U.S. patent application number 12/063371 was filed with the patent office on 2012-06-07 for antibody compositions, methods for treating neoplastic disease and methods for regulating fertility.
This patent application is currently assigned to Onconon, LLC. Invention is credited to Edwin Rock, Vernon Stevens, Pierre Triozzi.
Application Number | 20120141371 12/063371 |
Document ID | / |
Family ID | 37728017 |
Filed Date | 2012-06-07 |
United States Patent
Application |
20120141371 |
Kind Code |
A2 |
Rock; Edwin ; et
al. |
June 7, 2012 |
ANTIBODY COMPOSITIONS, METHODS FOR TREATING NEOPLASTIC DISEASE AND
METHODS FOR REGULATING FERTILITY
Abstract
Antibody compositions and methods for inhibition of the effects
of gonadotropin hormones are provided. Methods for treating cancer
and methods for regulating fertility are provided by administration
of the antibody compositions to a mammalian subject in need
thereof.
Inventors: |
Rock; Edwin; (Washington,
DC) ; Stevens; Vernon; (Washington, DC) ;
Triozzi; Pierre; (Washington, DC) |
Assignee: |
Onconon, LLC
Washington
DC
20007
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20100068135 A1 |
March 18, 2010 |
|
|
Family ID: |
37728017 |
Appl. No.: |
12/063371 |
Filed: |
August 8, 2006 |
PCT Filed: |
August 8, 2006 |
PCT NO: |
PCT/US06/30988 |
371 Date: |
September 24, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60/706,506 |
Aug 8, 2005 |
|
|
|
Current U.S.
Class: |
424/1.49 ;
424/139.1; 424/178.1; 435/325; 435/7.1; 514/44R; 530/387.3;
530/387.9; 536/23.53 |
Current CPC
Class: |
A61P 43/00 20180101;
C07K 16/26 20130101; C07K 16/241 20130101; C07K 2317/73 20130101;
A61P 31/04 20180101; C07K 2317/55 20130101; A61P 35/02 20180101;
C07K 2317/34 20130101; A61P 29/00 20180101; C07K 2317/52 20130101;
A61P 15/04 20180101; A61P 35/00 20180101; A61P 35/04 20180101; C07K
2319/00 20130101; A61P 15/08 20180101; A61P 5/24 20180101; A61P
25/28 20180101 |
Class at
Publication: |
424/001.49 ;
530/387.9; 530/387.3; 424/139.1; 435/007.1; 536/023.53;
514/044.00R; 435/325; 424/178.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; G01N 33/53 20060101 G01N033/53; A61P 35/00 20060101
A61P035/00; A61K 31/7088 20060101 A61K031/7088; C12N 5/10 20060101
C12N005/10; A61K 51/10 20060101 A61K051/10; C07K 16/00 20060101
C07K016/00; C07H 21/04 20060101 C07H021/04 |
Claims
1. An isolated monoclonal antibody which binds to human chorionic
gonadotropin comprising an amino acid sequence in its heavy chain
variable region as set forth in SEQ ID NO:2 or an amino acid
sequence which is at least 90% homologous to SEQ ID NO:2.
2. An isolated monoclonal antibody which binds to human chorionic
gonadotropin comprising an amino acid sequence in its heavy chain
variable region as set forth in SEQ ID NO:4 or an amino acid
sequence which is at least 90% homologous to SEQ ID NO:4.
3. The antibody of claim 2, wherein the antibody is an IgG.sub.1,
an IgG.sub.2, an IgG.sub.3, an IgG4, an IgM, an IgA.sub.1, an
IgA.sub.2, a secretory IgA, an IgD, or an IgE antibody.
4. The antibody of claim 1, wherein the antibody is an
IgG.sub.1.kappa. or IgG.sub.1.lamda. isotype.
5. The antibody of claim 1, wherein the antibody is an
IgG.sub.4.kappa. or IgG.sub.4.lamda. isotype.
6. The antibody of claim 1, wherein the antibody is an IgG.sub.1,
an IgG.sub.2, an IgG.sub.3, an IgG4, an IgM, an IgA.sub.1, an
IgA.sub.2, a secretory IgA, an IgD, or an IgE antibody.
7. The antibody of claim 2, wherein the antibody is an
IgG.sub.1.kappa.; or IgG.sub.1.lamda. isotype.
8. The antibody of claim 2, wherein the antibody is an
IgG.sub.4.kappa. or IgG.sub.4.lamda. isotype.
9. The antibody of claim 1, wherein the antibody is human,
non-human primate, rabbit, rat, or mouse, or a combination
thereof.
10. The antibody of claim 2, wherein the antibody is human,
non-human primate, rabbit, rat, or mouse, or a combination
thereof.
11. An isolated monoclonal antibody which binds to human chorionic
gonadotropin comprising an amino acid sequence in its light chain
variable region and a heavy chain variable region, wherein the
heavy chain variable region is set forth in SEQ ID NO:2 or SEQ ID
NO:4 or an amino acid sequence which is at least 90% homologous to
in SEQ ID NO:2 or SEQ ID NO:4.
12. The antibody of claim 11, wherein the antibody has one or more
of the following characteristics: (i) inhibits proliferation in
vitro of BXPC-3 pancreatic carcinoma cells; and (ii) does not
inhibit proliferation in vitro of MCF-7 breast carcinoma cells or
HeLa cells.
13. The antibody of claim 11 having a dissociation equilibrium
constant (K.sub.D) of approximately 10.sup.-8 M or less, when
determined by surface plasmon resonance (SPR) using recombinant
human chorionic gonadotropin as an analyte and the antibody as a
ligand.
14. The antibody of claim 11, wherein the antibody is capable of
binding human chorionic gonadotropin with a binding affinity of
about 10.sup.8 M.sup.-1 or greater.
15. The antibody of claim 11 which is an intact antibody, an intact
IgG.sub.1 antibody, an intact IgG.sub.2 antibody, an intact
IgG.sub.3 antibody, an intact IgG.sub.4 antibody, an intact IgM
antibody, an intact IgA.sub.1 antibody, an intact IgA.sub.2
antibody, an intact secretory IgA antibody, an intact IgD antibody,
or an intact IgE antibody, wherein the antibody is glycosylated in
a eukaryotic cell.
16. The antibody of claim 11 which is an antibody fragment or a
single chain antibody.
17. The antibody of claim 11 which is a binding-domain
immunoglobulin fusion protein comprising (i) a variable heavy chain
amino acid sequence as set forth in SEQ ID NO:2 or a variable heavy
chain sequence which is at least 90% homologous to SEQ ID NO:2,
fused to a variable light chain amino acid sequence via a linker
peptide, that is fused to an immunoglobulin hinge region
polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region
fused to the hinge region, and (iii) an immunoglobulin heavy chain
CH3 constant region fused to the CH2 constant region.
18. The antibody of claim 11 which is a binding-domain
immunoglobulin fusion protein comprising (i) a variable heavy chain
amino acid sequence as set forth in SEQ ID NO:4 or a variable heavy
chain sequence which is at least 90% homologous to SEQ ID NO:4,
fused to a variable light chain amino acid sequence via a linker
peptide, that is fused to an immunoglobulin hinge region
polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region
fused to the hinge region, and (iii) an immunoglobulin heavy chain
CH3 constant region fused to the CH2 constant region.
19. The antibody of claim 11, wherein the antibody binds to a
predetermined antigen with an equilibrium association constant (Ka)
of at least 10.sup.10 M.sup.-1.
20. The antibody of claim 11, wherein the antibody binds to a
predetermined antigen with an equilibrium association constant (Ka)
of at least 10.sup.9 M.sup.-1.
21. The antibody of claim 11, wherein the antibody binds to a
predetermined antigen with an equilibrium association constant (Ka)
of at least 10.sup.8 M.sup.-1.
22. The antibody of claim 11, wherein the antibody is
monoclonal.
23. The antibody of claim 11, wherein the antibody is a
F(ab').sub.2, Fab, Fv, or Fd fragment.
24. The antibody of claim 11, wherein the antibody is
antigen-specific.
25. An isolated human monoclonal antibody which binds to human
chorionic gonadotropin comprising an amino acid sequence in its
human heavy chain variable region as set forth in SEQ ID NO:2 or an
amino acid sequence which is at least 90% homologous to SEQ ID
NO:2.
26. An isolated human monoclonal antibody which binds to human
chorionic gonadotropin comprising an amino acid sequence in its
human heavy chain variable region as set forth in SEQ ID NO:4 or an
amino acid sequence which is at least 90% homologous to SEQ ID
NO:4.
27. A pharmaceutical composition comprising the antibody of claim 1
and a pharmaceutically acceptable carrier.
28. A pharmaceutical composition comprising the antibody of claim 2
and a pharmaceutically acceptable carrier.
29. A pharmaceutical composition comprising the antibody of claim
25 and a pharmaceutically acceptable carrier.
30. A pharmaceutical composition comprising the antibody of claim
26 and a pharmaceutically acceptable carrier.
31. An isolated recombinant anti-human chorionic gonadotropin
antibody or antigen-binding fragment thereof, said antibody
comprising a human constant region wherein said antibody or antigen
binding fragment (i) competitively inhibits binding of 2B2.6F5
antibody (ATCC Patent Deposit Designation No. PTA-7777) to human
chorionic gonadotropin, and (ii) binds to a neutralizing epitope of
human chorionic gonadotropin in vivo with an affinity of at least
1.times.10.sup.8 liter/mole, measured as an associate constant (Ka)
as determined by surface plasmon resonance.
32. The antibody or antigen-binding fragment of claim 31, wherein
the antibody or antigen-binding fragment comprises a human constant
region and a human variable region.
33. The antibody or antigen-binding fragment of claim 31, which
comprises at least one human light chain and at least one human
heavy chain.
34. The antibody or antigen-binding fragment of claim 33, wherein
the light chain comprises all antigen-binding regions of the light
chain of 2B2.6F5 antibody (ATCC Patent Deposit Designation No.
PTA-7777).
35. The antibody or antigen-binding fragment of claim 33, wherein
the heavy chain comprises all antigen-binding regions of the heavy
chain of 2B2.6F5 antibody (ATCC Patent Deposit Designation No.
PTA-7777).
36. The antibody or antigen-binding fragment of claim 33, wherein
the light chain comprises all antigen-binding regions of the light
chain of 2B2.6F5 antibody (ATCC Patent Deposit Designation No.
PTA-7777) and wherein the heavy chain comprises all antigen-binding
regions of the heavy chain of 2B2.6F5 antibody (ATCC Patent Deposit
Designation No. PTA-7777).
37. An isolated recombinant anti-human chorionic gonadotropin
antibody or antigen-binding fragment thereof, said antibody
comprising a human constant region wherein said antibody or antigen
binding fragment (i) competitively inhibits binding of 2B3.3E8
antibody (ATCC Patent Deposit Designation No. PTA-7775) to human
chorionic gonadotropin, and (ii) binds to a neutralizing epitope of
human chorionic gonadotropin in vivo with an affinity of at least
1.times.10.sup.8 liter/mole, measured as an associate constant (Ka)
as determined by surface plasmon resonance.
38. The antibody or antigen-binding fragment of claim 37, wherein
the antibody or antigen binding fragment comprises a human constant
region and a human variable region.
39. The antibody or antigen-binding fragment of claim 37, which
comprises at least one human light chain and at least one human
heavy chain.
40. The antibody or antigen-binding fragment of claim 39, wherein
the light chain comprises all antigen-binding regions of the light
chain of 2B3.3E8 antibody (ATCC Patent Deposit Designation No.
PTA-7775).
41. The antibody or antigen-binding fragment of claim 39, wherein
the heavy chain comprises all antigen-binding regions of the heavy
chain of 2B3.3E8 antibody (ATCC Patent Deposit Designation No.
PTA-7775).
42. The antibody or antigen-binding fragment of claim 39, wherein
the light chain comprises all antigen-binding regions of the light
chain of 2B3.3E8 antibody (ATCC Patent Deposit Designation No.
PTA-7775) and wherein the heavy chain comprises all antigen-binding
regions of the heavy chain of 2B3.3E8 antibody (ATCC Patent Deposit
Designation No. PTA-7775).
43. A method of detecting human chorionic gonadotropin in a sample,
the method comprising: (a) providing a sample; (b) contacting the
sample of (a) with a human monoclonal antibody 2B2.6F5 (ATCC Patent
Deposit Designation No. PTA-7777) or a human monoclonal antibody
2B3.3E8 (ATCC Patent Deposit Designation No. PTA-7775), which
specifically binds a polypeptide comprising human chorionic
gonadotropin under conditions which permit binding of the
polypeptide ligand to human chorionic gonadotropin; and (c)
detecting binding of the antibody 2B2.6F5 or antibody 2B3.3E8 with
human chorionic gonadotropin in the sample, wherein detection of
binding indicates the presence of human chorionic gonadotropin in
the sample; thereby detecting human chorionic gonadotropin in the
sample.
44. An isolated nucleic acid encoding the heavy chain
immunoglobulin variable domain sequence of the antibody of
claim.
45. A pharmaceutical composition comprising the nucleic acid of
claim 44 and a pharmaceutically acceptable carrier.
46. A recombinant cell that contains one or more nucleic acids that
encode the immunoglobulin variable domain sequences of the antibody
of claim 1.
47. A host cell that contains a first nucleic acid sequence
encoding a polypeptide comprising a HC variable domain of an
antibody and a second nucleic acid sequence encoding a polypeptide
comprising a LC variable domain of the antibody, wherein the
antibody is a protein according to claim 1.
48. A isolated human monoclonal antibody which specifically binds
to amino acids 38-57 of the .beta.-L2 loop of human chorionic
gonadotropin (SEQ ID NO:7) or an analog thereof.
49. A method for treating a neoplastic disease in a mammalian
subject comprising administering to the mammal subject a
pharmaceutical composition comprising an antibody which
specifically binds to .beta.-L2 loop of human chorionic
gonadotropin (SEQ ID NO:7), or an analog of the .beta.-L2 loop, in
an amount effective to reduce or eliminate the neoplastic disease
in the mammalian subject.
50. The method of claim 49 wherein the antibody comprises an amino
acid sequence of SEQ ID NO:2 or SEQ ID NO:4.
51. The method of claim 50 wherein the antibody is a human
monoclonal antibody 2B2.6F5 (ATCC Patent Deposit Designation No.
PTA-7777).
52. The method of claim 50 wherein the antibody is a human
monoclonal antibody 2B3.3E8 (ATCC Patent Deposit Designation No.
PTA-7775).
53. The method of claim 49 wherein the antibody blocks binding of
hCG to LH/hCG receptor.
54. The method of claim 49 wherein the antibody is linked to a
cytotoxic agent.
55. The method of claim 54, wherein said cytotoxic agent is a
cytotoxic drug.
56. The method of claim 54, wherein said cytotoxic agent is a
radioactive isotope.
57. The method of claim 54, further comprising administering a
pharmaceutical composition comprising a chemotherapeutic agent to
the mammalian subject.
58. The method of claim 49, wherein the neoplastic disease is solid
tumor, lung carcinoma, breast carcinoma, colorectal carcinoma,
prostate carcinoma, gastric carcinoma, pancreatic carcinoma, head
and neck carcinoma, renal cell carcinoma, ovarian carcinoma,
bladder carcinoma, melanoma, uterine cancer, uterine leiomyomas,
endometrial cancer, polycystic ovary syndrome, endometrial polyps,
pituitary cancer, adenomyosis, adenocarcinomas, meningioma, bone
cancer, hematological malignancy, leukemia, multiple myeloma,
glioma, glioblastoma or astrocytoma.
59. The method of claim 58, wherein the neoplastic disease is tumor
cell metastasis in said mammalian subject.
60. A method for diagnosing cancer in a mammalian subject suspected
of having neoplastic-disease or suspected of being at risk for
neoplastic disease comprising: obtaining a test sample from blood
or tissue of the subject, the test sample comprising a cell
population, providing a human monoclonal antibody 2B2.6F5 (ATCC
Patent Deposit Designation No. PTA-7777) or a human monoclonal
antibody 2B3.3E8 (ATCC Patent Deposit Designation No. PTA-7775) to
detect the presence or absence of an human chorionic gonadotropin
marker on the cells within the cell population, analyzing the cell
population detected by the human chorionic gonadotropin marker to
identify and characterize the cells, the presence of human
chorionic gonadotropin marker on or in the cells indicative of
neoplastic disease or risk of neoplastic disease in the mammalian
subject.
61. The method of claim 60 wherein the antibody comprises an amino
acid sequence of SEQ ID NO:2 or SEQ ID NO:4.
62. The method of claim 60 wherein the presence of human chorionic
gonadotropin marker on or in the cells in the specimen indicates
the presence of metastatic cancer in the mammalian subject.
63. The method of claim 60 wherein the presence of human chorionic
gonadotropin marker on or in the cells in the specimen indicates
the presence of early stage cancer in the mammalian subject.
64. The method of claim 60 wherein absence of human chorionic
gonadotropin marker on or in the cells in the specimen indicates
presence of a disease free state or a non-measurable disease state
in the mammalian subject.
65. The method of claim 60 wherein the presence or absence of human
chorionic gonadotropin marker on or in the cells in the specimen
monitors therapy management during cancer therapy or cancer
recovery.
66. The method of claim 60 further comprising an imaging moiety
associated with the antibody.
67. The method of claim 66, wherein the imaging moiety can be
imaged through magnetic resonance spectroscopy, X-ray spectroscopy,
or positron emission tomography (PET).
68. The method of claim 66, wherein the association is a covalent
bond.
69. The method of claim 66, wherein the association is a
non-covalent bond.
70. The method of claim 60, wherein the neoplastic disease is solid
tumor, lung carcinoma, breast carcinoma, colorectal carcinoma,
prostate carcinoma, gastric carcinoma, pancreatic carcinoma, head
and neck carcinoma, renal cell carcinoma, ovarian carcinoma,
bladder carcinoma, melanoma, uterine cancer, uterine leiomyomas,
endometrial cancer, polycystic ovary syndrome, endometrial polyps,
pituitary cancer, adenomyosis, adenocarcinomas, meningioma, bone
cancer, hematological malignancy, leukemia, multiple myeloma,
glioma, glioblastoma or astrocytoma.
71. A method for treating a disease caused by hormonal imbalance in
a mammalian subject comprising administering to the mammal subject
an antibody which comprises an amino acid sequence of SEQ ID NO: 2
or SEQ ID NO: 4 in a pharmaceutically acceptable carrier, wherein
said antibody specifically binds to human chorionic gonadotropin in
an amount effective to reduce or eliminate the hormonal imbalance
disease in the mammalian subject.
72. The method of claim 71 wherein the antibody is a human
monoclonal antibody 2B2.6F5 (ATCC Patent Deposit Designation No.
PTA-7777) or a human monoclonal antibody 283.3E8 (ATCC Patent
Deposit Designation No. PTA-7775).
73. The method of claim 71, wherein the disease is prostate cancer,
polycystic ovary disease, rheumatic disease, septic shock,
endometriosis, leiomyomatosis, ovarian degeneration during
cytotoxic chemotherapy, or Alzheimer's disease.
74. A method for inducing abortion in a mammalian subject
comprising administering to the mammal subject an antibody
comprising an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 in
a pharmaceutically acceptable carrier, wherein said antibody
specifically binds to human chorionic gonadotropin in an amount
effective to inducing abortion in the mammalian subject.
75. The method of claim 74 wherein the antibody is a human
monoclonal antibody 2B2.6F5 (ATCC Patent Deposit Designation No.
PTA-7777) or a human monoclonal antibody 2B3.3E8 (ATCC Patent
Deposit Designation No. PTA-7775).
76. A method for reducing fertility in a mammalian subject
comprising administering to the mammal subject an amino acid
sequence of SEQ ID NO: 2 or SEQ ID NO: 4 in a pharmaceutically
acceptable carrier, wherein said antibody specifically binds to
human chorionic gonadotropin in an amount effective to reduce
fertility in the mammalian subject.
77. The method of claim 76 wherein the antibody comprises a human
monoclonal antibody 2B2.6F5 (ATCC Patent Deposit Designation No.
PTA-7777) or a human monoclonal antibody 2B3.3E8 (ATCC Patent
Deposit Designation No. PTA-7775).
78. A method of screening a drug candidate compound for treatment
of cancer in a mammalian subject comprising, administering a
therapeutically effective amount of the drug candidate compound to
the subject suspected of having cancer, obtaining test samples from
blood or tissue of the subject before and after, treatment with the
drug candidate compound, the test samples comprising a cell
population suspected of containing tumor cells, providing a human
monoclonal antibody 2B2.6F5 (ATCC Patent Deposit Designation No.
PTA-7777) or a human monoclonal antibody 2B3.3E8 (ATCC Patent
Deposit Designation No. PTA-7775), to detect the presence or
absence of an human chorionic gonadotropin marker on the cells in
the test sample, analyzing the cell population detected by the
human chorionic gonadotropin marker to identify the tumor cells in
the test samples before treatment with the drug candidate compound
compared to after treatment with the drug candidate compound,
wherein the presence of a decreased number of the tumor cells in
the specimen after treatment compared to a number of the tumor
cells in a specimen before treatment indicating effectiveness of
the drug candidate compound in treating the cancer in the mammalian
subject.
79. The method of claim 78 wherein the antibody comprises an amino
acid sequence of SEQ ID NO:2 or SEQ ID NO:4.
80. The method of claim 78 wherein the cancer is metastatic cancer
or early stage cancer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is the National Phase of International
Application No. PCT/US2006/0030988, filed Aug. 8, 2006, which
claims the benefit of U.S. Provisional Application No. 60/706,506,
filed Aug. 8, 2005, the disclosures of which are incorporated
herein by reference in their entirety.
FIELD
[0002] The invention generally relates to antibody compositions and
methods for inhibition of the effects of gonadotropin hormones,
including methods for treating cancer and methods for regulating
fertility by administering the antibody compositions to a mammalian
subject in need thereof.
BACKGROUND
[0003] Research in human chorionic gonadotropin (hCG) relates to
three elements. A) structure of hCG protein chains and
carbohydrates; B) biology of hCG in fertility and cancer; and C)
vaccination strategies for immune targeting of hCG, including
against hCG peptides, beta chain, or carbohydrates to generate
either humoral or T cell-mediated immune responses.
[0004] Human chorionic gonadotropin is a 38 kD heterodimeric
glycoprotein. Morgan et al., J. Biol. Chem. 250: 5247, 1975; Hearn
and Gomme, J. Mol. Recognit. 13: 223, 2000. Key features of hCG's
structure can be seen on diagrams of the primary structure of hCG's
alpha and beta chains, as depicted in FIG. 1 A. Birken, et al.,
Clin Chem 49: 144, 2003. The alpha subunit of hCG (alpha-hCG, or
hCGa in FIG. 1A) is common to glycoprotein hormones including
follicle stimulating hormone (FSH), luteinizing hormone (LH), and
thyroid stimulating hormone (TSH). Alpha-hCG's protein chain
contains 92 amino acids and carries two N-linked oligosaccarides at
residues 52 and 78. hCG's hormone-specific beta chain (beta-hCG, or
hCGB in FIG. 1 A) contains 145 amino acids. Relative to the highly
homologous LH beta chain and unique among the glycoprotein
hormones, beta-hCG has an additional 31 amino acids at the carboxyl
terminus. 4 Morgan et al., J. Biol. Chem. 250: 5247, 1975. This
carboxyl terminal peptide (CTP) is both kinky and hydrophilic with
nine proline (29 mole %) and eight serine (26 mole %) residues.
Beta-hCG carries two N-linked oligosaccarides at residues 13 and
30, as well as four O-linked oligosaccharides at residues 121, 127,
132, and 138.
[0005] Tertiary structure of the hCG heterodimer is notable for
membership in the cystine knot growth factor family (CKGF) of
cytokines. Lapthom et al., Nature 369: 455, 1994; Wu et al.,
Structure 2: 545, 1994. The CKGF family includes glycoprotein
hormones, nerve growth factor (NGF), platelet derived growth factor
(PDGF), and transforming growth factor-beta (TGF-beta), among at
least forty other such proteins. Hearn and Gomme, J. Mol. Recognit.
