyr Val Phe Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp
Ile 35 40 45 Gly Asp Ile Asn Pro Val Asn Gly Asp Thr Asn Phe Asn
Glu Lys Phe 50 55 60 Lys Asn Arg Val Thr Ile Thr Ala Asp Lys 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 Tyr Thr Met
Asp Tyr Trp Gly Gln Gly 100 105 70109PRTArtificial Sequenceheavy
chain variable region 70Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Arg Ile Ser Cys Lys Gly Ser
Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Val Phe Trp Ile Arg Gln
Ser Pro Ser Arg Gly Leu Glu Trp Leu 35 40 45 Gly Asp Ile Asn Pro
Val Asn Gly Asp Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Asn Arg
Val Thr Ile Thr Ala Asp Lys 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 Tyr Thr Met Asp Tyr Trp Gly Gln Gly 100 105
71109PRTArtificial Sequenceheavy chain variable region 71Gln Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Thr Phe Thr Asn Tyr 20
25 30 Tyr Val Phe Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp
Ile 35 40 45 Gly Asp Ile Asn Pro Val Asn Gly Asp Thr Asn Phe Asn
Glu Lys Phe 50 55 60 Lys Asn Arg 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 Thr Met
Asp Tyr Trp Gly Gln Gly 100 105 72109PRTArtificial Sequenceheavy
chain variable region 72Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser
Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Val Phe Trp Val Arg Gln
Ala Arg Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Asp Ile Asn Pro
Val Asn Gly Asp Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Asn Arg
Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val 65 70 75 80 Leu
Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys 85 90
95 Ala Arg Gly Gly Tyr Thr Met Asp Tyr Trp Gly Gln Gly 100 105
73109PRTArtificial Sequenceheavy chain variable region 73Gln Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Thr Phe Thr Asn Tyr 20
25 30 Tyr Val Phe Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp
Ile 35 40 45 Gly Asp Ile Asn Pro Val Asn Gly Asp Thr Asn Phe Asn
Glu Lys Phe 50 55 60 Lys Asn Arg 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 Thr Met
Asp Tyr Trp Gly Gln Gly 100 105 74109PRTArtificial Sequenceheavy
chain variable region 74Glu 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 Tyr Val Phe Trp Ile Arg Gln
Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Asn Pro
Val Asn Gly Asp Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Asn Arg
Val Thr Ile Thr Ala Asp Lys 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 Tyr Thr Met Asp Tyr Trp Gly Gln Gly 100 105
75109PRTArtificial Sequenceheavy chain variable region 75Glu Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15
Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asn Tyr 20
25 30 Tyr Val Phe Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu Trp
Leu 35 40 45 Gly Asp Ile Asn Pro Val Asn Gly Asp Thr Asn Phe Asn
Glu Lys Phe 50 55 60 Lys Asn Arg Val Thr Ile Thr Ala Asp Lys 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 Tyr Thr Met
Asp Tyr Trp Gly Gln Gly 100 105 76109PRTArtificial Sequenceheavy
chain variable region 76Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Arg Ile Ser Cys Lys Gly Ser
Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Val Phe Trp Ile Arg Gln
Ser Pro Ser Arg Gly Leu Glu Trp Leu 35 40 45 Gly Asp Ile Asn Pro
Val Asn Gly Asp Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Asn Arg
Val Thr Ile Thr Ala Asp Lys 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 Tyr Thr Met Asp Tyr Trp Gly Gln Gly 100 105
77109PRTArtificial Sequenceheavy chain variable region 77Gln Ile
Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1 5 10 15
Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Tyr Thr Phe Thr Asn Tyr 20
25 30 Tyr Val Phe Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45 Gly Asp Ile Asn Pro Val Asn Gly Asp Thr Asn Phe Asn
Glu Lys Phe 50 55 60 Lys Asn Arg Val Thr Ile Thr Ala Asp Lys 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 Tyr Thr Met
Asp Tyr Trp Gly Gln Gly 100 105 78109PRTArtificial Sequenceheavy
chain variable region 78Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser
Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Val Phe Trp Val Arg Gln
Ala Arg Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Asp Ile Asn Pro
Val Asn Gly Asp Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Asn Arg
Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val 65 70 75 80 Leu
Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys 85 90
95 Ala Arg Gly Gly Tyr Thr Met Asp Tyr Trp Gly Gln Gly 100 105
79109PRTArtificial Sequenceheavy chain variable region 79Gln Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Thr Phe Thr Asn Tyr 20
25 30 Tyr Val Phe Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp
Ile 35 40 45 Gly Asp Ile Asn Pro Val Asn Gly Asp Thr Asn Phe Asn
Glu Lys Phe 50 55 60 Lys Asn Arg Leu Thr Ile Ser Lys Asp Thr Ser
Lys Asn Gln Val Val 65 70 75 80 Leu Thr Met Thr Asn Met Asp Pro Val
Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Thr Met
Asp Tyr Trp Gly Gln Gly 100 105 80109PRTArtificial Sequenceheavy
chain variable region 80Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser
Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Val Phe Trp Val Arg Gln
Ala Arg Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Asp Ile Asn Pro
Val Asn Gly Asp Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Asn Arg
Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val 65 70 75 80 Leu
Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys 85 90
95 Ala Arg Gly Gly Tyr Thr Met Asp Tyr Trp Gly Gln Gly 100 105
81109PRTArtificial Sequenceheavy chain variable region 81Gln Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val 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 Tyr Val Phe Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp
Ile 35 40 45 Gly Asp Ile Asn Pro Val Asn Gly Asp Thr Asn Phe Asn
Glu Lys Phe 50 55 60 Lys Asn Arg Val Thr Ile Thr Ala Asp Lys 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 Tyr Thr Met
Asp Tyr Trp Gly Gln Gly 100 105 82327DNAArtificial Sequenceheavy
chain variable region 82gaggtccagc tgcagcagtt tggggctgaa ctggtgaagc
ctggggcttc aatgaagttg 60tcctgcaagg cttctggcta caccttcacc aactactatg
tattctgggt gaaacagagg 120cctggacaag gccttgagtg gattggagac
attaatcctg tcaatggtga tactaacttc 180aatgagaaat tcaagaacaa
ggccacactg actgtagaca agtcctccac cacaacatac 240ttgcaactca
gcagcctgac atctgaggac tctgcggtct attactgtac aagagggggt
300tatactatgg actactgggg tcaagga 32783327DNAArtificial
Sequenceheavy chain variable sequence 83caggtgcagc tgcaggagtc
gggcccagga ctggtgaagc cttcacagac cctgtccctc 60acctgcactg tctctggcta
caccttcacc aactactatg tattctgggt gcgacaggct 120cgtggacaac
gccttgagtg gataggtgac attaatcctg tcaatggtga tactaacttc
180aatgagaaat tcaagaacag agtcaccatc tcagccgaca agtccatcag
caccgcctac 240ctgcagtgga gcagcctgaa ggcctcggac accgccatgt
attactgtgc gagagggggt 300tatactatgg actactgggg ccaggga
32784327DNAArtificial Sequenceheavy chain variable sequence
84caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc
60acctgcactg tctctggcta caccttcacc aactactatg tattctgggt gcgacaggct
120cgtggacaac gccttgagtg gataggtgac attaatcctg tcaatggtga
tactaacttc 180aatgagaaat tcaagaacag agtcaccatc tcagccgaca
agtccatcag caccgcctac 240ctgcagtgga gcagcctgaa ggcctcggac
accgccatgt attactgtgc gagagggggt 300tatactatgg actactgggg ccaggga
32785327DNAArtificial Sequenceheavy chain variable sequence
85gaagtgcagc tggtgcagtc tggagcagag gtgaaaaagc ccggggagtc tctgaggatc
