U.S. patent application number 15/907505 was filed with the patent office on 2018-07-12 for endoglin antibodies.
The applicant listed for this patent is Tracon Pharmaceuticals, Inc.. Invention is credited to Charles P. Theuer, Maximiliano Vasquez.
Application Number | 20180194854 15/907505 |
Document ID | / |
Family ID | 43780635 |
Filed Date | 2018-07-12 |
United States Patent
Application |
20180194854 |
Kind Code |
A1 |
Theuer; Charles P. ; et
al. |
July 12, 2018 |
Endoglin Antibodies
Abstract
The present application relates to compositions of humanized and
humanized/deimmunized anti-endoglin antibodies and antigen-binding
fragments thereof. One aspect relates to antibodies having one or
more modifications in at least one amino acid residue of at least
one of the framework regions of the variable heavy chain, the
variable light chain or both. Another aspect relates to antibodies
which bind endoglin and inhibit angiogenesis. Another aspect
relates to the deimmunization of humanized antibodies to reduce
immunogenicity. Another aspect relates to the use of humanized and
humanized/deimmunized antibodies which bind endoglin for the
detection, diagnosis or treatment of a disease or condition
associated with endoglin, angiogenesis or a combination
thereof.
Inventors: |
Theuer; Charles P.; (San
Diego, CA) ; Vasquez; Maximiliano; (Palo Alto,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Tracon Pharmaceuticals, Inc. |
La Jolla |
CA |
US |
|
|
Family ID: |
43780635 |
Appl. No.: |
15/907505 |
Filed: |
February 28, 2018 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15337113 |
Oct 28, 2016 |
9944714 |
|
|
15907505 |
|
|
|
|
14838006 |
Aug 27, 2015 |
9518122 |
|
|
15337113 |
|
|
|
|
14054446 |
Oct 15, 2013 |
9150652 |
|
|
14838006 |
|
|
|
|
13485702 |
May 31, 2012 |
8609094 |
|
|
14054446 |
|
|
|
|
12751907 |
Mar 31, 2010 |
8221753 |
|
|
13485702 |
|
|
|
|
12570918 |
Sep 30, 2009 |
|
|
|
12751907 |
|
|
|
|
61247290 |
Sep 30, 2009 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/282 20130101;
C07K 2317/24 20130101; C07K 2317/92 20130101; A61P 35/02 20180101;
C07K 16/30 20130101; C07K 2317/732 20130101; A61P 29/00 20180101;
A61P 19/02 20180101; A61K 45/06 20130101; A61K 47/6849 20170801;
G01N 2800/164 20130101; A61K 2039/505 20130101; A61K 31/4045
20130101; C07K 16/3046 20130101; C07K 2317/56 20130101; C07K
2317/73 20130101; A61K 39/39558 20130101; C07K 2317/565 20130101;
A61P 13/12 20180101; A61K 31/44 20130101; C07K 16/2863 20130101;
A61K 39/3955 20130101; A61P 9/10 20180101; A61P 35/00 20180101;
A61P 35/04 20180101; G01N 33/6872 20130101; G01N 2333/70596
20130101; A61P 1/00 20180101; A61P 1/04 20180101; G01N 33/57492
20130101; G01N 33/57496 20130101; C07K 2317/34 20130101; A61P 9/00
20180101; A61P 27/02 20180101; G01N 2800/16 20130101; A61K 31/69
20130101; A61K 31/704 20130101; C07K 16/2896 20130101; A61P 3/10
20180101; C07K 16/3015 20130101; A61K 31/337 20130101; C07K 16/3061
20130101; C07K 16/3069 20130101; G01N 2333/71 20130101; C07K
16/3038 20130101 |
International
Class: |
C07K 16/30 20060101
C07K016/30; A61K 31/282 20060101 A61K031/282; A61K 31/337 20060101
A61K031/337; A61K 31/4045 20060101 A61K031/4045; A61K 31/44
20060101 A61K031/44; A61K 31/69 20060101 A61K031/69; A61K 31/704
20060101 A61K031/704; A61K 39/395 20060101 A61K039/395; A61K 45/06
20060101 A61K045/06; C07K 16/28 20060101 C07K016/28; G01N 33/68
20060101 G01N033/68; G01N 33/574 20060101 G01N033/574; A61K 47/68
20170101 A61K047/68 |
Claims
1. An antibody, or antigen-binding fragment thereof, comprising a
heavy chain variable region having an amino acid sequence set forth
as SEQ ID NO: 89 and a light chain variable region having an amino
acid sequence set forth as SEQ ID NO: 93.
2. An antibody, or antigen-binding fragment thereof, that binds
endoglin, comprising a heavy chain variable region having an amino
acid sequence set forth as SEQ ID NO: 89 and a light chain variable
region having an amino acid sequence set forth as SEQ ID NO: 93,
wherein: said heavy chain variable region further comprises one or
more modifications selected from the group consisting of a
substitution of glycine (G) by alanine (A) or serine (S) at
position 49; a substitution of alanine (A) by isoleucine (I) at
position 51; a substitution of lysine (K) by arginine (R) or
glutamine (Q) at position 52b; a substitution of leucine (L) by
valine (V) at position 78 utilizing the Kabat numbering system; and
the light chain variable region further comprises one or more
modifications selected from the group consisting of a substitution
of methionine (M) by leucine (L) at position 4; a substitution of
alanine (A) by valine (V) at position 19; a substitution of
threonine (T) by serine (S) at position 22; a substitution of
alanine (A) by isoleucine (I) at position 48; and a substitution of
threonine (T) by serine (S) at position 51 utilizing the Kabat
numbering system.
3. An antibody, or antigen-binding fragment thereof, of claim 2
comprising a heavy chain variable region having an amino acid
sequence set forth as SEQ ID NO: 88, 89, 90, 91 or 92; and a light
chain variable region having an amino acid sequence set forth as
SEQ ID NO: 93, 94, 95, 96, 97, 100, 102, or 103.
4. An antigen-binding fragment of claim 1 wherein the
antigen-binding fragment is a Fab fragment, a Fab' fragment, a
F(ab')2 fragment, an Fv fragment, an scFv fragment, a single chain
binding polypeptide, a Fd fragment, a variable heavy chain, a
variable light chain or a dAb fragment.
5. A composition comprising an antibody or antigen-binding fragment
of claim 1, and an acceptable carrier or excipient.
6. A nucleic acid comprising a nucleotide sequence encoding an
antibody or antigen-binding fragment of claim 1.
7. An antibody or antigen-binding fragment thereof, of claim 1,
further labeled with a therapeutic label, a diagnostic label, or
both.
8. A method of treating an angiogenesis-related disease in a
subject comprising administering a composition of an antibody or
antigen-binding fragment thereof, of claim 1.
9. The method of claim 8, wherein said antibody or antigen-binding
fragment thereof is labeled with a therapeutic label, a detectable
label or both.
10. The method of claim 8, wherein the angiogenesis-related disease
is an ocular disease characterized by
angiogenesis/neovascularization, diabetic nephropathy, IBD,
rheumatoid arthritis, osteoarthritis, a cancer, or a
metastasis.
11. The method of claim 10, wherein the ocular disease is macular
degeneration.
12. The method of claim 10, wherein the ocular disease is diabetic
retinopathy.
13. The method of claim 10, wherein the cancer is a solid
tumor.
14. The method of claim 10, wherein the cancer is an epithelial
based tumor
15. The method of claim 10, wherein the cancer is selected from a
lung cancer, a gynecologic malignancy, a melanoma, a breast cancer,
a pancreatic cancer, an ovarian cancer, a uterine cancer, a
colorectal cancer, a prostate cancer, a kidney cancer, a head
cancer, a pancreatic cancer, a liver cancer (hepatocellular
cancer), a uterine cancer, a neck cancer, a kidney cancer (renal
cell cancer), a sarcoma, a myeloma, and a lymphoma.
16. The method of claim 8, further comprising administering one or
more angiogenesis inhibitors.
17. The method of claim 16, wherein the angiogenesis inhibitor is
chemotherapy, a VEGF receptor inhibitor, a VEGF inhibitor, or a
combination thereof.
18. The method of claim 8 wherein the composition of an antibody or
antigen-binding fragment thereof is administered in an amount of
about 0.01 mg/kg, about 0.05 mg/kg, about 0.1 mg/kg, about 0.5
mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 20
mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 60
mg/kg, about 70 mg/kg, about 80 mg/kg, about 90 mg/kg, about 100
mg/kg, about 125 mg/kg, about 150 mg/kg, about 175 mg/kg, or about
200 mg/kg.
19. A diagnostic method comprising: a) providing a sample of cancer
cells of a solid tumor or plasma from a patient to be tested; b)
detecting in the sample the expression of at least one gene or gene
product chosen from a panel of genes or gene products whose
expression has been correlated with sensitivity or resistance to an
angiogenesis inhibitor, wherein the at least one gene or gene
product is chosen from one or more genes or gene products selected
from the group consisting of VEGF, VEGF receptor, HIF-1.alpha.,
placental growth factor receptor, and CD105; and c) comparing the
level of expression of at least one gene or gene product detected
in the patient sample to a level of expression of at least one gene
or gene product that has been correlated with sensitivity or
resistance to the angiogenesis inhibitor.
20. The method of claim 19, wherein the angiogenesis inhibitor is
chosen from VEGF receptor inhibitors, VEGF inhibitors, and endoglin
inhibitors.
21. A kit comprising reagents for the detection of expression
levels that have been correlated with sensitivity or resistance to
an angiogenesis inhibitor of one or more genes selected from VEGF,
VEGF receptor, HIF-1.alpha., placental growth factor receptor, and
endoglin in a sample of cancer cells or human plasma.
22. The kit of claim 21, wherein said kit contains an antibody or
antigen-binding fragment thereof.
23. An antibody, or antigen-binding fragment thereof, that binds
endoglin, that comprises a heavy chain variable region having an
amino acid sequence set forth as SEQ ID NO: 88, 89, 90, 91 or 92
and a light chain variable region having an amino acid sequence set
forth as SEQ ID NO: 93, 94, 95, or 102, wherein the antibody, or
antigen-binding fragment thereof, has an affinity of from about 100
nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1
picomolar (pM) or from about 100 nM to about 1 femtomolar (fM).
24. The antibody, or antigen-binding fragment thereof, of claim 23,
wherein the antibody, or antigen-binding fragment thereof,
comprises a heavy chain variable region set forth as SEQ ID NO: 89
and a light chain variable region having an amino acid sequence set
forth as SEQ ID NO: 93, 94, 95, or 102.
25. The antibody, or antigen-binding fragment thereof, of claim 23,
wherein the antibody, or antigen-binding fragment thereof,
comprises a heavy chain variable region set forth as SEQ ID NO: 89
and a light chain variable region having an amino acid sequence set
forth as SEQ ID NO: 93.
26. The antibody, or antigen-binding fragment thereof, of claim 23,
wherein the antibody, or antigen-binding fragment thereof,
comprises a heavy chain variable region set forth as SEQ ID NO: 89
and a light chain variable region having an amino acid sequence set
forth as SEQ ID NO: 94.
27. The antibody, or antigen-binding fragment thereof, of claim 23,
wherein the antibody, or antigen-binding fragment thereof,
comprises a heavy chain variable region set forth as SEQ ID NO: 89
and a light chain variable region having an amino acid sequence set
forth as SEQ ID NO: 95.
28. The antibody, or antigen-binding fragment thereof, of claim 23,
wherein the antibody, or antigen-binding fragment thereof,
comprises a heavy chain variable region set forth as SEQ ID NO: 89
and a light chain variable region having an amino acid sequence set
forth as SEQ ID NO: 102.
29. A pharmaceutical composition that comprises the antibody, or
antigen-binding fragment thereof, of claim 23, and one or more
pharmaceutically acceptable carriers or excipients, wherein the one
or more pharmaceutically acceptable carriers or excipients comprise
a surfactant, a buffer, a polyol, an additive, an antibacterial
agent, an antifungal agent, a preservative, a stabilizer, an
antioxidant, or a combination thereof.
30. A method of treating an angiogenesis-related disease in a
subject in need thereof, comprising administering to the subject a
pharmaceutical composition of claim 29.
31. The method of claim 30, wherein the angiogenesis-related
disease comprises an ocular disease.
32. The method of claim 31, wherein the ocular disease comprises
age-related macular degeneration (AMD), choroidal
neovascularization, or diabetic retinopathy.
33. The method of claim 32, wherein the ocular disease comprises
age-related macular degeneration (AMD) that is wet AMD or dry
AMD.
34. The method of claim 30, wherein the angiogenesis-related
disease comprises diabetic nephropathy.
35. The method of claim 30, wherein the angiogenesis-related
disease comprises a cancer or a metastasis thereof.
36. The method of claim 35, wherein the cancer or a metastasis
thereof is a solid tumor.
37. The method of claim 35, wherein the cancer or a metastasis
thereof is an epithelial based tumor.
38. The method of claim 35, wherein the cancer or the metastasis
thereof is a lung cancer, a melanoma, a breast cancer, a pancreatic
cancer, an ovarian cancer, a colorectal cancer, a prostate cancer,
a kidney cancer, a liver cancer, a uterine cancer, a neck cancer, a
sarcoma, a myeloma, a brain cancer, or a lymphoma.
39. The method of claim 38, wherein the cancer or the metastasis
thereof is a brain cancer that is a glioblastoma multiforme or a
glioma.
40. The method of claim 35, wherein treatment results in partial
elimination of the cancer or the metastasis thereof, total
elimination of the cancer or the metastasis thereof, prolongation
of survival of the subject, or a combination thereof.
41. The method of claim 30, wherein the pharmaceutical composition
is administered to the subject one or more times.
42. The method of claim 30, wherein the pharmaceutical composition
is administered to the subject once per day, once per week, once
per month, once bi-monthly, once every two months, once every three
months, once every four months, once every 5 months, or once every
6 months.
43. An antibody, or antigen-binding fragment thereof, that binds
endoglin, that comprises a heavy chain variable region having an
amino acid sequence set forth as SEQ ID NO: 88, 89, 90, 91 or 92
and a light chain variable region having an amino acid sequence set
forth as SEQ ID NO: 93, 94, 95, or 102, wherein the antibody, or
antigen-binding fragment thereof, has a K.sub.D of from about
5.times.10.sup.-10 or greater.
44. The antibody, or antigen-binding fragment thereof, of claim 43,
wherein the antibody, or antigen-binding fragment thereof, has a
K.sub.D of from about 5.times.10.sup.-10 to about
9.times.10.sup.-10.
45. The antibody, or antigen-binding fragment thereof, of claim 43,
wherein the antibody, or antigen-binding fragment thereof,
comprises a heavy chain variable region having an amino acid
sequence set forth as SEQ ID NO: 89 and a light chain variable
region having an amino acid sequence set forth as SEQ ID NO: 93,
94, 95, or 102.
46. The antibody, or antigen-binding fragment thereof, of claim 43,
wherein the antibody, or antigen-binding fragment thereof,
comprises a heavy chain variable region set forth as SEQ ID NO: 89
and a light chain variable region having an amino acid sequence set
forth as SEQ ID NO: 93.
47. The antibody, or antigen-binding fragment thereof, of claim 43,
wherein the antibody, or antigen-binding fragment thereof,
comprises a heavy chain variable region set forth as SEQ ID NO: 89
and a light chain variable region having an amino acid sequence set
forth as SEQ ID NO: 94.
48. The antibody, or antigen-binding fragment thereof, of claim 43,
wherein the antibody, or antigen-binding fragment thereof,
comprises a heavy chain variable region set forth as SEQ ID NO: 89
and a light chain variable region having an amino acid sequence set
forth as SEQ ID NO: 95.
49. The antibody, or antigen-binding fragment thereof, of claim 43,
wherein the antibody, or antigen-binding fragment thereof,
comprises a heavy chain variable region set forth as SEQ ID NO: 89
and a light chain variable region having an amino acid sequence set
forth as SEQ ID NO: 102.
50. A pharmaceutical composition that comprises the antibody, or
antigen-binding fragment thereof, of claim 43, and one or more
pharmaceutically acceptable carriers or excipients, wherein the one
or more pharmaceutically acceptable carriers or excipients comprise
a surfactant, a buffer, a polyol, an additive, an antibacterial
agent, an antifungal agent, a preservative, a stabilizer, an
antioxidant, or a combination thereof.
51. A method of treating an angiogenesis-related disease in a
subject in need thereof, comprising administering to the subject a
pharmaceutical composition of claim 50.
52. The method of claim 51, wherein the angiogenesis-related
disease comprises an ocular disease.
53. The method of claim 52, wherein the ocular disease comprises
age-related macular degeneration (AMD), choroidal
neovascularization, or diabetic retinopathy.
54. The method of claim 53, wherein the ocular disease comprises
age-related macular degeneration (AMD) that is wet AMD or dry
AMD.
55. The method of claim 51, wherein the angiogenesis-related
disease comprises diabetic nephropathy.
56. The method of claim 51, wherein the angiogenesis-related
disease comprises a cancer or a metastasis thereof.
57. The method of claim 56, wherein the cancer or a metastasis
thereof is a solid tumor.
58. The method of claim 56, wherein the cancer or a metastasis
thereof is an epithelial based tumor.
59. The method of claim 56, wherein the cancer or the metastasis
thereof is a lung cancer, a melanoma, a breast cancer, a pancreatic
cancer, an ovarian cancer, a colorectal cancer, a prostate cancer,
a kidney cancer, a liver cancer, a uterine cancer, a neck cancer, a
sarcoma, a myeloma, a brain cancer, or a lymphoma.
60. The method of claim 59, wherein the cancer or the metastasis
thereof is a brain cancer that is a glioblastoma multiforme or a
glioma.
61. The method of claim 56, wherein treatment results in partial
elimination of the cancer or the metastasis thereof, total
elimination of the cancer or the metastasis thereof, prolongation
of survival of the subject, or a combination thereof.
62. The method of claim 51, wherein the pharmaceutical composition
is administered to the subject one or more times.
63. The method of claim 51, wherein the pharmaceutical composition
is administered to the subject once per day, once per week, once
per month, once bi-monthly, once every two months, once every three
months, once every four months, once every 5 months, or once every
6 months.
Description
CROSS-REFERENCE
[0001] This application is a continuation application of U.S.
application Ser. No. 15/337,133, filed Oct. 28, 2016, which is a
divisional application of U.S. application Ser. No. 14/838,006,
filed Aug. 27, 2015, now U.S. Pat. No. 9,518,122, which is a
continuation application of U.S. application Ser. No. 14/054,446,
filed on Oct. 15, 2013, now U.S. Pat. No. 9,150,652, which is a
continuation of U.S. application Ser. No. 13/485,702, filed on May
31, 2012, now U.S. Pat. No. 8,609,094, which is a divisional
application of U.S. application Ser. No. 12/751,907, filed Mar. 31,
2010, now U.S. Pat. No. 8,221,753, which claims the benefit of U.S.
Provisional Application No. 61/247,290, filed Sep. 30, 2009; and
which is a continuation application of U.S. Non-Provisional
application Ser. No. 12/570,918, filed Sep. 30, 2009, now
abandoned, the disclosures of all of which are hereby incorporated
by reference in their entireties.
BACKGROUND OF THE INVENTION
[0002] Endoglin, also known as, inter alia, CD105 or edg-1, is a
type I homodimeric membrane glycoprotein which is expressed at high
levels in proliferating vascular endothelial cells (Burrows et al.,
1995, Clin. Cancer Res. 1:1623-1634). Thus, endoglin is primarily a
proliferation-associated marker for endothelial cells undergoing
active angiogenesis. However, there is some expression of endoglin
by the vascular endothelium of normal tissues (Burrows et al.,
supra; Wang et al., 1993, Int. J. Cancer 54:363-370). Human
endoglin is known to specifically bind transforming growth
factor-.beta. (TGF-.beta.), and the deduced amino acid sequence of
endoglin has strong homology to .beta.-glycan, a type of TGF-.beta.
receptor.
[0003] Endoglin (EDG) has been targeted in antibody-based methods
of reducing tumor vasculature, as EDG is a proliferation-associated
antigen on endothelial and leukemia cells. Its expression is
up-regulated in tumor-associated vascular endothelium, and EDG is
essential for angiogenesis. Angiogenesis includes the formation of
new capillary blood vessels leading to neovascularization as well
as the maintenance of the existing vasculature. It is a complex
process which includes a series of sequential steps including
endothelial cell-mediated degradation of vascular basement membrane
and interstitial matrices, migration of endothelial cells,
proliferation of endothelial cells, and formation of capillary
loops by endothelial cells.
[0004] Several anti-endoglin antibodies, in particular
anti-endoglin monoclonal antibodies ("mAb"), have been described.
mAb SN6 is an antibody generated from immunization of mice with
glycoprotein mixtures of cell membranes of human leukemia cells
(Haruta and Seon, 1986, Proc. Natl. Acad. Sci. 83:7898-7902). SN6
is a murine mAb that recognizes human endoglin. mAb 44G4 is an
antibody generated from immunization of mice with whole cell
suspensions of human pre-B leukemia cells (Gougos and Letarte,
1988, J. Immunol. 141:1925-1933; 1990, J. Biol. Chem.
265:8361-8364). 44G4 is also a murine mAb that recognizes human
endoglin. mAb MJ7/18 is an antibody generated from immunization of
rats with inflamed mouse skins (Ge and Butcher, 1994, supra).
MJ7/18 is a mAb that recognizes murine endoglin. mAb Tec-11 is an
antibody generated from immunization of mice with human umbilical
vein endothelial cells (Burrows et al., 1995, Clin. Cancer Res.
1:1623-1634). Tec-11 is a murine mAb with reactivity restricted to
human endoglin. Antibodies against endoglin represent an important
area for the development of therapies for the treatment of a
variety of diseases and conditions which involve, are influenced
by, or affected by angiogenesis.
[0005] Angiogenesis is the physiological process by which new blood
vessels develop from pre-existing vessels (Varner, et al., Cell
Adh. Commun. 1995, 3:367-374; Blood, et al., Biochim. Biophys.
Acta. 1990, 1032:89-118; Weidner, et al., J. Natl. Cancer Inst.
1992, 84:1875-1887). Angiogenesis has been suggested to play a role
in both normal and pathological processes. For example, angiogenic
processes are involved in the development of the vascular systems
of animal organs and tissues. These processes are also involved in
transitory phases of angiogenesis, for example during the menstrual
cycle, in pregnancy, and in wound healing. On the other hand, a
number of diseases are known to be associated with deregulated
angiogenesis.
[0006] In certain pathological conditions, angiogenesis is
stimulated as a means to provide adequate blood and nutrient supply
to the cells within affected tissue. Many of these pathological
conditions involve aberrant cell proliferation and/or regulation.
Therefore, inhibition of angiogenesis is a potentially useful
approach to treating diseases that are characterized by new blood
vessel development. For example, angiogenesis is involved in
pathologic conditions including: various forms of ocular and
non-ocular diseases characterized by
angiogenesis/neovascularization (e.g., macular degeneration,
diabetic retinopathy), diabetic nephropathy, chronic inflammatory
diseases (e.g., IBD), rheumatoid arthritis, osteoarthritis, and
various forms of cancer, solid tumors, and metastases and the
like.
SUMMARY OF THE INVENTION
[0007] Provided herein are humanized antibodies or antigen-binding
fragments thereof that bind to endoglin. Such antibodies have in
vitro and in vivo purification, detection, diagnostic and
therapeutic uses. Also provided herein are humanized antibodies or
antigen-binding fragments thereof that bind to one or more species
or variants of endoglin and inhibit angiogenesis. Further provided
herein are methods of treating angiogenesis-associated diseases
with humanized antibodies or antigen-binding fragments thereof that
bind to endoglin.
[0008] The humanized antibodies and antigen-binding fragments which
bind endoglin and are described herein can be used to treat or
prevent macular degeneration, CNV, diabetic retinopathy, or
proliferative vitreoretinopathy. Described herein are methods of
treating or preventing macular degeneration, CNV, diabetic
retinopathy, or proliferative vitreoretinopathy via the
administration of the antibodies and antigen-binding fragments
described herein. The humanized antibodies and antigen-binding
fragments which bind endoglin and are described herein can also
shrink blood vessels, inhibit endothelial cell proliferation
associated with ocular disease, clear symptoms of bleeding, treat
cloudy vision, provide stasis of vision loss, and/or prevent
leakage of blood vessels. The humanized antibodies and
antigen-binding fragments described herein can also be used in
medicaments for the treatment of macular degeneration, CNV,
diabetic retinopathy or proliferative vitreoretinopathy. The
humanized antibodies and antigen-binding fragments described herein
can also be used in medicaments for the treatment of cancer.
[0009] Provided herein are antibodies, or antigen-binding fragments
thereof, having a heavy chain variable region having an amino acid
sequence set forth as SEQ ID NO: 41 and a light chain variable
region having an amino acid sequence set forth as SEQ ID NO: 3.
[0010] Provided herein are antibodies, or antigen-binding fragments
thereof that bind endoglin, having comprising a light chain
variable region having an amino acid sequence set forth as SEQ ID
NO: 3 and a heavy chain variable region having an amino acid
sequence set forth as SEQ ID NO: 41, wherein: said heavy chain
variable region further comprises one or more modifications
selected from the group consisting of a substitution of glycine (G)
by alanine (A) at position 49; a substitution of asparagine (N) by
serine (S) at position 76; a substitution of threonine (T) by
arginine (R) at position 77; a substitution of leucine (L) by
valine (V) at position 78; a substitution of asparagine (N) by
isoleucine (I) at position 82a; a substitution of valine (V) by
isoleucine (I) or leucine (L) at position 89; a substitution of
threonine (T) by arginine (R) or glycine (G) at position 94; a
substitution of leucine (L) by threonine (T) at position 108; a
substitution of valine (V) by leucine (L) at position 109; and a
substitution of serine (S) by alanine (A) at position 113 utilizing
the Kabat numbering system; and the light chain variable region
further comprises one or more modifications selected from the group
consisting of a substitution of aspartic acid (D) by glutamine (Q)
at position 1; a substitution of glutamine (Q) by valine (V) at
position 3; a substitution of methionine (M) by leucine (L) at
position 4; a substitution of threonine (T) by serine (S) at
position 5; a substitution of tyrosine (Y) by phenylalanine (F) at
position 36; a substitution of leucine (L) by proline (P) at
position 46; a substitution of leucine (L) by tryptophan (W) at
position 47; a substitution of serine (S) by valine (V) or alanine
(A) at position 60; a substitution of aspartic acid (D) by serine
(S) at position 70; a substitution of phenylalanine (F) by tyrosine
(Y) at position 71; a substitution of glutamine (G) by alanine (A)
at position 100; and a substitution of isoleucine (I) by leucine
(L) at position 106 utilizing the Kabat numbering system
[0011] Provided herein are antibodies, or antigen-binding fragments
thereof that bind endoglin, having a heavy chain variable region
and a light chain variable region,
[0012] wherein said heavy chain variable region comprises: [0013]
(i) a CDR1 of SEQ ID NO: 66, a CDR2 of SEQ ID NO: 67, and a CDR3 of
SEQ ID NO: 68; [0014] (ii) a heavy chain FR1 having the amino acid
sequence of SEQ ID NO: 44 or the amino acid sequence of SEQ ID NO:
44 except for one or more conservative substitutions; [0015] (iii)
a heavy chain FR2 having the amino acid sequence of SEQ ID NO: 45
or the amino acid sequence of SEQ ID NO: 45 except for a
substitution of glycine (G) by alanine (A) at position 49 utilizing
the Kabat numbering system; and [0016] (iv) a heavy chain FR3
having the amino acid sequence of SEQ ID NO: 47 or the amino acid
sequence of SEQ ID NO: 47 except for one or more substitutions
selected from the group consisting of: [0017] (a) a substitution of
asparagine (N) by serine (S) at position 76; [0018] (b) a
substitution of threonine (T) by arginine (R) at position 77;
[0019] (c) a substitution of leucine (L) by valine (V) at position
78; [0020] (d) a substitution of asparagine (N) by isoleucine (I)
at position 82a; [0021] (e) a substitution of valine (V) by
isoleucine (I) or leucine (L) at position 89; and [0022] (f) a
substitution of threonine (T) by arginine (R) or glycine (G) at
position 94 utilizing the Kabat numbering system; and [0023] (v) a
heavy chain FR4 having the amino acid sequence of SEQ ID NO: 56 or
the amino acid sequence of SEQ ID NO: 56 except for one or more
substitutions selected from the group consisting of: [0024] (a) a
substitution of leucine (L) by threonine (T) at position 108;
[0025] (b) a substitution of valine (V) by leucine (L) at position
109; and [0026] (c) a substitution of serine (S) by alanine (A) at
position 113 utilizing the Kabat numbering system;
[0027] and said light chain variable region comprises: [0028] (i) a
CDR1 of SEQ ID NO: 63, a CDR2 of SEQ ID NO: 64, and a CDR3 of SEQ
ID NO: 65; [0029] (ii) a light chain FR1 having the amino acid
sequence of SEQ ID NO: 6 or the amino acid sequence of SEQ ID NO: 6
except for one or more substitutions selected from the group
consisting of: [0030] (a) a substitution of aspartic acid (D) by
glutamine (Q) at position 1; [0031] (b) a substitution of glutamine
(Q) by valine (V) at position 3; [0032] (c) a substitution of
methionine (M) by leucine (L) at position 4; and [0033] (d) a
substitution of threonine (T) by serine (S) at position 5;
utilizing the Kabat numbering system; and [0034] (iii) a light
chain FR2 having the amino acid sequence of SEQ ID NO: 20 or the
amino acid sequence of SEQ ID NO: 20 except for one or more
substitutions selected from the group consisting of: [0035] (a) a
substitution of tyrosine (Y) by phenylalanine (F) at position 36;
[0036] (b) a substitution of leucine (L) by proline (P) at position
46; and [0037] (c) a substitution of leucine (L) by tryptophan (W)
at position 47 utilizing the Kabat numbering system; and [0038]
(iv) a light chain FR3 having the amino acid sequence of SEQ ID NO:
28 or the amino acid sequence of SEQ ID NO: 28 except for one or
more substitutions selected from the group consisting of: [0039]
(a) a substitution of serine (S) by valine (V) or alanine (A) at
position 60; [0040] (b) a substitution of aspartic acid (D) by
serine (S) at position 70; and [0041] (b) a substitution of
phenylalanine (F) by tyrosine (Y) at position 71 utilizing the
Kabat numbering system; and [0042] (v) a light chain FR4 having the
amino acid sequence of SEQ ID NO: 35 or the amino acid sequence of
SEQ ID NO: 35 except for one or more substitutions selected from
the group consisting of: [0043] (a) a substitution of glycine (G)
by alanine (A) at position 100; and [0044] (b) a substitution of
isoleucine (I) by leucine (L) at position 106 utilizing the Kabat
numbering system.
[0045] Provided herein is an antibody, or antigen-binding fragment
thereof, comprising a heavy chain variable region having an amino
acid sequence set forth as SEQ ID NO: 42 and a light chain variable
region having an amino acid sequence set forth as SEQ ID NO: 4.
[0046] Provided herein is an antibody, or antigen-binding fragment
thereof, that binds endoglin, comprising a light chain variable
region having an amino acid sequence set forth as SEQ ID NO: 4 and
a heavy chain variable region having an amino acid sequence set
forth as SEQ ID NO: 42, wherein: said heavy chain variable region
further comprises one or more modifications selected from the group
consisting of a substitution of glycine (G) by alanine (A) at
position 49; a substitution of asparagine (N) by serine (S) at
position 76; a substitution of threonine (T) by arginine (R) at
position 77; a substitution of leucine (L) by valine (V) at
position 78; a substitution of asparagine (N) by isoleucine (I) at
position 82a; a substitution of valine (V) by isoleucine (I) or
leucine (L) at position 89; a substitution of arginine (R) by
threonine (T) or glycine (G) at position 94; a substitution of
leucine (L) by threonine (T) at position 108; a substitution of
valine (V) by leucine (L) at position 109; and a substitution of
serine (S) by alanine (A) at position 113 utilizing the Kabat
numbering system; and the light chain variable region further
comprises one or more modifications selected from the group
consisting of a substitution of aspartic acid (D) by glutamine (Q)
at position 1; a substitution of glutamine (Q) by valine (V) at
position 3; a substitution of methionine (M) by leucine (L) at
position 4; a substitution of threonine (T) by serine (S) at
position 5; a substitution of tyrosine (Y) by phenylalanine (F) at
position 36; a substitution of proline (P) by leucine (L) at
position 46; a substitution of tryptophan (W) by leucine (L) at
position 47; a substitution of serine (S) by valine (V) or alanine
(A) at position 60; a substitution of aspartic acid (D) by serine
(S) at position 70; a substitution of tyrosine (Y) by phenylalanine
(F) at position 71; a substitution of glutamine (G) by alanine (A)
at position 100; and a substitution of isoleucine (I) by leucine
(L) at position 106 utilizing the Kabat numbering system
[0047] Provided herein is an antibody, or antigen-binding fragment
thereof, that binds endoglin, comprising a heavy chain variable
region and a light chain variable region,
[0048] wherein said heavy chain variable region comprises: [0049]
(i) a CDR1 of SEQ ID NO: 66, a CDR2 of SEQ ID NO: 67, and a CDR3 of
SEQ ID NO: 68; [0050] (ii) a heavy chain FR1 having the amino acid
sequence of SEQ ID NO: 44 or the amino acid sequence of SEQ ID NO:
44 except for one or more conservative substitutions; [0051] (iii)
a heavy chain FR2 having the amino acid sequence of SEQ ID NO: 45
or the amino acid sequence of SEQ ID NO: 45 except for a
substitution of glycine (G) by alanine (A) at position 49 utilizing
the Kabat numbering system; and [0052] (iv) a heavy chain FR3
having the amino acid sequence of SEQ ID NO: 47 or the amino acid
sequence of SEQ ID NO: 47 except for one or more substitutions
selected from the group consisting of: [0053] (a) a substitution of
asparagine (N) by serine (S) at position 76; [0054] (b) a
substitution of threonine (T) by arginine (R) at position 77;
[0055] (c) a substitution of leucine (L) by valine (V) at position
78; [0056] (d) a substitution of asparagine (N) by isoleucine at
position 82a; [0057] (e) a substitution of valine (V) by isoleucine
(I) or leucine (L) at position 89; and [0058] (f) a substitution of
arginine (R) by threonine (T) or glycine (G) at position 94
utilizing the Kabat numbering system; and [0059] (v) a heavy chain
FR4 having the amino acid sequence of SEQ ID NO: 56 or the amino
acid sequence of SEQ ID NO: 56 except for one or more substitutions
selected from the group consisting of: [0060] (a) a substitution of
leucine (L) by threonine (T) at position 108; [0061] (b) a
substitution of valine (V) by leucine (L) at position 109; and
[0062] (c) a substitution of serine (S) by alanine (A) at position
113 utilizing the Kabat numbering system;
[0063] and said light chain variable region comprises: [0064] (i) a
CDR1 of SEQ ID NO: 63, a CDR2 of SEQ ID NO: 64, and a CDR3 of SEQ
ID NO: 65; [0065] (ii) a light chain FR1 having the amino acid
sequence of SEQ ID NO: 6 or the amino acid sequence of SEQ ID NO: 6
except for one or more substitutions selected from the group
consisting of: [0066] (a) a substitution of aspartic acid (D) by
glutamine (Q) at position 1; [0067] (b) a substitution of glutamine
(Q) by valine (V) at position 3; [0068] (c) a substitution of
methionine (M) by leucine (L) at position 4; and [0069] (d) a
substitution of threonine (T) by serine (S) at position 5;
utilizing the Kabat numbering system; and [0070] (iii) a light
chain FR2 having the amino acid sequence of SEQ ID NO: 21 or the
amino acid sequence of SEQ ID NO: 20 except for one or more
substitutions selected from the group consisting of: [0071] (a) a
substitution of tyrosine (Y) by phenylalanine (F) at position 36;
[0072] (b) a substitution of proline (P) by leucine (L) at position
46; and [0073] (c) a substitution of tryptophan (W) by leucine (L)
at position 47 utilizing the Kabat numbering system; and [0074]
(iv) a light chain FR3 having the amino acid sequence of SEQ ID NO:
29 or the amino acid sequence of SEQ ID NO: 28 except for one or
more substitutions selected from the group consisting of: [0075]
(a) a substitution of serine (S) by valine (V) or alanine (A) at
position 60; [0076] (b) a substitution of aspartic acid (D) by
serine (S) at position 70; and [0077] (b) a substitution of
tyrosine (Y) by phenylalanine (F) at position 71 utilizing the
Kabat numbering system; and [0078] (v) a light chain FR4 having the
amino acid sequence of SEQ ID NO: 35 or the amino acid sequence of
SEQ ID NO: 35 except for one or more substitutions selected from
the group consisting of: [0079] (a) a substitution of glycine (G)
by alanine (A) at position 100; and [0080] (b) a substitution of
isoleucine (I) by leucine (L) at position 106 utilizing the Kabat
numbering system.
[0081] Provided herein is an antibody, or antigen-binding fragment
thereof, comprising a light chain variable region having an amino
acid sequence set forth as SEQ ID NO: 93 (VK1AA) and a heavy chain
variable region having an amino acid sequence set forth as SEQ ID
NO: 89 (VH1A2).
[0082] Provided herein is an antibody, or antigen-binding fragment
thereof, that binds endoglin, comprising a heavy chain variable
region having an amino acid sequence set forth as SEQ ID NO: 89 and
a light chain variable region having an amino acid sequence set
forth as SEQ ID NO: 93, wherein:
[0083] (i) the heavy chain variable region further comprises one or
more modifications selected from the group consisting of a
substitution of glycine (G) by alanine (A) or serine (S) at
position 49; a substitution of alanine (A) by isoleucine (I) at
position 51; a substitution of lysine (K) by arginine (R) or
glutamine (Q) at position 52b; a substitution of leucine (L) by
valine (V) at position 78 utilizing the Kabat numbering system;
and
[0084] (ii) the light chain variable region further comprises one
or more modifications selected from the group consisting of a
substitution of methionine (M) by leucine (L) at position 4; a
substitution of alanine (A) by valine (V) at position 19; a
substitution of threonine (T) by serine (S) at position 22; a
substitution of alanine (A) by isoleucine (I) at position 48; and a
substitution of threonine (T) by serine (S) at position 51
utilizing the Kabat numbering system.
[0085] Provided herein is an antibody, or antigen-binding fragment
thereof, of claim 2 comprising a heavy chain variable region having
an amino acid sequence set forth as SEQ ID NO: 88, 89, 90, 91 or
92; and a light chain variable region having an amino acid sequence
set forth as SEQ ID NO: 93, 94, 95, 96, 97, 100, 102, or 103.
[0086] In one aspect, the antibodies and antigen-binding fragments
described herein are humanized and can be any isotype. Also
encompassed herein are AVIMERs.RTM., diabodies, and heavy chain
dimers (including camelids and shark heavy chain constructs).
[0087] The terms "antigen-binding portion of an antibody,"
"antigen-binding fragment," "antigen-binding domain," "antibody
fragment" or a "functional fragment of an antibody" are used
interchangeably herein to refer to one or more fragments of an
antibody that retain the ability to specifically bind to an
antigen. Non-limiting examples of antibody fragments included
within such terms include, but are not limited to, (i) a Fab
fragment, a monovalent fragment consisting of the V.sub.L, V.sub.H,
C.sub.L and C.sub.H1 domains; (ii) a F(ab').sub.2 fragment, a
bivalent fragment containing two Fab fragments linked by a
disulfide bridge at the hinge region; (iii) a Fd fragment
consisting of the V.sub.H and C.sub.H1 domains; (iv) a Fv fragment
containing the V.sub.L and V.sub.H domains of a single arm of an
antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544
546), which containing a V.sub.H domain; and (vi) an isolated CDR.
Additionally included in this definition are "one-half" antibodies
comprising a single heavy chain and a single light chain. Other
forms of single chain antibodies, such as diabodies are also
encompassed herein.
[0088] An antigen-binding fragment can be any of those described
herein including, but not limited to, a Fab fragment, a Fab', a
F(ab').sub.2 fragment, an Fv fragment (including non-covalently and
covalently linked Fv fragments), an scFv fragment, a single chain
binding polypeptide, an Fd fragment, an Fv fragment or a dAb
fragment. In one non-limiting embodiment, the antigen-binding
fragment is a scFv which can, optionally, be further fused to a
human Fc portion of an antibody.
[0089] In one non-limiting embodiment, the antibody, or
antigen-binding fragment thereof that binds endoglin comprises a
heavy chain variable region having an amino acid sequence set forth
as SEQ ID NO: 41, 42, or 43, and a light chain variable region
having an amino acid sequence set forth as SEQ ID NO: 3, 4, or
5.
[0090] In another non-limiting embodiment, the antibody, or
antigen-binding fragment thereof comprises a heavy chain variable
region having an amino acid sequence set forth as SEQ ID NO: 41 and
a light chain variable region having an amino acid sequence set
forth as SEQ ID NO: 3.
[0091] In another non-limiting embodiment, the antibody, or
antigen-binding fragment thereof comprises a heavy chain variable
region having an amino acid sequence set forth as SEQ ID NO: 41 and
a light chain variable region having an amino acid sequence set
forth as SEQ ID NO: 4.
[0092] In another non-limiting embodiment, the antibody, or
antigen-binding fragment thereof comprises a comprising a heavy
chain variable region having an amino acid sequence set forth as
SEQ ID NO: 41 and a light chain variable region having an amino
acid sequence set forth as SEQ ID NO: 5.
[0093] In another non-limiting embodiment, the antibody, or
antigen-binding fragment thereof comprises a heavy chain variable
region having an amino acid sequence set forth as SEQ ID NO: 42 and
a light chain variable region having an amino acid sequence set
forth as SEQ ID NO: 3.
[0094] In another non-limiting embodiment, the antibody, or
antigen-binding fragment thereof comprises a heavy chain variable
region having an amino acid sequence set forth as SEQ ID NO: 42 and
a light chain variable region having an amino acid sequence set
forth as SEQ ID NO: 4.
[0095] In another non-limiting embodiment, the antibody, or
antigen-binding fragment thereof comprises a heavy chain variable
region having an amino acid sequence set forth as SEQ ID NO: 42 and
a light chain variable region having an amino acid sequence set
forth as SEQ ID NO: 5.
[0096] In another non-limiting embodiment, the antibody, or
antigen-binding fragment thereof comprises a heavy chain variable
region having an amino acid sequence set forth as SEQ ID NO: 43 and
a light chain variable region having an amino acid sequence set
forth as SEQ ID NO: 3.
[0097] In another non-limiting embodiment, the antibody, or
antigen-binding fragment thereof comprises a heavy chain variable
region having an amino acid sequence set forth as SEQ ID NO: 43 and
a light chain variable region having an amino acid sequence set
forth as SEQ ID NO: 4.
[0098] In another non-limiting embodiment, the antibody, or
antigen-binding fragment thereof comprises a heavy chain variable
region having an amino acid sequence set forth as SEQ ID NO: 43 and
a light chain variable region having an amino acid sequence set
forth as SEQ ID NO: 5.
[0099] In yet another non-limiting embodiment, the antibody, or
antigen-binding fragment thereof heavy chain variable region
further comprises one or more modifications selected from the group
consisting of: a substitution of asparagine (N) by serine (S) at
position 76; a substitution of threonine (T) by arginine (R) at
position 77; a substitution of asparagine (N) by isoleucine (I) at
position 82a; a substitution of valine (V) by isoleucine (I) or
leucine (L) at position 89; a substitution of threonine (T) by
glycine (G) at position 94; a substitution of leucine (L) by
threonine (T) at position 108; a substitution of valine (V) by
leucine (L) at position 109; and a substitution of serine (S) by
alanine (A) a position 113; and said light chain variable region
further comprises one or more modifications selected from the group
consisting of: a substitution of aspartic acid (D) by glutamine (Q)
at position 1; a substitution of glutamine (Q) by valine (V) at
position 3; a substitution of threonine (T) by serine (S) at
position 5; a substitution of tyrosine (Y) by phenylalanine (F) a
position 36; a substitution of serine (S) by valine (V) or alanine
(A) at position 60; a substitution of aspartic acid (D) by serine
(S) at position 70; a substitution of glycine (G) by alanine (A) at
position 100, and a substitution of isoleucine (I) by leucine (L)
at position 106 utilizing the Kabat numbering system.
[0100] In one aspect, the antibodies and antigen-binding fragments
described herein can be modified. For example, in one embodiment,
the compound can be modified to alter a pharmacokinetic property of
the compound such as, for example, in vivo stability, solubility,
bioavailability or half-life. Such modifications include, but are
not limited to, PEGylation and/or glycosylation.
[0101] The antibodies and antigen-binding fragments described
herein can be formulated for rapid or extended delivery using
conventional means. In one non-limiting embodiment, rapid delivery
is, for example, by intravenous injection. In another non-limiting
embodiment, extended delivery is, for example, by subcutaneous
deposition. In another non-limiting embodiment, delivery is
achieved via administration by aerosol.
[0102] Provided herein are compositions of the antibodies and
antigen-binding fragments described herein and an acceptable
carrier or excipient.
[0103] Provided herein are polynucleotides (nucleic acids)
comprising a nucleotide sequence encoding antibodies or
antigen-binding fragments described herein.
[0104] Antibodies and antigen-binding fragments thereof as
described herein can be used to treat various diseases and
conditions associated with angiogenesis, e.g., various forms of
ocular diseases characterized by angiogenesis/neovascularization
(e.g., macular degeneration, diabetic retinopathy), diabetic
nephropathy, chronic inflammatory diseases (e.g., IBD), rheumatoid
arthritis, osteoarthritis, and various forms of cancer, solid
tumors, and metastases. Additionally, these antibodies and
antigen-binding fragments thereof described herein can be used in
the formulation of a medicament for the prophylaxis, treatment, or
diagnosis of diseases and conditions associated with angiogenesis,
e.g., various forms of ocular and non-ocular diseases characterized
by angiogenesis/neovascularization (e.g., macular degeneration,
diabetic retinopathy), diabetic nephropathy, chronic inflammatory
diseases (e.g., IBD), rheumatoid arthritis, osteoarthritis, and
various forms of cancer, solid tumors, and metastases.
[0105] Provided herein is a method for inducing a host immune
response in a patient against endoglin, by administering to the
patient a composition, where the composition comprises a humanized
anti-endoglin antibody or antigen-binding fragment thereof that
induces an effective host immune response against the epitope
specifically recognized by said antibody or fragment thereof.
[0106] The host immune response can be a humoral immune response or
a cell-mediated immune response. If the immune response is a
humoral immune response, it can be a protective antibody response
that inhibits angiogenesis, an angiogenesis-dependent disease or an
angiogenesis-dependent disorder. Immune responses also include
induction or blockage of cell signaling pathways (e.g., Smad
signaling). The angiogenesis-dependent disease or disorder can be,
for example, various forms of ocular and non-ocular diseases
characterized by angiogenesis/neovascularization (e.g., macular
degeneration, diabetic retinopathy), diabetic nephropathy, chronic
inflammatory diseases (e.g., IBD), rheumatoid arthritis,
osteoarthritis, various forms of cancer (primary tumors and
metastases) and the like. In one embodiment, the protective
antibody response inhibits angiogenesis.
[0107] Provided herein is a method of affecting cell signaling
pathways associated with endoglin and angiogenesis. Angiogenic
cells can be contacted (in vitro, in vivo or ex vivo) with an
antibody or antigen-binding fragment thereof described herein in an
amount sufficient to alter cell signaling pathways. In one
non-limiting example, in response to antibody binding, Smad 1, 5
and/or 8 signaling is inhibited by about 1.5 fold or more in
angiogenic cells. In another non-limiting example, Smad 3 levels
increase by about 1.5 fold or more, indicating that cells are
returning to a quiescent state.
[0108] Provided herein is a method of inhibiting angiogenesis or an
angiogenesis-dependent disease or disorder in a subject by
administering a composition provided herein to a patient. The
angiogenesis-dependent disease or disorder can be any of the
following: various forms of ocular and non-ocular diseases
characterized by angiogenesis/neovascularization (e.g., macular
degeneration, diabetic retinopathy), diabetic nephropathy, chronic
inflammatory diseases (e.g., IBD), rheumatoid arthritis,
osteoarthritis, and various forms of cancer, solid tumors and
metastases. In one embodiment, inhibiting angiogenesis or an
angiogenesis-dependent disease or disorder alleviates symptoms
associated with the disease or disorder. In another embodiment,
inhibiting angiogenesis or an angiogenesis-dependent disease or
disorder results in decreased tumor size, prevention of tumor
progression, decreased cell proliferation, increased apoptosis, or
increased survival of a patient. Inhibiting in angiogenesis can
result in decreased tumor size or prevent tumor progression. The
method can further include surgical removal of a cancer, and/or
administration of one or more additional anti-cancer agents or
treatments to a patient suffering from cancer.
[0109] Provided herein is a method of preventing or treating a
cancer or metastasis in a subject by administering a composition
provided herein. In one embodiment, administration of the
pharmaceutical composition prolongs life of the subject being
treated. A cancer/tumor to be treated includes a solid tumor; a
tumor can be a primary tumor or a metastatic tumor. Exemplary solid
tumors are of a tissue or organ selected from among skin, melanoma,
lung, pancreas, breast, ovary, colon, rectum, stomach, thyroid,
laryngeal, ovarian, prostate, colorectal, head, neck, eye, mouth,
throat, esophagus, chest, bone, testicular, lymphoid, marrow, bone,
sarcoma, renal, sweat gland, liver, kidney, brain, e.g.,
glioblastoma multiforme and the like tissues. In one non-limiting
example a solid tumor is a colon tumor, a breast tumor, a kidney
tumor, a lung tumor, a prostate tumor, an ovarian tumor, or
metastasis of any of such tumors.
[0110] The method can further include surgical removal of the
cancer and/or administration of one or more anti-cancer agents. An
anti-cancer agent can be administered prior to, concomitant with,
or subsequent to, administration of the pharmaceutical composition.
An anti-cancer agent can be administered within a week before the
pharmaceutical composition, within a week after the pharmaceutical
composition, or the anti-cancer agent can be administered on the
same day as the pharmaceutical composition. If an anti-cancer agent
is administered on the same day as the pharmaceutical composition,
administration can be concomitant.
[0111] Provided herein is a method for preventing or treating a
cancer or a metastasis by surgical removal of the cancer/tumor and
concurrent administration of an anti-cancer agent or treatment and
a composition provided herein to a subject.
[0112] Provided herein is a method of inhibiting angiogenesis by
contacting a cell or tissue with a therapeutically effective amount
of an antibody or antigen-binding fragment thereof as described
herein sufficient to inhibit angiogenesis.
[0113] Provided herein is a method of inhibiting cancer cell growth
by contacting a therapeutically effective amount of an antibody or
antigen-binding fragment thereof as described herein sufficient to
inhibit cancer cell growth or cause apoptosis of the cancer
cell.
[0114] Provided herein is a method, comprising contacting a tissue
with an antibody or antigen-binding fragment thereof as described
herein, wherein contacting inhibits angiogenesis. The tissue can be
a cultured tissue biopsy sample or can be present in a subject.
[0115] Provided herein is a method of preventing or treating a cell
proliferative (e.g., angiogenic) disorder by administering to a
subject having or at risk of having a cell proliferative disorder
an effective amount of a composition provided herein effective to
treat the cell proliferative disorder. The cell proliferative
disorder can be, for example a benign or malignant solid or
non-solid tumor and the tumor can be metastatic or non-metastatic.
The treatment can result in improving the subject's condition and
can be assessed by determining if one or more of the following
factors has occurred: decreased cell proliferation, decreased
numbers of cells, increased apoptosis, or decreased survival of at
least a portion of the cells comprising the cell proliferative
disorder. One or more of these occurrences may, in some cases,
result in partial or total elimination of the cancer and
prolongation of survival of the patient. Optionally, the method can
further include administering an anti-cancer agent or treatment to
the subject.
[0116] Provided herein is a method for treating diabetic
retinopathy, macular degeneration, choroidal neovascularization or
neovascular glaucoma in a patient by administering to the patient a
therapeutically effective amount of a composition provided herein.
The treatment can result in improving the subject's condition and
can be assess by determining if one or more of the following
factors has occurred: decreased macular edema, decreased areas of
CNV, or increased visual acuity.
[0117] In the methods provided herein, the subject can be a human
or a non-human subject. Compositions and the anti-cancer agent or
treatments provided herein can be administered once or multiple
times depending on the health of the patient, the progression of
the disease or condition, and the efficacy of the treatment.
Adjustments to therapy and treatments can be made throughout the
course of treatment.
[0118] Compositions can be administered locally, regionally or
systemically, such as, for example, administration by subcutaneous,
subcutaneous, intravitreal, intradermal, intravenous,
intra-arterial, intraperitoneal or intramuscular injection.
[0119] Additionally, humanized antibodies and antigen-binding
fragments described herein can also be used in combination with
known therapies and/or compounds for the treatment of macular
degeneration, CNV, diabetic retinopathy or proliferative
vitreoretinopathy. Examples of such compounds include, but are not
limited to, bevacizumab (AVASTIN.RTM.), ranibizumab
(LUCENTIS.RTM.), aflibercept (VEGF-Trap), or Macugen. In addition
to the modes of administration described herein, the humanized
anti-endoglin antibodies and antigen-binding fragments can be
administered via intravitreal routes. Non-limiting examples of
intravitreal modes of administration include intravitreal injection
and the use of intravitreal implants.
[0120] Another aspect is the treatment of a chronic inflammatory
disease in a subject by administering a composition of an antibody
or antigen-binding fragment described herein. Non-limiting examples
of chronic inflammatory diseases include IBD, Crohn's disease, and
ulcerative colitis.
[0121] Another aspect is the treatment of rheumatoid arthritis in a
subject by administering a composition of an antibody or
antigen-binding fragment described herein.
[0122] Another aspect is the treatment of osteoarthritis in a
subject by administering a composition of an antibody or
antigen-binding fragment described herein.
[0123] Treatment of a subject with rheumatoid arthritis and/or
osteoarthritis can be assessed by various means, including
improvement in the appropriate categories of ACR scores as measured
according to published guidelines.
[0124] Provided herein is a method of monitoring the efficacy of
one or more of any of the methods provided herein. Increased levels
of soluble endoglin have been correlated with decreased survival in
cancer patients. Thus, in one aspect, levels of soluble endoglin
can be monitored prior to and during therapy. A decrease in the
levels of soluble endoglin can, therefore, be one indication that a
therapeutic regimen is effective in treating the patient.
[0125] One embodiment of the present invention contemplates the use
of any of the compositions of the present invention to formulate a
medicament for treating a disorder of the present invention.
Medicaments can be formulated based on the physical characteristics
of the patient/subject needing treatment, and can be formulated in
single or multiple formulations based on the stage of the cancerous
tissue. Medicaments of the present invention can be packaged in a
suitable pharmaceutical package with appropriate labels for the
distribution to hospitals and clinics wherein the label is for the
indication of treating a disorder as described herein in a subject.
Medicaments can be packaged as a single or multiple units.
Instructions for the dosage and administration of the
pharmaceutical compositions of the present invention can be
included with the pharmaceutical packages.
[0126] Provided herein is a diagnostic method for providing a
sample of cancer cells of a solid tumor or plasma from a patient to
be tested, detecting in the sample the expression of at least one
gene or gene product chosen from a panel of genes or gene products
whose expression has been correlated with sensitivity or resistance
to an angiogenesis inhibitor, wherein the at least one gene or gene
product is chosen from one or more genes or gene products selected
from the group consisting of VEGF, VEGF receptor, HIF-1.alpha.,
placental growth factor receptor, and CD105, and comparing the
level of expression of at least one gene or gene product detected
in the patient sample to a level of expression of at least one gene
or gene product that has been correlated with sensitivity or
resistance to the angiogenesis inhibitor. In one embodiment the
angiogenesis inhibitor is chosen from VEGF receptor inhibitors,
VEGF inhibitors, and endoglin inhibitors.
[0127] Provided herein is a kit for the detection of expression
levels of genes that have been correlated with sensitivity or
resistance to an angiogenesis inhibitor in a sample of cancer cells
or human plasma. In one embodiment, one or more genes are selected
from VEGF, VEGF receptor, HIF-1.alpha., placental growth factor
receptor, and endoglin.
INCORPORATION BY REFERENCE
[0128] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference in its entirety unless otherwise specifically noted.
[0129] This application contains references to amino acid sequences
which have been submitted concurrently herewith as the sequence
listing text file "35882-706-202-SeqList.txt", file size 67,843
KiloBytes (KB), created on Mar. 30, 2010. The aforementioned
sequence listing is hereby incorporated by reference in its
entirety pursuant to 37 C.F.R. .sctn. 1.52(e)(5).
BRIEF DESCRIPTION OF THE DRAWINGS
[0130] FIG. 1 provides a humanized O2-V.kappa.1-39 variable
(V.sub.L) light chain having the monoclonal murine chimeric TRC105
V.sub.L CDRs (underlined) grafted between the framework regions
(FRs) 1-3 of the human sequence O2-V.kappa.1-39 and a framework
region 4 from the human J.kappa.4 sequence (SEQ ID NO: 4) (all in
bold). Variations that can be made to the human FRs are indicated
at positions 1, 3, 4, 5, 36, 46, 47, 60, 70, 71, 100, and 106 of
the sequence (sequence disclosed at SEQ ID NO: 86) utilizing the
Kabat numbering system (shown in italics beneath the humanized
sequence).
[0131] FIG. 2 provides a humanized VH3-15 variable (V.sub.H) heavy
chain having the monoclonal murine monoclonal murine chimeric
TRC105 V.sub.H CDRs (underlined) grafted between the framework
regions (FRs) 1-3 of the human sequence VH3-15 and a framework
region 4 from the human JH4 sequence (SEQ ID NO: 42) (all in bold).
One or more variations that can be made to the human FRs are
indicated at positions 49, 76, 77, 78, 82a, 89, 94, 108, 109, and
113 of the sequence (sequence disclosed at SEQ ID NO: 87) utilizing
the Kabat numbering system (shown in italics beneath the humanized
sequence).
[0132] FIGS. 3A-B provide diagrams of the TGF-.beta./ALK5 signaling
pathway. The TGF-.beta./ALK5 pathway (FIG. 3A) leads to inhibition
of cell proliferation and migration. The TGF-.beta./ALK1 pathway
(FIG. 3B) induces endothelial cell proliferation and migration and
requires CD105 (endoglin) for ALK1 signaling. The dotted lines
indicate inactive or blocked pathways. The bolded arrow indicates
stimulation of a signaling pathway.
[0133] FIGS. 4A-4B provide an amino acid sequence alignment of
exemplary mouse and humanized VK chains (FIG. 4A; SEQ ID NOS 1-5,
respectively, in order of appearance) and V.sub.H chains (FIG. 4B;
SEQ ID NOS 39-43, respectively, in order of appearance) produced
according the invention described herein.
[0134] FIGS. 5A-5B provide an amino acid sequence alignment of
exemplary mouse and super-humanized VK chains (FIG. 5A; SEQ ID NOS
1 and 69-72, respectively, in order of appearance) and V.sub.H
chains (FIG. 5B; SEQ ID NOS 39 and 73-75, respectively, in order of
appearance) produced according the invention described herein.
[0135] FIG. 6 provides an amino acid sequence alignment and
comparison of exemplary mouse and humanized and super-humanized VK
chains (SEQ ID NOS 1, 3 and 70, respectively, in order of
appearance) and V.sub.H chains (SEQ ID NOS 39, 41 and 74,
respectively, in order of appearance) produced according the
invention described herein.
[0136] FIG. 7 illustrates binding of humanized variant constructs
to endoglin in a competition ELISA assay.
[0137] FIGS. 8A-8F. Anti-CD105 competition ELISA with humanized and
humanized/deimmunized antibodies. Varying concentrations of each
antibody were mixed with a fixed concentration of biotinylated
reference anti-CD105 antibody (6.25 ng/ml) and bound to CD105 (100
ng/ml) captured on a Nunc MaxiSorp plate. Binding was detected via
streptavidin-HRP and TMB substrate. Absorbance (OD) at 450 nm was
measured on a plate reader and this was plotted against the test
antibody concentration. FIG. 8A provides the results from the
chimeric control compared to VK1VH1, VK1VH2, VK2VH1 and VK2VH2.
FIG. 8B provides the results from the chimeric control compared to
VK1VH1, VK2AVH1A, VK2SAVH1Q, VK2SAVH1R and VK2SAVH1S. FIG. 8C
provides the results from the chimeric control compared to
VK2AAVH1A2, VK2ASVH1A2, VK2ASVH1Q, VK2SSVH1A and VK2SSVH1Q. FIG. 8D
provides the results from the chimeric control compared to VK2VH1,
VK2AAVH1A, VK2AAVH1Q, VK2AAVH1R and VK2AAVH1S. FIG. 8E provides the
results from the chimeric control compared to VK2VH1, VK2ASVH1A,
VK2ASVH1R, VK2ASVH1S, and VK2SAVH1A2. FIG. 8F provides the results
from the chimeric control compared to VK2VH1, VK2SSVH1Q,
VK2SSVH1A2, VK2SSVH1R and VK2SSVH1S.
[0138] FIG. 9 illustrates binding assay data for variant VK1AAVH1A
plus VK2 containing controls.
[0139] FIG. 10 illustrates binding assay data for chimeric compared
to VK1AAVH1A2 and VK2AAVH1A2.
[0140] FIG. 11 illustrates binding assay data for chimeric compared
to VK2AAVH1Q.
[0141] FIG. 12 illustrates binding assay data for chimeric compared
to VK2AAVH1R.
[0142] FIG. 13 illustrates binding assay data for chimeric compared
to VK1AAVH1A2.
[0143] FIG. 14 illustrates binding assay data for chimeric compared
to VK1AAVH1Q.
[0144] FIG. 15 illustrates binding assay data for chimeric compared
to VK1AAVH1R.
[0145] FIG. 16 illustrates binding assay data for chimeric compared
to VK2AAVH1A2.
[0146] FIG. 17 illustrates the lead humanized deimmunized heavy
chain variable region with CDRs in bold and underlined (sequence
disclosed at SEQ ID NO: 89). Variations that can be made are
indicated at the identified positions of the sequence utilizing the
Kabat numbering system (sequence disclosed at SEQ ID NO: 116)
(shown in italics beneath the humanized sequence). Variations may
be made as a single mutation or as more than one mutation, and
variations can be made with mutations in any combination.