13: 223, 2000. CKGF cytokines are characterized by strong,
specific, non-covalent dimerization of two subunits. Each subunit
features a remarkable, conserved configuration (knot) of three
cystine disulfide bonds in which two disulfides form a ring through
which the third disulfide bond passes. Secondary structure is
primarily of beta strands. Tertiary structure of each subunit is
highly elongated with a high surface:volume ratio and absence of
any defined hydrophobic core region. Quaternary structure of hCG
comprises head to tail association of subunits along their long
axes, involving approximately 25% of their surface area.
Dimerization is stabilized by a 21 amino acid loop that extends
from the cystine knot of beta-hCG and loops around alpha-hCG,
forming a disulfide bonded "seat belt". Beta-hCG may be
proteolytically nicked between residues 44 and 45 or 47 and 48 (hCG
.beta.n in FIG. 1 A). Nicking leads to deactivation of hCG and
hastens dissociation of subunits. Cole et al., J. Clin. Edocrinol.
Metab. 76: 704, 1993. A urinary metabolite, the core fragment of
beta-hCG (hCG .beta.cf in FIG. 1A), has no known function. Norman
et al., J. Endocrinol. 164: 299, 2000; Birken et al., Arch. Med.
Res. 32: 635, 2001. In addition to the alpha-beta heterodimer,
beta-hCG has been found in both monomeric and homodimeric forms.
Butler et al., J. Mol. Endocrinol. 22: 185, 1999.
[0006] Eight oligosaccharides comprise about 30% of hCG's molecular
weight. This is more carbohydrate than found on the closest
homolog; LH carries only three N-linked oligosaccharides, two on
the alpha and one on the beta chain. Each oligosaccharide carries
up to two negatively charged terminal sugars. Thus hCG carries a
noteworthy net negative charge. Oligosaccharides associated with
hCG are highly heterogeneous, accounting for a substantial
proportion of the hormone's size and charge heterogeneity.
[0007] Alpha-beta heterodimeric hCG binds to and activates the
LH/hCG receptor. Ascoli et al., Endocr. Rev. 23: 141, 2002. By
contrast, none of alpha-hCG, beta-hCG, nicked beta-hCG, or beta-hCG
core fragment bind to a recombinant human LH-hCG receptor. Ho et
al., Early Pregnancy 3: 204, 1997. The carboxyl terminal peptide
does not appear to be of any importance to receptor binding or
signaling since antibodies specific for this region of beta-hCG do
not interfere with LH/hCG receptor signaling. Iverson et al., Curr.
Opin. Mol. Ther. 5: 156, 2003; Dirnhofer et al., FAEB J. 7: 1381,
1993. Chemically deglycosylated hCG binds to but does not activate
the rat LH/hCG receptor. Chen et al., J. Biol. Chem. 257: 14446,
1982. Individual N-linked carbohydrate moieties likely do not
affect hCG function. Hearn and Gomme, J. Mol. Recognit. 13: 223,
2000.
[0008] Human chorionic gonadotropin has a demonstrated role in
reproduction. Ascheim and Zondek, Klin. Wochenschr 248, 1927. hCG
is obligately required for reproduction and appears to have myriad
roles in pregnancy given the expression in many tissues of LH/hCG
receptors. Rao, Semin. Reprod. Med. 19: 7, 2001; Filicori et al.,
Fertil. Steril 84: 275, 2005. Some of these proposed
receptor-mediated roles include facilitation of cytotrophoblast
invasion, angiogenesis, and immunosuppression (Islami et al.,
Semin. Reprod. Med. 19: 49, 2001; Licht et al., Semin. Reprod. Med.
19: 37, 2001), as well as inhibition of apoptosis. Kuroda et al.,
Int. J. Cancer 91: 309, 2001. In addition, the net negative charge
conferred by extensive sialylation of hCG on the
syncitiotrophoblast surface could by itself also be
immunosuppressive. Van et al., Int. J. Cancer 38: 915, 1986.
[0009] In non-pregnant states, serum hCG may be present at low
concentrations via pulsatile secretion of scant quantities from the
anterior pituitary. Birken et al., Endocrinology 137: 1402, 1996.
Yet hCG is also produced by cancer cells of many non-reproductive
tissues. Cosgrove et al., Biochim. Biophys. Acta. 1007: 44, 1989;
Stenman et al., Clin. Biochem. 37: 549, 2004. Given the parallels
between human reproduction and malignant transformation, hCG has
thus been proposed to be a marker of malignant transformation.
Acevedo, J. Exp. Ther. Oncol. 2: 133, 2002; Murray and Lessey,
Semin. Reprod. Endocrinol. 17: 275, 1999. Consistent with this,
alpha-beta heterodimeric hCG has been shown to block
cisplatin-induced apoptosis in ovarian carcinoma cells that express
the LH/hCG receptor. Kuroda et al., Int. J. Cancer 76: 571, 1998.
However, hCG is neither sensitive nor specific for malignancy.
[0010] Two surprising observations have been made concerning hCG's
putative role in cancer. First, membrane-bound hCG was found on the
surface of many different types of cultured cancer cells. Acevedo
et al., Cancer 69: 1829, 1992. This was noteworthy because hCG is a
secreted protein with no transmembrane domain. Second, serum
beta-hCG was noted to be associated with more aggressive,
metastatic presentations of bladder cancer. Iles et al., Br. J.
Urol. 64: 241, 1989. Also metastatic phenotype was found to
correlate with expression of beta-hCG in an animal model. Acevedo
and Hartsock, Cancer 78: 2388, 1996. These findings were of
uncertain significance because beta-hCG does not bind to the LH/hCG
receptor. Subsequently, hCG-beta has been found by multivariate
analysis to be an independent negative prognostic indicator in six
different epithelial cancers including colorectal, gastric, oral,
pancreatic, ovarian, and renal cell. Louhimo et al., Int. J. Cancer
101: 545, 2002; Louhimo et al., Int. J. Cancer 111: 929, 2004;
Hedstrom et al., Int. J. Cancer 84: 525, 1999; Louhimo et al.,
Oncology 66: 126, 2004; Vartianinen et al., Int. J. Cancer 95: 313,
2001; Hotakainen et al., Br. J. Cancer 86: 185, 2002. Curiously,
while tissue levels of beta-hCG by immunohistochemistry were also
negatively prognostic in colorectal cancer, only serum beta-hCG was
significantly prognostic in renal cell carcinoma. Lundin et al.,
Int. J. Cancer 95: 18, 2001; Hotakainen et al., Int. J. Cancer 104:
631, 2003. Thus soluble beta-hCG can now be presumed to play an
important role in cancer progression.
[0011] Beta-hCG was found to inhibit apoptosis of bladder cancer
cells in vitro as either a monomer or homodimer. Butler et al., Br.
J. Cancer 82: 1553, 2000; Butler and Iles, Tumour Biol. 25: 18,
2004. The authors propose a mechanism in which beta-hCG blocks
apoptosis mediated by TGF-beta via binding to without activating
TGF-beta receptors. Similarly, one can imagine that beta-hCG could
inhibit activity of another CKGF cytokine, PDGF. Pietras et al.,
Cancer Res. 62: 5476, 2002. Engineered expression of beta-hCG in
prostate cancer cells has been shown to down-regulate E-cadherin
and upregulate invasiveness. Wu and Walker, Cancer 106: 68, 2006.
In the latter experiments, conditioned medium was found to confer
the same effect, indicating that soluble beta-hCG in the culture
supernatant produced this effect. Although the receptor in this
instance is unknown, there are at least 40 CKGF cytokine family
members (Hearn and Gomme, J. Mol. Recognit. 13: 223, 2000), so
involvement of additional, as yet unidentified receptor(s) is
likely. The finding that hCG has been shown to inhibit Kaposi's
sarcoma has not been thought to be mutually exclusive with hCG's
role in diverse epithelial cancers of broader public health
significance. Butler and Iles, Clin Cancer Res. 9: 4666, 2003.
[0012] Vaccination targeting hCG to regulate fertility has been
pursued for decades. Naz et al., Hum. Reprod. 20: 3271, 2005. At
the outset these vaccines sought to generate active specific
humoral immunity either to the CTP of beta-hCG or to full-length
beta-hCG. Lee et al., Mol. Immunol. 17: 749, 1980; Talwar et al.,
Proc. Natl. Acad. Sci. U.S.A. 91: 8532, 1994. Efficacy of such
vaccines has in principle been demonstrated. Stevens et al.,
Fertil. Steril. 36: 98, 1981; Talwar et al., Proc. Natl. Acad. Sci.
U.S.A. 91: 8532, 1994. Embellishments of these approaches have
employed different beta-hCG components or recombinant antigen
expression as fusion proteins. Rock et al., Vaccine 14: 1560, 1996;
Rout and Vrati, Vaccine 15: 1503, 1997; Xu et al., Sheng Wu Gong.
Cheng. Xue. Bao. 20: 49, 2004; Yankai et al., Biochem. Biophys.
Res. Commun. 345: 1365, 2006; Geissler et al., Lab Invest. 76: 859,
1997.
[0013] Similar approaches have been pursued for cancer treatment.
Triozzi and Stevens, Oncol. Res. 6: 7, 1999; Moulton et al., Clin.
Cancer Res. 8: 2044, 2002. Additional refinements have included
passively administered antibodies (Butler et al., Oncol. Res. 14:
93, 2003), monoclonal antibodies specific for the CTP (Kalantarov
and Acevedo, Cancer 83: 783, 1998), genetic immunization (Geissler
et al., Lab Invest. 76: 859, 1997), and a targeted fusion protein
to generate active T cell mediated immunity against beta-hCG. He et
al., Clin. Cancer Res. 10: 1920, 2004. A further alternative seeks
to target via a monoclonal antibody the O-linked core 2
sugar-containing oligosaccharide isoforms displayed on the CTP of
hyperglycosylated hCG (H-hCG). Birken et al., Arch. Med. Res. 32:
635, 2001; Birken et al., Endocrine 10: 137, 1999; Birken, Tumour
Biol. 26: 131, 2005; Cole et al., Gynecol. Oncol. 102: 145, 2006;
U.S. Pat. No. 6,764,680; U.S. Patent Application No.
2005/0260196.
[0014] Most of the above approaches employ an active immunization
strategy. Thus months are required for either antibody or T cell
mediated immunity to develop, and some recipients will fail to
generate an adequate immune response. None of the above approaches
to immune targeting of beta-hCG or H-hCG seeks explicitly to block
binding of beta-hCG to the LH/hCG or any other receptor. In
particular, formulations targeting either the CTP or H-hCG don't
block receptor binding. Although experimental methods have been
developed to allow targeting of cancer cells that bear
surface-bound beta-hCG, the weight of data on beta-hCG's prognostic
significance argues that blockade of serum beta-hCG binding to
receptors mediating deleterious effects will be critically
important in treatment of cancer by immune targeting of hCG.
Furthermore, none of the above approaches have been shown to
synergize with cancer chemotherapy.
[0015] Thus a need exists in the art to generate more effective
treatment of cancers that secrete beta-hCG. To address this
problem, means are needed to target beta-hCG in a manner that
fulfills the following two criteria. First, the agent generated
should be able to target serum beta-hCG quickly following passive
administration. In practice this could be accomplished by use of
monoclonal antibodies or similar mediators of immune specificity.
Second, the method must generate treatment that blocks binding of
beta-hCG to its receptor(s) mediating deleterious effects
associated with cancer progression. In practice this implies
generation of immune specificity for an epitope that is both
conformationally defined and surface-accessible.
SUMMARY
[0016] The present invention generally relates to antibody
compositions and methods for inhibition of the effects of
gonadotropin hormones, including methods for treating cancer and
methods for regulating fertility by administering the antibody
compositions to a mammalian subject in need thereof. The invention
further relates to inhibition of binding of the human chorionic
gonadotropin beta chain to any of its cognate receptors and to
consequential inhibitory effects on growth of human cancers.
[0017] A method for treating a neoplastic disease in a mammalian
subject is provided which comprises administering to the mammal
subject a pharmaceutical composition comprising an antibody which
specifically binds to .beta.-L2 loop of human chorionic
gonadotropin (hCG) in an amount effective to reduce or eliminate
the neoplastic disease in the mammalian subject. In the method, the
antibody can comprise an amino acid sequence of SEQ ID NO:2 or SEQ
ID NO:4. The antibody can be a human monoclonal antibody 2B2.6F5
(ATCC Patent Deposit Designation No. PTA-7777) or a human
monoclonal antibody 2B3.3E8 (ATCC Patent Deposit Designation No.
PTA-7775). Antibody compositions are provided which comprise an
amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4. Antibody
compositions are provided which comprise a human monoclonal
antibody 2B2.6F5 (ATCC Patent Deposit Designation No. PTA-7777) or
a human monoclonal antibody 2B3.3E8 (ATCC Patent Deposit
Designation No. PTA-7775). In a further aspect, the antibody is
linked to a cytotoxic agent, e.g., a cytotoxic drug or a
radioactive isotope. In a further aspect, the method for treating
neoplastic disease further comprises administering a pharmaceutical
composition comprising a chemotherapeutic agent in combination with
the monoclonal antibody to the mammalian subject.
[0018] A method for inducing abortion in a mammalian subject is
provided which comprises administering to the mammal subject a
pharmaceutical composition comprising a human monoclonal antibody
2B2.6F5 (ATCC Patent Deposit Designation No. PTA-7777) or a human
monoclonal antibody 2B3.3E8 (ATCC Patent Deposit Designation No.
PTA-7775), which specifically binds to human chorionic gonadotropin
in an amount effective to inducing abortion in the mammalian
subject. A method for reducing fertility in a mammalian subject is
provided which comprises administering to the mammal subject a
pharmaceutical composition comprising a human monoclonal antibody
2B2.6F5 (ATCC Patent Deposit Designation No. PTA-7777) or a human
monoclonal antibody 2B3.3E8 (ATCC Patent Deposit Designation No.
PTA-7775), which specifically binds to human chorionic gonadotropin
in an amount effective to reduce fertility in the mammalian
subject. The antibody can further comprise an amino acid sequence
of SEQ ID NO:2 or SEQ ID NO:4.
[0019] An isolated monoclonal antibody is provided which binds to
human chorionic gonadotropin comprising an amino acid sequence in
its heavy chain variable region as set forth in SEQ ID NO:2 or an
amino acid sequence which is at least 90% homologous to SEQ ID
NO:2.
[0020] An isolated monoclonal antibody which binds to human
chorionic gonadotropin comprising an amino acid sequence in its
heavy chain variable region as set forth in SEQ ID NO:4 or an amino
acid sequence which is at least 90% homologous to SEQ ID NO:4.
[0021] An isolated monoclonal antibody is provided which binds to
human chorionic gonadotropin comprising an amino acid sequence in
its light chain variable region and a heavy chain variable region,
wherein the heavy chain variable region is set forth in SEQ ID NO:2
or SEQ ID NO:4 or an amino acid sequence which is at least 90%
homologous to in SEQ ID NO:2 or SEQ ID NO:4.
[0022] In one aspect, the isolated monoclonal antibody has one or
more of the following characteristics: (i) inhibits proliferation
in vitro of BXPC-3 pancreatic carcinoma cells; and (ii) does not
inhibit proliferation in vitro of MCF-7 breast carcinoma cells or
HeLa cells. In a further aspect, the antibody has a dissociation
equilibrium constant (K.sub.D) of approximately 10.sup.-8 M or
less, when determined by surface plasmon resonance (SPR) using
recombinant human chorionic gonadotropin as an analyte and the
antibody as a ligand the antibody is capable of binding human
chorionic gonadotropin with a binding affinity of about 10.sup.8
M.sup.-1 or greater. The isolated monoclonal antibody can be an
antibody fragment or a single chain antibody.
[0023] The isolated monoclonal antibody can be a binding-domain
immunoglobulin fusion protein comprising (i) a variable heavy chain
amino acid sequence as set forth in SEQ ID-NO:2 or a variable heavy
chain sequence which is at least 90% homologous to SEQ ID NO:2,
fused to a variable light chain amino acid sequence via a linker
peptide, that is fused to an immunoglobulin hinge region
polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region
fused to the hinge region, and (iii) an immunoglobulin heavy chain
CH3 constant region fused to the CH2 constant region. The isolated
monoclonal antibody can be a binding-domain immunoglobulin fusion
protein comprising (i) a variable heavy chain amino acid sequence
as set forth in SEQ ID NO:4 or a variable heavy chain sequence
which is at least 90% homologous to SEQ ID NO:4, fused to a
variable light chain amino acid sequence via a linker peptide, that
is fused to an immunoglobulin hinge region polypeptide, (ii) an
immunoglobulin heavy chain CH2 constant region fused to the hinge
region, and (iii) an immunoglobulin heavy chain CH3 constant region
fused to the CH2 constant region. The isolated monoclonal antibody
can bind to a predetermined antigen with an equilibrium association
constant (Ka) of at least 10.sup.10 M.sup.-1. The isolated
monoclonal antibody can be bind to a predetermined antigen with an
equilibrium association constant (Ka) of at least 10.sup.9
M.sup.-1. The antibody can bind to a predetermined antigen with an
equilibrium association constant (Ka) of at least 10.sup.8
M.sup.-1.
[0024] An isolated human monoclonal antibody is provided which
binds to human chorionic gonadotropin comprising an amino acid
sequence in its human heavy chain variable region as set forth in
SEQ ID NO:2 or an amino acid sequence which is at least 90%
homologous to SEQ ID NO:2. An isolated human monoclonal antibody is
provided which binds to human chorionic gonadotropin comprising an
amino acid sequence in its human heavy chain variable region as set
forth in SEQ ID NO:4 or an amino acid sequence which is at least
90% homologous to SEQ ID NO:4. A pharmaceutical composition is
provided which comprises the antibody and a pharmaceutically
acceptable carrier. An isolated recombinant anti-human chorionic
gonadotropin antibody or antigen-binding fragment thereof, said
antibody is provided which comprises a human constant region
wherein said antibody or antigen binding fragment (i) competitively
inhibits binding of 2B2.6F5 antibody (ATCC Patent Deposit
Designation No. PTA-7777) to human chorionic gonadotropin, and (ii)
binds to a neutralizing epitope of human chorionic gonadotropin in
vivo with an affinity of at least 1.times.10.sup.8 liter/mole,
measured as an associate constant (Ka) as determined by surface
plasmon resonance. An isolated recombinant anti-human chorionic
gonadotropin antibody or antigen-binding fragment thereof, said
antibody is provided which comprises a human constant region
wherein said antibody or antigen binding fragment (i) competitively
inhibits binding of 2B3.3E8 antibody (ATCC Patent Deposit
Designation No. PTA-7775) to human chorionic gonadotropin, and (ii)
binds to a neutralizing epitope of human chorionic gonadotropin in
vivo with an affinity of at least 1.times.10.sup.8 liter/mole,
measured as an associate constant (Ka) as determined by surface
plasmon resonance.
[0025] A method of detecting human chorionic gonadotropin in a
sample is provided, wherein the method comprises (a) providing a
sample; (b) contacting the sample of (a) with a human monoclonal
antibody 2B2.6F5 (ATCC Patent Deposit Designation No. PTA-7777) or
a human monoclonal antibody 2B3.3E8 (ATCC Patent Deposit
Designation No. PTA-7775), which specifically binds a polypeptide
comprising human chorionic gonadotropin under conditions which
permit binding of the polypeptide ligand to human chorionic
gonadotropin; and (c) detecting binding of the antibody 2B2.6F5 or
antibody 2B3.3E8 with human chorionic gonadotropin in the sample,
wherein detection of binding indicates the presence of human
chorionic gonadotropin in the sample; thereby detecting human
chorionic gonadotropin in the sample.
[0026] A isolated human monoclonal antibody is provided which
specifically binds to amino acids 38-57 of the .beta.-L2 loop of
human chorionic gonadotropin (SEQ ID NO:7) or an analog thereof. In
one aspect, the antibody blocks binding of hCG to LH/hCG
receptor.
[0027] A method for diagnosing cancer in a mammalian subject
suspected of having neoplastic disease or suspected of being at
risk for neoplastic disease is provided which comprises obtaining a
test sample from blood or tissue of the subject, the test sample
comprising a cell population providing a human monoclonal antibody
2B2.6F5 (ATCC Patent Deposit Designation No. PTA-7777) or a human
monoclonal antibody 2B3.3E8 (ATCC Patent Deposit Designation No.
PTA-7775) to detect the presence or absence of an human chorionic
gonadotropin marker on the cells within the cell population, and
analyzing the cell population detected by the human chorionic
gonadotropin marker to identify and characterize the cells, the
presence of human chorionic gonadotropin marker on or in the cells
indicative of neoplastic disease or risk of neoplastic disease in
the mammalian subject.
[0028] A method of screening a drug candidate compound for
treatment of cancer in a mammalian subject is provided which
comprises administering a therapeutically effective amount of the
drug candidate compound to the subject suspected of having cancer,
obtaining test samples from blood or tissue of the subject before
and after treatment with the drug candidate compound, the test
samples comprising a cell population suspected of containing tumor
cells, providing a human monoclonal antibody 2B2.6F5 (ATCC Patent
Deposit Designation No. PTA-7777) or a human monoclonal antibody
2B3.3E8 (ATCC Patent Deposit Designation No. PTA-7775), to detect
the presence or absence of an human chorionic gonadotropin marker
on the cells in the test sample, analyzing the cell population
detected by the human chorionic gonadotropin marker to identify the
tumor cells in the test samples before treatment with the drug
candidate compound compared to after treatment with the drug
candidate compound, wherein the presence of a decreased number of
the tumor cells in the specimen after treatment compared to a
number of the tumor cells in a specimen before treatment indicating
effectiveness of the drug candidate compound in treating the cancer
in the mammalian subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1A shows diagrams of structures of hCG and
metabolites.
[0030] FIG. 1B shows amino acid sequence in vicinity of beta-hCG L2
long loop.
[0031] FIG. 2 shows a schematic depiction of gonadotropin
heterodimer and hCG beta chain binding to respective receptors.
[0032] FIG. 3 shows a schematic depiction of generation of immune
specificity for gonadotropin L2 beta long loop.
[0033] FIG. 4 shows a schematic depiction of blockade of receptor
binding by monoclonal antibody specific for gonadotropin L2 beta
loop.
[0034] FIG. 5 shows a schematic depiction of immune effector
function targeted by antibody binding fragment that confers
specificity on gonadotropin beta chain.
[0035] FIG. 6 shows relative affinity of monoclonal antibodies for
hCG versus LH.
[0036] FIG. 7 shows monoclonal antibody blocking of hCG binding to
LH/hCG receptor.
[0037] FIG. 8 shows an effect of antibody on the growth of cancer
cells in vitro.
[0038] FIG. 9 shows synergy with chemotherapy of monoclonal
antibody to inhibit growth of cancer cells in vitro.
[0039] FIG. 10 shows an effect of monoclonal antibody on tumor
growth in vivo.
[0040] FIG. 11 shows DNA and amino acid sequences of mAb 2B2.6F5
heavy chain DNA and amino acid sequence, mAb 2B3.3E8 heavy chain
DNA and amino acid sequence, and mAb 2B3.3F5 heavy chain DNA and
amino acid sequence.
DETAILED DESCRIPTION
[0041] The invention relates to the field of vaccines including
antibodies that target human chorionic gonadotropin (hCG) or that
target a substituent component of hCG. The invention relates to
antibody compositions and methods for inhibition of the effects of
gonadotropin hormones, including methods for treating cancer and
methods for regulating fertility by administering the antibody
compositions to a mammalian subject in need thereof. The invention
further relates to inhibition of binding of the human chorionic
gonadotropin beta chain to any of its cognate receptors and to
consequential inhibitory effects on growth of human cancers. The
binding inhibition can be demonstrated with antibody compositions
of the present invention.
[0042] Antibody compositions of the invention are targeted to
beta-hCG to provide more effective methods for treatment of cancers
and methods for regulating fertility wherein the cells secrete
beta-hCG. The antibodies can target beta-hCG in a manner that
fulfills the following two criteria. First, the antibody
compositions should be able to target serum beta-hCG quickly
following passive administration. In practice this can be
accomplished by use of monoclonal antibodies or similar mediators
of immune specificity. Second, the method must generate a
therapeutic treatment that blocks binding of beta-hCG to its
receptor(s) mediating deleterious effects associated with cancer
progression or methods for regulating fertility. In practice this
implies generation of immune specificity for an epitope that is
both conformationally defined and surface-accessible.