60tcctgtaagg gttctggcta caccttcacc aactactatg tattctgggt gcgacaggct
120cgtggacaac gccttgagtg gataggtgac attaatcctg tcaatggtga
tactaacttc 180aatgagaaat tcaagaacag agtcacgatt accgcggaca
aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac
acggccgtgt attactgtgc gagagggggt 300tatactatgg actactgggg ccaggga
32786327DNAArtificial Sequenceheavy chain variable region
86caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc
60acctgcactg tctctggcta caccttcacc aactactatg tattctgggt ccgccaggct
120ccagggaagg ggctggagtg ggtcagtgac attaatcctg tcaatggtga
tactaacttc 180aatgagaaat tcaagaacag agtcaccata tcagtagaca
cgtccaagaa ccagttctcc 240ctgaagctga gctctgtgac cgccgcggac
acggctgtgt attactgtgc gagagggggt 300tatactatgg actactgggg ccaggga
32787327DNAArtificial Sequenceheavy chain variable region
87caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc
60acctgcactg tctctggcta caccttcacc aactactatg tattctgggt ccgccaggct
120ccagggaagg ggctggagtg ggtcagtgac attaatcctg tcaatggtga
tactaacttc 180aatgagaaat tcaagaacag agtcaccata tcagtagaca
cgtccaagaa ccagttctcc 240ctgaagctga gctctgtgac cgccgcggac
acggctgtgt attactgtgc gagagggggt 300tatactatgg actactgggg ccaggga
32788327DNAArtificial Sequenceheavy chain variable region
88caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc
60acctgcactg tctctggcta caccttcacc aactactatg tattctgggt gcgacaggct
120cgtggacaac gccttgagtg gataggtgac attaatcctg tcaatggtga
tactaacttc 180aatgagaaat tcaagaacag actcaccatc tccaaggaca
cctccaaaaa ccaggtggtc 240cttacaatga ccaacatgga ccctgtggac
acagccacgt attactgtgc aagagggggt 300tatactatgg actactgggg ccaggga
32789327DNAArtificial Sequenceheavy chain variable region
89caggtgcagc tgcaggagtc gggcccagga ctggtgaagc ctggggctac agtgaaaatc
60tcctgcaagg tttctggcta caccttcacc aactactatg tattctgggt gcgacaggct
120cgtggacaac gccttgagtg gataggtgac attaatcctg tcaatggtga
tactaacttc 180aatgagaaat tcaagaacag agtcacgatt accgcggaca
aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac
acggccgtgt attactgtgc gagagggggt 300tatactatgg actactgggg ccaggga
32790327DNAArtificial Sequenceheavy chain variable region
90cagatcacct tgaaggagtc tggtcctacg ctggtgaaac ccacacagac cctcacgctg
60acctgcacct tctctggcta caccttcacc aactactatg tattctggat caggcagtcc
120ccatcgagag gccttgagtg gctgggtgac attaatcctg tcaatggtga
tactaacttc 180aatgagaaat tcaagaacag attcaccatc tccagagaca
acgccaagaa ctcactgtat 240ctgcaaatga acagcctgag agccgaggac
acggctgtgt attactgtgc gagagggggt 300tatactatgg actactgggg ccaggga
32791327DNAArtificial Sequenceheavy chain variable region
91caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc
60acctgcactg tctctggcta caccttcacc aactactatg tattctgggt gcgacaggct
120cgtggacaac gccttgagtg gataggtgac attaatcctg tcaatggtga
tactaacttc 180aatgagaaat tcaagaacag actcaccatc tccaaggaca
cctccaaaaa ccaggtggtc 240cttacaatga ccaacatgga ccctgtggac
acagccacgt attactgtgc aagagggggt 300tatactatgg actactgggg ccaggga
32792327DNAArtificial Sequenceheavy chain variable region
92caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc
60acctgcactg tctctggcta caccttcacc aactactatg tattctgggt gcgacaggct
120cgtggacaac gccttgagtg gataggtgac attaatcctg tcaatggtga
tactaacttc 180aatgagaaat tcaagaacag actcaccatc tccaaggaca
cctccaaaaa ccaggtggtc 240cttacaatga ccaacatgga ccctgtggac
acagccacgt attactgtgc aagagggggt 300tatactatgg actactgggg ccaggga
32793327DNAArtificial Sequenceheavy chain variable region
93caggtgcagc tgcaggagtc gggcccagga ctggtgaagc ctggggctac agtgaaaatc
60tcctgcaagg tttctggcta caccttcacc aactactatg tattctgggt gcgacaggct
120cgtggacaac gccttgagtg gataggtgac attaatcctg tcaatggtga
tactaacttc 180aatgagaaat tcaagaacag agtcacgatt accgcggaca
aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac
acggccgtgt attactgtgc gagagggggt 300tatactatgg actactgggg ccaggga
32794327DNAArtificial Sequenceheavy chain variable region
94caggtgcagc tgcaggagtc gggcccagga ctggtgaagc ctggggctac agtgaaaatc
60tcctgcaagg tttctggcta caccttcacc aactactatg tattctgggt gcgacaggct
120cgtggacaac gccttgagtg gataggtgac attaatcctg tcaatggtga
tactaacttc 180aatgagaaat tcaagaacag agtcacgatt accgcggaca
aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac
acggccgtgt attactgtgc gagagggggt 300tatactatgg actactgggg ccaggga
32795327DNAArtificial Sequenceheavy chain variable region
95gaagtgcagc tggtgcagtc tggagcagag gtgaaaaagc ccggggagtc tctgaggatc
60tcctgtaagg gttctggcta caccttcacc aactactatg tattctggat caggcagtcc
120ccatcgagag gccttgagtg gctgggtgac attaatcctg tcaatggtga
tactaacttc 180aatgagaaat tcaagaacag agtcacgatt accgcggaca
aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac
acggccgtgt attactgtgc gagagggggt 300tatactatgg actactgggg ccaggga
32796327DNAArtificial Sequenceheavy chain variable region
96caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc
60acctgcactg tctctggcta caccttcacc aactactatg tattctgggt gcgacaggct
120cgtggacaac gccttgagtg gataggtgac attaatcctg tcaatggtga
tactaacttc 180aatgagaaat tcaagaacag agtcaccatc tcagccgaca
agtccatcag caccgcctac 240ctgcagtgga gcagcctgaa ggcctcggac
accgccatgt attactgtgc gagagggggt 300tatactatgg actactgggg ccaggga
32797327DNAArtificial Sequenceheavy chain variable region
97caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc
60acctgcactg tctctggcta caccttcacc aactactatg tattctgggt gcgacaggct
120cgtggacaac gccttgagtg gataggtgac attaatcctg tcaatggtga
tactaacttc 180aatgagaaat tcaagaacag actcaccatc tccaaggaca
cctccaaaaa ccaggtggtc 240cttacaatga ccaacatgga ccctgtggac
acagccacgt attactgtgc aagagggggt 300tatactatgg actactgggg ccaggga
32798327DNAArtificial Sequenceheavy chain variable region
98caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc
60acctgcactg tctctggcta caccttcacc aactactatg tattctgggt gcgacaggct
120cgtggacaac gccttgagtg gataggtgac attaatcctg tcaatggtga
tactaacttc 180aatgagaaat tcaagaacag agtcaccatc tcagccgaca
agtccatcag caccgcctac 240ctgcagtgga gcagcctgaa ggcctcggac
accgccatgt attactgtgc gagagggggt 300tatactatgg actactgggg ccaggga
32799327DNAArtificial Sequenceheavy chain variable region
99gaggtccagc tggtacagtc tggggctgag gtgaagaagc ctggggctac agtgaaaatc
60tcctgcaagg tttctggcta caccttcacc aactactatg tattctggat ccgccagccc
120ccagggaagg ggctggagtg gattggtgac attaatcctg tcaatggtga
tactaacttc 180aatgagaaat tcaagaacag agtcacgatt accgcggaca
aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac
acggccgtgt attactgtgc gagagggggt 300tatactatgg actactgggg ccaggga
327100327DNAArtificial Sequenceheavy chain variable region
100gaagtgcagc tggtgcagtc tggagcagag gtgaaaaagc ccggggagtc
tctgaggatc 60tcctgtaagg gttctggcta caccttcacc aactactatg tattctggat
caggcagtcc 120ccatcgagag gccttgagtg gctgggtgac attaatcctg
tcaatggtga tactaacttc 180aatgagaaat tcaagaacag agtcacgatt
accgcggaca aatccacgag cacagcctac 240atggagctga gcagcctgag
atctgaggac acggccgtgt attactgtgc gagagggggt 300tatactatgg
actactgggg ccaggga 327101327DNAArtificial Sequenceheavy chain
variable region 101gaagtgcagc tggtgcagtc tggagcagag gtgaaaaagc
ccggggagtc tctgaggatc 60tcctgtaagg gttctggcta caccttcacc aactactatg
tattctggat caggcagtcc 120ccatcgagag gccttgagtg gctgggtgac
attaatcctg tcaatggtga tactaacttc 180aatgagaaat tcaagaacag
agtcacgatt accgcggaca aatccacgag cacagcctac 240atggagctga
gcagcctgag atctgaggac acggccgtgt attactgtgc gagagggggt
300tatactatgg actactgggg ccaggga 327102327DNAArtificial
Sequenceheavy chain variable region 102cagatcacct tgaaggagtc
tggtcctacg ctggtgaaac ccacacagac cctcacgctg 60acctgcacct tctctggcta
caccttcacc aactactatg tattctgggt gcgacaggcc 120cctggacaag
ggcttgagtg gatgggtgac attaatcctg tcaatggtga tactaacttc
180aatgagaaat tcaagaacag agtcacgatt accgcggaca aatccacgag
cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt
attactgtgc gagagggggt 300tatactatgg actactgggg ccaggga
327103327DNAArtificial Sequenceheavy chain variable region
103caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac
cctgtccctc 60acctgcactg tctctggcta caccttcacc aactactatg tattctgggt
gcgacaggct 120cgtggacaac gccttgagtg gataggtgac attaatcctg
tcaatggtga tactaacttc 180aatgagaaat tcaagaacag actcaccatc
tccaaggaca cctccaaaaa ccaggtggtc 240cttacaatga ccaacatgga
ccctgtggac acagccacgt attactgtgc aagagggggt 300tatactatgg
actactgggg ccaggga 327104327DNAArtificial Sequenceheavy chain
variable region 104caggtgcagc tgcaggagtc gggcccagga ctggtgaagc
cttcacagac cctgtccctc 60acctgcactg tctctggcta caccttcacc aactactatg
tattctgggt gcgacaggct 120cgtggacaac gccttgagtg gataggtgac
attaatcctg tcaatggtga tactaacttc 180aatgagaaat tcaagaacag
actcaccatc tccaaggaca cctccaaaaa ccaggtggtc 240cttacaatga
ccaacatgga ccctgtggac acagccacgt attactgtgc aagagggggt
300tatactatgg actactgggg ccaggga 327105327DNAArtificial
Sequenceheavy chain variable region 105caggtgcagc tgcaggagtc
gggcccagga ctggtgaagc cttcacagac cctgtccctc 60acctgcactg tctctggcta
caccttcacc aactactatg tattctgggt gcgacaggct 120cgtggacaac
gccttgagtg gataggtgac attaatcctg tcaatggtga tactaacttc
180aatgagaaat tcaagaacag actcaccatc tccaaggaca cctccaaaaa
ccaggtggtc 240cttacaatga ccaacatgga ccctgtggac acagccacgt
attactgtgc aagagggggt 300tatactatgg actactgggg ccaggga
327106327DNAArtificial Sequenceheavy chain variable region
106caggtgcagc tgcaggagtc gggcccagga ctggtgaagc ctggggctac
agtgaaaatc 60tcctgcaagg tttctggcta caccttcacc aactactatg tattctgggt
gcgacaggct 120cgtggacaac gccttgagtg gataggtgac attaatcctg
tcaatggtga tactaacttc 180aatgagaaat tcaagaacag agtcacgatt
accgcggaca aatccacgag cacagcctac 240atggagctga gcagcctgag
atctgaggac acggccgtgt attactgtgc gagagggggt 300tatactatgg
actactgggg ccaggga 327107219PRTArtificial Sequencecomplete light
chain 107Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Ser
Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser
Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu
Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val
Ser Tyr 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 Phe Cys Ser Gln Asn 85 90 95 Thr His
Val Pro Arg 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 108660DNAArtificial
Sequencecomplete light chain 108gatgttgtta tgacccaaac tccactctcc
ctgtctgtca gtcttggaga tcaagcctcc 60atctcttgca gatctagtca gagccttgta
cacagtaatg gaaacaccta tttacattgg 120tacctgcaga agccaggcca
gtctccaaag ctcctgatct acaaagtttc ctaccgattt 180tctggggtcc
cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc
240agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaaatac
acatgttcct 300cggacgttcg gccaagggac caaggtggaa atcaaacgta
cggtggctgc accatctgtc 360ttcatcttcc cgccatctga tgagcagttg
aaatctggaa ctgcctctgt tgtgtgcctg 420ctgaataact tctatcccag
agaggccaaa gtacagtgga aggtggataa cgccctccaa 480tcgggtaact
cccaggagag tgtcacagag caggacagca aggacagcac ctacagcctc
540agcagcaccc tgacgctgag caaagcagac tacgagaaac acaaagtcta
cgcctgcgaa 600gtcacccatc agggcctgag ctcgcccgtc acaaagagct
tcaacagggg agagtgttga 660109446PRTArtificial Sequencecomplete heavy
chain 109Glu Val Gln Leu Gln Gln Phe Gly Ala Glu Leu Val Lys Pro
Gly Ala 1 5 10 15 Ser Met Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Asn Tyr 20 25 30 Tyr Val Phe Trp Val Lys Gln Arg Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Asn Pro Val Asn Gly
Asp Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Asn Lys Ala Thr Leu
Thr Val Asp Lys Ser Ser Thr Thr Thr Tyr 65 70 75 80 Leu Gln Leu Ser
Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg
Gly Gly Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115
120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235
240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360
365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys 435 440 445 1101341PRTArtificial
Sequencecomplete heavy chain 110Gly Ala Gly Gly Thr Cys Cys Ala Gly
Cys Thr Gly Cys Ala Gly Cys 1 5 10 15 Ala Gly Thr Thr Thr Gly Gly
Gly Gly Cys Thr Gly Ala Ala Cys Thr 20 25 30 Gly Gly Thr Gly Ala
Ala Gly Cys Cys Thr Gly Gly Gly Gly Cys Thr 35 40 45 Thr Cys Ala
Ala Thr Gly Ala Ala Gly Thr Thr Gly Thr Cys Cys Thr 50 55 60 Gly
Cys Ala Ala Gly Gly Cys Thr Thr Cys Thr Gly Gly Cys Thr Ala 65 70
75 80 Cys Ala Cys Cys Thr Thr Cys Ala Cys Cys Ala Ala Cys Thr Ala
Cys 85 90 95 Thr Ala Thr Gly Thr Ala Thr Thr Cys Thr Gly Gly Gly
Thr Gly Ala 100 105 110 Ala Ala Cys Ala Gly Ala Gly Gly Cys Cys Thr
Gly Gly Ala Cys Ala 115 120 125 Ala Gly Gly Cys Cys Thr Thr Gly Ala
Gly Thr Gly Gly Ala Thr Thr 130 135 140 Gly Gly Ala Gly Ala Cys Ala
Thr Thr Ala Ala Thr Cys Cys Thr Gly 145 150 155 160 Thr Cys Ala Ala
Thr Gly Gly Thr Gly Ala Thr Ala Cys Thr Ala Ala 165 170 175 Cys Thr
Thr Cys Ala Ala Thr Gly Ala Gly Ala Ala Ala Thr Thr Cys 180 185 190
Ala Ala Gly Ala Ala Cys Ala Ala Gly Gly Cys Cys Ala Cys Ala Cys 195
200 205 Thr Gly Ala Cys Thr Gly Thr Ala Gly Ala Cys Ala Ala Gly Thr
Cys 210 215 220 Cys Thr Cys Cys Ala Cys Cys Ala Cys Ala Ala Cys Ala
Thr Ala Cys 225 230 235 240 Thr Thr Gly Cys Ala Ala Cys Thr Cys Ala
Gly Cys Ala Gly Cys Cys 245 250 255 Thr Gly Ala Cys Ala Thr Cys Thr
Gly Ala Gly Gly Ala Cys Thr Cys 260 265 270 Thr Gly Cys Gly Gly Thr
Cys Thr Ala Thr Thr Ala Cys Thr Gly Thr 275 280 285 Ala Cys Ala Ala
Gly Ala Gly Gly Gly Gly Gly Thr Thr Ala Thr Ala 290 295 300 Cys Thr
Ala Thr Gly Gly Ala Cys Thr Ala Cys Thr Gly Gly Gly Gly 305 310 315
320 Cys Cys Ala Gly Gly Gly Ala Ala Cys Gly Cys Thr Gly Gly Thr Cys
325 330 335 Ala Cys Cys Gly Thr Cys Ala Gly Cys Thr Cys Ala Gly Cys
Cys Thr 340 345 350 Cys Cys Ala Cys Cys Ala Ala Gly Gly Gly Cys Cys
Cys Ala Thr Cys 355 360 365 Gly Gly Thr Cys Thr Thr Cys Cys Cys Cys
Cys Thr Gly Gly Cys Ala 370 375 380 Cys Cys Cys Thr Cys Cys Thr Cys
Cys Ala Ala Gly Ala Gly Cys Ala 385 390 395 400 Cys Cys Thr Cys Thr
Gly Gly Gly Gly Gly Cys Ala Cys Ala Gly Cys 405 410 415 Gly Gly Cys
Cys Cys Thr Gly Gly Gly Cys Thr Gly Cys Cys Thr Gly 420 425 430 Gly
Thr Cys Ala Ala Gly Gly Ala Cys Thr Ala Cys Thr Thr Cys Cys 435 440
445 Cys Cys Gly Ala Ala Cys Cys Gly Gly Thr Gly Ala Cys Gly Gly Thr
450 455 460 Gly Thr Cys Gly Thr Gly Gly Ala Ala Cys Thr Cys Ala Gly
Gly Cys 465 470 475 480 Gly Cys Cys Cys Thr Gly Ala Cys Cys Ala Gly
Cys Gly Gly Cys Gly 485 490 495 Thr Gly Cys Ala Cys Ala Cys Cys Thr
Thr Cys Cys Cys Gly Gly Cys 500 505 510 Thr Gly Thr Cys Cys Thr Ala
Cys Ala Gly Thr Cys Cys Thr Cys Ala 515 520 525 Gly Gly Ala Cys Thr
Cys Thr Ala Cys Thr Cys Cys Cys Thr Cys Ala 530 535 540 Gly Cys Ala
Gly Cys Gly Thr Gly Gly Thr Gly Ala Cys Cys Gly Thr 545 550 555 560
Gly Cys Cys Cys Thr Cys Cys Ala Gly Cys Ala Gly Cys Thr Thr Gly 565
570 575 Gly Gly Cys Ala Cys Cys Cys Ala Gly Ala Cys Cys Thr Ala Cys
Ala 580 585 590 Thr Cys Thr Gly Cys Ala Ala Cys Gly Thr Gly Ala Ala
Thr Cys Ala 595 600 605 Cys Ala Ala Gly Cys Cys Cys Ala Gly Cys Ala
Ala Cys Ala Cys Cys 610 615 620 Ala Ala Gly Gly Thr Gly Gly Ala Cys
Ala Ala Gly Ala Ala Ala Gly 625 630 635 640 Thr Thr Gly Ala Gly Cys
Cys Cys Ala Ala Ala Thr Cys Thr Thr Gly 645 650 655 Thr Gly Ala Cys
Ala Ala Ala Ala Cys Thr Cys Ala Cys Ala Cys Ala 660 665 670 Thr Gly
Cys Cys Cys Ala Cys Cys Gly Thr Gly Cys Cys Cys Ala Gly 675 680 685
Cys Ala Cys Cys Thr Gly Ala Ala Cys Thr Cys Cys Thr Gly Gly Gly 690
695 700 Gly Gly Gly Ala Cys Cys Gly Thr Cys Ala Gly Thr Cys Thr Thr
Cys 705 710 715 720 Cys Thr Cys Thr Thr Cys Cys Cys Cys Cys Cys Ala