[0147] FIG. 18 illustrates the lead humanized deimmunized light
chain variable region with CDRs in bold and underlined (sequence
disclosed at SEQ ID NO: 93). Variations that can be made are
indicated at the identified positions of the sequence utilizing the
Kabat numbering system (sequence disclosed at SEQ ID NO: 117)
(shown in italics beneath the humanized sequence). Variations may
be made as a single mutation or as more than one mutation, and
variations can be made with mutations in any combination.
[0148] FIGS. 19A-19C illustrate analysis of the regions of the
heavy chain (SEQ ID NO: 41) using iTope.TM.. Peptides spanning the
entire sequence were tested as 9mer peptides in one amino acid
increments. The predicted binding of each residue as the p1 anchor
of a core 9mer peptide to MHC class II alleles is indicated by a
"O" if the binding score was 0.55-0.6 and by a "X" if the binding
score was >0.6. Regions containing potentially immunogenic
peptides are indicated in the "iTope" column, dark grey indicates
promiscuous high affinity MHC class II binding peptides, light grey
indicates promiscuous moderate affinity MHC class II binding
peptides. The numbers of MHC class II alleles predicted to bind are
shown in the "total" and "high affinity" columns. Potential p1
anchor residues identified as germline sequences are shown in
reverse type in the "Sequence" column. FIG. 19A represents the
first part of the table of results.
[0149] FIG. 19B represents the second part of the table of results.
FIG. 19C represents the third part of the table of results.
[0150] FIG. 20 illustrates analysis of the variant regions of
variants of the heavy chain (SEQ ID NO: 118) using iTope.TM..
Peptides spanning the entire sequence were tested as 9mer peptides
in one amino acid increments. The predicted binding of each residue
as the p1 anchor of a core 9mer peptide to MHC class II alleles is
indicated by a "O" if the binding score was 0.55-0.6 and by a "X"
if the binding score was >0.6. Regions containing potentially
immunogenic peptides are indicated in the "iTope" column, dark grey
indicates promiscuous high affinity MHC class II binding peptides,
light grey indicates promiscuous moderate affinity MHC class II
binding peptides. The numbers of MHC class II alleles predicted to
bind are shown in the "total" and "high affinity" columns and the
difference to binding is shown. Potential p1 anchor residues
identified as germline sequences are shown in reverse type in the
"Sequence" column and the amino acid differences in the variants
are boxed.
[0151] FIGS. 21A-21C illustrate analysis of the regions of the
light chain (SEQ ID NO: 3) using iTope.TM.. Peptides spanning the
entire sequence were tested as 9mer peptides in one amino acid
increments. The predicted binding of each residue as the p1 anchor
of a core 9mer peptide to MHC class II alleles is indicated by a
"O" if the binding score was 0.55-0.6 and by a "X" if the binding
score was >0.6. Regions containing potentially immunogenic
peptides are indicated in the "iTope" column, dark grey indicates
promiscuous high affinity MHC class II binding peptides, light grey
indicates promiscuous moderate affinity MHC class II binding
peptides. The numbers of MHC class II alleles predicted to bind are
shown in the "total" and "high affinity" columns. Potential p1
anchor residues identified as germline sequences are shown in
reverse type in the "Sequence" column. FIG. 21A represents the
first part of the table of results.
[0152] FIG. 21B represents the second part of the table of results.
FIG. 21C represents the third part of the table of results.
[0153] FIG. 22 illustrates analysis of the variant regions of the
light chain (SEQ ID NO: 101) using iTope.TM.. Peptides spanning the
entire sequence were tested as 9mer peptides in one amino acid
increments. The predicted binding of each residue as the p1 anchor
of a core 9mer peptide to MHC class II alleles is indicated by a
"O" if the binding score was 0.55-0.6 and by a "X" if the binding
score was >0.6. Regions containing potentially immunogenic
peptides are indicated in the "iTope" column, dark grey indicates
promiscuous high affinity MHC class II binding peptides, light grey
indicates promiscuous moderate affinity MHC class II binding
peptides. The numbers of MHC class II alleles predicted to bind are
shown in the "total" and "high affinity" columns and the difference
to binding is shown. Potential p1 anchor residues identified as
germline sequences are shown in reverse type in the "Sequence"
column and the amino acid differences in the variants are
boxed.
[0154] FIG. 23 illustrates the frequency of MHC Class II allotypes
in the world population and the study population.
[0155] FIGS. 24A-C. Chimeric anti-endoglin antibody, humanized
anti-endoglin antibody VK1VH1 and humanized/deimmunized
anti-endoglin antibody VK1AAVH1A2 were tested in EPISCREEN.RTM.
time course T cell assays using PBMC from 20 donors. Bulk cultures
of PBMC incubated with test antibodies were sampled on days 5, 6, 7
and 8, and pulsed with .sup.3H-Thymidine. Cells were harvested and
incorporation of radioactivity measured by scintillation counting.
Results for each triplicate sample were averaged and normalized by
conversion to Stimulation Index (SI). The SI for each time point
with each donor is shown above for the chimeric antibody TRC105
(FIG. 24A), VK1VH1 (FIG. 24B) and VK1AAVH1A2 (FIG. 24C). The
cut-off for determining positive responses with an SI.gtoreq.2 is
highlighted by the dotted line and significant responses (p<0.05
in a student's t-test) are indicated (*).
DETAILED DESCRIPTION OF THE INVENTION
[0156] It is to be understood that this application is not limited
to particular formulations or process parameters, as these may, of
course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments
only, and is not intended to be limiting. Further, it is understood
that a number of methods and materials similar or equivalent to
those described herein can be used in the practice of the present
inventions.
[0157] In accordance with the present application, there may be
employed conventional molecular biology, microbiology, and
recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. See, e.g.,
Sambrook et al., "Molecular Cloning: A Laboratory Manual" (1989);
"Current Protocols in Molecular Biology" Volumes I-III [Ausubel, R.
M., ed. (1994)]; "Cell Biology: A Laboratory Handbook" Volumes
I-III [J. E. Celis, ed. (1994))]; "Current Protocols in Immunology"
Volumes I-III [Coligan, J. E., ed. (1994)]; "Oligonucleotide
Synthesis" (M. J. Gait ed. 1984); "Nucleic Acid Hybridization" [B.
D. Hames & S. J. Higgins eds. (1985)]; "Transcription And
Translation" [B. D. Hames & S. J. Higgins, eds. (1984)];
"Animal Cell Culture" [R. I. Freshney, ed. (1986)]; "Immobilized
Cells And Enzymes" [IRL Press, (1986)]; B. Perbal, "A Practical
Guide To Molecular Cloning" (1984), each of which is specifically
incorporated herein by reference in its entirety.
[0158] Murine monoclonal antibodies (mAbs) have been raised against
endoglin which modulate endoglin activity and thereby inhibit
angiogenesis and/or inhibit vasodilation of small blood vessels.
These murine antibodies are described in U.S. Pat. Nos. 5,928,641,
6,200,566, 6,190,660, and 7,097,836 which are hereby incorporated
in their entirety. Additionally, the ex vivo and in vivo efficiency
of a number of these antibodies has been demonstrated; these
monoclonal antibodies that bind endoglin are of interest as
endoglin modulating compounds. Therapeutic use of these murine
antibodies is not feasible, however, as their administration has a
number of limitations, including immunogenicity in, for example,
the form of human anti-mouse antibodies (HAMA). Humanized
antibodies are made to address these reactions.
[0159] Humanized antibodies that bind endoglin are described herein
that exhibit reduced immunogenicity while maintaining and/or
improving their specificity. Additionally, to address problems
associated with murine antibodies, humanized antibodies that bind
endoglin and decrease and/or inhibit angiogenesis are described
herein that exhibit reduced immunogenicity while maintaining and/or
improving their specificity. These humanized endoglin antibodies
are useful for the diagnosis and treatment of various conditions
and diseases as well as for purification and detection of
endoglin.
I. Anti-Endoglin Antibodies
[0160] Provided herein are humanized antibodies, and
antigen-binding fragments thereof that bind endoglin. Endoglin can
be found on cells that comprise and support existing vasculature as
well as cells that are promoting the growth of, and become part of,
new vasculature. These antibodies and antigen-binding fragments can
bind endoglin and thereby inhibit angiogenesis, inhibit the
existing vasculature or the maintenance of the existing
vasculature, and/or inhibit small vessel dilation. Hereinafter, a
reference to the terms "antibody" or "antibodies" are to be
considered inclusive of any of the antigen-binding fragments
described herein and the terms are to be interchangeable where
applicable. In addition to their use for purification of endoglin,
these antibodies are useful for purification, detection and
diagnostic purposes as well as therapeutic purposes. The antibodies
provided herein can be used for the formulation of medicaments for
the treatment a variety of conditions and diseases, methods to
treat said conditions and diseases and methods of detection or
diagnosis. As used herein, angiogenesis is inclusive of the growth
and/or development of new blood vessels (also referred to as
neovascularization), dilation of the small vessels, excessive or
prolonged vascular growth, and maintenance of the existing
vasculature. Angiogenesis conditions and diseases refers to those
diseases and conditions related to, caused by, or associated with
angiogenesis. Non-limiting examples of such diseases include, for
example, various forms of ocular diseases characterized by
angiogenesis/neovascularization (e.g., macular degeneration, CNV,
diabetic retinopathy), diabetic nephropathy, chronic inflammatory
diseases (e.g., IBD), rheumatoid arthritis, osteoarthritis, and
various forms of cancer (primary tumors and metastases).
[0161] A. Antibody Terminology
[0162] As used herein, the term "antibody" refers to an
immunoglobulin (Ig) whether natural or partly or wholly
synthetically produced. The term also covers any polypeptide or
protein having a binding domain which is, or is homologous to, an
antigen-binding domain. The term further includes "antigen-binding
fragments" and other interchangeable terms for similar binding
fragments such as described below. Complementarity determining
region (CDR) grafted antibodies and other humanized antibodies
(including CDR modifications and framework region modifications)
are also contemplated by this term.
[0163] Native antibodies and native immunoglobulins are usually
heterotetrameric glycoproteins of about 150,000 Daltons, composed
of two identical light (L) chains and two identical heavy (H)
chains. Each light chain is typically linked to a heavy chain by
one covalent disulfide bond, while the number of disulfide linkages
varies among the heavy chains of different immunoglobulin isotypes.
Each heavy and light chain also has regularly spaced intrachain
disulfide bridges. Each heavy chain has at one end a variable
domain ("V.sub.H") followed by a number of constant domains
("C.sub.H"). Each light chain has a variable domain at one end
("V.sub.L") and a constant domain ("C.sub.L") at its other end; the
constant domain of the light chain is aligned with the first
constant domain of the heavy chain, and the light-chain variable
domain is aligned with the variable domain of the heavy chain.
Particular amino acid residues are believed to form an interface
between the light- and heavy-chain variable domains.
[0164] The terms "synthetic polynucleotide," "synthetic gene" or
"synthetic polypeptide," as used herein, mean that the
corresponding polynucleotide sequence or portion thereof, or amino
acid sequence or portion thereof, is derived, from a sequence that
has been designed, or synthesized de novo, or modified, compared to
an equivalent naturally-occurring sequence. Synthetic
polynucleotides (antibodies or antigen binding fragments) or
synthetic genes can be prepared by methods known in the art,
including but not limited to, the chemical synthesis of nucleic
acid or amino acid sequences. Synthetic genes are typically
different from naturally-occurring genes, either at the amino acid,
or polynucleotide level, (or both) and are typically located within
the context of synthetic expression control sequences. For example,
synthetic gene sequences can include amino acid, or polynucleotide,
sequences that have been changed, for example, by the replacement,
deletion, or addition, of one or more, amino acids, or nucleotides,
thereby providing an antibody amino acid sequence, or a
polynucleotide coding sequence that is different from the source
sequence. Synthetic gene polynucleotide sequences, may not
necessarily encode proteins with different amino acids, compared to
the natural gene; for example, they can also encompass synthetic
polynucleotide sequences that incorporate different codons but
which encode the same amino acid (i.e., the nucleotide changes
represent silent mutations at the amino acid level).
[0165] With respect to antibodies, the term "variable domain"
refers to the variable domains of antibodies that are used in the
binding and specificity of each particular antibody for its
particular antigen. However, the variability is not evenly
distributed throughout the variable domains of antibodies. Rather,
it is concentrated in three segments called hypervariable regions
(also known as CDRs) in both the light chain and the heavy chain
variable domains. More highly conserved portions of variable
domains are called the "framework regions" or "FRs." The variable
domains of unmodified heavy and light chains each contain four FRs
(FR1, FR2, FR3 and FR4), largely adopting a .beta.-sheet
configuration interspersed with three CDRs which form loops
connecting and, in some cases, part of the .beta.-sheet structure.
The CDRs in each chain are held together in close proximity by the
FRs and, with the CDRs from the other chain, contribute to the
formation of the antigen-binding site of antibodies (see Kabat et
al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991), pages 647-669).
[0166] The terms "hypervariable region" and "CDR" when used herein,
refer to the amino acid residues of an antibody which are
responsible for antigen-binding. The CDRs comprise amino acid
residues from three sequence regions which bind in a complementary
manner to an antigen and are known as CDR1, CDR2, and CDR3 for each
of the V.sub.H and V.sub.L chains. In the light chain variable
domain, the CDRs typically correspond to approximately residues
24-34 (CDRL1), 50-56 (CDRL2) and 89-97 (CDRL3), and in the heavy
chain variable domain the CDRs typically correspond to
approximately residues 31-35 (CDRH1), 50-65 (CDRH2) and 95-102
(CDRH3) according to Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)). It is understood that
the CDRs of different antibodies may contain insertions, thus the
amino acid numbering may differ. The Kabat numbering system
accounts for such insertions with a numbering scheme that utilizes
letters attached to specific residues (e.g., 27A, 27B, 27C, 27D,
27E, and 27F of CDRL1 in the light chain) to reflect any insertions
in the numberings between different antibodies. Alternatively, in
the light chain variable domain, the CDRs typically correspond to
approximately residues 26-32 (CDRL1), 50-52 (CDRL2) and 91-96
(CDRL3), and in the heavy chain variable domain, the CDRs typically
correspond to approximately residues 26-32 (CDRH1), 53-55 (CDRH2)
and 96-101 (CDRH3) according to Chothia and Lesk, J. Mol. Biol.,
196: 901-917 (1987)).
[0167] As used herein, "framework region" or "FR" refers to
framework amino acid residues that form a part of the antigen
binding pocket or groove. In some embodiments, the framework
residues form a loop that is a part of the antigen binding pocket
or groove and the amino acids residues in the loop may or may not
contact the antigen. Framework regions generally comprise the
regions between the CDRs. In the light chain variable domain, the
FRs typically correspond to approximately residues 0-23 (FRL1),
35-49 (FRL2), 57-88 (FRL3), and 98-109 and in the heavy chain
variable domain the FRs typically correspond to approximately
residues 0-30 (FRH1), 36-49 (FRH2), 66-94 (FRH3), and 103-133
according to Kabat et al., Sequences of Proteins of Immunological
Interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda, Md. (1991)). As discussed above with the Kabat
numbering for the light chain, the heavy chain too accounts for
insertions in a similar manner (e.g., 35A, 35B of CDRH1 in the
heavy chain). Alternatively, in the light chain variable domain,
the FRs typically correspond to approximately residues 0-25 (FRL1),
33-49 (FRL2) 53-90 (FRL3), and 97-109 (FRL4), and in the heavy
chain variable domain, the FRs typically correspond to
approximately residues 0-25 (FRH1), 33-52 (FRH2), 56-95 (FRH3), and
102-113 (FRH4) according to Chothia and Lesk, J. Mol. Biol., 196:
901-917 (1987)).
[0168] The loop amino acids of a FR can be assessed and determined
by inspection of the three-dimensional structure of an antibody
heavy chain and/or antibody light chain. The three-dimensional
structure can be analyzed for solvent accessible amino acid
positions as such positions are likely to form a loop and/or
provide antigen contact in an antibody variable domain. Some of the
solvent accessible positions can tolerate amino acid sequence
diversity and others (e.g., structural positions) are, generally,
less diversified. The three dimensional structure of the antibody
variable domain can be derived from a crystal structure or protein
modeling.
[0169] Constant domains (Fc) of antibodies are not involved
directly in binding an antibody to an antigen but, rather, exhibit
various effector functions, such as participation of the antibody
in antibody-dependent cellular toxicity via interactions with, for
example, Fc receptors (FcR). Fc domains can also increase
bioavailability of an antibody in circulation following
administration to a patient.
[0170] Depending on the amino acid sequence of the constant domain
of their heavy chains, immunoglobulins can be assigned to different
classes. There are five major classes of immunoglobulins: IgA, IgD,
IgE, IgG, and IgM, and several of these can be further divided into
subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and
IgA2. The heavy-chain constant domains (Fc) that correspond to the
different classes of immunoglobulins are called .alpha., .delta.,
.epsilon., .gamma., and .mu., respectively. The subunit structures
and three-dimensional configurations of different classes of
immunoglobulins are well known.
[0171] The "light chains" of antibodies (immunoglobulins) from any
vertebrate species can be assigned to one of two clearly distinct
types, called kappa or (".kappa." or "K") and lambda or
(".lamda."), based on the amino acid sequences of their constant
domains.
[0172] The terms "antigen-binding portion of an antibody,"
"antigen-binding fragment," "antigen-binding domain," "antibody
fragment" or a "functional fragment of an antibody" are used
interchangeably herein to refer to one or more fragments of an
antibody that retain the ability to specifically bind to an
antigen. Non-limiting examples of antibody fragments included
within such terms include, but are not limited to, (i) a Fab
fragment, a monovalent fragment consisting of the V.sub.L, V.sub.H,
C.sub.L and C.sub.H1 domains; (ii) a F(ab').sub.2 fragment, a
bivalent fragment containing two Fab fragments linked by a
disulfide bridge at the hinge region; (iii) a Fd fragment
consisting of the V.sub.H and C.sub.H1 domains; (iv) a Fv fragment
containing the V.sub.L and V.sub.H domains of a single arm of an
antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544
546), which containing a V.sub.H domain; and (vi) an isolated CDR.
Additionally included in this definition are "one-half" antibodies
comprising a single heavy chain and a single light chain. Other
forms of single chain antibodies, such as diabodies are also
encompassed herein.
[0173] "F(ab').sub.2" and "Fab'" moieties can be produced by
treating an Ig with a protease such as pepsin and papain, and
include antibody fragments generated by digesting immunoglobulin
near the disulfide bonds existing between the hinge regions in each
of the two heavy chains. For example, papain cleaves IgG upstream
of the disulfide bonds existing between the hinge regions in each
of the two heavy chains to generate two homologous antibody
fragments in which an light chain composed of V.sub.L and C.sub.L
(light chain constant region), and a heavy chain fragment composed
of V.sub.H and C.sub.H.lamda.1 (.gamma.1) region in the constant
region of the heavy chain) are connected at their C terminal
regions through a disulfide bond. Each of these two homologous
antibody fragments is called Fab'. Pepsin also cleaves IgG
downstream of the disulfide bonds existing between the hinge
regions in each of the two heavy chains to generate an antibody
fragment slightly larger than the fragment in which the two
above-mentioned Fab' are connected at the hinge region. This
antibody fragment is called F(ab').sub.2.
[0174] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (C.sub.H1) of the heavy
chain. Fab' fragments differ from Fab fragments by the addition of
a few residues at the carboxyl terminus of the heavy chain C.sub.H1
domain including one or more cysteine(s) from the antibody hinge
region. Fab'-SH is the designation herein for Fab' in which the
cysteine residue(s) of the constant domains bear a free thiol
group. F(ab').sub.2 antibody fragments originally were produced as
pairs of Fab' fragments which have hinge cysteines between them.
Other chemical couplings of antibody fragments are also known.
[0175] "Fv" refers to an antibody fragment which contains a
complete antigen-recognition and antigen-binding site. This region
consists of a dimer of one heavy chain and one light chain variable
domain in tight, non-covalent or covalent association (disulfide
linked Fv's have been described in the art, Reiter et al. (1996)
Nature Biotechnology 14:1239-1245). It is in this configuration
that the three CDRs of each variable domain interact to define an
antigen-binding site on the surface of the V.sub.H-V.sub.L dimer.
Collectively, a combination of one or more of the CDRs from each of
the V.sub.H and V.sub.L chains confer antigen-binding specificity
to the antibody. For example, it would be understood that, for
example, the CDRH3 and CDRL3 could be sufficient to confer
antigen-binding specificity to an antibody when transferred to
V.sub.H and V.sub.L chains of a recipient antibody or
antigen-binding fragment thereof and this combination of CDRs can
be tested for binding, affinity, etc. using any of the techniques
described herein. Even a single variable domain (or half of an Fv
comprising only three CDRs specific for an antigen) has the ability
to recognize and bind antigen, although likely at a lower affinity
than when combined with a second variable domain. Furthermore,
although the two domains of a Fv fragment (V.sub.L and V.sub.H),
are coded for by separate genes, they can be joined using
recombinant methods by a synthetic linker that enables them to be
made as a single protein chain in which the V.sub.L and V.sub.H
regions pair to form monovalent molecules (known as single chain Fv
(scFv); Bird et al. (1988) Science 242:423-426; Huston et al.
(1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and Osbourn et al.
(1998) Nat. Biotechnol. 16:778). Such scFvs are also intended to be
encompassed within the term "antigen-binding portion" of an
antibody. Any V.sub.H and V.sub.L sequences of specific scFv can be
linked to an Fc region cDNA or genomic sequences, in order to
generate expression vectors encoding complete Ig (e.g., IgG)
molecules or other isotypes. V.sub.H and V.sub.L can also be used
in the generation of Fab, Fv or other fragments of Igs using either
protein chemistry or recombinant DNA technology.
[0176] "Single-chain Fv" or "sFv" antibody fragments comprise the
V.sub.H and V.sub.L domains of an antibody, wherein these domains
are present in a single polypeptide chain. In some embodiments, the
Fv polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the sFv to form the
desired structure for antigen binding. For a review of sFvs see,
e.g., Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol.
113, Rosenburg and Moore eds. Springer-Verlag, New York, pp.
269-315 (1994).
[0177] The term "AVIMER.RTM." refers to a class of therapeutic
proteins of human origin, which are unrelated to antibodies and
antibody fragments, and are composed of several modular and
reusable binding domains, referred to as A-domains (also referred
to as class A module, complement type repeat, or LDL-receptor class
A domain). They were developed from human extracellular receptor
domains by in vitro exon shuffling and phage display (Silverman et
al., 2005, Nat. Biotechnol. 23:1493-1494; Silverman et al., 2006,
Nat. Biotechnol. 24:220). The resulting proteins can contain
multiple independent binding domains that can exhibit improved
affinity (in some cases, sub-nanomolar) and specificity compared
with single-epitope binding proteins. See, for example, U.S. Patent
Application Publ. Nos. 2005/0221384, 2005/0164301, 2005/0053973 and
2005/0089932, 2005/0048512, and 2004/0175756, each of which is
hereby incorporated by reference herein in its entirety.
[0178] Each of the known 217 human A-domains comprises .about.35
amino acids (.about.4 kDa); and these domains are separated by
linkers that average five amino acids in length. Native A-domains
fold quickly and efficiently to a uniform, stable structure
mediated primarily by calcium binding and disulfide formation. A
conserved scaffold motif of only 12 amino acids is required for
this common structure. The end result is a single protein chain
containing multiple domains, each of which represents a separate
function. Each domain of the proteins binds independently and the
energetic contributions of each domain are additive. These proteins
were called "AVIMERs.RTM." from avidity multimers.
[0179] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy chain
variable domain (V.sub.H) connected to a light chain variable
domain (V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L).
By using a linker that is too short to allow pairing between the
two domains on the same chain, the domains are forced to pair with
the complementary domains of another chain and create two
antigen-binding sites. Diabodies are described more fully in, for
example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.
Acad. Sci. USA 90:6444 6448 (1993).
[0180] Antigen-binding polypeptides also include heavy chain dimers
such as, for example, antibodies from camelids and sharks. Camelid
and shark antibodies comprise a homodimeric pair of two chains of
V-like and C-like domains (neither has a light chain). Since the
V.sub.H region of a heavy chain dimer IgG in a camelid does not
have to make hydrophobic interactions with a light chain, the
region in the heavy chain that normally contacts a light chain is
changed to hydrophilic amino acid residues in a camelid. V.sub.H
domains of heavy-chain dimer IgGs are called V.sub.HH domains.
Shark Ig-NARs comprise a homodimer of one variable domain (termed a
V-NAR domain) and five C-like constant domains (C-NAR domains). In
camelids, the diversity of antibody repertoire is determined by the
CDRs 1, 2, and 3 in the V.sub.H or V.sub.HH regions. The CDR3 in
the camel V.sub.HH region is characterized by its relatively long
length, averaging 16 amino acids (Muyldermans et al., 1994, Protein
Engineering 7(9): 1129). This is in contrast to CDR3 regions of
antibodies of many other species. For example, the CDR3 of mouse
V.sub.H has an average of 9 amino acids. Libraries of
camelid-derived antibody variable regions, which maintain the in
vivo diversity of the variable regions of a camelid, can be made
by, for example, the methods disclosed in U.S. Patent Application
Ser. No. 20050037421.
[0181] "Humanized" forms of non-human (e.g., murine) antibodies
include chimeric antibodies which contain minimal sequence derived
from a non-human Ig. For the most part, humanized antibodies are
human Igs (recipient antibody) in which one or more of the CDRs of
the recipient are replaced by CDRs from a non-human species
antibody (donor antibody) such as mouse, rat, rabbit or non-human
primate having the desired specificity, affinity and binding
function. In some instances, one or more FR amino acid residues of
the human Ig are replaced by corresponding non-human amino acid
residues. Furthermore, humanized antibodies can contain residues
which are not found in the recipient antibody or in the donor
antibody. These modifications can be made to refine antibody
performance, if needed. A humanized antibody can comprise
substantially all of at least one and, in some cases two, variable
domains, in which all or substantially all of the hypervariable
regions correspond to those of a non-human immunoglobulin and all,
or substantially all, of the FRs are those of a human
immunoglobulin sequence. The humanized antibody optionally can also
include at least a portion of an immunoglobulin constant region
(Fc), typically that of a human immunoglobulin. For details, see
Jones et al., Nature 321: 522-525 (1986); Reichmann et al., Nature
332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596
(1992).
[0182] A humanized antibody also includes antibodies in which part,
or all of the CDRs of the heavy and light chain are derived from a
non-human monoclonal antibody, substantially all the remaining
portions of the variable regions are derived from human variable
region (both heavy and light chain), and the constant regions are
derived from a human constant region. In one embodiment, the CDR1,
CDR2 and CDR3 regions of the heavy and light chains are derived
from a non-human antibody. In yet another embodiment, at least one
CDR (e.g., a CDR3) of the heavy and light chains is derived from a
non-human antibody. Various combinations of CDR1, CDR2, and CDR3
can be derived from a non-human antibody and are contemplated
herein. In one non-limiting example, one or more of the CDR1, CDR2
and CDR3 regions of each of the heavy and light chains are derived
from a murine chimeric monoclonal antibody clone TRC105.
[0183] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to conventional
(polyclonal) antibody preparations, which can include different
antibodies directed against different determinants (epitopes), each
monoclonal antibody is directed against a single determinant on the
antigen. The modifier "monoclonal" indicates the character of the
antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
monoclonal antibodies can be made by the hybridoma method first
described by Kohler et al., Nature 256:495 (1975), or can be made
by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). In
certain embodiments, the monoclonal antibodies can be isolated from
phage antibody libraries using the techniques described in Clackson
et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol.
222:581-597 (1991), for example.
[0184] Antibodies can be isolated and purified from the culture
supernatant or ascites mentioned above by saturated ammonium
sulfate precipitation, euglobulin precipitation method, caproic
acid method, caprylic acid method, ion exchange chromatography
(DEAE or DE52), or affinity chromatography using anti-Ig column or
a protein A, G or L column such as described in more detail
below.
[0185] Exemplary antibodies for use in the compositions and methods
described herein are intact immunoglobulin molecules, such as, for
example, a humanized antibody or those portions of a humanized Ig
molecule that contain the antigen binding site (i.e., paratope) or
a single heavy chain and a single light chain, including those
portions known in the art as Fab, Fab', F(ab)', F(ab').sub.2, Fd,
scFv, a variable heavy domain, a variable light domain, a variable
NAR domain, bi-specific scFv, a bi-specific Fab.sub.2, a
tri-specific Fab.sub.3 and a single chain binding polypeptides and
others also referred to as antigen-binding fragments. When
constructing an immunoglobulin molecule or fragments thereof,
variable regions or portions thereof may be fused to, connected to,
or otherwise joined to one or more constant regions or portions
thereof to produce any of the antibodies or fragments thereof
described herein. This may be accomplished in a variety of ways
known in the art, including but not limited to, molecular cloning
techniques or direct synthesis of the nucleic acids encoding the
molecules. Exemplary non-limiting methods of constructing these
molecules can also be found in the examples described herein.
[0186] In one exemplary embodiment, the application contemplates a
single chain binding polypeptide having a heavy chain variable
region, and/or a light chain variable region which binds endoglin
and has, optionally, an immunoglobulin Fc region. In one exemplary
embodiment, the application contemplates a single chain binding
polypeptide having a heavy chain variable region, and/or a light
chain variable region which binds endoglin and inhibits
angiogenesis and has, optionally, an immunoglobulin Fc region. Such
a molecule is a single chain variable fragment optionally having
effector function or increased half-life through the presence of
the immunoglobulin Fc region. Methods of preparing single chain
binding polypeptides are known in the art (e.g., U.S. Patent
Application No. 2005/0238646).
[0187] The terms "germline gene segments" or "germline sequences"
refer to the genes from the germline (the haploid gametes and those
diploid cells from which they are formed). The germline DNA
contains multiple gene segments that encode a single Ig heavy or
light chain. These gene segments are carried in the germ cells but
cannot be transcribed and translated into heavy and light chains
until they are arranged into functional genes. During B-cell
differentiation in the bone marrow, these gene segments are
randomly shuffled by a dynamic genetic system capable of generating
more than 10.sup.8 specificities. Most of these gene segments are
published and collected by the germline database.
[0188] As used herein, "immunoreactive" refers to binding agents,
antibodies or fragments thereof that are specific to a sequence of
amino acid residues ("binding site" or "epitope"), yet if are
cross-reactive to other peptides/proteins, are not toxic at the
levels at which they are formulated for administration to human
use. The term "binding" refers to a direct association between two
molecules, due to, for example, covalent, electrostatic,
hydrophobic, and ionic and/or hydrogen-bond interactions under
physiological conditions, and including interactions such as salt
bridges and water bridges and any other conventional binding means.
The term "preferentially binds" means that the binding agent binds
to the binding site with greater affinity than it binds unrelated
amino acid sequences. Preferably such affinity is at least 1-fold
greater, at least 2-fold greater, at least 3-fold greater, at least
4-fold greater, at least 5-fold greater, at least 6-fold greater,
at least 7-fold greater, at least 8-fold greater, at least 9-fold
greater, 10-fold greater, at least 20-fold greater, at least
30-fold greater, at least 40-fold greater, at least 50-fold
greater, at least 60-fold greater, at least 70-fold greater, at
least 80-fold greater, at least 90-fold greater, at least 100-fold
greater, or at least 1000-fold greater than the affinity of the
binding agent for unrelated amino acid sequences. The terms
"immunoreactive" and "preferentially binds" are used
interchangeably herein.
[0189] As used herein, the term "affinity" refers to the
equilibrium constant for the reversible binding of two agents and
is expressed as K.sub.D. Affinity of a binding protein to a ligand
such as affinity of an antibody for an epitope can be, for example,
from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to
about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar
(fM). As used herein, the term "avidity" refers to the resistance
of a complex of two or more agents to dissociation after dilution.
Apparent affinities can be determined by methods such as an enzyme
linked immunosorbent assay (ELISA) or any other technique familiar
to one of skill in the art. Avidities can be determined by methods
such as a Scatchard analysis or any other technique familiar to one
of skill in the art.
[0190] "Epitope" refers to that portion of an antigen or other
macromolecule capable of forming a binding interaction with the
variable region binding pocket of an antibody. Such binding
interactions can be manifested as an intermolecular contact with
one or more amino acid residues of one or more CDRs. Antigen
binding can involve, for example, a CDR3 or a CDR3 pair or, in some
cases, interactions of up to all six CDRs of the V.sub.H and
V.sub.L chains. An epitope can be a linear peptide sequence (i.e.,
"continuous") or can be composed of noncontiguous amino acid
sequences (i.e., "conformational" or "discontinuous"). An antibody
can recognize one or more amino acid sequences; therefore an
epitope can define more than one distinct amino acid sequence.
Epitopes recognized by antibodies can be determined by peptide
mapping and sequence analysis techniques well known to one of skill
in the art. Binding interactions are manifested as intermolecular
contacts with one or more amino acid residues of a CDR. TRC105 is a
chimeric antibody which is the same variable amino acid sequence as
the murine antibody described as Y4-2F1 or SN6j in U.S. Pat. Nos.
5,928,641; 6,200,566; 6,190,660; and 7,097,836. Epitopes recognized
by Y4-2F1 and SN6j, and thus TRC105, have been previously
identified.
[0191] The term "specific" refers to a situation in which an
antibody will not show any significant binding to molecules other
than the antigen containing the epitope recognized by the antibody.
The term is also applicable where for example, an antigen binding
domain is specific for a particular epitope which is carried by a
number of antigens, in which case the antibody or antigen-binding
fragment thereof carrying the antigen binding domain will be able
to bind to the various antigens carrying the epitope. The terms
"preferentially binds" or "specifically binds" mean that the
antibodies or fragments thereof bind to an epitope with greater
affinity than it binds unrelated amino acid sequences, and, if
cross-reactive to other polypeptides containing the epitope, are
not toxic at the levels at which they are formulated for
administration to human use. In one aspect, such affinity is at
least 1-fold greater, at least 2-fold greater, at least 3-fold
greater, at least 4-fold greater, at least 5-fold greater, at least
6-fold greater, at least 7-fold greater, at least 8-fold greater,
at least 9-fold greater, 10-fold greater, at least 20-fold greater,
at least 30-fold greater, at least 40-fold greater, at least
50-fold greater, at least 60-fold greater, at least 70-fold
greater, at least 80-fold greater, at least 90-fold greater, at
least 100-fold greater, or at least 1000-fold greater than the
affinity of the antibody or fragment thereof for unrelated amino
acid sequences. The terms "immunoreactive," "binds,"
"preferentially binds" and "specifically binds" are used
interchangeably herein. The term "binding" refers to a direct
association between two molecules, due to, for example, covalent,
electrostatic, hydrophobic, and ionic and/or hydrogen-bond
interactions under physiological conditions, and includes
interactions such as salt bridges and water bridges, as well as any
other conventional means of binding.
[0192] The phrase "conservative amino acid substitution" refers to
grouping of amino acids on the basis of certain common properties.
A functional way to define common properties between individual
amino acids is to analyze the normalized frequencies of amino acid
changes between corresponding proteins of homologous organisms
(Schulz, G. E. and R. H. Schirmer, Principles of Protein Structure,
Springer-Verlag). According to such analyses, groups of amino acids
may be defined where amino acids within a group exchange
preferentially with each other, and therefore resemble each other
most in their impact on the overall protein structure (Schulz, G.
E. and R. H. Schirmer, Principles of Protein Structure,
Springer-Verlag). Examples of amino acid groups defined in this
manner include: [0193] (i) a charged group, consisting of Glu and
Asp, Lys, Arg and His, [0194] (ii) a positively-charged group,
consisting of Lys, Arg and His, [0195] (iii) a negatively-charged
group, consisting of Glu and Asp, [0196] (iv) an aromatic group,
consisting of Phe, Tyr and Trp, [0197] (v) a nitrogen ring group,
consisting of His and Trp, [0198] (vi) a large aliphatic non-polar
group, consisting of Val, Leu and Ile, [0199] (vii) a
slightly-polar group, consisting of Met and Cys, [0200] (viii) a
small-residue group, consisting of Ser, Thr, Asp, Asn, Gly, Ala,
Glu, Gin and Pro, [0201] (ix) an aliphatic group consisting of Val,
Leu, Ile, Met and Cys, and [0202] (x) a small hydroxyl group
consisting of Ser and Thr.
[0203] In addition to the groups presented above, each amino acid
residue may form its own group, and the group formed by an
individual amino acid may be referred to simply by the one and/or
three letter abbreviation for that amino acid commonly used in the
art as described above.
[0204] A "conserved residue" is an amino acid that is relatively
invariant across a range of similar proteins. Often conserved
residues will vary only by being replaced with a similar amino
acid, as described above for "conservative amino acid
substitution."
[0205] The letter "x" or "xaa" as used in amino acid sequences
herein is intended to indicate that any of the twenty standard
amino acids may be placed at this position unless specifically
noted otherwise. For the purposes of peptidomimetic design, an "x"
or a "xaa" in an amino acid sequence may be replaced by a mimic of
the amino acid present in the target sequence, or the amino acid
may be replaced by a spacer of essentially any form that does not
interfere with the activity of the peptidomimetic.
[0206] "Homology" or "identity" or "similarity" refers to sequence
similarity between two peptides or between two nucleic acid
molecules. Homology and identity can each be determined by
comparing a position in each sequence which may be aligned for
purposes of comparison. When an equivalent position in the compared
sequences is occupied by the same base or amino acid, then the
molecules are identical at that position; when the equivalent site
occupied by the same or a similar amino acid residue (e.g., similar
in steric and/or electronic nature), then the molecules can be
referred to as homologous (similar) at that position. Expression as
a percentage of homology/similarity or identity refers to a
function of the number of identical or similar amino acids at
positions shared by the compared sequences. A sequence which is
"unrelated" or "non-homologous" shares less than 40% identity,
though preferably less than 25% identity with a sequence of the
present invention. In comparing two sequences, the absence of
residues (amino acids or nucleic acids) or presence of extra
residues also decreases the identity and homology/similarity.
[0207] The term "homology" describes a mathematically based
comparison of sequence similarities which is used to identify genes
or proteins with similar functions or motifs. The nucleic acid
(nucleotide, oligonucleotide) and amino acid (protein) sequences of
the present invention may be used as a "query sequence" to perform
a search against public databases to, for example, identify other
family members, related sequences or homologs. Such searches can be
performed using the NBLAST and XBLAST programs (version 2.0) of
Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide
searches can be performed with the NBLAST program, score=100,
wordlength=12 to obtain nucleotide sequences homologous to nucleic
acid molecules of the invention. BLAST amino acid searches can be
performed with the XBLAST program, score=50, wordlength=3 to obtain
amino acid sequences homologous to protein molecules of the
invention. To obtain gapped alignments for comparison purposes,
Gapped BLAST can be utilized as described in Altschul et al.,
(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST
and Gapped BLAST programs, the default parameters of the respective
programs (e.g., XBLAST and BLAST) can be used (see,
www.ncbi.nlm.nih.gov).
[0208] As used herein, "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. Computer program methods to determine identity between
two sequences include, but are not limited to, the GCG program
package (Devereux, J., et al., Nucleic Acids Research 12(1): 387
(1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J.
Molec. Biol. 215: 403-410 (1990) and Altschul et al. Nuc. Acids
Res. 25: 3389-3402 (1997)). The BLAST X program is publicly
available from NCBI and other sources (BLAST Manual, Altschul, S.,
et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J.
Mol. Biol. 215: 403-410 (1990). The well known Smith Waterman
algorithm may also be used to determine identity.
[0209] "Isolated" (used interchangeably with "substantially pure")
when applied to polypeptides means a polypeptide or a portion
thereof which, by virtue of its origin or manipulation: (i) is
present in a host cell as the expression product of a portion of an
expression vector; or (ii) is linked to a protein or other chemical
moiety other than that to which it is linked in nature; or (iii)
does not occur in nature, for example, a protein that is chemically
manipulated by appending, or adding at least one hydrophobic moiety
to the protein so that the protein is in a form not found in
nature. By "isolated" it is further meant a protein that is: (i)
synthesized chemically; or (ii) expressed in a host cell and
purified away from associated and contaminating proteins. The term
generally means a polypeptide that has been separated from other
proteins and nucleic acids with which it naturally occurs.
Preferably, the polypeptide is also separated from substances such
as antibodies or gel matrices (polyacrylamide) which are used to
purify it.
[0210] As used herein, the terms "angiogenesis inhibitory,"
"angiogenesis inhibiting" or "anti-angiogenic" include
vasculogenesis, and are intended to mean effecting a decrease in
the extent, amount, or rate of neovascularization. Effecting a
decrease in the extent, amount, or rate of endothelial cell
proliferation or migration in the tissue is a specific example of
inhibiting angiogenesis.
[0211] The term "angiogenesis inhibitory composition" refers to a
composition which inhibits angiogenesis-mediated processes such as
endothelial cell migration, proliferation, tube formation and
subsequently leading to the inhibition of the generation of new
blood vessels from existing ones, and consequently affects
angiogenesis-dependent conditions.
[0212] As used herein, the term "angiogenesis-dependent condition"
is intended to mean a condition where the process of angiogenesis
or vasculogenesis sustains or augments a pathological condition or
beneficially influences normal physiological processes. Therefore,
treatment of an angiogenesis-dependent condition in which
angiogenesis sustains a pathological condition could result in
mitigation of disease, while treatment of an angiogenesis-dependent
condition in which angiogenesis beneficially influences normal
physiological processes could result in, e.g., enhancement of a
normal process.
[0213] Angiogenesis is the formation of new blood vessels from
pre-existing capillaries or post-capillary venules. Vasculogenesis
results from the formation of new blood vessels arising from
angioblasts which are endothelial cell precursors. Both processes
result in new blood vessel formation and are included in the
meaning of the term angiogenesis-dependent conditions. The term
"angiogenesis" as used herein is intended to include de novo
formation of vessels such as that arising from vasculogenesis as
well as those arising from branching and sprouting of existing
vessels, capillaries and venules. Angiogenesis can also be
inclusive of induction of ALK1 signaling and related Smad 1/5/8
phosphorylation and/or signaling. CD105 is also known to be
involved in the ALK1 signaling pathway and is thus also included
within the meaning of angiogenesis.
[0214] "Inducing a host immune response" means that a patient
experiences alleviation or reduction of signs or symptoms of
illness, and specifically includes, without limitation,
prolongation of survival. In certain preferred embodiments of the
methods according to the invention, a CD8+ IFN-.gamma. producing T
cell is activated to induce a cytotoxic T lymphocyte (CTL) immune
response in the patient administered the antagonist. In certain
embodiments of the methods according to the invention, a CD4+
IFN-.gamma. producing T cell is activated to induce a helper T cell
immune response in the patient administered with the composition.
These activated CD4+ IFN-.gamma. producing T cells (i.e., helper T
cells) provide necessary immunological help (e.g., by release of
cytokines) to induce and maintain not only CTL, but also a humoral
immune response mediated by B cells. Thus, in certain embodiments
of the methods according to the invention, a humoral response to
the antigen is activated in the patient administered with the
composition. In one aspect, an adjuvant may be added to the
composition to increase an immune response. Adjuvants are
well-known in the art.
[0215] Activation of a CD8+ and/or CD4+ T cells means causing T
cells that have the ability to produce cytokines (e.g.,
IFN-.gamma.) to actually produce one or more cytokine(s), or to
increase their production of one or more cytokine(s). "Induction of
CTL response" means causing potentially cytotoxic T lymphocytes to
exhibit antigen specific cytotoxicity. "Antigen specific
cytotoxicity" means cytotoxicity against a cell presenting an
antigen that is associated with the antigen associated with the
cancer that is greater than an antigen that is not associated with
a cancer. "Cytotoxicity" refers to the ability of the cytotoxic T
lymphocyte to kill a target cell. Such antigen-specific
cytotoxicity can be at least about 3-fold, at least about 10-fold
greater, at least about 100-fold greater or more than cytotoxicity
against a cell not presenting the antigen not associated with the
cancer. Antibody dependent cell-mediated cytotoxicity (ADCC) also
includes activation of natural killer cells ("NK cells") which
mediate cell killing via antibody binding. The antibodies and
antigen-binding fragments described herein can mediate ADCC via NK
cells through the binding of endoglin.
[0216] As used herein, the terms "proliferative disorder" and
"proliferative condition" mean any pathological or non-pathological
physiological condition characterized by aberrant or undesirable
proliferation. The terms "cell proliferative disorder" and "cell
proliferative condition" mean any pathological or non-pathological
physiological condition characterized by aberrant or undesirable
cell proliferation, as well as including conditions characterized
by undesirable or unwanted cell proliferation or cell survival
(e.g., due to deficient apoptosis), conditions characterized by
deficient or aberrant or deficient apoptosis, as well as conditions
characterized by aberrant or undesirable or unwanted cell survival.
The term "differentiative disorder" means any pathological or
non-pathological physiological condition characterized by aberrant
or deficient differentiation.
[0217] Proliferative or differentiative disorders amenable to
treatment include diseases and non-pathological physiological
conditions, benign and neoplastic, characterized by abnormal or
undesirable cell numbers, cell growth or cell survival. Such
disorders or conditions may therefore constitute a disease state
and include all types of cancerous growths or oncogenic processes,
metastatic tissues or malignantly transformed cells, tissues, or
organs, or may be non-pathologic, i.e., a deviation from normal but
which is not typically associated with disease. A specific example
of a non-pathologic condition that may be treated in accordance
with the invention is tissue re-growth from wound repair that
results in scarring.
[0218] Cells comprising the proliferative or differentiative
disorder may be aggregated in a cell mass or be dispersed. The term
"solid tumor" refers to neoplasias or metastases that typically
aggregate together and form a mass. Particular examples include
visceral tumors such as gastric or colon cancer, hepatomas, venal
carcinomas, lung and brain tumors/cancers. A "non-solid tumor"
refers to neoplasias of the hematopoietic system, such as
lymphomas, myelomas and leukemias, or neoplasias that are diffuse
in nature, as they do not typically form a solid mass. Particular
examples of leukemias include for example, acute and chronic
lymphoblastic, myeloblastic and multiple myeloma.
[0219] Such disorders include neoplasms or cancers, which can
affect virtually any cell or tissue type, e.g., carcinoma, sarcoma,
melanoma, metastatic disorders or hematopoietic neoplastic
disorders. A metastatic tumor can arise from a multitude of primary
tumor types, including but not limited to breast, lung, thyroid,
head and neck, brain, lymphoid, gastrointestinal (mouth, esophagus,
stomach, small intestine, colon, rectum), genito-urinary tract
(uterus, ovary, cervix, bladder, testicle, penis, prostate),
kidney, pancreas, liver, bone, muscle, skin, etc.
[0220] Carcinomas refer to malignancies of epithelial or endocrine
tissue, and include respiratory system carcinomas, gastrointestinal
system carcinomas, genitourinary system carcinomas, testicular
carcinomas, breast carcinomas, prostatic carcinomas, endocrine
system carcinomas, and melanomas. Exemplary carcinomas include
those forming from the cervix, lung, prostate, breast, head and
neck, colon, liver and ovary. The term also includes
carcinosarcomas, e.g., which include malignant tumors composed of
carcinomatous and sarcomatous tissues. Adenocarcinoma includes a
carcinoma of a glandular tissue, or in which the tumor forms a
gland like structure.
[0221] An ocular tissue to be treated is, for example, a retinal
tissue of a patient with diabetic retinopathy, macular degeneration
or neovascular glaucoma and the angiogenesis to be inhibited is
retinal tissue angiogenesis where there is neovascularization of
retinal tissue.
[0222] A cancerous tissue to be treated is, for example, an
endothelial tissue expressing an abnormal level of endoglin. As
used herein, the term "transformed cells" refers to cells that have
spontaneously converted to a state of unrestrained growth, i.e.,
they have acquired the ability to grow through an indefinite number
of divisions in culture. Transformed cells may be characterized by
such terms as neoplastic, anaplastic and/or hyperplastic, with
respect to their loss of growth control. For purposes of this
invention, the terms "transformed phenotype of malignant mammalian
cells" and "transformed phenotype" are intended to encompass, but
not be limited to, any of the following phenotypic traits
associated with cellular transformation of mammalian cells:
immortalization, morphological or growth transformation, and
tumorigenicity, as detected by prolonged growth in cell culture,
growth in semi-solid media, or tumorigenic growth in
immuno-incompetent or syngeneic animals.
[0223] The term "tumor cell antigen" is defined herein as an
antigen that is present in higher quantities on a tumor cell or in
body fluids than unrelated tumor cells, normal cells, or in normal
body fluid. The antigen presence may be tested by any number of
assays known to those skilled in the art and include without
limitation negative and/or positive selection with antibodies, such
as an ELISA assay, a radioimmunoassay, or by Western Blot.
[0224] The terms "apoptosis" or "programmed cell death," refers to
the physiological process by which unwanted or useless cells are
eliminated during development and other normal biological
processes. Apoptosis is a mode of cell death that occurs under
normal physiological conditions and the cell is an active
participant in its own demise ("cellular suicide"). It is most
often found during normal cell turnover and tissue homeostasis,
embryogenesis, induction and maintenance of immune tolerance,
development of the nervous system and endocrine-dependent tissue
atrophy. Cells undergoing apoptosis show characteristic
morphological and biochemical features. These features include
chromatin aggregation, nuclear and cytoplasmic condensation,
partition of cytoplasm and nucleus into membrane bound vesicles
(apoptotic bodies), which contain ribosomes, morphologically intact
mitochondria and nuclear material. In vivo, these apoptotic bodies
are rapidly recognized and phagocytized by macrophages, dendritic
cells or adjacent epithelial cells. Due to this efficient mechanism
for the removal of apoptotic cells in vivo no inflammatory response
is elicited. In vitro, the apoptotic bodies as well as the
remaining cell fragments ultimately swell and finally lyse. This
terminal phase of in vitro cell death has been termed "secondary
necrosis." Apoptosis can be measured by methods known to those
skilled in the art like DNA fragmentation, exposure of Annexin V,
activation of caspases, release of cytochrome c, etc. A cell that
has been induced to die is termed herein as an "apoptotic
cell."
[0225] "Apoptosis inducing agent" is defined herein to induce
apoptosis/programmed cell death, and include, for example,
irradiation, chemotherapeutic agents or receptor ligation agents,
wherein cells, for example, tumor cells are induced to undergo
programmed cell death. Exemplary apoptosis inducing agents are
described in more detail below.
[0226] Apoptosis can be tested using a standard Annexin V Apoptosis
Assay: NIH:OVCAR-3 cells are grown in 6-well plates (NUNC) and
irradiated or treated with an antagonist (or in combination with
another anti-cancer drug) for 4-48 hours, washed and stained with
Annexin V-FITC (BD-Pharmingen) for 1 hour. Cells are analyzed by
flow cytometry (Becton-Dickinson, CellQuest), counterstained with
Propidium Iodide and analyzed again in the flow cytometer.
[0227] B. Methods of Making and Expressing Humanized Anti-Endoglin
Antibodies
[0228] A chimeric monoclonal antibody has been developed that binds
endoglin. This antibody is designated TR105 (also known as
c-SN6j).
[0229] In one aspect, the antibodies and antigen-binding fragments
thereof described herein were created by humanization of the
V.sub.L and V.sub.H sequences of the chimeric monoclonal TRC105
antibody (SEQ ID NOS. 1 and 39, respectively).
[0230] Humanized immunoglobulins, including humanized antibodies,
have been constructed by means of genetic engineering. Most
humanized immunoglobulins that have been previously described have
comprised a framework that is identical to the framework of a
particular human immunoglobulin chain (i.e., an acceptor or
recipient), and three CDRs from a non-human (i.e., donor)
immunoglobulin chain. As described herein, humanization can also
include criteria by which a limited number of amino acids in the
framework of a humanized immunoglobulin chain are identified and
chosen to be the same as the amino acids at those positions in the
donor rather than in the acceptor, in order to increase or maintain
the affinity of an antibody comprising the humanized immunoglobulin
chain.
[0231] The present invention is based in part on the model that two
contributing causes of the loss of affinity in prior means of
producing humanized antibodies (using as examples mouse antibodies
as the source of CDRs) are: (1) when the mouse CDRs are combined
with a human framework, the amino acids in the frameworks close to
the CDRs become human instead of mouse. Without intending to be
bound by theory, these changed amino acids may slightly distort the
CDRs (e.g., they may create different electrostatic or hydrophobic
forces than in the donor mouse antibody, and the distorted-CDRs may
not make as effective contacts with the antigen as the CDRs did in
the donor antibody); (2) also, amino acids in the original mouse
antibody that are close to, but not part of, the CDRs (i.e., still
part of the framework), may make contacts with the antigen that
contribute to affinity. These amino acids are lost when the
antibody is humanized because, generally, all framework amino acids
are made human. To circumvent these issues, and to produce
humanized antibodies that have a very strong affinity for a desired
antigen, humanized antibodies and antigen-binging fragments thereof
can be constructed using one or more of the following
principles.
[0232] One non-limiting principle is that, for example, as
acceptor, a framework is used from a particular human
immunoglobulin that is unusually homologous to the donor
immunoglobulin to be humanized, or use a consensus framework from
many human antibodies is used as an acceptor. For example,
comparison of the sequence of a mouse heavy (or light) chain
variable region against human heavy (or light) variable regions in
a data bank (for example, the National Biomedical Research
Foundation Protein Identification Resource or the protein sequence
database of the National Center for Biotechnology
Information--NCBI) shows that the extent of homology to different
human regions can vary greatly, for example from about 40% to about
60%, about 70%, about 80% or higher. By choosing as the acceptor
immunoglobulin one of the human heavy chain variable regions that
is most homologous to the heavy chain variable region of the donor
immunoglobulin, fewer amino acids will be changed in going from the
donor immunoglobulin to the humanized immunoglobulin. By choosing
as the acceptor immunoglobulin one of the human light chain
variable regions that is most homologous to the light chain
variable region of the donor immunoglobulin, fewer amino acids will
be changed in going from the donor immunoglobulin to the humanized
immunoglobulin. Generally, using such techniques, there is a
reduced chance of changing an amino acid near one or more of the
CDRs that distorts their conformation. Moreover, the precise
overall shape of a humanized antibody comprising the humanized
immunoglobulin chain may more closely resemble the shape of the
donor antibody, thereby also reducing the chance of distorting the
CDRs.
[0233] One can also use light and heavy chains from the same human
antibody as acceptor sequences, to improve the likelihood that the
humanized light and heavy chains will make favorable contacts with
each other. Alternatively, one can also use light and heavy chains
from different human antibody germline sequences as acceptor
sequences; when such combinations are used, one can readily
determine whether the V.sub.H and V.sub.L bind an epitope of
interest using conventional assays (e.g., an ELISA). In one
example, the human antibody will be chosen in which the light and
heavy chain variable regions sequences, taken together, are overall
most homologous to the donor light and heavy chain variable region
sequences. Sometimes greater weight will be given to the heavy
chain sequence. Regardless of how the acceptor immunoglobulin is
chosen, higher affinity can, in some cases, be achieved by
selecting a small number of amino acids in the framework of the
humanized immunoglobulin chain to be the same as the amino acids at
those positions in the donor rather than in the acceptor. Methods
of affinity maturation are known in the art.
[0234] Humanized antibodies generally have at least three potential
advantages over mouse or chimeric antibodies for use in human
therapy. Because the effector portion of an antibody is human, it
is believed to interact better with the other parts of the human
immune system (e.g., destroy the target cells more efficiently by
complement-dependent cytotoxicity (CDC) or antibody-dependent
cellular cytotoxicity (ADCC)). Additionally, the human immune
system should not recognize the framework or constant region of the
humanized antibody as foreign, and therefore the antibody response
against such an injected antibody should be less than against a
totally foreign mouse antibody or a partially foreign chimeric
antibody. Finally, mouse antibodies are known to have a half-life
in the human circulation that is much shorter than the half-life of
human antibodies. Humanized antibodies can, presumably, have a
half-life more similar to naturally-occurring human antibodies,
allowing smaller and less frequent doses to be given.
[0235] Humanization of antibodies and antigen-binding fragments
thereof, can be accomplished via a variety of methods known in the
art and described herein. Similarly, production of humanized
antibodies can also be accomplished via methods known in the art
and described herein.
[0236] Methods for modifications of framework regions are known in
the art and are contemplated herein. Selection of one or more
relevant framework amino acid positions to altered depends on a
variety of criteria. One criterion for selecting relevant framework
amino acids to change can be the relative differences in amino acid
framework residues between the donor and acceptor molecules.
Selection of relevant framework positions to alter using this
approach has the advantage of avoiding any subjective bias in
residue determination or any bias in CDR binding affinity
contribution by the residue.
[0237] Another criterion that can be used for determining the
relevant amino acid positions to change can be, for example,
selection of framework residues that are known to be important or
to contribute to CDR conformation. For example, canonical framework
residues are important for CDR conformation and/or structure.
Targeting of a canonical framework residue as a relevant position
to change can be used to identify a more compatible amino acid
residue in context with its associated donor CDR sequence.
[0238] The frequency of an amino acid residue at a particular
framework position is another criterion which can be used for
selecting relevant framework amino acid positions to change. For
example, comparison of the selected framework with other framework
sequences within its subfamily can reveal residues that occur at
minor frequencies at a particular position or positions. Positions
harboring less abundant residues are similarly applicable for
selection as a position to alter in the acceptor variable region
framework.
[0239] The relevant amino acid positions to change also can be
selected, for example, based on proximity to a CDR. In certain
contexts, FR residues can participate in CDR conformation and/or
antigen binding. Moreover, this criterion can similarly be used to
prioritize relevant positions selected by other criteria described
herein. Therefore, differentiating between residues proximal and
distal to one or more CDRs represents one way to reduce the number
of relevant positions to change.
[0240] Other criteria for selecting relevant amino acid framework
positions to alter include, for example, residues that are known or
predicted to reside in a three dimensional space near the
antigen-CDR interface or predicted to modulate CDR activity.
Similarly, framework residues that are known to, or predicted to,
form contacts between the heavy (V.sub.H) and light (V.sub.L) chain
variable region interface can be selected. Such framework positions
can affect the conformation and/or affinity of a CDR by modulating
the CDR binding pocket, antigen (epitope) interaction or the
V.sub.H and V.sub.L interaction. Therefore, selection of these
amino acid positions for constructing a diverse population for
screening of binding activity can be used to identify framework
changes which replace residues having detrimental effects on CDR
conformation or compensate for detrimental effects of residues
occurring elsewhere in the framework.
[0241] Other framework residues that can be selected for alteration
include amino acid positions that are inaccessible to solvent. Such
residues are generally buried in the variable region and are,
therefore, capable of influencing the conformation of the CDR or
V.sub.H and V.sub.L interactions. Solvent accessibility can be
predicted, for example, from the relative hydrophobicity of the
environment created by the amino acid side chains of the
polypeptide and/or by known three-dimensional structural data.
[0242] Following selection of relevant amino acid positions in the
donor CDRs, as well as any relevant amino acid positions in the
framework regions desired to be varied, amino acid changes at some
or all of the selected positions can be incorporated into encoding
nucleic acids for the acceptor variable region framework and donor
CDRs. Altered framework or CDR sequences can be individually made
and tested, or can be sequentially or simultaneously combined and
tested.
[0243] The variability at any or all of the altered positions can
range from a few to a plurality of different amino acid residues,
including all twenty naturally occurring amino acids or functional
equivalents and analogues thereof. In some cases, non-naturally
occurring amino acids may also be considered and are known in the
art.
[0244] Selection of the number and location of the amino acid
positions to vary is flexible and can depend on the intended use
and desired efficiency for identification of the altered variable
region having a desirable activity such as substantially the same
or greater binding affinity compared to the donor variable region.
In this regard, the greater the number of changes that are
incorporated into an altered variable region population, the more
efficient it is to identify at least one species that exhibits a
desirable activity, for example, substantially the same or greater
binding affinity as the donor. Alternatively, where the user has
empirical or actual data to the affect that certain amino acid
residues or positions contribute disproportionally to binding
affinity, then it can be desirable to produce a limited population
of altered variable regions which focuses on changes within or
around those identified residues or positions.
[0245] For example, if CDR grafted variable regions are desired, a
large, diverse population of altered variable regions can include
all the non-identical framework region positions between the donor
and acceptor framework and all single CDR amino acid position
changes. Alternatively, a population of intermediate diversity can
include subsets, for example, of only the proximal non-identical
framework positions to be incorporated together with all single CDR
amino acid position changes to, for example, increase affinity of
the humanized antibodies or antigen binding fragments. The
diversity of the above populations can be further increased by, for
example, additionally including all pair-wise CDR amino acid
position changes. In contrast, populations focusing on
predetermined residues or positions which incorporate variant
residues at as few as one framework and/or one CDR amino acid
position can similarly be constructed for screening and
identification of an altered antibody variable region. As with the
above populations, the diversity of such focused populations can be
further increased by additionally expanding the positions selected
for change to include other relevant positions in either or both of
the framework and CDR regions. There are numerous other
combinations ranging from few changes to many changes in either or
both of the framework regions and CDRs that can additionally be
employed, all of which will result in a population of altered
variable regions that can be screened for the identification of at
least one CDR grafted altered variable region having desired
activity, for example, binding activity to endoglin. Those skilled
in the art will know, or can determine, which selected residue
positions in the framework or donor CDRs, or subsets thereof, can
be varied to produce a population for screening and identification
of an altered antibody of the invention given the teachings and
guidance provided herein. Codons encoding amino acids are known in
the art.
[0246] Another method of humanizing antibodies includes a method
termed "superhumanization." Superhumanization involves the steps of
obtaining a peptide sequence for a subject variable region encoded
by a non-human mature antibody gene and identifying a first set of
canonical CDR structure types for at least two CDRs within the
non-human antibody variable region. Canonical CDR structure types
are the structure types designated by Chothia (CITE). Chothia and
coworkers found that critical portions of the CDRs of many
antibodies adopt nearly identical peptide backbone conformations,
despite great diversity at the level of amino acid sequence.
Accordingly, Chothia defined for each CDR in each chain one or a
few "canonical structures." Each canonical structure specifies
primarily a set of peptide backbone torsion angles for a contiguous
segment of amino acid residues forming a loop.