[0043] Such a method might be accomplished by generation of immune
specificity for the beta-hCG L2 long loop, which includes
surface-accessible amino acids at positions 48 through 53. A cyclic
peptide of beta-hCG positions 38-57 inhibited binding of hCG to rat
ovarian membrane receptor and testosterone production by Leydig
cells. Keutmann, et al., Proc.Natl Acad.Sci. U.S.A. 84: 2038-2042,
1987. However, confirmation of binding inhibition could not be
replicated in two separate systems. Salesse, et al., Mol. Cell.
Endocrinol. 68: 113-119, 1990; Jagtap, et al., J Endocrinol. 172:
311-320, 2002. Polyclonal antisera raised against the
conformational epitope produced by this cyclic peptide were able to
neutralize the hormone in an in vivo assay, suggesting that the
epitope is in the vicinity of the receptor binding site. On the
other hand, this finding could also have followed from more rapid
clearance of the bound hCG. Finally, the L2 long loop is known to
be cleaved between residues 44 and 45, as well as residues 47 and
48. Such cleavage alters antibody binding and might interfere with
the approach proposed. In summary, blocking receptor binding by
.beta.-hCG would be useful for cancer treatment and fertility
regulation. The present invention provides antibody compositions
having immune specificity for the .beta.-hCG L2 long loop that have
been demonstrated to block LH/hCG receptor binding by
.beta.-hCG.
[0044] FIG. 1B shows amino acid sequences from the beta chains of
hCG, LH, and FSH that correspond to the L2 beta long loop of the
HCG and HCG beta chain protein structure. The L2 beta long loop
describes a region of the beta-hCG protein chain's known
three-dimensional structure that is composed of two strands of
amino acids that are joined by a hairpin turn. "L2" indicates that
this is the second of three hairpin turns between strands in the
HCG beta protein sequence. Number 2 in the figure refers to amino
acid numbering of the beta-hCG protein chain from amino acid
positions 38 to 57 in the L2 beta long loop. Number 4 indicates
amino acids comprising an immunogen that enables selection of
monoclonal antibodies or related proteins that specifically target
the L2 beta long loop of a particular gonadotropin beta chain, for
example hCG. Number 6 indicates surface-accessible amino acids in
the L2 beta long loop of the HCG protein crystal structure. Number
8 indicates a non-conservative amino acid (51 A.fwdarw.P)
difference between HCG and LH that enables specific targeting of
HCG vs. LH.
[0045] FIG. 2 shows a schematic depiction of binding of
heterodimeric gonadotropin and HCG beta chain to their respective
receptors. Number 10 refers to a heterodimeric gonadotropin, such
as HCG, LH, or FSH. Number 12 refers to a gonadotropin receptor,
including LH-HCG, FSH, and TSH receptors. Number 20 refers to the
hCG beta chain in a form without an associated common alpha chain
glycoprotein hormone chain, including monomeric, homodimeric, or
other multimeric forms. Number 22 depicts receptor(s) to which HCG
beta binds, separate and distinct from the LH-HCG, FSH, and TSH
receptors. Number 30 refers to the L2 beta hairpin loop of
gonadotropin beta chain. This is the specific region of
gonadotropin protein structure that is targeted by the invention
described here.
[0046] FIG. 3 shows the method described herein to induce immune
specificity for the L2 beta long loop of gonadotropins. Number 40
represents the beta-hCG L2 long loop amino acid sequence (38-57) of
the gonadotropin beta chain. This sequence is bounded by two
cysteine moieties that can be induced to form an intramolecular
cystine disulfide bond, causing cyclization of the peptide.
Although a 38-57 cystine disulfide bond is not formed in the native
gonadotropin protein structure, formation of the intramolecular
disulfide bond in the 38-57 sequence induces a three-dimensional
conformation that approximates the native L2 beta long loop. Such
cyclized peptide(s) can be covalently linked to a carrier protein
such as diphtheria toxoid in order to boost the anti-peptide immune
response in animal(s) or in vivo experimental system(s) that are
used to generate monoclonal antibodies or related proteins
conferring immune specificity. Formation of the 38-57 disulfide
bond thus enables selection of monoclonal antibodies or related
proteins that are specific for the gonadotropin L2 beta long loop.
Number 42 represents a monoclonal antibody that confers immune
specificity for the L2 beta long loop of the HCG beta chain and is
generated in response to Number 40. Number 44 represents any
covalently linked small molecule or macromolecule that influences
effector function(s). Such effector functions modify the outcome of
the immune specificity (Part 42) that targets the gonadotropin L2
beta long loop.
[0047] FIG. 4 shows schematically the action of a monoclonal
antibody (42) with immune specificity for the L2 beta long loop
(30) in preventing binding to receptors (12, 22) by heterodimeric
gonadotropin(s) (10) or the HCG beta chain (20) and any consequent
intracellular signalling events. Thus this invention blocks
receptor binding of the beta-hCG protein regardless of whether it
is in the form of heterodimeric hCG, monomeric beta-hCG, or
homodimeric beta-hCG. Alternative embodiments may be based on a
fragment of such a monoclonal antibody (42) and/or a recombinant
formulation of such a protein that provides immune specificity for
the L2 beta long loop (30). Additional alternative embodiments may
be derived by engineering an immune effector function (44) that is
different from the constant domains of the original monoclonal
antibody.
[0048] FIG. 5 shows potential immune effector function(s) (44) that
are separate, distinct, and/or additive to those inherent to a
monoclonal antibody or fragment that provides specificity for the
gonadotropin L2 beta long loop.
[0049] Reference numerals in FIGS. 1-5 refer to: Amino acid
numbering of HCG beta chain (2); Immunogen to induce immune
specificity for L2 beta long loop (4); Surface-accessible amino
acids of HCG in L2 beta long loop (6); Basis of LH versus HCG beta
chain immune specificity (8); Heterodimeric gonadotropin (10);
Heterodimeric gonadotropin recepto (12); HCG beta chain (20);
Receptor(s) to which HCG beta chain binds (22); L2 beta hairpin
loop of gonadotropin beta chain (30); 38-57 loop peptide of HCG
beta chain (40); Protein conferring humoral immune specificity to
gonadotropin beta chain (42); Protein conferring immune effector
function (44).
[0050] The present invention provides an antibody composition that
binds to the L2 long loop of beta-hCG protein. The antibody
compositions can be used in methods for treating neoplastic
disease, in methods for inducing abortions in a mammalian subject,
and in methods for reducing fertility in a mammalian subject. The
antibody compositions can comprise an amino acid sequence of SEQ ID
NO:2 or SEQ ID NO:4. The antibody can further comprise a human
monoclonal antibody 2B2.6F5 (ATCC Patent Deposit Designation No.
PTA-7777) or a human monoclonal antibody 2B3.3E8 (ATCC Patent
Deposit Designation No. PTA-7775).
[0051] In a further aspect, methods for treating disease in a
mammalian subject are provided which comprise administering
monoclonal antibodies directed to follicle stimulating hormone
(FSH), leutinizing hormone (LH) or thyroid stimulating hormone
(TSH), wherein the disease is reduced or eliminated in the
mammalian subject. In a detailed aspect the monoclonal antibodies
are directed to the L2 long loop of the beta subunit polypeptide of
FSH, LH or TSH.
[0052] It is to be understood that this invention is not limited to
particular methods, reagents, compounds, compositions or biological
systems, which can, 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. As
used in this specification and the appended claims, the singular
forms "a", "an" and "the" include plural referents unless the
content clearly dictates otherwise. Thus, for example, reference to
"a cell" includes a combination of two or more cells, and the
like.
[0053] The term "about" as used herein when referring to a
measurable value such as an amount, a temporal duration, and the
like, is meant to encompass variations of .+-.20% or .+-.10%, more
preferably .+-.5%, even more preferably .+-.1%, and still more
preferably .+-.0.1% from the specified value, as such variations
are appropriate to perform the disclosed methods.
[0054] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention pertains. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice for testing of the present
invention, the preferred materials and methods are described
herein. In describing and claiming the present invention, the
following terminology will be used.
[0055] "Patient", "subject" or "mammal" are used interchangeably
and refer to mammals such as human patients and non-human primates,
as well as experimental animals such as rabbits, rats, and mice,
and other animals. Animals include all vertebrates, e.g., mammals
and non-mammals, such as sheep, dogs, cows, chickens, amphibians,
and reptiles.
[0056] "Treating" or "treatment" includes the administration of the
antibody compositions, compounds or agents of the present invention
to prevent or delay the onset of the symptoms, complications, or
biochemical indicia of a disease, alleviating the symptoms or
arresting or inhibiting further development of the disease,
condition, or disorder (e.g., cancer, metastatic cancer, metastatic
epithelial cancer, colorectal carcinoma, gastric carcinoma, oral
carcinoma, pancreatic carcinoma, ovarian carcinoma, or renal cell
carcinoma). Treatment can be prophylactic (to prevent or delay the
onset of the disease, or to prevent the manifestation of clinical
or subclinical symptoms thereof) or therapeutic suppression or
alleviation of symptoms after the manifestation of the disease.
[0057] "Cancer" or "malignancy" are used as synonymous terms and
refer to any of a number of diseases that are characterized by
uncontrolled, abnormal proliferation of cells, the ability of
affected cells to spread locally or through the bloodstream and
lymphatic system to other parts of the body (i.e., metastasize) as
well as any of a number of characteristic structural and/or
molecular features. A "cancerous" or "malignant cell" is understood
as a cell having specific structural properties, lacking
differentiation and being capable of invasion and metastasis.
Examples of cancers are, breast, lung, brain, bone, liver, kidney,
colon, and prostate cancer. (see DeVita et al., Eds., Cancer
Principles and Practice of Oncology, 6th. Ed., Lippincott Williams
& Wilkins, Philadelphia, Pa., 2001; this reference is herein
incorporated by reference in its entirety for all purposes).
[0058] Cancer-associated" refers to the relationship of a nucleic
acid and its expression, or lack thereof, or a protein and its
level or activity, or lack thereof, to the onset of malignancy in a
subject cell. For example, cancer can be associated with expression
of a particular gene that is not expressed, or is expressed at a
lower level, in a normal healthy cell. Conversely, a
cancer-associated gene can be one that is not expressed in a
malignant cell (or in a cell undergoing transformation), or is
expressed at a lower level in the malignant cell than it is
expressed in a normal healthy cell.
[0059] In the context of the cancer, the term "transformation"
refers to the change that a normal cell undergoes as it becomes
malignant. In eukaryotes, the term "transformation" can be used to
describe the conversion of normal cells to malignant cells in cell
culture.
[0060] "Proliferating cells" are those which are actively
undergoing cell division and growing exponentially. "Loss of cell
proliferation control" refers to the property of cells that have
lost the cell cycle controls that normally ensure appropriate
restriction of cell division. Cells that have lost such controls
proliferate at a faster than normal rate, without stimulatory
signals, and do not respond to inhibitory signals.
[0061] "Advanced cancer" means cancer that is no longer localized
to the primary tumor site, or a cancer that is Stage III or IV
according to the American Joint Committee on Cancer (AJCC).
[0062] "Well tolerated" refers to the absence of adverse changes in
health status that occur as a result of the treatment and would
affect treatment decisions.
[0063] "Metastatic" refers to tumor cells, e.g., human epithelial
cancer, colorectal carcinoma, gastric carcinoma, oral carcinoma,
pancreatic carcinoma, ovarian carcinoma, or renal cell carcinoma,
that are able to establish secondary tumor lesions in the
alimentary tract, kidney, pancreae, ovaries, lungs, liver, bone or
brain of immune deficient mice upon injection into the mammary fat
pad and/or the circulation of the immune deficient mouse.
[0064] "Non-metastatic" refers to tumor cells, e.g., human
epithelial cancer cells, that are unable to establish secondary
tumor lesions in the lungs, liver, bone or brain or other target
organs of epithelial cell metastasis, e.g., colorectal carcinoma,
gastric carcinoma, oral carcinoma, pancreatic carcinoma, ovarian
carcinoma, or renal cell carcinoma in immune deficient mice upon
injection into the mammary fat pad and/or the circulation. The
human tumor cells used herein and addressed herein as
non-metastatic are able to establish primary tumors upon injection
into the mammary fat pad of the immune deficient mouse, but they
are unable to disseminate from those primary tumors.
[0065] "Lymphocyte" as used herein has the normal meaning in the
art, and refers to any of the mononuclear, nonphagocytic
leukocytes, found in the blood, lymph, and lymphoid tissues, e.g.,
B and T lymphocytes.
[0066] "Disease caused by hormonal imbalance" refers to diseases
caused by an imbalance of gonadotropin hormone, for example, human
chorionic gonadotropin, in the mammalian subject. Disease caused by
hormonal imbalance include, but are not limited to, prostate
cancer, polycystic ovary disease, rheumatic disease, septic shock,
endometriosis, leiomyomatosis, ovarian degeneration during
cytotoxic chemotherapy, or Alzheimer's disease.-"Disease caused by
hormonal imbalance" further refers to diseases caused by an
imbalance of gonadotropin hormone, for example, follicle
stimulating hormone (FSH), leutinizing hormone (LH) or thyroid
stimulating hormone (TSH), in the mammalian subject.
[0067] "Polypeptide fragment" as used herein refers to a
polypeptide that has an amino-terminal and/or carboxy-terminal
deletion, but where the remaining amino acid sequence is identical
to the corresponding positions in the naturally-occurring sequence
deduced, for example, from a full-length cDNA sequence. Fragments
typically are at least 5, 6, 8 or 10 amino acids long, preferably
at least 14 amino acids long, more preferably at least 20 amino
acids long, usually at least 50 amino acids long, and even more
preferably at least 70 amino acids long. The term "analog" as used
herein refers to polypeptides which are comprised of a segment of
at least 25 amino acids that has substantial identity to a portion
of a deduced amino acid sequence and which has at least one of the
following properties: (1) specific binding to human chorionic
gonadotropin .beta. chain (.beta.-hCG), under suitable binding
conditions, (2) ability to block .beta.-hCG binding to an
leutinizing hormone (LH)/hCG receptor, or (3) ability to
.beta.-hCG-expressing cell growth in vitro or in vivo. Typically,
polypeptide analogs comprise a conservative amino acid substitution
(or addition or deletion) with respect to the naturally-occurring
sequence. Analogs typically are at least 20 amino acids long,
preferably at least 50 amino acids long or longer, and can often be
as long as a full-length naturally-occurring polypeptide.
[0068] Peptide analogs are commonly used in the pharmaceutical
industry as non-peptide drugs with properties analogous to those of
the template peptide. These types of non-peptide compound are
termed "peptide mimetics" or "peptidomimetics". Fauchere, J. Adv.
Drug Res. 15: 29, 1986; Veber and Freidinger TINS p. 392 (1985);
and Evans et al. J. Med. Chem. 30: 1229, 1987, which are
incorporated herein by reference. Such compounds are often
developed with the aid of computerized molecular modeling. Peptide
mimetics that are structurally similar to therapeutically useful
peptides may be used to produce an equivalent therapeutic or
prophylactic effect. Generally, peptidomimetics are structurally
similar to a paradigm polypeptide (i.e., a polypeptide that has a
biochemical property or pharmacological activity), such as human
antibody, but have one or more peptide linkages optionally replaced
by a linkage selected from the group consisting of: --CH.sub.2NH--,
--CH.sub.2S--, --CH.sub.2--CH.sub.2--, --CH.dbd.CH--(cis and
trans), --COCH.sub.2--, --CH(OH)CH.sub.2--, and --CH.sub.2SO--, by
methods well known in the art. Systematic substitution of one or
more amino acids of a consensus sequence with D-amino acid of the
same type (e.g., D-lysine in place of L-lysine) may be used to
generate more stable peptides. In addition, constrained peptides
comprising a consensus sequence or a substantially identical
consensus sequence variation may be generated by methods known in
the art (Rizo and Gierasch Ann. Rev. Biochem. 61: 387, 1992,
incorporated herein by reference); for example, by adding internal
cysteine residues capable of forming intramolecular disulfide
bridges which cyclize the peptide.
[0069] As applied to polypeptides, the term "substantial identity"
means that two peptide sequences, when optimally aligned, such as
by the programs GAP or BESTFIT using default gap weights, share at
least 80 percent sequence identity, preferably at least 90 percent
sequence identity, more preferably at least 95 percent sequence
identity, and most preferably at least 99 percent sequence
identity. Preferably, residue positions which are not identical
differ by conservative amino acid substitutions. Conservative amino
acid substitutions refer to the interchangeability of residues
having similar side chains. For example, a group of amino acids
having aliphatic side chains is glycine, alanine, valine, leucine,
and isoleucine; a group of amino acids having aliphatic-hydroxyl
side chains is serine and threonine; a group of amino acids having
amide-containing side chains is asparagine and glutamine; a group
of amino acids having aromatic side chains is phenylalanine,
tyrosine, and tryptophan; a group of amino acids having basic side
chains is lysine, arginine, and histidine; and a group of amino
acids having sulfur-containing side chains is cysteine and
methionine. Preferred conservative amino acids substitution groups
are: valine-leucine-isoleucine, phenylalanine-tyrosine,
lysine-arginine, alanine-valine, glutamic-aspartic, and
asparagine-glutamine.
[0070] As discussed herein, minor variations in the amino acid
sequences of antibodies or immunoglobulin molecules are
contemplated as being encompassed by the present invention,
providing that the variations in the amino acid sequence maintain
at least 75%, more preferably at least 80%, 90%, 95%, and most
preferably 99% homology. In particular, conservative amino acid
replacements are contemplated. Conservative amino acid replacement
does not against the overall homology which can be maintained at
least 75%, more preferably at least 80%, 90%, 95%, and most
preferably 99% homology. Conservative replacements are those that
take place within a family of amino acids that are related in their
side chains. Genetically encoded amino acids are generally divided
into families: (1) acidic=aspartate, glutamate; (2) basic=lysine,
arginine, histidine; (3) non-polar=alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan; and (4)
uncharged polar=glycine, asparagine, glutamine, cysteine, serine,
threonine, tyrosine. More preferred families are: serine and
threonine are aliphatic-hydroxy family; asparagine and glutamine
are an amide-containing family; alanine, valine, leucine and
isoleucine are an aliphatic family; and phenylalanine, tryptophan,
and tyrosine are an aromatic family. For example, it is reasonable
to expect that an isolated replacement of a leucine with an
isoleucine or valine, an aspartate with a glutamate, a threonine
with a serine, or a similar replacement of an amino acid with a
structurally related amino acid will not have a major effect on the
binding or properties of the resulting molecule, especially if the
replacement does not involve an amino acid within a framework site.
Whether an amino acid change results in a functional peptide can
readily be determined by assaying the specific activity of the
polypeptide derivative. Assays are described in detail herein.
Fragments or analogs of antibodies or immunoglobulin molecules can
be readily prepared by those of ordinary skill in the art.
Preferred amino- and carboxy-termini of fragments or analogs occur
near boundaries of functional domains. Structural and functional
domains can be identified by comparison of the nucleotide and/or
amino acid sequence data to public or proprietary sequence
databases. Preferably, computerized comparison methods are used to
identify sequence motifs or predicted protein conformation domains
that occur in other proteins of known structure and/or function.
Methods to identify protein sequences that fold into a known
three-dimensional structure are known. Bowie et al. Science 253:
164, 1991. Thus, the foregoing examples demonstrate that those of
skill in the art can recognize sequence motifs and structural
conformations that may be used to define structural and functional
domains in accordance with the invention.
[0071] Preferred amino acid substitutions are those which: (1)
reduce susceptibility to proteolysis, (2) reduce susceptibility to
oxidation, (3) alter binding affinity for forming protein
complexes, (4) alter binding affinities, and (4) confer or modify
other physicochemical or functional properties of such analogs.
Analogs can include various muteins of a sequence other than the
naturally-occurring peptide sequence. For example, single or
multiple amino acid substitutions (preferably conservative amino
acid substitutions) may be made in the naturally-occurring sequence
(preferably in the portion of the polypeptide outside the domain(s)
forming intermolecular contacts. A conservative amino acid
substitution should not substantially change the structural
characteristics of the parent sequence (e.g., a replacement amino
acid should not tend to break a helix that occurs in the parent
sequence, or disrupt other types of secondary structure that
characterizes the parent sequence). Examples of art-recognized
polypeptide secondary and tertiary structures are described in
Proteins, Structures and Molecular Principles (Creighton, Ed., W.
H. Freeman and Company, New York (1984)); Introduction to Protein
Structure (C. Branden and J. Tooze, eds., Garland Publishing, New
York, N.Y. (1991)); and Thornton et at. Nature 354: 105, 1991,
which are each incorporated herein by reference.
[0072] "Antibody" or "antibody peptide(s)" refer to an intact
antibody, or a binding fragment thereof that competes with the
intact antibody for specific binding. Binding fragments are
produced by recombinant DNA techniques, or by enzymatic or chemical
cleavage of intact antibodies. Binding fragments include Fab, Fab',
F(ab').sub.2, Fv, and single-chain antibodies. An intact "antibody"
comprises at least two heavy (H) chains and two light (L) chains
interconnected by disulfide bonds. Each heavy chain is comprised of
a heavy chain variable region (abbreviated herein as HCVR or VH)
and a heavy chain constant region. The heavy chain constant region
is comprised of three domains, CH.sub.1, CH.sub.2 and CH.sub.3.
Each light chain is comprised of a light chain variable region
(abbreviated herein as LCVR or V.sub.L) and a light chain constant
region. The light chain constant region is comprised of one domain,
C.sub.L. The V.sub.H and V.sub.L regions can be further subdivided
into regions of hypervariability, termed complementarity
determining regions (CDR), interspersed with regions that are more
conserved, termed framework regions (FR). Each V.sub.H and V.sub.L
is composed of three CDRs and four FRs, arranged from
amino-terminus to carboxyl-terminus in the following order: FR1,
CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy
and light chains contain a binding domain that interacts with an
antigen. The constant regions of the antibodies can mediate the
binding of the immunoglobulin to host tissues or factors, including
various cells of the immune system (e.g., effector cells) and the
first component (Clq) of the classical complement system. The term
antibody includes antigen-binding portions of an intact antibody
that retain capacity to bind .beta.-hCG. Examples of binding
include (i) a Fab fragment, a monovalent fragment consisting of the
V.sub.L, V.sub.H, C.sub.L and CH1 domains; (ii) a F(ab').sub.2
fragment, a bivalent fragment comprising two Fab fragments linked
by a disulfide bridge at the hinge region; (iii) a Fd fragment
consisting of the VH and CH1 domains; (iv) a Fv fragment consisting
of the V.sub.L and V.sub.H domains of a single arm of an antibody,
(v) a dAb fragment (Ward et al., Nature 341: 544-546, 1989), which
consists of a VH domain; and (vi) an isolated complementarity
determining region (CDR).
[0073] An antibody other than a "bispecific" or "bifunctional"
antibody is understood to have each of its binding sites identical.
An antibody substantially inhibits adhesion of a receptor to a
counterreceptor when an excess of antibody reduces the quantity of
receptor bound to counterreceptor by at least about 20%, 40%, 60%
or 80%, and more usually greater than about 85% (as measured in an
in vitro competitive binding assay).
[0074] "Fab antibodies" or "Fab fragments" refers to antibody
fragments lacking all or part of an immunoglobulin constant region,
and containing the Fab regions of the antibodies. Fab antibodies
are prepared as described herein.
[0075] "Single chain antibodies" or "single chain Fv (scFv)" refers
to an antibody fusion molecule of the two domains of the Fv
fragment, V.sub.L, and V.sub.H. Although the two domains of the 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); see, e.g., Bird et al.,
Science 242: 423-426, 1988; and Huston et al., Proc. Natl. Acad.
Sci. USA, 85: 5879-5883, 1988). Such single chain antibodies are
included by reference to the term "antibody" fragments can be
prepared by recombinant techniques or enzymatic or chemical
cleavage of intact antibodies.