Ala Ala Ala Cys 725 730 735 Cys Cys Ala Ala Gly Gly Ala Cys Ala Cys
Cys Cys Thr Cys Ala Thr 740 745 750 Gly Ala Thr Cys Thr Cys Cys Cys
Gly Gly Ala Cys Cys Cys Cys Thr 755 760 765 Gly Ala Gly Gly Thr Cys
Ala Cys Ala Thr Gly Cys Gly Thr Gly Gly 770 775 780 Thr Gly Gly Thr
Gly Gly Ala Cys Gly Thr Gly Ala Gly Cys Cys Ala 785 790 795 800 Cys
Gly Ala Ala Gly Ala Cys Cys Cys Thr Gly Ala Gly Gly Thr Cys 805 810
815 Ala Ala Gly Thr Thr Cys Ala Ala Cys Thr Gly Gly Thr Ala Cys Gly
820 825 830 Thr Gly Gly Ala Cys Gly Gly Cys Gly Thr Gly Gly Ala Gly
Gly Thr 835 840 845 Gly Cys Ala Thr Ala Ala Thr Gly Cys Cys Ala Ala
Gly Ala Cys Ala 850 855 860 Ala Ala Gly Cys Cys Gly Cys Gly Gly Gly
Ala Gly Gly Ala Gly Cys 865 870 875 880 Ala Gly Thr Ala Cys Ala Ala
Cys Ala Gly Cys Ala Cys Gly Thr Ala 885 890 895 Cys Cys Gly Thr Gly
Thr Gly Gly Thr Cys Ala Gly Cys Gly Thr Cys 900 905 910 Cys Thr Cys
Ala Cys Cys Gly Thr Cys Cys Thr Gly Cys Ala Cys Cys 915 920 925 Ala
Gly Gly Ala Cys Thr Gly Gly Cys Thr Gly Ala Ala Thr Gly Gly 930 935
940 Cys Ala Ala Gly Gly Ala Gly Thr Ala Cys Ala Ala Gly Thr Gly Cys
945 950 955 960 Ala Ala Gly Gly Thr Cys Ala Gly Cys Ala Ala Cys Ala
Ala Ala Gly 965 970 975 Cys Cys Cys Thr Cys Cys Cys Ala Gly Cys Cys
Cys Cys Cys Ala Thr 980 985 990 Cys Gly Ala Gly Ala Ala Ala Ala Cys
Cys Ala Thr Cys Thr Cys Cys 995 1000 1005 Ala Ala Ala Gly Cys Cys
Ala Ala Ala Gly Gly Gly Cys Ala Gly 1010 1015 1020 Cys Cys Cys Cys
Gly Ala Gly Ala Ala Cys Cys Ala Cys Ala Gly 1025 1030 1035 Gly Thr
Gly Thr Ala Cys Ala Cys Cys Cys Thr Gly Cys Cys Cys 1040 1045 1050
Cys Cys Ala Thr Cys Cys Cys Gly Gly Gly Ala Thr Gly Ala Gly 1055
1060 1065 Cys Thr Gly Ala Cys Cys Ala Ala Gly Ala Ala Cys Cys Ala
Gly 1070 1075 1080 Gly Thr Cys Ala Gly Cys Cys Thr Gly Ala Cys Cys
Thr Gly Cys 1085 1090 1095 Cys Thr Gly Gly Thr Cys Ala Ala Ala Gly
Gly Cys Thr Thr Cys 1100 1105 1110 Thr Ala Thr Cys Cys Cys Ala Gly
Cys Gly Ala Cys Ala Thr Cys 1115 1120 1125 Gly Cys Cys Gly Thr Gly
Gly Ala Gly Thr Gly Gly Gly Ala Gly 1130 1135 1140 Ala Gly Cys Ala
Ala Thr Gly Gly Gly Cys Ala Gly Cys Cys Gly 1145 1150 1155 Gly Ala
Gly Ala Ala Cys Ala Ala Cys Thr Ala Cys Ala Ala Gly 1160 1165 1170
Ala Cys Cys Ala Cys Gly Cys Cys Thr Cys Cys Cys Gly Thr Gly 1175
1180 1185 Cys Thr Gly Gly Ala Cys Thr Cys Cys Gly Ala Cys Gly Gly
Cys 1190 1195 1200 Thr Cys Cys Thr Thr Cys Thr Thr Cys Cys Thr Cys
Thr Ala Cys 1205 1210 1215 Ala Gly Cys Ala Ala Gly Cys Thr Cys Ala
Cys Cys Gly Thr Gly 1220 1225 1230 Gly Ala Cys Ala Ala Gly Ala Gly
Cys Ala Gly Gly Thr Gly Gly 1235 1240 1245 Cys Ala Gly Cys Ala Gly
Gly Gly Gly Ala Ala Cys Gly Thr Cys 1250 1255 1260 Thr Thr Cys Thr
Cys Ala Thr Gly Cys Thr Cys Cys Gly Thr Gly 1265 1270 1275 Ala Thr
Gly Cys Ala Thr Gly Ala Gly Gly Cys Thr Cys Thr Gly 1280 1285 1290
Cys Ala Cys Ala Ala Cys Cys Ala Cys Thr Ala Cys Ala Cys Gly 1295
1300 1305 Cys Ala Gly Ala Ala Gly Ala Gly Cys Cys Thr Cys Thr Cys
Cys 1310 1315 1320 Cys Thr Gly Thr Cys Thr Cys Cys Gly Gly Gly Thr
Ala Ala Ala 1325 1330 1335 Thr Gly Ala 1340 111354PRTArtificial
Sequencecomplete heavy chain 111Glu Val Gln Leu Gln Gln Phe Gly Ala
Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Met Lys Leu Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Val Phe Trp Val
Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile
Asn Pro Val Asn Gly Asp Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys
Asn Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Thr Thr Thr Tyr 65 70
75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr
Cys 85 90 95 Thr Arg Gly Gly Tyr Thr Met Asp Tyr Trp Gly Gln Gly
Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195
200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val
260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Gln Ser Thr Tyr Arg
Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg
112442PRTArtificial Sequencecomplete heavy chain 112Gln Val Gln Leu
Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu
Ser Leu Thr Cys Thr Val Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30
Tyr Val Phe Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp Ile 35
40 45 Gly Asp Ile Asn Pro Val Asn Gly Asp Thr Asn Phe Asn Glu Lys
Phe 50 55 60 Lys Asn Arg 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 Thr Met Asp Tyr
Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Cys Ser Arg Ser
Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165
170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn
Phe 180 185 190 Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro
Ser Asn Thr 195 200 205 Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys
Val Glu Cys Pro Pro 210 215 220 Cys Pro Ala Pro Pro Val Ala Gly Pro
Ser Val Phe Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp 260 265 270 Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285
Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val 290
295 300 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn 305 310 315 320 Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Thr Lys Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu 340 345 350 Met Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Asn Thr
Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400 Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 405 410
415 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
420 425 430 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
113443PRTArtificial Sequencecomplete heavy chain 113Gln Val Gln Leu
Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu
Ser Leu Thr Cys Thr Val Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30
Tyr Val Phe Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp Ile 35
40 45 Gly Asp Ile Asn Pro Val Asn Gly Asp Thr Asn Phe Asn Glu Lys
Phe 50 55 60 Lys Asn Arg 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 Thr Met Asp Tyr
Trp Gly Gln Gly Thr Thr Val 100 105 110 Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Cys Ser Arg Ser
Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165
170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu 180 185 190 Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro
Ser Asn Thr 195 200 205 Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly
Pro Pro Cys Pro Pro 210 215 220 Cys Pro Ala Pro Glu Phe Leu Gly 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 Gln 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 Tyr Arg Val Val Ser Val Leu Thr Val 290
295 300 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser 305 310 315 320 Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile
Ser Lys Ala Lys 325 330 335 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Gln 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 Val Leu Asp Ser Asp Gly Ser Phe 385 390 395 400 Phe
Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 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 Leu Gly Lys 435 440
1141341PRTArtificial Sequencecomplete heavy chain 114Gly Ala Gly
Gly Thr Cys Cys Ala Gly Cys Thr Gly Cys Ala Gly Cys 1 5 10 15 Ala
Gly Thr Thr Thr Gly Gly Gly Gly Cys Thr Gly Ala Ala Cys Thr 20 25
30 Gly Gly Thr Gly Ala Ala Gly Cys Cys Thr Gly Gly Gly Gly Cys Thr
35 40 45 Thr Cys Ala Ala Thr Gly Ala Ala Gly Thr Thr Gly Thr Cys
Cys Thr 50 55 60 Gly Cys Ala Ala Gly Gly Cys Thr Thr Cys Thr Gly
Gly Cys Thr Ala 65 70 75 80 Cys Ala Cys Cys Thr Thr Cys Ala Cys Cys
Ala Ala Cys Thr Ala Cys 85 90 95 Thr Ala Thr Gly Thr Ala Thr Thr
Cys Thr Gly Gly Gly Thr Gly Ala 100 105 110 Ala Ala Cys Ala Gly Ala
Gly Gly Cys Cys Thr Gly Gly Ala Cys Ala 115 120 125 Ala Gly Gly Cys