[0247] After the identification of the canonical CDR structure
type, a library of peptide sequences for human antibody variable
regions for human antibodies is also obtained. This library
contains sequences for human germline variable regions as encoded
by germline nucleic acid segments, and may include mature human
antibody sequences. In either case, the method includes identifying
canonical CDR structure types (i.e., a second set of canonical CDR
structure types) for at least two CDRs for each sequence within the
library of human variable region sequences. From this library there
is selected a subset of candidate sequences by comparing the first
set of canonical CDR structure types to the second set of canonical
CDR structure types (i.e., comparing the mouse canonical CDR
structure types to the human canonical CDR structure types at
corresponding locations within the variable region) and selecting
those human sequences where the second set of canonical CDR
structure is the same as the first set of canonical CDR structure
types for the CDR sequences at corresponding locations within the
non-human and human variable regions, respectively. The method uses
these candidate human variable region sequences as a basis for
constructing a chimeric molecule that includes at least two of the
CDR sequences from the non-human variable region (e.g., of the
mouse CDRs) combined with the framework regions from candidate
human variable region sequences. The result of the construction is
that the chimeric antibody contains each of the non-human CDR
sequences substituted for each of the human CDR sequences at
corresponding locations in the variable regions so that the
framework sequences in the chimeric antibody differs from the
candidate human framework sequences.
[0248] The similarity to the subject CDRs of candidate human
antibody sequences is assessed for each domain at two levels.
Primarily, identical three-dimensional conformations of CDR peptide
backbones are sought. Experimentally determined atomic coordinates
of the subject CDRs are seldom available, hence three-dimensional
similarity is approximated by determining Chothia canonical
structure types of the subject CDRs and excluding from further
consideration candidates possessing different canonical structures.
Secondarily, residue-to-residue homology between subject CDRs and
the remaining human candidate CDRs is considered, and the candidate
with the highest homology is chosen.
[0249] Choosing highest homology is based on various criterion used
to rank candidate human variable regions having the same canonical
structure as the subject the non-human variable regions. The
criterion for ranking members of the selected set may be by amino
acid sequence identity or amino acid homology or both. Amino acid
identity is simple a score of position by position matches of amino
acid residues. Similarity by amino acid homology is position by
position similarity in residue structure of character. Homology may
be scored, for example, according to the tables and procedures
described by Henikoff and Henikoff, (1992) Amino acid substitution
matrices from protein blocks, Proc. Natl. Acad. Sci 89:
10915-10919, or by the BLOSUM series described by Henikoff and
Henikoff, (1996). The steps are as follows: [0250] a) Determine the
peptide sequences of the heavy and light chain variable domains of
the subject antibody. These can be determined by any of several
methods, such as DNA sequencing of the respective genes after
conventional cDNA cloning; DNA sequencing of cloning products that
have been amplified by the polymerase chain reaction from reverse
transcripts or DNA of the subject hybridoma line; or peptide
sequencing of a purified antibody protein. [0251] b) Apply the
Kabat numbering system (Kabat et al., Id. 1991) to the heavy and
light chain sequences of the subject non-human antibody. Determine
canonical structure types for each of the CDRs of the subject
non-human antibody. This determination is made from examination of
the peptide sequence in light of the guidelines discussed in
Chothia and Lesk (1987), Chothia et al (1992), Tomlinson et al
(1995), Martin and Thornton (1996), and Al-Lazikani et al
(1997).
[0252] The salient features of canonical structure determination
for each of the CDRs are as follows. For heavy chain CDR1, three
canonical structure types are currently known. Assignment of a new
sequence is straightforward because each canonical structure type
has a different number of residues. As described in Al-Lazikani et.
al (1997), when Kabat numbering is assigned to the sequence, the
numbering for residues 31-35 will be as follows for the respective
canonical structures.
[0253] Canonical structure type 1: 31, 32, 33, 34, 35.
[0254] Canonical structure type 2: 31, 32, 33, 34, 35, 35a.
[0255] Canonical structure type 3: 31, 32, 33, 34, 35, 35a,
35b.
[0256] For heavy chain CDR2, four canonical structure types are
currently known. Several have unique numbers of residues, and are
easily distinguished from their unique Kabat numbering of positions
52-56, viz.:
[0257] Canonical structure type 1: 52, 53, 54, 55, 56.
[0258] Canonical structure type 4: 52, 52a, 52b, 52c, 53, 54, 55,
56.
[0259] Canonical structure types 2 and 3 for heavy chain CDR2 have
equal numbers of residues, hence must be distinguished by clues
within their sequence, as discussed by Chothia et al (1992). The
Kabat numbering of the segment containing these clues is: 52, 52a,
53, 54, 55. Canonical structure type 2 has Pro or Ser at position
52a and Gly or Ser at position 55, with no restriction at the other
positions. Canonical structure type 3 has Gly, Ser, Asn, or Asp at
position 54, with no restriction at the other positions. These
criteria are sufficient to resolve the correct assignment in most
cases. Additionally, framework residue 71 is commonly Ala, Val,
Leu, Ile, or Thr for canonical structure type 2 and commonly Arg
for canonical structure type 3.
[0260] Heavy chain CDR3 is the most diverse of all the CDRs. It is
generated by genetic processes, some of a random nature, unique to
lymphocytes. Consequently, canonical structures for CDR3 have been
difficult to predict. In any case, human germline V gene segments
do not encode any part of CDR3; because the V gene segments end at
Kabat position 94, whereas positions 95 to 102 encode CDR3. For
these reasons, canonical structures of CDR3 are generally not
considered for choosing candidate human sequences.
[0261] For light chain CDR1, six canonical structure types are
currently known for CDR1 in kappa chains. Each canonical structure
type has a different number of residues, hence assignment of a
canonical structure type to a new sequence is apparent from the
Kabat numbering of residue positions 27-31.
[0262] Canonical structure type 1: 27, 29, 30, 31.
[0263] Canonical structure type 2: 27, 28, 29, 30, 31.
[0264] Canonical structure type 3: 27, 27a, 27b, 27c, 27d, 27e,
27f, 28, 29, 30, 31.
[0265] Canonical structure type 4: 27, 27a, 27b, 27c, 27d, 27e, 28,
29, 30, 31.
[0266] Canonical structure type 5: 27, 27a, 27b, 27c, 27d, 28, 29,
30, 31.
[0267] Canonical structure type 6: 27, 27a, 28, 29, 30, 31.
[0268] For light chain CDR2, only a single canonical structure type
is known for CDR2 in kappa chains, hence, barring exceptional
subject antibody sequences, assignment is automatic. For light
chain CDR3, up to six canonical structure types have been described
for CDR3 in kappa chains, but three of these are rare. The three
common ones can be distinguished by their length, reflected in
Kabat numbering of residue positions 91-97:
[0269] Canonical structure type 1: 91, 92, 93, 94, 95, 96, 97 (also
with an obligatory Pro at position 95 and Gln, Asn, or His at
position 90).
[0270] Canonical structure type 3: 91, 92, 93, 94, 95, 97.
[0271] Canonical structure type 5: 91, 92, 93, 94, 95, 96, 96a,
97.
[0272] After identifying the canonical CDR structure types of the
subject non-human antibody, human genes of the same chain type
(heavy or light) that have the same combination of canonical
structure types as the subject antibody are identified to form a
candidate set of human sequences. Most of these gene fragments have
been discovered and have already been assigned to a canonical
structure type (Chothia et al., 1992, Tomlinson et al., 1995).
[0273] For the heavy chain, conformity of CDR1 and CDR2 to the
mouse canonical structure types is assessed, and genes that do not
conform are excluded. For the light chain, conformity of CDR1 and
CDR2 of each human sequence to the canonical structure types of the
subject antibody is first assessed. The potential of residues 89-95
of a candidate Vk gene to form a CDR3 of the same canonical
structure type as the subject antibody is assessed, by positing a
fusion of the gene with a J region and applying criteria for CDR3
canonical CDR structure type determination to the fused sequence,
and non conforming sequences are excluded.
[0274] Alternatively, when a variable domain of the subject
antibody is of a canonical structure type not available in the
human genome, human germline V genes that have three-dimensionally
similar, but not identical, canonical structure types are
considered for comparison. Such a circumstance often occurs with
kappa chain CDR1 in murine antibodies, including two of the
examples described below. All 6 possible canonical structure types
have been observed at this CDR in murine antibodies, whereas the
human genome encodes only canonical types 2, 3, 4 and 6. In these
circumstances, a canonical CDR structure type having length of
amino acid residues within two of the length of the amino acid
residues of the subject non-human sequence may selected for the
comparison. For example, where a type 1 canonical structure is
found in the subject antibody, human Vk sequences with canonical
structure type 2 are used for comparison. Where a type 5 canonical
structure is found in the murine antibody, human Vk sequences with
either canonical structure type 3 or 4 are be used for
comparison.
[0275] Mature, rearranged human antibody sequences can be
considered for the sequence comparison. Such consideration might be
warranted under a variety of circumstances, including but not
limited to instances where the mature human sequence (1) is very
close to germline; (2) is known not to be immunogenic in humans; or
(3) contains a canonical structure type identical to that of the
subject antibody, but not found in the human germline.
[0276] For each of the candidate V genes with matching canonical
structure types, residue to residue sequence identity and/or
homology with the subject sequence is also evaluated to rank the
candidate human sequences. For example, the residues evaluated are
as follows: (1) Kappa (K) light chain CDR amino acid residue
positions are CDR1 (26-32), CDR2 (50-52), CDR3 (91-96); and (2)
heavy chain CDR amino acid residue positions are CDR1 (31-35) and
CDR2 (50-60). Additionally, heavy chain CDR3 amino acid residue
positions 95 to 102 can also be considered.
[0277] Residue-to-residue homology is first scored by the number of
identical amino acid residues between the subject and the candidate
human sequences. The human sequence used for subsequent
construction of a converted antibody is chosen from among the 25
percent of candidates with the highest score. When appropriate,
such as when several candidate sequences have similar identity
scores, similarity between non-identical amino acid residues may be
additionally considered as needed. Aliphatic-with-aliphatic,
aromatic-with-aromatic, or polar-with-polar matches between subject
and object residues are added to the scores. In another example,
quantitative evaluation of sequence homology may be performed using
amino acid substitution matrices such as the BLOSUM62 matrix of
Henikoff and Henikoff.
[0278] An object sequence for the framework region C-terminal to
CDR3 sequence can be selected from the set of known human germline
J segments. A J peptide sequence is selected by evaluating residue
to residue homology for each J segment for sequence positions for
which CDR3 and J overlap, using the scoring criteria specified for
the evaluation of candidate V genes as mentioned above. The J gene
segment peptide sequence used for subsequent construction of a
converted antibody is chosen from among the 25 percent of
candidates with the highest score.
[0279] As an example, the chimeric variable chain contains at least
two CDRs from a subject non-human sequence, and framework sequences
from a candidate human sequence. In another example, chimeric light
chain contains three CDRs from a subject non-human sequence and
framework sequences from a candidate human sequence. In additional
examples, a chimeric heavy chain contains at least two CDRs of a
subject heavy chain, and framework sequence of a candidate human
heavy chain, or a chimeric heavy chin contains each of the CDRs
from the subject heavy chain and framework sequences of a candidate
human heavy chain. In still another example, a chimeric antibody
heavy chain contains CDRs 1 and 2 from a subject non-human sequence
and residues 50-60 for CDR3 and residues 61-65 of a CDR from the
candidate human heavy chain, along with the framework sequences of
the candidate human sequence. In another example, a chimeric heavy
chain sequence contains each CDR from the subject non-human
sequence; frameworks sequences 27-30 form the subject sequence, and
the framework sequences from the candidate sequences. In all cases
however, the chimeric antibody molecule contains no more than 10
amino acid residues in the framework sequence that differ from
those in the framework sequence of the candidate human variable
ration.
[0280] When increased affinity of a humanized antibody is desired,
residues within the CDRs of a converted antibody may be
additionally substituted with other amino acids. Typically, no more
than four amino acid residues in a CDR are changed, and most
typically no more than two residues in the CDR will be changed,
except for heavy chain CDR2, where as many as 10 residues may be
changed. Changes in affinity can be measured by conventional
methods such as those described herein (e.g., Biacore).
[0281] The methods of superhumanizing antibodies are described in
more detail in U.S. Pat. No. 6,881,557 which is hereby incorporated
by reference in its entirety.
[0282] Humanized antibodies and antigen-binding fragments can be
constructed and produced using conventional techniques known in the
art. In addition, recombinantly prepared antibodies can often be
produced in large quantities, particularly when utilizing high
level expression vectors.
[0283] Antibodies can be sequenced using conventional techniques
known in the art. In one aspect, the amino acid sequences of one or
more of the CDRs is inserted into a synthetic sequence of, for
example, a human antibody (or antigen-binding fragment thereof)
framework to create a human antibody that could limit adverse side
reactions of treating a human patient with a non-human antibody.
The amino acid sequences of one or more of the CDRs can also be
inserted into a synthetic sequence of, for example, into a binding
protein such as an AVIMER.RTM. to create a construct for
administration to a human patient. Such techniques can be modified
depending on the species of animal to be treated. For example, for
veterinary uses, an antibody, antigen-binding fragment or binding
protein can be synthesized for administration of a non-human (e.g.,
a primate, a cow, a horse, etc.).
[0284] In another aspect, using art-recognized techniques such as
those provided and incorporated herein, nucleotides encoding amino
acid sequences of one or more of the CDRs can inserted, for
example, by recombinant techniques in restriction endonuclease
sites of an existing polynucleotide that encodes an antibody,
antigen-binding fragment or binding protein.
[0285] For expression, an expression system is one which utilizes
the GS system (Lonza) using a glutamine synthetase gene as the
selectable marker. Briefly, a transfection is performed in CHO
cells by electroporation (250V) using the GS system (Lonza) using
the glutamine synthetase gene as the selectable marker. Wild type
CHO cells are grown in DMEM (Sigma) containing 10% dialyzed Fetal
Calf Serum (FCS) with 2 mM glutamine. 6.times.10.sup.7 CHO cells
are transfected with 300 .mu.g of linearized DNA by
electroporation. After electroporation the cells are resuspended in
DMEM with glutamine and plated out into 36.times.96-well plates (50
.mu.l/well), and incubated at 37.degree. C. in 5% CO.sub.2. The
following day, 150 .mu.l/well of selective medium (DMEM without
glutamine) is added. After approximately 3 weeks the colonies are
screened by ELISA (see below) using an irrelevant antibody as a
negative control. All colonies producing >20 .mu.g/ml are
expanded into 24-well plates and then into duplicate T25
flasks.
[0286] For high level production, the most widely used mammalian
expression system is one which utilizes the gene amplification
procedure offered by dihydrofolate reductase deficient ("dhfr-")
Chinese hamster ovary cells. The system is well known to the
skilled artisan. The system is based upon the dehydrofolate
reductase "dhfr" gene, which encodes the DHFR enzyme, which
catalyzes conversion of dehydrofolate to tetrahydrofolate. In order
to achieve high production, dhfr- CHO cells are transfected with an
expression vector containing a functional DHFR gene, together with
a gene that encodes a desired protein. In this case, the desired
protein is recombinant antibody heavy chain and/or light chain.
[0287] By increasing the amount of the competitive DHFR inhibitor
methotrexate (MTX), the recombinant cells develop resistance by
amplifying the dhfr gene. In standard cases, the amplification unit
employed is much larger than the size of the dhfr gene, and as a
result the antibody heavy chain is co-amplified.
[0288] When large scale production of the protein, such as the
antibody chain, is desired, both the expression level and the
stability of the cells being employed are taken into account. In
long term culture, recombinant CHO cell populations lose
homogeneity with respect to their specific antibody productivity
during amplification, even though they derive from a single,
parental clone.
[0289] The present application provides an isolated polynucleotide
(nucleic acid) encoding an antibody or antigen-binding fragment as
described herein, vectors containing such polynucleotides, and host
cells and expression systems for transcribing and translating such
polynucleotides into polypeptides.
[0290] The present application also provides constructs in the form
of plasmids, vectors, transcription or expression cassettes which
comprise at least one polynucleotide as above.
[0291] The present application also provides a recombinant host
cell which comprises one or more constructs as above. A nucleic
acid encoding any antibody or antigen-binding fragments thereof
described herein as provided itself forms an aspect of the present
application, as does a method of production of the antibody or
antigen-binding fragments thereof described herein which method
comprises expression from encoding nucleic acid therefrom.
Expression can conveniently be achieved by culturing under
appropriate conditions recombinant host cells containing the
nucleic acid. Following production by expression, an antibody or
antigen-binding fragment can be isolated and/or purified using any
suitable technique, then used as appropriate.
[0292] Specific antibodies, antigen-binding fragments, and encoding
nucleic acid molecules and vectors described herein can be provided
isolated and/or purified, e.g., from their natural environment, in
substantially pure or homogeneous form. In the case of nucleic
acid, free or substantially free of nucleic acid or genes origin
other than the sequence encoding a polypeptide with the required
function. Nucleic acid can comprise DNA or RNA and can be wholly or
partially synthetic. Methods of purification are well known in the
art.
[0293] Systems for cloning and expression of a polypeptide in a
variety of different host cells are well known. Suitable host cells
include bacteria, mammalian cells, yeast and baculovirus systems.
Mammalian cell lines available in the art for expression of a
heterologous polypeptide include Chinese hamster ovary cells, HeLa
cells, baby hamster kidney cells, NS0 mouse myeloma cells and many
others. A common bacterial host is E. coli.
[0294] The expression of antibodies and antibody fragments in
prokaryotic cells such as E. coli is well established in the art.
For a review, see for example Pluckthun, A. Bio/Technology 9:
545-551 (1991). Expression in eukaryotic cells in culture is also
available to those skilled in the art as an option for production
of the antibodies and antigen-binding fragments described herein,
see for recent reviews, for example Raff, M. E. (1993) Curr.
Opinion Biotech. 4: 573-576; Trill J. J. et al. (1995) Curr.
Opinion Biotech 6: 553-560, each of which is which is incorporated
herein by reference in its entirety.
[0295] Suitable vectors can be chosen or constructed, containing
appropriate regulatory sequences, including promoter sequences,
terminator sequences, polyadenylation sequences, enhancer
sequences, marker genes and other sequences as appropriate. Vectors
can be plasmids, viral e.g., `phage, or phagemid, as appropriate.
For further details see, for example, Molecular Cloning: a
Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring
Harbor Laboratory Press. Many known techniques and protocols for
manipulation of nucleic acid, for example in preparation of nucleic
acid constructs, mutagenesis, sequencing, introduction of DNA into
cells and gene expression, and analysis of proteins, are described
in detail in Short Protocols in Molecular Biology, Second Edition,
Ausubel et al. eds., John Wiley & Sons, 1992. The methods
disclosures of Sambrook et al. and Ausubel et al. are incorporated
herein by reference in their entirety and are well known in the
art.
[0296] Thus, a further aspect provides a host cell containing
nucleic acid as disclosed herein. A still further aspect provides a
method comprising introducing such nucleic acid into a host cell.
The introduction can employ any available technique. For eukaryotic
cells, suitable techniques can include, for example, calcium
phosphate transfection, DEAE Dextran, electroporation,
liposome-mediated transfection and transduction using retrovirus or
other virus, e.g., vaccinia or, for insect cells, baculovirus. For
bacterial cells, suitable techniques can include, for example,
calcium chloride transformation, electroporation and transfection
using bacteriophage.
[0297] The introduction can be followed by causing or allowing
expression from the nucleic acid, e.g., by culturing host cells
under conditions for expression of the gene.
[0298] In one embodiment, the nucleic acid is integrated into the
genome (e.g., chromosome) of the host cell. Integration can be
promoted by inclusion of sequences which promote recombination with
the genome, in accordance with standard techniques. Ig enhances can
be initialized as needed to maximize expression.
[0299] The present application also provides a method which
comprises using a construct as stated above in an expression system
in order to express the antibodies or antigen-binding fragments
thereof as above.
[0300] The present application also relates to isolated nucleic
acids, such as recombinant DNA molecules or cloned genes, or
degenerate variants thereof, mutants, analogs, or fragments
thereof, which encode an antibody or antigen-binding sequence
described herein that binds endoglin.
[0301] In one aspect, the present application provides a nucleic
acid which codes for an antibody or antigen-binding fragment
thereof as described herein which binds endoglin.
[0302] In a further embodiment, the full DNA sequence of the
recombinant DNA molecule or cloned gene of an antibody or
antigen-binding fragment described herein can be operatively linked
to an expression control sequence which can be introduced into an
appropriate host. The application accordingly extends to
unicellular hosts transformed with the cloned gene or recombinant
DNA molecule comprising a DNA sequence encoding the V.sub.H and/or
V.sub.L, or portions thereof, of the antibody.
[0303] Another feature is the expression of the DNA sequences
disclosed herein. As is well known in the art, DNA sequences can be
expressed by operatively linking them to an expression control
sequence in an appropriate expression vector and employing that
expression vector to transform an appropriate unicellular host.
[0304] Such operative linking of a DNA sequence to an expression
control sequence, of course, includes, if not already part of the
DNA sequence, the provision of an initiation codon, ATG, in the
correct reading frame upstream of the DNA sequence.
[0305] Polynucleotides and vectors can be provided in an isolated
and/or a purified form (e.g., free or substantially free of
polynucleotides of origin other than the polynucleotide encoding a
polypeptide with the required function). As used herein,
"substantially pure," and "substantially free" refer to a solution
or suspension containing less than, for example, about 20% or less
extraneous material, about 10% or less extraneous material, about
5% or less extraneous material, about 4% or less extraneous
material, about 3% or less extraneous material, about 2% or less
extraneous material, or about 1% or less extraneous material.
[0306] A wide variety of host/expression vector combinations can be
employed in expressing the DNA sequences of this invention. Useful
expression vectors, for example, can consist of segments of
chromosomal, non-chromosomal and synthetic DNA sequences. Suitable
vectors include, but are not limited to, derivatives of SV40 and
known bacterial plasmids, e.g., E. coli plasmids col E1, Pcr1,
Pbr322, Pmb9 and their derivatives, plasmids such as RP4; phage
DNAs, e.g., the numerous derivatives of phage .lamda., e.g., NM989,
and other phage DNA, e.g., M13 and filamentous single stranded
phage DNA; yeast plasmids such as the 2u plasmid or derivatives
thereof; vectors useful in eukaryotic cells, such as vectors useful
in insect or mammalian cells; vectors derived from combinations of
plasmids and phage DNAs, such as plasmids that have been modified
to employ phage DNA or other expression control sequences; and the
like.
[0307] Also provided herein is a recombinant host cell which
comprises one or more polynucleotide constructs. A polynucleotide
encoding an antibody or antigen-binding fragment as provided herein
forms an aspect of the present application, as does a method of
production of the antibody or antigen-binding fragment which method
comprises expression from the polynucleotide. Expression can be
achieved, for example, by culturing under appropriate conditions
recombinant host cells containing the polynucleotide. An antibody
or antigen-binding fragment can then be isolated and/or purified
using any suitable technique, and used as appropriate.
[0308] Any of a wide variety of expression control
sequences--sequences that control the expression of a DNA sequence
operatively linked to it--can be used in these vectors to express
the DNA sequences. Such useful expression control sequences
include, for example, the early or late promoters of SV40, CMV,
vaccinia, polyoma or adenovirus, the lac system, the trp system,
the TAC system, the TRC system, the LTR system, the major operator
and promoter regions of phage .lamda., the control regions of fd
coat protein, the promoter for 3-phosphoglycerate kinase or other
glycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5),
the promoters of the yeast .alpha.-mating factors, and other
sequences known to control the expression of genes of prokaryotic
or eukaryotic cells or their viruses, and various combinations
thereof.
[0309] Systems for cloning and expression of a polypeptide in a
variety of different host cells are well known. Suitable host cells
include bacteria, mammalian cells, yeast and baculovirus systems.
Mammalian cell lines available in the art for expression of a
heterologous polypeptide include Chinese hamster ovary (CHO) cells,
HeLa cells, baby hamster kidney cells, NS0 mouse myeloma cells and
many others. A common, bacterial host can be, for example, E.
coli.
[0310] The expression of antibodies or antigen-binding fragments in
prokaryotic cells, such as E. coli, is well established in the art.
For a review, see for example Pluckthun, A. Bio/Technology 9:
545-551 (1991). Expression in eukaryotic cells in culture is also
available to those skilled in the art (Raff, M. E. (1993) Curr.
Opinion Biotech. 4: 573-576; Trill J. J. et al. (1995) Curr.
Opinion Biotech 6: 553-560).
[0311] A wide variety of unicellular host cells are also useful in
expressing the DNA sequences. These hosts include well-known
eukaryotic and prokaryotic hosts, such as strains of E. coli,
Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and
animal cells, such as CHO, YB/20, NS0, SP2/0, R1.1, B-W and L-M
cells, African Green Monkey kidney cells (e.g., COS 1, COS 7, BSC1,
BSC40, and BMT10), insect cells (e.g., Sf9), and human cells and
plant cells in tissue culture.
[0312] It will be understood that not all vectors, expression
control sequences and hosts will function equally well to express
the DNA sequences. Neither will all hosts function equally well
with the same expression system. However, one skilled in the art
will be able to select the proper vectors, expression control
sequences, and hosts without undue experimentation to accomplish
the desired expression without departing from the scope of this
application. For example, in selecting a vector, the host must be
considered because the vector must function in it. The vector's
copy number, the ability to control that copy number, and the
expression of any other proteins encoded by the vector, such as
antibiotic markers, will also be considered. One of ordinary skill
in the art can select the proper vectors, expression control
sequences, and hosts to accomplish the desired expression without
departing from the scope of this application. For example, in
selecting a vector, the host is considered because the vector
functions in it. The vector's copy number, the ability to control
that copy number, and the expression of any other proteins encoded
by the vector, such as antibiotic markers, can also be
considered.
[0313] The present application also provides constructs in the form
of plasmids, vectors, transcription or expression cassettes as
described elsewhere herein which comprise at least one
polynucleotide as above. Suitable vectors can be chosen or
constructed, containing appropriate regulatory sequences, including
promoter sequences, terminator sequences, polyadenylation
sequences, enhancer sequences, selectable markers and other
sequences as appropriate. Vectors can be plasmids, viral e.g.,
phage, phagemid, etc., as appropriate. For further details see, for
example, Molecular Cloning: a Laboratory Manual: 2nd edition,
Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many
known techniques and protocols for manipulation of nucleic acid,
for example in preparation of nucleic acid constructs, mutagenesis,
sequencing, introduction of DNA into cells and gene expression, and
analysis of proteins, are described in detail in Short Protocols in
Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley
& Sons, 1992. The methods and disclosures of Sambrook et al.
and Ausubel et al. are incorporated herein by reference.
[0314] In selecting an expression control sequence, a variety of
factors will normally be considered. These include, for example,
the relative strength of the system, its controllability, and its
compatibility with the particular DNA sequence or gene to be
expressed, particularly as regards potential secondary structures.
Suitable unicellular hosts will be selected by consideration of,
e.g., their compatibility with the chosen vector, their secretion
characteristics, their ability to fold proteins correctly, and
their fermentation requirements, as well as the toxicity to the
host of the product encoded by the DNA sequences to be expressed,
and the ease of purification of the expression products.
[0315] A further aspect provides a host cell containing one or more
polynucleotides as disclosed herein. Yet a further aspect provides
a method of introducing such one or more polynucleotides into a
host cell, any available technique. For eukaryotic cells, suitable
techniques can include, for example, calcium phosphate
transfection, DEAEDextran, electroporation, liposome-mediated
transfection and transduction using retrovirus or other virus
(e.g., vaccinia) or, for insect cells, baculovirus. For bacterial
cells, suitable techniques can include, for example calcium
chloride transformation, electroporation and transfection using
bacteriophages.
[0316] The introduction can be followed by causing or allowing
expression from the one or more polynucleotides, e.g., by culturing
host cells under conditions for expression of one or more
polypeptides from one or more polynucleotides. Inducible systems
can be used and expression induced by addition of an activator.
[0317] In one embodiment, the polynucleotides can be integrated
into the genome (e.g., chromosome) of the host cell. Integration
can be promoted by inclusion of sequences which promote
recombination with the genome, in accordance with standard
techniques. In another embodiment, the nucleic acid is maintained
on an episomal vector in the host cell.
[0318] Methods are provided herein which include using a construct
as stated above in an expression system in order to express a
specific polypeptide.
[0319] Considering these and other factors, a person skilled in the
art will be able to construct a variety of vector/expression
control sequence/host combinations that will express the DNA
sequences on fermentation or in large scale animal culture.
[0320] A polynucleotide encoding an antibody, antigen-binding
fragment, or a binding protein can be prepared
recombinantly/synthetically in addition to, or rather than, cloned.
The polynucleotide can be designed with the appropriate codons for
the antibody, antigen-binding fragment, or a binding protein. In
general, one will select preferred codons for an intended host if
the sequence will be used for expression. The complete
polynucleotide can be assembled from overlapping oligonucleotides
prepared by standard methods and assembled into a complete coding
sequence. See, e.g., Edge, Nature, 292:756 (1981); Nambair et al.,
Science, 223:1299 (1984); Jay et al., J. Biol. Chem., 259:6311
(1984).
[0321] A general method for site-specific incorporation of
unnatural amino acids into proteins is described in Christopher J.
Noren, Spencer J. Anthony-Cahill, Michael C. Griffith, Peter G.
Schultz, Science, 244:182-188 (April 1989). This method can be used
to create analogs with unnatural amino acids.
[0322] As mentioned above, a DNA sequence encoding an antibody or
antigen-binding fragment thereof can be prepared synthetically
rather than cloned. The DNA sequence can be designed with the
appropriate codons for the antibody or antigen-binding fragment
amino acid sequence. In general, one will select preferred codons
for the intended host if the sequence will be used for expression.
The complete sequence is assembled from overlapping
oligonucleotides prepared by standard methods and assembled into a
complete coding sequence. See, e.g., Edge, Nature, 292:756 (1981);
Nambair et al., Science, 223:1299 (1984); Jay et al., J. Biol.
Chem., 259:6311 (1984), each of which is which is incorporated
herein by reference in its entirety.
[0323] C. In Silico Analysis of Immunogenicity
[0324] If needed, an antibody or an antigen binding fragment
thereof described herein can be assessed for immunogenicity and, as
needed, be deimmunized (i.e., the antibody is made less immuno
reactive by altering one or more T cell epitopes). Analysis of
immunogenicity and T-cell epitopes present in the humanized
anti-endoglin antibodies and antigen-binding fragments described
herein can be carried out via the use of software and specific
databases. Exemplary software and databases include iTope.TM.
developed by Antitope of Cambridge, England. iTope.TM. is an in
silico technology for analysis of peptide binding to human MHC
class II alleles.
[0325] The iTope.TM. software predicts peptide binding to human MHC
class II alleles and thereby provides an initial screen for the
location of such "potential T cell epitopes." iTope.TM. software
predicts favorable interactions between amino acid side chains of a
peptide and specific binding pockets within the binding grooves of
34 human MHC class II allelles. The location of key binding
residues is achieved by the in silico generation of 9mer peptides
that overlap by one amino acid spanning the test antibody variable
region sequence. Each 9mer peptide can be tested against each of
the 34 MHC class II allotypes and scored based on their potential
"fit" and interactions with the MHC class II binding groove.
Peptides that produce a high mean binding score (>0.55 in the
iTope.TM. scoring function) against >50% of the MHC class II
alleles are considered as potential T cell epitopes. In such
regions, the core 9 amino acid sequence for peptide binding within
the MHC class II groove is analyzed to determine the MHC class II
pocket residues (P1, P4, P6, P7 and P9) and the possible T cell
receptor (TCR) contact residues (P-1, P2, P3, P5, P8).
[0326] After identification of any T-cell epitopes, amino acid
residue changes, substitutions, additions, and/or deletions can be
introduced to remove the identified T-cell epitope. Such changes
can be made so as to preserve antibody structure and function while
still removing the identified epitope. Exemplary changes can
include, but are not limited to, conservative amino acid
changes.
[0327] Techniques exploiting soluble complexes of recombinant MHC
molecules in combination with synthetic peptides have come into
use. These reagents and procedures may be used to identify the
presence of T-cell clones from peripheral blood samples from human
or experimental animal subjects that are able to bind particular
MHC-peptide complexes and are not adapted for the screening
multiple potential epitopes to a wide diversity of MHC
allotypes.
[0328] Biological assays of T-cell activation remain the best
practical option to providing a reading of the ability of a test
peptide/protein sequence to evoke an immune response. Examples of
this kind of approach include the use of T-cell proliferation
assays to the bacterial protein staphylokinase, followed by epitope
mapping using synthetic peptides to stimulate T-cell lines.
Similarly, T-cell proliferation assays using synthetic peptides of
the tetanus toxin protein have resulted in definition of
immunodominant epitope regions of the toxin. In one embodiment,
T-cell epitopes in a test protein may be determined using isolated
sub-sets of human immune cells, promoting their differentiation in
vitro and culture of the cells in the presence of synthetic
peptides of interest and measurement of any induced proliferation
in the cultured T-cells. Other techniques may also be used. Such a
technique involves careful application of cell isolation techniques
and cell culture with multiple cytokine supplements to obtain the
desired immune cell sub-sets (dendritic cells, CD4+ and or CD8+
T-cells). In another embodiment, the presence of T cell epitopes in
an antibody may be determined by adding the antibody to isolated
sub-sets of human immune cells, and assessing their differentiation
in vitro and measuring any induced proliferation in the cultured T
cells.
[0329] In silico techniques to define MHC class II ligands for
multiple proteins of therapeutic interest may also be utilized.
However, for reasons such as the requirement for proteolytic
processing and other physiologic steps leading to the presentation
of immunogenic peptides in vivo, a sub-set of the entire repertoire
of peptides definable by computer-based schemes may have ultimate
biological relevance. Thus, ex vivo human T-cell activation assays
may be used to identify the regions within the protein sequence of
a polypeptide that are able to support T-cell activation and are
thereby most biologically relevant to the problem of immunogenicity
in this protein. As used herein, "T-cell epitope" refers to an
amino acid sequence which is able to bind MHC class II, able to
stimulate T-cells and/or also to bind (without necessarily
measurably activating) T-cells in complex with MHC class II.
[0330] According to a method disclosed herein, synthetic peptides
or whole antibodies are tested for their ability to evoke a
proliferative response in human T-cells cultured in vitro. The
T-cells are present within a peripheral blood mononuclear cell
(PBMC) layer readily obtainable by well known means from whole
blood samples. Moreover, the PBMC preparation contains
physiological ratios of T-cells and antigen presenting cells and
is, therefore, a good source of materials with which to conduct a
surrogate immune reaction in vitro. In the operation of such an
assay, a stimulation index approaching or exceeding 2.0 is a useful
measure of induced proliferation. However, the stimulation index
may be different depending upon the antibody, or antigen-binding
fragment thereof, and may be established with reference to a
baseline for each antibody, or antigen-binding fragment thereof,
and corresponding peptide library. In one example of such testing,
the stimulation index (SI) may be conventionally derived by
division of the proliferation score (e.g., counts per minute of
radioactivity if using for example .sup.3H-thymidine incorporation)
measured to the test peptide by the score measured in cells not
contacted with a test peptide. Peptides which evoke no response may
give a SI=1.0 although SI values in the range 0.8-1.2 may also be
unremarkable. A number of technical procedures can be built into
the operation of such assays in order to ensure confidence in the
recorded scores. Typically all determinations are made at least in
triplicate and the mean score may be computed. Where a computed
SI=>2.0, individual scores of the triplicate can be examined for
evidence of outlying data. Test peptides are contacted with cells
in at least two different concentrations and the concentrations
would typically span a minimum two-fold concentration difference.
Such a concentration range provides an off-set to the kinetic
dimension to the assay and may be useful where a single time point
determination, for example at day plus 7, is being conducted. In
some assays, multiple time course determinations may be conducted
and these too may be made using peptide immunogen provided at a
minimum of two different concentrations. Similarly the inclusion of
control peptides for which there is expectation that the majority
of PBMC donor samples will be responsive may be included in each
assay plate. The influenza haemagglutinin peptide 307-309, sequence
PKYVKQNTLKLA (SEQ ID NO: 104); and the Chlamydia HSP 60 peptide
sequence KVVDQIKKISKPVQH (SEQ ID NO: 105) are examples of control
peptides to be used in such an assay. Alternatively, or in
addition, assays could also use a potent whole protein antigen,
such as hemocyanin from Keyhole Limpet, to which all PBMC samples
would be expected to exhibit an SI significantly greater than 2.0.
Other control antigens for such use will be well-known in the
art.
[0331] The methods disclosed herein can provide an epitope map of
antibodies, or antigen-binding fragments thereof, where the map has
relevance to a wide spectrum of possible MHC allotypes. The map may
be sufficiently representative to allow the design or selection of
a modified protein for which the ability of the protein to evoke a
T-cell driven immune response may be eliminated or at least
ameliorated for the majority of patients to whom the protein is
likely to be administered. Amelioration can refer to a reduction in
an immune response (i.e., reduced immunogenicity) compared to an
unmodified protein (e.g., about 1.5 fold less, about 2 fold less,
about 5 fold less, about 10 fold less, about 20 fold less, about 50
fold less, about 100 fold less, about 200 fold less, about 500 fold
less or more, or any range therein). Alternatively, antibodies, or
antigen-binding fragments thereof, with reduced immunogenicity can
refer to a percent reduction in its ability to elicit an immune
response compared to an unmodified protein (e.g., about 1% less,
about 2% less, about 3% less, about 4% less, about 5% less, about
10% less, about 20% less, about 50% less, about 100% less, and any
range therein). Accordingly in the practice of the screening
process, PBMC derived T-cells from naive donors are collected from
a pool of donors of sufficient immunological diversity to provide a
sample of at least greater than 90% of the MHC class II repertoire
(HLA-DR) extant in the human population. Where a naive T-cell
response is to be detected to a given synthetic peptide (or
antibody), the peptide (or antibody) in practice is contacted with
PBMC preparations derived from multiple donors in isolation; the
numbers of donors (or "donor pool" size), is for practical purposes
not likely to be less than 20 unrelated individuals and all samples
in the donor pool may be pre-selected according to their MHC class
II haplotype.
[0332] As used herein, the term "naive donor" refers to a subject
that has not been previously exposed to antibodies, or
antigen-binding fragments thereof, described herein either
environmentally, by vaccination, or by other means such as, for
example, blood transfusions.
[0333] When screening for T-cell epitopes, T-cells can be provided
from a peripheral blood sample from a multiplicity of different
healthy donors but who have not been in receipt of the protein
therapeutically. If needed, patient blood samples can be tested for
the presence of a particular polypeptide using conventional assays
such as an ELISA which uses antibodies to identify the presence or
absence of one or more polypeptides. The assay is conducted using
PBMC cultured in vitro using conventional procedures known in the
art and involves contacting the PBMC with synthetic peptide species
representative of the protein of interest (i.e., a library), or a
whole protein, such as an antibody and following a suitable period
of incubation, measurement of induced T cell activation such as
cellular proliferation. Measurement can be by any suitable means
and may, for example, be conducted using H.sup.3-thymidine
incorporation whereby the accumulation of H.sup.3 into cellular
material is readily measured using laboratory instruments. The
degree of cellular proliferation for each combination of PBMC
sample and synthetic peptide or whole protein can be examined
relative to that seen in an untreated PBMC sample. Reference may
also be made to the proliferative response seen following treatment
with a peptide or peptides or whole proteins for which there is an
expected proliferative effect. In this regard, it is advantageous
to use a peptide or whole protein with known broad MHC restriction
and especially peptide epitopes with MHC restriction to the DP or
DQ isotypes, although the invention is not limited to the use of
such restricted peptides or proteins. Such peptides have been
described above, for example, with respect to influenza
haemagglutinin and chlamydia HSP60.
[0334] In one non-limiting example, T-cell epitopes are mapped and
subsequently modified using the methods described herein. To
facilitate assembly of an epitope map, a library of synthetic
peptides is produced. Each of the peptides is 15 amino acid
residues in length and each overlapped the next peptide in the
series by 12 amino acid residues; i.e., each successive peptide in
the series incrementally added a further 3 amino acids to the
analysis. In this way, any given adjacent pair of peptides mapped
18 amino acids of contiguous sequence. One method for defining a
T-cell map using naive T-cell assays is illustrated in the Examples
below. Each of the peptides identified via the method to define a
T-cell map are suggested to be able to bind MHC class II and engage
at least one cognate TCR with sufficient affinity to evoke a
proliferative burst detectable in the assay system.
[0335] In another non-limiting example, the potential of an
antibody to be processed to generate T-cell epitopes that bind MHC
class II and engage at least one cognate TCR with sufficient
affinity to evoke a proliferative burst detectable in the assay
system is assessed.
[0336] The molecules described herein can be prepared in any of
several ways including the use of recombinant methods. The protein
sequences and information provided herein can be used to deduce a
polynucleotide (DNA) encoding an amino acid sequence. This can be
achieved for example using computer software tools such as the
DNAstar software suite [DNAstar Inc, Madison, Wis., USA] or
similar. Any such polynucleotide encoding the polypeptides or
significant homologues, variants, truncations, elongations, or
further modifications thereof, are contemplated herein.
[0337] Provided herein are methods of mapping (identifying) T-cell
epitopes and modifying the epitopes such that the modified sequence
reduces (partially or completely) induction of a T-helper response.
Modification includes amino acid substitutions, deletions, or
insertion made in codons of a polynucleotide encoding modified
polypeptides to affect similar changes. Codons encoding amino acid
residues are well known in the art. It is possible to use
recombinant DNA methods to achieve directed mutagenesis of the
target sequences and many such techniques are available, described
herein, and known in the art such as described above. In general,
the technique of site-specific mutagenesis is well known. Briefly,
a bacteriophage vector that produces a single stranded template for
oligonucleotide directed PCR mutagenesis is employed. Phage vectors
(e.g., M13) are commercially available and their use is generally
well known in the art. Similarly, double stranded plasmids are also
routinely employed in site directed mutagenesis, which eliminates
the step of transferring the polynucleotide of interest from a
phage to a plasmid. Synthetic oligonucleotide primers bearing the
desired mutated sequence can be used to direct the in vitro
synthesis of modified (desired mutant) DNA from this template and
the heteroduplex DNA is used to transform competent E. coli for the
growth selection and identification of desired clones.
Alternatively, a pair of primers can be annealed to two separate
strands of a double stranded vector to simultaneously synthesize
both corresponding complementary strands with the desired
mutation(s) in a PCR reaction.
[0338] In one embodiment, the Quick Change site-directed
mutagenesis method using plasmid DNA templates may be employed. PCR
amplification of the plasmid template containing the insert target
gene of insert is achieved using two synthetic oligonucleotide
primers containing the desired mutation. The oligonucleotide
primers, each complementary to opposite strands of the vector, are
extended during temperature cycling by mutagenesis-grade PfuTurbo
DNA polymerase. On incorporation of the oligonucleotide primers, a
mutated plasmid containing staggered nicks is generated. Amplified
un-methylated products are treated with Dpn I to digest methylated
parental DNA template and select for the newly synthesized DNA
containing mutations. Since DNA isolated from most E. coli strains
is dam methylated, it is susceptible to Dpn I digestion, which is
specific for methylated and hemimethylated DNA. The reaction
products are transformed into high efficiency strains of E. coli to
obtain plasmids containing the desired modifications. Additional
methods for introducing amino acid modifications into a polypeptide
are well known in the art and can also be used herein.
[0339] Suitable modifications to a protein may include amino acid
substitution of particular residues or combinations of residues.
For the elimination of T-cell epitopes, amino acid substitutions
are made at appropriate points or amino acid residues within an
amino acid sequence predicted to achieve reduction or elimination
of the activity of the T-cell epitope. In practice, an appropriate
point or amino acid residue will preferably equate to an amino acid
residue binding within one of the pockets provided within the MHC
class II binding groove. Such modifications may alter binding
within the first pocket of the cleft at the so-called "P1" or "P1
anchor" position of the peptide. The quality of binding interaction
between the P1 anchor residue of the peptide and the first pocket
of the MHC class II binding groove is recognized as being a major
determinant of overall binding affinity for the whole peptide. An
appropriate substitution at this position of the amino acid
sequence will generally incorporate an amino acid residue less
readily accommodated within the pocket (e.g., substitution to a
more hydrophilic residue). Amino acid residues in the peptide at
positions equating to binding within other pocket regions within
the MHC binding cleft are also considered and fall under the scope
of the present.
[0340] It is understood that single amino acid modifications within
a given potential T-cell epitope represent one route by which one
or more T-cell epitopes may be eliminated. Combinations of
modifications within a single epitope may be contemplated and can
be appropriate where individually defined epitopes are in overlap
with each other. Moreover, amino acid modifications (either singly
within a given epitope or in combination within a single epitope)
may be made at positions not equating to the "pocket residues" with
respect to the MHC class II binding groove, but at any point within
the amino acid sequence. Modifications may be made with reference
to a homologous structure or structural method produced using in
silico techniques known in the art and described herein may be
based on known structural features of the polypeptide. A change
(modification) may be contemplated to restore structure or
biological activity of the variant molecule. Such compensatory
changes and changes may also include deletion or addition
(insertion) of particular amino acid residues from a polypeptide.
Additionally, modifications can be made that alter the structure
and/or reduce the biological activity of the molecule and also
eliminate a T-cell epitope, thus reducing the immunogenicity of the
molecule. All types of modifications are contemplated herein.
[0341] An additional means of removing epitopes from protein
molecules is the concerted use of a naive T-cell activation assay
scheme as outlined herein together with an in silico tool developed
according to the scheme described in WO 02/069232 which is also
incorporated fully herein by reference. The software simulates the
process of antigen presentation at the level of the polypeptide-MHC
class II binding interaction to provide a binding score for any
given polypeptide sequence. Such a score is determined for many of
the predominant MHC class II allotypes extant in the population. As
this scheme is able to test any polypeptide sequence, the
consequences of amino acid substitutions additions or deletions
with respect to the ability of a polypeptide to interact with a MHC
class II binding groove can be predicted. Consequently new sequence
compositions can be designed which contain reduced numbers of amino
acids able to interact with a MHC class II and thereby function as
immunogenic T-cell epitopes. Where the biological assay using any
one given donor sample can assess binding to a maximum of four DR
allotypes, the in silico process can test a same polypeptide
sequence using >40 allotypes simultaneously. In practice this
approach is able to direct the design of new sequence variants
which are altered in their ability to interact with multiple MHC
allotypes. As will be clear to one in the art, multiple alternative
sets of substitutions could be arrived at which achieve the
objective of removing undesired epitopes. The resulting sequences
would however be recognized to be closely homologous with the
specific compositions disclosed herein and therefore fall within
the scope of the present application.
[0342] A combined approach of using an in silico tool for the
identification of MHC class II ligands and design of sequence
analogues lacking MHC class II ligands, in concert with epitope
mapping and re-testing optionally using biologically based assays
of T-cell activation is an additional method and embodiment of the
present application. The general method according to this
embodiment comprises the following steps:
i) use of naive T-cell activation assays and synthetic peptides
collectively encompassing the protein sequence of interest to
identify epitope regions capable of activating T-cells; ii) use of
a computational scheme simulating the binding of the peptide ligand
with one or more MHC allotypes to analyze the epitope regions
identified in step (i) and thereby identify MHC class II ligands
within the epitope region; iii) use of a computational scheme
simulating the binding of the peptide ligand with one or more MHC
allotypes to identify sequence analogues of the MHC ligands
encompassed within the epitope region(s) which no longer bind MHC
class II or bind with lowered affinity to a lesser number of MHC
allotypes and optionally, iv) use of naive T-cell activation assays
and synthetic peptides encompassing entirely or in collection
encompassing the epitope regions identified within the protein of
interest and testing the sequence analogues in naive T-cell
activation assay in parallel with the wild-type (parental)
sequences.
[0343] In one embodiment, a method of making a modified antibody,
or antigen-binding fragment thereof, exhibiting reduced
immunogenicity compared to an unmodified antibody, or
antigen-binding fragment thereof, comprises identifying at least
one T-cell epitope within the amino acid sequence of an antibody,
or antigen-binding fragment thereof, and modifying at least one
amino acid residue within at least one identified T-cell
epitope.
[0344] In another embodiment, a modified antibody, or
antigen-binding fragment thereof, exhibiting reduced immunogenicity
compared to an unmodified antibody, or antigen-binding fragment
thereof, is produced by a process of identifying at least one
T-cell epitope within the amino acid sequence of a antibody, or
antigen-binding fragment thereof, and modifying at least one amino
acid residue within at least one identified T-cell epitope.
[0345] In yet another embodiment, a method of selecting a modified
antibody, or antigen-binding fragment thereof, that exhibits
reduced immunogenicity compared to an unmodified antibody, or
antigen-binding fragment thereof, comprises identifying at least
one T-cell epitope within the amino acid sequence of a antibody, or
antigen-binding fragment thereof, modifying at least one amino acid
residue within at least one identified T-cell epitope, and
selecting a modified antibody, or antigen-binding fragment thereof,
that exhibits reduced immunogenicity compared to an unmodified
antibody, or antigen-binding fragment thereof.
[0346] T-cell epitopes described herein can be further
characterized by the regions of the epitope. Such regions include
the epitope core, the N-terminus and the C-terminus. As used herein
"epitope core" refers to the core 9-mer amino acid sequences of the
T-cell epitopes. The epitope core can further include 0, 1, 2, or 3
amino acid residues adjacent to the core 9-mer amino acid sequence
on the N-terminus and/or the C-terminus. Thus the epitope core, in
certain embodiments, can range in length from about 9 amino acids
up to about 15 amino acids.
[0347] As used herein, "N-terminus" refers to the amino acids
adjacent to the N-terminus of the epitope core and includes at
least 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids adjacent to and
upstream of the N-terminus of the epitope core.
[0348] As used herein, "C-terminus" refers to the amino acids
adjacent to the C-terminus of the epitope core and includes at
least 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids adjacent to and
downstream of the C-terminus of the epitope core.
[0349] In one embodiment, a modified antibody, or antigen-binding
fragment thereof, contains one or more modifications.
[0350] In one embodiment, a modified antibody, or antigen-binding
fragment thereof, contains two modifications.
[0351] In one embodiment, a modified antibody, or antigen-binding
fragment thereof, contains three modifications.
[0352] In one embodiment, a modified antibody, or antigen-binding
fragment thereof, contains four modifications.
[0353] In one embodiment, a modified antibody, or antigen-binding
fragment thereof, contains five modifications.
[0354] In one embodiment, a modified antibody, or antigen-binding
fragment thereof, contains six modifications.
[0355] In one embodiment, a modified antibody, or antigen-binding
fragment thereof, contains seven modifications.
[0356] In one embodiment, a modified antibody, or antigen-binding
fragment thereof, contains eight modifications.
[0357] In one embodiment, a modified antibody, or antigen-binding
fragment thereof, contains nine modifications.
[0358] In one embodiment, a modified antibody, or antigen-binding
fragment thereof, contains ten modifications.
[0359] In one embodiment, a modified antibody, or antigen-binding
fragment thereof, contains up to twenty modifications.
[0360] Provided herein is an antibody, or antigen-binding fragment
thereof, comprising a light chain variable region having an amino
acid sequence set forth as SEQ ID NO: 93--(VK1AA) and a heavy chain
variable region having an amino acid sequence set forth as SEQ ID
NO: 89 (VH1A2).
[0361] Provided herein is an antibody, or antigen-binding fragment
thereof, comprising a heavy chain variable region having an amino
acid sequence set forth as any one of SEQ ID NOS: 88, 89, 90, 91
and 92.
[0362] Provided herein is an antibody, or antigen-binding fragment
thereof, comprising a light chain variable region having an amino
acid sequence set forth as any one of SEQ ID NOS: 93, 94, 95, 96,
97, 100, 102, and 103.
[0363] Provided herein is an antibody, or antigen-binding fragment
thereof, that binds endoglin, comprising a heavy chain variable
region having an amino acid sequence set forth as SEQ ID NO: 89 and
a light chain variable region having an amino acid sequence set
forth as SEQ ID NO: 93, wherein:
[0364] (i) the heavy chain variable region further comprises one or
more modifications selected from the group consisting of a
substitution of glycine (G) by alanine (A) or serine (S) at
position 49; a substitution of alanine (A) by isoleucine (I) at
position 51; a substitution of lysine (K) by arginine (R) or
glutamine (Q) at position 52b; a substitution of leucine (L) by
valine (V) at position 78 utilizing the Kabat numbering system;
and
[0365] (ii) the light chain variable region further comprises one
or more modifications selected from the group consisting of a
substitution of methionine (M) by leucine (L) at position 4; a
substitution of alanine (A) by valine (V) at position 19; a
substitution of threonine (T) by serine (S) at position 22; a
substitution of alanine (A) by isoleucine (I) at position 48; and a
substitution of threonine (T) by serine (S) at position 51
utilizing the Kabat numbering system
[0366] Provided herein is an antibody, or antigen-binding fragment
thereof comprising a heavy chain variable region having an amino
acid sequence set forth as SEQ ID NO: 88, 89, 90, 91 and 92; and a
light chain variable region having an amino acid sequence set forth
as SEQ ID NO: 93, 95, 96, 97, 100, 102, or 103.
[0367] In addition to the aforementioned examples and embodiments,
a modified antibody, or antigen-binding fragment thereof, with one
or more amino acid modifications in one or more T-cell epitopes are
contemplated herein. In one non-limiting example, provided herein
are antibodies, or antigen-binding fragments thereof, having at
least one modification in at least one T-cell epitope. In another
non-limiting example, provided herein are antibodies, or
antigen-binding fragments thereof, having at least one amino acid
modification in 1, 2, 3, 4, 5, 6, or 7 of the T-cell epitopes
described herein. Additional non-limiting examples include
antibodies, or antigen-binding fragments thereof, having more than
one amino acid modification in more than one T-cell epitope. Any
combination of the amino acid modifications in any number of the
antibodies, or antigen-binding fragments thereof, T-cell epitopes
described above are contemplated herein.
T-Cell Epitopes and Allotype Frequency
[0368] Individual epitopes found within antigens can be
preferentially presented by specific MHC class II allotypes, and
similarly other specific epitopes within the same antigen may not
be presented on MHC class II molecules at all. Such associations of
particular epitopes with specific MCH class II molecules have been
shown to depend upon the MHC class II allotype of the individual.
The association of a specific epitope with a specific allotype can
also be considered when modifying antibodies, or antigen-binding
fragments thereof, for the removal of T-cell epitopes. Such
considerations can allow for the highly specific modification of an
antibody, or antigen-binding fragment thereof, for specific
allotypes (e.g., for specific populations of subjects having
certain MHC class II allotypes). The MHC class II allotype of a
subject or subjects can be easily determined by genotyping methods
known in the art, and the association of T-cell epitopes with the
given allotype thus easily identified, for consideration in
modification of antibodies, or antigen-binding fragments thereof,
tailored to that allotype. Identification of associations between
T-cell epitopes and MHC class II allotypes are described in more
detail in the examples below. Contemplated herein are modified
antibodies, or antigen-binding fragments thereof, that have T-cell
epitope modifications tailored to the MHC class II associations
identified for the given epitopes.
[0369] D. Anti-Endoglin Antibodies
[0370] Simultaneous incorporation of all of the FR and/or CDR
encoding nucleic acids and all of the selected amino acid position
changes can be accomplished by a variety of methods known to those
skilled in the art, including for example, recombinant and chemical
synthesis. For example, simultaneous incorporation can be
accomplished by, for example, chemically synthesizing the
nucleotide sequence for the acceptor variable region, fused
together with the donor CDR encoding nucleic acids, and
incorporating at the positions selected for harboring variable
amino acid residues a plurality of corresponding amino acid
codons.
[0371] Provided herein are antibodies and antigen-binding fragments
thereof that bind to endoglin. Also provided are antibodies and
antigen-binding fragments thereof that bind endoglin and inhibit
(partially or fully) or manage/treat (partially or fully)
angiogenesis/neovascularization, dilation of small vessels, and/or
diseases associated with excessive angiogenesis. Similarly,
inhibition of endoglin function (e.g., signaling, binding,
activation, and the like) is also included within the meaning of
inhibiting or binding endoglin. In yet another embodiment, an
antibody or antigen-binding fragment inhibits angiogenesis by
binding to endoglin. The application also provides cell lines which
can be used to produce the antibodies, methods for producing the
cell lines, methods for expressing antibodies or antigen-binding
fragments and purifying the same.
[0372] One can recognize that the antibodies and antigen-binding
fragments thereof that specifically bind endoglin generated using
the methods described herein can be tested using the assays
provided herein or known in the art for the ability to bind to
endoglin using conventional methods including, but not limited to,
ELISA. Affinity of antibodies described herein can also be
determined using conventional methods including, but not limited
to, Biacore or surface plasmon resonance.
[0373] The antibodies and antigen binding fragments thereof
described herein were constructed by humanization of the V.sub.H
and V.sub.L sequences of the TRC105 antibody. To accomplish this
humanization, a 3-dimensional model of the V.sub.H and V.sub.L
chains of TRC105 was created and analyzed. The V.sub.H and V.sub.L
sequences were then compared individually to a database of human
germline sequences, from which human V.sub.H and V.sub.L sequences
were chosen based on their homology to the V.sub.H and V.sub.L
sequences of TRC105. The human V.sub.L sequence chosen for
humanization was O2/O12 (VK1-39) (SEQ ID NO. 2). O2/O12 has a
sequence identity with TRC105 of 65% and the gene is highly
expressed in the human germline repertoire. The human V.sub.H
sequence chosen for humanization was VH3-15 (SEQ ID NO. 40). VH3-15
has sequence identity with TRC105 of 70% and is expressed with
reasonable frequency in the human germline repertoire. The amino
acid positions which were different between TRC105 and the human
sequences were examined in the 3D model of TRC105 to determine
which substitutions would be considered for modification. Amino
acid selection criteria based on the 3D model analysis included,
but was not limited to, for example, steric effects related to the
amino acid, relative charge of the amino acid, and the location of
the amino acid within the variable heavy and/or light chains. The
identified and proposed substitutions for the human framework
regions are incorporated into the O2 and VH3-15 human framework
regions, and the CDRs of TRC105 are grafted into the corresponding
O2 and VH3-15 human framework regions resulting in a multitude of
humanized antibodies or antigen-binding fragments. Additionally,
the FR-4 of the light chain is derived from human J germline
sequence Jk4. Similarly, the FR-4 of the heavy chain is derived
from human J germline sequence JH4.
[0374] Described herein are humanized antibodies and
antigen-binding fragments that bind endoglin. Also described herein
are humanized antibodies and antigen-binding fragments that bind
endoglin and inhibit angiogenesis. The antibodies and
antigen-binding fragments described herein were generated as
described above.
[0375] Antibodies and antigen-binding fragments thereof can have a
variable heavy (V.sub.H) chain, a variable light (V.sub.L) chain,
both, or binding portions thereof. In one embodiment, the V.sub.H
chain has an amino acid sequence set forth as any of SEQ ID NOS:
41-43, or a binding portion thereof. Such V.sub.H chains can have
framework regions sequences set forth as any of SEQ ID NOS: 44-62.
In another embodiment, the V.sub.L chain has an amino acid sequence
set forth as any of SEQ ID NOS: 3-5, or a binding portion thereof.
Such V.sub.L chains can have framework regions sequences set forth
as any of SEQ ID NOS: 6-38.
[0376] Provided herein is an antibody, or antigen-binding fragment
thereof, comprising a light chain variable region having an amino
acid sequence set forth as SEQ ID NO: 3 and a heavy chain variable
region having an amino acid sequence set forth as SEQ ID NO:
41.
[0377] Provided herein is an antibody, or antigen-binding fragment
thereof, comprising a light chain variable region having an amino
acid sequence set forth as SEQ ID NO: 3 and a heavy chain variable
region having an amino acid sequence set forth as SEQ ID NO: 41,
wherein: the heavy chain variable region further comprises one or
more modifications selected from the group consisting of a
substitution of glycine (G) by alanine (A) at position 49; a
substitution of asparagine (N) by serine (S) at position 76; a
substitution of threonine (T) by arginine (R) at position 77; a
substitution of leucine (L) by valine (V) at position 78; a
substitution of asparagine (N) by isoleucine (I) at position 82a; a
substitution of valine (V) by isoleucine (I) or leucine (L) at
position 89; a substitution of threonine (T) by arginine (R) or
glycine (G) at position 94; a substitution of leucine (L) by
threonine (T) at position 108; a substitution of valine (V) by
leucine (L) at position 109; and a substitution of serine (S) by
alanine (A) at position 113 utilizing the Kabat numbering system;
and the light chain variable region further comprises one or more
modifications selected from the group consisting of a substitution
of aspartic acid (D) by glutamine (Q) at position 1; a substitution
of glutamine (Q) by valine (V) at position 3; a substitution of
methionine (M) by leucine (L) at position 4; a substitution of
threonine (T) by serine (S) at position 5; a substitution of
tyrosine (Y) by phenylalanine (F) at position 36; a substitution of
leucine (L) by proline (P) at position 46; a substitution of
leucine (L) by tryptophan (W) at position 47; a substitution of
serine (S) by valine (V) or alanine (A) at position 60; a
substitution of aspartic acid (D) by serine (S) at position 70; a
substitution of phenylalanine (F) by tyrosine (Y) at position 71; a
substitution of glutamine (G) by alanine (A) at position 100; and a
substitution of isoleucine (I) by leucine (L) at position 106
utilizing the Kabat numbering system.