[0076] "Human sequence antibody" includes antibodies having
variable and constant regions (if present) derived from human
germline immunoglobulin sequences. The human sequence antibodies of
the invention can include amino acid residues not encoded by human
germline immunoglobulin sequences (e.g., mutations introduced by
random or site-specific mutagenesis in vitro or by somatic mutation
in vivo). Such antibodies can be generated in non-human transgenic
animals, e.g., as described in PCT Publication Nos. WO 01/14424 and
WO 00/37504. However, the term "human sequence antibody", as used
herein, is not intended to include antibodies in which CDR
sequences derived from the germline of another mammalian species,
such as a mouse, have been grafted onto human framework sequences
(e.g., humanized antibodies).
[0077] Also, recombinant immunoglobulins may be produced. See,
Cabilly, U.S. Pat. No. 4,816,567, incorporated herein by reference
in its entirety and for all purposes; and Queen et al., Proc. Nat'l
Acad. Sci. USA 86: 10029-10033, 1989.
[0078] "Monoclonal antibody" refer to a preparation of antibody
molecules of single molecular composition. A monoclonal antibody
composition displays a single binding specificity and affinity for
a particular epitope. Accordingly, the term "human monoclonal
antibody" refers to antibodies displaying a single binding
specificity which have variable and constant regions (if present)
derived from human germline immunoglobulin sequences. In one
embodiment, the human monoclonal antibodies are produced by a
hybridoma which includes a B cell obtained from a transgenic
non-human animal, e.g., a transgenic mouse, having a genome
comprising a human heavy chain transgene and a light chain
transgene fused to an immortalized cell.
[0079] "Polyclonal antibody" refers to a preparation of more than 1
(two or more) different antibodies to a cell surface receptor or a
ligand, e.g., .beta.-hCG binding to LH/hCG receptor. Such a
preparation includes antibodies binding to a range of .beta.-hCG
binding to LH/hCG receptor. Similarly antibodies to .beta.-hCG can
act as peptidomimetics that bind to LH/hCG receptor and thus
inhibit .beta.-hCG binding to LH/hCG receptor. These and other
antibodies suitable for use in the present invention can be
prepared according to methods that are well known in the art and/or
are described in the references cited here. In preferred
embodiments, anti-.beta.-hCG antibodies used in the invention are
"human antibodies"--e.g., antibodies isolated from a human--or they
are "human sequence antibodies".
[0080] "Immune cell response" refers to the response of immune
system cells to external or internal stimuli (e.g., antigen, cell
surface receptors, .beta.-hCG, LH/hCG receptor, cytokines,
chemokines, and other cells) producing biochemical changes in the
immune cells that result in immune cell migration, killing of
target cells, phagocytosis, production of antibodies, other soluble
effectors of the immune response, and the like.
[0081] "Immune response" refers to the concerted action of
lymphocytes, antigen presenting cells, phagocytic cells,
granulocytes, and soluble macromolecules produced by the above
cells or the liver (including antibodies, cytokines, and
complement) that results in selective damage to, destruction of, or
elimination from the human body of cancerous cells, metastatic
tumor cells, metastatic epithelial cancer, colorectal carcinoma,
gastric carcinoma, oral carcinoma, pancreatic carcinoma, ovarian
carcinoma, or renal cell carcinoma, invading pathogens, cells or
tissues infected with pathogens, or, in cases of autoimmunity or
pathological inflammation, normal human cells or tissues.
[0082] "T lymphocyte response" and "T lymphocyte activity" are used
here interchangeably to refer to the component of immune response
dependent on T lymphocytes (e.g., the proliferation and/or
differentiation of T lymphocytes into helper, cytotoxic killer, or
suppressor T lymphocytes, the provision of signals by helper T
lymphocytes to B lymphocytes that cause or prevent antibody
production, the killing of specific target cells by cytotoxic T
lymphocytes, and the release of soluble factors such as cytokines
that modulate the function of other immune cells).
Cancer Treatment
[0083] Blockade of .beta.-hCG binding to LH/hCG receptor by
antibody compositions, for example, an antibody which specifically
binds to .beta.-L2 loop of human chorionic gonadotropin (hCG), can
enhance the memory or secondary immune response to cancerous cells
in the patient. Antibodies to hCG can be combined with an
immunogenic agent, such as cancerous cells, purified tumor antigens
(including recombinant proteins, peptides, and carbohydrate
molecules), cells, and cells transfected with genes encoding immune
stimulating cytokines and cell surface antigens, or used alone, to
stimulate immunity.
[0084] Antibodies to .beta.-hCG are effective when following a
vaccination protocol. Many experimental strategies for vaccination
against tumors have been devised (see Rosenberg, ASCO Educational
Book Spring: 60-62, 2000; Logothetis, ASCO Educational Book Spring:
300-302, 2000; Khayat, ASCO Educational Book Spring: 414-428, 2000;
Foon, ASCO Educational Book Spring: 730-738, 2000; see also Restifo
et al., Cancer: Principles and Practice of Oncology, 61: 3023-3043,
1997. In one of these strategies, a vaccine is prepared using
autologous or allogeneic tumor cells. These cellular vaccines have
been shown to be most effective when the tumor cells are transduced
to express GM-CSF. GM-CSF has been shown to be a potent activator
of antigen presentation for tumor vaccination. Dranoff et al.,
Proc. Natl. Acad. Sci. U.S.A., 90: 3539-43, 1993.
[0085] Antibodies to .beta.-hCG can boost GM-CSF-modified tumor
cell vaccines improves efficacy of vaccines in a number of
experimental tumor models such as mammary carcinoma (Hurwitz et
al., 1998, supra), primary prostate cancer (Hurwitz et al., Cancer
Research, 60: 2444-8, 2000) and melanoma (van Elsas et al., J. Exp.
Med., 190: 355-66, 1999). In these instances, non-immunogenic
tumors, such as the B 16 melanoma, have been rendered susceptible
to destruction by the immune system. The tumor cell vaccine can
also be modified to express other immune activators such as IL2,
and costimulatory molecules, among others.
[0086] "Antineoplastic agent" is used herein to refer to agents
that have the functional property of inhibiting a development or
progression of a neoplasm in a human, particularly a malignant
(cancerous) lesion, such as a carcinoma, sarcoma, lymphoma, or
leukemia. Inhibition of metastasis is frequently a property of
antineoplastic agents.
[0087] Chemotherapeutic agents can be used in combination with
monoclonal antibodies to .beta.-hCG, e.g., an antibody which
specifically binds to .beta.-L2 loop of hCG, in methods for
treatment of neoplastic disease. An antibody-cytotoxin conjugate
comprising antibodies to (.beta.-hCG can also be used to boost
immunity induced through standard cancer treatments. In these
instances, it can be possible to reduce the dose of
chemotherapeutic reagent administered (Mokyr et al., Cancer
Research 58: 5301-5304, 1998). The scientific rationale behind the
combined use of antibodies to .beta.-hCG and chemotherapy is that
cell death, that is a consequence of the cytotoxic action of most
chemotherapeutic compounds, should result in increased levels of
tumor antigen in the antigen presentation pathway. Thus, antibodies
to .beta.-hCG can boost an immune response primed to chemotherapy
release of tumor cells. Examples of chemotherapeutic agents
combined with treatment with antibodies to .beta.-hCG can include,
but are not limited to, Actinomycetes or Streptomyces antibiotics,
duocarmycin, aldesleukin, altretamine, amifostine, asparaginase,
bleomycin, capecitabine, carboplatin, carmustine, cladribine,
cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine
(DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol,
duocarmycin, epoetin alpha, etoposide, filgrastim, fludarabine,
fluorouracil, gemcitabine, granisetron, hydroxyurea, idarubicin,
ifosfamide, interferon alpha, irinotecan, lansoprazole, levamisole,
leucovorin, megestrol, mesna, methotrexate, metoclopramide,
mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron,
paclitaxel (Taxol.TM.), pilocarpine, prochloroperazine, rituximab,
saproin, tamoxifen, taxol, topotecan hydrochloride, trastuzumab,
vinblastine, vincristine and vinorelbine tartrate. For prostate
cancer treatment, a preferred chemotherapeutic agent with which
anti-.beta.-hCG can be combined is paclitaxel (Taxol.TM.). For
melanoma cancer treatment, a preferred chemotherapeutic agent with
which anti-.beta.-hCG can be combined is dacarbazine (DTIC).
[0088] A "solid tumor" includes, but is not limited to, sarcoma,
melanoma, carcinoma, or other solid tumor cancer.
[0089] "Sarcoma" refers to a tumor which is made up of a substance
like the embryonic connective tissue and is generally composed of
closely packed cells embedded in a fibrillar or homogeneous
substance. Sarcomas include, but are not limited to,
chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma,
myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma,
liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma,
botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal
sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal
sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma,
giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma,
idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic
sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells,
Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma,
angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma,
parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic
sarcoma, synovial sarcoma, and telangiectaltic sarcoma.
[0090] "Melanoma" refers to a tumor arising from the melanocytic
system of the skin and other organs. Melanomas include, for
example, acral-lentiginous melanoma, amelanotic melanoma, benign
juvenile melanoma, Cloudman's melanoma, S91 melanoma,
Harding-Passey melanoma, juvenile melanoma, lentigo maligna
melanoma, malignant melanoma, nodular melanoma, subungal melanoma,
and superficial spreading melanoma.
[0091] "Carcinoma" refers to a malignant new growth made up of
epithelial cells tending to infiltrate the surrounding tissues and
give rise to metastases. Exemplary carcinomas further include, for
example, epithelial cancer, colorectal carcinoma, gastric
carcinoma, oral carcinoma, pancreatic carcinoma, ovarian carcinoma,
or renal cell carcinoma. Exemplary carcinomas further include, for
example, acinar carcinoma, acinous carcinoma, adenocystic
carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum,
carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell
carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid
carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma,
bronchiolar carcinoma, bronchogenic carcinoma, cerebriform
carcinoma, cholangiocellular carcinoma, chorionic carcinoma,
colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform
carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical
carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma
durum, embryonal carcinoma, encephaloid carcinoma, epiermoid
carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma,
carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma,
gelatinous carcinoma, giant cell carcinoma, carcinoma
gigantocellulare, glandular carcinoma, granulosa cell carcinoma,
hair-matrix carcinoma, hematoid carcinoma, hepatocellular
carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid
carcinoma, infantile embryonal carcinoma, carcinoma in situ,
intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's
carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma,
lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma,
lymphoepithelial carcinoma, carcinoma medullare, medullary
carcinoma, melanotic carcinoma, carcinoma molle, mucinous
carcinoma, carcinoma muciparum, carcinoma mucocellulare,
mucoepidemoid carcinoma, carcinoma mucosum, mucous carcinoma,
carcinoma myxomatodes, naspharyngeal carcinoma, oat cell carcinoma,
carcinoma ossificans, osteoid carcinoma, papillary carcinoma,
periportal carcinoma, preinvasive carcinoma, prickle cell
carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney,
reserve cell carcinoma, carcinoma sarcomatodes, schneiderian
carcinoma, scirrhous carcinoma, carcinoma scroti, signet-ring cell
carcinoma, carcinoma simplex, small-cell carcinoma, solanoid
carcinoma, spheroidal cell carcinoma, spindle cell carcinoma,
carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma,
string carcinoma, carcinoma telangiectaticum, carcinoma
telangiectodes, transitional cell carcinoma, carcinoma tuberosum,
tuberous carcinoma, verrucous carcinoma, and carcinoma
viflosum.
[0092] "Leukemia" refers to progressive, malignant diseases of the
blood-forming organs and is generally characterized by a distorted
proliferation and development of leukocytes and their precursors in
the blood and bone marrow. Leukemia is generally clinically
classified on the basis of (1) the duration and character of the
disease--acute or chronic; (2) the type of cell involved; myeloid
(myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the
increase or non-increase in the number of abnormal cells in the
blood--leukemic or aleukemic (subleukemic). Leukemia includes, for
example, acute nonlymphocytic leukemia, chronic lymphocytic
leukemia, acute granulocytic leukemia, chronic granulocytic
leukemia, acute promyelocytic leukemia, adult T-cell leukemia,
aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia,
blast cell leukemia, bovine leukemia, chronic myelocytic leukemia,
leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross'
leukemia, hairy-cell leukemia, hemoblastic leukemia,
hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia,
acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia,
lymphoblastic leukemia, lymphocytic leukemia, lymphogenous
leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell
leukemia, megakaryocytic leukemia, micromyeloblastic leukemia,
monocytic leukemia, myeloblastic leukemia, myelocytic leukemia,
myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli
leukemia, plasma cell leukemia, plasmacytic leukemia, promyelocytic
leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell
leukemia, subleukemic leukemia, and undifferentiated cell
leukemia.
[0093] Additional cancers include, for example, Hodgkin's Disease,
Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast
cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary
thrombocytosis, primary macroglobulinemia, small-cell lung tumors,
primary brain tumors, stomach cancer, colon cancer, malignant
pancreatic insulanoma, malignant carcinoid, urinary bladder cancer,
premalignant skin lesions, testicular cancer, lymphomas, thyroid
cancer, neuroblastoma, esophageal cancer, genitourinary tract
cancer, malignant hypercalcemia, cervical cancer, endometrial
cancer, adrenal cortical cancer, and prostate cancer.
Antibody Structure
[0094] The basic antibody structural unit is known to comprise a
tetramer. Each tetramer is composed of two identical pairs of
polypeptide chains, each pair having one "light"-(about 25 kDa) and
one "heavy" chain (about 50-70 kDa). The amino-terminal portion of
each chain includes a variable region of about 100 to 110 or more
amino acids primarily responsible for antigen recognition. The
carboxy-terminal portion of each chain defines a constant region
primarily responsible for effector function. Human light chains are
classified as kappa and lambda light chains. Heavy chains are
classified as mu, delta, gamma, alpha, or epsilon, and define the
antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
Within light and heavy chains, the variable and constant regions
are joined by a "J" region of about 12 or more amino acids, with
the heavy chain also including a "D" region of about 10 more amino
acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed.,
2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its
entirety for all purposes). The variable regions of each
light/heavy chain pair form the antibody binding site.
[0095] Thus, an intact IgG antibody has two binding sites. Except
in bifunctional or bispecific antibodies, the two binding sites are
the same.
[0096] The chains all exhibit the same general structure of
relatively conserved framework regions (FR) joined by three hyper
variable regions, also called complementarity determining regions
or CDRs. The CDRs from the two chains of each pair are aligned by
the framework regions, enabling binding to a specific epitope. From
N-terminal to C-terminal, both light and heavy chains comprise the
domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of
amino acids to each domain is in accordance with the definitions of
Kabat Sequences of Proteins of Immunological Interest (National
Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia
& Lesk J. Mol. Biol. 196: 901-917, 1987; Chothia et al., Nature
342: 878-883, 1989.
[0097] A bispecific or bifunctional antibody is an artificial
hybrid antibody having two different heavy/light chain pairs and
two different binding sites. Bispecific antibodies can be produced
by a variety of methods including fusion of hybridomas or linking
of Fab' fragments. See, e.g., Songsivilai and Lachmann, Clin. Exp.
Immunol. 79: 315-321, 1990, Kostelny et al., J. Immunol. 148:
1547-1553, 1992. In addition, bispecific antibodies may be formed
as "diabodies" (Holliger et al., PNAS USA 90: 6444-6448, 1993 or
"Janusins" (Traunecker et al., EMBO J. 10: 3655-3659, 1991 and
Traunecker et al., Int J Cancer 7:51-52, 1992). Production of
bispecific antibodies can be a relatively labor intensive process
compared with production of conventional antibodies and yields and
degree of purity are generally lower for bispecific antibodies.
Bispecific antibodies do not exist in the form of fragments having
a single binding site (e.g., Fab, Fab', and Fv).
FAB or scFV Phage Libraries
[0098] An approach for a phage display library to identify an
antibody composition which binds to .beta.-hCG, e.g., an antibody
which specifically binds to .beta.-L2 loop of hCG, or that
specifically binds to a ligand or a cell surface receptor on a
metastatic cell, for example, LH/hCG receptor, has been the use of
Fab or single-chain Fv (scFv) phage-libraries. See, e.g., Huston et
al., Proc. Natl. Acad. Sci. U.S.A., 85: 5879-5883, 1988; Chaudhary
et al., Proc. Natl. Acad. Sci. U.S.A., 87: 1066-1070, 1990; Zhang
et al., J. Virol. 78: 9233-9242, 2004. Various embodiments of Fab
or scFv libraries displayed on bacteriophage coat proteins have
been described. Refinements of phage display approaches are also
known, for example as described in WO96/06213 and WO92/01047
(Medical Research Council et al.) and WO97/08320 (Morphosys), which
are incorporated herein by reference. The display of Fab libraries
is known, for instance as described in WO92/01047 (CAT/MRC) and
WO91/17271 (Affymax).
[0099] Hybrid antibodies or hybrid antibody fragments that are
cloned into a display vector can be selected against the
appropriate antigen associated with a metastatic cell, e.g., a cell
surface receptor or ligand to a cell surface receptor on a
metastatic tumor cell, in order to identify variants that
maintained good binding activity because the antibody or antibody
fragment will be present on the surface of the phage or phagemid
particle. See for example Barbas III et al., Phage Display, A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 2001, the contents of which are incorporated herein
by reference. For example, in the case of Fab fragments, the light
chain and heavy chain Fd products are under the control of a lac
promoter, and each chain has a leader signal fused to it in order
to be directed to the periplasmic space of the bacterial host. It
is in this space that the antibody fragments will be able to
properly assemble. The heavy chain fragments are expressed as a
fusion with a phage coat protein domain which allows the assembled
antibody fragment to be incorporated into the coat of a newly made
phage or phagemid particle. Generation of new phagemid particles
requires the addition of helper phage which contain all the
necessary phage genes. Once a library of antibody fragments is
presented on the phage or phagemid surface, a process termed
panning follows. This is a method whereby i) the antibodies
displayed on the surface of phage or phagemid particles are bound
to the desired antigen, ii) non-binders are washed away, iii) bound
particles are eluted from the antigen, and iv) eluted particles are
exposed to fresh bacterial hosts in order to amplify the enriched
pool for an additional round of selection. Typically three or four
rounds of panning are performed prior to screening antibody clones
for specific binding. In this way phage/phagemid particles allow
the linkage of binding phenotype (antibody) with the genotype (DNA)
making the use of antibody display technology very successful.
However, other vector formats could be used for this humanization
process, such as cloning the antibody fragment library into a lytic
phage vector (modified T7 or Lambda Zap systems) for selection
and/or screening.
[0100] After selection of desired hybrid antibodies and/or hybrid
antibody fragments, it is contemplated that they can be produced in
large volume by any technique known to those skilled in the art,
e.g., prokaryotic or eukaryotic cell expression and the like. For
example, hybrid antibodies or fragments may be produced by using
conventional techniques to construct an expression vector that
encodes an antibody heavy chain in which the CDRs and, if
necessary, a minimal portion of the variable region framework, that
are required to retain original species antibody binding
specificity (as engineered according to the techniques described
herein) are derived from the originating species antibody and the
remainder of the antibody is derived from a target species
immunoglobulin which may be manipulated as described herein,
thereby producing a vector for the expression of a hybrid antibody
heavy chain.
[0101] In a detailed embodiment, a Fab or single-chain Fv (scFv)
antibody library can be prepared from the peripheral blood
lymphocytes of 5, 10, 15, or 20 or more patients with various
cancer diseases. Completely human high-affinity Fab or scFv
antibodies can then be selected by using synthetic sialyl
Lewis.sup.x and Lewis.sup.x BS A conjugates. In one study, these
human scFv antibodies were specific for sialyl Lewis.sup.x and
Lewis.sup.x, as demonstrated by ELISA, BIAcore, and flow cytometry
binding to the cell surface of pancreatic adenocarcinoma cells.
Nucleotide sequencing revealed that at least four unique scFv genes
were obtained. The K.sub.d values ranged from 1.1 to
6.2.times.10.sup.-7 M that were comparable to the affinities of
mAbs derived from the secondary immune response. These antibodies
could be valuable reagents for probing the structure and function
of carbohydrate antigens and in the treatment of human tumor
diseases. Mao et al., Proc. Natl. Acad. Sci. U.S.A. 96: 6953-6958,
1999.
[0102] In a further detailed embodiment, phage displayed
combinatorial antibody libraries can be used to generate and select
a wide variety of antibodies to an appropriate antigen associated
with a metastatic cell, e.g., a cell surface receptor or a ligand
to a cell surface receptor on a metastatic tumor cell. The phage
coat proteins pVII and pIX can be used to display the heterodimeric
structure of the antibody Fv region. Aspects of this technology
have been extended to construct a large, human Fab or single-chain
Fv (scFv) library of 4.5.times.10.sup.9 members displayed on pIX of
filamentous bacteriophage. Furthermore, the diversity, quality, and
utility of the library were demonstrated by the selection of Fab or
scFv clones against six different protein antigens. Notably, more
than 90% of the selected clones showed positive binding for their
respective antigens after as few as three rounds of panning.
Analyzed Fabs or scFvs were also found to be of high affinity. For
example, kinetic analysis (BIAcore) revealed that Fabs or scFvs
against staphylococcal enterotoxin B and cholera toxin B subunit
had a nanomolar and subnanomolar dissociation constant,
respectively, affording affinities comparable to, or exceeding
that, of mAbs obtained from immunization. High specificity was also
attained, not only between very distinct proteins, but also in the
case of more closely related proteins, e.g., Ricinus communis
("ricin") agglutinins (RCA.sub.60 and RCA.sub.120), despite>80%
sequence homology between the two. The results suggested that the
performance of pIX-display libraries can potentially exceed that of
the pIII-display format and make it ideally suited for panning a
wide variety of target antigens. Gao et al., Proc. Natl. Acad. Sci.
U.S.A. 99: 12612-12616, 2001.
[0103] Specific binding between an antibody or other binding agent
and an antigen means a binding affinity of at least 10.sup.-6 M.
Preferred binding agents bind with affinities of at least about
10.sup.-7 M, and preferably 10.sup.-8 M to 10.sup.-9 M, 10.sup.-10
M, 10.sup.-11 M, or 10.sup.-12 M. The term epitope means an
antigenic determinant capable of specific binding to an antibody.
Epitopes usually consist of chemically active surface groupings of
molecules such as amino acids or sugar side chains and usually have
specific three dimensional structural characteristics, as well as
specific charge characteristics. Conformational and
nonconformational epitopes are distinguished in that the binding to
the former but not the latter is lost in the presence of denaturing
solvents.
[0104] "Epitope" refers to that portion of any molecule capable of
being recognized by and bound by an antibody or T-cell receptor at
one or more of the antibody's or T cell receptor's antigen binding
region. Epitopes usually consist of chemically active surface
groupings of molecules such as amino acids or sugar side chains and
have specific three dimensional structural characteristics as well
as specific charge characteristics. By "inhibiting and/or
neutralizing epitope" is intended an epitope, which, when bound by
an antibody, results in loss of biological activity of the molecule
or organism containing the epitope, in vivo, in vitro or in situ,
more preferably in vivo, including binding of HCG to an LH/hCG
receptor. An antibody is said to specifically bind an antigen when
the dissociation constant is less than 1 .mu.M, preferably less
than 100 nM and most preferably less than 10 nM. Conformational and
nonconformational epitopes are distinguished in that the binding to
the former but not the latter is lost in the presence of denaturing
solvents.
[0105] Epitopes recognized by antibodies, and fragments and regions
thereof, of the present invention can include 5 or more amino acids
comprising at least one amino acid of each or both of the following
amino acid sequences of .beta.-hCG, which provide a topographical
or three dimensional epitope of .beta.-hCG which is recognized by,
and/or binds with anti-.beta.-hCG activity, an antibody, and
fragments, and variable regions thereof, of the present invention:
Screening Methods for determining .beta.-hCG neutralizing and/or
inhibiting activity are also provided in the present invention. In
the context of the present invention, anti-.beta.-hCG neutralizing
activity or .beta.-hCG inhibiting activity refers to the ability of
a .beta.-hCG neutralizing compound to block at least one biological
activity of .beta.-hCG, such as preventing .beta.-hCG from binding
to a LH/hCG receptor, blocking production of .beta.-hCG by
intracellular processing, such as transcription, translation or
post-translational modification, expression on the cell surface,
secretion or assembly of the bioactive .beta.-hCG. Additionally,
.beta.-hCG neutralizing compounds can act by inducing regulation of
metabolic pathways such as those involving the up or down
regulation of .beta.-hCG production. Alternatively .beta.-hCG
neutralizing compounds can modulate cellular sensitivity to
.beta.-hCG by decreasing such sensitivity. .beta.-hCG neutralizing
compounds can be selected from the group consisting of antibodies,
or fragments or portions thereof, peptides, peptido mimetic
compounds or organo mimetic compounds that neutralizes .beta.-hCG
activity in vitro, in situ or in vivo is considered a .beta.-hCG
neutralizing compound if used according to the present invention.