Cys Thr Thr Gly Ala Gly Thr Gly Gly Ala Thr Thr 130 135 140 Gly Gly
Ala Gly Ala Cys Ala Thr Thr Ala Ala Thr Cys Cys Thr Gly 145 150 155
160 Thr Cys Ala Ala Thr Gly Gly Thr Gly Ala Thr Ala Cys Thr Ala Ala
165 170 175 Cys Thr Thr Cys Ala Ala Thr Gly Ala Gly Ala Ala Ala Thr
Thr Cys 180 185 190 Ala Ala Gly Ala Ala Cys Ala Ala Gly Gly Cys Cys
Ala Cys Ala Cys 195 200 205 Thr Gly Ala Cys Thr Gly Thr Ala Gly Ala
Cys Ala Ala Gly Thr Cys 210 215 220 Cys Thr Cys Cys Ala Cys Cys Ala
Cys Ala Ala Cys Ala Thr Ala Cys 225 230 235 240 Thr Thr Gly Cys Ala
Ala Cys Thr Cys Ala Gly Cys Ala Gly Cys Cys 245 250 255 Thr Gly Ala
Cys Ala Thr Cys Thr Gly Ala Gly Gly Ala Cys Thr Cys 260 265 270 Thr
Gly Cys Gly Gly Thr Cys Thr Ala Thr Thr Ala Cys Thr Gly Thr 275 280
285 Ala Cys Ala Ala Gly Ala Gly Gly Gly Gly Gly Thr Thr Ala Thr Ala
290 295 300 Cys Thr Ala Thr Gly Gly Ala Cys Thr Ala Cys Thr Gly Gly
Gly Gly 305 310 315 320 Cys Cys Ala Gly Gly Gly Ala Ala Cys Gly Cys
Thr Gly Gly Thr Cys 325 330 335 Ala Cys Cys Gly Thr Cys Ala Gly Cys
Thr Cys Ala Gly Cys Cys Thr 340 345 350 Cys Cys Ala Cys Cys Ala Ala
Gly Gly Gly Cys Cys Cys Ala Thr Cys 355 360 365 Gly Gly Thr Cys Thr
Thr Cys Cys Cys Cys Cys Thr Gly Gly Cys Ala 370 375 380 Cys Cys Cys
Thr Cys Cys Thr Cys Cys Ala Ala Gly Ala Gly Cys Ala 385 390 395 400
Cys Cys Thr Cys Thr Gly Gly Gly Gly Gly Cys Ala Cys Ala Gly Cys 405
410 415 Gly Gly Cys Cys Cys Thr Gly Gly Gly Cys Thr Gly Cys Cys Thr
Gly 420 425 430 Gly Thr Cys Ala Ala Gly Gly Ala Cys Thr Ala Cys Thr
Thr Cys Cys 435 440 445 Cys Cys Gly Ala Ala Cys Cys Gly Gly Thr Gly
Ala Cys Gly Gly Thr 450 455 460 Gly Thr Cys Gly Thr Gly Gly Ala Ala
Cys Thr Cys Ala Gly Gly Cys 465 470 475 480 Gly Cys Cys Cys Thr Gly
Ala Cys Cys Ala Gly Cys Gly Gly Cys Gly 485 490 495 Thr Gly Cys Ala
Cys Ala Cys Cys Thr Thr Cys Cys Cys Gly Gly Cys 500 505 510 Thr Gly
Thr Cys Cys Thr Ala Cys Ala Gly Thr Cys Cys Thr Cys Ala 515 520 525
Gly Gly Ala Cys Thr Cys Thr Ala Cys Thr Cys Cys Cys Thr Cys Ala 530
535 540 Gly Cys Ala Gly Cys Gly Thr Gly Gly Thr Gly Ala Cys Cys Gly
Thr 545 550 555 560 Gly Cys Cys Cys Thr Cys Cys Ala Gly Cys Ala Gly
Cys Thr Thr Gly 565 570 575 Gly Gly Cys Ala Cys Cys Cys Ala Gly Ala
Cys Cys Thr Ala Cys Ala 580 585 590 Thr Cys Thr Gly Cys Ala Ala Cys
Gly Thr Gly Ala Ala Thr Cys Ala 595 600 605 Cys Ala Ala Gly Cys Cys
Cys Ala Gly Cys Ala Ala Cys Ala Cys Cys 610 615 620 Ala Ala Gly Gly
Thr Gly Gly Ala Cys Ala Ala Gly Ala Ala Ala Gly 625 630 635 640 Thr
Thr Gly Ala Gly Cys Cys Cys Ala Ala Ala Thr Cys Thr Thr Gly 645 650
655 Thr Gly Ala Cys Ala Ala Ala Ala Cys Thr Cys Ala Cys Ala Cys Ala
660 665 670 Thr Gly Cys Cys Cys Ala Cys Cys Gly Thr Gly Cys Cys Cys
Ala Gly 675 680 685 Cys Ala Cys Cys Thr Gly Ala Ala Cys Thr Cys Cys
Thr Gly Gly Gly 690 695 700 Gly Gly Gly Ala Cys Cys Gly Thr Cys Ala
Gly Thr Cys Thr Thr Cys 705 710 715 720 Cys Thr Cys Thr Thr Cys Cys
Cys Cys Cys Cys Ala Ala Ala Ala Cys 725 730 735 Cys Cys Ala Ala Gly
Gly Ala Cys Ala Cys Cys Cys Thr Cys Ala Thr 740 745 750 Gly Ala Thr
Cys Thr Cys Cys Cys Gly Gly Ala Cys Cys Cys Cys Thr 755 760 765 Gly
Ala Gly Gly Thr Cys Ala Cys Ala Thr Gly Cys Gly Thr Gly Gly 770 775
780 Thr Gly Gly Thr Gly Gly Ala Cys Gly Thr Gly Ala Gly Cys Cys Ala
785 790 795 800 Cys Gly Ala Ala Gly Ala Cys Cys Cys Thr Gly Ala Gly
Gly Thr Cys 805 810 815 Ala Ala Gly Thr Thr Cys Ala Ala Cys Thr Gly
Gly Thr Ala Cys Gly 820 825 830 Thr Gly Gly Ala Cys Gly Gly Cys Gly
Thr Gly Gly Ala Gly Gly Thr 835 840 845 Gly Cys Ala Thr Ala Ala Thr
Gly Cys Cys Ala Ala Gly Ala Cys Ala 850 855 860 Ala Ala Gly Cys Cys
Gly Cys Gly Gly Gly Ala Gly Gly Ala Gly Cys 865 870 875 880 Ala Gly
Thr Ala Cys Cys Ala Gly Ala Gly Cys Ala Cys Gly Thr Ala 885 890 895
Cys Cys Gly Thr Gly Thr Gly Gly Thr Cys Ala Gly Cys Gly Thr Cys 900
905 910 Cys Thr Cys Ala Cys Cys Gly Thr Cys Cys Thr Gly Cys Ala Cys
Cys 915 920 925 Ala Gly Gly Ala Cys Thr Gly Gly Cys Thr Gly Ala Ala
Thr Gly Gly 930 935 940 Cys Ala Ala Gly Gly Ala Gly Thr Ala Cys Ala
Ala Gly Thr Gly Cys 945 950 955 960 Ala Ala Gly Gly Thr Cys Ala Gly
Cys Ala Ala Cys Ala Ala Ala Gly 965 970 975 Cys Cys Cys Thr Cys Cys
Cys Ala Gly Cys Cys Cys Cys Cys Ala Thr 980 985 990 Cys Gly Ala Gly
Ala Ala Ala Ala Cys Cys Ala Thr Cys Thr Cys Cys 995 1000 1005 Ala
Ala Ala Gly Cys Cys Ala Ala Ala Gly Gly Gly Cys Ala Gly 1010 1015
1020 Cys Cys Cys Cys Gly Ala Gly Ala Ala Cys Cys Ala Cys Ala Gly
1025 1030 1035 Gly Thr Gly Thr Ala Cys Ala Cys Cys Cys Thr Gly Cys
Cys Cys 1040 1045 1050 Cys Cys Ala Thr Cys Cys Cys Gly Gly Gly Ala
Thr Gly Ala Gly 1055 1060 1065 Cys Thr Gly Ala Cys Cys Ala Ala Gly
Ala Ala Cys Cys Ala Gly 1070 1075 1080 Gly Thr Cys Ala Gly Cys Cys
Thr Gly Ala Cys Cys Thr Gly Cys 1085 1090 1095 Cys Thr Gly Gly Thr
Cys Ala Ala Ala Gly Gly Cys Thr Thr Cys 1100 1105 1110 Thr Ala Thr
Cys Cys Cys Ala Gly Cys Gly Ala Cys Ala Thr Cys 1115 1120 1125 Gly
Cys Cys Gly Thr Gly Gly Ala Gly Thr Gly Gly Gly Ala Gly 1130 1135
1140 Ala Gly Cys Ala Ala Thr Gly Gly Gly Cys Ala Gly Cys Cys Gly
1145 1150 1155 Gly Ala Gly Ala Ala Cys Ala Ala Cys Thr Ala Cys Ala
Ala Gly 1160 1165 1170 Ala Cys Cys Ala Cys Gly Cys Cys Thr Cys Cys
Cys Gly Thr Gly 1175 1180 1185 Cys Thr Gly Gly Ala Cys Thr Cys Cys
Gly Ala Cys Gly Gly Cys 1190 1195 1200 Thr Cys Cys Thr Thr Cys Thr
Thr Cys Cys Thr Cys Thr Ala Cys 1205 1210 1215 Ala Gly Cys Ala Ala
Gly Cys Thr Cys Ala Cys Cys Gly Thr Gly 1220 1225 1230 Gly Ala Cys
Ala Ala Gly Ala Gly Cys Ala Gly Gly Thr Gly Gly 1235 1240 1245 Cys
Ala Gly Cys Ala Gly Gly Gly Gly Ala Ala Cys Gly Thr Cys 1250 1255
1260 Thr Thr Cys Thr Cys Ala Thr Gly Cys Thr Cys Cys Gly Thr Gly
1265 1270 1275 Ala Thr Gly Cys Ala Thr Gly Ala Gly Gly Cys Thr Cys
Thr Gly 1280 1285 1290 Cys Ala Cys Ala Ala
Cys Cys Ala Cys Thr Ala Cys Ala Cys Gly 1295 1300 1305 Cys Ala Gly
Ala Ala Gly Ala Gly Cys Cys Thr Cys Thr Cys Cys 1310 1315 1320 Cys
Thr Gly Thr Cys Thr Cys Cys Gly Gly Gly Thr Ala Ala Ala 1325 1330
1335 Thr Gly Ala 1340 1151326PRTArtificial Sequencecomplete heavy
chain 115Cys Ala Gly Gly Thr Gly Cys Ala Gly Cys Thr Gly Cys Ala
Gly Gly 1 5 10 15 Ala Gly Thr Cys Gly Gly Gly Cys Cys Cys Ala Gly
Gly Ala Cys Thr 20 25 30 Gly Gly Thr Gly Ala Ala Gly Cys Cys Thr
Thr Cys Ala Cys Ala Gly 35 40 45 Ala Cys Cys Cys Thr Gly Thr Cys
Cys Cys Thr Cys Ala Cys Cys Thr 50 55 60 Gly Cys Ala Cys Thr Gly
Thr Cys Thr Cys Thr Gly Gly Cys Thr Ala 65 70 75 80 Cys Ala Cys Cys
Thr Thr Cys Ala Cys Cys Ala Ala Cys Thr Ala Cys 85 90 95 Thr Ala
Thr Gly Thr Ala Thr Thr Cys Thr Gly Gly Gly Thr Gly Cys 100 105 110
Gly Ala Cys Ala Gly Gly Cys Thr Cys Gly Thr Gly Gly Ala Cys Ala 115
120 125 Ala Cys Gly Cys Cys Thr Thr Gly Ala Gly Thr Gly Gly Ala Thr
Ala 130 135 140 Gly Gly Thr Gly Ala Cys Ala Thr Thr Ala Ala Thr Cys
Cys Thr Gly 145 150 155 160 Thr Cys Ala Ala Thr Gly Gly Thr Gly Ala
Thr Ala Cys Thr Ala Ala 165 170 175 Cys Thr Thr Cys Ala Ala Thr Gly
Ala Gly Ala Ala Ala Thr Thr Cys 180 185 190 Ala Ala Gly Ala Ala Cys
Ala Gly Ala Gly Thr Cys Ala Cys Cys Ala 195 200 205 Thr Cys Thr Cys
Ala Gly Cys Cys Gly Ala Cys Ala Ala Gly Thr Cys 210 215 220 Cys Ala
Thr Cys Ala Gly Cys Ala Cys Cys Gly Cys Cys Thr Ala Cys 225 230 235
240 Cys Thr Gly Cys Ala Gly Thr Gly Gly Ala Gly Cys Ala Gly Cys Cys
245 250 255 Thr Gly Ala Ala Gly Gly Cys Cys Thr Cys Gly Gly Ala Cys
Ala Cys 260 265 270 Cys Gly Cys Cys Ala Thr Gly Thr Ala Thr Thr Ala
Cys Thr Gly Thr 275 280 285 Gly Cys Gly Ala Gly Ala Gly Gly Gly Gly
Gly Thr Thr Ala Thr Ala 290 295 300 Cys Thr Ala Thr Gly Gly Ala Cys