[0378] Provided herein is an antibody, or antigen-binding fragment
thereof, that binds endoglin comprising a heavy chain variable
region having an amino acid sequence set forth as SEQ ID NO: 41,
42, or 43; and a light chain variable region having an amino acid
sequence set forth as SEQ ID NO: 3, 4, or 5. An antibody, or
antigen-binding fragment thereof, can comprise a heavy chain
variable region having an amino acid sequence set forth as SEQ ID
NO: 41 and a light chain variable region having an amino acid
sequence set forth as SEQ ID NO: 3. An antibody, or antigen-binding
fragment thereof, can comprise a heavy chain variable region having
an amino acid sequence set forth as SEQ ID NO: 41 and a light chain
variable region having an amino acid sequence set forth as SEQ ID
NO: 4. An antibody, or antigen-binding fragment thereof, can
comprise a heavy chain variable region having an amino acid
sequence set forth as SEQ ID NO: 41 and a light chain variable
region having an amino acid sequence set forth as SEQ ID NO: 5. An
antibody, or antigen-binding fragment thereof, can comprise a heavy
chain variable region having an amino acid sequence set forth as
SEQ ID NO: 42 and a light chain variable region having an amino
acid sequence set forth as SEQ ID NO: 3. An antibody, or
antigen-binding fragment thereof, can comprise a heavy chain
variable region having an amino acid sequence set forth as SEQ ID
NO: 42 and a light chain variable region having an amino acid
sequence set forth as SEQ ID NO: 4. An antibody, or antigen-binding
fragment thereof, can comprise a heavy chain variable region having
an amino acid sequence set forth as SEQ ID NO: 42 and a light chain
variable region having an amino acid sequence set forth as SEQ ID
NO: 5. An antibody, or antigen-binding fragment thereof, can
comprise a heavy chain variable region having an amino acid
sequence set forth as SEQ ID NO: 43 and a light chain variable
region having an amino acid sequence set forth as SEQ ID NO: 3. An
antibody, or antigen-binding fragment thereof, can comprise a heavy
chain variable region having an amino acid sequence set forth as
SEQ ID NO: 43 and a light chain variable region having an amino
acid sequence set forth as SEQ ID NO: 4. An antibody, or
antigen-binding fragment thereof, can comprise a heavy chain
variable region having an amino acid sequence set forth as SEQ ID
NO: 43 and a light chain variable region having an amino acid
sequence set forth as SEQ ID NO: 5. Such antibodies can bind to
endoglin and inhibit angiogenesis.
[0379] In any of such embodiments, a heavy chain variable region
can further comprise one or more modifications selected from the
group consisting of: a substitution of asparagine (N) by serine (S)
at position 76; a substitution of threonine (T) by arginine (R) at
position 77; a substitution of asparagine (N) by isoleucine (I) at
position 82a; a substitution of valine (V) by isoleucine (I) or
leucine (L) at position 89; a substitution of threonine (T) by
glycine (G) at position 94; a substitution of leucine (L) by
threonine (T) at position 108; a substitution of valine (V) by
leucine (L) at position 109; and a substitution of serine (S) by
alanine (A) a position 113; and the light chain variable region can
further comprise one or more modifications selected from the group
consisting of: a substitution of aspartic acid (D) by glutamine (Q)
at position 1; a substitution of glutamine (Q) by valine (V) at
position 3; a substitution of threonine (T) by serine (S) at
position 5; a substitution of tyrosine (Y) by phenylalanine (F) a
position 36; a substitution of serine (S) by valine (V) or alanine
(A) at position 60; a substitution of aspartic acid (D) by serine
(S) at position 70; a substitution of glycine (G) by alanine (A) at
position 100, and a substitution of isoleucine (I) by leucine (L)
at position 106 utilizing the Kabat numbering system.
[0380] Provided herein is an antibody, or antigen-binding fragment
thereof, that binds endoglin, comprising a heavy chain variable
region and a light chain variable region, wherein said heavy chain
variable region comprises: [0381] (i) a CDR1 of SEQ ID NO: 66, a
CDR2 of SEQ ID NO: 67, and a CDR3 of SEQ ID NO: 68; [0382] (ii) a
heavy chain FR1 having the amino acid sequence of SEQ ID NO: 44 or
the amino acid sequence of SEQ ID NO: 44 except for one or more
conservative substitutions; [0383] (iii) a heavy chain FR2 having
the amino acid sequence of SEQ ID NO: 45 or the amino acid sequence
of SEQ ID NO: 45 except for a substitution of glycine (G) by
alanine (A) at position 49 utilizing the Kabat numbering system;
and [0384] (iv) a heavy chain FR3 having the amino acid sequence of
SEQ ID NO: 47 or the amino acid sequence of SEQ ID NO: 47 except
for one or more substitutions selected from the group consisting
of: [0385] (a) a substitution of asparagine (N) by serine (S) at
position 76; [0386] (b) a substitution of threonine (T) by arginine
(R) at position 77; [0387] (c) a substitution of leucine (L) by
valine (V) at position 78; [0388] (d) a substitution of asparagine
(N) by isoleucine (I) at position 82a; [0389] (e) a substitution of
valine (V) by isoleucine (I) or leucine (L) at position 89; and
[0390] (f) a substitution of threonine (T) by arginine (R) or
glycine (G) at position 94 utilizing the Kabat numbering system;
and [0391] (v) a heavy chain FR4 having the amino acid sequence of
SEQ ID NO: 56 or the amino acid sequence of SEQ ID NO: 56 except
for one or more substitutions selected from the group consisting
of: [0392] (a) a substitution of leucine (L) by threonine (T) at
position 108; [0393] (b) a substitution of valine (V) by leucine
(L) at position 109; and [0394] (c) a substitution of serine (S) by
alanine (A) at position 113 utilizing the Kabat numbering
system;
[0395] and said light chain variable region comprises: [0396] (i) a
CDR1 of SEQ ID NO: 63, a CDR2 of SEQ ID NO: 64, and a CDR3 of SEQ
ID NO: 65; [0397] (ii) a light chain FR1 having the amino acid
sequence of SEQ ID NO: 6 or the amino acid sequence of SEQ ID NO: 6
except for one or more substitutions selected from the group
consisting of: [0398] (a) a substitution of aspartic acid (D) by
glutamine (Q) at position 1; [0399] (b) a substitution of glutamine
(Q) by valine (V) at position 3; [0400] (c) a substitution of
methionine (M) by leucine (L) at position 4; and [0401] (d) a
substitution of threonine (T) by serine (S) at position 5;
utilizing the Kabat numbering system; and [0402] (iii) a light
chain FR2 having the amino acid sequence of SEQ ID NO: 20 or the
amino acid sequence of SEQ ID NO: 20 except for one or more
substitutions selected from the group consisting of: [0403] (a) a
substitution of tyrosine (Y) by phenylalanine (F) at position 36;
[0404] (b) a substitution of leucine (L) by proline (P) at position
46; and [0405] (c) a substitution of leucine (L) by tryptophan (W)
at position 47 utilizing the Kabat numbering system; and [0406]
(iv) a light chain FR3 having the amino acid sequence of SEQ ID NO:
28 or the amino acid sequence of SEQ ID NO: 28 except for one or
more substitutions selected from the group consisting of: [0407]
(a) a substitution of serine (S) by valine (V) or alanine (A) at
position 60; [0408] (b) a substitution of aspartic acid (D) by
serine (S) at position 70; and [0409] (b) a substitution of
phenylalanine (F) by tyrosine (Y) at position 71 utilizing the
Kabat numbering system; and [0410] (v) a light chain FR4 having the
amino acid sequence of SEQ ID NO: 35 or the amino acid sequence of
SEQ ID NO: 35 except for one or more substitutions selected from
the group consisting of: [0411] (a) a substitution of glycine (G)
by alanine (A) at position 100; and [0412] (b) a substitution of
isoleucine (I) by leucine (L) at position 106 utilizing the Kabat
numbering system.
[0413] An antibody, or antigen-binding fragment thereof, provided
herein can comprise a heavy chain variable region CDR1 having an
amino acid sequence as set forth in SEQ ID NO: 66, a heavy chain
variable region CDR2 having an amino acid sequence as set forth in
SEQ ID NO: 67, a heavy chain variable region CDR3 having an amino
acid sequence as set forth in SEQ ID NO: 68, a light chain variable
region CDR1 having an amino acid sequence as set forth in SEQ ID
NO: 63, a light chain variable region CDR2 having an amino acid
sequence as set forth in SEQ ID NO: 64, and a light chain variable
region CDR3 having an amino acid sequence as set forth in SEQ ID
NO: 65.
[0414] In one embodiment, the antibody, or antigen-binding fragment
thereof binds endoglin and comprises a heavy chain variable region
FR1 having an amino acid sequence as set forth in SEQ ID NO: 44; a
heavy chain variable region FR2 having an amino acid sequence as
set forth in SEQ ID NO: 45; a heavy chain variable region FR3
having an amino acid sequence as set forth in SEQ ID NO: 47; a
heavy chain variable region FR4 having an amino acid sequence as
set forth in SEQ ID NO: 56.
[0415] In another embodiment, the antibody, or antigen-binding
fragment thereof binds endoglin and comprises a heavy chain
variable region FR1 having an amino acid sequence as set forth in
SEQ ID NO: 44; a heavy chain variable region FR2 having an amino
acid sequence as set forth in SEQ ID NO: 46; a heavy chain variable
region FR3 having an amino acid sequence as set forth in SEQ ID NO:
48; a heavy chain variable region FR4 having an amino acid sequence
as set forth in SEQ ID NO: 56.
[0416] In another embodiment, the antibody, or antigen-binding
fragment thereof, comprises a light chain variable region FR1
having an amino acid sequence as set forth in SEQ ID NO: 6; a light
chain variable region FR2 having an amino acid sequence as set
forth in SEQ ID NO: 20; a light chain variable region FR3 having an
amino acid sequence as set forth in SEQ ID NO: 28; and a light
chain variable region FR4 having an amino acid sequence as set
forth in SEQ ID NO: 35.
[0417] In another embodiment, the antibody, or antigen-binding
fragment thereof binds endoglin and comprises a light chain
variable region FR1 having an amino acid sequence as set forth in
SEQ ID NO: 6; a light chain variable region FR2 having an amino
acid sequence as set forth in SEQ ID NO: 21; a light chain variable
region FR3 having an amino acid sequence as set forth in SEQ ID NO:
29; and a light chain variable region FR4 having an amino acid
sequence as set forth in SEQ ID NO: 35.
[0418] In another embodiment, the antibody, or antigen-binding
fragment thereof binds endoglin and comprises a light chain
variable region FR1 having an amino acid sequence as set forth in
SEQ ID NO: 7; a light chain variable region FR2 having an amino
acid sequence as set forth in SEQ ID NO: 21; a light chain variable
region FR3 having an amino acid sequence as set forth in SEQ ID NO:
29; and a light chain variable region FR4 having an amino acid
sequence as set forth in SEQ ID NO: 35.
[0419] Provided herein is an antibody, or antigen-binding fragment
thereof, comprising a heavy chain variable region having an amino
acid sequence set forth as SEQ ID NO: 42 and a light chain variable
region having an amino acid sequence set forth as SEQ ID NO: 4.
[0420] Provided herein is an antibody, or antigen-binding fragment
thereof, that binds endoglin, comprising a light chain variable
region having an amino acid sequence set forth as SEQ ID NO: 4 and
a heavy chain variable region having an amino acid sequence set
forth as SEQ ID NO: 42, wherein: said heavy chain variable region
further comprises one or more modifications selected from the group
consisting of a substitution of glycine (G) by alanine (A) at
position 49; a substitution of asparagine (N) by serine (S) at
position 76; a substitution of threonine (T) by arginine (R) at
position 77; a substitution of leucine (L) by valine (V) at
position 78; a substitution of asparagine (N) by isoleucine (I) at
position 82a; a substitution of valine (V) by isoleucine (I) or
leucine (L) at position 89; a substitution of arginine (R) by
threonine (T) or glycine (G) at position 94; a substitution of
leucine (L) by threonine (T) at position 108; a substitution of
valine (V) by leucine (L) at position 109; and a substitution of
serine (S) by alanine (A) at position 113 utilizing the Kabat
numbering system; and the light chain variable region further
comprises one or more modifications selected from the group
consisting of a substitution of aspartic acid (D) by glutamine (Q)
at position 1; a substitution of glutamine (Q) by valine (V) at
position 3; a substitution of methionine (M) by leucine (L) at
position 4; a substitution of threonine (T) by serine (S) at
position 5; a substitution of tyrosine (Y) by phenylalanine (F) at
position 36; a substitution of proline (P) by leucine (L) at
position 46; a substitution of tryptophan (W) by leucine (L) at
position 47; a substitution of serine (S) by valine (V) or alanine
(A) at position 60; a substitution of aspartic acid (D) by serine
(S) at position 70; a substitution of tyrosine (Y) by phenylalanine
(F) at position 71; a substitution of glutamine (G) by alanine (A)
at position 100; and a substitution of isoleucine (I) by leucine
(L) at position 106 utilizing the Kabat numbering system
[0421] Provided herein is an antibody, or antigen-binding fragment
thereof, that binds endoglin, comprising a heavy chain variable
region and a light chain variable region,
[0422] wherein said heavy chain variable region comprises: [0423]
(i) a CDR1 of SEQ ID NO: 66, a CDR2 of SEQ ID NO: 67, and a CDR3 of
SEQ ID NO: 68; [0424] (ii) a heavy chain FR1 having the amino acid
sequence of SEQ ID NO: 44 or the amino acid sequence of SEQ ID NO:
44 except for one or more conservative substitutions; [0425] (iii)
a heavy chain FR2 having the amino acid sequence of SEQ ID NO: 45
or the amino acid sequence of SEQ ID NO: 45 except for a
substitution of glycine (G) by alanine (A) at position 49 utilizing
the Kabat numbering system; and [0426] (iv) a heavy chain FR3
having the amino acid sequence of SEQ ID NO: 47 or the amino acid
sequence of SEQ ID NO: 47 except for one or more substitutions
selected from the group consisting of: [0427] (a) a substitution of
asparagine (N) by serine (S) at position 76; [0428] (b) a
substitution of threonine (T) by arginine (R) at position 77;
[0429] (c) a substitution of leucine (L) by valine (V) at position
78; [0430] (d) a substitution of asparagine (N) by isoleucine (I)
at position 82a; [0431] (e) a substitution of valine (V) by
isoleucine (I) or leucine (L) at position 89; and [0432] (f) a
substitution of arginine (R) by threonine (T) or glycine (G) at
position 94 utilizing the Kabat numbering system; and [0433] (v) a
heavy chain FR4 having the amino acid sequence of SEQ ID NO: 56 or
the amino acid sequence of SEQ ID NO: 56 except for one or more
substitutions selected from the group consisting of: [0434] (a) a
substitution of leucine (L) by threonine (T) at position 108;
[0435] (b) a substitution of valine (V) by leucine (L) at position
109; and [0436] (c) a substitution of serine (S) by alanine (A) at
position 113 utilizing the Kabat numbering system;
[0437] and said light chain variable region comprises: [0438] (i) a
CDR1 of SEQ ID NO: 63, a CDR2 of SEQ ID NO: 64, and a CDR3 of SEQ
ID NO: 65; [0439] (ii) a light chain FR1 having the amino acid
sequence of SEQ ID NO: 6 or the amino acid sequence of SEQ ID NO: 6
except for one or more substitutions selected from the group
consisting of: [0440] (a) a substitution of aspartic acid (D) by
glutamine (Q) at position 1; [0441] (b) a substitution of glutamine
(Q) by valine (V) at position 3; [0442] (c) a substitution of
methionine (M) by leucine (L) at position 4; and [0443] (d) a
substitution of threonine (T) by serine (S) at position 5;
utilizing the Kabat numbering system; and [0444] (iii) a light
chain FR2 having the amino acid sequence of SEQ ID NO: 21 or the
amino acid sequence of SEQ ID NO: 20 except for one or more
substitutions selected from the group consisting of: [0445] (a) a
substitution of tyrosine (Y) by phenylalanine (F) at position 36;
[0446] (b) a substitution of proline (P) by leucine (L) at position
46; and [0447] (c) a substitution of tryptophan (W) by leucine (L)
at position 47 utilizing the Kabat numbering system; and [0448]
(iv) a light chain FR3 having the amino acid sequence of SEQ ID NO:
29 or the amino acid sequence of SEQ ID NO: 28 except for one or
more substitutions selected from the group consisting of: [0449]
(a) a substitution of serine (S) by valine (V) or alanine (A) at
position 60; [0450] (b) a substitution of aspartic acid (D) by
serine (S) at position 70; and [0451] (b) a substitution of
tyrosine (Y) by phenylalanine (F) at position 71 utilizing the
Kabat numbering system; and [0452] (v) a light chain FR4 having the
amino acid sequence of SEQ ID NO: 35 or the amino acid sequence of
SEQ ID NO: 35 except for one or more substitutions selected from
the group consisting of: [0453] (a) a substitution of glycine (G)
by alanine (A) at position 100; and [0454] (b) a substitution of
isoleucine (I) by leucine (L) at position 106 utilizing the Kabat
numbering system.
[0455] A substantial portion of a variable domain will include
three CDR regions, together with their intervening framework
regions. The portion can also include at least about 50% of either
or both of the first and fourth framework regions, the 50% being
the C-terminal 50% of the first framework region and the N-terminal
50% of the fourth framework region. Additional residues at the
N-terminal or C-terminal end of the substantial part of the
variable domain may be those not normally associated with naturally
occurring variable domain regions. For example, construction of
humanized endoglin antibodies and antigen-binding fragments
described herein made by recombinant DNA techniques can result in
the introduction of N- or C-terminal residues encoded by linkers
introduced to facilitate cloning or other manipulation steps. Other
manipulation steps include the introduction of linkers to join
variable domains to further protein sequences including
immunoglobulin heavy chains, other variable domains (for example in
the production of diabodies) or protein labels as discussed in more
detail below.
[0456] Humanized endoglin CDR3 regions having amino acid sequences
substantially as set out as the CDR3 regions of the antibodies
described herein will be carried in a structure which allows for
binding of the CDR3 regions to endoglin. The structure for carrying
the CDR3s can be of an antibody heavy or light chain sequence or
substantial portion thereof in which the CDR3 regions are located
at locations corresponding to the CDR3 region of
naturally-occurring V.sub.H and V.sub.L antibody variable domains
encoded by rearranged immunoglobulin genes.
[0457] In one non-limiting example, provided herein are antibodies
or antigen binding fragments thereof containing a variable heavy
chain having a CDR3 which has an amino acid sequence set forth as
SEQ ID NO: 68 and/or a variable light chain having a CDR3 which has
an amino acid sequence set forth as SEQ ID NO: 65. In one
embodiment, the variable heavy chain has an amino acid sequence set
forth as SEQ ID NO: 40 except for a substitution of the CDR3 by the
CDR3 amino sequence set forth as SEQ ID NO: 68. In another
embodiment, the variable light chain has an amino acid sequence set
forth as SEQ ID NO: 2 except for a substitution of the CDR3 by the
CDR3 amino acid sequence set forth as SEQ ID NO: 65. Additionally,
such CDR3 containing variable regions/chains can comprise one or
more FR amino acid sequences set forth as, for example, described
above (or such FRs containing one or more additional
modifications), where the antibodies or antigen binding fragments
have 3 CDRs and 4 FRs in each of the VH and VL regions, have
specific binding activity for endoglin and which are able to
inhibit angiogenesis. Additionally, various antibody J segments can
also be substituted within these variable regions for further
variation within the variable region chains.
[0458] In one aspect, variable heavy and light chains described
herein can also be created by further replacing FR4 sequences. In
one embodiment, heavy chain FR4 sequences can be substituted for
one of the following:
TABLE-US-00001 SEQ ID Kabat- NO: Number 103 104 105 106 107 108 109
110 111 112 113 76 FRM4 from W G Q G T L V T V S S JH1, JH4 or JH5
77 FRM4 from W G R G T L V T V S S JH2 78 FRM4 from W G Q G T M V T
V S S JH3 79 FRM4 from W G Q G T T V T V S S JH6
[0459] In one embodiment, light chain FR4 sequences can be
substituted for one of the following:
TABLE-US-00002 SEQ ID NO Kabat Number 98 99 100 101 102 103 104 105
106 107 80 JK1 F G Q G T K V E I K 81 JK2 F G Q G T K L E I K 82
JK3 F G P G T K V D I K 83 JK4 F G G G T K V E I K 84 JK5 F G Q G T
R L E I K
[0460] Further provided herein are humanized versions of
anti-endoglin antibodies alternatively named "superhumanized"
anti-endoglin antibodies or antigen-binding fragments thereof. Such
superhumanized antibodies, or antigen-binding fragments thereof,
can comprise a light chain variable region having an amino acid
sequence set forth as SEQ ID NOS: 71 or 72 and a heavy chain
variable region having an amino acid sequence set forth as SEQ ID
NO: 75.
[0461] In another aspect, the present application provides a
humanized antibody capable of competing with a humanized
anti-endoglin antibody or antigen-binding described herein under
conditions in which at least 5% of an antibody having the V.sub.H
and V.sub.L sequences of the antibody is blocked from binding to
endoglin by competition with such an antibody in an ELISA
assay.
[0462] Provided herein are neutralizing antibodies or
antigen-binding fragments that bind to endoglin and modulate the
activity of endoglin. The neutralizing antibody can for example,
inhibit angiogenesis by binding to endoglin.
[0463] Percentage of (%) inhibition of angiogenesis by an humanized
anti-endoglin antibody or antigen-binding fragment thereof of at
least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at
least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at
least 10-fold, at least 20-fold, at least 30-fold, at least
40-fold, at least 50-fold, at least 60-fold, or greater than
negative controls is indicative of a antibody or antigen-binding
fragment thereof inhibits angiogenesis. Percentage (%) of
inhibition of angiogenesis by a humanized anti-endoglin antibody or
antigen-binding fragment thereof of less than 2-fold greater than
negative controls is indicative of an antibody or antigen-binding
fragment thereof that does not inhibit angiogenesis.
[0464] Binding of an antibody or antigen-binding fragment to
endoglin can partially (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 95%, 98%, 99% or any number therein) or completely
inhibit angiogenesis. The neutralizing or inhibiting activity of an
antibody or antigen-binding fragment can be determined using an in
vitro assay and/or in vivo using art-recognized assays such as
those described herein or otherwise known in the art.
[0465] In one aspect, the antigen-binding fragment of any one of
the humanized antibodies described above is a Fab, a Fab', a Fd, a
F(ab').sub.2, a Fv, a scFv, a single chain binding polypeptide
(e.g., a scFv with Fc portion) or any other functional fragment
thereof as described herein.
[0466] Antibodies or antigen-binding fragments described herein are
useful in detection or diagnostic applications as described in more
detail below. Antibodies or antigen-binding fragments described
herein are useful for binding to endoglin, which, in turn, can
inhibit angiogenesis as described herein.
[0467] Antibodies, or antigen-binding fragments thereof, described
herein can be further modified to alter the specific properties of
the antibody while retaining the desired functionality, if needed.
For example, in one embodiment, the compound can be modified to
alter a pharmacokinetic property of the compound, such as in vivo
stability, solubility, bioavailability or half-life. Antibodies, or
antigen-binding fragments thereof, described herein can further
comprise a therapeutic moiety, a detectable moiety, or both, for
use in diagnostic and/or therapeutic applications.
[0468] Antibodies, or antigen-binding fragments thereof, described
herein can also be used as immunoconjugates. As used herein, for
purposes of the specification and claims, immunoconjugates refer to
conjugates comprised of the humanized anti-endoglin antibodies or
fragments thereof according to the present invention and at least
one therapeutic label. Therapeutic labels include antitumor agents
and angiogenesis-inhibitors. Such antitumor agents are known in the
art and include, but not limited to, toxins, drugs, enzymes,
cytokines, radionuclides, photodynamic agents, and angiogenesis
inhibitors. Toxins include, but are not limited to, ricin A chain,
mutant Pseudomonas exotoxins, diphtheria toxoid, streptonigrin,
boamycin, saporin, gelonin, and pokeweed antiviral protein. Drugs
include daunorubicin, methotrexate, and calicheamicins.
Radionuclides include radiometals. Cytokines include, but are not
limited to, transforming growth factor (TGF)-.beta., interleukins,
interferons, and tumor necrosis factors. Photodynamic agents
include, but are not limited to, porphyrins and their derivatives.
Additional therapeutic labels will be known in the art and are also
contemplated herein. The methods for complexing the anti-endoglin
mAbs or a fragment thereof with at least one antitumor agent are
well known to those skilled in the art (i.e., antibody conjugates
as reviewed by Ghetie et al., 1994, Pharmacol. Ther. 63:209-34).
Such methods may utilize one of several available
heterobifunctional reagents used for coupling or linking molecules.
Additional radionuclides are further described herein along with
additional methods for linking molecules, such as therapeutic and
diagnostic labels.
[0469] Antibodies, or antigen-binding fragments thereof, can be
modified using techniques known in the art for various purposes
such as, for example, by addition of polyethylene glycol (PEG). PEG
modification (PEGylation) can lead to one or more of improved
circulation time, improved solubility, improved resistance to
proteolysis, reduced antigenicity and immunogenicity, improved
bioavailability, reduced toxicity, improved stability, and easier
formulation (for a review, see, Francis et al., International
Journal of Hematology 68:1-18, 1998).
[0470] In the case of an antigen-binding fragment which does not
contain an Fc portion, an Fc portion can be added to (e.g.,
recombinantly) the fragment, for example, to increase half-life of
the antigen-binding fragment in circulation in blood when
administered to a patient. Choice of an appropriate Fc region and
methods of to incorporate such fragments are known in the art.
Incorporating a Fc region of an IgG into a polypeptide of interest
so as to increase its circulatory half-life, but so as not to lose
its biological activity can be accomplished using conventional
techniques known in the art such as, for example, described in U.S.
Pat. No. 6,096,871, which is hereby incorporated by reference in
its entirety. Fc portions of antibodies can be further modified to
increase half-life of the antigen-binding fragment in circulation
in blood when administered to a patient. Modifications can be
determined using conventional means in the art such as, for
example, described in U.S. Pat. No. 7,217,798, which is hereby
incorporated by reference in its entirety.
[0471] Other methods of improving the half-life of antibody-based
fusion proteins in circulation are also known such as, for example,
described in U.S. Pat. Nos. 7,091,321 and 6,737,056, each of which
is hereby incorporated by reference. Additionally, antibodies and
antigen-binding fragments thereof may be produced or expressed so
that they do not contain fucose on their complex N-glycoside-linked
sugar chains. The removal of the fucose from the complex
N-glycoside-linked sugar chains is known to increase effector
functions of the antibodies and antigen-binding fragments,
including but not limited to, antibody dependent cell-mediated
cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC).
Similarly, antibodies or antigen-binding fragments thereof that can
bind endoglin can be attached at their C-terminal end to all or
part of an immunoglobulin heavy chain derived from any antibody
isotype, e.g., IgG, IgA, IgE, IgD and IgM and any of the isotype
sub-classes, particularly IgG1, IgG2b, IgG2a, IgG3 and IgG4.
[0472] Additionally, the antibodies or antigen-binding fragments
described herein can also be modified so that they are able to
cross the blood-brain barrier. Such modification of the antibodies
or antigen-binding fragments described herein allows for the
treatment of brain diseases such as glioblastoma multiforme (GBM).
Exemplary modifications to allow proteins such as antibodies or
antigen-binding fragments to cross the blood-brain barrier are
described in US Patent Application Publication 2007/0082380 which
is hereby incorporated by reference in its entirety.
[0473] Glycosylation of immunoglobulins has been shown to have
significant effects on their effector functions, structural
stability, and rate of secretion from antibody-producing cells
(Leatherbarrow et al., Mol. Immunol. 22:407 (1985)). The
carbohydrate groups responsible for these properties are generally
attached to the constant (C) regions of the antibodies. For
example, glycosylation of IgG at asparagine 297 in the C.sub.H 2
domain is required for full capacity of IgG to activate the
classical pathway of complement-dependent cytolysis (Tao and
Morrison, J. Immunol. 143:2595 (1989)). Glycosylation of IgM at
asparagine 402 in the C.sub.H 3 domain is necessary for proper
assembly and cytolytic activity of the antibody (Muraoka and
Shulman, J. Immunol. 142:695 (1989)). Removal of glycosylation
sites as positions 162 and 419 in the C.sub.H 1 and C.sub.H3
domains of an IgA antibody led to intracellular degradation and at
least 90% inhibition of secretion (Taylor and Wall, Mol. Cell.
Biol. 8:4197 (1988)). Additionally, antibodies and antigen-binding
fragments thereof may be produced or expressed so that they do not
contain fucose on their complex N-glycoside-linked sugar chains.
The removal of the fucose from the complex N-glycoside-linked sugar
chains is known to increase effector functions of the antibodies
and antigen-binding fragments, including but not limited to,
antibody dependent cell-mediated cytotoxicity (ADCC) and complement
dependent cytotoxicity (CDC). These "defucosylated" antibodies and
antigen-binding fragments may be produced through a variety of
systems utilizing molecular cloning techniques known in the art,
including but not limited to, transgenic animals, transgenic
plants, or cell-lines that have been genetically engineered so that
they no longer contain the enzymes and biochemical pathways
necessary for the inclusion of a fucose in the complex
N-glycoside-linked sugar chains (also known as fucosyltransferase
knock-out animals, plants, or cells). Non-limiting examples of
cells that can be engineered to be fucosyltransferase knock-out
cells include CHO cells, SP2/0 cells, NS0 cells, and YB2/0
cells.
[0474] Glycosylation of immunoglobulins in the variable (V) region
has also been observed. Sox and Hood reported that about 20% of
human antibodies are glycosylated in the V region (Proc. Natl.
Acad. Sci. USA 66:975 (1970)). Glycosylation of the V domain is
believed to arise from fortuitous occurrences of the N-linked
glycosylation signal Asn-Xaa-Ser/Thr in the V region sequence and
has not been recognized in the art as playing a role in
immunoglobulin function.
[0475] Glycosylation at a variable domain framework residue can
alter the binding interaction of the antibody with antigen. The
present invention includes criteria by which a limited number of
amino acids in the framework or CDRs of a humanized immunoglobulin
chain are chosen to be mutated (e.g., by substitution, deletion, or
addition of residues) in order to increase the affinity of an
antibody.
[0476] Affinity for binding a pre-determined polypeptide antigen
can, generally, be modulated by introducing one or more mutations
into the V region framework, typically in areas adjacent to one or
more CDRs and/or in one or more framework regions. Typically, such
mutations involve the introduction of conservative amino acid
substitutions that either destroy or create the glycosylation site
sequences but do not substantially affect the hydropathic
structural properties of the polypeptide. Typically, mutations that
introduce a proline residue are avoided. Glycosylation of
antibodies and antigen-binding fragments thereof is further
described in U.S. Pat. No. 6,350,861, which is incorporated by
reference herein with respect to glycosylation.
[0477] Antibodies, or antigen-binding fragments thereof, can be
formulated for short-term delivery or extended (long term)
delivery.
[0478] Antibodies, or antigen-binding fragments thereof, that bind
to endoglin can also be used for purification of endoglin and/or to
detect endoglin levels in a sample or patient to detect or diagnose
a disease or disorder associated with endoglin as described in more
detail below.
[0479] Humanized antibodies, antigen-binding fragments, and binding
proteins which bind endoglin generated using such methods can be
tested for one or more of their binding affinity, avidity, and
neutralizing capabilities. Useful humanized antibodies,
antigen-binding fragments, and binding proteins can be used to
administer a patient to prevent, inhibit, manage or treat a
condition disease or disorder associated with angiogenesis.
[0480] Provided herein are methods of identifying humanized
antibodies or antigen-binding fragments thereof that bind to
endoglin. Antibodies and antigen-binding fragments can be evaluated
for one or more of binding affinity, association rates,
disassociation rates and avidity. In one aspect, antibodies can be
evaluated for their ability to neutralize the activity of endoglin
or a polypeptide in which the endoglin binding sequence is present.
Measurement binding affinity, association rates, disassociation
rates and avidity can be accomplished using art-recognized assays
including (Surface Plasmon Resonance), but not limited to, an
enzyme-linked-immunosorbent assay (ELISA), Scatchard Analysis,
BIACORE analysis, etc., as well as other assays commonly used and
known to those of ordinary skill in the art.
[0481] Measurement of binding of antibodies to endoglin and/or the
ability of the antibodies and antigen-binding fragments thereof,
for example, to inhibit angiogenesis, can be determined using, for
example, an enzyme-linked-immunosorbent assay (ELISA), a
competitive binding assay, an ELISPOT assay, or any other useful
assay known in the art. These assays are commonly used and
well-known to those of ordinary skill in the art.
[0482] In one non-limiting embodiment, an ELISA assay can be used
to measure the binding capability of specific antibodies or
antigen-binding fragments that bind to endoglin.
[0483] Assays, such as an ELISA, also can be used to identify
antibodies or antigen-binding fragments thereof which exhibit
increased specificity for endoglin in comparison to other
antibodies or antigen-binding fragments thereof. Assays, such as an
ELISA, also can be used to identify antibodies or antigen-binding
fragments thereof with bind to epitopes across one or more
polypeptides and across one or more species of endoglin. The
specificity assay can be conducted by running parallel ELISAs in
which a test antibodies or antigen-binding fragments thereof is
screened concurrently in separate assay chambers for the ability to
bind one or more epitopes on different species of the polypeptide
containing the endoglin epitopes to identify antibodies or
antigen-binding fragments thereof that bind to endoglin. Another
technique for measuring apparent binding affinity familiar to those
of skill in the art is a surface plasmon resonance technique
(analyzed on a BIACORE 2000 system) (Liljeblad, et al., Glyco. J.
2000, 17:323-329). Standard measurements and traditional binding
assays are described by Heeley, R. P., Endocr. Res. 2002,
28:217-229.
[0484] Humanized antibodies to endoglin can also be assayed for
their ability to treat various diseases and conditions associated
with angiogenesis, e.g., various forms of ocular diseases
characterized by angiogenesis/neovascularization (e.g., macular
degeneration, diabetic retinopathy), diabetic nephropathy, chronic
inflammatory diseases (e.g., IBD), rheumatoid arthritis,
osteoarthritis, and various forms of cancer (primary tumors and
metastases). Any suitable assay known to one of skill in the art
can be used to monitor such effects. Several such techniques are
described herein. In one example, the antibodies and
antigen-binding fragments described herein are assayed for their
ability to bind endoglin. In another example, affinity constants
for the antibodies and antigen-binding fragments described herein
are determined by surface plasmon resonance (SPR). In yet another
example, the antibodies and antigen-binding fragments described
herein are assayed for their effect on the inhibition of
angiogenesis.
II. Compositions
[0485] Each of the compounds described herein can be used as a
composition when combined with an acceptable carrier or excipient.
Such compositions are useful for in vitro or in vivo analysis or
for administration to a subject in vivo or ex vivo for treating a
subject with the disclosed compounds.
[0486] Thus pharmaceutical compositions can include, in addition to
active ingredient, a pharmaceutically acceptable excipient,
carrier, buffer, stabilizer or other materials well known to those
skilled in the art. Such materials should be non-toxic and should
not interfere with the efficacy of the active ingredient. The
precise nature of the carrier or other material will depend on the
route of administration.
[0487] Pharmaceutical formulations comprising a protein of
interest, e.g., an antibody or antigen-binding fragment, identified
by the methods described herein can be prepared for storage by
mixing the protein having the desired degree of purity with
optional physiologically acceptable carriers, excipients or
stabilizers (Remington's Pharmaceutical Sciences 16th edition,
Osol, A. Ed. (1980)), in the form of lyophilized formulations or
aqueous solutions. Acceptable carriers, excipients, or stabilizers
are those that are non-toxic to recipients at the dosages and
concentrations employed, and include buffers such as phosphate,
citrate, and other organic acids; antioxidants including ascorbic
acid and methionine; preservatives (such as octadecyldimethylbenzyl
ammonium chloride; hexamethonium chloride; benzalkonium chloride,
benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl
parabens such as methyl or propyl paraben; catechol; resorcinol;
cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less
than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g., Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.RTM., PLURONICS.RTM. or
polyethylene glycol (PEG).
[0488] Acceptable carriers are physiologically acceptable to the
administered patient and retain the therapeutic properties of the
compounds with/in which it is administered. Acceptable carriers and
their formulations are and generally described in, for example,
Remington' pharmaceutical Sciences (18th Edition, ed. A. Gennaro,
Mack Publishing Co., Easton, Pa. 1990). One exemplary carrier is
physiological saline. The phrase "pharmaceutically acceptable
carrier" as used herein means a pharmaceutically acceptable
material, composition or vehicle, such as a liquid or solid filler,
diluent, excipient, solvent or encapsulating material, involved in
carrying or transporting the subject compounds from the
administration site of one organ, or portion of the body, to
another organ, or portion of the body, or in an in vitro assay
system. Each carrier is acceptable in the sense of being compatible
with the other ingredients of the formulation and not injurious to
a subject to whom it is administered. Nor should an acceptable
carrier alter the specific activity of the subject compounds.
[0489] In one aspect, provided herein are pharmaceutically
acceptable or physiologically acceptable compositions including
solvents (aqueous or non-aqueous), solutions, emulsions, dispersion
media, coatings, isotonic and absorption promoting or delaying
agents, compatible with pharmaceutical administration.
Pharmaceutical compositions or pharmaceutical formulations
therefore refer to a composition suitable for pharmaceutical use in
a subject. The pharmaceutical compositions and formulations include
an amount of a compound described herein and a pharmaceutically or
physiologically acceptable carrier.
[0490] Compositions can be formulated to be compatible with a
particular route of administration (i.e., systemic or local). Thus,
compositions include carriers, diluents, or excipients suitable for
administration by various routes.
[0491] In another embodiment, the compositions can further
comprise, if needed, an acceptable additive in order to improve the
stability of the compounds in composition and/or to control the
release rate of the composition. Acceptable additives do not alter
the specific activity of the subject compounds. Exemplary
acceptable additives include, but are not limited to, a sugar such
as mannitol, sorbitol, glucose, xylitol, trehalose, sorbose,
sucrose, galactose, dextran, dextrose, fructose, lactose and
mixtures thereof. Acceptable additives can be combined with
acceptable carriers and/or excipients such as dextrose.
Alternatively, exemplary acceptable additives include, but are not
limited to, a surfactant such as polysorbate 20 or polysorbate 80
to increase stability of the peptide and decrease gelling of the
solution. The surfactant can be added to the composition in an
amount of 0.01% to 5% of the solution. Addition of such acceptable
additives increases the stability and half-life of the composition
in storage.
[0492] The pharmaceutical composition can be administered, for
example, by injection, including, but not limited to, subcutaneous,
subcutaneous, intravitreal, intradermal, intravenous,
intra-arterial, intraperitoneal, or intramuscular injection.
Excipients and carriers for use in formulation of compositions for
each type of injection are contemplated herein. The following
descriptions are by example only and are not meant to limit the
scope of the compositions. Compositions for injection include
aqueous solutions (where water soluble) or dispersions and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersion. For intravenous administration, suitable
carriers include physiological saline, bacteriostatic water,
Cremophor EL.TM. (BASF, Parsippany, N.J.) or phosphate buffered
saline (PBS). The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyetheylene glycol, and
the like), and suitable mixtures thereof. Fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. Antibacterial and
antifungal agents include, for example, parabens, chlorobutanol,
phenol, ascorbic acid and thimerosal. Isotonic agents, for example,
sugars, polyalcohols such as manitol, sorbitol, and sodium chloride
may be included in the composition. The resulting solutions can be
packaged for use as is, or lyophilized; the lyophilized preparation
can later be combined with a sterile solution prior to
administration. For intravenous, injection, or injection at the
site of affliction, the active ingredient will be in the form of a
parenterally acceptable aqueous solution which is pyrogen-free and
has suitable pH, isotonicity and stability. Those of relevant skill
in the art are well able to prepare suitable solutions using, for
example, isotonic vehicles such as Sodium Chloride Injection,
Ringer's Injection, Lactated Ringer's Injection. Preservatives,
stabilizers, buffers, antioxidants and/or other additives may be
included, as needed. Sterile injectable solutions can be prepared
by incorporating an active ingredient in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
ingredient into a sterile vehicle which contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze drying which yields a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
[0493] Compositions can be conventionally administered
intravitreally, sub-cutaneous, or via intravitreal implant.
[0494] Compositions can be conventionally administered
intravenously, such as by injection of a unit dose, for example.
For injection, an active ingredient can be in the form of a
parenterally acceptable aqueous solution which is substantially
pyrogen-free and has suitable pH, isotonicity and stability. One
can prepare suitable solutions using, for example, isotonic
vehicles such as Sodium Chloride Injection, Ringer's Injection,
Lactated Ringer's Injection. Preservatives, stabilizers, buffers,
antioxidants and/or other additives may be included, as required.
Additionally, compositions can be administered via aerosolization.
(Lahn et al., Aerosolized Anti-T-cell-Receptor Antibodies Are
Effective against Airway Inflammation and Hyperreactivity, Int.
Arch. Allegery Immuno., 134: 49-55 (2004)).
[0495] In one embodiment, the composition is lyophilized, for
example, to increase shelf-life in storage. When the compositions
are considered for use in medicaments or any of the methods
provided herein, it is contemplated that the composition can be
substantially free of pyrogens such that the composition will not
cause an inflammatory reaction or an unsafe allergic reaction when
administered to a human patient. Testing compositions for pyrogens
and preparing compositions substantially free of pyrogens are well
understood to one or ordinary skill of the art and can be
accomplished using commercially available kits.
[0496] Acceptable carriers can contain a compound that stabilizes,
increases or delays absorption or clearance. Such compounds
include, for example, carbohydrates, such as glucose, sucrose, or
dextrans; low molecular weight proteins; compositions that reduce
the clearance or hydrolysis of peptides; or excipients or other
stabilizers and/or buffers. Agents that delay absorption include,
for example, aluminum monostearate and gelatin. Detergents can also
be used to stabilize or to increase or decrease the absorption of
the pharmaceutical composition, including liposomal carriers. To
protect from digestion the compound can be complexed with a
composition to render it resistant to acidic and enzymatic
hydrolysis, or the compound can be complexed in an appropriately
resistant carrier such as a liposome. Means of protecting compounds
from digestion are known in the art (see, e.g., Fix (1996) Pharm
Res. 13:1760 1764; Samanen (1996) J. Pharm. Pharmacol. 48:119 135;
and U.S. Pat. No. 5,391,377, describing lipid compositions for oral
delivery of therapeutic agents).
[0497] The phrase "pharmaceutically acceptable" refers to molecular
entities and compositions that are physiologically tolerable and do
not typically produce an allergic or similar untoward reaction,
such as gastric upset, dizziness and the like, when administered to
a human.
[0498] The term "unit dose" when used in reference to a therapeutic
composition refers to physically discrete units suitable as unitary
dosage for humans, each unit containing a predetermined quantity of
active material calculated to produce the desired therapeutic
effect in association with the required diluent; i.e., carrier, or
vehicle.
[0499] The compositions can be administered in a manner compatible
with the dosage formulation, and in a therapeutically effective
amount. The quantity to be administered depends on the subject to
be treated, capacity of the subject's immune system to utilize the
active ingredient, and degree of binding capacity desired. Precise
amounts of active ingredient required to be administered depend on
the judgment of the practitioner and are peculiar to each
individual. Suitable regimes for initial administration and booster
shots are also variable, but are typified by an initial
administration followed by repeated doses at one or more hour
intervals by a subsequent injection or other administration.
Alternatively, continuous intravenous infusion sufficient to
maintain concentrations in the blood are contemplated.
[0500] One embodiment contemplates the use of the compositions
described herein to make a medicament for treating a condition,
disease or disorder described herein. Medicaments can be formulated
based on the physical characteristics of the patient/subject
needing treatment, and can be formulated in single or multiple
formulations based on the stage of the condition, disease or
disorder. Medicaments can be packaged in a suitable package with
appropriate labels for the distribution to hospitals and clinics
wherein the label is for the indication of treating a subject
having a disease described herein. Medicaments can be packaged as a
single or multiple units. Instructions for the dosage and
administration of the compositions can be included with the
packages as described below. The invention is further directed to
medicaments of a humanized anti-endoglin antibody or antigen
binding fragment thereof described hereinabove and a
pharmaceutically acceptable carrier.
[0501] Provided herein are compositions of humanized antibodies and
antigen-binding fragments thereof that bind endoglin and include
those such as described elsewhere herein. Humanized antibodies and
antigen-binding fragments thereof that bind endoglin as described
herein can be used for the treatment of various forms of ocular
diseases characterized by angiogenesis/neovascularization (e.g.,
macular degeneration, diabetic retinopathy), diabetic nephropathy,
chronic inflammatory diseases (e.g., IBD), rheumatoid arthritis,
osteoarthritis, and various forms of cancer (primary tumors and
metastases).
[0502] A composition (an antibody or an antigen-binding fragment
described herein) can be administered alone or in combination with
a second composition either simultaneously or sequentially
dependent upon the condition to be treated. In one embodiment, a
second therapeutic treatment is an angiogenesis inhibitor (as
described herein). When two or more compositions are administered,
the compositions can be administered in combination (either
sequentially or simultaneously). A composition can be administered
in a single dose or multiple doses.
[0503] In one embodiment of the present invention, the compositions
are formulated to be free of pyrogens such that they are acceptable
for administration to human patients. Testing compositions for
pyrogens and preparing pharmaceutical compositions free of pyrogens
are well understood to one of ordinary skill in the art.
[0504] One embodiment of the present invention contemplates the use
of any of the compositions of the present invention to make a
medicament for treating a disorder of the present invention.
Medicaments can be formulated based on the physical characteristics
of the patient/subject needing treatment, and can be formulated in
single or multiple formulations based on the disorder. Medicaments
of the present invention can be packaged in a suitable
pharmaceutical package with appropriate labels for the distribution
to hospitals and clinics wherein the label is for the indication of
treating a disorder as described herein in a subject. Medicaments
can be packaged as a single or multiple units. Instructions for the
dosage and administration of the pharmaceutical compositions of the
present invention can be included with the pharmaceutical
packages.
III. Methods of Use
[0505] Provided herein is a method of inducing a response in a
patient (human or non-human) by administering to the patient a
composition of an antibody or antigen-binding fragment thereof that
preferentially binds to endoglin. The binding site to which the
antibody binds can be a continuous or conformation/dis-continuous
epitope.
[0506] An effective response of the present invention is achieved
when the patient experiences partial or total alleviation or
reduction of signs or symptoms of illness, and specifically
includes, without limitation, prolongation of survival and/or
visual acuity. The expected progression-free survival times may be
measured in months to years, depending on prognostic factors
including the number of relapses, stage of disease, and other
factors. Prolonging survival includes without limitation times of
at least 1 month (mo), about at least 2 mos., about at least 3
mos., about at least 4 mos., about at least 6 mos., about at least
1 year, about at least 2 years, about at least 3 years, etc.
Overall survival can be also measured in months to years.
Alternatively, an effective response may be that a patient's
symptoms remain static. Further indications of treatment of
indications are described in more detail below.
[0507] Compositions of antibodies and antigen-binding fragments
described herein can be used as non-therapeutic agents (e.g., as
affinity purification agents). Generally, in one such embodiment, a
protein of interest is immobilized on a solid phase such a Sephadex
resin or filter paper, using conventional methods known in the art.
The immobilized protein is contacted with a sample containing the
target of interest (or fragment thereof) to be purified, and
thereafter the support is washed with a suitable solvent that will
remove substantially all the material in the sample except the
target protein, which is bound to the immobilized antibody.
Finally, the support is washed with another suitable solvent, such
as glycine buffer, pH 5.0, which will release the target protein.
In addition to purification, compositions can be used for
detection, diagnosis and therapy of diseases and disorders
associated with endoglin and angiogenesis.
[0508] The term "contacting" as used herein refers to adding
together a solution or composition of a compound with a liquid
medium bathing the polypeptides, cells, tissue or organ from an
organism. Alternately, "contacting" refers to mixing together a
solution or composition of a compound, with a liquid such as blood,
serum, or plasma derived from an organism. For in vitro
applications, a composition can also comprise another component,
such as dimethyl sulfoxide (DMSO). DMSO facilitates the uptake of
the compounds or solubility of the compounds. The solution
comprising the test compound may be added to the medium bathing the
cells, tissues, or organs, or mixed with another liquid such as
blood, by utilizing a delivery apparatus, such as a pipette-based
device or syringe-based device. For in vivo applications,
contacting can occur, for example, via administration of a
composition to a patient by any suitable means; compositions with
pharmaceutically acceptable excipients and carriers have been
described in more detail above.
[0509] A "patient" (e.g., a mammal such as a human or a non-human
animal such as a primate, rodent, cow, horse, pig, sheep, etc.)
according to one embodiment of the present application, is a mammal
who exhibits one or more clinical manifestations and/or symptoms of
a disease or disorder described herein. In certain situations, the
patient may be asymptomatic and yet still have clinical
manifestations of the disease or disorder. An antibody or
antigen-binding fragment thereof can be conjugated to a therapeutic
moiety or be a fusion protein containing a therapeutic moiety. An
antibody or antigen-binding fragment thereof can be conjugated to a
detectable moiety or be a fusion protein containing a detectable
moiety. In one embodiment, the antibody or antigen-binding fragment
thereof can be conjugated to both a therapeutic moiety and a
detectable moiety. An antibody or antigen-binding fragment thereof
can be conjugated to, or recombinantly engineered with, an affinity
tag (e.g., a purification tag). Affinity tags such as, for example,
His6 tags (SEQ ID NO: 85) are conventional in the art.
[0510] Antibodies or antigen-binding fragments thereof provided
herein are such that they can be conjugated or linked to a
therapeutic moiety and/or an imaging or a detectable moiety and/or
an affinity tag. Methods for conjugating or linking polypeptides
are well known in the art. Associations (binding) between compounds
and labels include any means known in the art including, but not
limited to, covalent and non-covalent interactions, chemical
conjugation as well as recombinant techniques.
[0511] A. Binding of Endoglin and Angiogenesis
[0512] Endoglin (CD105) is expressed on the cell surface as a 180
kDa homodimeric transmembrane protein. The external domain binds
TGF-.beta.1 and -3 isoforms with high affinity (50 nM), and the
transmembrane and the intracellular domains of CD105 share a 71%
sequence similarity with betaglycan. The human CD105 gene is
located on chromosome 9q34, identified using fluorescence in situ
hybridization, and the coding region contains 14 exons, and two
different isoforms (L and S) of CD105 with capacity to bind
TGF-.beta. have been characterized. The L-CD105 consists of 633
amino acid residues with 47 amino acid residues in the cytoplasmic
tail as opposed to the S-CD105, which consists of 600 amino acid
residues with a 14 amino acid cytoplasmic tail. However, L-CD105 is
the predominant form. CD105 is constitutively phosphorylated in
endothelial cells, mainly on serine and threonine residues, and
this phosphorylation is due to the constitutively active TGF-.beta.
RII within the cell. TGF-.beta. binding to CD105 results in
down-regulation of phosphorylation, similar to effects seen with
protein kinase C inhibitors. The human CD105 amino acid sequence
contains the tripeptide arginine-glycine-aspartic acid (RGD)
located in an exposed region of the extracellular domain. The RGD
peptide is a key recognition structure found on ECM proteins such
as fibronectin, vitronectin, von Willebrand factor (vWF), type I
collagen, and fibrinogen and is recognized by cell surface
integrins. Integrin adhesion has been implicated in hemostasis,
thrombosis, angiogenesis and inflammation, processes in which the
endothelium plays a critical role. (Duff et al., FASEB J.,
17:984-992 (2003)).
[0513] CD105 is a member of the TGF-.beta. receptor family that is
expressed by proliferating endothelial cells. Normal levels of
CD105 are needed for endothelial cell proliferation. CD105
expression is increased by cellular hypoxia through the production
of hypoxia-inducible factor-1-.alpha. (HIF-1-.alpha.) and protects
hypoxic cells from apoptosis. Several functions of CD105 are
associated with TGF-.beta. signaling. TGF-.beta. signals through
heterodimeric receptors consisting of serine kinases, receptor I
(RI), and receptor II (RII). Binding of TGF-.beta. to the external
domains of the receptor unmasks the cytoplasmic RII kinase activity
that phosphorylates the TGF-.beta. RI, which can then interact with
downstream signalers such as the Smad proteins. CD105 forms part of
the TGF-.beta. receptor complex but it can exist independently on
the cell surface. In many cells in vitro, CD105 suppresses
TGF-.beta. signaling.
[0514] CD105 also binds other growth factors such as activin A and
bone morphogenic proteins (BMP) -10, -9, -7 and -2. Binding of
TGF-.beta. or other growth factor ligands to CD105 requires the
presence of at least the receptor RII, and it cannot bind ligands
by itself. CD105 association with receptors does not alter their
affinity for the ligand itself. Upon association, the cytoplasmic
domain of CD105 is phosphorylated by TGF-.beta. RI and TGF-.beta.
RII; then TGF-.beta. RI, but not TGF-.beta. RII, kinase dissociates
from the receptor complex.
[0515] CD105 expression inhibits phosphorylation levels of
TGF-.beta. RII but increases that of TGF-.beta. RI, resulting in
increased phosphorylation of Smad 2 but not Smad 3. Since Smad 2
can interact with a variety of transcription factors,
co-activators, and suppressors, phosphorylated Smad 2 may act as an
integrator of multiple signals to modulate gene transcription.
Thus, CD105 modulates TGF-.beta. functions via interaction with
TGF-.beta. RI and TGF-.beta. RII and modifies the phosphorylation
of downstream Smad proteins.
[0516] CD105 acts to modulate signaling of multiple kinase receptor
complexes of the TGF-.beta. superfamily, including TGF-.beta.
receptors (TGF-.beta.R), activin receptor-like kinases (ALK) and
activin receptors. In the absence of CD105, activation of
TGF-.beta. receptors results in phosphorylation of SMAD proteins
(SMAD 2 and 3) that inhibit endothelial cell growth. However,
activation of CD105 by TGF-.beta. modulates SMAD protein
phosphorylation (including the phosphorylation of SMAD 1, 5 and 8).
The end result is release of the growth inhibitory effects of
TGF-.beta. receptor activation on endothelial cells (see FIG. 3).
Not surprisingly, prevention of CD105 activation by anti-CD105
antibody or antisense oligonucleotide acts synergistically with
TGF-.beta. to suppress endothelial cell growth.
[0517] The CD105 promoter is 2.6 kb in length but does not contain
TATA or CAAT transcription initiation boxes. However, it has two
GC-rich regions, consensus motifs for Sp1, ets, GATA, AP-2,
NGF-.beta., and Mad, as well as TGF-.beta. response elements.
Nonetheless, CD105 has a relatively restricted cellular
distribution. The basal level of transcription appears to require
an ets site at position -68 and the Sp1 sites, but the relative
restriction of expression, for example, to endothelial cells,
appears to involve multiple regulatory regions, in particular, one
at -1294 to -932 and another very close to the transcription
initiation site. CD105 is up-regulated by TGF-.beta., and this has
been shown to require a Sp1 site at -37 to -29, also involving one
or more juxtaposed upstream SBE sites binding Smads 3 and/or 4
(which are activated by TGF-.beta. signaling). Hypoxia is a common
feature of ischemic tissues and tumors, and is a potent stimulator
for CD105 gene expression in vascular endothelial cells (ECs). Such
an effect is potentiated in combination with TGF-.beta. 1. The
up-regulated CD105 can exert a self-protective role in ECs under
hypoxic stress.
[0518] Vascular EC are the major source of CD105. Other cell types
including vascular smooth muscle cells, fibroblasts, macrophages,
leukemic cells of pre-B and myelomonocytic origin, and erythroid
precursors express CD105 to a lesser extent.
[0519] CD105 is involved in angiogenesis. Antisense experiments
have demonstrated that suppression of CD105 expression in HUVEC
results in marked inhibition of in vitro angiogenesis in
combination with TGF-.beta.1, indicating that CD105 is a
proangiogenic component in the endothelial cells. Further evidence
of the important role of CD105 in angiogenesis comes from CD105
knockout mice. The CD105 null mice exhibit multiple vascular and
cardiac defects leading to death at an early embryonic stage.
Severe vascular impairments observed in CD105 null mice indicate
that CD105 is required for the formation of mature blood vessels in
the extraembryonic vasculature, further confirming the direct role
of endoglin in angiogenesis.
[0520] Endoglin, also known as, inter alia, CD105 or edg-1, is a
type I homodimeric membrane glycoprotein which is expressed at high
levels in proliferating vascular endothelial cells. Thus, endoglin
is primarily a proliferation-associated marker for endothelial
cells undergoing active angiogenesis. However, there may be limited
expression of endoglin by the vascular endothelium of normal
tissues. Human endoglin is known to specifically bind transforming
growth factor-.beta. (TGF-.beta.), and the deduced amino acid
sequence of endoglin has strong homology to .beta.-glycan, a type
of TGF-.beta. receptor.
[0521] Endoglin (EDG) has been targeted in antibody-based methods
of reducing tumor vasculature, as EDG is a proliferation-associated
antigen on endothelial and leukemia cells. Its expression is
up-regulated in tumor-associated vascular endothelium, and EDG is
essential for angiogenesis. Angiogenesis includes the formation of
new capillary blood vessels leading to neovascularization as well
as the maintenance of the existing vasculature. It is a complex
process which includes a series of sequential steps including
endothelial cell-mediated degradation of vascular basement membrane
and interstitial matrices, migration of endothelial cells,
proliferation of endothelial cells, and formation of capillary
loops by endothelial cells.
[0522] Provided herein are humanized antibodies that bind endoglin.
Endoglin can be found on cells that comprise and support existing
vasculature as well as cells that are promoting the growth of, and
become part of, new vasculature. The antibodies can bind endoglin
and thereby inhibit angiogenesis, inhibit the existing vasculature
or the maintenance of the existing vasculature, and/or inhibit
small vessel dilation. In addition to their use for purification of
endoglin, these antibodies are useful for purification, detection
and diagnostic purposes as well as therapeutic purposes. The
antibodies provided herein can be used for the formulation of
medicaments for the treatment a variety of conditions and diseases,
methods to treat said conditions and diseases and methods of
detection or diagnosis.
[0523] Murine monoclonal antibodies (mAbs) have been raised against
endoglin which modulate endoglin activity and thereby inhibit
angiogenesis and/or inhibit vasodilation of small blood vessels.
These murine antibodies are described in U.S. Pat. Nos. 5,928,641,
6,200,566, 6,190,660, and 7,097,836, each of which is hereby
incorporated in their entirety. Additionally, the ex vivo and in
vivo efficiency of a number of these antibodies has been
demonstrated; monoclonal antibodies that bind endoglin are of
interest as endoglin modulating compounds. Therapeutic use of
murine antibodies is not feasible, however, as administration of
the murine antibodies has a number of limitations, including
immunogenicity in, for example, the form of human anti-mouse
antibodies (HAMA).
[0524] "Angiogenesis" is used herein to include all aspects of
blood vessel maintenance and development. Thus, angiogenesis
includes the formation of new capillary blood vessels leading to
neovascularization as well as the maintenance and control of the
existing vasculature and small blood vessels. Angiogenesis is a
complex process which includes a series of sequential steps
including endothelial cell-mediated degradation of vascular
basement membrane and interstitial matrices, migration of
endothelial cells, proliferation of endothelial cells, and
formation of capillary loops by endothelial cells. Angiogenesis is
inclusive of the growth and/or development of new blood vessels
(also referred to as neovascularization), dilation of the small
vessels, excessive or prolonged vascular growth, and maintenance of
the existing vasculature. Endoglin is known to be involved in the
regulation of angiogenesis and is believed to be involved in
multiple biochemical pathways related to the induction of
angiogenesis. (Duff et al., FASEB J., 17:984-992 (2003); Bernabeu
et al., J. Cell. Biochem., 102(6):1375-1388 (2007)).
[0525] As used herein, the terms "angiogenesis inhibitory,"
"angiogenesis inhibiting" or "anti-angiogenic" include inhibition
of vasculogenesis, and are intended to mean affecting a decrease in
the extent, amount, or rate of neovascularization. Effecting a
decrease in the extent, amount, or rate of endothelial cell
proliferation or migration in the tissue is a specific example of
inhibiting angiogenesis.
[0526] The term "angiogenesis inhibitory composition" refers to a
composition which inhibits angiogenesis-mediated processes such as
endothelial cell migration, proliferation, tube formation and
subsequently leading to the inhibition of the generation of new
blood vessels from existing ones, and consequently affects
angiogenesis-dependent conditions.
[0527] The term "angiogenesis-associated disease" is used herein,
for purposes of the specification and claims, to mean certain
pathological processes in humans where angiogenesis is abnormally
prolonged. This further includes angiogenesis conditions and
diseases, such as those diseases and conditions related to, caused
by, or associated with angiogenesis. Non-limiting examples of such
diseases include various forms of ocular diseases characterized by
angiogenesis/neovascularization (e.g., macular degeneration,
diabetic retinopathy), diabetic nephropathy, chronic inflammatory
diseases (e.g., IBD), rheumatoid arthritis, osteoarthritis, and
various forms of cancers and metastasis. The antibodies and
antigen-binding fragments thereof described herein can be used to
treat an angiogenesis-associated disease by binding endoglin and
inhibiting angiogenesis.
[0528] The term "anti-angiogenic therapy" is used herein, for
purposes of the specification and claims, to mean therapy targeted
to cells and/or vasculature expressing endoglin (expressed at
higher levels on proliferating vasculature as compared to quiescent
vasculature); this further includes therapy that is directed
against angiogenesis (i.e., the formation of new capillary blood
vessels leading to neovascularization), therapy that is directed
against existing vasculature and/or excessive vascularization or
blood vessel growth, therapy directed towards the dilation of small
vessels, and therapy directed to a disease or condition (e.g.,
vascular targeting therapy). Exemplary diseases or conditions
contemplated within the invention include, but are not limited to,
various forms of ocular diseases characterized by
angiogenesis/neovascularization (e.g., macular degeneration,
diabetic retinopathy), diabetic nephropathy, chronic inflammatory
diseases (e.g., IBD), rheumatoid arthritis, osteoarthritis, and
various forms of cancer, solid tumors, and metastases.
[0529] "Ocular disease characterized by neovascularization" is used
herein, for purposes of the specification and claims, to mean any
ocular disease caused by, or resulting in, increased angiogenesis
within any portion of the eye, including the retina, cornea, pupil,
iris, vitreous humor or aqueous humor. Such diseases include for
example, age-related macular degeneration, diabetic retinopathy,
non-diabetic retinopathy, choroidal neovascularization (CNV) and
subretinal neovascularization (SRN or SRNV) and neoplasms of the
eye.
[0530] B. Diagnostic Applications
[0531] Humanized anti-endoglin antibodies and fragments thereof can
be used for in vivo and in vitro detection, diagnostic and/or
monitoring purposes. Endoglin is believed to be involved in
multiple diseases and disorders as described further below.