Screening methods which can be used to determine .beta.-hCG
neutralizing activity of a .beta.-hCG neutralizing compound can
include in vitro or in vivo assays. Such in vitro assays can
include an assay for (i) inhibition of proliferation in vitro of
BXPC-3 pancreatic carcinoma cells; and (ii) no inhibition of
proliferation in vitro of MCF-7 breast carcinoma cells or HeLa
cells at an antibody concentration about 4 nM or greater; (ii)
inhibition of .beta.-hCG binding to LH/hCG receptor; or (iii)
inhibition of cell migration in a cell migration assay.
Alternatively or additionally, in vivo testing of .beta.-hCG
neutralizing activity of .beta.-hCG neutralizing compounds can be
tested using an in vitro assay for inhibition of proliferation in
vitro of BXPC-3 pancreatic carcinoma cells at an antibody
concentration about 4 nM or greater, as described herein.
[0106] "Neutralizing" refers to an antibody that inhibits
.beta.-hCG activity by preventing the binding of human .beta.-hCG
to its specific receptor, LH/hCG receptor, or by inhibiting the
signaling of .beta.-hCG through its receptor, should binding occur.
A monoclonal antibody is neutralizing if it is 90% effective,
preferably 95% effective and most preferably 100% effective in
inhibiting .beta.-hCG activity, for example, as measured by in
vitro cell assay, such as (i) inhibition of proliferation in vitro
of BXPC-3 pancreatic carcinoma cells; and (ii) no inhibition of
proliferation in vitro of MCF-7 breast carcinoma cells or HeLa
cells.
[0107] "Agent" is used herein to denote a chemical compound, a
mixture of chemical compounds, a biological macromolecule, or an
extract made from biological materials.
[0108] "Altered antibody" refers to a protein encoded by an altered
immunoglobulin coding region, which may be obtained by expression
in a selected host cell. Such altered antibodies are engineered
antibodies (e.g., chimeric or humanized antibodies) or antibody
fragments lacking all or part of an immunoglobulin constant region,
e.g., Fv, Fab, or F(ab).sub.2 and the like.
[0109] "Altered immunoglobulin coding region" refers to a nucleic
acid sequence encoding altered antibody of the invention. When the
altered antibody is a CDR-grafted or humanized antibody, the
sequences that encode the complementarity determining regions
(CDRs) from a non-human immunoglobulin are inserted into a first
immunoglobulin partner comprising human variable framework
sequences. Optionally, the first immunoglobulin partner is
operatively linked to a second immunoglobulin partner.
[0110] "High affinity" refers to an antibody having a binding
affinity characterized by a Kd equal to or less than
3.5.times.10.sup.-11 M for human .beta.-hCG as determined by
surfact plasmon resonance.
[0111] By "binding specificity for human .beta.-hCG" is meant a
high affinity for human chorionic gonadotropin. Monoclonal
antibodies have a high binding specificity for .beta.-hCG and do
not bind with high affinity to other associated hCG subunits or
receptors. Monoclonal antibodies mAb 2B2.6F5 and 2B3.3E8 have a
high binding specificity for .beta.-hCG, and do not bind to
.alpha.-hCG or to LH/hCG receptor.
[0112] The terms Fv, Fc, Fd, Fab, or F(ab).sub.2 are used with
their standard meanings (see, e.g., Harlow et al., Antibodies A
Laboratory Manual, Cold Spring Harbor Laboratory, (1988)).
[0113] "Engineered antibody" describes a type of altered antibody,
i.e., a full-length synthetic antibody (e.g., a chimeric or
humanized antibody as opposed to an antibody fragment) in which a
portion of the light and/or heavy chain variable domains of a
selected acceptor antibody are replaced by analogous parts from one
or more donor antibodies which have specificity for the selected
epitope. For example, such molecules may include antibodies
characterized by a humanized heavy chain associated with an
unmodified light chain (or chimeric light chain), or vice versa.
Engineered antibodies may also be characterized by alteration of
the nucleic acid sequences encoding the acceptor antibody light
and/or heavy variable domain framework regions in order to retain
donor antibody binding specificity. These antibodies can comprise
replacement of one or more CDRs (preferably all) from the acceptor
antibody with CDRs from a donor antibody described herein.
[0114] A "chimeric antibody" refers to a type of engineered
antibody which contains naturally-occurring variable region (light
chain and heavy chains) derived from a donor antibody in
association with light and heavy chain constant regions derived
from an acceptor antibody.
[0115] A "humanized antibody" refers to a type of engineered
antibody having its CDRs derived from a non-human donor
immunoglobulin, the remaining immunoglobulin-derived parts of the
molecule being derived from one (or more) human immunoglobulin(s).
In addition, framework support residues may be altered to preserve
binding affinity. See, e.g., Queen et al., Proc. Natl. Acad Sci
USA, 86: 10029-10032, 1989, Hodgson et al., Bio/Technology, 2:-421,
1991.
[0116] "Donor antibody" refers to an antibody (monoclonal, or
recombinant) which contributes the nucleic acid sequences of its
variable regions, CDRs, or other functional fragments or analogs
thereof to a first immunoglobulin partner, so as to provide the
altered immunoglobulin coding region and resulting expressed
altered antibody with the antigenic specificity and neutralizing
activity characteristic of the donor antibody.
[0117] "Acceptor antibody" refers to an antibody (monoclonal, or
recombinant) heterologous to the donor antibody, which contributes
all (or any portion, but preferably all) of the nucleic acid
sequences encoding its heavy and/or light chain framework regions
and/or its heavy and/or light chain constant regions to the first
immunoglobulin partner. Preferably a human antibody is the acceptor
antibody.
[0118] "CDRs" are defined as the complementarity determining region
amino acid sequences of an antibody which are the hypervariable
regions of immunoglobulin heavy and light chains. See, e.g., Kabat
et al., Sequences of Proteins of Immunological Interest, 4th Ed.,
U.S. Department of Health and Human Services, National Institutes
of Health (1987). There are three heavy chain and three light chain
CDRs (or CDR regions) in the variable portion of an immunoglobulin.
Thus, "CDRs" as used herein refers to all three heavy chain CDRs,
or all three light chain CDRs (or both all heavy and all light
chain CDRs, if appropriate).
[0119] CDRs provide the majority of contact residues for the
binding of the antibody to the antigen or epitope. CDRs of interest
in this invention are derived from donor antibody variable heavy
and light chain sequences, and include analogs of the naturally
occurring CDRs, which analogs also share or retain the same antigen
binding specificity and/or neutralizing ability as the donor
antibody from which they were derived.
[0120] By "sharing the antigen binding specificity or neutralizing
ability" is meant, for example, that although mAb 2B2.6F5 or
2B3.3E8 can be characterized by a certain level of antigen
affinity, a CDR encoded by a nucleic acid sequence of mAb 2B2.6F5
or 2B3.3E8 in an appropriate structural environment may have a
lower, or higher affinity. It is expected that CDRs of mAb 2B2.6F5
or 2B3.3E8 in such environments will nevertheless recognize the
same epitope(s) as the original monoclonal antibodies. Exemplary
heavy chain CDRs include SEQ ID NO:1; SEQ ID NO:3; and SEQ ID NO:5.
See, for example, FIG. 11.
[0121] A "functional fragment" is a partial heavy or light chain
variable sequence (e.g., minor deletions at the amino or carboxy
terminus of the immunoglobulin variable region) which retains the
same antigen binding specificity and/or neutralizing ability as the
antibody from which the fragment was derived.
[0122] An "analog" is an amino acid sequence modified by at least
one amino acid, wherein said modification can be chemical or a
substitution or a rearrangement of a few amino acids (i.e., no more
than 10), which modification permits the amino acid sequence to
retain the biological characteristics, e.g., antigen specificity
and high affinity, of the unmodified sequence. For example,
(silent) mutations can be constructed, via substitutions, when
certain endonuclease restriction sites are created within or
surrounding CDR-encoding regions.
[0123] Analogs may also arise as allelic variations. An "allelic
variation or modification" is an alteration in the nucleic acid
sequence encoding the amino acid or peptide sequences of the
invention. Such variations or modifications may be due to
degeneracy in the genetic code or may be deliberately engineered to
provide desired characteristics. These variations or modifications
may or may not result in alterations in any encoded amino acid
sequence.
[0124] "Carrier agents" or "effector agents" refers to non-protein
carrier molecules to which the altered antibodies, and/or natural
or synthetic light or heavy chains of the donor antibody or other
fragments of the donor antibody may be associated by conventional
means. Such non-protein carriers can include conventional carriers
used in the diagnostic field, e.g., polystyrene or other plastic
beads, polysaccharides, e.g., as used in the BIAcore.RTM.
[Pharmacia] system, or other non-protein substances useful in the
medical field and safe for administration to humans and animals.
Other effector agents may include a macrocycle, for chelating a
heavy metal atom, or radioisotopes. Such effector agents may also
be useful to increase the half-life of the altered antibodies,
e.g., polyethylene glycol.
[0125] Components of an immune response can be detected in vitro by
various methods that are well known to those of ordinary skill in
the art. For example, (1) cytotoxic T lymphocytes can be incubated
with radioactively labeled target cells and the lysis of these
target cells detected by the release of radioactivity; (2) helper T
lymphocytes can be incubated with antigens and antigen presenting
cells and the synthesis and secretion of cytokines measured by
standard methods (Windhagen et al., Immunity, 2: 373-80, 1995); (3)
antigen presenting cells can be incubated with whole protein
antigen and the presentation of that antigen on MHC detected by
either T lymphocyte activation assays or biophysical methods
(Harding et al., Proc. Natl. Acad. Sci., 86: 4230-4, 1989); (4)
mast cells can be incubated with reagents that cross-link their
Fc-epsilon receptors and histamine release measured by enzyme
immunoassay (Siraganian et al., TIPS, 4: 432-437, 1983).
[0126] Similarly, products of an immune response in either a model
organism (e.g., mouse) or a human patient can also be detected by
various methods that are well known to those of ordinary skill in
the art. For example, (1) the production of antibodies in response
to vaccination can be readily detected by standard methods
currently used in clinical laboratories, e.g., an ELISA; (2) the
migration of immune cells to sites of inflammation can be detected
by scratching the surface of skin and placing a sterile container
to capture the migrating cells over scratch site (Peters et al.,
Blood, 72: 1310-5, 1988); (3) the proliferation of peripheral blood
mononuclear cells in response to mitogens or mixed lymphocyte
reaction can be measured using .sup.3H-thymidine; (4) the
phagocytic capacity of granulocytes, macrophages, and other
phagocytes in PBMCs can be measured by placing PMBCs in wells
together with labeled particles (Peters et al., Blood, 72: 1310-5,
1988); and (5) the differentiation of immune system cells can be
measured by labeling PBMCs with antibodies to CD molecules such as
CD4 and CD8 and measuring the fraction of the PBMCs expressing
these markers.
[0127] For convenience, immune responses are often described in the
present invention as being either "primary" or "secondary" immune
responses. A primary immune response, which is also described as a
"protective" immune response, refers to an immune response produced
in an individual as a result of some initial exposure (e.g. the
initial "immunization") to a particular antigen, e.g., cell surface
receptor, ligand, .beta.-hCG, or LH/hCG receptor. Such an
immunization can occur, for example, as the result of some natural
exposure to the antigen (for example, from initial infection by
some pathogen that exhibits or presents the antigen) or from
antigen presented by cancer cells of some tumor in the individual
(for example, a metastatic epithelial cancer, colorectal carcinoma,
gastric carcinoma, oral carcinoma, pancreatic carcinoma, ovarian
carcinoma, or renal cell carcinoma). Alternatively, the
immunization can occur as a result of vaccinating the individual
with a vaccine containing the antigen. For example, the vaccine can
be a cancer vaccine comprising one or more antigens from a cancer
cell e.g., cells from a metastatic epithelial cancer, colorectal
carcinoma, gastric carcinoma, oral carcinoma, pancreatic carcinoma,
ovarian carcinoma, or renal cell carcinoma.
[0128] A primary immune response can become weakened or attenuated
over time and can even disappear or at least become so attenuated
that it cannot be detected. Accordingly, the present invention also
relates to a "secondary" immune response, which is also described
here as a "memory immune response." The term secondary immune
response refers to an immune response elicited in an individual
after a primary immune response has already been produced. Thus, a
secondary or immune response can be elicited, e.g., to enhance an
existing immune response that has become weakened or attenuated, or
to recreate a previous immune response that has either disappeared
or can no longer be detected. An agent that can be administrated to
elicit a secondary immune response is after referred to as a
"booster" since the agent can be said to "boost" the primary immune
response.
[0129] As an example, and not by way of limitation, a secondary
immune response can be elicited by re-introducing to the individual
an antigen that elicited the primary immune response (for example,
by re-administrating a vaccine). However, a secondary immune
response to an antigen can also be elicited by administrating other
agents that can not contain the actual antigen. For example, the
present invention provides methods for potentiating a secondary
immune response by administrating an antibody to .beta.-hCG to an
individual. In such methods the actual antigen need not necessarily
be administered with the antibody to .beta.-hCG and the composition
containing the antibody need not necessarily contain the antigen.
The secondary or memory immune response can be either a humoral
(antibody) response or a cellular response. A secondary or memory
humoral response occurs upon stimulation of memory B cells that
were generated at the first presentation of the antigen. Delayed
type hypersensitivity (DTH) reactions are a type of cellular
secondary or memory immune response that are mediated by CD4.sup.+
cells. A first exposure to an antigen primes the immune system and
additional exposure(s) results in a DTH.
[0130] "Immunologically cross-reactive" or "immunologically
reactive" refers to an antigen which is specifically reactive with
an antibody which was generated using the same ("immunologically
reactive") or different ("immunologically cross-reactive") antigen.
Generally, the antigen is .beta.-hCG or LH/hCG receptor, or
subsequence thereof.
[0131] "Immunologically reactive conditions" refers to conditions
which allow an antibody, generated to a particular epitope of an
antigen, to bind to that epitope to a detectably greater degree
than the antibody binds to substantially all other epitopes,
generally at least two times above background binding, preferably
at least five times above background. Immunologically reactive
conditions are dependent upon the format of the antibody binding
reaction and typically are those utilized in immunoassay protocols.
See, Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring
Harbor Publications, New York, 1988 for a description of
immunoassay formats and conditions.
[0132] "Cell surface receptor" refers to molecules and complexes of
molecules capable of receiving a signal and the transmission of
such a signal across the plasma membrane of a cell. An example of a
"cell surface receptor"-of the present invention is an LH/hCG
receptor on a metastatic cell.
[0133] "Nonspecific T cell activation" refers to the stimulation of
T cells independent of their antigenic specificity.
[0134] "Effector cell" refers to an immune cell which is involved
in the effector phase of an immune response, as opposed to the
cognitive and activation phases of an immune response. Exemplary
immune cells include a cell of a myeloid or lymphoid origin, e.g.,
lymphocytes (e.g., B cells and T cells including cytolytic T cells
(CTLs)), killer cells, natural killer cells, macrophages,
monocytes, eosinophils, neutrophils, polymorphonuclear cells,
granulocytes, mast cells, and basophils. Effector cells express
specific Fe receptors and carry out specific immune functions. An
effector cell can induce antibody-dependent cell-mediated
cytotoxicity (ADCC), e.g., a neutrophil capable of inducing ADCC.
For example, monocytes, macrophages, neutrophils, eosinophils, and
lymphocytes which express Fc.alpha.R are involved in specific
killing of target cells and presenting antigens to other components
of the immune system, or binding to cells that present antigens. An
effector cell can also phagocytose a target antigen, target cell,
metastatic cancer cell, or microorganism.
[0135] "Target cell" refers to any undesirable cell in a subject
(e.g., a human or animal) that can be targeted by the Ab or Ab
composition of the invention. The target cell can be a cell
expressing or overexpressing human LH/hCG receptor. Cells
expressing human LH/hCG receptor can include tumor cells, e.g. a
metastatic epithelial cancer, colorectal carcinoma, gastric
carcinoma, oral carcinoma, pancreatic carcinoma, ovarian carcinoma,
or renal cell carcinoma.
[0136] Targets of interest for antibody compositions metastatic
cancer cells, e.g., metastatic epithelial cancer cells, include,
but are not limited to, cell surface receptors, growth factor
receptors, .beta.-hCG, LH/hCG receptor, (See, for example, Burtrum
D., et al, Cancer Res., 63: 8912-8921, 2003; Lu et al., J. Biol.
Chem. 279: 2856-2865, 2004; Miyamoto et al., Clin. Cancer Res. 11:
3494-3502, 2005; Goya et al., Cancer Research 64: 6252-6258, 2004.)
antibodies, including anti-idiotypic antibodies and autoantibodies
present in cancer, such as metastatic cancer, metastatic epithelial
cancer, colorectal carcinoma, gastric carcinoma, oral carcinoma,
pancreatic carcinoma, ovarian carcinoma, or renal cell carcinoma.
Other targets are adhesion proteins such as integrins, selectins,
and immunoglobulin superfamily members. Springer, Nature, 346:
425-433, 1990; Osborn, Cell, 62: 3, 1990; Hynes, Cell, 69: 11,
1992. Other targets of interest are growth factor receptors (e.g.,
FGFR, PDGFR, EGF, her/neu, NGFR, and VEGF) and their ligands. Other
targets are G-protein receptors and include substance K receptor,
the angiotensin receptor, the .alpha.- and .beta.-adrenergic
receptors, the serotonin receptors, and PAF receptor. See, e.g.,
Gilman, Ann. Rev. Biochem. 56: 625-649, 1987. Other targets include
ion channels (e.g., calcium, sodium, potassium channels, channel
proteins that mediate multidrug resistance), muscarinic receptors,
acetylcholine receptors, GABA receptors, glutamate receptors, and
dopamine receptors (see Harpold, U.S. Pat. No. 5,401,629 and U.S.
Pat. No. 5,436,128). Other targets are cytokines, such as
interleukins IL-1 through IL-13, tumor necrosis factors .alpha.-
and .beta., interferons .alpha.-, .beta.- and .gamma., tumor growth
factor Beta (TGF-.beta.), colony stimulating factor (CSF) and
granulocyte monocyte colony stimulating factor (GM-CSF). See
Aggrawal et al., eds., Human Cytokines: Handbook for Basic &
Clinical Research, Blackwell Scientific, Boston, Mass., 1991. Other
targets are hormones, enzymes, and intracellular and intercellular
messengers, such as adenyl cyclase, guanyl cyclase, and
phospholipase C. Drugs are also targets of interest. Target
molecules can be human, mammalian or bacterial. Other targets are
antigens, such as proteins, glycoproteins and carbohydrates from
microbial pathogens, both viral and bacterial, and tumors. Still
other targets are described in U.S. Pat. No. 4,366,241,
incorporated herein by reference in its entirety and for all
purposes. Some agents screened by the target merely bind to a
target. Other agents agonize or antagonize the target.
Recombinant Expression of Anti-Human-.beta.-CG Antibodies
[0137] Recombinant human antibodies that bind to .beta.-hCG, e.g.,
an antibody which specifically binds to .beta.-L2 loop of hCG,
inhibit .beta.-hCG binding to LH/hCG receptor, are provided
according to the present invention using known techniques based on
the teaching provided herein. See, e.g., Ausubel et al., eds.
Current Protocols in Molecular Biology, Wiley Interscience, N.Y.
(1987, 1992, 1993); and Sambrook et at. Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), the
entire contents of which are incorporated herein by reference.
[0138] The DNA encoding an anti-hCG antibody of the present
invention can be genomic DNA or cDNA which encodes at least one of
the heavy chain constant region (C.sub.H), the heavy chain variable
region (V.sub.H), the light chain variable region (V.sub.L) and the
light chain constant regions (C.sub.L). A convenient alternative to
the use of chromosomal gene fragments as the source of DNA encoding
the murine V region antigen-binding segment is the use of cDNA for
the construction of chimeric immunoglobulin genes, e.g., as
reported by Liu et al., Proc. Natl. Acad. Sci., USA 84:3439 (1987)
and J. Immunology 139: 3521 (1987), which references are hereby
entirely incorporated herein by reference. The use of cDNA requires
that gene expression elements appropriate for the host cell be
combined with the gene in order to achieve synthesis of the desired
protein. The use of cDNA sequences is advantageous over genomic
sequences (which contain introns), in that cDNA sequences can be
expressed in bacteria or other hosts which lack appropriate RNA
splicing systems.
[0139] Such techniques for synthesizing such oligonucleotides are
well known and disclosed by, for example, Wu, et al., Prog. Nucl.
Acid. Res. Molec. Biol. 21:101-141 (1978)), and Ausubel et al.,
eds. Current Protocols in Molecular Biology, Wiley Interscience
(1987, 1993), the entire contents of which are herein incorporated
by reference.
[0140] Because the genetic code is degenerate, more than one codon
can be used to encode a particular amino acid (Watson, et al.,
infra). Using the genetic code, one or more different
oligonucleotides can be identified, each of which would be capable
of encoding the amino acid. The probability that a particular
oligonucleotide will, in fact, constitute the actual
anti-.beta.-hCG antibody encoding sequence can be estimated by
considering abnormal base pairing relationships and the frequency
with which a particular codon is actually used (to encode a
particular amino acid) in eukaryotic or prokaryotic cells
expressing an anti-.beta.-hCG antibody or fragment. Such "codon
usage rules" are disclosed by Lathe, et al., J. Molec. Biol.
183:1-12 (1985). Using the "codon usage rules" of Lathe, a single
oligonucleotide, or a set of oligonucleotides, that contains a
theoretical "most probable" nucleotide sequence capable of encoding
anti-.beta.-hCG variable or constant region sequences is
identified.
[0141] Although occasionally an amino acid sequence can be encoded
by only a single oligonucleotide, frequently the amino acid
sequence can be encoded by any of a set of similar
oligonucleotides. Importantly, whereas all of the members of this
set contain oligonucleotides which are capable of encoding the
peptide fragment and, thus, potentially contain the same
oligonucleotide sequence as the gene which encodes the peptide
fragment, only one member of the set contains the nucleotide
sequence that is identical to the nucleotide sequence of the gene.
Because this member is present within the set, and is capable of
hybridizing to DNA even in the presence of the other members of the
set, it is possible to employ the unfractionated set of
oligonucleotides in the same manner in which one would employ a
single oligonucleotide to clone the gene that encodes the
protein.
[0142] The oligonucleotide, or set of oligonucleotides, containing
the theoretical "most probable" sequence capable of encoding an
anti-.beta.-hCG antibody or fragment including a variable or
constant region is used to identify the sequence of a complementary
oligonucleotide or set of oligonucleotides which is capable of
hybridizing to the "most probable" sequence, or set of sequences.
An oligonucleotide containing such a complementary sequence can be
employed as a probe to identify and isolate the variable or
constant region anti-.beta.-hCG gene (Sambrook et al., infra).
[0143] A suitable oligonucleotide, or set of oligonucleotides,
which is capable of encoding a fragment of the variable or constant
anti-.beta.-hCG region (or which is complementary to such an
oligonucleotide, or set of oligonucleotides) is identified (using
the above-described procedure), synthesized, and hybridized by
means well known in the art, against a DNA or, more preferably, a
cDNA preparation derived from cells which are capable of expressing
anti-.beta.-hCG antibodies or variable or constant regions thereof.
Single stranded oligonucleotide molecules complementary to the
"most probable" variable or constant anti-.beta.-hCG region peptide
coding sequences can be synthesized using procedures which are well
known to those of ordinary skill in the art (Belagaje, et al., J.