Thr Ala Cys Thr Gly Gly Gly Gly 305 310 315 320 Cys Cys Ala Gly Gly
Gly Ala Ala Cys Gly Cys Thr Gly Gly Thr Cys 325 330 335 Ala Cys Cys
Gly Thr Cys Ala Gly Cys Thr Cys Ala Gly Cys Cys Thr 340 345 350 Cys
Cys Ala Cys Cys Ala Ala Gly Gly Gly Cys Cys Cys Ala Thr Cys 355 360
365 Gly Gly Thr Cys Thr Thr Cys Cys Cys Cys Cys Thr Gly Gly Cys Ala
370 375 380 Cys Cys Cys Thr Gly Cys Thr Cys Cys Ala Gly Gly Ala Gly
Cys Ala 385 390 395 400 Cys Cys Thr Cys Cys Gly Ala Gly Ala Gly Cys
Ala Cys Ala Gly Cys 405 410 415 Gly Gly Cys Cys Cys Thr Gly Gly Gly
Cys Thr Gly Cys Cys Thr Gly 420 425 430 Gly Thr Cys Ala Ala Gly Gly
Ala Cys Thr Ala Cys Thr Thr Cys Cys 435 440 445 Cys Cys Gly Ala Ala
Cys Cys Gly Gly Thr Gly Ala Cys Gly Gly Thr 450 455 460 Gly Thr Cys
Gly Thr Gly Gly Ala Ala Cys Thr Cys Ala Gly Gly Cys 465 470 475 480
Gly Cys Thr Cys Thr Gly Ala Cys Cys Ala Gly Cys Gly Gly Cys Gly 485
490 495 Thr Gly Cys Ala Cys Ala Cys Cys Thr Thr Cys Cys Cys Gly Gly
Cys 500 505 510 Thr Gly Thr Cys Cys Thr Ala Cys Ala Gly Thr Cys Cys
Thr Cys Ala 515 520 525 Gly Gly Ala Cys Thr Cys Thr Ala Cys Thr Cys
Cys Cys Thr Cys Ala 530 535 540 Gly Cys Ala Gly Cys Gly Thr Gly Gly
Thr Gly Ala Cys Cys Gly Thr 545 550 555 560 Gly Cys Cys Cys Thr Cys
Cys Ala Gly Cys Ala Ala Cys Thr Thr Cys 565 570 575 Gly Gly Cys Ala
Cys Cys Cys Ala Gly Ala Cys Cys Thr Ala Cys Ala 580 585 590 Cys Cys
Thr Gly Cys Ala Ala Cys Gly Thr Ala Gly Ala Thr Cys Ala 595 600 605
Cys Ala Ala Gly Cys Cys Cys Ala Gly Cys Ala Ala Cys Ala Cys Cys 610
615 620 Ala Ala Gly Gly Thr Gly Gly Ala Cys Ala Ala Gly Ala Cys Ala
Gly 625 630 635 640 Thr Thr Gly Ala Gly Cys Gly Cys Ala Ala Ala Thr
Gly Thr Thr Gly 645 650 655 Thr Gly Thr Cys Gly Ala Gly Thr Gly Cys
Cys Cys Ala Cys Cys Gly 660 665 670 Thr Gly Cys Cys Cys Ala Gly Cys
Ala Cys Cys Ala Cys Cys Thr Gly 675 680 685 Thr Gly Gly Cys Ala Gly
Gly Ala Cys Cys Gly Thr Cys Ala Gly Thr 690 695 700 Cys Thr Thr Cys
Cys Thr Cys Thr Thr Cys Cys Cys Cys Cys Cys Ala 705 710 715 720 Ala
Ala Ala Cys Cys Cys Ala Ala Gly Gly Ala Cys Ala Cys Cys Cys 725 730
735 Thr Cys Ala Thr Gly Ala Thr Cys Thr Cys Cys Cys Gly Gly Ala Cys
740 745 750 Cys Cys Cys Thr Gly Ala Gly Gly Thr Cys Ala Cys Gly Thr
Gly Cys 755 760 765 Gly Thr Gly Gly Thr Gly Gly Thr Gly Gly Ala Cys
Gly Thr Gly Ala 770 775 780 Gly Cys Cys Ala Cys Gly Ala Ala Gly Ala
Cys Cys Cys Cys Gly Ala 785 790 795 800 Gly Gly Thr Cys Cys Ala Gly
Thr Thr Cys Ala Ala Cys Thr Gly Gly 805 810 815 Thr Ala Cys Gly Thr
Gly Gly Ala Cys Gly Gly Cys Gly Thr Gly Gly 820 825 830 Ala Gly Gly
Thr Gly Cys Ala Thr Ala Ala Thr Gly Cys Cys Ala Ala 835 840 845 Gly
Ala Cys Ala Ala Ala Gly Cys Cys Ala Cys Gly Gly Gly Ala Gly 850 855
860 Gly Ala Gly Cys Ala Gly Thr Thr Cys Ala Ala Cys Ala Gly Cys Ala
865 870 875 880 Cys Gly Thr Thr Cys Cys Gly Thr Gly Thr Gly Gly Thr
Cys Ala Gly 885 890 895 Cys Gly Thr Cys Cys Thr Cys Ala Cys Cys Gly
Thr Cys Gly Thr Gly 900 905 910 Cys Ala Cys Cys Ala Gly Gly Ala Cys
Thr Gly Gly Cys Thr Gly Ala 915 920 925 Ala Cys Gly Gly Cys Ala Ala
Gly Gly Ala Gly Thr Ala Cys Ala Ala 930 935 940 Gly Thr Gly Cys Ala
Ala Gly Gly Thr Gly Thr Cys Cys Ala Ala Cys 945 950 955 960 Ala Ala
Ala Gly Gly Cys Cys Thr Cys Cys Cys Ala Gly Cys Cys Cys 965 970 975
Cys Cys Ala Thr Cys Gly Ala Gly Ala Ala Ala Ala Cys Cys Ala Thr 980
985 990 Cys Thr Cys Cys Ala Ala Ala Ala Cys Cys Ala Ala Ala Gly Gly
Gly 995 1000 1005 Cys Ala Gly Cys Cys Cys Cys Gly Ala Gly Ala Ala
Cys Cys Ala 1010 1015 1020 Cys Ala Gly Gly Thr Gly Thr Ala Cys Ala
Cys Cys Cys Thr Gly 1025 1030 1035 Cys Cys Cys Cys Cys Ala Thr Cys
Cys Cys Gly Gly Gly Ala Gly 1040 1045 1050 Gly Ala Gly Ala Thr Gly
Ala Cys Cys Ala Ala Gly Ala Ala Cys 1055 1060 1065 Cys Ala Gly Gly
Thr Cys Ala Gly Cys Cys Thr Gly Ala Cys Cys 1070 1075 1080 Thr Gly
Cys Cys Thr Gly Gly Thr Cys Ala Ala Ala Gly Gly Cys 1085 1090 1095
Thr Thr Cys Thr Ala Cys Cys Cys Cys Ala Gly Cys Gly Ala Cys 1100
1105 1110 Ala Thr Cys Gly Cys Cys Gly Thr Gly Gly Ala Gly Thr Gly
Gly 1115 1120 1125 Gly Ala Gly Ala Gly Cys Ala Ala Thr Gly Gly Gly
Cys Ala Gly 1130 1135 1140 Cys Cys Gly Gly Ala Gly Ala Ala Cys Ala
Ala Cys Thr Ala Cys 1145 1150 1155 Ala Ala Cys Ala Cys Cys Ala Cys
Ala Cys Cys Thr Cys Cys Cys 1160 1165 1170 Ala Thr Gly Cys Thr Gly
Gly Ala Cys Thr Cys Cys Gly Ala Cys 1175 1180 1185 Gly Gly Cys Thr
Cys Cys Thr Thr Cys Thr Thr Cys Cys Thr Cys 1190 1195 1200 Thr Ala
Cys Ala Gly Cys Ala Ala Gly Cys Thr Cys Ala Cys Cys 1205 1210 1215
Gly Thr Gly Gly Ala Cys Ala Ala Gly Ala Gly Cys Ala Gly Gly 1220
1225 1230 Thr Gly Gly Cys Ala Gly Cys Ala Gly Gly Gly Gly Ala Ala
Cys 1235 1240 1245 Gly Thr Cys Thr Thr Cys Thr Cys Ala Thr Gly Cys
Thr Cys Cys 1250 1255 1260 Gly Thr Gly Ala Thr Gly Cys Ala Thr Gly
Ala Gly Gly Cys Thr 1265 1270 1275 Cys Thr Gly Cys Ala Cys Ala Ala
Cys Cys Ala Cys Thr Ala Cys 1280 1285 1290 Ala Cys Gly Cys Ala Gly
Ala Ala Gly Ala Gly Cys Cys Thr Cys 1295 1300 1305 Thr Cys Cys Cys
Thr Gly Thr Cys Thr Cys Cys Gly Gly Gly Thr 1310 1315 1320 Ala Ala
Ala 1325 1161329PRTArtificial Sequencecomplete heavy chain 116Cys
Ala Gly Gly Thr Gly Cys Ala Gly Cys Thr Gly Cys Ala Gly Gly 1 5 10
15 Ala Gly Thr Cys Gly Gly Gly Cys Cys Cys Ala Gly Gly Ala Cys Thr
20 25 30 Gly Gly Thr Gly Ala Ala Gly Cys Cys Thr Thr Cys Ala Cys
Ala Gly 35 40 45 Ala Cys Cys Cys Thr Gly Thr Cys Cys Cys Thr Cys
Ala Cys Cys Thr 50 55 60 Gly Cys Ala Cys Thr Gly Thr Cys Thr Cys
Thr Gly Gly Cys Thr Ala 65 70 75 80 Cys Ala Cys Cys Thr Thr Cys Ala
Cys Cys Ala Ala Cys Thr Ala Cys 85 90 95 Thr Ala Thr Gly Thr Ala
Thr Thr Cys Thr Gly Gly Gly Thr Gly Cys 100 105 110 Gly Ala Cys Ala
Gly Gly Cys Thr Cys Gly Thr Gly Gly Ala Cys Ala 115 120 125 Ala Cys
Gly Cys Cys Thr Thr Gly Ala Gly Thr Gly Gly Ala Thr Ala 130 135 140
Gly Gly Thr Gly Ala Cys Ala Thr Thr Ala Ala Thr Cys Cys Thr Gly 145
150 155 160 Thr Cys Ala Ala Thr Gly Gly Thr Gly Ala Thr Ala Cys Thr
Ala Ala 165 170 175 Cys Thr Thr Cys Ala Ala Thr Gly Ala Gly Ala Ala
Ala Thr Thr Cys 180 185 190 Ala Ala Gly Ala Ala Cys Ala Gly Ala Gly
Thr Cys Ala Cys Cys Ala 195 200 205 Thr Cys Thr Cys Ala Gly Cys Cys
Gly Ala Cys Ala Ala Gly Thr Cys 210 215 220 Cys Ala Thr Cys Ala Gly
Cys Ala Cys Cys Gly Cys Cys Thr Ala Cys 225 230 235 240 Cys Thr Gly
Cys Ala Gly Thr Gly Gly Ala Gly Cys Ala Gly Cys Cys 245 250 255 Thr
Gly Ala Ala Gly Gly Cys Cys Thr Cys Gly Gly Ala Cys Ala Cys 260 265
270 Cys Gly Cys Cys Ala Thr Gly Thr Ala Thr Thr Ala Cys Thr Gly Thr
275 280 285 Gly Cys Gly Ala Gly Ala Gly Gly Gly Gly Gly Thr Thr Ala
Thr Ala 290 295 300 Cys Thr Ala Thr Gly Gly Ala Cys Thr Ala Cys Thr
Gly Gly Gly Gly 305 310 315 320 Cys Cys Ala Gly Gly Gly Cys Ala Cys
Cys Ala Cys Cys Gly Thr Gly 325 330 335 Ala Cys Cys Gly Thr Gly Thr
Cys Cys Thr Cys Cys Gly Cys Thr Thr 340 345 350 Cys Cys Ala Cys Cys
Ala Ala Gly Gly Gly Cys Cys Cys Ala Thr Cys 355 360 365 Cys Gly Thr
Cys Thr Thr Cys Cys Cys Cys Cys Thr Gly Gly Cys Gly 370 375 380 Cys
Cys Cys Thr Gly Cys Thr Cys Cys Ala Gly Gly Ala Gly Cys Ala 385 390
395 400 Cys Cys Thr Cys Cys Gly Ala Gly Ala Gly Cys Ala Cys Ala Gly
Cys 405 410 415 Cys Gly Cys Cys Cys Thr Gly Gly Gly Cys Thr Gly Cys
Cys Thr Gly 420 425 430 Gly Thr Cys Ala Ala Gly Gly Ala Cys Thr Ala
Cys Thr Thr Cys Cys 435 440 445 Cys Cys Gly Ala Ala Cys Cys Gly Gly
Thr Gly Ala Cys Gly Gly Thr 450 455 460 Gly Thr Cys Gly Thr Gly Gly
Ala Ala Cys Thr Cys Ala Gly Gly Cys 465 470 475 480 Gly Cys Cys Cys
Thr Gly Ala Cys Cys Ala Gly Cys Gly Gly Cys Gly 485 490 495 Thr Gly
Cys Ala Cys Ala Cys Cys Thr Thr Cys Cys Cys Gly Gly Cys 500 505 510
Thr Gly Thr Cys Cys Thr Ala Cys Ala Gly Thr Cys Cys Thr Cys Ala 515