Treatment of endoglin related diseases and conditions depends, in
part, upon their diagnosis, and the antibodies and antigen-binding
fragments thereof described herein are useful for the diagnosis of
excess endoglin or for diagnosis for diseases and conditions
associated with endoglin activity.
[0532] Provided herein is method of detecting levels of endoglin in
a sample or a subject comprising (i) contacting an antibody or
antigen binding fragment described herein with the sample or
subject, and (ii) detecting a complex of the antibody or
antigen-binding fragment thereof and endoglin.
[0533] Provided herein is a method of imaging or diagnosing
angiogenesis or an angiogenic-dependent disease or disorder,
comprising contacting a composition of an antibody or
antigen-binding fragment thereof as described herein with a sample.
The sample can be, for example, blood, serum, plasma, platelets,
biopsy fluid, spinal tap fluid, meninges and urine. Imaging or
diagnosis method can occur in an in vitro assay. Alternatively,
when contacting is by administration of the composition to a
patient, the angiogenesis or angiogenic-dependent disease or
disorder is imaged or diagnosed in vivo.
[0534] In one embodiment, the antibody or antigen-binding fragment
further comprises a detectable moiety. Detection can occur in
vitro, in vivo or ex vivo. In vitro assays for the detection and/or
determination (quantification, qualification, etc.) of endoglin
with the antibodies or antigen-binding fragments thereof include
but are not limited to, for example, ELISAs, RIAs and western
blots. In vitro detection, diagnosis or monitoring of endoglin can
occur by obtaining a sample (e.g., a blood sample) from a patient
and testing the sample in, for example, a standard ELISA assay. For
example, a 96-well microtiter plate can be coated with an antibody
or antigen-binding fragment thereof described herein, washed and
coating with PBS-Tween/BSA to inhibit non-specific binding. The
blood sample can be serially diluted and placed in duplicate wells
compared to a serially-diluted standard curve of endoglin. After
incubating and washing the wells, an anti-endoglin antibody labeled
with biotin can be added, followed by addition of
streptavidin-alkaline phosphatase. The wells can be washed and a
substrate (horseradish peroxidase) added to develop the plate. The
plate can be read using a conventional plate reader and
software.
[0535] When detection occurs in vivo, contacting occurs via
administration of the antibody or antigen binding fragment using
any conventional means such as those described elsewhere herein. In
such methods, detection of endoglin in a sample or a subject can be
used to diagnose a disease or disorder associated with, or
correlated with the activity of endoglin such as those diseases and
disorders described herein.
[0536] In the in vivo detection, diagnosis or monitoring of
endoglin, a patient is administered an antibody or antigen-binding
fragment that binds to endoglin, which antibody or antigen-binding
fragment is bound to a detectable moiety. The detectable moiety can
be visualized using art-recognized methods such as, but not limited
to, magnetic resonance imaging (MRI), fluorescence, radioimaging,
light sources supplied by endoscopes, laparoscopes, or
intravascular catheter (i.e., via detection of photoactive agents),
photoscanning, positron emission tomography (PET) scanning, whole
body nuclear magnetic resonance (NMR), radioscintography, single
photon emission computed tomography (SPECT), targeted near infrared
region (NIR) scanning, X-ray, ultrasound, etc. such as described,
for example, in U.S. Pat. No. 6,096,289, U.S. Pat. No. 7,115,716,
U.S. Pat. No. 7,112,412, U.S. Patent Application No. 20030003048
and U. S. Patent Application No. 20060147379, each of which is
incorporated herein in its entirety by reference. Labels for
detecting compounds using such methods are also known in the art
and described in such patents and applications and are incorporated
herein by reference. Visualization of the detectable moiety can
allow for detection, diagnosis, and/or monitoring of a condition or
disease associated with endoglin and/or angiogenesis.
[0537] Additional diagnostic assays that utilize antibodies
specific to the desired target protein, i.e., endoglin, are known
in the art and are also contemplated herein.
[0538] Non-limiting conditions, diseases and disorders to be
considered for these methods include, but are not limited to, those
associated with angiogenesis such as, for example, various forms of
ocular diseases characterized by angiogenesis/neovascularization
(e.g., macular degeneration, diabetic retinopathy), diabetic
nephropathy, chronic inflammatory diseases (e.g., IBD), rheumatoid
arthritis, osteoarthritis, and various forms of cancer (primary
tumors and metastases). In the detection, diagnosis or monitoring
of such diseases, the subject patient is administered a composition
of an antibody or antigen-binding fragment thereof described
herein, which antibody or antigen-binding fragment thereof is
conjugated to a detectable moiety. The moiety can be visualized
using art-recognized methods such as those described above.
Visualization of the detectable moiety can allow for detection,
diagnosis, and/or monitoring of such conditions and diseases.
[0539] For in vitro detection methods, samples to be obtained from
a patient include, but are not limited to, blood, tissue biopsy
samples and fluid therefrom.
[0540] Thus, the present invention provides humanized antibodies
and antigen-binding fragments thereof against endoglin which are
useful for detecting or diagnosing levels of endoglin associated
with a disease or disorder, potentially indicating need for
therapeutic treatment. In certain embodiments, the antibodies
comprise a humanized anti-endoglin antibody described herein. In
other embodiments the antibody further comprises a second agent.
Such an agent can be a molecule or moiety such as, for example, a
reporter molecule or a detectable label. Detectable labels/moieties
for such detection methods are known in the art and are described
in more detail below. Reporter molecules are any moiety which can
be detected using an assay. Non-limiting examples of reporter
molecules which have been conjugated to polypeptides include
enzymes, radiolabels, haptens, fluorescent labels, phosphorescent
molecules, chemiluminescent molecules, chromophores, luminescent
molecules, photoaffinity molecules, colored particles or ligands,
such as biotin. Detectable labels include compounds and/or elements
that can be detected due to their specific functional properties,
and/or chemical characteristics, the use of which allows the
polypeptide to which they are attached to be detected, and/or
further quantified if desired. Many appropriate detectable
(imaging) agents are known in the art, as are methods for their
attachment to polypeptides (see, for e.g., U.S. Pat. Nos.
5,021,236; 4,938,948; and 4,472,509, each of which is hereby
incorporated by reference).
[0541] Methods of joining polypeptides such as antibodies with
detectable moieties are known in the art and include, for example,
recombinant DNA technology to form fusion proteins and conjugation
(e.g., chemical conjugation). Methods for preparing fusion proteins
by chemical conjugation or recombinant engineering are well-known
in the art. Methods of covalently and non-covalently linking
components are also known in the art. See, e.g., Williams (1995)
Biochemistry 34:1787 1797; Dobeli (1998) Protein Expr. Purif.
12:404-414; and Kroll (1993) DNA Cell. Biol. 12: 441-453.
[0542] It may be necessary, in some instances, to introduce an
unstructured polypeptide linker region between a label or a moiety
and one or more portion of the antibodies, antigen-binding
fragments or binding proteins described herein. A linker can
facilitate enhanced flexibility, and/or reduce steric hindrance
between any two fragments. The linker can also facilitate the
appropriate folding of each fragment to occur. The linker can be of
natural origin, such as a sequence determined to exist in random
coil between two domains of a protein. One linker sequence is the
linker found between the C-terminal and N-terminal domains of the
RNA polymerase a subunit. Other examples of naturally occurring
linkers include linkers found in the 1CI and LexA proteins.
[0543] Within a linker, an amino acid sequence can be varied based
on the characteristics of the linker as determined empirically or
as revealed by modeling. Considerations in choosing a linker
include flexibility of the linker, charge of the linker, and
presence of some amino acids of the linker in the
naturally-occurring subunits. The linker can also be designed such
that residues in the linker contact deoxyribose nucleic acid (DNA),
thereby influencing binding affinity or specificity, or to interact
with other proteins. In some cases, such as when it is necessary to
span a longer distance between subunits or when the domains must be
held in a particular configuration, the linker can, optionally,
contain an additional folded domain. In some embodiments, the
design of a linker can involve an arrangement of domains which
requires the linker to span a relatively short distance, e.g., less
than about 10 Angstroms (.ANG.). However, in certain embodiments,
linkers span a distance of up to about 50 Angstroms.
[0544] Within the linker, the amino acid sequence can be varied
based on the characteristics of the linker as determined
empirically or as revealed by modeling. Considerations in choosing
a linker include flexibility of the linker, charge of the linker,
and presence of some amino acids of the linker in the
naturally-occurring subunits. The linker can also be designed such
that residues in the linker contact DNA, thereby influencing
binding affinity or specificity, or to interact with other
proteins. In some cases, when it is necessary to span a longer
distance between subunits or when the domains must be held in a
particular configuration, the linker can optionally contain an
additional folded domain.
[0545] Methods for coupling polypeptides (free or cell-bound) to
beads are known in the art. Methods for selecting coupled
polypeptides or cells displaying a polypeptide are also known in
the art. Briefly, paramagnetic polystyrene microparticles are
commercially available (Spherotech, Inc., Libertyville, Ill.;
Invitrogen, Carlsbad, Calif.) that couple peptides to microparticle
surfaces that have been modified with functional groups or coated
with various antibodies or ligands such as, for example, avidin,
streptavidin or biotin.
[0546] The paramagnetic property of microparticles allows them to
be separated from solution using a magnet. The microparticles can
be easily re-suspended when removed from the magnet. Polypeptides
can be coupled to paramagnetic polystyrene microparticles coated
with a polyurethane layer in a tube. The hydroxy groups on the
microparticle surface are activated by reaction with
p-toluensulphonyl chloride (Nilsson K and Mosbach K.
"p-Toluenesulfonyl chloride as an activating agent of agarose for
the preparation of immobilized affinity ligands and proteins." Eur.
J. Biochem. 1980:112: 397-402). Alternatively, paramagnetic
polystyrene microparticles containing surface carboxylic acid can
be activated with a carbodiimide followed by coupling to a
polypeptide, resulting in a stable amide bond between a primary
amino group of the polypeptide and the carboxylic acid groups on
the surface of the microparticles (Nakajima N and Ikade Y,
Mechanism of amide formation by carbodiimide for bioconjugation in
aqueous media, Bioconjugate Chem. 1995, 6(1): 123-130; Gilles M A,
Hudson A Q and Borders C L Jr, Stability of water-soluble
carbodiimides in aqueous solution, Anal Biochem. 1990 Feb. 1;
184(2):244-248; Sehgal D and Vijay I K, a method for the high
efficiency of water-soluble carbodiimide-mediated amidation, Anal
Biochem. 1994 April; 218(1):87-91; Szajani B et al., Effects of
carbodiimide structure on the immobilization of enzymes, Appl
Biochem Biotechnol. 1991 August; 30(2): 225-231). Another option is
to couple biotinylated polypeptides to paramagnetic polystyrene
microparticles whose surfaces have been covalently linked with a
monolayer of streptavidin. (Argarana C E, Kuntz I D, Birken S, Axel
R, Cantor C R. Molecular cloning and nucleotide sequence of the
streptavidin gene. Nucleic Acids Res. 1986; 14(4):1871-82; Pahler
A, Hendrickson W A, Gawinowicz Kolks M A, Aragana C E, Cantor C R.
Characterization and crystallization of core streptavidin. J Biol
Chem 1987:262(29):13933-13937).
[0547] Polypeptides can be conjugated to a wide variety of
fluorescent dyes, quenchers and haptens such as fluorescein,
R-phycoerythrin, and biotin. Conjugation can occur either during
polypeptide synthesis or after the polypeptide has been synthesized
and purified. Biotin is a small (244 kilodaltons) vitamin that
binds with high affinity to avidin and streptavidin proteins and
can be conjugated to most peptides without altering their
biological activities. Biotin-labeled polypeptides are easily
purified from unlabeled polypeptides using immobilized streptavidin
and avidin affinity gels, and streptavidin or avidin-conjugated
probes can be used to detect biotinylated polypeptides in, for
example, ELISA, dot blot or Western blot applications.
N-hydroxysuccinimide esters of biotin are the most commonly used
type of biotinylation agent. N-hydroxysuccinimide-activated biotins
react efficiently with primary amino groups in physiological
buffers to form stable amide bonds. Polypeptides have primary
amines at the N-terminus and can also have several primary amines
in the side chain of lysine residues that are available as targets
for labeling with N-hydroxysuccinimide-activated biotin reagents.
Several different N-hydroxysuccinimide esters of biotin are
available, with varying properties and spacer arm length (Pierce,
Rockford, Ill.). The sulfo-N-hydroxysuccinimide ester reagents are
water soluble, enabling reactions to be performed in the absence of
organic solvents.
[0548] The mole-to-mole ratio of biotin to polypeptide can be
estimated using a 2-(4'-Hydroxyazobenzene-2-carboxylic acid) assay
using art-recognized techniques (Green, N M, (1975) "Avidin. In
Advances in Protein Chemistry." Academic Press, New York. 29,
85-133; Green, N M, (1971) "The use of bifunctional biotinyl
compounds to determine the arrangement of subunits in avidin."
Biochem J. 125, 781-791; Green, N M., (1965) "A spectrophotometric
assay for avidin and biotin based on binding of dyes by avidin."
Biochem. J. 94: 23c-24c). Several biotin molecules can be
conjugated to a polypeptide and each biotin molecule can bind one
molecule of avidin. The biotin-avidin bond formation is very rapid
and stable in organic solvents, extreme pH and denaturing reagents.
To quantitate biotinylation, a solution containing the biotinylated
polypeptide is added to a mixture of
2-(4'-Hydroxyazobenzene-2-carboxylic acid) and avidin. Because
biotin has a higher affinity for avidin, it displaces the
2-(4'-Hydroxyazobenzene-2-carboxylic acid) and the absorbance at
500 nanometers decreases proportionately. The amount of biotin in a
solution can be quantitated in a single cuvette by measuring the
absorbance of the 2-(4'-Hydroxyazobenzene-2-carboxylic acid)-avidin
solution before and after addition of the biotin-containing
peptide. The change in absorbance relates to the amount of biotin
in the sample by the extinction coefficient of the
2-(4'-Hydroxyazobenzene-2-carboxylic acid)-avidin complex.
[0549] Alternatively, an antibody, antigen-binding fragment or
binding protein can be conjugated with a fluorescent moiety.
Conjugating polypeptides with fluorescent moieties (e.g.,
R-Phycoerythrin, fluorescein isothiocyanate (FITC), etc.) can be
accomplished using art-recognized techniques described in, for
example, Glazer, A N and Stryer L. (1984). Trends Biochem. Sci.
9:423-7; Kronick, M N and Grossman, P D (1983) Clin. Chem.
29:1582-6; Lanier, L L and Loken, M R (1984) J. Immunol.,
132:151-156; Parks, D R et al. (1984) Cytometry 5:159-68; Hardy, R
R et al. (1983) Nature 306:270-2; Hardy R R et al. (1984) J. Exp.
Med. 159:1169-88; Kronick, M N (1986) J. Immuno. Meth. 92:1-13;
Der-Balian G, Kameda, N and Rowley, G. (1988) Anal. Biochem.
173:59-63.
[0550] In one non-limiting embodiment, an antibody antigen-binding
fragment can be associated with (conjugated to) a detectable label,
such as a radionuclide, iron-related compound, a dye, an imaging
agent or a fluorescent agent for immunodetection of endoglin which
can be used to visualize binding of the antibodies to endoglin in
vitro and/or in vivo.
[0551] Non-limiting examples of radiolabels include, for example,
.sup.32P, .sup.33P, .sup.43K, .sup.52Fe, .sup.57C, .sup.64Cu,
.sup.67Ga, .sup.67Cu, .sup.68Ga, .sup.71Ge, .sup.75Br, .sup.76Br,
.sup.77Br, .sup.77As, .sup.77Br, .sup.81Rb/.sup.81MKr, .sup.87MSr,
.sup.90Y, .sup.97Ru, .sup.99Tc, .sup.100Pd, .sup.101Rh, .sup.103Pb,
.sup.105Rh, .sup.109Pd, .sup.111Ag, .sup.111In, .sup.113In,
.sup.119Sb, .sup.121Sn, .sup.123I, .sup.125I, .sup.127Cs,
.sup.128Ba .sup.129Cs, .sup.131I, .sup.131Cs, .sup.143Pr,
.sup.153Sm, .sup.161Tb, .sup.166Ho, .sup.169Eu, .sup.177Lu,
.sup.186Re, .sup.188Re, .sup.189Re, .sup.191Os, .sup.193Pt,
.sup.194Ir, .sup.197Hg, .sup.199Au, .sup.203Pb, .sup.211At,
.sup.212Pb, .sup.212Bi and .sup.213Bi. Radiolabels can be attached
to compounds using conventional chemistry known in the art of
antibody imaging. Radiolabeled compounds are useful in in vitro
diagnostics techniques and in in vivo radioimaging techniques and
in radioimmunotherapy. For example, in the instance of in vivo
imaging, the antibodies and antigen-binding fragments thereof can
be conjugated to an imaging agent rather than a radioisotope(s),
including but not limited to a magnetic resonance image enhancing
agent, wherein for instance an antibody molecule is loaded with a
large number of paramagnetic ions through chelating groups.
Examples of chelating groups include EDTA, porphyrins, polyamines
crown ethers and polyoximes. Examples of paramagnetic ions include
gadolinium, iron, manganese, rhenium, europium, lanthanium, holmium
and ferbium. Such detectable moieties also include: metals; metal
chelators; lanthanides; lanthanide chelators; radiometals;
radiometal chelators; positron-emitting nuclei; microbubbles (for
ultrasound); liposomes; molecules microencapsulated in liposomes or
nanosphere; monocrystalline iron oxide nanocompounds; magnetic
resonance imaging contrast agents; light absorbing, reflecting
and/or scattering agents; colloidal particles; fluorophores, such
as near-infrared fluorophores. In many embodiments, such secondary
functionality/moiety will be relatively large, e.g., at least 25
amu in size, and in many instances can be at least 50, 100 or 250
amu in size. In certain embodiments, the secondary functionality is
a chelate moiety for chelating a metal, e.g., a chelator for a
radiometal or paramagnetic ion. In embodiments, it is a chelator
for a radionuclide useful for radiotherapy or imaging
procedures.
[0552] Antagonists of the invention also can be assayed for their
ability to modulate angiogenesis in a tissue. Any suitable assay
known to one of skill in the art can be used to monitor such
effects. Several such techniques are described herein.
[0553] One measures angiogenesis is an in vivo rabbit eye model and
is referred to as the rabbit eye assay. The rabbit eye assay has
been described in detail by others, and further has been used to
measure both angiogenesis and neovascularization in the presence of
angiogenic inhibitors such as thalidomide. See D'Amato et al.
(1994) Proc. Natl. Acad. Sci. 91:4082-4085.
[0554] The rabbit eye assay is a well recognized assay model for in
vivo angiogenesis because the neovascularization process,
exemplified by rabbit blood vessels growing from the rim of the
cornea into the cornea, is easily visualized through the naturally
transparent cornea of the eye. Additionally, both the extent and
the amount of stimulation or inhibition of neovascularization or
regression of neovascularization can easily be monitored over
time.
[0555] Finally, the rabbit is exposed to any test reagent, and
therefore the health of the rabbit is an indication of toxicity of
the test reagent.
[0556] Another assay measures angiogenesis in the chimeric
mouse:human mouse model and is referred to as the chimeric mouse
assay. The assay has been described in detail by others, and
further has been described herein to measure angiogenesis,
neovascularization, and regression of tumor tissues. See Yan, et
al. (1993) J. Clin. Invest. 91:986-996.
[0557] The chimeric mouse assay is a useful assay model for in vivo
angiogenesis because the transplanted skin grafts closely resemble
normal human skin histologically and neovascularization of whole
tissue is occurring wherein actual human blood vessels are growing
from the grafted human skin into the human tumor tissue on the
surface of the grafted human skin. The origin of the
neovascularization into the human graft can be demonstrated by
immunohistochemical staining of the neovasculature with
human-specific endothelial cell markers.
[0558] The chimeric mouse assay demonstrates regression of
neovascularization based on both the amount and extent of
regression of new vessel growth. Furthermore, it is easy to monitor
effects on the growth of any tissue transplanted upon the grafted
skin, such as a tumor tissue. Finally, the assay is useful because
there is an internal control for toxicity in the assay system. The
chimeric mouse is exposed to any test reagent, and therefore the
health of the mouse is an indication of toxicity. Other animal
models described herein and known in the art can also be utilized
in the methods described herein.
[0559] C. Treatment with Humanized Endoglin Antibodies
[0560] Provided herein are methods of preventing or treating one or
more diseases or disorders associated with
angiogenesis/neovascularization, excessive vascularization, or
small vessel dilation comprising administering a composition
comprising a humanized antibody or antigen-binding fragment
described herein that binds to endoglin associated with the disease
or disorder and prevents angiogenesis, thereby preventing,
treating, ameliorating, or lessening the disease or its
severity.
[0561] Provided herein are methods of preventing or treating one or
more diseases or disorders associated with
angiogenesis/neovascularization comprising administering a
composition comprising a humanized antibody or antigen-binding
fragment described herein that binds to endoglin associated with
the disease or disorder, decreases angiogenesis, or prevents
excessive angiogenesis.
[0562] As used herein, "prevention" refers to prophylaxis,
prevention of onset of symptoms, prevention of progression of a
disease or disorder associated with angiogenesis or correlated with
endoglin activity. As used herein, "inhibition," "treatment" and
"treating" are used interchangeably and refer to, for example,
stasis of symptoms, prolongation of survival, partial or full
amelioration of symptoms, and partial or full eradication of a
condition, disease or disorder associated with angiogenesis or
correlated with endoglin activity.
[0563] Compositions can be administered to a patient in a
therapeutically effective amount which are effective for producing
some desired therapeutic effect by inhibiting a disease or disorder
such as described herein which can be associated with endoglin, at
a reasonable benefit/risk ratio applicable to any medical
treatment. For the administration of the present compositions to
human patients, the compositions can be formulated by methodology
known by one of ordinary skill in the art. A therapeutically
effective amount is an amount achieves at least partially a desired
therapeutic or prophylactic effect in an organ or tissue. The
amount of a humanized anti-endoglin antibody or antigen binding
fragment thereof necessary to bring about prevention and/or
therapeutic treatment of a disease or disorder is not fixed per se.
The amount of humanized anti-endoglin antibody or antigen binding
fragment thereof administered may vary with the type of disease,
extensiveness of the disease, and size of the mammal suffering from
the disease or disorder. In one embodiment, two or more humanized
anti-endoglin antibodies described herein are administered to a
patient in combination. Combination includes concomitant or
subsequent administration of the antibodies.
[0564] "Administering" is defined herein as a means providing the
composition to the patient in a manner that results in the
composition being inside the patient's body. Such an administration
can be by any route including, without limitation, locally,
regionally or systemically by subcutaneous, intravitreal,
intradermal, intravenous, intra-arterial, intraperitoneal, or
intramuscular administration (e.g., injection). "Concurrent
administration" means administration within a relatively short time
period from each other; such time period can be less than 2 weeks,
less than 7 days, less than 1 day and could even be administered
simultaneously.
[0565] Actual dosage levels of the active ingredients in the
compositions can be varied so as to obtain an amount of the active
ingredient that is effective to achieve the desired therapeutic
response for a particular patient, composition, and mode of
administration, without being toxic to the patient. The selected
dosage level will depend upon a variety of factors including the
activity of the particular compound employed, the route of
administration, the time of administration, the rate of excretion
of the particular compound being employed, the duration of the
treatment, other drugs, compounds and/or materials used in
combination with the particular composition employed, the age, sex,
weight, condition, general health and prior medical history of the
patient being treated, and like factors well known in the medical
arts. The antibodies and antigen-binding fragments described herein
can be administered to a subject in various dosing amounts and over
various time frames. Non-limiting doses include about 0.01 mg/kg,
about 0.05 mg/kg, about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg,
about 5 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg,
about 40 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70 mg/kg,
about 80 mg/kg, about 90 mg/kg, about 100 mg/kg, about 125 mg/kg,
about 150 mg/kg, about 175 mg/kg, about 200 mg/kg, or any integer
in between. Additionally, the dose(s) of an antibody or
antigen-binding fragment can be administered twice a week, weekly,
every two weeks, every three weeks, every 4 weeks, every 6 weeks,
every 8 weeks, every 12 weeks, or any combination of weeks therein.
Dosing cycles are also contemplated such as, for example,
administering antibodies or antigen-binding fragments thereof once
or twice a week for 4 weeks, followed by two weeks without therapy.
Additional dosing cycles including, for example, different
combinations of the doses and weekly cycles described herein are
also contemplated within the invention.
[0566] "Contacting" is defined herein as a means of bringing a
composition as provided herein in physical proximity with a cell,
organ, tissue or fluid as described herein. Contacting encompasses
systemic or local administration of any of the compositions
provided herein and includes, without limitation, in vitro, in vivo
and/or ex vivo procedures and methods. "Combining" and "contacting"
are used interchangeably herein and are meant to be defined in the
same way.
[0567] A response is achieved when the patient experiences partial
or total alleviation, or reduction of signs or symptoms of illness,
and specifically includes, without limitation, prolongation of
survival. The expected progression-free survival times can be
measured in months to years, depending on prognostic factors
including the number of relapses, stage of disease, and other
factors. Prolonging survival includes without limitation times of
at least 1 month (mo), about at least 2 months (mos.), about at
least 3 mos., about at least 4 mos., about at least 6 mos., about
at least 1 year, about at least 2 years, about at least 3 years, or
more. Overall survival can also be measured in months to years. The
patient's symptoms can remain static or can decrease.
[0568] A physician or veterinarian having ordinary skill in the art
can readily determine and prescribe the effective amount (ED50) of
the composition required. For example, the physician or
veterinarian could start doses of the compounds employed in the
composition at levels lower than that required in order to achieve
the desired therapeutic effect and gradually increase the dosage
until the desired effect is achieved. Alternatively, a dose can
remain constant.
[0569] Compositions can be administered to a patient by any
convenient route such as described above. Regardless of the route
of administration selected, the compounds of the present invention,
which can be used in a suitable hydrated form, and/or the
compositions, are formulated into acceptable dosage forms such as
described below or by other conventional methods known to those of
skill in the art.
[0570] Antibodies can be combined with a therapeutic moiety or to a
detectable (imaging) moiety using methods known in the art such as,
for example, chemical conjugation, covalent or non-covalent bonds
or recombinant techniques to create conjugates or fusion proteins
such as described in more detail below. Alternatively, antibodies
and/or other agents can be combined in separate compositions for
simultaneous or sequential administration.
[0571] Toxicity and therapeutic efficacy of such ingredient can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (the
dose lethal to 50% of the population) and the ED.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD.sub.50/ED.sub.50. While
compounds that exhibit toxic side effects may be used, care should
be taken to design a delivery system that targets such compounds to
the site of affected tissue in order to minimize potential damage
to healthy cells and, thereby, reduce side effects.
[0572] Data obtained from cell culture assays and animal studies
can be used in formulating a range of dosage for use in humans. The
dosage of such compounds lies preferably within a range of
circulating concentrations that include the ED.sub.50 with little
or no toxicity. The dosage may vary within this range depending
upon the dosage form employed and the route of administration
utilized. For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. A dose can be formulated in animal models to
achieve a circulating plasma concentration arrange that includes
the IC.sub.50 (i.e., the concentration of the test compound which
achieves a half-maximal inhibition) as determined in cell culture.
Levels in plasma can be measured, for example, by high performance
liquid chromatography. Such information can be used to more
accurately determine useful doses in humans.
[0573] The pro-angiogenic role of endoglin has been established in
many models including endothelial cell culture and knock-out mouse
models. Endothelial and the associated cells are well known to
express endoglin (CD105), and the role of endoglin in angiogenesis,
generally, as well as cardiac development has also been confirmed
in numerous studies, culture models, and animal models. (Duff et
al., FASEB J., 17:984-992 (2003); Bernabeu et al., J. Cell.
Biochem., 102(6): 1375-1388 (2007); U.S. Pat. No. 7,097,836).
[0574] Thus, methods which inhibit angiogenesis in a diseased
tissue ameliorate symptoms of the disease and, depending upon the
disease, can contribute to cure of the disease. In one embodiment,
the invention contemplates inhibition of angiogenesis in a tissue.
The extent of angiogenesis in a tissue, and therefore the extent of
inhibition achieved by the present methods, can be evaluated by a
variety of methods, such as are described herein.
[0575] The unique specificity of the antibodies which recognize
(e.g., bind) an epitope on endoglin and inhibits angiogenesis,
provides diagnostic and therapeutic uses for diseases characterized
by angiogenesis (neovascularization), small vessel dilation, and/or
excessive vascularization such as described herein. Humanized
anti-endoglin antibodies and fragments thereof can be administered
to a subject such as a mammal (e.g., a human), suffering from a
medical disorder, e.g., various forms of ocular diseases
characterized by angiogenesis/neovascularization (e.g., macular
degeneration, diabetic retinopathy), diabetic nephropathy, chronic
inflammatory diseases (e.g., IBD), rheumatoid arthritis,
osteoarthritis, and various forms of cancer (primary tumors and
metastases). Provided herein is a method for treating a subject
having an ocular disease characterized by angiogenesis by
administering a humanized antibody or fragment thereof described
herein that binds endoglin and inhibits angiogenesis. Also provided
herein is a method for treating a subject having a chronic
inflammatory disease by administering a humanized antibody or
fragment thereof described herein that binds endoglin and inhibits
angiogenesis. Examples of such chronic inflammatory diseases
include, but are not limited to, Crohn's disease and ulcerative
colitis. Further provided herein is a method for treating a subject
having diabetic nephropathy by administering a humanized antibody
or fragment thereof described herein. Provided herein is a method
for treating a subject having rheumatoid arthritis or
osteoarthritis by administering a humanized antibody or fragment
thereof described herein.
[0576] One would understand that the anti-endoglin antibodies can
be effective for treating angiogenesis, it is contemplated herein
that a subject can also be treated with one or more additional
angiogenesis inhibitors.
[0577] The term "angiogenesis inhibitor" is used herein, for
purposes of the specification and claims, to mean a compound or
molecule including, but not limited to, peptides, proteins,
enzymes, polysaccharides, oligonucleotides, DNA, RNA, recombinant
vectors, and drugs which function to inhibit angiogenesis.
Angiogenesis inhibitors are known in the art and all types are
contemplated herein. Non-limiting examples of compounds and
molecules include natural and synthetic biomolecules such as
paclitaxel, O-(chloroacetyl-carbomyl) fumagillol ("TNP-470" or "AGM
1470"), thrombospondin-1, thrombospondin-2, angiostatin, human
chondrocyte-derived inhibitor of angiogenesis ("hCHIAMP"),
cartilage-derived angiogenic inhibitor, platelet factor-4,
gro-beta, human interferon-inducible protein 10 ("IP10"),
interleukin 12, Ro 318220, tricyclodecan-9-yl xanthate ("D609"),
irsogladine, 8,9-dihydroxy-7-methyl-benzo[b]quinolizinium bromide
("GPA 1734"), medroxyprogesterone, a combination of heparin and
cortisone, glucosidase inhibitors, genistein, thalidomide,
diamino-antraquinone, herbimycin, ursolic acid, and oleanolic acid.
Non-limiting examples of antibodies include those directed towards
molecules such as VEGF, VEGF receptor, or different epitopes of
endoglin. Additionally, small molecular inhibitors of VEGF receptor
are known and contemplated herein. Non-limiting examples of VEGF
receptor inhibitors include bevacizumab (AVASTIN.RTM.), ranibizumab
(Lucentis), aflibercept (VEGF-Trap), sunitinib (Sutent), sorafenib
(Nexavar), axitinib, pegaptanib and pazopanib.
[0578] The antibodies and antigen-binding fragments described
herein can be administered in combination with VEGF receptor
inhibitors for combination therapy of any of the
angiogenesis-related conditions or diseases described herein. In
one non-limiting embodiment, the VEGF receptor inhibitor is
bevacizumab. Exemplary dosage for bevacizumab are about 7.5, about
10, or about 15 mg/kg, administered every 2 or 3 weeks.
[0579] In one non-limiting embodiment, the VEGF receptor inhibitor
is ranibizumab. Exemplary ocular dosages for ranibizumab include
about 0.5 mg, administered intravitreally monthly. In one
non-limiting embodiment, the VEGF receptor inhibitor is aflibercept
(VEGF-Trap). Exemplary dosages for VEGF-Trap include about
0.5-about 10 mg/kg administered every 2 or 3 weeks. Exemplary
ocular dosages for VEGF-Trap include about 0.5-about 2.0 mg
administered intravitreally monthly or quarterly.
[0580] In another non-limiting embodiment, the VEGF receptor
inhibitor is sunitinib. Exemplary regimens for sunitinib include
about 50 mg administered for 4 weeks, followed by 2 weeks of no
drug. Treatment regimens can be repeated in a cyclic or acyclic
basis.
[0581] In another non-limiting embodiment, the VEGF receptor
inhibitor is sorafenib. Exemplary dosages for sorafenib include
about 400 mg administered daily.
[0582] In another non-limiting embodiment the VEGF receptor
inhibitor is axitinib. Exemplary dosages for axitinib include about
3, about 5, or about 10 mg administered twice daily.
[0583] In another non-limiting embodiment the VEGF receptor
inhibitor is pegaptanib. Exemplary dosages for pegaptanib include
about 0.3-about 3 mg administered intravitreally every 6 weeks.
[0584] In yet another non-limiting embodiment, the VEGF receptor
inhibitor is pazopanib. Exemplary dosages for pazopanib include
about 200-about 1000 mg administered daily.
[0585] Multiple combinations of these VEGF receptor inhibitors can
be administered with the antibodies and antigen-binding fragments
described herein. In one embodiment, combinations may result in the
use of lower doses for the antibodies or antigen binding fragments
described herein, the VEGF receptor inhibitors, or both. Such
alterations in dosing may result from synergistic effects of the
combinations of the antibodies and antigen-binding fragments
described herein with the VEGF receptor inhibitors.
[0586] Ocular Conditions Involving Angiogenesis
[0587] Macular Degeneration Conditions and Diabetic Retinopathy
[0588] In one aspect, the present invention provides a method for
treating diabetic retinopathy, macular degeneration, choroidal
neovascularization or neovascular glaucoma in a patient by
administering to the patient a therapeutically effective amount of
any of the compositions provided herein.
[0589] Endoglin is a receptor associated with angiogenesis and the
extracellular matrix. Macular degeneration (AMD) is the loss of
photoreceptors in the portion of the central retina, termed the
macula, responsible for high-acuity vision. Degeneration of the
macula is associated with abnormal deposition of extracellular
matrix components and other debris in the membrane between the
retinal pigment epithelium and the vascular choroid. This
debris-like material is termed drusen. Drusen is observed with a
funduscopic eye examination. Normal eyes may have maculas free of
drusen, yet drusen may be abundant in the retinal periphery. The
presence of soft drusen in the macula, in the absence of any loss
of macular vision, is considered an early stage of AMD. Macular
degeneration is characterized by choroidal neovascularization
(CNV), the development of abnormal blood vessels beneath the
retinal pigment epithelium (RPE) layer of the retina. These vessels
break through the Bruch's membrane, disrupting the retinal
pigmented epithelium, bleed, and eventually cause macular scarring
which results in profound loss of central vision (disciform
scarring).
[0590] Choroidal neovascularization (CNV) commonly occurs in
macular degeneration in addition to other ocular disorders and is
associated with proliferation of choroidal endothelial cells,
overproduction of extracellular matrix, and formation of a
fibrovascular subretinal membrane. Retinal pigment epithelium cell
proliferation and production of angiogenic factors appears to
effect choroidal neovascularization.
[0591] Diabetic retinopathy (DR) is an ocular disorder
characterized by excessive angiogenesis that develops in diabetes
due to thickening of capillary basement membranes, and lack of
contact between pericytes and endothelial cells of the capillaries.
Loss of pericytes increases leakage of the capillaries and leads to
breakdown of the blood-retina barrier. Diabetic retinopathy is the
result of microvascular retinal changes. Hyperglycemia-induced
pericyte death and thickening of the basement membrane lead to
incompetence of the vascular walls. These damages change the
formation of the blood-retinal barrier and also make the retinal
blood vessels become more permeable. Small blood vessels--such as
those in the eye--are especially vulnerable to poor blood sugar
(blood glucose) control. An over-accumulation of glucose and/or
fructose damages the tiny blood vessels in the retina. Macular
edema can also develop when the damaged blood vessels leak fluid
and lipids onto the macula. These fluids make the macula swell,
which blurs vision. This damage also results in a lack of oxygen at
the retina.
[0592] As the disease progresses, the lack of oxygen in the retina
stimulates angiogenesis along the retina and in the clear, gel-like
vitreous humour that fills the inside of the eye. Without timely
treatment, these new blood vessels can bleed, cloud vision, and
destroy the retina. Fibrovascular proliferation can also cause
tractional retinal detachment. The new blood vessels can also grow
into the angle of the anterior chamber of the eye and cause
neovascular glaucoma.
[0593] Proliferative vitreoretinopathy is associated with cellular
proliferation of cellular and fibrotic membranes within the
vitreous membranes and on the surfaces of the retina. Retinal
pigment epithelium cell proliferation and migration is common with
this ocular disorder. The membranes associated with proliferative
vitreoretinopathy contain extracellular matrix components such as
collagen types I, II, and IV and fibronectin, and become
progressively fibrotic.
[0594] Age-related macular degeneration (AMD) and diabetic
retinopathy are the two leading causes of blindness in the
developed world. The recent approval of the macromolecules
LUCENTIS.RTM., AVASTIN.RTM., and MACUGEN.RTM. have improved the
treatment options available for AMD patients. LUCENTIS.RTM. is a
Fab and AVASTIN.RTM. is a monoclonal antibody. They both bind
vascular endothelial growth factor (VEGF) and have demonstrated the
most impressive results to date treating AMD; however, only a
minority of treated patients experience a significant improvement
in visual acuity. Anti-angiogenic therapy focused on a target other
than VEGF may overcome some of the limitations associated with
agents that target the VEGF pathway.
[0595] The humanized antibodies and antigen-binding fragments which
bind endoglin and are described herein can be used to treat or
prevent macular degeneration, CNV, diabetic retinopathy, or
proliferative vitreoretinopathy. Described herein are methods of
treating or preventing macular degeneration, CNV, diabetic
retinopathy, or proliferative vitreoretinopathy via the
administration of the antibodies and antigen-binding fragments
described herein. The humanized antibodies and antigen-binding
fragments which bind endoglin and are described herein can also
shrink blood vessels, inhibit endothelial cell proliferation
associated with ocular disease, clear symptoms of bleeding, treat
cloudy vision, provide stasis of vision loss, and/or prevent
leakage of blood vessels. The humanized antibodies and
antigen-binding fragments described herein can also be used in
medicaments for the treatment of macular degeneration, CNV,
diabetic retinopathy or proliferative vitreoretinopathy.
[0596] Additionally, humanized antibodies and antigen-binding
fragments described herein can also be used in combination with
known therapies and/or compounds for the treatment of macular
degeneration, CNV, diabetic retinopathy or proliferative
vitreoretinopathy. Examples of such compounds include, but are not
limited to, bevacizumab (AVASTIN.RTM.), ranibizumab
(LUCENTIS.RTM.), aflibercept (VEGF-Trap), sunitinib (SUTENT.RTM.),
sorafenib (NEXAVAR.RTM.), axitinib, pegaptanib, pazopanib or
MACUGEN.RTM.. In addition to the modes of administration described
herein, the humanized anti-endoglin antibodies and antigen-binding
fragments can be administered via intravitreal routes. Non-limiting
examples of intravitreal modes of administration include
intravitreal injection and the use of intravitreal implants.
[0597] Patients can be assessed for improvement and responsiveness
to treatment. Treatment includes, but is not limited to, decreasing
the macular edema, decreased areas of CNV, and increased visual
acuity. Measurements of symptoms are as known in the art and are
further described in the examples below.
[0598] Chronic Inflammatory Diseases
[0599] Any of a variety of tissues or organs comprised of organized
tissues, can support angiogenesis in disease conditions including
skin, muscle, gut, connective tissue, joints, bones and the like
tissue in which blood vessels can invade upon angiogenic stimuli.
Thus, in one embodiment, a tissue to be treated is an inflamed
tissue and the angiogenesis to be inhibited is inflamed tissue
angiogenesis where there is neovascularization of inflamed
tissue.
[0600] Inflammatory Bowel Diseases
[0601] Angiogenesis plays an important role in inflammatory bowel
disease (IBD). IBD is an umbrella term for a set of bowel and
intestinal diseases or conditions including Crohn's disease and
ulcerative colitis. Crohn's disease is typically characterized by
inflammation of the small and large bowel, whereas ulcerative
colitis is generally localized to the colon. Abnormal or
pathological angiogenesis is central to both Crohn's disease and
ulcerative colitis. Both diseases involve increased microvascular
density and microvascular dysfunction, and this angiogenesis is
temporally related with tissue pathology and the inflammatory
cycles found in both diseases. Endoglin is known to be expressed in
these tissues and to play a role in the dysregulation of
angiogenesis during IBD. (Chidlow et al., Am. J. Physiol.
Gastrointest. Liver Physiol., 293:5-18 (2007)).
[0602] The humanized antibodies and antigen-binding fragments which
bind endoglin and are described herein can be used to treat IBD.
Additionally, humanized antibodies and antigen-binding fragments
which bind endoglin can be used for the treatment of Crohn's
disease or ulcerative colitis. The humanized anti-endoglin
antibodies and antigen-binding fragments can also be used in
combination with surgery and/or known therapies for IBD, Crohn's
disease or ulcerative colitis. Examples of such known therapies
include, but are not limited to, Aminosalicylates (e.g.,
Mesalamine), corticosteroids (e.g., budesonide, prednisone, etc.),
antibiotics (e.g., metronidizole, etc.), immunosuppresive drugs
(e.g., azathioprine, 6-mercaptopurine, methotrexate, Tacrolimus,
and cyclosporine, etc.), and biologic drugs such as proteins and
antibodies (e.g., infliximab, etc.).
[0603] Treatment of IBD can be assessed by decreased
vascularization of the inflamed tissue. Treatment can also be
assessed by stasis, resolution, and/or healing of the ulcerative
lesions which characterize IBD.
[0604] Diabetic Nephropathy & Renal Transplantation
Ischemia
[0605] Diabetic nephropathy is a major cause of morbidity and
mortality in both type 1 and type 2 diabetics. It is the leading
cause of end-stage renal disease world-wide. Diabetic nephropathy
is characterized by glomerular microvascular injury due to the
increased synthesis of pro-angiogenic factors. These pro-angiogenic
factors cause increased endothelial cell proliferation and
subsequent angiogenesis, and endoglin is known to be upregulated in
chronic renal disease. This angiogenesis results in destruction of
the glomeruli and finally renal failure. (Zent et al., Seminars in
Nephrology, 27(2): 161-171 (2007); Roy-Chaudhury et al., Exp.
Nephrol., 5:55-60 (1997)).
[0606] Similar effects are seen in renal transplantation resulting
in ischemia and failure of the transplanted organ. The upregulation
of endoglin results in upregulated angiogenesis and inflammation in
the kidney. Conversely, studies with endoglin null mice show
significantly reduced renal damage after transplantation/ischemia
and increased organ survival. (Docherty et al., Nephol. Dial.
Transplant., 21:2106-2119 (2006)).
[0607] The humanized antibodies and antigen-binding fragments which
bind endoglin and are described herein can be used to treat or
prevent diabetic nephropathy, renal failure following
transplantation, and/or ischemic renal injury following
transplantation.
[0608] Described herein are methods of treating or preventing
diabetic nephropathy, renal failure following transplantation,
and/or ischemic renal injury following transplantation via the
administration of the antibodies and antigen-binding fragments
described herein. The humanized antibodies and antigen-binding
fragments described herein can also be used in medicaments for the
treatment of diabetic nephropathy, renal failure following
transplantation, and/or ischemic renal injury following
transplantation. Additionally, humanized antibodies and
antigen-binding fragments described herein can also be used in
combination with known therapies and/or compounds for the treatment
of diabetic nephropathy, renal failure following transplantation,
and/or ischemic renal injury following transplantation.
[0609] Patients can be assessed with respect to the efficacy of
treatment by, for example, improvement in renal function.
[0610] Rheumatoid Arthritis & Osteoarthritis
[0611] Rheumatoid arthritis is characterized by excessive
angiogenesis, and is well understood in this regard. The
inflammation and destruction found in the synovial fluids is
directly related to the increased angiogenesis found surrounding
and in the synovial tissues. Numerous pro-angiogenic factors are
present in the affected tissues of rheumatoid arthritis patients.
(Koch and Distler, Arthritis Res. & Ther., 9(Suppl. 2): S3, 1-9
(2007).
[0612] Osteoarthritis is a group of chronic disabling conditions
that affects the synovial joints. Angiogenesis and inflammation are
integral processes in the pathophysiology of the disease, and they
contribute to joint damage through a variety of mechanisms,
including but not limited to, stimulation of MMP production and
endochondral ossification. Additionally, angiogenesis in
osteoarthritis induces further innervation, which develops into a
feedback loop where each continues to stimulate the other. (Bonnet
& Walsh, Rheumatology, 44:7-16 (2005)).
[0613] The humanized antibodies and antigen-binding fragments which
bind endoglin and are described herein can be used to treat or
prevent rheumatoid arthritis and osteoarthritis. Described herein
are methods of treating or preventing rheumatoid arthritis and
osteoarthritis via the administration of the antibodies and
antigen-binding fragments described herein. The humanized
antibodies and antigen-binding fragments described herein can also
be used in medicaments for the treatment of rheumatoid arthritis
and osteoarthritis.
[0614] Two well accepted composite measures of improvement of RA in
trials are: the Paulus Criteria and The American College of
Rheumatology Criteria (ACR). Paulus Criteria is defined as
improvement in 4 of the following: tender and swollen joint counts,
morning stiffness, patient assessment of disease activity,
physician assessment of disease activity and erythrocyte
sedimentation rate (ESR) rage. The level of improvement is set as a
percentage improvement of each of these variables i.e., a Paulus 20
classification indicates a responder who has shown 20% improvement
in 4 of the 6 parameters.
[0615] Rheumatoid arthritis can also be assessed using American
College of Rheumatology (ACR) Scoring. Briefly, ACR Classification
Criteria for Determining Clinical Remission in Rheumatoid Arthritis
is assessed by the presence of 5 or more of the following factors
present at least two consecutive months:
[0616] a. Morning stiffness <15 minutes;
[0617] b. No fatigue;
[0618] c. No joint pain;
[0619] d. No joint tenderness or pain on motion;
[0620] e. No soft tissue swelling in joints or tendon sheaths;
and
[0621] f. ESR (Westergren method) <30 mm/hour for a female or 20
mm/hour for a male.
[0622] Exclusions may occur and include: clinical manifestations of
active vasculitis, pericarditis, pleuritis or myositis, and
unexplained recent weight loss or fever attributable to rheumatoid
arthritis will prohibit a designation of complete clinical
remission (Pinals R S, et. al.: Arthritis Rheum 24:1308, 1981).
Additionally, ACR Classification Criteria of Functional Status in
Rheumatoid Arthritis includes classification based on the following
patient abilities: [0623] Class I: Completely able to perform usual
activities of daily living (self-care, vocational, and
avocational); [0624] Class II: Able to perform usual self-care and
vocational activities, but limited in avocational activities;
[0625] Class III: Able to perform usual self-care activities, but
limited in vocational and avocational activities; and [0626] Class
IV: Limited ability to perform usual self-care, vocational, and
avocational activities.
[0627] Osteoarthritis can also be assessed using ACR scoring. ACR
Clinical Classification Criteria for Osteoarthritis of the hip is
assessed utilizing patient history, physical examination and
laboratory findings: a patient is assessed for pain in the hip and
one of the following: [0628] (1) Internal hip rotation of less than
15 degrees and ESR less than or equal to 45 degrees mm/hour or hip
flexion less than or equal to 115 degrees if ESR is unavailable; or
[0629] (2) Internal hip rotation of less than 15 degrees, pain
associated with internal hip, morning stiffness of the hip for less
than or equal to 60 minutes and the patient is over 50 years of
age.
[0630] Using history, physical examination, laboratory and
radiographic findings, traditional format is pain in the hip and
two of the following indications: ESR less than 20 mm/hour,
radiographic femoral and/or acetabular osteophytes, or radiographic
joint space narrowing (superior, axial, and/or medial). A
classification tree is as follows: pain in the hip in association
with (1) radiographic femoral and/or acetabular osteophytes or (2)
ESR less than or equal to 20 mm/hour and radiographic axial joint
space narrowing (Altman, R, et al.: Arthritis Rheum 34:505,
1991).
[0631] ACR Clinical Classification Criteria for Osteoarthritis of
the Knee
[0632] ACR Clinical Classification Criteria for Osteoarthritis of
the knee is assessed using history and physical examination
utilizing the following criteria: pain in the knee in connection
with three (3) of the following:
[0633] (1) a patient is over 50 years of age;
[0634] (2) less than 30 minutes of morning stiffness;
[0635] (3) Crepitus on active motion;
[0636] (4) bony tenderness;
[0637] (5) bony enlargement; and
[0638] (6) no palpable warmth of synovium.
[0639] Using patient history, physical examination and radiographic
findings, pain in the knee can be assessed in connection with one
of the following patient characteristics: (1) a patient is over 50
years of age; (2) less than 30 minutes of morning stiffness; and
(3) Crepitus on active motion and osteophytes. Using history,
physical examination and laboratory findings: pain in the knee can
be assessed in connection with five (5) of the following
characteristics:
[0640] (1) a patient is over 50 years of age;
[0641] (2) less than 30 minutes of morning stiffness;
[0642] (3) Crepitus on active motion;
[0643] (4) bony tenderness;
[0644] (5) bony enlargement;
[0645] (6) No palpable warmth of synovium;
[0646] (7) ESF is less than 40 mm/hour;
[0647] (8) Rheumatoid factor (RF) of less than 1:40; and
[0648] (9) Synovial Fluid (SF) signs of osteoarthritis.
See, e.g., Altman, R, et al.: Arthritis Rheum 29:1039, 1986.
[0649] ACR Clinical Classification Criteria for Osteoarthritis of
the hand can be assessed as follows: pain, aching or stiffness in
the hand in connection with three (3) of the following: (1) hard
tissue enlargement of two or more of the following joints (second
and third distal interphalangeal, the second and third proximal
interphalangeal, and the first carpometacarpal joints of both
hands; (2) hard tissue enlargement of two or more distal
interphalangeal joints; (3) less than three swollen MCP joints and
(4) deformity of at least one of the joints listed above in
(1).
[0650] Cancer
[0651] CD105 is associated with tumor angiogenesis and is strongly
up-regulated in the endothelium of various tumor tissues compared
with that in normal tissues. CD105 is up-regulated in a wide range
of tumor endothelia including, for example, colon, breast, brain,
lung, prostate, endometrial, kidney, liver, gastric, head &
neck and cervical cancer. Additionally, it is known that there is
stronger expression of CD105 in tumor endothelium than
corresponding normal tissues. (Duff et al., FASEB J., 17:984-992
(2003); Bernabeu et al., J. Cell. Biochem., 102(6): 1375-1388
(2007); U.S. Pat. No. 7,097,836). Thus, the inhibition of
angiogenesis with anti-endoglin humanized antibodies represents a
treatment option for cancerous tumors. The humanized antibodies and
antigen-binding fragments which bind endoglin and are described
herein can be used to treat cancerous tumors. The humanized
antibodies and antigen-binding fragments described herein can also
be used in medicaments for the treatment cancerous tumors.
[0652] The term "tumor" is used herein to refer to a cancerous
tissue expressing endoglin (as compared to expression by normal
tissue of the same type). Tumors can include solid tumors and
semi-solid tumors. Non-limiting examples of tumors include human
leukemias, including non-T-cell-type (non-T) acute lymphoblastic
leukemia (ALL), myelo-monocytic leukemia; and human solid and
semi-solid tumors, with its surrounding vasculature expressing
endoglin at moderate to high levels (as compared to expression by
normal tissue of the same type) including angiosarcoma, breast
carcinoma, stomach cancer, colon carcinoma, Hodgkins lymphoma,
lymphoma, glioblastoma multiforme (GBM), lung carcinoma, melanoma,
myeloma, lymphoma, osteosarcoma, ovarian carcinoma, parotid tumor,
pharyngeal carcinoma, prostate carcinoma, hepatocellular carcinoma,
renal carcinoma, and rectosigmoid carcinoma.
[0653] A cancerous tissue to be treated is, for example, an
endothelial tissue expressing an abnormal level of endoglin.
[0654] In the absence of neovascularization of tumor tissue, the
tumor tissue does not obtain the required nutrients, slows in
growth, ceases additional growth, regresses and ultimately becomes
necrotic resulting in killing of the tumor. The present invention
provides for a method of inhibiting tumor neovascularization by
inhibiting tumor angiogenesis according to the present methods.
Similarly, the invention provides a method of inhibiting tumor
growth by practicing the angiogenesis-inhibiting methods.
[0655] The methods are also particularly effective against the
formation of metastases because their formation requires
vascularization of a primary tumor so that the metastatic cancer
cells can exit the primary tumor and their establishment in a
secondary site requires neovascularization to support growth of the
metastases.
[0656] It will be appreciated that a "patient suffering from a
cancer/metastasis" of the invention may express a mutant protein
(tumor associated antigen) or a mutant gene and not yet be
symptomatic for the disease. In one non-limiting example, where the
cancer is colon cancer (which is associated with the mutant K-ras
protein), a patient with a mutant K-ras protein in some cells of
the colon is a patient according to the invention even though that
patient may not yet be symptomatic for colon cancer. "Signs or
symptoms of illness" are clinically recognized manifestations or
indications of disease.
[0657] By "treating" a patient suffering from cancer it is meant
that the patient's symptoms are partially or totally alleviated, or
remain static following treatment according to the invention. A
patient that has been treated can exhibit a partial or total
alleviation of symptoms and/or tumor load. This is intended to
encompass prophylaxis, therapy and cure. In one non-limiting
example, a patient suffering from a highly metastatic cancer (e.g.,
breast cancer) is treated where additional metastasis either do not
occur, or are reduced in number as compared to a patient who does
not receive treatment. In another non-limiting example, a patient
is treated where the patient's solid cancer either becomes reduced
in size or does not increase in size as compared to a patient who
does not receive treatment. In yet another non-limiting example,
the number of cancer cells in a treated patient either does not
increase or is reduced as compared to the number of cancer cells in
a patient who does not receive treatment. Improvement can also be
defined, for example, as decreased cell proliferation, decreased
numbers of cells, increased apoptosis, and/or increased survival of
the patient being treated.
[0658] As further used herein, treatment of cancer includes stasis,
partial or total elimination of a cancerous growth or tumor.
Treatment or partial elimination includes, for example, a fold
reduction in growth or tumor size and/or volume such as about
2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold,
about 20-fold, about 50-fold, or any fold reduction in between.
Similarly, treatment or partial elimination can include a percent
reduction in growth or tumor size and/or volume of about 1%, 2%,
3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or any
percentage reduction in between.
[0659] A tumor or cancer to be treated in the methods described
herein includes, but is not limited to, a lung cancer, a
gynecologic malignancy, a melanoma, a breast cancer, a brain cancer
(e.g., glioblastoma multiforme, "GBM") a pancreatic cancer, an
ovarian cancer, a uterine cancer, a colorectal cancer, a prostate
cancer, a kidney cancer, a head cancer, a liver cancer
(hepatocellular cancer), a uterine cancer, a neck cancer, a kidney
cancer (renal cell cancer), a sarcoma, a myeloma, and lymphoma. In
one embodiment, a tumor to be treated is a solid or semi-solid
tumor. In another embodiment, a tumor to be treated is a primary
tumor. In another embodiment, a tumor to be treated is a metastatic
tumor. In one embodiment, a tumor or cancer to be treated is of
epithelial origin. In another embodiment, the cancer to be treated
is myeloma. In another embodiment, the cancer to be treated is
ovarian cancer. In another embodiment, the cancer to be treated is
kidney/renal cancer. In yet another embodiment, the cancer to be
treated is hepatocellular/liver cancer.
[0660] Lung Cancer
[0661] In one aspect, provided herein is a method to treat lung
cancer. The most common type of lung cancer is non-small cell lung
cancer (NSCLC), which accounts for approximately 80-85% of lung
cancers and is divided into squamous cell carcinomas,
adenocarcinomas, and large cell undifferentiated carcinomas. Small
cell lung cancer accounts for 15-20% of lung cancers.
[0662] Lung cancer staging is an assessment of the degree of spread
of the cancer from its original source. It is an important factor
affecting the prognosis and potential treatment of lung cancer.
Non-small cell lung carcinoma is staged from IA ("one A"; best
prognosis) to IV ("four"; worst prognosis). Small cell lung
carcinoma is classified as limited stage if it is confined to one
half of the chest and within the scope of a single radiotherapy
field; otherwise, it is extensive stage.
[0663] Non-small cell lung cancer may be staged using EUS
(endoscopic ultrasound) or CT or MRI scan or at surgery to classify
the extent of disease according to the TNM system. These subjects
undergo staging as part of the process of considering prognosis and
treatment. The AJCC recommends TNM staging followed by further
grouping.
[0664] Primary tumor (T): TX: The primary tumor cannot be assessed,
or there are malignant cells in the sputum or bronchoalveolar
lavage but not seen on imaging or bronchoscopy; Tis: Carcinoma in
situ. TO: No evidence of primary tumor. T1: Tumor less than 3 cm in
its greatest dimension, surrounded by lung or visceral pleura and
without bronchoscopic invasion into the main bronchus. T2: A tumor
with any of: more than 3 cm in greatest dimension; extending into
the main bronchus (but more than 2 cm distal to the carina), and
obstructive pneumonitis (but not involving the entire lung). T3: A
tumor with any of: invasion of the chest wall, diaphragm,
mediastinal pleura, or parietal pericardium; extending into the
main bronchus, within 2 cm of the carina, but not involving the
carina; and obstructive pneumonitis of the entire lung. T4: A tumor
with any of: invasion of the mediastinum, heart, great vessels,
trachea, esophagus, vertebra, or carina; separate tumor nodules in
the same lobe; and malignant pleural effusion. Lymph nodes (N): NX:
Lymph nodes cannot be assessed; N0: No lymph nodes involved; N1:
Metastasis to ipsilateral peribronchial or ipsilateral hilar lymph
nodes; N2: Metastasis to ipsilateral mediastinal or subcarinal
lymph nodes; and N3: Metastasis to any of: ipsilateral
supraclavicular lymph nodes; ipsilateral scalene lymph nodes; and
contralateral lymph nodes. Distant metastasis (M): MX: Distant
metastasis cannot be assessed; M0: No distant metastasis; and M1:
Distant metastasis is present.
[0665] Uterine Cancers/Gynecologic Malignancy
[0666] Uterine cancers may refer to any of several different types
of cancer which occur in the uterus, namely: uterine sarcomas
(e.g., sarcomas of the myometrium, or muscular layer of the uterus,
are most commonly leiomyosarcomas); endometrial cancer; and
cervical cancer.
[0667] In another aspect, provided herein is a method to treat
endometrium cancer. Endometrial cancer is a cancer that starts in
the endometrium, the inner lining of the uterus. Some of the
examples of the cancer of uterus and endometrium include, but are
not limited to, adenocarcinomas, adenoacanthomas, adenosquamous
carcinomas, papillary serous adenocarcinomas, clear cell
adenocarcinomas, uterine sarcomas, stromal sarcomas, malignant
mixed mesodermal tumors, and leiomyosarcomas.
[0668] In another aspect, the method treats cervical cancer,
preferably an adenocarcinoma in the cervix epithelial. Two main
types of this cancer exist: squamous cell carcinoma and
adenocarcinomas. The former constitutes about 80-90% of all
cervical cancers and develops where the ectocervix (portion closest
to the vagina) and the endocervix (portion closest to the uterus)
join. The latter develop in the mucous-producing gland cells of the
endocervix. Some cervical cancers have characteristics of both of
these and are called adenosquamous carcinomas or mixed
carcinomas.
[0669] Ovarian Cancer
[0670] In another aspect, provided herein is a method of treating
ovarian cancer, including epithelial ovarian tumors.
[0671] Ovarian cancer is classified according to the histology of
the tumor, obtained in a pathology report. Surface
epithelial-stromal tumor, also known as ovarian epithelial
carcinoma, is the most typical type of ovarian cancer. It includes
serous tumor, endometrioid tumor and mucinous cystadenocarcinoma.
Sex cord-stromal tumor, including estrogen-producing granulosa cell
tumor and virilizing Sertoli-Leydig cell tumor or arrhenoblastoma,
accounts for 8% of ovarian cancers. Germ cell tumor accounts for
approximately 30% of ovarian tumors but only 5% of ovarian cancers
because most germ cell tumors are teratomas and most teratomas are
benign. Germ cell tumor tends to occur in young women and girls.
The prognosis depends on the specific histology of germ cell tumor,
but overall is favorable. Mixed tumors contain elements of more
than one of the above classes of tumor histology.
[0672] Ovarian cancer can also be a secondary cancer, the result of
metastasis from a primary cancer elsewhere in the body. Common
primary cancers are breast cancer and gastrointestinal cancer (in
which case the ovarian cancer is a Krukenberg cancer). Surface
epithelial-stromal tumor can originate in the peritoneum (the
lining of the abdominal cavity), in which case the ovarian cancer
is secondary to primary peritoneal cancer, but treatment is
basically the same as for primary surface epithelial-stromal tumor
involving the peritoneum.
[0673] Ovarian cancer staging is by the FIGO staging system and
uses information obtained after surgery, which can include a total
abdominal hysterectomy, removal of both ovaries and fallopian
tubes, the omentum, and pelvic (peritoneal) washings for cytology.
The AJCC stage is the same as the FIGO stage.
[0674] Stage I refers to ovarian cancer limited to one or both
ovaries: IA--involves one ovary; capsule intact; no tumor on
ovarian surface; no malignant cells in ascites or peritoneal
washings; IB--involves both ovaries; capsule intact; no tumor on
ovarian surface; negative washings; and IC--tumor limited to
ovaries with any of the following: capsule ruptured, tumor on
ovarian surface, positive washings.