Biol. Chem. 254: 5765-5780 (1979); Maniatis, et al., In: Molecular
Mechanisms in the Control of Gene Expression, Nierlich, et al.,
Eds., Acad. Press, NY (1976); Wu, et al., Prog. Nucl. Acid Res.
Molec. Biol. 21: 101-141 (1978); Khorana, Science 203: 614-625
(1979)). Additionally, DNA synthesis can be achieved through the
use of automated synthesizers. Techniques of nucleic acid
hybridization are disclosed by Sambrook et al. (infra), and by
Hayrnes, et al. (In: Nucleic Acid Hybridization, A Practical
Approach, IRL Press, Washington, D.C. (1985)), which references are
herein incorporated by reference. Techniques such as, or similar
to, those described above have successfully enabled the cloning of
genes for human aldehyde dehydrogenases (Hsu, et al., Proc. Natl.
Acad. Sci. USA 82: 3771-3775 (1985)), fibronectin (Suzuki, et al.,
Bur. Mol. Biol. Organ. J. 4: 2519-2524 (1985)), the human estrogen
receptor gene (Walter, et al., Proc. Natl. Acad. Sci. USA 82:
7889-7893 (1985)), tissue-type plasminogen activator (Pennica, et
al., Nature 301: 214-221 (1983)) and human term placental alkaline
phosphatase complementary DNA (Keun, et al., Proc. Natl. Acad. Sci.
USA 82: 8715-8719 (1985)).
[0144] In an alternative way of cloning a polynucleotide encoding
an anti-.beta.-hCG antibody variable or constant region, a library
of expression vectors is prepared by cloning DNA or, more
preferably, cDNA (from a cell capable of expressing an
anti-.beta.-hCG antibody or variable or constant region) into an
expression vector. The library is then screened for members capable
of expressing a protein which competitively inhibits the binding of
an anti-.beta.-hCG antibody, such as mAb 2B2.6F5 or 2B3.3E8, to
LH/hCG receptor and which has a nucleotide sequence that is capable
of encoding polypeptides that have the same amino acid sequence as
anti-.beta.-hCG antibodies or fragments thereof. In this
embodiment, DNA, or more preferably cDNA, is extracted and purified
from a cell which is capable of expressing an anti-.beta.-hCG
antibody or fragment. The purified cDNA is fragmentized (by
shearing, endonuclease digestion, etc.) to produce a pool of DNA or
cDNA fragments. DNA or cDNA fragments from this pool are then
cloned into an expression vector in order to produce a genomic
library of expression vectors whose members each contain a unique
cloned DNA or cDNA fragment such as in a lambda phage library,
expression in prokaryotic cell (e.g., bacteria) or eukaryotic
cells, (e.g., mammalian, yeast, insect or, fungus). See, e.g.,
Ausubel, infra, Harlow, infra, Colligan, infra; Nyyssonen et al.
Bio/Technology 11: 591-595 (Can 1993); Marks et al., Bio/Technology
11: 1145-1149, 1993. Once nucleic acid encoding such variable or
constant anti-.beta.-hCG regions is isolated, the nucleic acid can
be appropriately expressed in a host cell, along with other
constant or variable heavy or light chain encoding nucleic acid, in
order to provide recombinant MAbs that bind .beta.-hCG with
inhibitory activity. Such antibodies preferably include a murine or
human anti-.beta.-hCG variable region which contains a framework
residue having complimentarily determining residues which are
responsible for antigen binding. In a preferred embodiment, an
anti-.beta.-hCG variable light or heavy chain encoded by a nucleic
acid as described above binds an epitope of at least 5 amino acids
of, SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.
[0145] Human genes which encode the constant (C) regions of the
murine and chimeric antibodies, fragments and regions of the
present invention can be derived from a human fetal liver library,
by known methods. Human C regions genes can be derived from any
human cell including those which express and produce human
immunoglobulins. The human C.sub.H region can be derived from any
of the known classes or isotypes of human H chains, including
.gamma., .mu., .alpha., .delta., or .epsilon., and subtypes
thereof, such as G1, G2, G3 and G4. Since the H chain isotype is
responsible for the various effector functions of an antibody, the
choice of C.sub.H region will be guided by the desired effector
functions, such as complement fixation, or activity in
antibody-dependent cellular cytotoxicity (ADCC). Preferably, the
C.sub.H region is derived from gamma 1 (IgG1), gamma 3 (IgG3),
gamma 4 (IgG4), or mu (IgM).
Human Antibodies and Humanization of Antibodies
[0146] Human antibodies avoid certain of the problems associated
with antibodies that possess murine or rat variable and/or constant
regions. The presence of such murine or rat derived proteins can
lead to the rapid clearance of the antibodies or can lead to the
generation of an immune response against the antibody by a patient.
In order to avoid the utilization of murine or rat derived
antibodies, it has been postulated that one can develop humanized
antibodies or generate fully human antibodies through the
introduction of human antibody function into a rodent so that the
rodent would produce antibodies having fully human sequences.
[0147] The ability to clone and reconstruct megabase-sized human
loci in YACs and to introduce them into the mouse germline provides
a powerful approach to elucidating the functional components of
very large or crudely mapped loci as well as generating useful
models of human disease. Furthermore, the utilization of such
technology for substitution of mouse loci with their human
equivalents could provide unique insights into the expression and
regulation of human gene products during development, their
communication with other systems, and their involvement in disease
induction and progression.
[0148] An important practical application of such a strategy is the
"humanization" of the mouse humoral immune system. Introduction of
human immunoglobulin (Ig) loci into mice in which the endogenous Ig
genes have been inactivated offers the opportunity to study the
mechanisms underlying programmed expression and assembly of
antibodies as well as their role in B-cell development.
Furthermore, such a strategy could provide an ideal source for
production of fully human monoclonal antibodies (Mabs) an important
milestone towards fulfilling the promise of antibody therapy in
human disease. Fully human antibodies are expected to minimize the
immunogenic and allergic responses intrinsic to mouse or
mouse-derivatized Mabs and thus to increase the efficacy and safety
of the administered antibodies. The use of fully human antibodies
can be expected to provide a substantial advantage in the treatment
of chronic and recurring human diseases, such as inflammation,
autoimmunity, and cancer, which require repeated antibody
administrations.
[0149] One approach towards this goal was to engineer mouse strains
deficient in mouse antibody production with large fragments of the
human Ig loci in anticipation that such mice would produce a large
repertoire of human antibodies in the absence of mouse antibodies.
Large human Ig fragments would preserve the large variable gene
diversity as well as the proper regulation of antibody production
and expression. By exploiting the mouse machinery for antibody
diversification and selection and the lack of immunological
tolerance to human proteins, the reproduced human antibody
repertoire in these mouse strains should yield high affinity
antibodies against any antigen of interest, including human
antigens. Using the hybridoma technology, antigen-specific human
Mabs with the desired specificity could be readily produced and
selected.
[0150] Such approach is further discussed and delineated in U.S.
patent application Ser. No. 07/466,008, filed Jan. 12, 1990, Ser.
No. 07/610,515, filed Nov. 8, 1990, Ser. No. 07/919,297, filed Jul.
24, 1992, Ser. No. 07/922,649, filed Jul. 30, 1992, filed Ser. No.
08/031,801, filed Mar. 15, 1993, Ser. No. 08/112,848, filed Aug.
27, 1993, Ser. No. 08/234,145, filed Apr. 28, 1994, Ser. No.
08/376,279, filed Jan. 20, 1995, Ser. No. 08/430, 938, Apr.
27,-1995, Ser. No. 08/464,584, filed Jun. 5, 1995, Ser. No.
08/464,582, filed Jun. 5, 1995, Ser. No. 08/463,191, filed Jun. 5,
1995, Ser. No. 08/462,837, filed Jun. 5, 1995, Ser. No. 08/486,853,
filed Jun. 5, 1995, Ser. No. 08/486,857, filed Jun. 5, 1995, Ser.
No. 08/486,859, filed Jun. 5, 1995, Ser. No. 08/462,513, filed Jun.
5, 1995, Ser. No. 08/724,752, filed Oct. 2, 1996, and Ser. No.
08/759,620, filed Dec. 3, 1996. See also Mendez et al., Nature
Genetics 15: 146-156, 1997 and Green and Jakobovits, J. Exp. Med.
188: 483-495, 1998. See also European Patent No., EP 0 463 151 B1,
grant published Jun. 12, 1996, International Patent Application
No., WO 94/02602, published Feb. 3, 1994, International Patent
Application No., WO 96/34096, published Oct. 31, 1996, and WO
98/24893, published Jun. 11, 1998. The disclosures of each of the
above-cited patents, applications, and references are hereby
incorporated by reference in their entirety.
[0151] In an alternative approach, others, including GenPharm
International, Inc., have utilized a "minilocus" approach. In the
minilocus approach, an exogenous Ig locus is mimicked through the
inclusion of pieces (individual genes) from the Ig locus. Thus, one
or more V.sub.H genes, one or more DH genes, one or more J.sub.H
genes, a mu constant region, and a second constant region
(preferably a gamma constant region) are formed into a construct
for insertion into an animal. This approach is described in U.S.
Pat. No. 5,545,807 to Surani et al. and U.S. Pat. Nos. 5,545,806,
5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650,
and 5,814,318 each to Lonberg and Kay, U.S. Pat. No. 5,591,669 to
Krimpenfort and Berns, U.S. Pat. Nos. 5,612,205, 5,721,367,
5,789,215 to Berns et al., and U.S. Pat. No. 5,643,763 to Choi and
Dunn, and GenPharm International U.S. patent application Ser. No.
07/574,748, filed Aug. 29, 1990, Ser. No. 07/575,962, filed Aug.
31, 1990, Ser. No. 07/810,279, filed Dec. 17, 1991, Ser. No.
07/853,408, filed Mar. 18, 1992, Ser. No. 07/904,068, filed Jun.
23, 1992, Ser. No. 07/990,860, filed Dec. 16, 1992, Ser. No.
08/053,131, filed Apr. 26, 1993, Ser. No. 08/096,762, filed Jul.
22, 1993, Ser. No. 08/155,301, filed Nov. 18, 1993, Ser. No.
08/161,739, filed Dec. 3, 1993, Ser. No. 08/165,699, filed Dec. 10,
1993, Ser. No. 08/209,741, filed Mar. 9, 1994, the disclosures of
which are hereby incorporated by reference. See also European
Patent No. 0 546 073 B 1, International Patent Application Nos. WO
92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO
94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO 98/24884,
the disclosures of which are hereby incorporated by reference in
their entirety. See further Taylor et al., 1992, Chen et al., 1993,
Tuaillon et al., 1993, Choi et al., 1993, Lonberg et al., 1994,
Taylor et al., 1994, and Tuaillon et al., 1995, Fishwild et al.,
1996, the disclosures of which are hereby incorporated by reference
in their entirety.
[0152] A transgenic mouse possessing an Ig locus has been produced
through use of the minilocus approach. An advantage of the
minilocus approach is the rapidity with which constructs including
portions of the Ig locus can be generated and introduced into
animals. Commensurately, however, a significant disadvantage of the
minilocus approach is that, in theory, insufficient diversity is
introduced through the inclusion of small numbers of V, D, and J
genes. Indeed, the published work appears to support this concern.
B-cell development and antibody production of animals produced
through use of the minilocus approach appear stunted. Therefore,
research surrounding the present invention has consistently been
directed towards the introduction of large portions of the Ig locus
in order to achieve greater diversity and in an effort to
reconstitute the immune repertoire of the animals.
[0153] Human anti-mouse antibody (HAMA) responses have led the
industry to prepare chimeric or otherwise humanized antibodies.
While chimeric antibodies have a human constant region and a murine
variable region, it is expected that certain human anti-chimeric
antibody (HACA) responses will be observed, particularly in chronic
or multi-dose utilizations of the antibody. Thus, it would be
desirable to provide fully human antibodies against .beta.-hCG in
order to vitiate concerns and/or effects of HAMA or HACA
response.
Humanization and Display Technologies
[0154] As was discussed above in connection with human antibody
generation, there are advantages to producing antibodies with
reduced immunogenicity. To a degree, this can be accomplished in
connection with techniques of humanization and display techniques
using appropriate libraries. It will be appreciated that murine
antibodies or antibodies from other species can be humanized or
primatized using techniques well known in the art. See e.g., Winter
and Harris, Immunol Today 14: 43-46, 1993 and Wright et al., Crit.
Reviews in Immunol. 12:125-168, 1992. The antibody of interest may
be engineered by recombinant DNA techniques to substitute the
C.sub.H1, C.sub.H2, C.sub.H3, hinge domains, and/or the framework
domain with the corresponding human sequence (see WO 92/02190 and
U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,792,
5,714,350, and 5,777,085). Also, the use of Ig cDNA for
construction of chimeric immunoglobulin genes is known in the art
(Liu et al., PNAS USA 84: 3439, 1987 and J. Immunol. 139: 3521,
1987). mRNA is isolated from a hybridoma or other cell producing
the antibody and used to produce cDNA. The cDNA of interest may be
amplified by the polymerase chain reaction using specific primers
(U.S. Pat. Nos. 4,683,195 and 4,683,202). Alternatively, a library
is made and screened to isolate the sequence of interest. The DNA
sequence encoding the variable region of the antibody is then fused
to human constant region sequences. The sequences of human constant
regions genes may be found in Kabat et al. (1991) Sequences of
Proteins of Immunological Interest, NIH publication no. 91-3242.
Human C region genes are readily available from known clones. The
choice of isotype will be guided by the desired effector functions,
such as complement fixation, or activity in antibody-dependent
cellular cytotoxicity. Preferred isotypes are IgG1, IgG2, IgG3 and
IgG4. Particularly preferred isotypes for antibodies of the
invention are IgG2 and IgG4. Either of the human light chain
constant regions, kappa or lambda, may be used. The chimeric,
humanized antibody is then expressed by conventional methods.
[0155] Antibody fragments, such as Fv, F(ab').sub.2 and Fab may be
prepared by cleavage of the intact protein, e.g. by protease or
chemical cleavage. Alternatively, a truncated gene is designed. For
example, a chimeric gene encoding a portion of the F(ab').sub.2
fragment would include DNA sequences encoding the CH1 domain and
hinge region of the H chain, followed by a translational stop codon
to yield the truncated molecule.
[0156] In one approach, consensus sequences encoding the heavy and
light chain J regions may be used to design oligonucleotides for
use as primers to introduce useful restriction sites into the J
region for subsequent linkage of V region segments to human C
region segments. C region cDNA can be modified by site directed
mutagenesis to place a restriction site at the analogous position
in the human sequence.
[0157] Expression vectors include plasmids, retroviruses, cosmids,
YACs, EBV derived episomes, and the like. A convenient vector is
one that encodes a functionally complete human C.sub.H or C.sub.L
immunoglobulin sequence, with appropriate restriction sites
engineered so that any V.sub.H or V.sub.L sequence can be easily
inserted and expressed. In such vectors, splicing usually occurs
between the splice donor site in the inserted J region and the
splice acceptor site preceding the human C region, and also at the
splice regions that occur within the human C.sub.H exons.
Polyadenylation and transcription termination occur at native
chromosomal sites downstream of the coding regions. The resulting
chimeric antibody may be joined to any strong promoter, including
retroviral LTRs, e.g. SV-40 early promoter, (Okayama et al., Mol.
Cell. Bio. 3: 280, 1983), Rous sarcoma virus LTR (Gorman et al.,
P.N.A.S. 79: 6777, 1982), and moloney murine leukemia virus LTR
(Grosschedl et al., Cell 41: 885, 1985); native 1 g promoters,
etc.
[0158] Further, human antibodies or antibodies from other species
can be generated through display-type technologies, including,
without limitation, phage display, retroviral display, ribosomal
display, and other techniques, using techniques well known in the
art and the resulting molecules can be subjected to additional
maturation, such as affinity maturation, as such techniques are
well known in the art. Wright and Harris, supra., Hanes and
Plucthau, PNAS USA 94: 4937-4942, 1997 (ribosomal display), Parmley
and Smith, Gene 73: 305-318, 1988 (phage display), Scott, TIBS 17:
241-245, 1992, Cwirla et al., PNAS USA 87: 6378-6382, 1990, Russel
et al., Nucl. Acids Research 21: 1081-1085, 1993, Hoganboom et al.,
Immunol. Reviews 130: 43-68, 1992, Chiswell and McCafferty, TIBTECH
10: 80-84, 1992, and U.S. Pat. No. 5,733,743. If display
technologies are utilized to produce antibodies that are not human,
such antibodies can be humanized as described above.
[0159] Using these techniques, antibodies can be generated to
.beta.-hCG expressing cells, .beta.-hCG, hCG or forms of hCG,
epitopes or peptides thereof, and expression libraries thereto (see
e.g. U.S. Pat. No. 5,703,057) which can thereafter be screened as
described above for the activities described above.
Design and Generation of Other Therapeutics
[0160] In accordance with the present invention and based on the
activity of the antibodies that are produced and characterized
herein, such as antibodies to .beta.-hCG, or an antibody which
specifically binds to .beta.-L2 loop of hCG, the design of other
therapeutic modalities including other antibodies, other
antagonists, or chemical moieties other than antibodies is
facilitated. Such modalities include, without limitation,
antibodies having similar binding activity or functionality,
advanced antibody therapeutics, such as bispecific antibodies,
immunotoxins, and radiolabeled therapeutics, generation of peptide
therapeutics, gene therapies, particularly intrabodies, antisense
therapeutics, and small molecules. Furthermore, as discussed above,
the effector function of the antibodies of the invention may be
changed by isotype switching to an IgG1, IgG2, IgG3, IgG4, IgD,
IgA, IgE, or IgM for various therapeutic uses.
[0161] In connection with the generation of advanced antibody
therapeutics, where complement fixation is a desirable attribute,
it may be possible to sidestep the dependence on complement for
cell killing through the use of bispecifics, immunotoxins, or
radiolabels, for example.
[0162] In connection with bispecific antibodies, bispecific
antibodies can be generated that comprise (i) two antibodies one
with a specificity to .beta.-hCG and another to a second molecule
that are conjugated together, (ii) a single antibody that has one
chain specific to .beta.-hCG and a second chain specific to a
second molecule, or (iii) a single chain antibody that has
specificity to .beta.-hCG and the other molecule. Such bispecific
antibodies can be generated using techniques that are well known
for example, in connection with (i) and (ii) see e.g., Fanger et
al., Immunol Methods 4: 72-81, 1994 and Wright and Harris, supra.,
and in connection with (iii) see e.g., Traunecker et al., Int. J.
Cancer 7: 51-52, 1992.
[0163] In addition, "Kappabodies" (Ill et al., Protein Eng 10:
949-57, 1997), "Minibodies" (Martin et al., EMBO J. 13: 5303-9,
1994), "Diabodies" (Holliger et al., PNAS USA 90: 6444-6448, 1993),
or "Janusins" (Traunecker et al., EMBO J. 10: 3655-3659, 1991) and
Traunecker et al., Int J Cancer 7:51-52, 1992) may also be
prepared.
[0164] In connection with immunotoxins, antibodies can be modified
to act as immunotoxins utilizing techniques that are well known in
the art. See e.g., Vitetta, Immunol Today 14: 252, 1993. See also
U.S. Pat. No. 5,194,594. In connection with the preparation of
radiolabeled antibodies, such modified antibodies can also be
readily prepared utilizing techniques that are well known in the
art. See e.g., Junghans et al., Cancer Chemotherapy and Biotherapy
655-686 (2d edition, Chafier and Longo, eds., Lippincott Raven,
1996). See also U.S. Pat. Nos. 4,681,581, 4,735,210, 5,101,827,
5,102,990 (RE 35,500), 5,648,471, and 5,697,902. Each of
immunotoxins and radiolabeled molecules would be likely to kill
cells expressing .beta.-hCG, and particularly those cells in which
the antibodies of the invention are effective.
[0165] In connection with the generation of therapeutic peptides,
through the utilization of structural information related to
.beta.-hCG and antibodies thereto, such as the antibodies of the
invention (as discussed below in connection with small molecules)
or screening of peptide libraries, therapeutic peptides can be
generated that are directed against .beta.-hCG. Design and
screening of peptide therapeutics is discussed in connection with
Houghten et al., Biotechniques 13: 412-421, 1992, Houghten PNAS USA
82: 5131-5135, 1985, Pinalla et al., Biotechniques 13: 901-905,
1992, Blake and Litzi-Davis, BioConjugate Chem. 3: 510-513, 1992.
Immunotoxins and radiolabeled molecules can also be prepared, and
in a similar manner, in connection with peptidic moieties as
discussed above in connection with antibodies.
[0166] Important information related to the binding of an antibody
to an antigen can be gleaned through phage display experimentation.
Such experiments are generally accomplished through panning a phage
library expressing random peptides for binding with the antibodies
of the invention to determine if peptides can be isolated that
bind. If successful, certain epitope information can be gleaned
from the peptides that bind.
[0167] In general, phage libraries expressing random peptides can
be purchased from New England Biolabs (7-mer and 12-mer libraries,
Ph.D.-7 Peptide 7-mer Library Kit and Ph.D.-12 Peptide 12-mer
Library Kit, respectively) based on a bacteriophage M13 system. The
7-mer library represents a diversity of approximately 2.0.times.
10.sup.9 independent clones, which represents most, if not all, of
the 20.sup.7=1.28.times.10.-sup.9 possible 7-mer sequences. The
12-mer library contains approximately 1.9.times.10.sup.9
independent clones and represents only a very small sampling of the
potential sequence space of 20.sup. 12=4.1.times.10.sup.15 12-mer
sequences. Each of 7-mer and 12-mer libraries are panned or
screened in accordance with the manufacturer's recommendations in
which plates were coated with an antibody to capture the
appropriate antibody (a goat anti-human IgG Fc for an IgG antibody
for example) followed by washing. Bound phage are eluted with 0.2 M
glycine-HC1, pH 2.2. After 3 rounds of selection/amplification at
constant stringency (0.5% Tween), through use of DNA sequencing,
one can characterize clones from the libraries that are reactive
with one or more of the antibodies. Reactivity of the peptides can
be determined by ELISA. For an additional discussion of epitope
analysis of peptides see also Scott and Smith, Science 249:
386-390, 1990; Cwirla et al., PNAS USA 87: 6378-6382, 1990; Felici
et al., J. Mol. Biol. 222: 301-310, 1991, and Kuwabara et al.,
Nature Biotechnology 15: 74-78, 1997.
[0168] The design of gene and/or antisense therapeutics through
conventional techniques is also facilitated through the present
invention. Such modalities can be utilized for modulating the
function of .beta.-hCG. In connection therewith the antibodies of
the present invention facilitate design and use of functional
assays related thereto. A design and strategy for antisense
therapeutics is discussed in detail in International Patent
Application No. WO 94/29444. Design and strategies for gene therapy
are well known. However, in particular, the use of gene therapeutic
techniques involving intrabodies could prove to be particularly
advantageous. See e.g., Chen et al., Human Gene Therapy 5: 595-601,
1994 and Marasco, Gene Therapy 4: 11-15, 1997. General design of
and considerations related to gene therapeutics is also discussed
in International Patent Application No. WO 97/38137. Genetic
materials encoding an antibody of the invention (such as mAb
2B2.6F5 or 2B3.3E8, or others) may be included in a suitable
expression system (whether viral, attenuated viral, non-viral,
naked, or otherwise) and administered to a host for in vivo
generation of the antibody in the host.
[0169] Small molecule therapeutics can also be envisioned in
accordance with the present invention. Drugs can be designed to
modulate the activity of hCG based upon the present invention.