520 525 Gly Gly Ala Cys Thr Cys Thr Ala Cys Thr Cys Cys Cys Thr Cys
Ala 530 535 540 Gly Cys Ala Gly Cys Gly Thr Gly Gly Thr Gly Ala Cys
Cys Gly Thr 545 550 555 560 Gly Cys Cys Cys Thr Cys Cys Ala Gly Cys
Ala Gly Cys Thr Thr Gly 565 570 575 Gly Gly Cys Ala Cys Gly Ala Ala
Gly Ala Cys Cys Thr Ala Cys Ala 580 585 590 Cys Cys Thr Gly Cys Ala
Ala Cys Gly Thr Ala Gly Ala Thr Cys Ala 595 600 605 Cys Ala Ala Gly
Cys Cys Cys Ala Gly Cys Ala Ala Cys Ala Cys Cys 610 615 620 Ala Ala
Gly Gly Thr Gly Gly Ala Cys Ala Ala Gly Ala Gly Ala Gly 625 630 635
640 Thr Thr Gly Ala Gly Thr Cys Cys Ala Ala Ala Thr Ala Thr Gly Gly
645 650 655 Thr Cys Cys Cys Cys Cys Ala Thr Gly Cys Cys Cys Ala Cys
Cys Gly 660 665 670 Thr Gly Cys Cys Cys Ala Gly Cys Ala Cys Cys Thr
Gly Ala Gly Thr 675 680 685 Thr Cys Cys Thr Gly Gly Gly Gly Gly Gly
Ala Cys Cys Ala Thr Cys 690 695 700 Ala Gly Thr Cys Thr Thr Cys Cys
Thr Gly Thr Thr Cys Cys Cys Cys 705 710 715 720 Cys Cys Ala Ala Ala
Ala Cys Cys Cys Ala Ala Gly Gly Ala Cys Ala 725 730 735 Cys Thr Cys
Thr Cys Ala Thr Gly Ala Thr Cys Thr Cys Cys Cys Gly 740 745 750 Gly
Ala Cys Cys Cys Cys Thr Gly Ala Gly Gly Thr Cys Ala Cys Gly 755 760
765 Thr Gly Cys Gly Thr Gly Gly Thr Gly Gly Thr Gly Gly Ala Cys Gly
770 775 780 Thr Gly Ala Gly Cys Cys Ala Gly Gly Ala Ala Gly Ala Cys
Cys Cys 785 790 795 800 Cys Gly Ala Gly Gly Thr Cys Cys Ala Gly Thr
Thr Cys Ala Ala Cys 805 810 815 Thr Gly Gly Thr Ala Cys Gly Thr Gly
Gly Ala Thr Gly Gly Cys Gly 820 825 830 Thr Gly Gly Ala Gly Gly Thr
Gly Cys Ala Thr Ala Ala Thr Gly Cys 835 840 845 Cys Ala Ala Gly Ala
Cys Ala Ala Ala Gly Cys Cys Gly Cys Gly Gly 850 855 860 Gly Ala Gly
Gly Ala Gly Cys Ala Gly Thr Thr Cys Ala Ala Cys Ala 865 870 875 880
Gly Cys Ala Cys Gly Thr Ala Cys Cys Gly Thr Gly Thr Gly Gly Thr 885
890 895 Cys Ala Gly Cys Gly Thr Cys Cys Thr Cys
Ala Cys Cys Gly Thr Cys 900 905 910 Cys Thr Gly Cys Ala Cys Cys Ala
Gly Gly Ala Cys Thr Gly Gly Cys 915 920 925 Thr Gly Ala Ala Cys Gly
Gly Cys Ala Ala Gly Gly Ala Gly Thr Ala 930 935 940 Cys Ala Ala Gly
Thr Gly Cys Ala Ala Gly Gly Thr Gly Thr Cys Cys 945 950 955 960 Ala
Ala Cys Ala Ala Ala Gly Gly Cys Cys Thr Cys Cys Cys Gly Thr 965 970
975 Cys Cys Thr Cys Cys Ala Thr Cys Gly Ala Gly Ala Ala Ala Ala Cys
980 985 990 Cys Ala Thr Cys Thr Cys Cys Ala Ala Ala Gly Cys Cys Ala
Ala Ala 995 1000 1005 Gly Gly Gly Cys Ala Gly Cys Cys Cys Cys Gly
Ala Gly Ala Gly 1010 1015 1020 Cys Cys Ala Cys Ala Gly Gly Thr Gly
Thr Ala Cys Ala Cys Cys 1025 1030 1035 Cys Thr Gly Cys Cys Cys Cys
Cys Ala Thr Cys Cys Cys Ala Gly 1040 1045 1050 Gly Ala Gly Gly Ala
Gly Ala Thr Gly Ala Cys Cys Ala Ala Gly 1055 1060 1065 Ala Ala Cys
Cys Ala Gly Gly Thr Cys Ala Gly Cys Cys Thr Gly 1070 1075 1080 Ala
Cys Cys Thr Gly Cys Cys Thr Gly Gly Thr Cys Ala Ala Ala 1085 1090
1095 Gly Gly Cys Thr Thr Cys Thr Ala Cys Cys Cys Cys Ala Gly Cys
1100 1105 1110 Gly Ala Cys Ala Thr Cys Gly Cys Cys Gly Thr Gly Gly
Ala Gly 1115 1120 1125 Thr Gly Gly Gly Ala Gly Ala Gly Cys Ala Ala
Thr Gly Gly Gly 1130 1135 1140 Cys Ala Gly Cys Cys Gly Gly Ala Gly
Ala Ala Cys Ala Ala Cys 1145 1150 1155 Thr Ala Cys Ala Ala Gly Ala
Cys Cys Ala Cys Gly Cys Cys Thr 1160 1165 1170 Cys Cys Cys Gly Thr
Gly Cys Thr Gly Gly Ala Cys Thr Cys Cys 1175 1180 1185 Gly Ala Cys
Gly Gly Cys Thr Cys Cys Thr Thr Cys Thr Thr Cys 1190 1195 1200 Cys
Thr Cys Thr Ala Cys Ala Gly Cys Ala Gly Gly Cys Thr Ala 1205 1210
1215 Ala Cys Cys Gly Thr Gly Gly Ala Cys Ala Ala Gly Ala Gly Cys
1220 1225 1230 Ala Gly Gly Thr Gly Gly Cys Ala Gly Gly Ala Gly Gly
Gly Gly 1235 1240 1245 Ala Ala Thr Gly Thr Cys Thr Thr Cys Thr Cys
Ala Thr Gly Cys 1250 1255 1260 Thr Cys Cys Gly Thr Gly Ala Thr Gly
Cys Ala Thr Gly Ala Gly 1265 1270 1275 Gly Cys Thr Cys Thr Gly Cys
Ala Cys Ala Ala Cys Cys Ala Cys 1280 1285 1290 Thr Ala Cys Ala Cys
Ala Cys Ala Gly Ala Ala Gly Ala Gly Cys 1295 1300 1305 Cys Thr Cys
Thr Cys Cys Cys Thr Gly Thr Cys Thr Cys Thr Gly 1310 1315 1320 Gly
Gly Thr Ala Ala Ala 1325 117113PRTArtificial Sequencelight chain
constant region 117Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro
Ser Val Phe Ile 1 5 10 15 Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
Gly Thr Ala Ser Val Val 20 25 30 Cys Leu Leu Asn Asn Phe Tyr Pro
Arg Glu Ala Lys Val Gln Trp Lys 35 40 45 Val Asp Asn Ala Leu Gln
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu 50 55 60 Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu 65 70 75 80 Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr 85 90 95
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu 100
105 110 Cys 118337PRTArtificial Sequenceheavy chain constant region
118Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
1 5 10 15 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
Ala Leu 20 25 30 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp 35 40 45 Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu 50 55 60 Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser 65 70 75 80 Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro 85 90 95 Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 100 105 110 Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 115 120 125
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 130
135 140 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp 145 150 155 160 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 165 170 175 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val 180 185 190 Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu 195 200 205 Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 210 215 220 Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 225 230 235 240 Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 245 250
255 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
260 265 270 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu 275 280 285 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys 290 295 300 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu 305 310 315 320 Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly 325 330 335 Lys
119245PRTArtificial Sequenceheavy chain constant region 119Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 1 5 10 15
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 20
25 30 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp 35 40 45 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu 50 55 60 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser 65 70 75 80 Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro 85 90 95 Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp Lys 100 105 110 Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 115 120 125 Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 130 135 140 Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 145 150
155 160 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn 165 170 175 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Gln Ser Thr
Tyr Arg Val 180 185 190 Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu 195 200 205 Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys 210 215 220 Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr 225 230 235 240 Leu Pro Pro Ser
Arg 245 120337PRTArtificial Sequenceheavy chain constant region
120Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
1 5 10 15 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser
Glu Ser 20 25 30 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val 35 40 45 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe 50 55 60 Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val 65 70 75 80 Thr Val Pro Ser Ser Asn
Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val 85 90 95 Asp His Lys Pro
Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys 100 105 110 Cys Cys
Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro 115 120 125
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 130
135 140 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp 145 150 155 160 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 165 170 175 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
Asn Ser Thr Phe Arg Val 180 185 190 Val Ser Val Leu Thr Val Val His
Gln Asp Trp Leu Asn Gly Lys Glu 195 200 205 Tyr Lys Cys Lys Val Ser
Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys 210 215 220 Thr Ile Ser Lys
Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 225 230 235 240 Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 245 250
255 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
260 265 270 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro
Met Leu 275 280 285 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys 290 295 300 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu 305 310 315 320 Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly 325 330 335 Lys
121334PRTArtificial Sequenceheavy chain constant region 121Thr Thr
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 1 5 10 15
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 20
25 30 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp 35 40 45 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu 50 55 60 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser 65 70 75 80 Ser Ser Leu Gly Thr Lys Thr Tyr Thr
Cys Asn Val Asp His Lys Pro 85 90 95 Ser Asn Thr Lys Val Asp Lys
Arg Val Glu Ser Lys Tyr Gly Pro Pro 100 105 110 Cys Pro Pro Cys Pro
Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe 115 120 125 Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 130 135 140 Glu
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 145 150
155 160 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr 165 170 175 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val
Val Ser Val 180 185 190 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys 195 200 205 Lys Val Ser Asn Lys Gly Leu Pro Ser
Ser Ile Glu Lys Thr Ile Ser 210 215 220 Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro 225 230 235 240 Ser Gln Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 245 250 255 Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 260 265 270
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 275
280 285 Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg
Trp 290 295 300 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His 305 310 315 320 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Leu Gly Lys 325 330 122443PRTArtificial Sequencefull length
heavy chain variable domain and a constant domain amino acid
sequence. 122Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr
Thr Phe Thr Asn Tyr 20 25 30 Tyr Val Phe Trp Val Arg Gln Ala Arg
Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Asp Ile Asn Pro Val Asn
Gly Asp Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Asn Arg 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 Thr Met Asp Tyr Trp Gly Gln Gly Thr Thr Val 100 105
110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125 Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Lys Thr Tyr
Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp
Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro 210 215 220 Cys
Pro Ala Pro Glu Phe Glu Gly 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 Gln 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 Tyr Arg
Val Val Ser Val Leu Thr Val 290 295 300 Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser 305 310 315 320 Asn Lys Gly Leu
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys 325 330 335 Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 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 Val Leu Asp Ser Asp
Gly Ser Phe 385 390 395 400 Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys
Ser Arg Trp Gln Glu 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 Leu Gly Lys 435 440
1231329DNAArtificial SequenceIgG4 S228P L235E Heavy Chain Nucleic
Acid Sequence 123caggtgcagc tgcaggagtc gggcccagga ctggtgaagc
cttcacagac cctgtccctc 60acctgcactg tctctggcta caccttcacc aactactatg
tattctgggt gcgacaggct 120cgtggacaac gccttgagtg gataggtgac
attaatcctg tcaatggtga tactaacttc 180aatgagaaat tcaagaacag
agtcaccatc tcagccgaca agtccatcag caccgcctac 240ctgcagtgga
gcagcctgaa ggcctcggac accgccatgt attactgtgc gagagggggt
300tatactatgg actactgggg ccagggcacc accgtgaccg tgtcctccgc
ttccaccaag 360ggcccatccg tcttccccct ggcgccctgc tccaggagca
cctccgagag cacagccgcc 420ctgggctgcc tggtcaagga ctacttcccc
gaaccggtga cggtgtcgtg gaactcaggc 480gccctgacca gcggcgtgca
caccttcccg gctgtcctac agtcctcagg actctactcc 540ctcagcagcg
tggtgaccgt gccctccagc agcttgggca cgaagaccta cacctgcaac
600gtagatcaca agcccagcaa caccaaggtg gacaagagag ttgagtccaa
atatggtccc 660ccatgcccac cgtgcccagc acctgagttc gaggggggac
catcagtctt cctgttcccc 720ccaaaaccca aggacactct catgatctcc
cggacccctg aggtcacgtg cgtggtggtg 780gacgtgagcc aggaagaccc
cgaggtccag ttcaactggt acgtggatgg cgtggaggtg 840cataatgcca
agacaaagcc gcgggaggag cagttcaaca gcacgtaccg tgtggtcagc
900gtcctcaccg tcctgcacca ggactggctg aacggcaagg agtacaagtg
caaggtgtcc 960aacaaaggcc tcccgtcctc catcgagaaa accatctcca
aagccaaagg gcagccccga 1020gagccacagg tgtacaccct gcccccatcc
caggaggaga tgaccaagaa ccaggtcagc 1080ctgacctgcc tggtcaaagg
cttctacccc agcgacatcg ccgtggagtg ggagagcaat 1140gggcagccgg
agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc
1200ttcctctaca gcaggctaac cgtggacaag agcaggtggc aggaggggaa
tgtcttctca 1260tgctccgtga tgcatgaggc tctgcacaac cactacacac
agaagagcct ctccctgtct 1320ctgggtaaa 1329124334PRTArtificial
SequenceFramework 4 + Heavy Chain Constant IgG4 S228P L235E Domain
124Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
1 5 10 15 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala
Ala Leu 20 25 30 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp 35 40 45 Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu 50 55 60 Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser 65 70 75 80 Ser Ser Leu Gly Thr Lys
Thr Tyr Thr Cys Asn Val Asp His Lys Pro 85 90 95 Ser Asn Thr Lys
Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 100 105 110 Cys Pro
Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe 115 120 125
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 130
135 140 Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu
Val 145 150 155 160 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr 165 170 175 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
Tyr Arg Val Val Ser Val 180 185 190 Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys 195 200 205 Lys Val Ser Asn Lys Gly
Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 210 215 220 Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 225 230 235 240 Ser
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 245 250
255 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
260 265 270 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp 275 280 285 Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
Lys Ser Arg Trp 290 295 300 Gln Glu Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His 305 310 315 320 Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Leu Gly Lys 325 330
* * * * *