[0675] Stage II refers to pelvic extension or implants:
IIA--extension or implants onto uterus or fallopian tube; negative
washings; IIB--extension or implants onto other pelvic structures;
negative washings; and IIC--pelvic extension or implants with
positive peritoneal washings
[0676] Stage III refers to microscopic peritoneal implants outside
of the pelvis; or limited to the pelvis with extension to the small
bowel or omentum: IIIA--microscopic peritoneal metastases beyond
pelvis; IIIB--macroscopic peritoneal metastases beyond pelvis less
than 2 cm in size; and IIIC--peritoneal metastases beyond pelvis
>2 cm or lymph node metastases
[0677] Stage IV refers to distant metastases to the liver or
outside the peritoneal cavity.
[0678] Para-aortic lymph node metastases are considered regional
lymph nodes (Stage IIIC).
[0679] In some embodiments, the methods described herein treat an
ovarian cancer selected from the following: an adenocarcinoma in
the ovary and an adenocarcinoma that has migrated from the ovary
into the abdominal cavity.
[0680] Melanoma
[0681] A melanoma is a malignant tumor of melanocytes which are
found predominantly in skin but also in the bowel and the eye
(uveal melanoma). It is one of the rarer types of skin cancer but
causes the majority of skin cancer related deaths. Malignant
melanoma is a serious type of skin cancer caused by uncontrolled
growth of pigment cells, called melanocytes. Melanomas also
include, but are not limited to, a choroidea melanoma, malignant
melanomas, cutaneous melanomas and intraocular melanomas.
[0682] Melanoma may be divided into the following types: Lentigo
maligna, Lentigo maligna melanoma, superficially spreading
melanoma, acral lentiginous melanoma, mucosal melanoma, nodular
melanoma, polypoid melanoma, desmoplastic melanoma, amelanotic
melanoma, soft-tissue melanoma, and uveal melanoma. Melanoma stages
are as follows:
[0683] Stage 0--melanoma in situ (Clark Level I).
[0684] Stage I/II--invasive melanoma: T1a: less than 1.00 mm
primary, without ulceration, Clark Level II-III; T1b: less than
1.00 mm primary, with ulceration or Clark Level IV-V; and T2a:
1.00-2.00 mm primary, without ulceration.
[0685] Stage II--High Risk Melanoma: T2b: 1.00-2.00 mm primary,
with ulceration; T3a: 2.00-4.00 mm primary, without ulceration;
T3b: 2.00-4.00 mm primary, with ulceration; T4a: 4.00 mm or greater
primary without ulceration; and T4b: 4.00 mm or greater primary
with ulceration.
[0686] Stage III--Regional Metastasis: N1: single positive lymph
node; N2: 2-3 positive lymph nodes or regional skin/in-transit
metastasis; and N3: 4 positive lymph nodes or lymph node and
regional skin/in transit metastases.
[0687] Stage IV--Distant Metastasis: M1a: Distant Skin Metastasis,
Normal LDH; M1b: Lung Metastasis, Normal LDH; and M1c: Other
Distant Metastasis OR Any Distant Metastasis with Elevated LDH.
[0688] In one embodiment, the methods described herein treat a
melanoma.
[0689] Colon Cancer and Colorectal Cancer
[0690] Colorectal cancer (also called colon cancer or large bowel
cancer) includes cancerous growths in the colon, rectum (anus) and
appendix. With 655,000 deaths worldwide per year, it is the third
most common form of cancer and the second leading cause of
cancer-related death in the Western world. Many colorectal cancers
are thought to arise from adenomatous polyps in the colon. These
mushroom-like growths are usually benign, but some may develop into
cancer over time.
[0691] In another embodiment, Dukes classification may be used to
classify colorectal cancer based on stages A-D. Stage A refers to
colorectal cancer that is limited to mucosa (i.e., has not invaded
through the bowel wall). Stage B1 refers to extending into
muscularis propria, but not penetrating through it (i.e., lymph
nodes have not been invaded); whereas Stage B2 cancer has
penetrated through the muscularis propria, but not penetrating
through it (i.e., lymph nodes have not been invaded). Stage C1
refers to cancer that extends into the muscularis propria, but not
penetrating through it (i.e., lymph nodes are involved); whereas
Stage C2 refers to cancer that extends into the muscularis propria
and penetrating through it (i.e., lymph nodes are involved). Stage
D refers to distant metastatic spread. The TNM system may also be
used to stage colorectal cancer according to conventional means
known in the art.
[0692] Breast Cancer
[0693] Several types of breast cancer exist that may be treated by
the methods described herein. A lobular carcinoma in situ and a
ductal carcinoma in situ are breast cancers that have developed in
the lobules and ducts, respectively, but have not spread to the
fatty tissue surrounding the breast or to other areas of the body.
Infiltrating (or invasive) lobular and ductal carcinoma are cancers
that have developed in the lobules and ducts, respectively, and
have spread to either the breast's fatty tissue and/or other parts
of the body. In one aspect, provided herein is a method of treating
breast cancer, such as a ductal carcinoma in duct tissue in a
mammary gland, a breast cancer that is Her2- and/or ER- and/or
PR-.Other cancers of the breast that would benefit from treatment
by the methods are medullary carcinomas, colloid carcinomas,
tubular carcinomas, and inflammatory breast cancer.
[0694] In one embodiment, breast cancer is staged according to the
TNM system. Prognosis is closely linked to results of staging, and
staging is also used to allocate patients to treatments both in
clinical trials and clinical practice.
[0695] Briefly, the information for staging is as follows: TX:
Primary tumor cannot be assessed. T0: No evidence of tumor. Tis:
Carcinoma in situ, no invasion; T1: Tumor is 2 cm or less; T2:
Tumor is more than 2 cm but not more than 5 cm; T3: Tumor is more
than 5 cm; T4: Tumor of any size growing into the chest wall or
skin, or inflammatory breast cancer. NX: Nearby lymph nodes cannot
be assessed N0: cancer has not spread to regional lymph nodes. N1:
cancer has spread to 1 to 3 maxillary or one internal mammary lymph
node N2: cancer has spread to 4 to 9 maxillary lymph nodes or
multiple internal mammary lymph nodes N3: One of the following
applies: cancer has spread to 10 or more maxillary lymph nodes, or
cancer has spread to the lymph nodes under the clavicle (collar
bone), or cancer has spread to the lymph nodes above the clavicle,
or cancer involves maxillary lymph nodes and has enlarged the
internal mammary lymph nodes, or cancer involves 4 or more
maxillary lymph nodes, and tiny amounts of cancer are found in
internal mammary lymph nodes on sentinel lymph node biopsy. MX:
presence of distant spread (metastasis) cannot be assessed. M0: no
distant spread. M1: spread to distant organs (not including the
supraclavicular lymph node) has occurred.
[0696] Pancreatic Cancer
[0697] In another aspect, provided herein is a method of treating
pancreatic cancer selected from the following: an epithelial
carcinoma in the pancreatic duct tissue and an adenocarcinoma in a
pancreatic duct. The most common type of pancreatic cancer is an
adenocarcinoma, which occurs in the lining of the pancreatic
duct.
[0698] In one embodiment, the methods described herein treat a
pancreatic cancer.
[0699] Prostate Cancer
[0700] In one other aspect, provided herein is a method to treat
prostate cancer selected from the following: an adenocarcinoma or
an adenocarcinoma that has migrated to the bone. Prostate cancer
develops in the prostate organ in men, which surrounds the first
part of the urethra. The prostate has several cell types but 99% of
tumors are adenocarcinomas that develop in the glandular cells
responsible for generating seminal fluid.
[0701] There are two schemes commonly used to stage prostate
cancer. The most common is the TNM system, which evaluates the size
of the tumor, the extent of involved lymph nodes, and any
metastasis (distant spread). As with many other cancers, these are
often grouped into four stages (I-IV). Another scheme, used less
commonly, is the Whitmore-Jewett stage.
[0702] Briefly, Stage I disease is cancer that is found
incidentally in a small part of the sample when prostate tissue was
removed for other reasons, such as benign prostatic hypertrophy,
and the cells closely resemble normal cells and the gland feels
normal to the examining finger. In Stage II more of the prostate is
involved and a lump can be felt within the gland. In Stage III, the
tumor has spread through the prostatic capsule and the lump can be
felt on the surface of the gland. In Stage IV disease, the tumor
has invaded nearby structures, or has spread to lymph nodes or
other organs. Grading is based on cellular content and tissue
architecture from biopsies (Gleason) which provides an estimate of
the destructive potential and ultimate prognosis of the
disease.
[0703] In one embodiment, the methods described herein treat a
prostate cancer.
[0704] Head and Neck Cancers
[0705] Head and neck cancers (e.g., oral, laryngeal,
nasopharyngeal, esophageal, etc.), refer to a group of biologically
similar cancers originating from the upper aerodigestive tract,
including the lip, oral cavity (mouth), nasal cavity, paranasal
sinuses, pharynx, and larynx. Most head and neck cancers are
squamous cell carcinomas, originating from the mucosal lining
(epithelium) of these regions. Head and neck cancers often spread
to the lymph nodes of the neck, and this is often the first (and
sometimes only) manifestation of the disease at the time of
diagnosis. Head and neck cancer is strongly associated with certain
environmental and lifestyle risk factors, including tobacco
smoking, alcohol consumption, and certain strains of the sexually
transmitted human papillomavirus. Management of patients with head
and neck cancers remains a formidable task. Cancers such as,
hypopharyngeal cancer, laryngeal cancer, nasopharyngeal cancer,
oropharyngeal cancer, may be treated using the compounds described
herein.
[0706] In one embodiment, the methods described herein treat a head
or neck cancer.
[0707] Kidney Cancer
[0708] In another aspect, provided herein is a method to treat
kidney cancer. Kidney cancer (also called renal cell cancer, renal
cell carcinoma, renal adenocarcinoma, and hypernephroma) is a
disease in which malignant cells are found in the lining of tubules
in the kidney. Renal cell carcinoma is the most common form of
kidney cancer arising from the proximal renal tubule. It is the
most common type of kidney cancer in adults, responsible for
approximately 80% of cases.
[0709] In one embodiment, the methods described herein treat a
kidney cancer.
[0710] Liver Cancer
[0711] In another aspect, provided herein is a method to treat
primary liver cancer (cancer that begins in the liver). Primary
liver cancer can occur in both adults and children. Liver cancer is
characterized by the presence of malignant hepatic tumors--tumors
or growths on or in the liver. They may be discovered on medical
imaging (even for a different reason than the cancer itself), or
may be present in patients as an abdominal mass, abdominal pain,
jaundice, or some other liver dysfunction. There are several types
of liver cancer.
[0712] Hemangiomas: These are the most common type of benign liver
tumor. They start in blood vessels. Most of these tumors do not
cause symptoms, they do not need treatment. Some may bleed and need
to be removed if it is mild to severe.
[0713] Hepatic adenomas: These benign epithelial liver tumors
develop in the liver. They are, in most cases, located in the right
hepatic lobe and are frequently seen as solitary. The size of
adenomas range from 1 to 30 cm. Symptoms associated with hepatic
adenomas are all associated with large lesions which can cause
intense abdominal pain.
[0714] Focal nodular hyperplasia: Focal nodular hyperplasia (FNH)
is the second most common tumor of the liver. This tumor is the
result of a congenital arteriovenous malformation hepatocyte
response. This process is one in which all normal constituents of
the liver are present, but the pattern by which they are presented
is abnormal. Even though those conditions exist the liver still
seems to perform in the normal range.
[0715] Hepatocellular Cancer: Hepatocellular cancer (HCC) is the
most common cancer of the liver. It is associated with alcohol
abuse and hepatitis B infection and is particularly prevalent in
Asia. The majority of HCC is detected at a time when cure by
surgical resection is not possible; systemic treatment of
un-resectable HCC is associated with survival of less than one
year.
[0716] In one embodiment, the methods described herein treat a
liver cancer.
[0717] Lymphoma
[0718] Lymphoma is a type of cancer that originates in lymphocytes
of the immune system. They often originate in lymph nodes,
presenting as an enlargement of the node (a tumor). Lymphomas are
closely related to lymphoid leukemias, which also originate in
lymphocytes but typically involve only circulating blood and the
bone marrow (where blood cells are generated in a process termed
haematopoesis) and do not usually form tumors. There are many types
of lymphomas, and in turn, lymphomas are a part of the broad group
of diseases called hematological neoplasms. Some forms of lymphoma
are indolent (e.g., small lymphocytic lymphoma), compatible with a
long life even without treatment, whereas other forms are
aggressive (e.g., Burkitt's lymphoma), causing rapid deterioration
and death.
[0719] The WHO Classification, published in 2001 and updated in
2008;
en.wikipedia.org/wiki/Lymphoma--cite_note-isbn92-832-2411-6-2#cite_note-i-
sbn92-832-2411-6-2 is the latest classification of lymphoma and is
based upon the foundations laid within the "Revised
European-American Lymphoma classification" (REAL). This system
groups lymphomas by cell type (i.e., the normal cell type that most
resembles the tumor) and defining phenotypic, molecular or
cytogenetic characteristics. There are three large groups: the B
cell, T cell, and natural killer cell tumors. Other less common
groups are also recognized. Hodgkin's lymphoma, although considered
separately within the WHO (and preceding) classifications, is now
recognized as being a tumor of, albeit markedly abnormal,
lymphocytes of mature B cell lineage.
[0720] In one embodiment, the methods described herein treat a
lymphoma.
[0721] Sarcoma
[0722] A sarcoma is a cancer of the connective tissue (bone,
cartilage, fat) resulting in mesoderm proliferation.
[0723] This is in contrast to carcinomas, which are of epithelial
origin (breast, colon, pancreas, and others). However, due to an
evolving understanding of tissue origin, the term "sarcoma" is
sometimes applied to tumors now known to arise from epithelial
tissue. The term soft tissue sarcoma is used to describe tumors of
soft tissue, which includes elements that are in connective tissue,
but not derived from it (such as muscles and blood vessels).
[0724] Sarcomas are given a number of different names, based on the
type of tissue from which they arise. For example, osteosarcoma
arises from bone, chondrosarcoma arises from cartilage, and
leiomyosarcoma arises from smooth muscle. Sarcomas strike people in
all age ranges, but they are very rare, accounting for only 1% of
all cases of cancer. GIST is the most common form of sarcoma, with
approximately 3000-3500 cases per year in the United States. This
should be compared with breast cancer, with approximately 200,000
cases per year in North America.
[0725] Approximately 50% of bone sarcomas and 20% of soft tissue
sarcomas are diagnosed in people under the age of 35. Some
sarcomas, such as leiomyosarcoma, chondrosarcoma, and
gastrointestinal stromal tumor (GIST), are more common in adults
than in children. Most high grade bone sarcomas, including Ewing's
sarcoma and osteosarcoma, are much more common in children and
young adults.
[0726] In one embodiment, the methods described herein treat a
sarcoma.
[0727] Carcinoma
[0728] A carcinoma is any malignant cancer that arises from
epithelial cells. Carcinomas invade surrounding tissues and organs
and may metastasize, or spread, to lymph nodes and other sites.
[0729] Carcinoma, like all neoplasia, is classified by its
histopathological appearance. Adenocarcinoma and squamous cell
carcinoma, two common descriptive terms for tumors, reflect the
fact that these cells may have glandular or squamous cell
appearances respectively. Severely anaplastic tumors might be so
undifferentiated that they do not have a distinct histological
appearance (undifferentiated carcinoma).
[0730] Sometimes a tumor is referred to by the presumptive organ of
the primary (e.g., carcinoma of the prostate) or the putative cell
of origin (hepatocellular carcinoma, renal cell carcinoma).
[0731] Adenocarcinoma is a malignant tumor originating in the
epithelial cells of glandular tissue and forming glandular
structures. This is common in the lung (forming 30-40% of all lung
carcinomas). It is found peripherally, arising from goblet cells or
type II pneumocytes.
[0732] Squamous cell carcinoma results from squamous metaplasia.
This accounts for 20-30 percent of lung tumors and is usually hilar
in origin.
[0733] Small cell carcinoma is almost certainly due to smoking.
These metastasize early, and may secrete ADH (lowering patient
sodium concentration).
[0734] Large cell undifferentiated carcinomas account for 10-15
percent of lung neoplasms. These are aggressive and difficult to
recognize due to the undifferentiated nature. These are most
commonly central in the lung.
[0735] Sinonasal undifferentiated carcinoma.
[0736] In one embodiment, the methods described herein treat a
carcinoma.
[0737] Myeloma
[0738] Multiple myeloma (also known as MM, myeloma, plasma cell
myeloma, or as Kahler's disease after Otto Kahler) is a cancer of
plasma cells. These immune cells are formed in bone marrow, are
numerous in lymphatics and produce antibodies. Myeloma is regarded
as incurable, but remissions may be induced with steroids,
chemotherapy, thalidomide and stem cell transplants. Myeloma is
part of the broad group of diseases called hematological
malignancies.
[0739] Multiple myeloma develops in post-germinal center B
lymphocytes. A chromosomal translocation between the immunoglobulin
heavy chain gene (on the fourteenth chromosome, locus 14q32) and an
oncogene (often 11q13, 4p16.3, 6p21, 16q23 and 20q11) is frequently
observed in patients with multiple myeloma. This mutation results
in dysregulation of the oncogene which is thought to be an
important initiating event in the pathogenesis of myeloma. The
result is proliferation of a plasma cell clone and genomic
instability that leads to further mutations and translocations. The
chromosome 14 abnormality is observed in about 50% of all cases of
myeloma. Deletion of (parts of) the thirteenth chromosome is also
observed in about 50% of cases.
[0740] Production of cytokines (especially IL-6) by the plasma
cells causes much of their localized damage, such as osteoporosis,
and creates a microenvironment in which the malignant cells thrive.
Angiogenesis (the attraction of new blood vessels) is
increased.
[0741] In one embodiment, the methods described herein treat a
myeloma.
[0742] Stomach Cancer
[0743] Stomach or gastric cancer can develop in any part of the
stomach and may spread throughout the stomach and to other organs;
particularly the esophagus, lungs and the liver. Stomach cancer
causes about 800.000 deaths worldwide per year.
[0744] Metastasis occurs in 80-90% of individuals with stomach
cancer, with a six month survival rate of 65% in those diagnosed in
early stages and less than 15% of those diagnosed in late
stages.
[0745] Stomach cancer is often asymptomatic or causes only
nonspecific symptoms in its early stages. By the time symptoms
occur, the cancer has generally metastasized to other parts of the
body, one of the main reasons for its poor prognosis.
[0746] In one embodiment, the methods described herein treat a
stomach cancer.
[0747] Thyroid Cancer
[0748] Thyroid neoplasm or thyroid cancer usually refers to any of
four kinds of malignant tumors of the thyroid gland: papillary,
follicular, medullary or anaplastic. Papillary and follicular
tumors are the most common. They grow slowly and may recur, but are
generally not fatal in patients under 45 years of age. Medullary
tumors have a good prognosis if restricted to the thyroid gland and
a poorer prognosis if metastasis occurs. Anaplastic tumors are
fast-growing and respond poorly to therapy.
[0749] Thyroid cancer is usually found in a euthyroid patient, but
symptoms of hyperthyroidism or hypothyroidism may be associated
with a large or metastatic well-differentiated tumor. Nodules are
of particular concern when they are found in those under the age of
20. The presentation of benign nodules at this age is less likely,
and thus the potential for malignancy is far greater.
[0750] Thyroid cancers can be classified according to their
pathological characteristics. The following variants can be
distinguished (distribution over various subtypes may show regional
variation): papillary thyroid cancer (up to 75%); follicular
thyroid cancer (up to 15%); medullary thyroid cancer (up to 8%);
and anaplastic thyroid cancer (less than 5%). The follicular and
papillary types together can be classified as "differentiated
thyroid cancer". These types have a more favorable prognosis than
the medullary and undifferentiated types. Thyroid adenoma is a
benign neoplasm of the thyroid.
[0751] In one embodiment, the methods described herein treat a
thyroid cancer.
[0752] Bladder Cancer
[0753] Bladder cancer refers to any of several types of malignant
growths of the urinary bladder. It is a disease in which abnormal
cells multiply without control in the bladder. The bladder is a
hollow, muscular organ that stores urine; it is located in the
pelvis. The most common type of bladder cancer begins in cells
lining the inside of the bladder and is called transitional cell
carcinoma (sometimes urothelial cell carcinoma).
[0754] 90% of bladder cancers are transitional cell carcinoma. The
other 10% are squamous cell carcinoma, adenocarcinoma, sarcoma,
small cell carcinoma and secondary deposits from cancers elsewhere
in the body.
[0755] The following stages are used to classify the location,
size, and spread of the cancer, according to the TNM (tumor, lymph
node, and metastasis) staging system: Stage 0: Cancer cells are
found only on the inner lining of the bladder. Stage I: Cancer
cells have proliferated to the layer beyond the inner lining of the
urinary bladder but not to the muscles of the urinary bladder.
Stage II: Cancer cells have proliferated to the muscles in the
bladder wall but not to the fatty tissue that surrounds the urinary
bladder. Stage III: Cancer cells have proliferated to the fatty
tissue surrounding the urinary bladder and to the prostate gland,
vagina, or uterus, but not to the lymph nodes or other organs.
Stage IV: Cancer cells have proliferated to the lymph nodes, pelvic
or abdominal wall, and/or other organs. Recurrent: Cancer has
recurred in the urinary bladder or in another nearby organ after
having been treated.
[0756] Bladder TCC is staged according to the 1997 TNM system: Ta
Non-invasive papillary tumor; T1 Invasive but not as far as the
muscular bladder layer; T2 Invasive into the muscular layer; T3
Invasive beyond the muscle into the fat outside the bladder; and T4
Invasive into surrounding structures like the prostate, uterus or
pelvic wall.
[0757] In one embodiment, the methods described herein treat a
bladder cancer.
[0758] In accordance with the invention, the humanized endoglin
antibodies or fragments thereof can be administered alone or in
combination with active or inactive agents. When combinations are
used, the invention contemplates simultaneous or sequential
administration of the humanized endoglin antibodies or
antigen-binding fragments and the active or inactive agents.
[0759] Compounds of the present invention can be, as needed,
administered in combination with one or more therapeutic treatments
including, but not limited to, adriamycin, cyclophosphamide,
paclitaxel, pemetrexed, temozolomide, oxaliplatin, bevacizumab,
erbitux, vectibix, sorafenib, sunitinib, gefitinib, erlotinib,
5-fluorouracil (5-FU) irinotecan, topotecan, leucovorin,
VELCADE.RTM., lenalidomide, thalidomide, xeloda, taxotere and many
other conventional cancer therapies described herein. One would
understand that the listing of therapeutic regimens listed below
represents conventional therapies, but the present invention
encompasses other known therapeutic regimens which are not
specifically disclosed herein
[0760] As used herein, "radiation" refers to, for example,
microwaves, ultraviolet (UV), infrared (IR), or alpha-, beta- or
gamma-radiation. Radiation can be "focused" or locally delivered
using conventional techniques to target radiation to the site of
one or more tumors without radiating the entire body.
[0761] In one embodiment, the cancer is ovarian cancer and the one
or more therapeutic treatments is surgery, chemotherapy (e.g.,
doxorubicin, doxil, gemcitabine, Rubitecan, and platinum-based
chemotherapeutics such as cisplatin, carboplatin and oxaliplatin),
melphalan, paclitaxel, topoisomerase I inhibitors such as topotecan
and irinotecan, taxane-based therapy, hormones, radiation therapy,
whole body hypothermia, isoflavone derivatives such as Phenoxodial,
cytotoxic macrolides such as Epothilones, angiogenesis inhibitors
such as bevacizumab, signal transduction inhibitors such as
trastuzumab, gene therapy, RNAi therapy, immunotherapy, monoclonal
antibodies, phosphatidylinositol-like kinase inhibitors such as
rapamycin, or any combination thereof. In one embodiment the
combination is a humanized anti-endoglin antibody or
antigen-binding fragment thereof and doxil. In another embodiment,
the combination is a humanized anti-endoglin antibody or
antigen-binding fragment thereof and topotecan. In yet another
embodiment, the combination is a humanized anti-endoglin antibody
or antigen-binding fragment thereof and a VEGF receptor inhibitor.
Non-limiting examples of VEGF receptor inhibitors include
bevacizumab (AVASTIN.RTM.), ranibizumab (LUCENTIS.RTM.),
aflibercept (VEGF-Trap), sunitinib (SUTENT.RTM.), sorafenib
(NEXAVAR.RTM.), axitinib, pegaptanib and pazopanib. The combination
therapy of the antibodies and antigen-binding fragments described
herein with the ovarian cancer therapies may also provide for lower
doses of either therapy, or both, due to a synergistic effect from
the co-administration of the therapies.
[0762] In one embodiment, the cancer is renal/kidney cancer and the
one or more therapeutic treatments is surgery, chemotherapy,
sunitinib, sorafenib, pazopanib, AVASTIN.RTM., interferon-alpha, or
IL-2. In one embodiment the combination is a humanized
anti-endoglin antibody or antigen-binding fragment thereof and
sorafenib. In one embodiment the combination is a humanized
anti-endoglin antibody or antigen-binding fragment thereof and
sunitinib. In one embodiment the combination is a humanized
anti-endoglin antibody or antigen-binding fragment thereof and
AVASTIN.RTM.. In yet another embodiment, the combination is a
humanized anti-endoglin antibody or antigen-binding fragment
thereof and a VEGF receptor inhibitor. Non-limiting examples of
VEGF receptor inhibitors include bevacizumab (AVASTIN.RTM.),
ranibizumab (LUCENTIS.RTM.), aflibercept (VEGF-Trap), sunitinib
(SUTENT.RTM.), sorafenib (NEXAVAR.RTM.), axitinib, pegaptanib and
pazopanib. The combination therapy of the antibodies and
antigen-binding fragments described herein with the kidney cancer
therapies may also provide for lower doses of either therapy, or
both, due to a synergistic effect from the co-administration of the
therapies.
[0763] In one embodiment, the cancer is myeloma and the one or more
therapeutic treatments is surgery, radiotherapy, VELCADE.RTM.,
lenalidomide, or thalidomide. In one embodiment the combination is
a humanized anti-endoglin antibody or antigen-binding fragment
thereof and VELCADE.RTM.. The dosages for any of these therapies
are known in the art and can be adjusted with combination therapy
accordingly.
[0764] In one embodiment, the cancer is prostate cancer and the one
or more therapeutic treatments is surgery, radiotherapy (e.g.,
external beam or brachytherapy), hormonal deprivation (androgen
suppression), heat shock protein 90 (HSP90) inhibitors,
chemotherapy (e.g., docetaxel, platinum-based chemotherapy such as
cisplatin, carboplatin, satraplatin and oxaliplatin, taxane,
estramustine), prednisone or prednisolone, cholesterol-lowering
drugs such as statins, leutinizing hormone-releasing hormone (LHRH)
agonists, RNAi therapy, whole tumor cells genetically modified to
secrete granulocyte macrophage--colony stimulating factor (GM-CSF)
(also known as GVAX), or any combination thereof. In yet another
embodiment, the combination is a humanized anti-endoglin antibody
or antigen-binding fragment thereof and a VEGF receptor inhibitor.
Non-limiting examples of VEGF receptor inhibitors include
bevacizumab (AVASTIN.RTM.), ranibizumab (LUCENTIS.RTM.),
aflibercept (VEGF-Trap), sunitinib (SUTENT.RTM.), sorafenib
(NEXAVAR.RTM.), axitinib, pegaptanib and pazopanib.
[0765] In one embodiment, the cancer is lung cancer and the one or
more therapeutic treatments is surgery, radiotherapy (e.g.,
thoracic radiotherapy, radiation therapy with charged particles,
Uracil-tegafur and Platinum-based chemotherapy (e.g., cisplatin,
carboplatin, oxaliplatin, etc.) and vinorelbine, Erlotinib
(TARCEVA.RTM.), Gefitinib (IRESSA.RTM.), anti-epidermal growth
factor receptor antibodies (e.g., Cetuximab), anti-vascular
endothelial growth factor antibodies (e.g., Bevacizumab), small
molecule inhibitors of tyrosine kinases, direct inhibitors of
proteins involved in lung cancer cell proliferation, Aurora kinase
inhibitors, laser-induced thermotherapy, RNAi therapy, whole tumor
cells genetically modified to secrete granulocyte
macrophage--colony stimulating factor (GM-CSF) (also known as
GVAX), or any combination thereof. Additional therapeutic
treatments include Taxol and pemetrexed. In one embodiment the
combination is a humanized anti-endoglin antibody or
antigen-binding fragment thereof and erlotinib. In one embodiment
the combination is a humanized anti-endoglin antibody or
antigen-binding fragment thereof and gefitinib. In one embodiment
the combination is a humanized anti-endoglin antibody or
antigen-binding fragment thereof and pemetrexed. In yet another
embodiment, the combination is a humanized anti-endoglin antibody
or antigen-binding fragment thereof and a VEGF receptor inhibitor.
Non-limiting examples of VEGF receptor inhibitors include
bevacizumab (AVASTIN.RTM.), ranibizumab (LUCENTIS.RTM.),
aflibercept (VEGF-Trap), sunitinib (SUTENT.RTM.), sorafenib
(NEXAVAR.RTM.), axitinib, pegaptanib and pazopanib. The dosages for
any of these therapies are known in the art and can be adjusted
with combination therapy accordingly.
[0766] In one embodiment, the cancer is breast cancer and the one
or more therapeutic treatments is surgery, monoclonal antibodies
(e.g., Her-2 antibodies, herceptin), adjuvant chemotherapy such as
single agent chemotherapy or combination chemotherapy (e.g.,
anthracycline- and taxane-based polychemotherapies, taxol, or
target-specific trastuzumab with or without endocrine manipulation
with or without PMRT, vinorelbine), adriamycin, cyclophosphamide,
xeloda, taxotere, selective estrogen receptor modulators such as
Tamoxifen and Raloxifene, allosteric estrogen receptor modulators
such as Trilostane, radiation (e.g., interstitial brachytherapy,
Mammosite device, 3-dimensional conformal external radiation and
intraoperative radiotherapy), Aromatase inhibitors that suppress
total body synthesis (e.g., anastrozole, exemestane and letrozole),
RNAi therapy, intravenous analogs of rapamycin that are
immunosuppressive and anti-proliferative such as Temsirolimus
(CCI779), or any combination thereof. A review of methods for
conducting three-dimensional in vitro tissue culture models of
breast cancer are described by Kim et al., Breast Cancer Research
Treatment 85(3): 281-91 (2004). Other in vivo and in vitro models
for testing cancers are known and can be used to test anti-endoglin
antibodies described herein. In one embodiment the combination is a
humanized anti-endoglin antibody or antigen-binding fragment
thereof, taxol, and AVASTIN.RTM.. In one embodiment the combination
is a humanized anti-endoglin antibody or antigen-binding fragment
thereof and adriamycin. In one embodiment the combination is a
humanized anti-endoglin antibody or antigen-binding fragment
thereof and xeloda. In one embodiment the combination is a
humanized anti-endoglin antibody or antigen-binding fragment
thereof and taxotere. In yet another embodiment, the combination is
a humanized anti-endoglin antibody or antigen-binding fragment
thereof and a VEGF receptor inhibitor. Non-limiting examples of
VEGF receptor inhibitors include bevacizumab (AVASTIN.RTM.),
ranibizumab (LUCENTIS.RTM.), aflibercept (VEGF-Trap), sunitinib
(SUTENT.RTM.), sorafenib (NEXAVAR.RTM.), axitinib, pegaptanib and
pazopanib. The dosages for any of these therapies are known in the
art and can be adjusted with combination therapy accordingly.
[0767] In one embodiment, the cancer is colon cancer and the one or
more therapeutic treatments is surgery, radiation therapy, and
chemotherapy (e.g., 5-fluorouracil, levamisole, leucovorin or
semustine (methyl CCNU)),
N-[2-(dimethylamino)ethyl]acridine-4-carboxamide and other related
carboxamide anticancer drugs; non-topoisomerase II inhibitors,
irinotecan, liposomal topotecan, taxane class of anticancer agents
(e.g., paclitaxel or docetaxel), a compound of the xanthenone
acetic acid class (e.g., 5,6-dimethylanthenone-4-acetic acid PMAA),
laminarin, site-selective cyclic AMP Analogs (e.g.,
8-chloroadenosine 3',5'-cyclic phosphate), pyranoindole inhibitors
of Cox-2, carbazole inhibitors of Cox-2, tetrahydrocarbazole
inhibitors of Cox-2, indene inhibitors of Cox-2, localized
inhibitors of NSAIDS (e.g., anthranilic acids, aspirin
(5-acetylsalicylic acid), azodisal sodium, carboheterocyclic acids,
carprofen, chlorambucil, diclophenac, fenbufen, fenclofenac,
fenoprofen, flufenamic acid, flurbiprofen, fluprofen, furosemide,
gold sodium thiomalate, ibuprofen, indomethacin, indoprofen,
ketoprofen, lonazolac, loxoprofen, meclofenamic acid, mefanamic
acid, melphalan, naproxen, penicillamine, phenylacetic acids,
proprionic acids, salicylic acids, salazosulfapyridine, sulindac,
tolmetin, a pyrazolone butazone propazone NSAID, meloxicam,
oxicams, piroxicam, feldene, piroxicam beta cyclodextran,
tenoxicam, etodolac, and oxaprozin), an inhibitor of HER-2/neu,
RNAi therapy, GM-CSF, monoclonal antibodies (e.g., anti-Her-2/neu
antibodies, anti-CEA antibodies, A33 (HB 8779), 100-210 (HB 11764)
and 100-310 (HB 11028)), erbitux, vectibix, hormonal therapy,
pyrimidineamines, camptothecin derivatives (e.g., CPT-11), folinic
acid (FA), Gemcitabine, Ara-C, platinum-based chemotherapeutics
such as cisplatin, carboplatin and oxaliplatin, a cGMP-specific
phosphodiesterase inhibitor, or any combination thereof. In one
embodiment the combination is a humanized anti-endoglin antibody or
antigen-binding fragment thereof and a combination of 5-FU,
leucovorin and oxaliplatin (FOLFOX). In one embodiment the
combination is a humanized anti-endoglin antibody or
antigen-binding fragment thereof and a combination of 5-FU,
irinotecan and leucovorin (IFL). In one embodiment the combination
is a humanized anti-endoglin antibody or antigen-binding fragment
thereof and erbitux. In one embodiment the combination is a
humanized anti-endoglin antibody or antigen-binding fragment
thereof and a vectibix. In yet another embodiment, the combination
is a humanized anti-endoglin antibody or antigen-binding fragment
thereof and a VEGF receptor inhibitor. Non-limiting examples of
VEGF receptor inhibitors include bevacizumab (AVASTIN.RTM.),
ranibizumab (LUCENTIS.RTM.), aflibercept (VEGF-Trap), sunitinib
(SUTENT.RTM.), sorafenib (NEXAVAR.RTM.), axitinib, pegaptanib and
pazopanib. The dosages for any of these therapies are known in the
art and can be adjusted with combination therapy accordingly.
[0768] In one embodiment, the cancer is pancreatic cancer and the
one or more therapeutic treatments is surgery, radiation therapy
(RT), Fluorouracil (5-FU) and RT, systemic therapy, stenting,
Gemcitabine (GEMZAR.RTM.), Gemcitabine and RT, Cetuximab, erlotinib
(TARCEVA.RTM.), chemoradiation, bevacizumab (AVASTIN.RTM.), or any
combination thereof. In yet another embodiment, the combination is
a humanized anti-endoglin antibody or antigen-binding fragment
thereof and a VEGF receptor inhibitor. Non-limiting examples of
VEGF receptor inhibitors include bevacizumab (AVASTIN.RTM.),
ranibizumab (LUCENTIS.RTM.), aflibercept (VEGF-Trap), sunitinib
(SUTENT.RTM.), sorafenib (NEXAVAR.RTM.), axitinib, pegaptanib and
pazopanib.
[0769] Patients can be assessed with respect to symptoms at one or
more multiple time points including prior to, during, and after
treatment regimens. Treatment can result in improving the subject's
condition and can be assessed by determining if one or more of the
following factors has occurred: decreased tumor size, decreased
cell proliferation, decreased numbers of cells, decreased
neovascularization, increased apoptosis, or decreased survival of
at least a portion of the cells comprising the cell proliferative
disorder. One or more of these occurrences may, in some cases,
result in partial or total elimination of the cancer and
prolongation of survival of the patient. Alternatively, for
terminal stage cancers, treatment may result in stasis of disease,
better quality of life and/or prolongation of survival.
Biomarker Assessment
[0770] Certain genes can be expressed at increased or decreased
levels in cancers. Changes in expression levels of genes in cancers
can be indicative of resistance or sensitivity to a cancer therapy
or treatment.
[0771] Provided herein is a diagnostic method for detecting the
expression of at least one gene chosen from a panel of genes whose
expression has been correlated with sensitivity or resistance to an
angiogenesis inhibitor, wherein the at least one gene is: VEGF,
VEGF receptor, HIF-1.alpha., placental growth factor receptor or
endoglin (CD105). The method can further include the step of
comparing the level of expression of at least one gene detected in
the patient sample to a level of expression of at least one gene
that has been correlated with sensitivity or resistance to the
angiogenesis inhibitor. In one non-limiting embodiment, the
angiogenesis inhibitor is a humanized anti-endoglin antibody. In
another embodiment, the angiogenesis inhibitor is a VEGF receptor
inhibitor or a VEGF inhibitor.
[0772] As used herein, the term "expression," when used in
connection with detecting the expression of a gene, can refer to
detecting transcription of the gene and/or to detecting translation
of the gene. To detect expression of a gene refers to the act of
actively determining whether a gene is expressed or not. This can
include determining whether the gene expression is upregulated as
compared to a control, downregulated as compared to a control, or
unchanged as compared to a control. Therefore, the step of
detecting expression does not require that expression of the gene
actually is upregulated or downregulated, but rather, can also
include detecting that the expression of the gene has not changed
(i.e., detecting no expression of the gene or no change in
expression of the gene).
[0773] Biomarkers to be assessed in connection with the present
invention include VEGF receptor, placental growth factor,
HIF-1.alpha. and endoglin (CD105).
[0774] For assessment of biomarker expression, patient samples
containing endothelial tissue, tumor cells, or proteins or nucleic
acids produced by tumor cells, can be used in methods described
herein and further known in the art. Briefly, the level of
expression of the biomarker can be assessed by assessing the amount
(e.g., absolute amount or concentration) of the marker in a sample,
e.g., a tumor biopsy obtained from a patient, or other patient
sample containing material derived from the tumor (e.g., blood,
serum, urine, or other bodily fluids or excretions as described
herein above). The cell sample can, of course, be subjected to a
variety of well-known post-collection preparative and storage
techniques (e.g., nucleic acid and/or protein extraction, fixation,
storage, freezing, ultrafiltration, concentration, evaporation,
centrifugation, etc.) prior to assessing the amount of the marker
in the sample. Likewise, tumor biopsies can also be subjected to
post-collection preparative and storage techniques, e.g.,
fixation.
[0775] One can detect expression of biomarker proteins having at
least one portion which is displayed on the surface of cells which
express it. One can determine whether a marker protein, or a
portion thereof, is exposed on the cell surface. For example,
immunological methods can be used to detect such proteins on whole
cells, or well known computer-based sequence analysis methods can
be used to predict the presence of at least one extracellular
domain (i.e., including both secreted proteins and proteins having
at least one cell-surface domain). Expression of a marker protein
having at least one portion which is displayed on the surface of a
cell which expresses it can be detected without necessarily lysing
the tumor cell (e.g., using a labeled antibody which binds
specifically with a cell-surface domain of the protein).
[0776] Expression of biomarkers can be assessed by any of a wide
variety of well known methods for detecting expression of a
transcribed nucleic acid or protein. Non-limiting examples of such
methods include, for example, immunological methods for detection
of secreted, cell-surface, cytoplasmic, or nuclear proteins,
protein purification methods, protein function or activity assays,
nucleic acid hybridization methods, nucleic acid reverse
transcription methods, and nucleic acid amplification methods or
any other method known in the art.
[0777] A mixture of transcribed polynucleotides obtained from the
sample can be contacted with a substrate having fixed thereto a
polynucleotide complementary to or homologous with at least a
portion (e.g., at least 7, 10, 15, 20, 25, 30, 40, 50, 100, 500, or
more nucleotide residues) of a biomarker nucleic acid. If
polynucleotides complementary to, or homologous with, are
differentially detectable on the substrate (e.g., detectable using
different chromophores or fluorophores, or fixed to different
selected positions), then the levels of expression of a plurality
of biomarkers can be assessed simultaneously using a single
substrate (e.g., a "gene chip" microarray of polynucleotides fixed
at selected positions). When a method of assessing biomarker
expression is used which involves hybridization of one nucleic acid
with another, hybridization can be performed under stringent
hybridization conditions.
[0778] When a plurality of biomarkers of the invention are used in
the methods of the invention, the level of expression of each
biomarker in a patient sample can be compared with the normal level
of expression of each of the plurality of biomarkers in
non-cancerous samples of the same type, either in a single reaction
mixture (i.e., using reagents, such as different fluorescent
probes, for each biomarker) or in individual reaction mixtures
corresponding to one or more of the biomarkers.
[0779] The level of expression of a biomarker in normal (i.e.,
non-cancerous) human tissue can be assessed in a variety of ways.
This normal level of expression can be assessed by assessing the
level of expression of the biomarker in a portion of cells which
appears to be non-cancerous, and then comparing the normal level of
expression with the level of expression in a portion of the tumor
cells. As further information becomes available as a result of
routine performance of the methods described herein,
population-average values for normal expression of the biomarkers
can be used. Alternatively, the normal level of expression of a
biomarker can be determined by assessing expression of the
biomarker in a patient sample obtained from a non-cancer-afflicted
patient, from a patient sample obtained from a patient before the
suspected onset of cancer in the patient, from archived patient
samples, and the like.
[0780] An exemplary method for detecting the presence or absence of
a biomarker protein or nucleic acid in a biological sample involves
obtaining a biological sample from a test subject and contacting
the biological sample with a compound or an agent capable of
detecting the polypeptide or nucleic acid (e.g., mRNA, genomic DNA,
or cDNA). The detection methods can, thus, be used to detect mRNA,
protein, cDNA, or genomic DNA, for example, in a biological sample
in vitro as well as in vivo. In vitro techniques for detection of
mRNA include, for example, reverse transcriptase-polymerase chain
reaction (RT-PCR; e.g., the experimental embodiment set forth in
Mullis, 1987, U.S. Pat. No. 4,683,202), Northern hybridizations and
in situ hybridizations. In vitro techniques for detection of a
biomarker protein include, but are not limited to, enzyme linked
immunosorbent assays (ELISAs), Western blots, immunoprecipitations
and immunofluorescence. In vitro techniques for detection of
genomic DNA include, for example, Southern hybridizations. In vivo
techniques for detection of mRNA include, for example, polymerase
chain reaction (PCR), quantitative PCR, Northern hybridizations and
in situ hybridizations. Furthermore, in vivo techniques for
detection of a biomarker protein include introducing into a subject
a labeled antibody directed against the protein or fragment
thereof. For example, the antibody can be labeled with a
radioactive marker whose presence and location in a subject can be
detected by standard imaging techniques.
[0781] A general principle of such diagnostic and prognostic assays
involves preparing a sample or reaction mixture that may contain a
biomarker, and a probe, under appropriate conditions and for a time
sufficient to allow the biomarker and probe to interact and bind,
thus forming a complex that can be removed and/or detected in the
reaction mixture. These assays can be conducted in a variety of
ways using a variety of methods.
[0782] It is also possible to directly detect biomarker/probe
complex formation without further manipulation or labeling of
either component (biomarker or probe), for example by utilizing the
technique of fluorescence energy transfer (i.e., FET, see for
example, Lakowicz et al., U.S. Pat. No. 5,631,169; and
Stavrianopoulos, et al., U.S. Pat. No. 4,868,103).
[0783] In another embodiment, determination of the ability of a
probe to recognize a biomarker can be accomplished without labeling
either assay component (probe or biomarker) by utilizing a
technology such as real-time Biomolecular Interaction Analysis
(BIA; see, e.g., Sjolander, S. and Urbaniczky, C., 1991, Anal.
Chem. 63:2338-2345 and Szabo et al., 1995, Curr. Opin. Struct.
Biol. 5:699-705). As used herein, "BIA" or "surface plasmon
resonance" refer to a technology for studying biospecific
interactions in real time, without labeling any of the interactants
(e.g., BIAcore). Changes in the mass at the binding surface
(indicative of a binding event) result in alterations of the
refractive index of light near the surface (the optical phenomenon
of surface plasmon resonance (SPR)), resulting in a detectable
signal which can be used as an indication of real-time reactions
between biological molecules.
[0784] As an alternative to making determinations based on the
absolute expression level of the biomarker, determinations can be
based on the normalized expression level of the biomarker.
Expression levels are normalized by correcting the absolute
expression level of a biomarker by comparing its expression to the
expression of a gene that is not a biomarker, e.g., a housekeeping
gene that is constitutively expressed. Suitable genes for
normalization include housekeeping genes such as the actin gene, or
epithelial cell-specific genes. This normalization allows the
comparison of the expression level in one sample, e.g., a patient
sample, to another sample, e.g., a non-tumor sample, or between
samples from different sources.
[0785] Alternatively, the expression level can be provided as a
relative expression level. To determine a relative expression level
of a biomarker (e.g., VEGF receptor, placental growth factor,
Hif-1.alpha. and endoglin (CD105)), the level of expression of the
biomarker is determined for 10 or more, 20 or more, 30 or more, 40
or more, or 50 or more samples of normal versus cancer cell
isolates prior to the determination of the expression level for the
sample in question. The mean expression level of each of the genes
assayed in the larger number of samples is determined and this is
used as a baseline expression level for the biomarker. The
expression level of the biomarker determined for the test sample
(absolute level of expression) is then divided by the mean
expression value obtained for that biomarker. This provides a
relative expression level.
[0786] In another embodiment of the present invention, a biomarker
protein is detected. One type of agent for detecting biomarker
protein of the invention is an antibody capable of binding to such
a protein or a fragment thereof such as, for example, a detectably
labeled antibody. Antibodies can be polyclonal or monoclonal. An
intact antibody, or an antigen binding fragment thereof (e.g., Fab,
F(ab')2, Fv, scFv, single binding chain polypeptide) can be used.
The term "labeled," with regard to the probe or antibody, is
intended to encompass direct labeling of the probe or antibody by
coupling (i.e., physically linking) a detectable substance to the
probe or antibody, as well as indirect labeling of the probe or
antibody by reactivity with another reagent that is directly
labeled. Examples of indirect labeling include detection of a
primary antibody using a fluorescently labeled secondary antibody
and end-labeling of a DNA probe with biotin such that it can be
detected with fluorescently labeled streptavidin. A variety of
formats can be employed to determine whether a sample contains a
protein that binds to a given antibody. Examples of such formats
include, but are not limited to, enzyme immunoassay (EIA),
radioimmunoassay (RIA), Western blot analysis and enzyme linked
immunosorbant assay (ELISA). A skilled artisan can readily adapt
known protein/antibody detection methods for use in determining
whether tumor cells express a biomarker of the present invention. A
combination of two or more of the assays for the detection of
biomarkers (non-limiting examples include those described above)
can also be used to assess one or more biomarkers.
[0787] Also provided herein is a method of selecting a cancer
patient for treatment with an angiogenesis inhibitor. The method
comprises providing a sample of the cancer from the patient,
detecting the expression of one or more genes whose expression has
been correlated with sensitivity or resistance to an angiogenesis
inhibitor, comparing the level of expression of the gene or genes
detected in the patient sample to a level of expression of the gene
or genes that have been correlated with sensitivity or resistance
to the angiogenesis inhibitor. Non-limiting examples of genes that
have been correlated with sensitivity or resistance to the
angiogenesis inhibitor include VEGF, VEGF receptor, HIF-1.alpha.,
placental growth factor receptor, and endoglin (CD105). In a
further embodiment, a patient is selected as being predicted to
benefit from administration of the angiogenesis inhibitor if the
expression of the gene or genes is similar to the expression of the
gene or genes that have been correlated with sensitivity to the
angiogenesis inhibitor. In one non-limiting embodiment, the
angiogenesis inhibitor for which the subject or the subject's
cancer is tested for sensitivity or resistance is an endoglin
(CD105) inhibitor (e.g., humanized anti-endoglin antibodies). In
another embodiment, the angiogenesis inhibitor for which the
subject or the subject's cancer is tested for sensitivity or
resistance is a VEGF receptor inhibitor or a VEGF inhibitor.
IV. Functional Assays
[0788] Antibodies and antigen binding fragments thereof can be
tested for a variety of functions using a variety of in vitro and
in vivo methods including, but not limited to those known in the
art and those described herein.
[0789] Methods of Assaying CD105 Signaling and Function
[0790] CD105 (endoglin) is a member of the TGF-.beta. receptor
family that is expressed by proliferating endothelial cells, and
normal levels of CD105 are needed for endothelial cell
proliferation. CD105 expression is increased by cellular hypoxia
through the production of hypoxia-inducible factor-1-.alpha.
(HIF-1-.alpha.) and protects hypoxic cells from apoptosis. CD105
acts to modulate signaling of multiple kinase receptor complexes of
the TGF-.beta. superfamily, including TGF-.beta. receptors
(TGF-.beta.R), activin receptor-like kinases (ALK) and activin
receptors. In the absence of CD105, activation of TGF-.beta.
receptors results in phosphorylation of SMAD proteins that inhibit
endothelial cell growth. However, activation of CD105 by TGF-.beta.
modulates SMAD protein phosphorylation. The end result is release
of the growth inhibitory effects of TGF-.beta. receptor activation
on endothelial cells. Prevention of CD105 activation by anti-CD105
antibody acts synergistically with TGF-.beta. to suppress
endothelial cell growth. TGF-.beta. can stimulate two distinct type
I receptor/SMAD signaling pathways with opposite effects in
endothelial cells. The TGF-.beta./ALK5 signaling pathway (A) leads
to inhibition of cell proliferation and migration, whereas the
TGF-.beta./ALK1 pathway (B) induces endothelial cell proliferation
and migration. CD105, an accessory TGF-.beta. receptor, highly
expressed during angiogenesis, is essential for ALK1 signaling. In
the absence of CD105, TGF-.beta./ALK5 signaling is predominant and
maintains quiescent endothelium. High CD105 expression stimulates
the ALK1 pathway and indirectly inhibits ALK5 signaling, thus
promoting the activation state of angiogenesis.
[0791] In one non-limiting embodiment, the antibodies and
antigen-binding fragments provided herein block angiogenesis by
blocking the TGF-.beta./ALK1 signaling pathway. In another
embodiment, the antibodies and antigen-binding fragments provided
herein block angiogenesis by preventing Smad1/5/8 phosphorylation
and/or signaling. CD105 participates in the promotion of
angiogenesis through signaling of the TGF-.beta./ALK1, which in
turn involves the decrease and/or blockage of the phosphorylation
of Smad2/3 proteins. In yet another embodiment, the antibodies and
antigen-binding fragments provided herein block angiogenesis by
enhancing Smad2/3 phosphorylation and/or signaling. Methods and
techniques to assay the blocking or inhibitory effect of the
antibodies and antigen-binding fragments provided herein on the
TGF-.beta./ALK1 signaling pathway and/or the phosphorylation of
Smad1/5 include, but are not limited to, known molecular
techniques. By way of example, western blotting with antibodies
specific to any of the proteins in the TGF-.beta./ALK5 or
TGF-.beta./ALK1 pathways can be used to determine the inhibitory
and/or stimulatory effect of the antibodies and antigen-binding
fragments disclosed herein on the TGF-.beta./ALK5 or
TGF-.beta./ALK1 pathways. Similarly, detection of mRNA or
regulation of the mRNA for the proteins involved in the
TGF-.beta./ALK5 or TGF-.beta./ALK1 pathways can be used to assay
the inhibitory and/or stimulatory effect of the antibodies and
antigen-binding fragments disclosed herein. Additional methods for
the assaying cell signaling for the TGF-.beta./ALK5 or
TGF-.beta./ALK1 pathways are known in the art and are contemplated
herein.
[0792] In one non-limiting embodiment, the antibodies can be
assessed with respect to inhibiting angiogenesis and endothelial
cell proliferation. Binding of anti-endoglin antibodies to HUVECs
does not prevent subsequent binding of TGF-.beta. to HUVECs. Thus,
direct suppression of the endothelial cell growth by anti-endoglin
antibodies represents one of the underlying mechanisms by which
anti-angiogenic and tumor-suppressive effects are observed in vivo.
In another embodiment, the antibodies can be assessed with respect
to blocking angiogenesis by preventing Smad1/5/8 phosphorylation
and/or signaling. CD105 participates in the promotion of
angiogenesis through signaling of the TGF-.beta./ALK1, which in
turn involves the decrease and/or blockage of the phosphorylation
of Smad2/3 proteins. In yet another embodiment, the antibodies can
be assessed with respect to blocking angiogenesis by enhancing
Smad2/3 phosphorylation and/or signaling.
[0793] Methods and techniques to assay the blocking or inhibitory
effect of the antibodies provided herein on the TGF-.beta./ALK1
signaling pathway and/or the phosphorylation of Smad1/5 include,
but are not limited to, known molecular techniques. By way of
example, western blotting with antibodies specific to any of the
proteins in the TGF-.beta./ALK5 or TGF-.beta./ALK1 pathways can be
used to determine the inhibitory and/or stimulatory effect of the
anti-endoglin antibodies disclosed herein on the TGF-.beta./ALK5 or
TGF-.beta./ALK1 pathways. Similarly, detection of mRNA or
regulation of the mRNA for the proteins involved in the
TGF-.beta./ALK5 or TGF-.beta./ALK1 pathways can be used to assay
the inhibitory and/or stimulatory effect of the antibodies
disclosed herein. Additional methods for the assaying cell
signaling for the TGF-.beta./ALK5 or TGF-.beta./ALK1 pathways are
known in the art and are contemplated herein.
[0794] Activity of the anti-endoglin antibodies disclosed herein
can be assessed using art recognized assays by, for example,
binding assays such ELISAs, competitive ELISAs, surface plasmon
resonance, and effect on HUVEC cells as described in more detail
below.
[0795] Methods of Assaying Cell Adhesion
[0796] Cell adhesion can be measured by methods known to those of
skill in the art. Assays have been described previously, e.g., by
Lebrin, et al., J. Clin. Invest 1997, 99:1390-1398. Briefly, cells
can be allowed to adhere to substrate (i.e., an ECM component) on
coated wells. Non-attached cells are removed by washing, and
non-specific binding sites are blocked by incubation with BSA. The
attached cells are stained with crystal violet, and cell adhesion
is quantified by measuring the optical density of eluted crystal
violet from attached cells at a wavelength of 600 nm.
[0797] Methods of Assaying Cell Migration
[0798] Assays for cell migration have been described in the
literature, e.g., by Brooks, et al., J. Clin. Invest 1997,
99:1390-1398 and methods for measuring cell migration are known to
those of skill in the art. In one method for measuring cell
migration described herein, membranes from transwell migration
chambers are coated with substrate, the transwells washed, and
non-specific binding sites blocked with BSA. Tumor cells from
sub-confluent cultures are harvested, washed, and resuspended in
migration buffer in the presence or absence of assay antibodies.
After the tumor cells are allowed to migrate to the underside of
the coated transwell membranes, the cells remaining on the top-side
of the membrane are removed and cells that migrate to the
under-side are stained with crystal violet. Cell migration is then
quantified by direct cell counts per microscopic field.
[0799] SCID/Nude Mice
[0800] One method for assaying tumor growth makes use of SCID
mouse, as follows: subconfluent human M21 melanoma cells are
harvested, washed, and resuspended in sterile PBS
(20.times.10.sup.6 per mL). SCID mice are injected subcutaneously
with 100 .mu.L of M21 human melanoma cell (2.times.10.sup.6)
suspension. Three days after tumor cell injection, mice are either
untreated or treated intravenously or intraperitoneally (for
example, 100 .mu.g/mouse) with one or more control or test
compositions. The mice are treated daily for 24 days. Tumor size is
measured with calipers and the volume estimated using the formula
V=(L=W.sup.2)/2, where V is equal to the volume, L is equal to the
length, and W is equal to the width.
[0801] One method for assaying tumor growth makes use of nude
mouse, as follows: MDA-MB-435 tumor cells (0.4.times.10.sup.6
cells/mouse) in 50 .mu.l PBS are orthotopically implanted in the
mammary fat pad of female nude mice (five to six weeks old). When
tumors reached a mean volume of approximately 50-80 mm.sup.3, mice
are randomized (at least 10/group) and intravenous or
intraperitoneal treatment with one or more antibodies at 1 .mu.g
(0.05 mg/kg) per dose, 10 .mu.g (0.5 mg/kg), 100 .mu.g (5 mg/kg) or
200 .mu.g (10 mg/kg), or 100 .mu.g control antibody in 100 .mu.l
PBS, or vehicle PBS 100 .mu.l twice per week is initiated; in some
studies, an untreated group can also be valuated. Tumor size is
measured with calipers and the volume estimated using the formula
V=(L=W.sup.2)/2, where V is equal to the volume, L is equal to the
length, and W is equal to the width.
[0802] BALB/c Syngeneic Mouse Models
[0803] Alternatively, BALB/c syngeneic mouse models can also be
utilized to assess tumor growth and inhibition thereof by the
antibodies or described herein as exemplified by, for example,
Tsujie et al., Int. J. Oncology, 29: 1087-1094 (2006).
[0804] Chimeric Mice
[0805] Another assay measures angiogenesis in a chimeric
mouse:human mouse model and is referred to as the chimeric mouse
assay. The assay has been described in detail by others, and
further has been described herein to measure angiogenesis,
neovascularization, and regression of tumor tissues. See Yan, et
al. (1993) J. Clin. Invest. 91:986-996.
[0806] The chimeric mouse assay is a useful assay model for in vivo
angiogenesis because the transplanted skin grafts closely resemble
normal human skin histologically and neovascularization of whole
tissue is occurring wherein actual human blood vessels are growing
from the grafted human skin into the human tumor tissue on the
surface of the grafted human skin. The origin of the
neovascularization into the human graft can be demonstrated by
immunohistochemical staining of the neovasculature with
human-specific endothelial cell markers.
[0807] The chimeric mouse assay demonstrates regression of
neovascularization based on both the amount and extent of
regression of new vessel growth. Furthermore, it is easy to monitor
effects on the growth of any tissue transplanted upon the grafted
skin, such as a tumor tissue. Finally, the assay is useful because
there is an internal control for toxicity in the assay system. The
chimeric mouse is exposed to any test reagent, and therefore the
health of the mouse is an indication of toxicity. Other animal
models described herein and known in the art can also be utilized
in the methods described herein.
[0808] Rabbit Eye Assay
[0809] Another measure of angiogenesis is an in vivo rabbit eye
model and is referred to as the rabbit eye assay. The rabbit eye
assay has been described in detail by others, and has been used to
measure both angiogenesis and neovascularization in the presence of
angiogenic inhibitors as exemplified by D'Amato et al. (1994) Proc.
Natl. Acad. Sci. USA, 91(9): 4082-4085.
[0810] The rabbit eye assay is a recognized assay model for in vivo
angiogenesis because the neovascularization process, exemplified by
rabbit blood vessels growing from the rim of the cornea into the
cornea, is easily visualized through the naturally transparent
cornea of the eye. Additionally, both the extent and the amount of
stimulation or inhibition of neovascularization or regression of
neovascularization can easily be monitored over time.
[0811] Finally, the rabbit is exposed to any test reagent, and
therefore the health of the rabbit is an indication of toxicity of
the test reagent.
[0812] Briefly, chicken chorioallantoic membrane (CAM) assays are
performed and the effects on the developing vasculature are
recorded at 48 hours after implantation of a 0.5%
carboxymethylcellulose pellet containing one or more control or
test compounds. Corneal neovascularization is induced by an
implanted pellet of poly(hydroxyethyl methacrylate) (Hydron;
Interferon Sciences, New Brunswick, N.J.) containing 650 ng of the
potent angiogenic protein basic fibroblast growth factor (bFGF)
bound to sucralfate (sucrose aluminum sulfate; Bukh Meditec,
Copenhagen). The addition of sucralfate to the pellet protects the
bFGF from degradation and provides for its slow release, thus
producing consistent aggressive angiogenesis that is more
pronounced than that induced by bFGF/Hydron alone. Release of bFGF
from pellets containing sucralfate/Hydron can be detected in vitro
for up to 4 days after the pellets are formed compared to just 1
day for pellets with Hydron alone. Pellets are made by mixing 110
.mu.l of saline containing 12 .mu.g of recombinant bFGF (Takeda,
Osaka) with 40 mg of sucralfate; this suspension is added to 80
.mu.l of 12% (wt/vol) Hydron in ethanol. Aliquots (10 .mu.l) of
this mixture are then pipetted onto Teflon pegs and allowed to dry
producing approximately 17 pellets.
[0813] A pellet is implanted into corneal micropockets of each eye
of an anesthetized female New Zealand White rabbit, 2 mm from the
limbus, followed by a single topical application of erythromycin
ointment on the surface of the cornea. Histologic examination on
consecutive days demonstrates progressive blood vessel growth into
the cornea toward the pellet with only rare inflammatory cells
seen. This angiogenic response is not altered by severe immune
suppression with total body irradiation, and pellets with
sucralfate alone do not induce angiogenesis. New vessels are
primarily induced by the bFGF rather than by inflammation. The
animals are fed daily from 2 days after implantation by gastric
lavage with one or more compounds suspended in 0.5%
carboxymethylcellulose or vehicle alone. Immunosuppressed animals
receive total body radiation of 6 Gy for 6 minutes immediately
prior to implantation of the pellets. This dose of radiation
results in a marked leukocytopenia with >80% reduction in the
leukocyte count by day 2 and >90% reduction by day 3, results
that are consistent with previous reports.
[0814] Animals are examined with a slit lamp every other day in a
masked manner by the same corneal specialist (M.S.L.). The area of
corneal neovascularization is determined by measuring with a
reticule the vessel length (L) from the limbus and the number of
clock hours (C) of limbus involved. A formula is used to determine
the area of a circular band segment: C/12.times.3.1416 [r.sup.2-(r
-L).sup.2], where r=6 mm, the measured radius of the rabbit cornea.