Knowledge gleaned from the structure of the .beta.-hCG molecule and
its interactions with other molecules in accordance with the
present invention, such as the antibodies of the invention, LH/hCG
receptor, and others can be utilized to rationally design
additional therapeutic modalities. In this regard, rational drug
design techniques such as X-ray crystallography, computer-aided (or
assisted) molecular modeling (CAMM), quantitative or qualitative
structure-activity relationship (QSAR), and similar technologies
can be utilized to focus drug discovery efforts. Rational design
allows prediction of protein or synthetic structures which can
interact with the molecule or specific forms thereof which can be
used to modify or modulate the activity of hCG. Such structures can
be synthesized chemically or expressed in biological systems. This
approach has been reviewed in Capsey et al., Genetically Engineered
Human Therapeutic Drugs (Stockton Press, NY, 1988). Indeed, the
rational design of molecules (either peptides, peptidomimetics,
small molecules, or the like) based upon known, or delineated,
structure-activity relationships with other molecules (such as
antibodies in accordance with the invention) has become generally
routine. See, e.g., Fry et al., Proc NatlAcad Sci USA 95: 12022-7,
1998; Hoffman et al., J Mol Biol 282: 195-208, 1998; Ginalski et
al., Acta Biochim Pol 44: 557-64, 1997; Jouko et al., Biochem J
322: 927-35, 1997; Singh et al., J Med Chem 40: 1130-5, 1997;
Mandel et al., Nat Biotechnol 14: 323-8, 1996; Monfardini et al.,
Proc Assoc Am Physicians 108: 420-31, 1996; Furet et al., J Comput
Aided Mol Des 9: 465-72, 1995.
[0170] Further, combinatorial libraries can be designed and
synthesized and used in screening programs, such as high throughput
screening efforts.
Preparation of Antibodies in Transgenic Mice
[0171] Antibodies in accordance with the invention are preferably
prepared through the utilization of a transgenic mouse that has a
substantial portion of the human antibody producing genome inserted
but that is rendered deficient in the production of endogenous,
murine, antibodies. Such mice, then, are capable of producing human
immunoglobulin molecules and antibodies and are deficient in the
production of murine immunoglobulin molecules and antibodies. In
particular, however, a preferred embodiment of transgenic
production of mice and antibodies therefrom is disclosed in U.S.
patent application Ser. No. 08/759,620, filed Dec. 3, 1996, the
disclosure of which is hereby incorporated by reference. See also
Mendez et al., Nature Genetics 15: 146-156, 1997, the disclosure of
which is hereby incorporated by reference.
[0172] Through use of such technology, we have produced fully human
monoclonal antibodies to a variety of antigens. Essentially, we
immunize lines of mice with an antigen of interest, recover
lymphatic cells (such as B-cells) from the mice that express
antibodies, fuse such recovered cells with a myeloid-type cell line
to prepare immortal hybridoma cell lines, and such hybridoma cell
lines are screened and selected to identify hybridoma cell lines
that produce antibodies specific to the antigen of interest. We
utilized these techniques in accordance with the present invention
for the preparation of antibodies specific to .beta.-hCG. Herein,
we describe the production of multiple hybridoma cell lines that
produce antibodies specific to .beta.-hCG. Further, we provide a
characterization of the antibodies produced by such cell lines,
including nucleotide and amino acid sequence analyses of the heavy
and light chains of such antibodies.
[0173] The antibodies derived from hybridoma cell lines for mAb
2B2.6F5 and 2B3.3E8 were expressed as discussed herein. Each of the
antibodies produced by the aforementioned cell lines are either
fully human IgG1 heavy chains and human IgG1 light chains. In
general, antibodies in accordance with the invention possess very
high affinities, typically possessing Kd's of from about 10.sup.-9
through about 10.sup.-11 M, when measured by either solid phase or
solution phase.
[0174] As will be appreciated, antibodies in accordance with the
present invention can be expressed in cell lines other than
hybridoma cell lines. Sequences encoding the cDNAs or genomic
clones for the particular antibodies can be used for transformation
of a suitable mammalian or nonmammalian host cells. Transformation
can be by any known method for introducing polynucleotides into a
host cell, including, for example packaging the polynucleotide in a
virus (or into a viral vector) and transducing a host cell with the
virus (or vector) or by transfection procedures known in the art,
as exemplified by U.S. Pat. Nos. 4,399,216, 4,912,040, 4,740,461,
and 4,959,455 (which patents are hereby incorporated herein by
reference). The transformation procedure used depends upon the host
to be transformed. Methods for introduction of heterologous
polynucleotides into mammalian cells are well known in the art and
include, but are not limited to, dextran-mediated transfection,
calcium phosphate precipitation, polybrene mediated transfection,
protoplast fusion, electroporation, particle bombardment,
encapsulation of the polynucleotide(s) in liposomes, peptide
conjugates, dendrimers, and direct microinjection of the DNA into
nuclei.
[0175] Mammalian cell lines available as hosts for expression are
well known in the art and include many immortalized cell lines
available from the American Type Culture Collection (ATCC),
including but not limited to Chinese hamster ovary (CHO) cells,
NSO.sub.o, HeLa cells, baby hamster kidney (BHK) cells, monkey
kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep
G2), and a number of other cell lines. Non-mammalian cells
including but not limited to bacterial, yeast, insect, and plants
can also be used to express recombinant antibodies. Site directed
mutagenesis of the antibody CH2 domain to eliminate glycosylation
may be preferred in order to prevent changes in either the
immunogenicity, pharmacokinetic, and/or effector functions
resulting from non-human glycosylation. The expression methods are
selected by determining which system generates the highest
expression levels and produce antibodies with constitutive
.beta.-hCG binding properties.
[0176] Further, expression of antibodies of the invention (or other
moieties therefrom) from production cell lines can be enhanced
using a number of known techniques. For example, the glutamine
sythetase and DHFR gene expression systems are common approaches
for enhancing expression under certain conditions. High expressing
cell clones can be identified using conventional techniques, such
as limited dilution cloning and Microdrop technology. The GS system
is discussed in whole or part in connection with European Patent
Nos. 0 216 846, 0 256 055, and 0 323 997 and European Patent
Application No. 89303964.4.
[0177] Antibodies of the invention can also be produced
transgenically through the generation of a mammal or plant that is
transgenic for the immunoglobulin heavy and light chain sequences
of interest and production of the antibody in a recoverable form
therefrom. In connection with the transgenic production in mammals,
antibodies can be produced in, and recovered from, the milk of
goats, cows, or other mammals. See, e.g., U.S. Pat. Nos. 5,827,690,
5,756,687, 5,750,172, and 5,741,957.
[0178] In connection with functional analysis of antibodies in
accordance with the present invention, such antibodies proved to be
potent inhibitors of .beta.-hCG and its binding to its LH/hCG
receptor. For example, antibodies in accordance with the present
invention, e.g., mAb 2B2.6F5 and 2B3.3E8 were demonstrated to bind
to .beta.-hCG and block binding of hCG to LH/hCG receptor. See
FIGS. 6 and 7. For example, antibodies in accordance with the
present invention, e.g., mAb 2B2.6F5 and 2B3.3E8, were shown to
inhibit (ii) inhibit proliferation in vitro of BXPC-3 pancreatic
carcinoma cells; and (ii) to not inhibit proliferation in vitro of
MCF-7 breast carcinoma cells or HeLa cells.
[0179] The results demonstrated in accordance with the present
invention indicate that antibodies of the present invention possess
certain qualities that may make the present antibodies more
efficacious than current therapeutic antibodies against .beta.-hCG,
for treatment of neoplastic disease.
[0180] In particular, the antibodies mAb 2B2.6F5 or 2B3.3E8 of the
invention possess highly desirable properties. Their structural
characteristics, functions, or activities provide criteria that
facilitate the design or selection of additional antibodies or
other molecules as discussed above.
[0181] Treatment Regimes
[0182] The invention provides pharmaceutical compositions
comprising one or a combination of antibodies, e.g., antibodies to
.beta.-hCG (monoclonal, polyclonal or single chain Fv; intact or
binding fragments thereof) formulated together with a
pharmaceutically acceptable carrier. Some compositions include a
combination of multiple (e.g., two or more) monoclonal antibodies
or antigen-binding portions thereof of the invention. In some
compositions, each of the antibodies or antigen-binding portions
thereof of the composition is a monoclonal antibody or a human
sequence antibody that binds to a distinct, pre-selected epitope of
an antigen.
[0183] In prophylactic applications, pharmaceutical compositions or
medicaments are administered to a patient susceptible to, or
otherwise at risk of a disease or condition (i.e., a neoplastic
disease) in an amount sufficient to eliminate or reduce the risk,
lessen the severity, or delay the outset of the disease, including
biochemical, histologic and/or behavioral symptoms of the disease,
its complications and intermediate pathological phenotypes
presenting during development of the disease. In therapeutic
applications, compositions or medicants are administered to a
patient suspected of, or already suffering from such a disease in
an amount sufficient to cure, or at least partially arrest, the
symptoms of the disease (biochemical, histologic and/or
behavioral), including its complications and intermediate
pathological phenotypes in development of the disease. An amount
adequate to accomplish therapeutic or prophylactic treatment is
defined as a therapeutically- or prophylactically-effective dose.
In both prophylactic and therapeutic regimes, agents are usually
administered in several dosages until a sufficient immune response
has been achieved. Typically, the immune response is monitored and
repeated dosages are given if the immune response starts to
wane.
[0184] Effective Dosages
[0185] Effective doses of the antibody compositions of the present
invention, e.g., antibodies to .beta.-hCG, for the treatment of
cancer-related conditions and diseases, e.g., metastic cancer,
described herein vary depending upon many different factors,
including means of administration, target site, physiological state
of the patient, whether the patient is human or an animal, other
medications administered, and whether treatment is prophylactic or
therapeutic. Usually, the patient is a human but nonhuman mammals
including transgenic mammals can also be treated. Treatment dosages
need to be titrated to optimize safety and efficacy.
[0186] For administration with an antibody, the dosage ranges from
about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the
host body weight. For example dosages can be 1 mg/kg body weight or
10 mg/kg body weight or within the range of 1-10 mg/kg. An
exemplary treatment regime entails administration once per every
two weeks or once a month or once every 3 to 6 months. In, some
methods, two or more monoclonal antibodies with different binding
specificities are administered simultaneously, in which case the
dosage of each antibody administered falls within the ranges
indicated. Antibody is usually administered on multiple occasions.
Intervals between single dosages can be weekly, monthly or yearly.
Intervals can also be irregular as indicated by measuring blood
levels of antibody in the patient. In some methods, dosage is
adjusted to achieve a plasma antibody concentration of 1-1000
.mu.g/ml and in some methods 25-300 .mu.g/ml. Alternatively,
antibody can be administered as a sustained release formulation, in
which case less frequent administration is required. Dosage and
frequency vary depending on the half-life of the antibody in the
patient. In general, human antibodies show the longest half life,
followed by humanized antibodies, chimeric antibodies, and nonhuman
antibodies. The dosage and frequency of administration can vary
depending on whether the treatment is prophylactic or therapeutic.
In, prophylactic applications, a relatively low dosage is
administered at relatively infrequent intervals over a long period
of time. Some patients continue to receive treatment for the rest
of their lives. In therapeutic applications, a relatively high
dosage at relatively short intervals is sometimes required until
progression of the disease is reduced or terminated, and preferably
until the patient shows partial or complete amelioration of
symptoms of disease. Thereafter, the patent can be administered a
prophylactic regime.
[0187] Doses for nucleic acids encoding immunogens range from about
10 ng to 1 g, 100 ng to 100 mg, 1 .mu.g to 10 mg, or 30-300 .mu.g
DNA per patient. Doses for infectious viral vectors vary from
10-100, or more, virions per dose.
[0188] Routes of Administration
[0189] Antibody compositions for inducing an immune response, e.g.,
antibodies to .beta.-hCG, for the treatment of cancer-related
conditions and diseases, e.g., metastic cancer, can be administered
by parenteral, topical, intravenous, oral, subcutaneous,
intraarterial, intracranial, intraperitoneal, intranasal or
intramuscular means for prophylactic as inhalants for antibody
preparations targeting brain lesions, and/or therapeutic treatment.
The most typical route of administration of an immunogenic agent is
subcutaneous although other routes can be equally effective. The
next most common route is intramuscular injection. This type of
injection is most typically performed in the arm or leg muscles. In
some methods, agents are injected directly into a particular tissue
where deposits have accumulated, for example intracranial
injection. Intramuscular injection on intravenous infusion are
preferred for administration of antibody. In some methods,
particular therapeutic antibodies are injected directly into the
cranium. In some methods, antibodies are administered as a
sustained release composition or device, such as a Medipad.TM.
device.
[0190] Agents of the invention can optionally be administered in
combination with other agents that are at least partly effective in
treating various diseases including various cancer-related
diseases. In the case of tumor metastasis to the brain, agents of
the invention can also be administered in conjunction with other
agents that increase passage of the agents of the invention across
the blood-brain barrier (BBB).
[0191] Formulation
[0192] Antibody compositions for inducing an immune response, e.g.,
antibodies to .beta.-hCG for the treatment of cancer-related
conditions and diseases, e.g., metastic cancer, are often
administered as pharmaceutical compositions comprising an active
therapeutic agent, i.e., and a variety of other pharmaceutically
acceptable components. (See Remington's Pharmaceutical Science,
15th ed., Mack Publishing Company, Easton, Pa., 1980). The
preferred form depends on the intended mode of administration and
therapeutic application. The compositions can also include,
depending on the formulation desired, pharmaceutically-acceptable,
non-toxic carriers or diluents, which are defined as vehicles
commonly used to formulate pharmaceutical compositions for animal
or human administration. The diluent is selected so as not to
affect the biological activity of the combination. Examples of such
diluents are distilled water, physiological phosphate-buffered
saline, Ringer's solutions, dextrose solution, and Hank's solution.
In addition, the pharmaceutical composition or formulation may also
include other carriers, adjuvants, or nontoxic, nontherapeutic,
nonimmunogenic stabilizers and the like.
[0193] Pharmaceutical compositions can also include large, slowly
metabolized macromolecules such as proteins, polysaccharides such
as chitosan, polylactic acids, polyglycolic acids and copolymers
(such as latex functionalized Sepharose.TM., agarose, cellulose,
and the like), polymeric amino acids, amino acid copolymers, and
lipid aggregates (such as oil droplets or liposomes). Additionally,
these carriers can function as immunostimulating agents (i.e.,
adjuvants).
[0194] For parenteral administration, compositions of the invention
can be administered as injectable dosages of a solution or
suspension of the substance in a physiologically acceptable diluent
with a pharmaceutical carrier that can be a sterile liquid such as
water oils, saline, glycerol, or ethanol. Additionally, auxiliary
substances, such as wetting or emulsifying agents, surfactants, pH
buffering substances and the like can be present in compositions.
Other components of pharmaceutical compositions are those of
petroleum, animal, vegetable, or synthetic origin, for example,
peanut oil, soybean oil, and mineral oil. In general, glycols such
as propylene glycol or polyethylene glycol are preferred liquid
carriers, particularly for injectable solutions. Antibodies can be
administered in the form of a depot injection or implant
preparation which can be formulated in such a manner as to permit a
sustained release of the active ingredient. An exemplary
composition comprises monoclonal antibody at 5 mg/mL, formulated in
aqueous buffer consisting of 50 mM L-histidine, 150 mM NaCl,
adjusted to pH 6.0 with HCl.
[0195] Typically, compositions are prepared as injectables, either
as liquid solutions or suspensions; solid forms suitable for
solution in, or suspension in, liquid vehicles prior to injection
can also be prepared. The preparation also can be emulsified or
encapsulated in liposomes or micro particles such as polylactide,
polyglycolide, or copolymer for enhanced adjuvant effect, as
discussed above. Langer, Science 249: 1527, 1990 and Hanes,
Advanced Drug Delivery Reviews 28: 97-119, 1997. The agents of this
invention can be administered in the form of a depot injection or
implant preparation which can be formulated in such a manner as to
permit a sustained or pulsatile release of the active
ingredient.
[0196] Additional formulations suitable for other modes of
administration include oral, intranasal, and pulmonary
formulations, suppositories, and transdermal applications.
[0197] For suppositories, binders and carriers include, for
example, polyalkylene glycols or triglycerides; such suppositories
can be formed from mixtures containing the active ingredient in the
range of 0.5% to 10%, preferably 1%-2%. Oral formulations include
excipients, such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, and
magnesium carbonate. These compositions take the form of solutions,
suspensions, tablets, pills, capsules, sustained release
formulations or powders and contain 10%-95% of active ingredient,
preferably 25%-70%.
[0198] Topical application can result in transdermal or intradermal
delivery. Topical administration can be facilitated by
co-administration of the agent with cholera toxin or detoxified
derivatives or subunits thereof or other similar bacterial toxins.
Glenn et al., Nature 391: 851, 1998. Co-administration can be
achieved by using the components as a mixture or as linked
molecules obtained by chemical crosslinking or expression as a
fusion protein.
[0199] Alternatively, transdermal delivery can be achieved using a
skin patch or using transferosomes. Paul et al., Eur. J. Immunol.
25: 3521-24, 1995; Cevc et al., Biochem. Biophys. Acta 1368:
201-15, 1998.
[0200] The pharmaceutical compositions are generally formulated as
sterile, substantially isotonic and in full compliance with all
Good Manufacturing Practice (GMP) regulations of the U.S. Food and
Drug Administration.
[0201] Diagnostic Uses
[0202] Characteristics of Antibodies and Antibody Compositions for
Use as Diagnostic Reagents. Human antibodies for use in diagnostic
methods to identify metastatic tumor cells, e.g., cells from
metastatic epithelial cancer, colorectal carcinoma, gastric
carcinoma, oral carcinoma, pancreatic carcinoma, ovarian carcinoma,
or renal cell carcinoma., are preferably produced using the methods
described above. The methods result in virtually unlimited numbers
of antibodies and antibody compositions of the invention of any
epitope binding specificity and very high binding affinity to any
desired antigen. In general, the higher the binding affinity of an
antibody for its target, the more stringent wash conditions can be
performed in an immunoassay to remove nonspecifically bound
material without removing target antigen. Accordingly, antibodies
and antibody compositions of the invention used in the above assays
usually have binding affinities of at least 10.sup.8, 10.sup.9,
10.sup.10, 10.sup.11 or 10.sup.12 M.sup.-1. Further, it is
desirable that antibodies used as diagnostic reagents have a
sufficient on-rate to reach equilibrium under standard conditions
in at least 12 hours, preferably at least five hours and more
preferably at least one hour.
[0203] Antibodies and antibody compositions of the invention used
in the claimed methods preferably have a high immunoreactivity,
that is, percentages of antibodies molecules that are correctly
folded so that they can specifically bind their target antigen.
Such can be achieved by expression of sequences encoding the
antibodies in E. coli as described above. Such expression usually
results in immunoreactivity of at least 80%, 90%, 95% or 99%.
[0204] Some methods of the invention employ polyclonal preparations
of antibodies and antibody compositions of the invention as
diagnostic reagents, and other methods employ monoclonal isolates.
The use of polyclonal mixtures has a number of advantages with
respect to compositions made of one monoclonal antibody. By binding
to multiple sites on a target, polyclonal antibodies or other
polypeptides can generate a stronger signal (for diagnostics) than
a monoclonal that binds to a single site. Further, a polyclonal
preparation can bind to numerous variants of a prototypical target
sequence (e.g., allelic variants, species variants, strain
variants, drug-induced escape variants) whereas a monoclonal
antibody may bind only to the prototypical sequence or a narrower
range of variants thereto. However, monoclonal antibodies are
advantageous for detecting a single antigen in the presence or
potential presence of closely related antigens.
[0205] In methods employing polyclonal human antibodies prepared in
accordance with the methods described above, the preparation
typically contains an assortment of antibodies with different
epitope specificities to the intended target antigen. In some
methods employing monoclonal antibodies, it is desirable to have
two antibodies of different epitope binding specificities. A
difference in epitope binding specificities can be determined by a
competition assay.
[0206] Samples and Target. Although human antibodies can be used as
diagnostic reagents for any kind of sample, they are most useful as
diagnostic reagents for human samples. Samples can be obtained from
any tissue or body fluid of a patient. Preferred sources of samples
include, whole blood, plasma, semen, saliva, tears, urine, fecal
material, sweat, buccal, skin and hair. Samples can also be
obtained from biopsies of internal organs or from cancers. Samples
can be obtained from clinical patients for diagnosis or research or
can be obtained from undiseased individuals, as controls or for
basic research.
[0207] The methods can be used for detecting any type of target
antigen. Exemplary target antigens including tumor antigens, for
example, tumor antigens for metastatic epithelial cancer,
colorectal carcinoma, gastric carcinoma, oral carcinoma, pancreatic
carcinoma, ovarian carcinoma, or renal cell carcinoma. Other target
antigens are human proteins whose expression levels or compositions
have been correlated with human disease or other phenotype.
Examples of such antigens include adhesion proteins, hormones,
growth factors, cellular receptors, autoantigens, autoantibodies,
and amyloid deposits. Other targets of interest include tumor cell
antigens, such as carcinoembryonic antigen. Other antigens of
interest are class I and class II MHC antigens.
[0208] Formats for Diagnostic Assays. Human antibodies can be used
to detect a given target in a variety of standard assay formats.
Such formats include immunoprecipitation, Western blotting, ELISA,
radioimmunoassay, and immunometric assays. See Harlow & Lane,
supra; U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,879,262;
4,034,074; 3,791,932; 3,817,837; 3,839,153; 3,850,752; 3,850,578;
3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;
3,996,345; 4,034,074; and 4,098,876, each incorporated herein by
reference in their entirety and for all purposes.
[0209] Immunometric or sandwich assays are a preferred format. See
U.S. Pat. Nos. 4,376,110; 4,486,530; 5,914,241; and 5,965,375, each
incorporated herein by reference in their entirety and for all
purposes. Such assays use one antibody or population of antibodies
immobilized to a solid phase, and another antibody or population of
antibodies in solution. Typically, the solution antibody or
population of antibodies is labelled. If an antibody population is
used, the population typically contains antibodies binding to
different epitope specificities within the target antigen.
Accordingly, the same population can be used for both solid phase
and solution antibody. If monoclonal antibodies are used, first and
second monoclonal antibodies having different binding specificities
are used for the solid and solution phase. Solid phase and solution
antibodies can be contacted with target antigen in either order or
simultaneously. If the solid phase antibody is contacted first, the
assay is referred to as being a forward assay. Conversely, if the
solution antibody is contacted first, the assay is referred to as
being a reverse assay. If target is contacted with both antibodies
simultaneously, the assay is referred to as a simultaneous assay.
After contacting the target with antibody, a sample is incubated
for a period that usually varies from about 10 min to about 24 hr
and is usually about 1 hr. A wash step is then performed to remove
components of the sample not specifically bound to the antibody
being used as a diagnostic reagent. When solid phase and solution
antibodies are bound in separate steps, a wash can be performed
after either or both binding steps. After washing, binding is
quantified, typically by detecting label linked to the solid phase
through binding of labelled solution antibody. Usually for a given
pair of antibodies or populations of antibodies and given reaction
conditions, a calibration curve is prepared from samples containing
known concentrations of target antigen. Concentrations of antigen
in samples being tested are then read by interpolation from the
calibration curve. Analyte can be measured either from the amount
of labelled solution antibody bound at equilibrium or by kinetic
measurements of bound labelled solution antibody at a series of
time points before equilibrium is reached. The slope of such a
curve is a measure of the concentration of target in a sample.
[0210] Suitable supports for use in the above methods include, for
example, nitrocellulose membranes, nylon membranes, and derivatized
nylon membranes, and also particles, such as agarose, a
dextran-based gel, dipsticks, particulates, microspheres, magnetic
particles, test tubes, microtiter wells, SEPHADEX.TM.. (Amersham
Pharmacia Biotech, Piscataway N.J.) Imobilization can be by
absorption or by covalent attachment. Optionally, antibodies can be
joined to a linker molecule, such as biotin for attachment to a
surface bound linker, such as avidin.