The uniform contiguous band of neovascularization adjacent to the
pellet is measured, thus, the total inhibition of
neovascularization can be assessed.
[0815] Mouse Matrigel Pug Angiogenesis Assays
[0816] To confirm the effects of a composition on angiogenesis, a
mouse Matrigel plug angiogenesis assay can be used. Various growth
factors (e.g., IGF-1, bFGF or VEGF) (250 ng) and Heparin (0.0025
units per/mL) are mixed with growth factor reduced Matrigel as
previously described (Montesano, et al., J. Cell Biol. 1983,
97:1648-1652; Stefansson, et al., J. Biol. Chem. 2000,
276:8135-8141). Compositions described herein or control antibodies
can be included in the Matrigel preparations utilizing one or more
dosage groups of animals. In control experiments, Matrigel is
prepared in the absence of growth factors. Mice are injected
subcutaneously with 0.5 mL of the Matrigel preparation and allowed
to incubate for one week. Following the incubation period, the mice
are sacrificed and the polymerized Matrigel plugs surgically
removed. Angiogenesis within the Matrigel plugs is quantified by
two established methods, including immunohistochemical analysis and
hemoglobin content (Furstenberger, et al., Lancet. 2002, 3:298-302;
Volpert, et al., Cancer Cell 2002, 2(6):473-83; and Su, et al.,
Cancer Res. 2003, 63:3585-3592). For immunohistochemical analysis,
the Matrigel plugs are embedded in OCT, snap frozen and 4 am
sections prepared. Frozen sections are fixed in methanol/acetone
(1:1). Frozen sections are stained with polyclonal antibody
directed to CD31. Angiogenesis is quantified by microvascular
density counts within 20 high powered (200.times.) microscopic
fields.
[0817] Hemoglobin content can be quantified as described previously
(Schnaper, et al., J. Cell Physiol. 1993, 256:235-246; Montesano,
et al., J. Cell Biol. 1983, 97:1648-1652; Stefansson, et al., J.
Biol. Chem. 2000, 276:8135-8141; and Gigli, et al., J. Immunol.
1986, 100:1154-1164). The Matrigel implants are snap frozen on dry
ice and lyophilized overnight. The dried implants are resuspended
in 0.4 mL of 1.0% saponin (Calbiochem) for one hour, and disrupted
by vigorous pipetting. The preparations are centrifuged at
14,000.times.g for 15 minutes to remove any particulates. The
concentration of hemoglobin in the supernatant is then determined
directly by measuring the absorbency at 405 nm and compared to a
standard concentration of purified hemoglobin.
[0818] Methods of Assaying Tumor Growth
[0819] Tumor growth can be assayed by methods known to those of
skill in the art, e.g., the SCID mouse model, the nude mouse model,
and BALB/c mice with syngeneic tumors. SCID mouse models for tumor
growth are carried out as follows: subconfluent human M21 melanoma
cells (or any desired tumor cell type) are harvested, washed, and
resuspended in sterile PBS (20.times.106 per mL). SCID mice are
injected subcutaneously with 100 .mu.L of M21 human melanoma cell
(2.times.10.sup.6) suspension. Three days after tumor cell
injection, mice are either untreated or treated intraperitoneally
with an antagonist in the desired dose ranges. The mice are treated
daily for 24 days. Tumor size is measured with calipers and the
volume estimated using the formula V=(L.times.W.sup.2)/2, where V
is equal to the volume, L is equal to the length, and W is equal to
the width.
[0820] Alternatively, nude mouse models, SCID mouse models and/or
BALB/c syngeneic mouse models can also be utilized to assess tumor
growth and inhibition thereof by the humanized anti-endoglin
antibodies or antigen-binding fragments described herein. (Tsujie
et al., Int. J. Oncology, 29: 1087-1094 (2006)).
[0821] Methods of Assaying Cell Proliferation
[0822] Cell proliferation can be assayed by methods known to those
of skill in the art. As described herein, subconfluent human
endothelial cells (HUVECs) can be resuspended in proliferation
buffer containing low (5.0%) serum in the presence or absence of CM
(25 .mu.L) from ECV or ECVL cells, and endothelial cells allowed to
proliferate for 24 hours. Proliferation can be quantified by
measuring mitochondrial dehydrogenase activity using a commercially
available WST-1 assay kit (Chemicon). Also, as described herein,
proliferation can be quantified by measuring .sup.3H incorporation
using standard methods. (She et al., Int. J. Cancer, 108: 251-257
(2004)).
[0823] Other methods of assessing cell proliferation are known in
the art and are contemplated herein. Further non-limiting examples
are described in more detail in the examples.
[0824] Methods of Inducing CDC, ADCC and Opsonization
[0825] Various therapies have been directed to augmenting the
body's natural immune response to transformed cells. Conventional
effector methods include complement dependent cytolysis ("CDC"),
antibody dependent cellular cytotoxicity ("ADCC") and phagocytosis
(clearance by reticuloendothelial system after the target cell is
coated with immunoglobulin). It is known that in the presence of
antibodies, certain effector cells, such as lymphoid cells having
surface bound receptors for the Fc regions of antibodies, mediate
an antibody dependent cellular cytoxicity ("ADCC") reaction against
target cells. By means of ADCC, these effector cells exert
cytolytic activity against such target cells.
[0826] Two types of ADCC reactions have been demonstrated in vitro.
In classical ADCC reactions, effector cells attach to
antibody-coated target cells and subsequently cause cytolysis of
the target cells (A. H. Greenberg et al., "Characteristics Of The
Effector Cells Mediating Cytotoxicity Against Antibody-Coated
Target Cells." I., Immunology, 21, p. 719 (1975)). This attachment
between effector and target cell results from the interaction of
the Fc region of the antibody coating the target cell and the Fc
receptor of the effector cell. One disadvantage of this type of
ADCC reaction is that it may be hampered by circulating
antigen-antibody complexes, often associated with various diseases,
which compete with the target-cell bound antibody for the Fc
receptors of the effector cells (I. C. M. MacLennan, "Competition
For Receptors For Immunoglobulin On Cytotoxic Lymphocytes," Clin.
Exp. Immunol., 10, p. 275 (1972)). Due to this drawback of
classical ADCC, a second type of ADCC reaction--antibody-directed
ADCC--has been proposed. In antibody-directed ADCC, the
target-specific antibody is first attached to the effector cell and
the resulting complex is then "directed," via the antibody, to its
specific antigen on the target cell surface. Advantageously,
antibody-directed ADCC may not be affected by the presence of
antigen-antibody complexes circulating in the host system. The
interaction between antibodies and effector cells via Fc region/Fc
receptor attachment is normally weak. And, in some instances,
antibodies do not remain associated with effector cells for a
period of time sufficient to permit lysis of target cells. In view
of this potential problem, antibodies have been attached to the
effector cells using pre-treatment with polyethylene glycol and a
mixture of phthalate oils (J. F. Jones and D. M. Segal,
"Antibody-Dependent Cell Mediated Cytolysis (ADCC) With
Antibody-Coated Effectors: New Methods For Enhancing Antibody
Binding And Cytolysis," J. Immunol., 125, pp. 926-33 (1980)). The
applicability of this method for in vivo treatments, however, may
be diminished by the toxic effects that any polyethylene glycol and
phthalate oil residues on the antibody-effector cell complex may
have on the body.
[0827] Alternatively, a method has been proposed for enhancing
antibody-directed ADCC by adjuvant chemotherapy with cytotoxic
drugs (I. R. Mackay et al., "Effect On Natural Killer And
Antibody-Dependent Cellular Cytotoxicity Of Adjuvant Cytotoxic
Chemotherapy Including Melphalan In Breast Cancer," Cancer Immunol.
Immunother., 16, pp. 98-100 (1983)). Assays for testing for ADCC
are well-known in the art, such as for example, U.S. Pat. No.
5,756,097.
[0828] Accordingly, the present invention provides antibodies
(e.g., humanized anti-endoglin antibodies) that can bind to cells
having a role in neovascularization or angiogenesis of that can
enhance phagocytosis and killing of the cells and thereby enhance
protection in vivo. Also provided are other antibodies and
functional fragments thereof that immunoreact, specifically bind
to, or preferentially bind to a binding site or epitope to which
such antibodies can bind and which have the same effect.
[0829] The antibodies of the invention can also be opsonic, or
exhibit opsonic activity, for cells having a role in
neovascularization or angiogenesis (e.g., endothelial cells). As
those in the art recognize, "opsonic activity" refers to the
ability of an opsonin (generally either an antibody or the serum
factor C3b) to bind to an antigen or cell receptor to promote
attachment of the antigen or cell receptor to a phagocyte and
thereby enhance phagocytosis. Certain cells become extremely
attractive to phagocytes such as neutrophils and macrophages when
coated with an opsonic antibody and their rate of clearance from
the bloodstream is strikingly enhanced. Opsonic activity may be
measured in any conventional manner as described, for example, in
U.S. Pat. No. 6,610,293.
[0830] In another non-limiting embodiment, a patient having a
neovascular disorder or an angiogenesis dependent disorder sheds
antigens/peptides (e.g., endoglin) from the angiogenesis. These
antigens/peptides can be "tumor associated antigens." Such patients
can be systemically administered an antibody to the antigen/peptide
(e.g., endoglin) and can initiate any of the pathways described
herein to induce CDC, ADCC, opsonization, or any other form of
cell-mediated killing.
V. Packages and Kits
[0831] In still further embodiments, the present application
concerns kits for use with the compounds described above. Humanized
antibodies or antigen-binding fragments that bind endoglin can be
provided in a kit. The kits will thus comprise, in suitable
container means, a composition comprising an antibody or
antigen-binding fragment thereof that binds endoglin. The kit may
comprise an antibody or antigen-binding fragment thereof that binds
endoglin in suitable container means.
[0832] The container means of the kits will generally include at
least one vial, test tube, flask, bottle, syringe and/or other
container means, into which the at least one polypeptide can be
placed, and/or preferably, suitably aliquoted. The kits can include
a means for containing at least one fusion protein, detectable
moiety, reporter molecule, and/or any other reagent containers in
close confinement for commercial sale. Such containers may include
injection and/or blow-molded plastic containers into which the
desired vials are retained. Kits can also include printed material
for use of the materials in the kit.
[0833] Packages and kits can additionally include a buffering
agent, a preservative and/or a stabilizing agent in a
pharmaceutical formulation. Each component of the kit can be
enclosed within an individual container and all of the various
containers can be within a single package. Invention kits can be
designed for cold storage or room temperature storage.
[0834] Additionally, the preparations can contain stabilizers to
increase the shelf-life of the kits and include, for example,
bovine serum albumin (BSA). Where the compositions are lyophilized,
the kit can contain further preparations of solutions to
reconstitute the lyophilized preparations. Acceptable
reconstitution solutions are well known in the art and include, for
example, pharmaceutically acceptable phosphate buffered saline
(PBS).
[0835] Additionally, the packages or kits provided herein can
further include any of the other moieties provided herein such as,
for example, one or more reporter molecules and/or one or more
detectable moieties/agents.
[0836] Packages and kits can further include one or more components
for an assay, such as, for example, an ELISA assay. Samples to be
tested in this application include, for example, blood, plasma, and
tissue sections and secretions, urine, lymph, and products thereof.
Packages and kits can further include one or more components for
collection of a sample (e.g., a syringe, a cup, a swab, etc.).
[0837] Packages and kits can further include a label specifying,
for example, a product description, mode of administration and/or
indication of treatment. Packages provided herein can include any
of the compositions as described herein. The package can further
include a label for treating an ocular diseases characterized by
angiogenesis/neovascularization (e.g., macular degeneration, CNV,
diabetic retinopathy), diabetic nephropathy, a chronic inflammatory
disease (e.g., IBD), rheumatoid arthritis, osteoarthritis, a forms
of cancer and their metastases.
[0838] The term "packaging material" refers to a physical structure
housing the components of the kit. The packaging material can
maintain the components sterilely and can be made of material
commonly used for such purposes (e.g., paper, corrugated fiber,
glass, plastic, foil, ampules, etc.). The label or packaging insert
can include appropriate written instructions. Kits, therefore, can
additionally include labels or instructions for using the kit
components in any method of the invention. A kit can include a
compound in a pack, or dispenser together with instructions for
administering the compound in a method described herein.
[0839] The invention further provides kits which utilize the
diagnostic methods and assays described herein. In some
embodiments, a kit according to the invention comprises reagents
for the detection of a gene or genes whose expression levels have
been correlated with sensitivity or resistance to an angiogenesis
inhibitor in a sample of cancer cells from a patient. In some
embodiments, the gene or genes are selected from VEGF, VEGF
receptor, HIF-1.alpha., placental growth factor receptor, and
CD105. In some embodiments, the kit comprises VEGF. In some
embodiments, the kit comprises VEGF receptor. In some embodiments,
the kit comprises HIF-1.alpha.. In some embodiments, the kit
comprises placental growth factor receptor. In some embodiments,
the kit comprises CD105. In some embodiments, the kit comprises at
least two of VEGF, VEGF receptor, HIF-1.alpha., placental growth
factor receptor and CD105. In some embodiments, the kit comprises
at least two genes that have been correlated with sensitivity to an
angiogenesis inhibitor. In some embodiments, the kit comprises at
least two genes that have been correlated with resistance to an
angiogenesis inhibitor. In some embodiments, the kit comprises at
least one gene that has been correlated with sensitivity to an
angiogenesis inhibitor and one gene that has been correlated with
resistance to an angiogenesis inhibitor.
[0840] In still further embodiments, a kit according to the
invention comprises reagents for the detection of VEGF, VEGF
receptor, HIF-1.alpha., placental growth factor receptor, and CD105
expression levels in a sample of tumor cells from a patient to be
treated; and a dose or doses an inhibitor, including but not
limited to humanized anti-endoglin antibodies or antigen-binding
fragments described herein, in a variety of dosage forms, such as
capsules, caplets, gel caps, powders for suspension, etc. It is
further contemplated within the invention that kit comprising
reagents for the detection of VEGF, VEGF receptor, HIF-1.alpha.,
placental growth factor receptor, and CD105 expression levels in a
sample of tumor cells from a patient to be treated will further
comprise any of the aforementioned embodiments of the kits for
co-administration of at least one additional angiogenesis
inhibitor.
[0841] Instructions can include instructions for practicing any of
the methods described herein including treatment methods.
Instructions can additionally include indications of a satisfactory
clinical endpoint or any adverse symptoms that may occur, or
additional information required by regulatory agencies such as the
Food and Drug Administration for use on a human subject.
[0842] The instructions may be on "printed matter," e.g., on paper
or cardboard within or affixed to the kit, or on a label affixed to
the kit or packaging material, or attached to a vial or tube
containing a component of the kit. Instructions may additionally be
included on a computer readable medium, such as a disk (floppy
diskette or hard disk), optical CD such as CD- or DVD-ROM/RAM,
magnetic tape, electrical storage media such as RAM and ROM, IC tip
and hybrids of these such as magnetic/optical storage media.
[0843] The embodiments of the compounds and methods of the present
application are intended to be illustrative and not limiting.
Modifications and variations can be made by persons skilled in the
art in light of the above teachings specifically those that may
pertain to alterations in the antibodies or antigen-binding
fragments which bind endoglin surrounding the described
modifications while maintaining near native functionally with
respect to binding of endoglin. Therefore, it should be understood
that changes may be made in the particular embodiments disclosed
which are within the scope of what is described.
EXAMPLES
[0844] The application may be better understood by reference to the
following non-limiting examples, which are provided as exemplary
embodiments of the application. The following examples are
presented in order to more fully illustrate embodiments of the
invention and should in no way be construed, however, as limiting
the broad scope of the application. While certain embodiments of
the present application have been shown and described herein, it
will be obvious that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions may
occur to those skilled in the art without departing from the
invention; it should be understood that various alternatives to the
embodiments described herein may be employed in practicing the
methods described herein.
Example 1
Generation and Binding of Anti-CD105 Humanized and
Humanized/Deimmunized Antibodies
[0845] Construction, Expression and Purification of Antibodies
[0846] All humanized and humanized/deimmunized VH and VK region
genes were synthesized using a series of overlapping
oligonucleotides that were annealed, ligated and PCR amplified to
give full length synthetic V regions. The assembled variants were
then cloned directly into Antitope Ltd.'s pANT expression vector
system for IgG1 heavy chains and kappa light chains.
[0847] All combinations of humanized heavy and light chains (i.e.,
a total of 4 pairings) and combinations of humanized/deimmunized
heavy and light chains (i.e., a total of 24 pairings) were stably
transfected into NS0 cells via electroporation and selected using
200 nM methotrexate (Sigma Cat. No. M8407). Methotrexate-resistant
colonies for each construct were tested for IgG expression levels
using an IgG1 ELISA. The best expressing lines were selected and
frozen under liquid nitrogen. Successful transfection and clone
selection was achieved for all variants and expression levels of
humanized and humanized/deimmunized antibody variants in saturated
static cultures are shown in Table 1.
[0848] Twenty-four IgG variants were therefore purified from NS0
cell culture supernatants on a Protein A sepharose column (GE
Healthcare Cat. No. 110034-93) and quantified by OD.sub.280 nm
using an extinction coefficient, Ec.sub.(0.1%)=1.62, based on the
predicted amino acid sequence. Approximately 500 .mu.g of each
antibody variant was purified and lead variants were analyzed by
reducing SDS-PAGE. Briefly, Coomassie blue stained reducing
SDS-PAGE gel of lead antibody variants. 1 .mu.g of each sample was
loaded on a NuPage 4-12% Bis-Tris gel (Invitrogen Cat. No.
NP0322BOX) and run at 200 V for 30 minutes. Marker was Bio-Rad
Precision Plus (Cat. No. 161-073). Bands corresponding to the
predicted sizes of the heavy and light chains were observed with no
evidence of any contamination in any lane (data not shown).
[0849] ELISA Methodology
[0850] An ELISA was used to assay binding of humanized and
humanized/deimmunized anti-endoglin antibodies to endoglin.
Briefly, an ELISA was performed according to the following steps:
[0851] 1. Coat a Nunc Maxisorp plate with MAB9811-01 (polyclonal
anti-endoglin antibody) at 1500 ng/ml in PBS, 100 .mu.l/well. Cover
the plate with a sealer and incubate overnight (16-24 hours) at
4.degree. C. [0852] 2. Wash the plate 2.times. with -200 .mu.l of
PBS (without Tween). [0853] 3. Add 200 .mu.l/well of BSA blocking
solution (1% BSA) and incubate 60 minutes at room temperature.
[0854] 4. Wash the plate 3.times. with PBS containing Tween (PBS-T)
using the BioTek plate washer. [0855] 5. Add 100 .mu.l/well of
CD105 (R&D Systems Cat 1097-EN) at 100 ng/ml in PBS-T with 0.1%
BSA and incubate 60 minutes at room temperature. [0856] 6. Wash the
plate 3.times. with PBS-T using the BioTek plate washer. [0857] 7.
In test wells: add 100 .mu.l/well of anti-endoglin antibodies at
20, 10, 4, 2, 1, 0.5 and 0.2 ng/ml (diluted in PBS-T with 0.1% BSA)
and incubate 60 minutes at room temperature. In negative control
wells: add 100 .mu.l/well of isotype matched control antibody.
[0858] 8. Wash the plate 3.times. with PBS-T using the BioTek plate
washer. [0859] 9. Add 100 .mu.l/well of Goat anti-Human IgG
conjugated to HRP (Jackson Immunoresearch), diluted 1:10000 in
PBS-T with 0.1% BSA to all wells; incubate 30-60 minutes at room
temperature. [0860] 10. Wash the plate 5.times. with PBS-T using
the BioTek plate washer. [0861] 11. Add 100 .mu.l/well of TMB
substrate solution and incubate uncovered in the dark for 15
minutes. [0862] 12. Stop the reaction by addition of 100 .mu.l/well
of TMB Stop Solution.
[0863] Samples are run in triplicate and the optical density is
read to construct a standard curve and determine the binding
constant. Statistical analysis is conducted using the Student's
t-test or another standard test.
Competition ELISA
[0864] Antibodies were tested in a competition ELISA for binding to
CD105 against biotinylated chimeric anti-CD105. Briefly, chimeric
anti-CD105 was biotinylated using a micro-biotinylation kit (Sigma,
Catalog No. BTAG-1KT) following the manufacturer's instructions.
Nunc Immuno MaxiSorp 96-well flat-bottom microtiter plates were
coated with mouse anti-human CD105 (Southern Biotechnologies,
Catalog No. 9811-01) at 1.5 .mu.g/mL in phosphate buffered saline
(PBS) overnight at 4.degree. C. The following day, 100 ng/ml human
CD105 (R&D Systems, Catalog No. 1097-EN) in PBS/2% BSA was
added to the pre-coated plate and incubated at room temperature for
1 hour. Varying concentrations of either chimeric, humanized or
humanized/deimmunized anti-CD105 antibodies (4 .mu.g/mL to 0.0018
.mu.g/mL in three-fold dilutions) were mixed with a fixed
concentration of biotinylated chimeric anti-CD105 antibody (6.25
ng/ml) and added to the plate. Binding of the biotinylated chimeric
antibody was detected via streptavidin-HRP (Sigma, Catalog No.
S5512) and TMB substrate (Sigma, Catalog No. T0440). OD450 nm
values were measured on a Dynex MRX TCII plate reader. The results
of the competition analysis are illustrated in FIGS. 7 and 8.
Curves were fitted through the straight line portion of each of the
plots of absorbance against the log sample concentration and the
equations of the lines were used to calculate the concentrations of
humanized or humanized/deimmunized antibody required to inhibit
biotinylated chimeric antibody binding to CD105 by 50% (IC50). To
allow for comparisons within and between experiments, IC50 values
of humanized or humanized/deimmunized variants were normalized
against the reference antibody that was included on each plate to
give a value for the fold difference. IC50 values are relative to
chimeric anti-CD105 and are representative of three experiments.
Summary ELISA data are presented in Table 1 and include antibody
expression levels (.mu.g/ml) as assayed in saturated static
cultures.
TABLE-US-00003 TABLE 1 Characteristics of humanized and
humanized/deimmunized antibody variants. IC50 values are relative
to chimeric anti-endoglin antibody and are representative of three
experiments. Antibody expression levels (.mu.g/ml) were assayed in
saturated static cultures. The level of deimmunization is
represented by an arbitrary scale based upon the location in the
epitopes of the mutations. Expression Levels Level of Construct
Relative IC50 (.mu.g/ml) Deimmunization VK1VH1 1.51 10.2 n/a VK1VH2
1.15 12.9 n/a VK2VH1 0.93 11.1 n/a VK2VH2 1.19 15.8 n/a VK2AAVH1A
0.79 10.8 ++++ VK2AAVH1A2 0.99 6.6 ++++++ VK2AAVH1Q 0.76 7.5 +++++
VK2AAVH1R 0.61 10.0 +++++ VK2AAVH1S 1.27 9.7 ++++ VK2ASVH1A 1.41
6.6 +++ VK2ASVH1A2 0.85 5.9 +++++ VK2ASVH1Q 1.04 8.7 ++++ VK2ASVH1R
1.02 7.9 ++++ VK2ASVH1S 1.31 8.8 +++ VK2SAVH1A 0.49 7.5 +++
VK2SAVH1A2 0.84 10.3 +++++ VK2SAVH1Q 0.87 11.5 ++++ VK2SAVH1R 0.77
8.8 ++++ VK2SAVH1S 0.96 34.6 +++ VK2SSVH1A 1.06 9.7 ++ VK2SSVH1A2
1.03 17.4 ++++ VK2SSVH1Q 1.21 14.7 +++ VK2SSVH1R 0.62 13.7 +++
VK2SSVH1S 1.21 16.2 +++ VK1AAVH1A 2.52 6.9 ++++ VK1AAVH1A2 1.12
13.5 ++++++ VK1AAVH1Q 1.30 7.6 +++++ VK1AAVH1R 1.05 11.5 +++++
Example 2
BIAcore (Surface Plasmon Resonance: SPR) Analysis of Humanized and
Humanized/Deimmunized Anti-Endoglin Antibody Binding
[0865] Affinity of antibodies can be assessed using, for example,
BIAcore analysis using standard protocols. Briefly, protein A is
chemically coupled to a BIAcore CM5 chip, with the amount of
protein A immobilized corresponding to .about.2000 RU. Subsequent
steps are performed in a running buffer of 10 mM HEPES, 150 mM
NaCl, 3 mM EDTA, 0.05% TWEEN, pH=7.4, at 25 degrees Celsius using a
10 Hz data collection rate. Anti-endoglin antibody (10 nM) is
captured at a 10 .mu.L/min flow rate by the immobilized protein A
on the BIAcore chip: typically, capture times of 20, 40 and 80
seconds allow the capture of antibody densities corresponding to
130 RU, 330 RU and 570 RU, respectively. Start-up cycles are
performed using running buffer at a flow rate of 40 .mu.L/min, a
contact time of 90 seconds and a dissociation time of 90 seconds.
Sample cycles are performed using recombinant endoglin at
concentrations ranging from 0 to 40 nM. Endoglin is passed over the
BIAcore chip containing captured antibody at a flow rate of 40
.mu.L/min with a contact time of 525 seconds and dissociation time
of 2500 seconds. Eight sample cycles are typically performed at
each antibody capture density Regeneration of the chip is
accomplished using 10 mM glycine pH=1.7. Data analysis is performed
using BIAcore T100 Evaluation Software v1.1 Signals generated using
BIAcore chips with different captured antibody densities are
compared and data generated in the absence of recombinant endoglin
are used to adjust for the intraassay blank signal. For fitting of
the data, the R.sub.max is allowed to float to account for
variation in capture levels of each antibody in each cycle. Data
from each capture density is fitted simultaneously during analysis
of each antibody. BIAcore data are presented in Table 2 for
chimeric, humanized and humanized/deimmunized anti-endoglin
antibodies, including k.sub.a (1/Ms), k.sub.d (1/s), K.sub.D (M)
and Chi.sup.2 (RU.sup.2).
TABLE-US-00004 TABLE 2 BIAcore binding data for chimeric
anti-endoglin antibody, humanized anti-endoglin antibody VK1VH1 and
humanized/ deimmunized anti-endoglin antibodies VK1AAVH1R,
VK1AAVH1Q and VK1AAVKVH1A2. Anti-endoglin k.sub.a k.sub.d K.sub.D
Chi.sup.2 Antibody (.times.10.sup.4) (.times.10.sup.-5)
(.times.10.sup.-10) (.times.10.sup.-2) VK1AAVH1R 6.40 3.41 5.33
6.84 VK1VH1 4.19 3.46 8.25 5.53 Chimeric 5.47 3.28 6.00 6.78
VK1AAVH1Q 3.56 3.03 8.50 7.07 VK1AAVH1A2 4.60 3.25 7.06 5.52
Example 3
Antibody Avidity and Number of Available Epitopes on
Endoglin-Expressing Cells
[0866] Antibody avidity and number of available epitopes on
endoglin-expressing cells can be assessed utilizing Scatchard plot
analyses using standard protocols.
[0867] Briefly, Scatchard plot analyses of direct binding of
radiolabeled humanized anti-endoglin antibodies to
endoglin-expressing KM-3 leukemia cells and sub-confluent
proliferating HUVECs are carried out. The purified anti-endoglin
antibodies are individually radiolabeled with .sup.125I using
Iodo-Gen and according to standard methods known to those skilled
in the art. The radiolabeled humanized anti-endoglin antibodies are
assayed for the mean number of iodine atoms per IgG molecule.
Titration experiments are carried out using a fixed amount (0.1
.mu.g) of each .sup.125I-labeled mAb and 2-fold serial increments
of endoglin-expressing HUVEC cells to determine antigen-binding
activity. Analysis of Scatchard plot of binding data is carried out
according to known methods. An equilibrium constant and an average
maximal number of mAb bound/cell are estimated by this
analysis.
Example 4
Western Blot Assay for Anti-Endoglin Antibody Activity
[0868] The ability of humanized anti-endoglin antibodies to modify
intracellular signaling in proliferating endothelial cells that
express CD105 can be assayed via western blots to detect the
phosphorylation of the proteins involved in the CD105 signaling
pathway.
[0869] Western blot analyses are performed to identify
phosphorylated Smad1/5/8 or Smad2/3 according to known western
blotting techniques in untransfected endothelial cells. Primary
antibodies against phosphorylated Smad1, Smad2, Smad5, Id1 (Santa
Cruz) and endoglin are utilized to detect molecules in samples.
Detection is performed by enhanced chemoluminescence (ECL).
Example 5
Inhibition of HUVEC Growth and .sup.3H-Thymidine Incorporation
Assay
[0870] A number of assays are available to assess inhibition of
cell growth.
[0871] In one example, the HUVEC cell line E6/E7 P3-17 was cultured
in EBM2 media with supplements (Lonza-Clonetics) containing 5%
fetal calf serum. Cells were cultured in 75-cm.sup.2 flasks
(Falcon, Becton-Dickinson, Franklin Lakes, N.J.) in a CO.sub.2
incubator at 37.degree. C. under subconfluent conditions. Cells are
detached by incubating with Hanks' balanced salt solution with 15
mM EDTA in 25 mM HEPES buffer, pH=7.3, at 37.degree. C. for 15 min.
After washing twice with ice-cold PBS, cells are re-suspended in
endothelial cell growth medium at a concentration of 25,000
cells/mL. In additional experiments, human umbilical vein
endothelial cells (HUVECs) are suspended and cultured in an
endothelial cell growth medium free of FBS and bovine brain
extracts. A 200 .mu.l aliquot of cell suspension containing 2,500
cells is seeded to each well of 96-well culture plates. Cells are
cultured at 37.degree. C. in a CO.sub.2 incubator overnight before
100 .mu.g/mL of humanized anti-endoglin antibody VK1AAVH1A2 or,
control IgG or PBS are added in triplicate. Culture plates are kept
in the incubator for 72 hr, during which fresh media and humanized
anti-endoglin antibody, control IgG or PBS are replaced every 24
hr. .sup.3H-thymidine (1 .mu.Ci) is added into each well and the
plates are incubated for 20 hr. Cells are washed with PBS followed
by treatment with 100 .mu.l/well trypsin-EDTA (0.05% trypsin, 0.53
mM EDTA) at 37.degree. C. for 15 min. Cells are harvested onto
glass fiber filters (Wallac Printed FiltermatA) using Harvester 96
(TOMTEC, Hamden, Conn.) and .sup.3H-radioactivity is determined in
a Trilux 1540 MicroBeta Liquid Scintillation and Luminescence
Counter (Wallac, Turku, Finland). In a second example, the cells
used were a primary culture of HUVEC cells, HUVEC 2517C, Humanized
TRC105 antibody inhibited the growth of the HUVEC cell line E6/E7
and the primary HUVEC culture, HUVEC 2517C derived from a single
donor, compared to antibody control and PBS (Table 3).
TABLE-US-00005 TABLE 3 Inhibition of human endothelial cell growth
by humanized/ deimmunized anti-endoglin antibody VK1AAVH1A2. E6/E7
cells Mean counts per Standard % Inhibition Condition minute
deviation versus PBS PBS 18320 173 -- Control IgG 18061 172 0.9
Chimeric 14452 1348 20.7 Humanized/deimmunized 14025 983 23.1
anti-endoglin antibody VK1AAVH1A2
Example 6
Assay for Inhibition of Cell Migration by Anti-Endoglin
Antibodies
[0872] Migration (chemokinesis) as a measure of cell proliferation
and activation is measured using a Boyden chamber.
[0873] Briefly, cell migration is assessed as follows: a Costar
nucleopore filter (8 mm pore) is coated with fibronectin overnight
at 4.degree. C. The chamber is washed with phosphate-buffered
saline (PBS) and the lower chamber was filled with DMEM with or
without serum and with or without TGF-.beta.3. Cells are
trypsinized and suspended at a final concentration of 50,000
cells/ml in DMEM with anti-endoglin antibody. A 150 .mu.l aliquot
of the cell suspension is added to the upper chamber and incubated
at 37.degree. C. After 16 hrs, the cells are washed and the upper
surface is wiped to remove the non-migrating cells. The membranes
are fixed in methanol, washed with water, stained and the numbers
of cells present on the lower surface are counted.
Example 7
ADCC Assay for Humanized/Deimmunized Anti-Endoglin Antibodies
[0874] The anti-endoglin antibodies described herein can be
assessed with respect to their ability to bind to IL-2 activated
natural killer (NK) cells and to induce antibody dependent
cell-mediated cytotoxicity (ADCC) of HUVECs using, for example, the
following protocols.
[0875] NK Isolation and Generation of IL-2 Activated NK Cells
[0876] PBMC are isolated and allowed to rest for 24 hrs at
4.degree. C. in RPMI with 10% FBS. PBMC are then placed in RPMI
with 2% FBS (Total Volume=50 mL), and O1 mL of the cell suspension
are plated in a petri dish. PBMC are incubated for 2 hrs at
37.degree. C. and the non-adherent cells are collected. NK cells
are cultured at 8.times.10.sup.6/mL with 1000 U/mL IL-2 for 48 hrs,
followed by normal culturing for 5-8 days before use in an ADCC
assay.
[0877] Cytotoxicity and ADCC Assays
[0878] NK cells are scraped from the culture and collected in a 50
mL conical tube. Cells are washed once with RPMI Complete media and
spun at 1200 rpm for 10 minutes. NK cells are then resuspended in 5
mL RPMI Complete media and counted. Prior to performing the assay,
the NK cell count is normalized to an effector: target ratio of
10:1. Normalized NK cells are plated and 10 .mu.L of anti-endoglin
antibody is added into designated wells and incubated for 30
minutes at 37.degree. C. Control samples include untreated or
control-antibody treated cell populations. All samples and controls
are tested in quintiplicate.
[0879] Target cells of interest are collected (HUVEC cells),
washed, spun at 1200 rpm for 10 minutes, and re-suspended in 5 mL
RPMI Complete media. Target cells are washed again and re-suspended
in Serum Free RPMI to a final concentration of 1.times.10.sup.6
cells/mL. Target cells are then labeled with a final concentration
of 5 .mu.g/mL Calcein AM for 1 hr at 37.degree. C., followed by two
washes with RPMI Complete media. Target cells are then re-suspended
and added to the NK cell wells. The target cell/NK cell combination
is incubated at 37.degree. C. for 4 hours. After incubation, the
plates are spun at 1200 rpm for 5 minutes and cells are washed and
resuspended in DPBS. The fluorescence is read using
Excitation/Emission of 450/530 nm and the emission is a measure of
the cell killing mediated by the antibodies. The mean fluorescence
intensity and standard deviation are calculated and used to
calculate % ADCC according the following formula:
% ADCC=100%*[(f.sub.sample-f.sub.media)-(f.sub.isotype
control-f.sub.Triton)],
where:
[0880] f.sub.sample=mean fluorescence in wells containing
anti-endoglin antibody
[0881] f.sub.media=mean fluorescence in wells containing media
without antibody
[0882] f.sub.isotype control=mean fluorescence in wells containing
isotype control IgG
[0883] f.sub.Triton=mean fluorescence in wells containing Triton
detergent (to lyse target cells)
[0884] Humanized/deimmunized antibody VK1AAVH1A2 demonstrated dose
dependent ADCC of HUVECs that was significantly greater than
isotype control antibody (Table 4).
TABLE-US-00006 TABLE 4 ADCC of humanized/deimmunized anti-endoglin
antibody VK1AAVH1A2 versus isotype matched control IgG on HUVEC
cells. ADCC of HUVECs Standard Test Condition (%) Deviation Isotype
Control (2 .mu.g/mL) 3.4 4.7 VK1AAVH1A2 (0.4 .mu.g/mL) 14.6 2.1
VK1AAVH1A2 (0.08 .mu.g/mL) 14.6 2.1 VK1AAVH1A2 (0.016 .mu.g/mL)
12.6 4.1 VK1AAVH1A2 (0.0032 .mu.g/mL) 8.4 4.1 VK1AAVH1A2 (0.00064
.mu.g/mL) 7.6 2.0
Example 8
Effect of Humanized/Deimmunized Anti-Endoglin Antibodies on a
Murine Model of Choroidal Neovascularization
[0885] The effect of humanized/deimmunized anti-endoglin antibodies
can be assessed in a murine model of choroidal
neovascularization.
[0886] Briefly, 4 to 5 week old C57BL/6 mice are anesthetized with
ketamine hydrochloride (100 mg/kg) and the pupils dilated with 1%
tropicamide (Alcon Laboratories, Inc Fort Worth, Tex.). Three burns
of a 532-nm diode laser photocoagulation (75-pm spot size,
01-second duration, 120 mW) are delivered to each retina using the
slit lamp delivery system of a photocoagulator (OcuLight; Iridex,
Mountain View, Calif.) and a handheld cover slip as a contact lens.
Burns are performed in the 9, 12 and 3 o'clock positions of the
posterior pole of the retina. Production of a bubble at the time of
lasering, which indicates rupture of Bruch's membrane, is an
important factor in obtaining choroidal neovascularization (CNV);
thus only burns in which a bubble is produced are included in the
study.
[0887] Four independent experiments are performed to investigate
the effect of intraocular injections on day 0 after rupture of
Bruch's membrane. Mice in Group 1 are given an intraocular
injection of about 0.5 to about 5 .mu.g of a anti-endoglin antibody
or antigen-binding fragment in 1 .mu.L of PBS in one eye and 1
.mu.L of PBS in the fellow eye. Group 2 mice are given an
intraocular injection of about 1.5 to about 10 .mu.g of
anti-endoglin antibody or antigen-binding fragment in 1 .mu.L of
PBS in one eye and 1 .mu.L of PBS in the fellow eye. Group 3 mice
are given an intraocular injection of about 5 to about 25 .mu.g of
anti-endoglin antibody or antigen-binding fragment in one eye and 1
.mu.L of PBS in the fellow eye. Group 4 receive PBS in both
eyes.
[0888] After 14 days, mice are anesthetized and perfused with
fluorescein-labeled dextran (2.times.10.sup.6 average molecular
weight, Sigma-Aldrich) and choroidal flat mounts are prepared.
Briefly, the eyes are removed, fixed for 1 hour in 10%
phosphate-buffered formalin, and the cornea and lens are removed.
The entire retina is carefully dissected from the eyecup, radial
cuts are made from the edge of the eyecup to the equator in all
four quadrants, and the retina is flat-mounted in aqueous mounting
medium (Aquamount; BDH, Poole, UK). Flat mounts are examined by
fluorescence microscopy (Axioskop; Carl Zeiss Meditec, Thornwood,
N.Y.), and the images are digitized with a three charge-coupled
device (CCD) color video camera (1K-TU40A, Toshiba, Tokyo, Japan).
Frame grabber image-analysis software is used to measure the area
of each CNV lesion. Statistical comparisons are made using ANOVA
with Dunnett's correction for multiple comparisons.
Example 9
Anti-Angiogenic Therapy of Preformed Human Breast Cancer Tumors in
Human Skin Grafted into SCID Mice
[0889] The effect of the humanized/deimmunized anti-endoglin
antibodies described herein can be assessed with respect to their
anti-angiogenic effect on preformed human breast cancer tumors
grown in human skin grafted into SCID mice.
[0890] Briefly, MCF-7 cells (8.times.10.sup.6 cells in 0.1 ml PBS)
are transplanted intradermally into human full-thickness skin
grafted into SCID mice when the grafts showed no signs of
inflammation, contraction or rejection. The mice are left untreated
until distinct palpable tumors (3 to 6 mm in diameter in most
cases) appear. Mice with distinct tumors are divided into groups
for the therapeutic studies. Humanized/deimmunized anti-endoglin
monoclonal antibody (mAb) and an isotype-matched control IgG are
diluted with sterile PBS containing mouse serum albumin (0.05%
final concentration). For the antibody therapy, 1 to 20 mg/kg
anti-endoglin antibody or control IgG is intravenously (i.v.)
administered via the tail vein of mice. The administration is given
every two to three days.
[0891] During the treatment, mice are monitored daily for tumor
size and morbidity. Mice are weighed twice a week using an
electronic balance (OHAUS.TM. Model GT210). Tumor size is measured
three times a week using an electronic caliper (PRO-MAX 6 inch
caliper; Fowler Co., Newton, Mass.) connected to a computer using
OptoDemo.TM. software (Fowler Co.). The measured tumor diameters
are converted to tumor volumes using the following formula:
V=length.times.width.times.height.times.pi/6. Statistical analysis
of the data for the comparison of different groups of mice is
carried out using Student's t-test.
Example 10
Mouse Model of Ovarian Cancer
[0892] To determine the ability of humanized/deimmunized
anti-endoglin antibodies, or antigen-binding fragments thereof, to
treat ovarian cancer, an ovarian cancer cell line can be used in
SCID or nude mice.
[0893] Briefly, ovarian cancer cells are implanted into SCID or
nude mice to generate ovarian tumors. Groups of mice bearing
established tumors are treated by i.v. administration of escalating
doses (starting at 1.8 mg/kg body weight) of humanized/deimmunized
anti-endoglin antibody or control IgG. The treatment is performed 2
or 3 times per week. A VEGF inhibitor and/or other anticancer agent
may be used in some or all groups. The mice are monitored and tumor
growth is measured 2 or 3 times per week.
Example 11
Mouse Model of Colorectal Cancer
[0894] To determine the ability of humanized/deimmunized
anti-endoglin antibodies, or antigen-binding fragments thereof, to
treat colorectal cancer, a colorectal cancer cell line can be used
in SCID, nude or immunocompetent mice.
[0895] Briefly, colorectal cancer cells are implanted into SCID,
nude or immunocompetent mice to generate colorectal tumors. Groups
of mice bearing established tumors are treated by i.v.
administration of escalating doses (starting at 1.8 mg/kg body
weight) of humanized/deimmunized anti-endoglin antibody or control
IgG. The treatment is performed 2 or 3 times per week. A VEGF
inhibitor and/or other anticancer agent may be used in some or all
groups. The mice are monitored and tumor growth is measured 2 or 3
times per week. Tumors may be imaged by standard imaging test,
including PET and ultrasound. Treated tumors may be explanted to
assess intracellular signaling pathways or vascularity by
immunohistochemistry.
Example 12
Mouse Model of Kidney Cancer
[0896] To determine the ability of humanized/deimmunized
anti-endoglin antibodies or antigen-binding fragments thereof to
treat kidney cancer, a kidney cancer cell line is used in SCID or
nude mice.
[0897] Briefly, kidney cancer cells are implanted into SCID or nude
mice to generate kidney tumors. Groups of mice bearing established
tumors are treated by i.v. administration of escalating doses
(starting at 1.8 .mu.g/g body weight) of humanized/deimmunized
anti-endoglin antibody or control IgG. The treatment is performed
at 3-day intervals for the first three injections and at a 7-day
interval for the fourth injection. A VEGF inhibitor and/or other
anticancer agent may be used in some or all groups. The mice are
monitored and tumor growth is measured via sacrifice of animals on
a weekly basis.
Example 13
Mouse Model of Myeloma
[0898] To determine the ability of humanized/deimmunized
anti-endoglin antibodies or antigen-binding fragments thereof to
treat myeloma, a myeloma cell line is used in SCID or nude
mice.
[0899] Briefly, myeloma cancer cells are implanted into SCID or
nude mice to generate myeloma tumors. Groups of mice bearing
established tumors are treated by i.v. administration of escalating
doses (starting at 1.8 mg/kg body weight) of humanized/deimmunized
anti-endoglin antibody or control IgG. The treatment is performed 2
or 3 times per week. A VEGF inhibitor and/or other anticancer agent
may be used in some or all groups. The mice are monitored and tumor
growth is measured 2 or 3 times per week.
Example 14
Mouse Model of Sarcoma
[0900] To determine the ability of humanized/deimmunized
anti-endoglin antibodies or antigen-binding fragments thereof to
treat sarcoma, a sarcoma cell line is used in SCID or nude
mice.
[0901] Briefly, sarcoma cancer cells are implanted into SCID or
nude mice to generate sarcoma tumors. Groups of mice bearing
established tumors are treated by i.v. administration of escalating
doses (starting at 1.8 mg/kg body weight) of humanized/deimmunized
anti-endoglin antibody or control IgG. The treatment is performed 2
or 3 times per week. A VEGF inhibitor and/or other anticancer agent
may be used in some or all groups. The mice are monitored and tumor
growth is measured 2 or 3 times per week.
Example 15
Mouse Model of Breast Cancer
[0902] To determine the ability of a humanized/deimmunized
anti-endoglin antibody to treat breast cancer, a breast cancer cell
line is used in SCID or nude mice.
[0903] Briefly, breast cancer cells are implanted into SCID or nude
mice to generate breast tumors. Groups of mice bearing established
tumors are treated by i.v. administration of escalating doses
(starting at 1.8 mg/kg body weight) of a humanized/deimmunized
anti-endoglin antibody. Control animals are administered a control
IgG. The treatment is performed 2 or 3 times per week. A VEGF
inhibitor and/or other anticancer agent may be used in some or all
groups. The mice are monitored and tumor growth is measured 2 to 3
times per week.
Example 16
Clinical Trial of Combination Therapy for Colorectal Cancer
[0904] This example describes a randomized, blinded,
placebo-controlled, multicenter, Phase 2 study designed to provide
a preliminary assessment of the safety and efficacy of a
humanized/deimmunized anti-endoglin antibody in patients with
colorectal cancer. Approximately about 100-about 800 patients are
enrolled, with about 50-about 400 patients being assigned to a
treatment group and about 50-about 400 patients assigned to a
placebo group. The trial will consist of the administration of
intravenous repeating doses of humanized/deimmunized anti-endoglin
antibody at about 0.1-about 20 mg/kg or placebo every one, two or
three weeks for 6-10 cycles. A VEGF inhibitor and/or other
anticancer agent may be used in all groups. The time frame of the
study is estimated at about 6 months to about 5 years, with
continued therapy for responders as indicated at the end of the
initial study. Additional outcome measures are as follows:
[0905] Primary outcome measure: overall response rate. One goal of
the study is to demonstrate an increase progression-free survival
by 35% with humanized/deimmunized anti-endoglin antibody.
[0906] Secondary outcome measures that can be assessed include
overall response rate, duration of response, overall survival,
serious and non-serious adverse events. For example, a treatment
may prevent progression of the disease (i.e., stasis) or may result
in an improvement. Alternately, or in addition, other goals can be
measured with respect to one or more of the following: decreased
tumor burden, decreased vascularity, reduced side effects,
decreased adverse reactions, and/or increased patient
compliance.
Example 17
Clinical Trial of Combination Therapy for Myeloma
[0907] This example describes a randomized, blinded,
placebo-controlled, multicenter, Phase 2 study designed to provide
a preliminary assessment of the safety and efficacy of combining
humanized/deimmunized anti-endoglin antibody with bortezomib in
patients with myeloma. Approximately about 100-about 800 patients
are enrolled, with about 50-about 400 patients being assigned to a
treatment group and about 50-about 400 patients assigned to a
placebo group. The trial will consist of the administration of
intravenous repeating doses of humanized/deimmunized anti-endoglin
antibody at about 1-about 20 mg/kg or placebo every one, two or
three weeks combined with bortezomib at about 1.3 mg/kg weekly. The
time frame of the study is estimated at about 6 months to about 5
years, with continued therapy for responders as indicated at the
end of the initial study. Additional outcome measures are as
follows:
[0908] Primary outcome measure: overall response rate. One goal of
the study is to demonstrate an increase overall response rate from
about 40% with bortezomib plus placebo to about 60% (or more) with
bortezomib plus humanized/deimmunized anti-endoglin antibody.
[0909] Secondary outcome measures that can be assessed include
duration of response, progression free survival, overall survival,
serious and non-serious adverse events. For example, a treatment
may prevent progression of the disease (i.e., stasis) or may result
in an improvement. Alternately, or in addition, other goals can be
measured with respect to one or more of the following: decreased
tumor burden, decreased vascularity, reduced side effects,
decreased adverse reactions, and/or increased patient
compliance.
Example 18
Clinical Trial of Combination Therapy for Kidney Cancer
[0910] This example describes a randomized, blinded,
placebo-controlled, multicenter, Phase 2 study designed to provide
a preliminary assessment of the safety and efficacy of combining
humanized/deimmunized anti-endoglin antibody with sunitinib
(Sutent.RTM.) in patients with renal cell cancer (kidney cancer).
Approximately about 100-about 800 patients are enrolled, with about
50-about 400 patients being assigned to a treatment group and about
50-about 400 patients assigned to a placebo group. The trial will
consist of the administration of intravenous repeating doses of
humanized/deimmunized anti-endoglin antibody at about 0.1-about 20
mg/kg or placebo every one, two or three weeks combined with
sunitinib at about 5-about 50 mg administered daily for 4 weeks,
with 2 weeks off prior to repeating the 4 week dosage cycle. The
time frame of the study is estimated at about 6 months-about 5
years, with continued therapy for responders as indicated at the
end of the initial study. Additional outcome measures are as
follows:
[0911] Primary outcome measure: progression-free survival. One goal
of the study is to demonstrate an increase in progression free
survival from about 9-13 months in the sunitinib plus placebo arm
to about 14-18 months (or more) in the sunitinib plus
humanized/deimmunized anti-endoglin antibody arm.
[0912] Secondary outcome measures that can be assessed include
response rate, duration of response, time to tumor progression,
overall survival, serious and non-serious adverse events. For
example, a treatment may prevent progression of the disease (i.e.,
stasis) or may result in an improvement. Alternately, or in
addition, other goals can be measured with respect to one or more
of the following: decreased tumor burden, decreased vascularity,
reduced side effects, decreased adverse reactions, and/or increased
patient compliance.
Example 19
Clinical Trial of Combination Therapy for Hepatocellular Cancer
[0913] This example describes a randomized, blinded,
placebo-controlled, multicenter, Phase 2 study designed to provide
a preliminary assessment of the safety and efficacy of combining
humanized/deimmunized anti-endoglin antibody with sorafenib
(NEXAVAR.RTM.) in patients with hepatocellular cancer (liver
cancer). Approximately about 100-about 800 patients are enrolled,
with about 50-about 400 patients being assigned to a treatment
group and about 50-about 400 patients assigned to a placebo group.
The trial will consist of the administration of intravenous
repeating doses of humanized/deimmunized anti-endoglin antibody at
about 0.1-about 20 mg/kg or placebo every one, two or three weeks
combined with sorafenib at about 400 mg daily for 3-6 cycles or
until progression. The time frame of the study is estimated at
about 6 months to about 5 years, with continued therapy for
responders as indicated at the end of the initial study. Additional
outcome measures are as follows:
[0914] Primary outcome measures: progression-free survival. One
goal of the study is to demonstrate an increase in progression free
survival from about 3-9 months in the sorafenib plus placebo arm to
about 6-12 months (or more) in the sorafenib plus
humanized/deimmunized anti-endoglin antibody arm.
[0915] Secondary outcome measures that can be assessed include
duration of response, time to tumor progression, overall survival,
serious and non-serious adverse events. For example, a treatment
may prevent progression of the disease (i.e., stasis) or may result
in an improvement. Alternately, or in addition, other goals can be
measured with respect to one or more of the following: decreased
tumor burden, decreased vascularity, reduced side effects,
decreased adverse reactions, and/or increased patient
compliance.
Example 20
Clinical Trial of Combination Therapy for Kidney Cancer
[0916] This example describes a randomized, blinded,
placebo-controlled, multicenter, Phase 2 study designed to provide
a preliminary assessment of the safety and efficacy of combining
humanized/deimmunized anti-endoglin antibody with bevacizumab
(AVASTIN.RTM.) in patients with renal cell cancer (kidney cancer).
Approximately about 100-about 800 patients are enrolled, with about
50-about 400 patients being assigned to a treatment group and about
50-about 400 patients assigned to a placebo group. The trial will
consist of the administration of intravenous repeating doses of
humanized/deimmunized anti-endoglin antibody at about 0.1-about 20
mg/kg or placebo every one, two or three weeks combined with
bevacizumab at about 7.5, about 10, or about 15 mg/kg administered
intravenously every two weeks. The time frame of the study is
estimated at about 6 months to about 5 years, with continued
therapy for positive responders as indicated at the end of the
initial study. Additional outcome measures are as follows:
[0917] Primary outcome measure: progression-free survival. One goal
of the study is to demonstrate an increase in progression free
survival from about 8-12 months in the bevacizumab plus placebo arm
to about 13-18 months (or more) in the bevacizumab plus
humanized/deimmunized anti-endoglin antibody arm.
[0918] Secondary outcome measures that can be assessed include
overall response rate, duration of response, time to tumor
progression, overall survival, serious and non-serious adverse
events. For example, a treatment may prevent progression of the
disease (i.e., stasis) or may result in an improvement.
Alternately, or in addition, other goals can be measured with
respect to one or more of the following: decreased tumor burden,
decreased vascularity, reduced side effects, decreased adverse
reactions, and/or increased patient compliance.
Example 21
Clinical Trial of Combination Therapy for Ovarian Cancer
[0919] This example describes a randomized, blinded,
placebo-controlled, multicenter, Phase 2 study designed to provide
a preliminary assessment of the safety and efficacy of combining
humanized/deimmunized anti-endoglin antibody with Doxil.RTM. in
patients with ovarian cancer. Approximately about 100-about 800
patients are enrolled, with about 50-about 400 patients being
assigned to a treatment group and about 50-about 400 patients
assigned to a placebo group. The trial will consist of the
administration of intravenous repeating doses of
humanized/deimmunized anti-endoglin antibody at about 0.1-about 20
mg/kg or placebo every one, two or four weeks combined with Doxil
at about 5 to about 50 mg/m.sup.2 administered once every 4 weeks
The time frame of the study is estimated at 6 months to about 5
years, with continued therapy for responders as indicated at the
end of the initial study. Additional outcome measures are as
follows:
[0920] Primary outcome measure: progression-free survival. One goal
of the study is to demonstrate an increase in progression free
survival from about 3-6 months in the Doxil.RTM. plus placebo arm
to about 4-12 months (or more) in the Doxil.RTM. plus
humanized/deimmunized anti-endoglin antibody arm.
[0921] Secondary outcome measures that can be assessed include
duration of response, time to tumor progression, overall survival,
serious and non-serious adverse events. For example, a treatment
may prevent progression of the disease (i.e., stasis) or may result
in an improvement. Alternately, or in addition, other goals can be
measured with respect to one or more of the following: decreased
tumor burden, decreased vascularity, reduced side effects,
decreased adverse reactions, and/or increased patient
compliance.
Example 22
Clinical Trial of Platinum Based Combination Therapy for Ovarian
Cancer
[0922] This example describes a randomized, blinded,
placebo-controlled, multicenter, Phase 2 study designed to provide
a preliminary assessment of the safety and efficacy of combining
humanized/deimmunized anti-endoglin antibody with platinum based
chemotherapy in patients with ovarian cancer. Approximately about
100-about 800 patients are enrolled, with about 50-about 400
patients being assigned to a treatment group and about 50-about 400
patients assigned to a placebo group. The trial will consist of the
administration of intravenous repeating doses of
humanized/deimmunized anti-endoglin antibody at about 0.1-about 20
mg/kg or placebo every one, two or three weeks combined with a
platinum based chemotherapy regimen (e.g., carboplatin and
paclitaxel) by intravenous infusion with courses repeating
throughout the study. The time frame of the study is estimated at
about 6 months-about 5 years, with continued therapy for responders
as indicated at the end of the initial study. Additional outcome
measures are as follows:
[0923] Primary outcome measure: progression-free survival. One goal
of the study is to demonstrate an increase in progression free
survival from about 12-18 months in the topotecan plus placebo arm
to about 12-24 months (or more) in platinum-based chemotherapy plus
humanized/deimmunized anti-endoglin antibody arm.
[0924] Secondary outcome measures that can be assessed include
duration of response, time to tumor progression, overall survival,
serious and non-serious adverse events. For example, a treatment
may prevent progression of the disease (i.e., stasis) or may result
in an improvement. Alternately, or in addition, other goals can be
measured with respect to one or more of the following: decreased
tumor burden, decreased vascularity, reduced side effects,
decreased adverse reactions, and/or increased patient
compliance.
Example 23
Use of Anti-Endoglin Antibodies for the Treatment of Diabetic
Retinopathy
Study Design
[0925] To evaluate the biologic activity of multiple intravitreal
injections of humanized/deimmunized anti-endoglin antibodies in
patients with center-involving clinically significant diabetic
macular edema (DME) and to report any associated adverse events, a
single-center, open-label, dose-escalating pilot study is
initiated. Patients with DME involving the center of the macula and
best-corrected visual acuity (BCVA) in the study eye between 20/40
and 20/400 are enrolled.
Study Treatment
[0926] Eligible patients are randomly assigned in a 1:1 ratio to
receive three intravitreal injections of humanized/deimmunized
anti-endoglin antibodies (about 0.25 to 2.5 mg per each injection)
administered monthly and observations are continued until month 24.
Primary end points are the frequency and severity of ocular and
systemic adverse events. Secondary end points are 1) best corrected
visual assessment as assessed with the Early Treatment Diabetic
Retinopathy Study (ETDRS) chart, with the use of standardized
refraction and testing protocol at a starting test distance of 2 m
and 2) measurement of retinal thickness by optical coherence
tomography. The evaluating physician is unaware of the patient's
treatment assignment; the physician who administers the injection
is aware of the patient's treatment assignment regarding
humanized/deimmunized anti-endoglin antibody or sham treatment but
is unaware of the dose of humanized/deimmunized anti-endoglin
antibody. Other personnel at each study site (except for those
assisting with injections), patients, and personnel at the central
reading center are unaware of the patient's treatment
assignment.
Efficacy and Safety Analyses
[0927] Efficacy analyses are performed on an intention-to-treat
basis among all patients with the use of a
last-observation-carried-forward method for missing data. For all
pairwise comparisons, the statistical model is adjusted for
baseline score for visual acuity (<55 letters vs. 255 letters).
Between-group comparisons for dichotomous end points are performed
with the use of the Cochran chi-square test. Change from baseline
visual acuity is analyzed with the use of analysis-of-variance
models. For end points for lesion characteristics,
analysis-of-covariance models adjusting for the baseline value are
used. The Hochberg-Bonferroni multiple-comparison procedure is used
to adjust for the two pairwise treatment comparisons for the
primary end point. Safety analyses include all treated
patients.
Conclusion
[0928] Humanized anti-endoglin antibodies will be a well-tolerated
therapy for patients with DME. This pilot study demonstrates that
humanized/deimmunized anti-endoglin antibody therapy has the
potential to maintain or improve best corrected visual acuity and
reduce retinal thickness in patients with center-involved
clinically significant DME.
Example 24
Clinical Trial of Anti-Endoglin Antibodies and Age-Related Macular
Degeneration
Study Design
[0929] At multiple sites in the United States, patients are
enrolled in a 2-year, prospective, randomized, double-blind,
sham-controlled study of the safety and efficacy of repeated
intravitreal injections of humanized/deimmunized anti-endoglin
antibodies among patients with choroidal neovascularization
associated with age-related macular degeneration. Primary efficacy
analysis is performed at 12 months. The primary efficacy end point
is the proportion of patients who had lost fewer than 15 letters
(approximately 3 lines) from baseline visual acuity, as assessed
with the Early Treatment Diabetic Retinopathy Study (ETDRS) chart,
with the use of standardized refraction and testing protocol at a
starting test distance of 2 m. The eligibility of lesions is
confirmed by an independent central reading center with the use of
standardized criteria and trained graders who are unaware of
patients' treatment assignments. Patients provide written informed
consent before determination of their full eligibility. Screening
may last as long as 28 days.
[0930] To be included in the study, patients must be at least 50
years old; have a best corrected visual acuity of 20/40 to 20/320
(Snellen equivalent determined with the use of an ETDRS chart);
have primary or recurrent choroidal neovascularization associated
with age-related macular degeneration, involving the foveal center;
have a type of lesion that had been assessed with the use of
fluorescein angiography and fundus photography as minimally classic
or occult with no classic choroidal neovascularization; have a
maximum lesion size of 12 optic-disk areas (1 optic-disk area
equals 2.54 mm.sup.2 on the basis of 1 optic-disk diameter of 1.8
mm), with neovascularization composing 50% or more of the entire
lesion; and have presumed recent progression of disease, as
evidenced by observable blood, recent vision loss, or a recent
increase in a lesion's greatest linear diameter of 10% or more.
There are no exclusion criteria regarding preexisting
cardiovascular, cerebrovascular, or peripheral vascular
conditions.
First Study
[0931] Fifty to 500 patients (50 to 500 eyes) with AMD will
participated in the study at multiple sites. Eligible patients are
randomly assigned in a 1:1:1 ratio to receive humanized/deimmunized
anti-endoglin antibodies at a dose of about 0.25 mg to 2.5 mg or a
sham injection monthly (within 23 to 37 days) for 2 years (24
injections) in one eye. The evaluating physician is unaware of the
patient's treatment assignment; the physician who administers the
injection is aware of the patient's treatment assignment regarding
humanized/deimmunized anti-endoglin antibody or sham treatment but
is unaware of the dose of humanized/deimmunized anti-endoglin
antibody. Other personnel at each study site (except for those
assisting with injections), patients, and personnel at the central
reading center are unaware of the patient's treatment assignment.
Intervention therapy (e.g., Verteporfin photodynamic therapy) is
allowed if the choroidal neovascularization in the study eye
becomes predominantly classic.
[0932] As the first step of treatment, the patients are to receive
a full ophthalmic examination to establish a baseline of ocular
health. The ophthalmic examination includes indirect
ophthalmoscopy, slit-lamp biomicroscopy, peripheral retinal
examination, intraocular pressure measurements, visual acuity
(unaided and best corrected) symptomatology, fundus photography,
fluorescein angiography, optical coherence tomography,
electroretinography and A-scan measurements.
[0933] Following the preliminary examination, an intravitreal
injection as described above is given to a patient's affected eye
manifesting AMD. If both eyes are affected, they may be treated
separately. The eye to be treated is injected with an ophthalmic
solution.
[0934] After treatment, the patients' eyes are to be examined on
days one (1), two (2), seven (7), fifteen (15), thirty (30) and
sixty (60) and every month thereafter for 2 years. Because of the
possibility of reoccurrence, the patients should return for
periodic examinations on a monthly basis thereafter. On each
examination day the patient is monitored for vitreous liquefaction.
Additionally, the patients are monitored for posterior vitreous
detachments using indirect ophthalmoscopy with scleral depression.
Finally, the extent of AMD presented by the patients is
continuously monitored through periodic retinal examinations,
optical coherence tomography and fluorescein angiograms to monitor
for the presence of subretinal fluid, blood, exudates, RPE
detachment, cystic retinal changes, or the presence of grayish
green subretinal neovascular membrane. Additional treatments may be
required if indicia of reoccurring neovascularization are observed.
Additional treatments may be given on weekly or monthly basis. In a
preferred embodiment, an initial treatment is followed by
subsequent treatments between 1-6 months apart.
[0935] Efficacy analyses are performed on an intention-to-treat
basis among all patients with the use of a
last-observation-carried-forward method for missing data. For all
pairwise comparisons, the statistical model is adjusted for
baseline score for visual acuity (<55 letters vs. .gtoreq.55
letters) and subtype of choroidal neovascularization (minimally
classic vs. occult with no classic disease). Between-group
comparisons for dichotomous end points are performed with the use
of the Cochran chi-square test. Change from baseline visual acuity
is analyzed with the use of analysis-of-variance models. For end
points for lesion characteristics, analysis-of-covariance models
adjusting for the baseline value are used. The Hochberg-Bonferroni
multiple-comparison procedure is used to adjust for the two
pairwise treatment comparisons for the primary end point. Safety
analyses include all treated patients.
Second Study
[0936] Patients manifesting age-related macular degeneration are
treated according to the methods of the First Study (see above)
with an intravitreal injection of (1) humanized/deimmunized
anti-endoglin antibody alone, (2) ranibizumab alone, (3)
humanized/deimmunized anti-endoglin antibody in combination with
ranibizumab in the same composition or different compositions or
(4) control antibody to reduce or prevent the development of
neovascularization, macular disease, and retinal damage.
Conclusion
[0937] Humanized/deimmunized anti-endoglin antibodies will be a
well-tolerated therapy for patients with AMD. This clinical trial
demonstrates that humanized/deimmunized anti-endoglin antibody
therapy has the potential to maintain or improve best corrected
visual acuity and reduce choroidal neovascularization in patients
with AMD. Further, humanized/deimmunized anti-endoglin antibodies
will demonstrate superior activity compared to ranibizumab and the
combination of humanized/deimmunized anti-endoglin antibodies and
ranibizumab therapy and will demonstrate increased activity versus
either antibody alone.
Example 25
Systemic Toxicology in Cynomolgus Monkeys
[0938] Cynomolgus monkeys are utilized in a study to address the
systemic toxicology of humanized/deimmunized anti-endoglin
antibodies.
[0939] Briefly, monkeys are dosed weekly for three weeks with 10.0
mg/kg, 30.0 mg/kg or 100.0 mg/kg of the humanized/deimmunized
anti-endoglin antibody. Placebo animals are dosed on the same
schedule with an appropriate solution in the absence of antibody.
The doses are administered as an intravenous bolus over 30 to 60
minutes and at least six animals are dosed at each dose level.
Toxicology is assessed via one or more of the following
indications: body weight measurements, basic physiologic clinical
measurements, serial serum chemistry, hematologic evaluations and
histopathological evaluations.
Example 26
Systemic Toxicology in Combination with Bevacizumab in Cynomolgus
Monkeys
[0940] Cynomolgus monkeys are utilized in a study to address the
systemic toxicology of humanized/deimmunized anti-endoglin
antibodies in combination with ranibizumab (LUCENTIS.RTM.).
[0941] Briefly, monkeys are dosed weekly for three weeks with 10.0
mg/kg, 30.0 mg/kg or 100.0 mg/kg of the humanized/deimmunized
anti-endoglin antibody in combination with about 10 mg/kg to 100
mg/kg of bevacizumab. Other animals receive either
humanized/deimmunized anti-endoglin antibody or bevacizumab alone.
Placebo animals are dosed on the same schedule with an appropriate
solution in the absence of antibody. The doses are administered as
an intravenous bolus over 30 to 60 minutes and at least six animals
are dosed at each dose level. Toxicology is assessed via one or
more of the following indications: body weight measurements, basic
physiologic clinical measurements, serial serum chemistry,
hematologic evaluations and histopathological evaluations.
Example 27
Regional Toxicology Study in Cynomolgus Monkeys
[0942] Cynomolgus monkeys are utilized in a study to address the
regional toxicology of humanized/deimmunized anti-endoglin
antibodies.
[0943] Briefly, monkeys are dosed by intravitreal injection weekly
for six weeks with 0.25, 1.25 and 2.5 mg of humanized/deimmunized
anti-endoglin antibody. Placebo animals are dosed on the same
schedule with an appropriate solution in the absence of antibody.
The doses are administered as intravitreal injections and at least
six animals are dosed at each dose level. Toxicology is assessed
via one or more of the following indications: body weight
measurements, basic physiologic clinical measurements, serial serum
chemistry, hematologic evaluations and histopathological
evaluations.
Combination Regional Toxicology Study
[0944] Cynomolgus monkeys are utilized in a study to address the
toxicology of humanized/deimmunized anti-endoglin antibodies in
combination with ranibizumab (LUCENTIS.RTM.) when given by
intravitreal injection.
[0945] Briefly, monkeys are dosed by intravitreal injection weekly
for six weeks with 0.25, 1.25 and 2.5 mg of humanized/deimmunized
anti-endoglin antibody and 0.5 mg of ranibizumab (LUCENTIS.RTM.).
Other animals receive either antibody alone, at the same dose and
schedule. Placebo animals are dosed on the same schedule with an
appropriate solution in the absence of antibody. The doses are
administered as intravitreal injections and at least six animals
are dosed at each dose level. Toxicology is assessed via one or
more of the following indications: body weight measurements, basic
physiologic clinical measurements, serial serum chemistry,
hematologic evaluations and histopathological evaluations.
Example 28
Sprouting Assays
[0946] Angiogenesis can be tested in a three-dimensional in vitro
model of sprouting. HUVECs are isolated from umbilical cords and
grown in M199 supplemented with 10% fetal bovine serum (FBS)
(GIBCO, Carlsbad, Calif.) and endothelial cell growth supplement
(ECGS) (BD Biosciences, Bedford, Mass.) at 3.degree. C. and 5%
CO.sub.2, Passage 2 to 4 HUVEC are used for all experiments
(Passage 0 being the primary culture). Lung fibroblasts (LF) are
routinely grown in DMEM (GIBCO, Carlsbad, Calif.) supplemented with
10% FBS at 37.degree. C. and 5% CO.sub.2 and used between P10 and
P15. Other fibroblast lines, obtainable from ATCC, can also be
used.
Preparing the Cells
[0947] HUVEC and fibroblasts are expanded in M199/10% FBS/Pen-Strep
(1:100) 1 to 2 days before beading. For HUVEC, medium is switched
to EGM-2 (Clonetics, Walkersville, Md.) the day before beading. For
fibroblasts, medium is switched to EGM-2 the day before embedding.
Beading requires approximately 400 HUVEC per bead. Fibroblasts are
used at 20,000 cells per well for a 24-well plate. Ninety-six-well
plates can also be used with quantities scaled accordingly.
Cytodex 3 Bead Preparation
[0948] Cytodex 3 microcarrier beads, for example, can be used in
the assay (Amersham Pharmacia Biotech, Piscataway, N.J.).
[0949] Dry beads (0.5 g) are hydrated and swollen in 50 ml PBS
(pH=7.4) for at least 3 hours at room temperature (RT) in a 50-ml
tube and placed it on a rocker.
[0950] The beads are allowed to settle (about 15 min). The
supernatant is discarded and the beads are washed for a few minutes
in fresh PBS (50 ml).
[0951] The wash PBS is discarded and replaced with fresh PBS:
[0952] The bead suspension is placed in a siliconized glass bottle
(from e.g., Windshield Wiper or Sigmacote). The beads are
sterilized by autoclaving for 15 min at 115.degree. C. and then
stored at 4.degree. C.
Reagents
[0953] Fibrinogen Solution
[0954] A fibrinogen solution is made by dissolving 2 mg/ml
fibrinogen in DPBS in a 37.degree. C. waterbath. The solution is
then mixed by inverting the tube rather than vortexing. The
percentage of clottable protein can be determined and adjusted
accordingly. The solution is then passed through a 0.22-.mu.m
filter to sterilize.
[0955] Aprotinin
[0956] Lyophilized aprotinin can be reconstituted at 4 U/ml in DI
water and sterile filtered. Aliquots of 1 ml each are made and
stored at -20.degree. C.
[0957] Thrombin
[0958] Thrombin is reconstituted in sterile water at 50 U/ml.
Aliquots of 0.5 ml are made and stored at -20.degree. C.
[0959] Coating the Beads with HUVEC (Day 1)
[0960] HUVEC cells are trypsinized. Beads are allowed to settle (do
not centrifuge), the supernatant is aspirated, and the beads are
briefly washed in 1 ml of warm EGM-2 medium. Beads (2500) are mixed
with 1.times.10.sup.6 HUVEC in 1.5 ml of warm EGM-2 medium in a
FACS tube and placed vertically in the incubator. (This will be
enough for approximately 10 wells; scale up if needed).
[0961] The mixture is incubated for 4 hours at 37.degree. C.,
inverting and mixing the tube every 20 min. (beads should look like
mini golf balls after beading which indicates sufficient coating
for sprouting).
[0962] After 4 hours, the coated beads are transferred to a T25
tissue culture flask (Falcon, Bedford, Mass.) and incubated
overnight in 5 ml of EGM-2 medium at 37.degree. C. and 5%
CO.sub.2.
[0963] Embedding Coated Beads in Fibrin Gel (Day 0)
[0964] A 2.0 mg/ml fibrinogen solution is prepared as described
above and 0.15 Units/ml of aprotinin are added to the fibrinogen
solution.
[0965] Coated beads are transferred to a 15 mL conical tube and the
beads are allowed to settle.
[0966] Beads are resuspended in 1 ml of EGM-2 medium and
transferred to a 1.5-ml centrifuge tube. The beads are washed three
times with 1 ml of EGM-2 medium, mixing by pipetting up and down
slowly with a P1000 pipette. The beads are counted on a coverslip
and resuspended in a fibrinogen solution at a concentration of 500
beads/ml.
[0967] Thrombin (0.625 Units/ml) is added to each well of a 24-well
plate. The fibrinogen/bead suspension (0.5 ml) to each well
changing the pipette tip for each well.
[0968] The thrombin and the fibrinogen/beads are mixed by pipetting
up and down gently about four to five times; avoid creating bubbles
in the fibrin gel. Control samples either are treated in the
absence of antibodies or one or more control antibodies. Test
samples are treated with anti-endoglin antibodies alone, anti-VEGF
antibodies alone, or a combination thereof. Multiple concentrations
of agents can be tested. The fibrinogen/bead solution is allowed to
clot for 5 minutes at room temperature and then at 37.degree. C./5%
CO.sub.2 for 15 min. It is important that the plate not be
disturbed during the first 5 min of clotting to minimize shearing
fibrin, which can result in reduced sprouting.
[0969] EGM-2 (1 mL) is added to each well in a drop-wise fashion.
Lung fibroblasts are seeded on top of the clot at a concentration
of 20,000 cells/well. Replace culture medium with fresh EGM-2
medium every other day until desired growth is achieved.
[0970] When the fibrin gel is formed, tiny bubbles may be present
in the gel; they will disappear in 3 to 4 days. Sprouting should be
apparent between day 2 and 4. Lumen formation begins around day 4
to 5 and sprouts continue to elongate. Newly formed tubes begin to
branch around day 4 to 6. By day 6 to 7, the microvessel-like
structures begin to anastomose with adjoining tubes; increasing the
number of beads per well results in earlier anastomosis. Sprouting
distance is measured by standard techniques.
Example 29
Immunocytochemistry of Angiogenic Sprouts In Vitro
[0971] For endothelial cell (EC) nuclei staining, fibrin gels are
washed twice with 1.times. PBS and then fixed overnight in 2%
paraformaldehyde. After two more washes with 1.times.PBS, gels are
then stained with 4', 6-diamidino-2-phenylindole (DAPI) (Sigma, St.
Louis, Mo.).
[0972] For immunostaining, lung fibroblasts (LF) are first removed
through a brief treatment of the gels with 10.times. trypsin.
Digestion is stopped with serum as soon as all fibroblasts are
removed. Gels are then extensively washed with HBSS, 1.times.
(Cellgro, Herndon, Va.). Cultures are then fixed for 10 minutes in
10% formalin and permeabilized with 0.5% Triton X-100 for 5
minutes. Non-specific binding is blocked with a solution of 5% BSA
in PBS for 2 hours.
[0973] Primary antibodies are used at a 1/100 dilution in blocking
buffer and incubated overnight at 4.degree. C. After extensive
washing, bound antibody is detected by species-specific Alexa Fluor
488-conjugated or Alexa Fluor 568-conjugated secondary antibodies
at a 1/1000 dilution (Molecular Probes, Carlsbad, Calif.).
Isotype-specific non-binding antibodies are used as a control. If
high background occurs, the concentration of primary or secondary
antibody can be reduced and, if necessary, incubation and/or
washing times can be increased. F-actin is stained with
TRITC-phalloidin (Sigma, St. Louis, Mo.) at a concentration of 0.2
.mu.M.
[0974] Phase-contrast and fluorescent images are captured on an
IX70 Olympus microscope coupled with a digital camera. Fluorescent
Z-series image stacks are captured on a two-photon Carl Zeiss
MicroImaging LSM 510 Meta microscope and compiled into
three-dimensional renderings with Metamorph software (Universal
Imaging Corporation, Downingtown Pa.). Thus, expression of various
markers can be readily detected.
[0975] Fluorescent optical image stacks along the z-axis of the
cultures can be captured to create 3D representations of the
vessels. The nuclei are stained by DAPI (green), and vessel walls
are stained for vimentin (orange). Wide, hollow lumens are clearly
visible, surrounded by a single layer of endothelial cells. These
images confirm that the lumens present in the in vitro assay are
intercellular and not intracellular slits as is often seen in
Matrigel assays. Furthermore, it can be confirmed that the HUVECs
are polarized, in that they have an apical membrane, facing the
lumen, and a basal membrane, apposed to a collagen IV-rich basement
membrane and the fibrin gel.
Example 30
Suppression of Choroidal Neovascularization in Cynomolgus
Monkeys
[0976] The effect of compositions described herein on laser-induced
choroidal neovascularization is evaluated in adult cynomolgus
monkeys.
[0977] In this experiment, (1) humanized/deimmunized anti-endoglin
antibody alone, (2) anti-VEGF antibody alone, (3)
humanized/deimmunized anti-endoglin antibody in combination with
anti-VEGF antibody in the same composition or different
compositions or (4) control antibody is administered by intravenous
or intravitreal injection. Each animal receives nine or ten laser
burns to each retina, and the development of active choroidal
neovascular lesions is assessed by fluorescein angiography, once
before the initiation of treatment and 15, 20 and 29 days
post-laser treatment. Compositions are administered intravenously
once per week, beginning one week before laser injury. Intravitreal
injections are made once every two weeks beginning one week before
laser, or once, two weeks following laser, at which time active CNV
lesions have already formed. Control animals receive weekly
intravenous or biweekly intravitreal injections of placebo,
beginning one week before laser.
[0978] CNV lesions are visualized by fluorescein angiography and
graded according to standard procedures.
Example 31
Inhibition of Injury-Induced Corneal Neovascularization
[0979] Corneal neovascularization is induced in male C57BL/6 mice
by intrastromal placement of 3 nylon sutures, or by chemical injury
(NaOH) and mechanical debridement of the corneal epithelium.
Multiple experiments are conducted in which (1)
humanized/deimmunized anti-endoglin antibody alone, (2) anti-VEGF
antibody alone, (3) humanized/deimmunized anti-endoglin antibody in
combination with anti-VEGF antibody alone in the same composition
or different compositions or (4) control antibody is administered
intraperitoneally once or at multiple time points immediately
before or following injury.
[0980] The growth of corneal neovessels is evaluated by slit-lamp
microscopy and histological evaluation. The vasculature is labeled
with an endothelial cell specific fluorescein-conjugated lectin,
and neovascularization is evaluated in corneal flat-mounts, as well
as in cross sections using PECAM immunohistochemistry. The presence
of corneal edema is evaluated, using slit lamp microscopy, and
corneal thickness is measured in cross-sections; increases in
corneal thickness reflect the amount of edema. The numbers of
polymorphonuclear leukocytes (PMN) and macrophages are determined
by staining cross-sections with HEMA-3 or rat anti-mouse F4/80
monoclonal antibody, respectively.
Example 32
Identification of T-Cell Epitopes in Humanized Anti-Endoglin
Antibodies
[0981] Sequences of humanized variable regions were tested by
iTope.TM. analysis. Humanized variable region sequences were
divided into overlapping 9-15mer peptides. The variable region
sequences were analyzed for promiscuous high affinity binding to
human MHC class II (potential T cell epitopes) using iTope.TM., an
in silico analytic tool that determines the affinity of peptides
for MHC Class II by computational analysis. Sequences with the
lowest frequencies of potential T cell epitopes from the iTope.TM.
analysis are identified as leads for generation of a humanized
antibody. The selected humanized variable region sequences may
redesigned through inclusion of mutations in order to remove
potential T cell epitopes. Mutations are designed using iTope.TM.
to reduce or eliminate MHC class II binding. Alternatively,
germline human sequences can be substituted at sites of potential T
cell epitopes or alternative sequences may be substituted.
[0982] FIGS. 19-23 present the predicted binding of 9mer peptides
for the humanized anti-endoglin antibody containing the light chain
HuVK_v0 and the heavy chain HuVH_v0, noted in FIG. 4.
Example 33
Design of Anti-CD105 Humanized/Deimmunized Antibodies
[0983] This example describes the design of therapeutic monoclonal,
humanized-deimmunized antibodies targeting human CD105 that exhibit
reduced immunogenicity.
[0984] The promiscuous high affinity MHC class II binding sequences
identified using iTope.TM. (see Example 31) were further analyzed
by iTope.TM. in order to identify amino acid substitutions at key
MHC class II pocket positions that would reduce or eliminate
peptide binding to MHC class II. Since all the sequences overlapped
CDRs, consideration was also given to the CDR location of the
changes (potential antigen contact residues) and the
physicochemical characteristics of the original and replacement
amino acids. TCR contact residues and residues outside the main
binding groove involved in the stabilization of peptide/MHC class
II-TCR interactions were also considered for replacement.
[0985] In VHV1, a 9-mer peptide lying completely within CDR2 and
starting at residue 51 was identified as a promiscuous high
affinity MHC class II binding peptide. The most successful method
for elimination of MHC class II binding is to target the first
amino acid of the 9-mer (the pocket 1 or p1 position) where removal
of the hydrophobic side-chain or replacement with a hydrophilic
side-chain eliminates MHC class II binding. However, this type of
radical amino acid replacement may not always be successful in
retaining antibody affinity, hence secondary pocket positions (p4,
p6, p7 or p9), alone or in combination, were also assessed.
iTope.TM. analysis revealed that targeting the p4 position of this
peptide by changing K52b to Q or R is predicted to significantly
reduce MHC class II binding while replacement of I51 at p1 with A
is predicted to remove binding entirely (Table 5).
TABLE-US-00007 TABLE 5 B1* B1* B1* B1* B1* B1* B1* B1* B1* B* B* B*
B* B1* B1* B1* B1* B1* Sequence 0101 0102 0501 0502 0301 0305 0306
0309 0401 0405 0404 0405 0408 0410 0421 1101 1102 1104 WVGEIRSK X X
X .largecircle. X X X X X X X X X X X X ASNHAT WVGEIRSQ X X
.largecircle. X X X ASNHAT WVGEIRSR X X X X ASNHAT WVGEARSQ ASNHAT
Total High B1* B1* B1* B1* B1* B1* B1* B1* B1* B1* B1* B1* B1* B1*
B1* B5* Allelles Affi- Sequence 1107 1114 1120 1128 1301 1304 1307
1321 0701 0801 0802 0804 0806 0813 0817 0101 Binding nity WVGEIRSK
X X X X X X X .largecircle. .largecircle. X X X X X X 31 26 ASNHAT
WVGEIRSQ X .largecircle. .largecircle. .largecircle. .largecircle.
11 6 ASNHAT WVGEIRSR .largecircle. .largecircle. 6 4 ASNHAT
WVGEARSQ 0 0 ASNHAT
Table 5: Analysis of the effect of substitutions on the immunogenic
region of VHV1 using iTope.TM.. The core 9-mer peptide is
underlined in the "sequence" column and substitutions are
highlighted in bold type. Flanking residues are not underlined. The
predicted binding of each core 9-mer peptide to each MHC class II
alleles is indicated by "O" if the binding score was 0.55-0.6 and
"X" if the binding score was >0.6. The numbers of MHC class II
alleles predicted to bind are shown in the "total" and "high
affinity" columns. Table 5 discloses SEQ ID NOS 106-109,
respectively, in order of appearance.
[0986] Crystal structures of antibody/antigen complexes suggest
that 151 may infrequently contact with antigen; however a radical
change of I to A (a substitution for disrupting a p1 anchor
position) at this position could affect the overall conformation of
the CDR. Therefore, relatively conservative changes at K52b (p4
anchor position) were also included since this residue is solvent
exposed but may not contact antigen. Finally, additional mutations
lying outside the CDR were also designed (G49 to A or S) to assess
the destabilizing effect on peptide/MHC class II/TCR interactions.
Table 6 lists the humanized and humanized/deimmunized variant VH
regions that were constructed; the SEQ ID NOS for the corresponding
nucleotide and amino acid sequences are indicated next to the
constructs.
TABLE-US-00008 TABLE 6 Construct Parental Amino Acid Name Sequence
Substitutions SEQ ID NO VH1 VHV1 N/A 42 VH2 VHV2 N/A 43 VK1 VKV1
N/A 4 VK2 VKV2 N/A 5 VH1A2 VHV1 I51A 89 VH1Q VHV1 K52bQ 90 VH1R
VHV1 K52bR 91 VH1S VHV1 G49S 92 VH1A VHV1 G49A 88 VK2AA VKV2 V19A +
I48A 94 VK2AS VKV2 V19A + T51S 95 VK2SA VKV2 T22S + I48A 96 VK2SS
VKV2 T22S + T51S 97 VK1AA VKV1 V19A + I48A 93 VK1AS VKV1 V19A +
T51S 102 VK1SA VKV1 T22S + I48A 103 VK1SS VKV1 T22S + T51S 100
[0987] Two promiscuous high affinity MHC class II binding peptides
were identified in VKV2 and VKV1. The first, with a p1 anchor at
V19, partially overlaps CDR1 and the second, with a p1 anchor at
148, overlaps CDR2. Both p1 anchors lie outside the CDRs and were
targeted by mutation to A, which may completely remove MHC class II
binding (Table 7). However, both these residues may be involved in
the maintenance of the conformations of CDRs 1 and 2; therefore,
additional mutations were designed that significantly reduced MHC
class II binding (Table 7). In both cases, p4 residues were
targeted by mutation of T to S. T22S also lies outside the CDR and
is less likely to affect CDR conformation than V19A. T51 lies
inside CDR2; however evidence from crystal structures of antibodies
complexed with antigen suggests that this residue rarely contacts
antigen. Table 6 lists the humanized and humanized/deimmunized VK
regions that were constructed.
TABLE-US-00009 TABLE 7 B1* B1* B1* B1* B1* B1* B1* B1* B1* B* B* B*
B* B1* B1* B1* B1* Sequence 0101 0102 0501 0502 0301 0305 0306 0309
0401 0405 0404 0405 0408 0410 0421 1101 B1*1102 1104 DRVTITCR
.largecircle. X X X X .largecircle. .largecircle. .largecircle.
.largecircle. X X X ASSSV DRVTISCR X X X X ASSSV DRATITCR ASSSV
PWIYATSN .largecircle. X X .largecircle. .largecircle. X X X X X X
X X .largecircle. X LASGV PWIYASSN .largecircle. X .largecircle.
.largecircle. X LASGV PWAYATSN LASGV Total High B1* B1* B1* B1* B1*
B1* B1* B1* B1* B1* B1* B1* B1* B1* B1* B5* Allelles Affi- Sequence
1107 1114 1120 1128 1301 1304 1307 1321 0701 0801 0802 0804 0806
0813 0817 0101 Binding nity DRVTITCR X X X X X X X X X X X X X
.largecircle. .largecircle. 27 20 ASSSV DRVTISCR X 5 5 ASSSV
DRATITCR 0 0 ASSSV PWIYATSN X .largecircle. X X X X .largecircle.
.largecircle. 23 16 LASGV PWIYASSN X .largecircle. 7 3 LASGV
PWAYATSN 0 0 LASGV
[0988] Table 7: Analysis of the effect of substitutions on the
immunogenic regions of VKV2 or VKV1 using iTope.TM.. The core 9-mer
peptide is underlined in the "sequence" column and substitutions
are highlighted in bold type. Flanking residues are not underlined.
The predicted binding of each core 9-mer peptide to each MHC class
II alleles is indicated by "O" if the binding score was 0.55-0.6
and "X" if the binding score was >0.6. The numbers of MHC class
II alleles predicted to bind are shown in the "total" and "high
affinity" columns. Table 7 discloses SEQ ID NOS 110-115,
respectively, in order of appearance.
Example 34
[0989] This example describes a method of screening anti-endoglin
antibodies for T-cell epitopes. The interaction between MHC,
polypeptide and T cell receptor (TCR) provides the structural basis
for the antigen specificity of T cell recognition. T cell
proliferation assays test the binding of polypeptides processed
from antibodies to MHC and the recognition of MHC/polypeptide
complexes by the TCR. In vitro T cell proliferation assays of the
present example, involve the stimulation of peripheral blood
mononuclear cells (PBMCs), containing antigen presenting cells
(APCs) and T cells. Stimulation is conducted in vitro using intact
anti-endoglin antibodies. Stimulated T cell proliferation is
measured using .sup.3H-thymidine (.sup.3H-Thy) and the presence of
incorporated .sup.3H-Thy is assessed using scintillation counting
of washed fixed cells.
[0990] All humanized and humanized/deimmunized VH and VK region
genes were synthesized using a series of overlapping
oligonucleotides that were annealed, ligated and PCR amplified to
give full length synthetic V regions. The assembled variants were
then cloned directly into Antitope Ltd.'s pANT expression vector
system for IgG1 heavy chains and kappa light chains.
[0991] Purification of Antibodies
[0992] Anti-endoglin antibodies were purified from the supernatants
of mammalian cultures by protein A chromatography. Buffer exchange
and protein concentration was done using PBS pH=7.4. Anti-endoglin
antibody was further purified by size exclusion chromatography
using a Sephacryl S200 column (GE Healthcare, AMersham, UK). The
major peak is collected, filter sterilized and shown to have
endotoxin levels <5 EU/mg using an Endosafe-PTS (Charles River,
Margate, UK). The purified antibodies are stored at 4 degrees
Celsius. Final concentrations were determined by UV absorption
using calculated molar extinction coefficients, where A280 1.0=1.62
mg/mL. Each antibody was then diluted to 100 .mu.g/mL in AIMV
culture medium.
[0993] Preparation and Selection of Donor PBMC
[0994] Peripheral Blood Mononuclear cells (PBMC) are isolated from
healthy community donor buffy coats (from blood drawn within 24
hours) which are obtained from the UK National Blood Transfusion
Service (Addenbrooke's Hospital, Cambridge, UK) according to
approval granted by Addenbrooke's Hospital Local Research Ethics
Committee. PBMC are isolated from buffy coats by Lymphoprep
(Axis-shield, Dundee, Scotland) density centrifugation and CD8+ T
cells are depleted using CD8+ RossetteSep.TM. (StemCell
Technologies, Inc.). Donors are characterized by identifying HLA-DR
haplotypes using a Biotest HLA SSP-PCR based tissue-typing kit
(Biotest, Landsteinerstra.beta.e, Denmark). T cell responses to a
control antigen, Keyhole Limpet Haemocyanin (KLH) (Pierce,
Rockford, Ill., USA) are determined for a positive control. PBMC
were then frozen and stored in liquid nitrogen until required. When
required for use, cells are thawed rapidly in a water bath at
37.degree. C. before transferring to 10 ml pre-warmed AIM V
medium.
[0995] A cohort of 20 donors is selected to best represent the
number and frequency of HLA-DR allotypes expressed in the world
population. Analysis of the allotypes expressed in the cohort
against those expressed in the world population revealed that
coverage of >80% is achieved and that all major HLA-DR alleles
(individual allotypes with a frequency >5% expressed in the
world population) are well represented. A summary of donor
haplotypes is provided in FIG. 23, and a comparison of the
frequency of donor allotypes used in the study versus those present
in the world population is made.
[0996] PBMCs from each donor are thawed, counted and viability
assessed. Cells were revived and resuspended in AIMV culture medium
to 4-6.times.10.sup.6 PBMC/mL. For each donor, bulk cultures were
established in which a total of 1 mL proliferation cell stock was
added to a 24-well plate. A total of 1 mL of each diluted test
sample was added to the PBMC to give a final concentration of 50
.mu.g/mL per antibody sample. For each donor, a positive control
(cells incubated with 100 .mu.g/mL KLH) and a negative control
(cells incubated with culture media only) were also included. For
the first 4 donors, an additional control was included to test for
modulation of T cell responses by the test samples, where test
sample and KLH were added to the PBMC. Comparison of these samples
with KLH alone can be used to assess the effects of the test
samples on proliferation. Cultures were incubated for a total of 8
days at 37 degrees Celsius with 5% carbon dioxide. On days 5, 6. 7
and 8, the cells in each well are gently resuspended and three 100
.mu.L aliquots are transferred to individual wells of a round
bottom 96 well plate. The cultures are pulsed with 1 .mu.Ci
.sup.3[H]-Thy (Perkin Elmer, Waltham, Mass.) in 100 .mu.L AIMV
culture medium and incubated for a further 18 hours before
harvesting onto filter mats using a TomTec Mach III cell harvester.
Counts per minute (cpm) for each well are determined by
Meltilex.TM. (Perkin Elmer.RTM., Waltham, Mass., USA) scintillation
counting on a Microplate Beta Counter (Perkin Elmer.RTM., Waltham,
Mass., USA) in paralux, low background counting mode.
[0997] Results are expressed as stimulation indices, where the
stimulation index (SI) is derived by division of the proliferation
score (e.g., counts per minute of radioactivity) measured to the
test anti-endoglin antibody by the score measured in cells not
contacted with a test anti-endoglin antibody. All basal cpm for the
control wells are above the minimum threshold for the assay of 150
cpm.
[0998] For proliferation assays, an empirical threshold of a
stimulation index (SI) equal to or greater than 2 (SI.gtoreq.2) has
been previously established whereby samples inducing proliferative
responses above this threshold are deemed positive (where included,
borderline SIs .gtoreq.1.90 are highlighted). Extensive assay
development and previous studies have shown that this is the
minimum signal to noise threshold allowing maximum sensitivity
without detecting large numbers of false positive responses.
Positive responses are defined by the following statistical and
empirical thresholds: [0999] 1. Significance (p<0.05) of the
response by comparing cpm of test wells against medium control
wells using unpaired two sample student's t-test. [1000] 2.
Stimulation index greater than 2 (SI.gtoreq.2), where SI=mean of
test wells (cpm)/mean medium control wells (cpm).
[1001] In addition, intra-assay variation is assessed by
calculating the coefficient of variance and standard deviation (SD)
of the raw data from replicate cultures.
[1002] Results for the EPISCREEN.RTM. time course proliferation
assay with the anti-endoglin antibodies are shown in FIG. 24 and
summarized in tabular form (Table 8). The chimeric antibody
stimulated responses in 4 of 20 donors (20% of the study cohort)
and, although two of the donor responses were borderline (1.92 and
1.95 for donors 11 and 17, respectively), they were significantly
different from background (p<0.05). The humanized antibody
VK1VH1 stimulated responses in 2 of 20 donors (10% of the study
cohort) including one borderline response (1.91 for donor 20) that
was significantly different from background (p<0.05). It is
noteworthy that donors 11 and 20 responded to both of these
antibodies suggesting that there could be a shared T cell epitope.
In contrast, none of the donors in the study cohort responded
positively to the deimmunized anti-endoglin antibody VK1AA VH1A2.
Results with the control antigen KLH show that there was a good
correlation between positive and negative results, indicating a
high level of reproducibility in the assay.
TABLE-US-00010 TABLE 8 T cells stimulation, as a measure of
immunogenicity, induced culture with anti-endoglin antibodies and
KLH, where "P*" indicate borderline stimulation above baseline and
"P" indicates a stimulation index greater than 2. VK1AA Basal Donor
No. Chimeric VK1VH1 VH1A2 KLH CPM 1 P 7456 2 P* 2272 3 P 3943 4 P
2827 5 P 2029 6 P 1918 7 3870 8 P 3110 9 P 1242 10 6042 11 P* P P
1696 12 P 3275 13 P 4644 14 P 1993 15 P 2727 16 P 1781 17 P* P 3681
18 P 893 19 P P 1705 20 P P* P 2901 Positive 4(20%) 2(10%) 0(0%) 92
Responses:
Example 35
EPISCREEN.RTM. T Cell Epitope Mapping
[1003] EPISCREEN.RTM. is an ex vivo technology for measurement of T
cell epitopes in whole antibodies or for mapping the sequence
location of such T cell epitopes as described in more detail
below.
EPISCREEN.RTM. Donor Selection
[1004] Peripheral Blood Mononuclear cells (PBMC) are isolated from
healthy community donor buffy coats (from blood drawn within 24
hours) which are obtained from the UK National Blood Transfusion
Service (Addenbrooke's Hospital, Cambridge, UK) according to
approval granted by Addenbrooke's Hospital Local Research Ethics
Committee. PBMC are isolated from buffy coats by Lymphoprep
(Axis-shield, Dundee, Scotland) density centrifugation and CD8+ T
cells are depleted using CD8+ RossetteSep.TM. (StemCell
Technologies, Inc.). Donors are characterized by identifying HLA-DR
haplotypes using a Biotest HLA SSP-PCR based tissue-typing kit
(Biotest, Landsteinerstra.beta.e, Denmark). T cell responses to a
control antigen, e.g., Keyhole Limpet Haemocyanin (KLH) (Pierce,
Rockford, USA) are also determined for a positive control. A cohort
of 54 donors is selected to best represent the number and frequency
of HLA-DR allotypes expressed in the world population. Analysis of
the allotypes expressed in the cohort against those expressed in
the world population revealed that coverage of >80% is achieved
and that all major HLA-DR alleles (individual allotypes with a
frequency >5% expressed in the world population) are well
represented. A summary of donor haplotypes is provided, and a
comparison of the frequency of donor allotypes used in the study
versus those present in the world population is made.
[1005] Donor details and haplotypes. Donor responses (SI) to KLH
are tested in two independent experiments. Test 1 is performed on
freshly isolated PBMC and an antibody is the re-test in the current
study. Responses that did not produce the same result (i.e.,
positive or negative) in both tests are highlighted. Donors with
very low basal cpm (<150 cpm) are excluded from the
analysis.
EPISCREEN.RTM. Analysis: Proliferation Assays
[1006] EPISCREEN.RTM. is used to test overlapping peptides derived
from the sequence of chimeric, humanized and humanized/deimmunized
antibodies. Overlapping peptides are designed. A series of
128.times.15-mer peptides overlapping by 12 amino acids are
synthesized together with 1.times.14-mer and 1.times.11-mer and
used to stimulate peripheral blood mononuclear cells (PBMC) derived
from a cohort of 51 healthy donors using EPISCREEN.RTM. T cell
epitope mapping. Individual peptides are tested in replicate
cultures and responses are assessed using T cell proliferation
assays to identify the precise location of epitopes. PBMC from each
donor are thawed, counted and assessed for viability. Cells are
revived in room temperature AIM V culture medium (Invitrogen,
Carlsbad, Calif.) before adjusting the cell density to
2.5.times.10.sup.6 PBMC/ml (proliferation cell stock). Peptides are
dissolved in DMSO (Sigma-Aldrich, St Louis, Mo., USA) to a final
concentration of 10 mM. Peptide culture stocks are then prepared by
diluting into AIM V culture medium to a final concentration of 5
.mu.M. For each peptide and each donor, sextuplicate cultures are
established by adding 100 .mu.l of the peptide culture stocks to
100 .mu.l of proliferation cell stock in a flat bottomed 96 well
plate. Both positive and negative control cultures are also
established in sextuplicate. A total of 9.times.96 well plates are
used for each donor, and each plate is sufficient to test 15
peptides with one negative control (carrier alone) in sextuplicate.
On the final plate, a positive control is added.
[1007] Cultures are incubated for a total of 6 days before adding
0.5 .mu.Ci .sup.3[H]-Thymidine (Perkin Elmer.RTM., Waltham, Mass.,
USA) to each well. Cultures are incubated for a further 18 hours
before harvesting onto filter mats using a TomTec Mach III cell
harvester. Counts per minute (cpm) for each well are determined by
Meltilex.TM. (Perkin Elmer.RTM., Waltham, Mass., USA) scintillation
counting on a Microplate Beta Counter (Perkin Elmer.RTM., Waltham,
Mass., USA) in paralux, low background counting mode.
[1008] For proliferation assays, an empirical threshold of a
stimulation index (SI) equal to or greater than 2 (SI.gtoreq.2) has
been previously established whereby samples inducing proliferative
responses above this threshold are deemed positive (where included,
borderline SIs .gtoreq.1.90 are highlighted). Extensive assay
development and previous studies have shown that this is the
minimum signal to noise threshold allowing maximum sensitivity
without detecting large numbers of false positive responses.
Positive responses are defined by the following statistical and
empirical thresholds: [1009] 1. Significance (p<0.05) of the
response by comparing cpm of test wells against medium control
wells using unpaired two sample student's t-test. [1010] 2.
Stimulation index greater than 2 (SI.gtoreq.2), where SI=mean of
test wells (cpm)/mean medium control wells (cpm).
[1011] In addition, intra-assay variation is assessed by
calculating the coefficient of variance and standard deviation (SD)
of the raw data from replicate cultures.
[1012] Proliferation assays are set up in sextuplicate cultures
("non adjusted data"). To ensure that intra assay variability is
low, data is also analyzed after removing the maximum and minimum
cpm values ("adjusted data") and the SI of donor responses are
compared using both data sets. Details of donor SIs from both
adjusted and non-adjusted data sets are prepared. T cell epitopes
are identified by calculating the average frequency of responses to
all peptides in the study+2.times.SD (background response rate).
Any peptide(s) that induced proliferation above this threshold is
considered to contain a T cell epitope.
In Silico iTope.TM. Analysis of Peptides
[1013] The sequences of peptides that are positive in the
proliferation assay are analyzed using Antitope's predictive
iTope.TM. software. This software predicts favorable interactions
between amino acid side chains of the peptide and specific binding
pockets within the MHC class II binding groove. The location of key
binding residues is determined by generating 10-mer peptides that
overlapped by one amino acid spanning the long peptide sequence.
Each 10-mer is tested against Antitope's database of MHC class II
allotypes and scored based on their fit and interactions with the
MHC class II molecules. Peptides that produced a high binding score
against a large number of alleles are considered to contain the
core 9mer.
Identification of T Cell Epitopes
[1014] All peptides identified using the EPISCREEN.RTM. Analysis
described above are successfully synthesized for testing against 51
healthy donors (54 donors are originally selected; donors may be
excluded from the analysis due to low basal cpm, i.e., below the
cut off value of 150 cpm). Positive responses are defined by donors
that produced a significant (p<0.05) response with a SI.gtoreq.2
to any given peptide. Borderline responses (a significant
(p<0.05) response with an SI.gtoreq.1.90) are also included. The
outputs from non-adjusted and adjusted data analyses are compared
to ensure that intra-assay variability is low and that positive
responses are not the result of spurious proliferation in
individual wells. The results from each analysis showed little
difference between the methods; consequently, the T cell epitope
map is compiled using the adjusted data analysis. Donor stimulation
indices from both non-adjusted and adjusted analyses are prepared.
T cell epitopes are identified by calculating the average frequency
of the responses to all peptides in the study plus twice the
standard deviation (termed `background response rate`). This is
calculated to be 5.6% and is the equivalent of inducing a positive
response in three or more donors. Peptides inducing proliferative
responses above this threshold are considered to contain a T cell
epitope.
Immunogenicity Testing of Lead Variants Using EPISCREEN.RTM.
[1015] Lead variants are purified and compared against the
wild-type polypeptide using EPISCREEN.RTM. time course T cell
assays. A large number of healthy donors representing the world
population according to expression of HLA allotypes are selected
from a donor library as described above. Donors are stimulated with
each protein in separate bulk cultures containing
2-4.times.10.sup.6 CD8.sup.+ T cell depleted PBMC. Replicate
samples (of T blasts) are removed from bulk cultures on days 5-8,
and proliferation along with IL-2 secretion (ELISPOT) is assessed.
To further validate the assessment between wild type and variants,
the study cohort is supplemented with responding donors from the
EPISCREEN.RTM. T cell epitope mapping study (provided sufficient
numbers of CD8.sup.+ T cell depleted PBMC remain).
[1016] In order to confirm loss of immunogenicity in lead variants,
an analysis of T cell immunogenicity by EPISCREEN.RTM. time course
T cell assays is undertaken as follows: [1017] (i) Buffy coats from
healthy donors (with >80% DRB 1 allotypic coverage for world
population) are used to isolate PBMC which contain physiological
levels of APC and CD4.sup.+ T cells; [1018] (ii) Each donor is
tested against positive control antigens including keyhole limpet
haemocyanin (a potent neoantigen); [1019] (iii) CD8.sup.+ T cells
are depleted to exclude the detection of MHC class I restricted T
cell responses; [1020] (iv) Lead variants and wild-type
polypeptides are compared against each other to evaluate relative
capacity to activate T cells CD4.sup.+ T cells; [1021] (v) Data is
analyzed using previously validated assay parameters with positive
responses of SI>2 supported by additional information including
statistical and frequency analysis; [1022] (vi) Data from
EPISCREEN.RTM. time course T cell assays provides information on
the magnitude and kinetics of T cell responses to individual
molecules; [1023] (vii) Any remaining PBMC from donors that produce
positive responses is archived and is available for use in repeat
testing studies; and [1024] (viii) An assessment is made of
association between donor allotype and responses to wild-type
polypeptide and any responses to variant leads.
[1025] Aspects of this invention may be embodied in other forms or
carried out in other ways without departing from the spirit or
essential characteristics thereof. The present disclosure is
therefore to be considered as in all aspects illustrated and not
restrictive, and all changes which come within the meaning and
range of equivalency are intended to be embraced therein.
Example 36
Cross Reactivity of Anti-Endoglin Antibodies
[1026] Anti-endoglin antibodies have been demonstrated to be cross
reactive with endothelial cells from human and mouse (Matsuno et
al., 1999). Humanized/deimmunized anti-endoglin antibodies are
tested for their ability to bind to human and human and murine
endothelium by the radioiummunoassay (RIA) according to the method
of Haruta et al., 1986. Briefly, purified anti-endoglin antibodies
are individually radiolabeled with .sup.125I using Iodo-Gen and
according to standard methods known to those skilled in the art.
The radiolabeled humanized/deimmunized anti-endoglin antibodies are
assayed for the mean number of iodine atoms per IgG molecule.
Counts per minute are compared by testing anti-endoglin antibodies
or isotype matched control IgG on cultures of human and murine
endothelial cells. Binding to subconfluent murine and human
endothlial cells may also be demonstrated using with FITC labeled
anti-endoglin antibody and analyzed by Becton Dickinson FACScan to
compare mean fluorescence intensity according to the method of
Matsuno et al., 1999. Binding to murine endothelium may also be
demonstrated by imaging the biodistribution of radiolabelled
anti-endoglin antibody in mice bearing syngeneic tumors. Briefly,
immunocompetent mice are implanted with syngeneic 4T1 breast
carcinomas. Tumors are allowed to grow to palpable size and animals
are treated with antibody chelated to a radioisotope, such as
.sup.64Cu. The distribution of labeled anti-endoglin antibody in
tumor-bearing BALB/c mice by autoradiography or PET scanning is
compared to the distribution of similarly labeled isotype
controlled antibody. Tumor uptake of labeled antibody is reported
relative to uptake in solid organs and the blood.
Sequence CWU 1
1
1181106PRTMus sp. 1Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser
Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser
Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly
Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Ala Thr Ser Asn Leu Ala
Ser Gly Val Pro Val Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr
Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu 65 70 75 80 Asp Ala
Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95
Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 2107PRTHomo sapiens
2Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser
Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys 100 105 3106PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
3Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr
Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile Tyr 35 40 45 Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser
Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys 100 105 4106PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 4Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Ile Tyr 35 40
45 Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln
Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser
Asn Pro Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 5106PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 5Asp 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 Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Pro Trp Ile Tyr 35 40 45 Ala Thr Ser
Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly
Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65 70
75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu
Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
623PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 6Asp 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 20
723PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 7Asp 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 20
823PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 8Gln 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 20
923PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 9Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys 20
1023PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 10Asp Ile Gln Met Ser Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys 20
1123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 11Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys 20
1223PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 12Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys 20
1323PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 13Asp Ile Val Leu Ser Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys 20
1423PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 14Asp Ile Gln Leu Ser Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys 20
1523PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 15Gln Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys 20
1623PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 16Gln 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 20
1723PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 17Gln Ile Gln Met Ser Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys 20
1823PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 18Gln Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys 20
1923PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 19Gln Ile Gln Leu Ser Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys 20
2015PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 20Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile Tyr 1 5 10 15 2115PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 21Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Pro Trp Ile Tyr 1 5 10 15 2215PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 22Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr 1 5 10 15
2315PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 23Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Trp Ile Tyr 1 5 10 15 2415PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 24Trp Phe Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile Tyr 1 5 10 15 2515PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 25Trp
Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr 1 5 10 15
2615PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 26Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Trp Ile Tyr 1 5 10 15 2715PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 27Trp Phe Gln Gln Lys Pro Gly
Lys Ala Pro Lys Pro Trp Ile Tyr 1 5 10 15 2832PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
28Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1
5 10 15 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
Cys 20 25 30 2932PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 29Gly Val Pro Ser Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Tyr Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25 30 3032PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
30Gly Val Pro Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1
5 10 15 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
Cys 20 25 30 3132PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 31Gly Val Pro Ala Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25 30 3232PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
32Gly Val Pro Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr 1
5 10 15 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
Cys 20 25 30 3332PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 33Gly Val Pro Ala Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Tyr Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25 30 3432PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
34Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Thr 1
5 10 15 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
Cys 20 25 30 3510PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 35Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys 1 5 10 3610PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 36Phe Gly Ala Gly Thr Lys Val Glu Ile
Lys 1 5 10 3710PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 37Phe Gly Gly Gly Thr Lys Val Glu Leu
Lys 1 5 10 3810PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 38Phe Gly Ala Gly Thr Lys Val Glu Leu
Lys 1 5 10 39118PRTMus sp. 39Glu Val Lys Leu Glu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Met Lys Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Asp Ala 20 25 30 Trp Met Asp Trp Val
Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Glu Ile
Arg Ser Lys Ala Ser Asn His Ala Thr Tyr Tyr Ala Glu 50 55 60 Ser
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70
75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Ile
Tyr 85 90 95 Tyr Cys Thr Arg Trp Arg Arg Phe Phe Asp Ser Trp Gly
Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115 40115PRTHomo
sapiens 40Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Asn Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Lys Ser Lys Thr Asp
Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60 Pro Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln
Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys
Thr Thr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110
Val Ser Ser 115 41118PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 41Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala 20 25 30 Trp Met
Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Gly Glu Ile Arg Ser Lys Ala Ser Asn His Ala Thr Tyr Tyr Ala Glu 50
55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn
Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr
Ala Val Tyr 85 90 95 Tyr Cys Thr Thr Trp Arg Arg Phe Phe Asp Ser
Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115
42118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 42Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Asp Ala 20 25 30 Trp Met Asp Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Glu Ile Arg Ser
Lys Ala Ser Asn His Ala Thr Tyr Tyr Ala Glu 50 55 60 Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu
Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90
95 Tyr Cys Thr Arg Trp Arg Arg Phe Phe Asp Ser Trp Gly Gln Gly Thr
100 105 110 Leu Val Thr Val Ser Ser 115 43118PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
43Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp
Ala 20 25 30 Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Glu Ile Arg Ser Lys Ala Ser Asn His Ala
Thr Tyr Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ser
Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Arg Trp
Arg Arg Phe Phe Asp Ser Trp Gly Gln Gly Thr 100
105 110 Leu Val Thr Val Ser Ser 115 4430PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
44Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20
25 30 4514PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 45Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val Gly 1 5 10 4614PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 46Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val Ala 1 5 10 4732PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 47Arg Phe Thr Ile Ser Arg
Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu
Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Thr Thr 20 25 30
4832PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 48Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn
Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Thr Glu Asp Thr
Ala Val Tyr Tyr Cys Thr Arg 20 25 30 4932PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
49Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Val Tyr Leu Gln 1
5 10 15 Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Thr
Thr 20 25 30 5032PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 50Arg Phe Thr Ile Ser Arg Asp Asp
Ser Lys Ser Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Thr
Glu Asp Thr Ala Val Tyr Tyr Cys Thr Thr 20 25 30 5132PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
51Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Arg Leu Tyr Leu Gln 1
5 10 15 Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Thr
Thr 20 25 30 5232PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 52Arg Phe Thr Ile Ser Arg Asp Asp
Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Ile Ser Leu Lys Thr
Glu Asp Thr Ala Val Tyr Tyr Cys Thr Thr 20 25 30 5332PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
53Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln 1
5 10 15 Met Arg Phe Thr Ile Ser Arg Asp Asp Ser Ile Tyr Tyr Cys Thr
Thr 20 25 30 5432PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 54Arg Phe Thr Ile Ser Arg Asp Asp
Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Arg Phe Thr Ile Ser
Arg Asp Asp Ser Leu Tyr Tyr Cys Thr Thr 20 25 30 5532PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
55Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln 1
5 10 15 Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Thr
Gly 20 25 30 5611PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 56Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 1 5 10 5711PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 57Trp Gly Gln Gly Thr Thr Val Thr Val
Ser Ser 1 5 10 5811PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 58Trp Gly Gln Gly Thr Leu Leu Thr Val
Ser Ser 1 5 10 5911PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 59Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ala 1 5 10 6011PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 60Trp Gly Gln Gly Thr Thr Val Thr Val
Ser Ala 1 5 10 6111PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 61Trp Gly Gln Gly Thr Leu Leu Thr Val
Ser Ala 1 5 10 6211PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 62Trp Gly Gln Gly Thr Thr Leu Thr Val
Ser Ser 1 5 10 6310PRTMus sp. 63Arg Ala Ser Ser Ser Val Ser Tyr Met
His 1 5 10 647PRTMus sp. 64Ala Thr Ser Asn Leu Ala Ser 1 5
659PRTMus sp. 65Gln Gln Trp Ser Ser Asn Pro Leu Thr 1 5 665PRTMus
sp. 66Asp Ala Trp Met Asp 1 5 6719PRTMus sp. 67Glu Ile Arg Ser Lys
Ala Ser Asn His Ala Thr Tyr Tyr Ala Glu Ser 1 5 10 15 Val Lys Gly
687PRTMus sp. 68Trp Arg Arg Phe Phe Asp Ser 1 5 69107PRTHomo
sapiens 69Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser
Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr
Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu 85 90 95 Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 70106PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
70Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1
5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Ser Tyr
Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile Tyr 35 40 45 Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala
Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys 100 105 71106PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 71Glu Ile Val Leu Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Pro Trp Ile Tyr 35 40
45 Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser
50 55 60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu
Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser
Asn Pro Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 72106PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 72Glu Ile Val Leu Thr Gln Ser Pro
Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser
Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Arg Pro Trp Ile Tyr 35 40 45 Ala Thr
Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60
Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65
70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro
Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
73115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 73Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Gly Ser 20 25 30 Ala Met His Trp Val Arg Gln
Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser
Lys Ala Asn Ser Tyr Ala Thr Ala Tyr Ala Ala 50 55 60 Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala
Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90
95 Tyr Cys Thr Arg Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110 Val Ser Ser 115 74118PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 74Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala 20 25 30 Trp
Met Asp Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40
45 Gly Glu Ile Arg Ser Lys Ala Ser Asn His Ala Thr Tyr Tyr Ala Glu
50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys
Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp
Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Arg Trp Arg Arg Phe Phe Asp
Ser Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115
75118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 75Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Asp Ala 20 25 30 Trp Met Asp Trp Val Arg Gln
Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Glu Ile Arg Ser
Lys Ala Ser Asn His Ala Thr Tyr Tyr Ala Glu 50 55 60 Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Val
Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90
95 Tyr Cys Thr Arg Trp Arg Arg Phe Phe Asp Ser Trp Gly Gln Gly Thr
100 105 110 Leu Val Thr Val Ser Ser 115 7611PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 76Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 7711PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 77Trp
Gly Arg Gly Thr Leu Val Thr Val Ser Ser 1 5 10 7811PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 78Trp
Gly Gln Gly Thr Met Val Thr Val Ser Ser 1 5 10 7911PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 79Trp
Gly Gln Gly Thr Thr Val Thr Val Ser Ser 1 5 10 8010PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 80Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 1 5 10 8110PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 81Phe
Gly Gln Gly Thr Lys Leu Glu Ile Lys 1 5 10 8210PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 82Phe
Gly Pro Gly Thr Lys Val Asp Ile Lys 1 5 10 8310PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 83Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys 1 5 10 8410PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 84Phe
Gly Gln Gly Thr Arg Leu Glu Ile Lys 1 5 10 856PRTArtificial
SequenceDescription of Artificial Sequence Synthetic 6xHis tag
85His His His His His His 1 5 86106PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
polypeptideMOD_RES(1)..(1)Asp or GlnMOD_RES(3)..(3)Gln or
ValMOD_RES(4)..(4)Met or LeuMOD_RES(5)..(5)Thr or
SerMOD_RES(35)..(35)Tyr or PheMOD_RES(45)..(45)Pro or
LeuMOD_RES(46)..(46)Trp or LeuMOD_RES(59)..(59)Ser, Val or
AlaMOD_RES(69)..(69)Asp or SerMOD_RES(70)..(70)Tyr or
PheMOD_RES(99)..(99)Gly or AlaMOD_RES(105)..(105)Ile or Leu 86Xaa
Ile Xaa Xaa Xaa Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met
20 25 30 His Trp Xaa Gln Gln Lys Pro Gly Lys Ala Pro Lys Xaa Xaa
Ile Tyr 35 40 45 Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Xaa Arg
Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Xaa Xaa Thr Leu Thr Ile
Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Xaa Gly Thr Lys
Val Glu Xaa Lys 100 105 87118PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptideMOD_RES(49)..(49)Gly or
AlaMOD_RES(79)..(79)Asn or SerMOD_RES(80)..(80)Thr or
ArgMOD_RES(81)..(81)Leu or ValMOD_RES(86)..(86)Asn or
IleMOD_RES(95)..(95)Val, Ile or LeuMOD_RES(100)..(100)Arg, Thr or
GlyMOD_RES(113)..(113)Leu or ThrMOD_RES(114)..(114)Val or
LeuMOD_RES(118)..(118)Ser or Ala 87Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala 20 25 30 Trp Met Asp Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Xaa Glu
Ile Arg Ser Lys Ala Ser Asn His Ala Thr Tyr Tyr Ala Glu 50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Xaa Xaa 65
70 75 80 Xaa Tyr Leu Gln Met Xaa Ser Leu Lys Thr Glu Asp Thr Ala
Xaa Tyr 85 90 95 Tyr Cys Thr Xaa Trp Arg Arg Phe Phe Asp Ser Trp
Gly Gln Gly Thr 100 105 110 Xaa Xaa Thr Val Ser Xaa 115
88118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic VH_v1(A) polypeptide 88Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala 20 25 30 Trp Met Asp Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Glu
Ile Arg Ser Lys Ala Ser Asn His Ala Thr Tyr Tyr Ala Glu 50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65
70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala
Val Tyr 85 90 95 Tyr Cys Thr Arg Trp Arg Arg Phe Phe Asp Ser Trp
Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115
89118PRTArtificial SequenceDescription of Artificial
Sequence Synthetic VH_v1(A2) polypeptide 89Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala 20 25 30 Trp Met
Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Gly Glu Ala Arg Ser Lys Ala Ser Asn His Ala Thr Tyr Tyr Ala Glu 50
55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn
Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr
Ala Val Tyr 85 90 95 Tyr Cys Thr Arg Trp Arg Arg Phe Phe Asp Ser
Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115
90118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic VH_v1(Q) polypeptide 90Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala 20 25 30 Trp Met Asp Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Glu
Ile Arg Ser Gln Ala Ser Asn His Ala Thr Tyr Tyr Ala Glu 50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65
70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala
Val Tyr 85 90 95 Tyr Cys Thr Arg Trp Arg Arg Phe Phe Asp Ser Trp
Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115
91118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic VH_v1(R) polypeptide 91Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala 20 25 30 Trp Met Asp Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Glu
Ile Arg Ser Arg Ala Ser Asn His Ala Thr Tyr Tyr Ala Glu 50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65
70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala
Val Tyr 85 90 95 Tyr Cys Thr Arg Trp Arg Arg Phe Phe Asp Ser Trp
Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115
92118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic VH_v1(S) polypeptide 92Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala 20 25 30 Trp Met Asp Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Glu
Ile Arg Ser Lys Ala Ser Asn His Ala Thr Tyr Tyr Ala Glu 50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65
70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala
Val Tyr 85 90 95 Tyr Cys Thr Arg Trp Arg Arg Phe Phe Asp Ser Trp
Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115
93106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic VK_v1(AA) polypeptide 93Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Ala Thr Ile Thr
Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Ala Tyr 35 40 45 Ala Thr
Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60
Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65
70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro
Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
94106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic VK_v2(AA) polypeptide 94Asp Ile Gln Leu Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Ala Thr Ile Thr
Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Ala Tyr 35 40 45 Ala Thr
Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60
Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65
70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro
Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
95106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic VK_v2(AA) polypeptide 95Asp Ile Gln Leu Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Ala Thr Ile Thr
Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Ile Tyr 35 40 45 Ala Ser
Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60
Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65
70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro
Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
96106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic VK_v2(SA) polypeptide 96Asp Ile Gln Leu Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Ser
Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Ala Tyr 35 40 45 Ala Thr
Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60
Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65
70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro
Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
97106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic VK_v2(SS) polypeptide 97Asp Ile Gln Leu Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Ser
Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Ile Tyr 35 40 45 Ala Ser
Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60
Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65
70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro
Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
98330PRTArtificial SequenceDescription of Artificial Sequence
Synthetic IgG1(m3)_constant_region polypeptide 98Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35
40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165
170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290
295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
99107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic CK_constant_region polypeptide 99Arg Thr Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50
55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105 100106PRTArtificial SequenceDescription of Artificial
Sequence Synthetic 97 with L4M polypeptide 100Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Ser Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Ile Tyr 35 40
45 Ala Ser Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln
Pro Glu 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser
Asn Pro Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 10132PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 101Asp Ile Gln Leu Thr Gln Ser Pro
Ser Ser Leu Ser Pro Trp Ile Tyr 1 5 10 15 Ala Thr Ser Asn Leu Ala
Ser Tyr Thr Leu Thr Ile Ser Ser Leu Gln 20 25 30
102106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic 95 with L4M polypeptide 102Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Ala Thr Ile
Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Ile Tyr 35 40 45 Ala
Ser Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55
60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu
65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro
Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
103106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic 96 with L4M polypeptide 103Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile
Ser Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Ala Tyr 35 40 45 Ala
Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55
60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu
65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro
Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
10412PRTInfluenza A virus 104Pro Lys Tyr Val Lys Gln Asn Thr Leu
Lys Leu Ala 1 5 10 10515PRTUnknownDescription of Unknown Chlamydia
HSP 60 peptide 105Lys Val Val Asp Gln Ile Lys Lys Ile Ser Lys Pro
Val Gln His 1 5 10 15 10614PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 106Trp Val Gly Glu Ile Arg
Ser Lys Ala Ser Asn His Ala Thr 1 5 10 10714PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 107Trp
Val Gly Glu Ile Arg Ser Gln Ala Ser Asn His Ala Thr 1 5 10
10814PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 108Trp Val Gly Glu Ile Arg Ser Arg Ala Ser Asn
His Ala Thr 1 5 10 10914PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 109Trp Val Gly Glu Ala Arg
Ser Lys Ala Ser Asn His Ala Thr 1 5 10 11013PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 110Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val 1 5 10
11113PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 111Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Ser
Ser Val 1 5 10 11213PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 112Asp Arg Ala Thr Ile Thr Cys Arg Ala
Ser Ser Ser Val 1 5 10 11313PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 113Pro Trp Ile Tyr Ala Thr
Ser Asn Leu Ala Ser Gly Val 1 5 10 11413PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 114Pro
Trp Ile Tyr Ala Ser Ser Asn Leu Ala Ser Gly Val 1 5 10
11513PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 115Pro Trp Ala Tyr Ala Thr Ser Asn Leu Ala Ser
Gly Val 1 5 10 116118PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptideMOD_RES(49)..(49)Gly, Ala
or SerMOD_RES(51)..(51)Ala or
IleMOD_RES(54)..(54)Lys, Gln or ArgMOD_RES(81)..(81)Leu or Val
116Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Asp Ala 20 25 30 Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Xaa Glu Xaa Arg Ser Xaa Ala Ser Asn His
Ala Thr Tyr Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Xaa Tyr Leu Gln Met Asn
Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Arg
Trp Arg Arg Phe Phe Asp Ser Trp Gly Gln Gly Thr 100 105 110 Leu Val
Thr Val Ser Ser 115 117106PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptideMOD_RES(19)..(19)Ala or
ValMOD_RES(22)..(22)Thr or SerMOD_RES(47)..(47)Ala or
IleMOD_RES(50)..(50)Thr or Ser 117Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Xaa Thr Ile Xaa
Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Xaa Tyr 35 40 45 Ala Xaa
Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60
Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65
70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro
Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
11834PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 118Trp Val Ala Glu Ile Arg Ser Lys Ala Ser
Asn Val Tyr Leu Gln Met 1 5 10 15 Asn Ser Leu Lys Val Tyr Tyr Cys
Thr Arg Trp Arg Arg Phe Phe Asp 20 25 30 Ser Trp
* * * * *
References