[0211] Labels
[0212] The particular label or detectable group used in the assay
is not a critical aspect of the invention, so long as it does not
significantly interfere with the specific binding of the antibody
used in the assay. The detectable group can be any material having
a detectable physical or chemical property. Such detectable labels
have been well-developed in the field of immunoassays and, in
general, most any label useful in such methods can be applied to
the present invention. Thus, a label is any composition detectable
by spectroscopic, photochemical, biochemical, immunochemical,
electrical, optical or chemical means. Useful labels in the present
invention include magnetic beads (e.g., Dynabeads.TM.), fluorescent
dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and
the like), radiolabels (e.g., .sup.3H, .sup.14C, .sup.35S,
.sup.125I, .sup.121I, .sup.112In, .sup.99mTc), other imaging agents
such as microbubbles (for ultrasound imaging), .sup.18F, .sup.11C,
.sup.15O, (for Positron emission tomography), .sup.99mTC,
.sup.111In (for Single photon emission tomography), enzymes (e.g.,
horse radish peroxidase, alkaline phosphatase and others commonly
used in an ELISA), and calorimetric labels such as colloidal gold
or colored glass or plastic (e.g. polystyrene, polypropylene,
latex, and the like) beads. Patents that described the use of such
labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;
3,996,345; 4,277,437; 4,275,149; and 4,366,241, each incorporated
herein by reference in their entirety and for all purposes. See
also Handbook of Fluorescent Probes and Research Chemicals,
6.sup.th Ed., Molecular Probes, Inc., Eugene Oreg.).
[0213] The label may be coupled directly or indirectly to the
desired component of the assay according to methods well known in
the art. As indicated above, a wide variety of labels may be used,
with the choice of label depending on sensitivity required, ease of
conjugation with the compound, stability requirements, available
instrumentation, and disposal provisions.
[0214] Non-radioactive labels are often attached by indirect means.
Generally, a ligand molecule (e.g., biotin) is covalently bound to
the molecule. The ligand then binds to an anti-ligand (e.g.,
streptavidin) molecule which is either inherently detectable or
covalently bound to a signal system, such as a detectable enzyme, a
fluorescent compound, or a chemiluminescent compound. A number of
ligands and anti-ligands can be used. Where a ligand has a natural
anti-ligand, for example, biotin, thyroxine, and cortisol, it can
be used in conjunction with the labeled, naturally occurring
anti-ligands. Alternatively, any haptenic or antigenic compound can
be used in combination with an antibody.
[0215] The molecules can also be conjugated directly to signal
generating compounds, e.g., by conjugation with an enzyme or
fluorophore. Enzymes of interest as labels will primarily be
hydrolases, particularly phosphatases, esterases and glycosidases,
or oxidoreductases, particularly peroxidases. Fluorescent compounds
include fluorescein and its derivatives, rhodamine and its
derivatives, dansyl, umbelliferone, and the like Chemiluminescent
compounds include luciferin, and 2,3-dihydrophthalazinediones,
e.g., luminol. For a review of various labeling or signal producing
systems which may be used, see, U.S. Pat. No. 4,391,904,
incorporated herein by reference in its entirety and for all
purposes.
[0216] Means of detecting labels are well known to those of skill
in the art. Thus, for example, where the label is a radioactive
label, means for detection include a scintillation counter or
photographic film as in autoradiography. Where the label is a
fluorescent label, it may be detected by exciting the fluorochrome
with the appropriate wavelength of light and detecting the
resulting fluorescence. The fluorescence may be detected visually,
by means of photographic film, by the use of electronic detectors
such as charge coupled devices (CCDs) or photomultipliers and the
like. Similarly, enzymatic labels may be detected by providing the
appropriate substrates for the enzyme and detecting the-resulting
reaction product. Finally simple calorimetric labels may be
detected simply by observing the color associated with the label.
Thus, in various dipstick assays, conjugated gold often appears
pink, while various conjugated beads appear the color of the
bead.
[0217] Some assay formats do not require the use of labeled
components. For instance, agglutination assays can be used to
detect the presence of the target antibodies. In, this case,
antigen-coated particles are agglutinated by samples comprising the
target antibodies. In, this format, none of the components need be
labeled and the presence of the target antibody is detected by
simple visual inspection.
[0218] Frequently, the .beta.-hCG proteins and antibodies to
.beta.-hCG will be labeled by joining, either covalently or
non-covalently, a substance which provides for a detectable
signal.
[0219] Toxicity
[0220] Preferably, a therapeutically effective dose of the antibody
compositions described herein will provide therapeutic benefit
without causing substantial toxicity.
[0221] Toxicity of the proteins described herein can be determined
by standard pharmaceutical procedures in cell cultures or
experimental animals, e.g., by determining the LD.sub.50 (the dose
lethal to 50% of the population) or the LD.sub.100 (the dose lethal
to 100% of the population). The dose ratio between toxic and
therapeutic effect is the therapeutic index. The data obtained from
these cell culture assays and animal studies can be used in
formulating a dosage range that is not toxic for use in human. The
dosage of the proteins described herein lies preferably within a
range of circulating concentrations that include the effective dose
with little or no toxicity. The dosage can vary within this range
depending upon the dosage form employed and the route of
administration utilized. The exact formulation, route of
administration and dosage can be chosen by the individual physician
in view of the patient's condition. (See, e.g., Fingl et al., 1975,
In: The Pharmacological Basis of Therapeutics, Ch. 1.
[0222] Kits
[0223] Also within the scope of the invention are kits comprising
the compositions (e.g., monoclonal antibodies, human sequence
antibodies, human antibodies, multispecific and bispecific
molecules) of the invention and instructions for use. The kit can
further contain a least one additional reagent, or one or more
additional human antibodies of the invention (e.g., a human
antibody having a complementary activity which binds to an epitope
in the antigen distinct from the first human antibody). Kits
typically include a label indicating the intended use of the
contents of the kit. The term label includes any writing, or
recorded material supplied on or with the kit, or which otherwise
accompanies the kit.
[0224] The following cDNA clones described in the specification and
further described in the examples below will be deposited with the
American Type Culture Collection, 10801 University Boulevard,
Manassas, Va. 20110-2209 under the Budapest Treaty on Aug. 8, 2006.
The hybridoma cell line for mAb 2B2.6F5 has the ATCC Patent Deposit
Designation No. indicated: PTA-7777. The hybridoma cell line for
mAb 2B3.3E8 has the ATCC Patent Deposit Designation No. indicated:
PTA-7775.
[0225] Other embodiments and uses will be apparent to one skilled
in the art in light of the present disclosures.
EXEMPLARY EMBODIMENTS
Example 1
Antigens
[0226] The immunogen for generation of monoclonal antibodies was
the conjugate of a peptide and a carrier protein. The peptide was
based on beta-hCG L2 Long Loop amino acids 38-57 (LP, Loop
Peptide). The carrier protein was diphtheria toxoid (DT, Sanofi
Aventis, Toronto, Canada). The chemical name of this antigenic
formulation is Ala-(Pro6)-beta-hCG(38-57)-Gly[Hyp.sup.39]-DT
conjugate. Hydroxyproline was substituted for proline at position
39 of the beta-hCG protein sequence. Amino acid sequence of the
beta-hCG(38-57) LP is
Ala-Pro-Pro-Pro-Pro-Pro-Pro-Cys-Hyp-Thr-Met-Thr-Arg-Val-Leu-Gln-Gly-Val-L-
eu-Pro-Ala-Leu-Pro-Gln-Val-Val-Cys. The homologous L2 Long Loop of
any other glycoprotein cystine knot growth factor could be
substituted for residues 38-57 of beta-hCG with or without
hydroxyproline substitution at position 39. Alternative carrier
proteins would also be amenable to this procedure. See U.S. Pat.
No. 6,716,428.
[0227] Loop Peptide (LP) was synthesized by standard Fmoc synthesis
methods using an automated process on an ACT synthesizer and the
manufacturer's directions (Advanced ChemTech, Louisville, Ky.).
Dimethylformamide (DMF), Dichloromethane (DCM), Trifluoroacetic
acid (TFA), 1,2 Ethanedithiol (EDT), N,N-Diisopropylethylamine
(DIEA), and Benzotriazolyl N-oxy-trisdimethylaminophosphonium
hexafluorophosphate (BOP) were obtained from Sigma (St. Louis,
Mo.). Fmoc-protected-glycine-p-alkoxybenzyl alcohol resin (Wang
resin) served as the solid support. This was prepared by washing in
DMF and DCM. Fmoc-Gly was deprotected with 20% piperidine in DMF.
After coupling, the resin was washed in DMF. Fmoc cysteine, the
next amino acid residue in the LP was coupled using DIEA in the
presence of BOP. Coupling efficiency was checked with ninhydrin. If
the coupling was incomplete, up to two additional coupling cycles
were repeated. After completion of 27 standard cycles of
deprotection, neutralisation, coupling, and washing, a sample of
peptide resin was evaluated for amino acid composition prior to
cleavage of the completed peptide mixture from the resin. Protected
peptide-resin was exposed to a Cleavage reagent consisting of
p-cresol: TFA: EDT: H.sub.2O for two hours to cleave peptide from
resin and to remove side chain protecting groups. The peptide/resin
mixture was washed successively with ethyl ether over a sintered
glass funnel. This was followed by 70% acetic acid in order to
dissolve the peptide and separate it from the resin. The crude
peptide was cyclized via formation of an intra-chain disulfide bond
by treatment with potassium ferricyanide (K.sub.3Fe(CN.sub.6)) for
20 hours at room temperature. Cyclized peptide was removed from
non-cyclized peptide by ion exchange chromatography over a
Biorex-70 cation exchange resin (Bio-Rad, Hercules, Calif.) with
70% acetic acid. Chromatographed pools of peptide were checked by
reverse phase high performance liquid chromatography (RP HPLC) to
assess peptide admixture. Peptide was purified from crude cyclized
mixture in two stages. First, low-pressure reversed phase
chromatography was performed on a C.sub.18 silica resin with a 0.1%
trifluoroacetic acid plus acetonitrile gradient. This was followed
by anion exchange chromatography over an AG-1.times.8 anion
exchange column (Bio-Rad, Hercules, Calif.) with elution using 10%
acidic acid. Aliquots were tested for purity by thin layer
chromatography and reverse phase HPLC. Peptide aliquots of adequate
purity were lyophilized to remove remaining solvent. Dry aliquots
of peptide were dissolved in USP Purified Water and shell frozen,
then lyophilized. Aliquots were then pooled, and yield was
established by weighing.
[0228] Diphtheria toxoid (DT, Sanofi-Aventis, Toronto, Canada) was
used as the carrier in the examples described. However, as is known
to those skilled in the art, many different carrier proteins could
be employed for this purpose. DT manufacture is based on toluene
treatment of a culture of Corynebacterium diphtheriae strain L34T1.
The toxin is purified, dialysed, detoxified with formaldehyde, and
concentrated by ultrafiltration. After ammonium sulfate
precipitation, toxoid is dissolved and diafiltered to remove
ammonium sulfate.
[0229] LP-DT conjugate was produced by a double two-stage process
using two different heterobifunctional linker reagents,
N-succinimidyl-3(2-pyridylthio) propionate (SPDP, Pierce Chemical,
Rockford, Ill.) and .epsilon.-maleimidocaproic acid
N-hydroxysuccinimide ester (eMCS, Sigma Aldrich Fine Chemicals, St.
Louis, Mo.). The process resulted in conjugation of lysine amino
groups in DT (via SPDP) to the amino terminus of the peptide (via
eMCS). Purified DT was reacted with SPDP to form an intermediate,
SPDP-DT. LP was reacted with eMCS to form another intermediate,
maleimido-LP (M-LP). SPDP-DT and M-LP were then reacted with each
other to form LP-DT conjugate via a thioether bond. Removal of
unreacted reagents, purification of intermediates, and buffer
exchanges were accomplished via sequential diafiltration steps.
Purified DT was adjusted to 20 mg/ml with Sodium Borate buffer, pH
9.2. SPDP was added at 10 ml/min sufficient to activate DT for
coupling at 18 moles of peptide per mole DT. The mixture was
stirred for one hour at room temperature to allow coupling of SPDP
via its activated N-hydroxysuccinimide ester to amino groups of the
DT to produce SPDP-DT. This reaction mixture was concentrated then
purified by diafiltration against 30 volumes of Citrate Coupling
Buffer (CCB, pH 6.0). Samples were assessed for pH (6.0.+-.0.2),
purity by size exclusion chromatography HPLC, protein concentration
by Lowry, and thiol quantification by
5,5'-dithio-bis-2-nitrobenzoic acid (Ellman's reagent,
Sigma-Aldrich, St. Louis, Mo.) to confirm 15-21 moles per mole of
DT. LP was reacted with the N-hydroxysuccinimidyl ester of eMCS
sufficient to produce a molar quantity of the M-LP intermediate to
react with one mole of SPDP-DT. M-LP was purified over a column of
Sephadex G10 (Pharmacia, Uppsala, Sweden). Maleimido content was
quantitated using Ellman's Reagent. SPDP-DT was reacted with M-LP
to produce LP-DT conjugate. Purified SPDP-DT solution was adjusted
with CCB to approximately 20 mg/ml. Sufficient M-LP Peptide at 50
mg/ml in CCB was added at 10 ml/min to couple 18 moles of peptide
per mole of DT. The reaction mixture was stirred for at least 6
hours at RT to allow coupling of LP via the maleimido of its
C-terminal glycine residue to the thiol moiety of SPDP-DT to
produce the LP-DT conjugate. The conjugate reaction mixture was
then concentrated to approximately 30 mg/ml and purified by
diafiltration against 15 volumes of PBS (pH 7.2). pH was tested
(7.2.+-.0.2). Purity was confirmed by SEC HPLC. Purified LP DT was
then filtered through a sterile 0.22 .mu.m filter (Millipore,
Billerica, Mass.) and adjusted with sterile PBS (pH 7.2) to produce
a final bulk LP-DT concentrate that was lyophilized prior to
storage.
Example 2
Immunizations
[0230] As is known to those skilled in the art, female C57BL/6 mice
(Charles River Laboratories, Wilmington, Mass.) were selected for
immunizations due to the known propensity of this inbred mouse
strain to generate a humoral response and hybridomas specific for
beta-hCG. All procedures involving animals were reviewed by an
Institutional Animal Care and Use Committee. Mice were immunized in
groups of three. All immunizations were subcutaneous or
intramuscular in either one or two sites. LP-DT conjugate was
solubilized in sterile water then thoroughly emulsified in either
Complete Freund's Adjuvant (CFA; Sigma-Aldrich, St. Louis, Mo.) or
Incomplete Freund's Adjuvant (IFA; Sigma-Aldrich, St. Louis, Mo.).
CFA was vortexed prior to use. Emulsification was performed by
mixing the aqueous immunogen solution and either CFA or IFA between
two 1 ml glass syringes connected by a luer lock, approximately 20
times, until the mixture became milky white and became difficult to
push. Final immunogen concentration was 0.5 milligrams per
milliliter.
[0231] In the first hybridoma generation (Fusion 1), initial
immunizations were with 0.1 mg of LP-DT conjugate in CFA. Each of
two sites were injected subcutaneously with 0.1 milliliter of
emulsified immunogen via a 25 gauge needle (Becton-Dickinson,
Franklin Lakes, N.J.). Subsequent immunizations were performed at
two-week intervals with 0.05 milligrams of loop-DT conjugate in
IFA, each time injected to two sites. Two weeks after the fourth
immunization, tail bleeds were performed to assess binding capacity
of serum from each mouse. Mice were mobilized individually in a
restraining device, and the tail was heated for a minute or so
under an infrared lamp. After swabbing with alcohol, the mouse tail
was lanced with a scalpel and several drops of blood were obtained.
Blood was incubated for one hour at 37.degree. C. Each tube was
then flicked to dislodge the blood clot prior to storage overnight
at 4.degree. C. Tubes were spun at 10,000 g, and serum was
transferred from each tube to a separate container. Sera were
frozen at -20.degree. C. prior to screening.
[0232] In a second experiment (Fusion 2), mice were immunized on
three occasions separated by four week intervals. As previously,
the initial immunization was with 0.10 milligrams per mouse,
whereas subsequent immunizations were with 0.5 milligrams per
mouse. Priming and other procedures were the same.
Example 3
Radioimmunoassays
[0233] Serum, culture supernatant, or purified antibody samples
were diluted in Phophate Buffered Saline (PBS) containing 10% mouse
serum (MS) and 1 millimolar ethylenediaminetetraacetate (EDTA).
Initial dilutions for screening purposes were 1:10. Other reagents
include PBS containing 1% (w/v) bovine serum albumin (BSA);
.sup.125I hCG at 2.5 ng/ml; assay controls with representative
rabbit antisera specific for hCG having high, medium, and low
antibody levels revealed by prior testing; non-specific monoclonal
antibody serum; PBS-EDTA containing 40% calf serum (40% CS); and
25% (w/v) polyethylene glycol (PEG). .sup.125I hCG was prepared by
the Chloramine T method.
[0234] 100 microliters of PBS containing 1% (w/v) bovine serum
albumin (BSA) was added in quadruplicate to sets of four
10.times.75 mm disposable glass tubes. 100 microliters of .sup.125I
hCG at 2.5 nanograms/milliliter was then dispensed into all sets of
tubes, as well as to an additional single set of four empty tubes
(to serve as total count tubes). 100 microliters of diluted sample
was then added to quadruplicate sets of tubes. Sets of tubes were
included with non-specific monoclonal antibody serum as a negative
control and the three representative anti-hCG antisera as positive
controls. Tubes were shaken gently to mix, placed in plastic finger
racks, then covered with parafilm, followed by aluminum foil. Racks
of covered plastic tubes were incubated for 16-24 hours at
4.degree. C. Tubes were then uncovered, and to each tube was added
100 microliters of PBS-EDTA-40% CS, followed by 400 microliters of
25% PEG. Subsequent vortexing of tubes was followed by incubation
for 15 minutes at room temperature. Tubes were then centrifuged for
20 minutes at 4.degree. C. and 1500.times.g. Liquid was decanted,
and radioactivity remaining within tubes was counted in a gamma
spectrometer for at least one minute per tube.
[0235] Mean counts per minute were calculated for each
quadruplicate set of tubes. This was corrected by subtraction of
the mean count from the tubes with non-specific monoclonal antibody
serum. The bound/free (B/F) ratio was calculated for each dilution.
(Total Counts-Bound=Free counts)
[0236] Receptor Binding Assay. Testes from adult male rats were
decapsulated and torn apart in PBS using 19 gauge needles on 2.0 ml
syringes. The mass of dispersed material was stirred for five
minutes, filtered through nylon mesh and cotton wool, then
centrifuged at 120 g for twenty minutes. Homogenate equivalent to
100 micrograms protein was transferred a 10.times.75 mm tube. 100
microliters of .sup.125I hCG at 2.5 nanograms/milliliter was then
dispensed into all sets of tubes, as well as to an additional
single set of four empty tubes (to serve as total count tubes). 100
microliters of diluted sample was then added to quadruplicate sets
of tubes. A standard curve was constructed with 5, 10, 25, 50, and
100 ng unlabeled hCG. Additional sets of tubes were prepared with
increasing concentrations of monoclonal antibody. Tubes were shaken
gently to mix, placed in plastic finger racks, then covered with
parafilm, followed by aluminum foil. Racks of covered plastic tubes
were incubated for 16-24 hours at 4.degree. C. 100 microliters of
PBS-EDTA-40% CS was then added to each tube, followed by 400
microliters of 25% PEG. Tubes were vortexed then incubated for 15
minutes at room temperature. After centrifugation at 1500.times.g
for 20 minutes at 4.degree. C., liquid was decanted. Pellet
radioactivity was counted in a gamma spectrometer for at least one
minute per tube per tube. Mean counts per minute were calculated
for each quadruplicate set of tubes.
Example 4
Cell Lines and Culture and Hybridoma Generation
[0237] NS-1 murine myeloma cells and three human cell lines with
the following phenotypic characteristics were obtained from the
American Type Culture Collection (ATCC), Manassas, Va. First,
BXPC-3 human pancreatic carcinoma cells produce beta-hCG protein
but do not express the LH/hCG receptor to which the hCG heterodimer
binds. Second, MCF-7 human breast carcinoma cells by contrast
produce little or no beta-hCG protein but do express the LH/hCG
receptor to which the hCG heterodimer (but not the beta-hCG chain
alone) binds. Finally, HeLa human cervical carcinoma cells produce
both the common alpha chain of the human heterodimeric glycoprotein
family and the LH/hCG receptor.
[0238] NS-1 myeloma and hybridoma cells were cultured in RPMI-1640
(Sigma-Aldrich, St. Louis, Mo.). Media was supplemented with 10%
fetal bovine serum (FBS), 100 units/milliliter penicillin, 0.1
milligrams/milliliter streptomycin, and 2 mM L-glutamine. For
initial hybridoma culture following fusion and for limiting
dilution subcloning, 20% FBS was employed. RPMI was also
supplemented with penicillin at a final concentration of 100 units
per milliliter; streptomycin at 100 micrograms per milliliter.
Hybridoma selection media was made by addition of Hybri-Max HAT
Media Supplement (Sigman-Aldrich Biotechnology, St. Louis, Mo.) to
RPMI 1640 with 10% FBS, penicillinj, streptomycin, and L-glutamine
to obtain a final working concentration of 100 .mu.M hypoxanthine,
0.4 .mu.M aminopterin, and 16 .mu.M thymidine. BXPC-3 and MCF-7
cells were cultured in RPMI 1640 with 10% FBS,
penicillin-streptomycin, and L-glutamine. HeLa cells were cultured
in DMEM with 10% FBS and penicillin-streptomycin. Cell culture
media were obtained from Mediatech, Inc. (Herndon, Va.) or
Sigma-Aldrich (St. Louis, Mo.).
[0239] Cell culture was performed at 37.degree. C. in a humidified
atmosphere of 5% CO.sub.2. Once cell concentration in culture
reached approximately 10.sup.6 cells per milliliter, a 1:10 or 1:20
dilution with fresh media was performed. Cell freezing was
performed as follows. Rapidly dividing cells in good health were
transferred to a sterile, chilled centrifuge tube and spun at 400 g
for five minutes at 4.degree. C. Supernatant was decanted, and the
pellet was resuspended with 75% RPMI/20% FBS/5% DMSO sufficient to
generate a final cell concentration of approximately 10.sup.7 cells
per milliliter. 0.5 ml aliquots of this suspension were distributed
to freezing vials on ice. Vials were stored in a freezing rack at
-70.degree. C. overnight, followed by long term storage at
-185.degree. C. in liquid nitrogen. Cells are thawed by warming a
frozen vial in a 37.degree. C. water bath. After washing with RPMI
1640/10% FBS at room temperature, cells are resuspended 10 ml of
the same media and cultured at 37.degree. C.
[0240] Hybridoma Generation. Polyethylene glycol 1500 (PEG 1500,
Boehringer Mannheim, Indianapolis, Ind.) and fetal bovine serum
(FBS, Invitrogen/GIBCO, Carlsbad, Calif.) were pre-screened for
capacity to support cell fusion and hybridoma growth, respectively.
NS-1 myeloma cells were confirmed to be free of mycoplasma via a
mycoplasma testing service (Bionique Testing Laboratories, Saranac
Lake, N.Y.).
[0241] Mice received an antigenic boost with 0.05 .mu.g LP-DT
conjugate via the intravenous (tail) route four days prior to
fusion. This was intended to induce B lymphocyte cell cycling, as
well as to promote migration of B lymphocytes to the spleen. On the
day of cell fusion, 0.5 gram PEG 1500 was melted in a 50.degree. C.
water bath, combined with 0.5 ml of unsupplemented RPMI, and
maintained in a 37.degree. C. water bath. Animals were sacrificed
by cervical dislocation. The spleen was removed aseptically and
placed in a 100 mm tissue culture plate that contained 10 ml of
unsupplemented RPMI 1640 at 37.degree. C. The spleen was torn apart
into small pieces using 19-gauge needles on 2.0 ml syringes until
most cells were released. Cell clumps were disrupted by pipetting.
Cells and media were transferred to a sterile 50-ml-polystyrene
centrifuge tube. The tissue culture plate was washed with an
additional 10 ml of unsupplemented RPMI 1640, which was then added
to the contents of the centrifuge tube. After three minutes,
supernatant was pipetted away from settled debris to a new sterile
50-ml-centrifuge tube. Splenocytes so obtained were washed twice in
unsupplemented, prewarmed RPMI 1640 and centrifuged at 400 g for 5
minutes. Log phase myeloma cells were washed once in unsupplemented
RPMI 1640. Washed splenocytes and myeloma cells were counted
visually using a Neubauer chamber hemocytometer (Reichert
Scientific Instruments, Buffalo, N.Y.). Up to 1,000,000,000
splenocytes were then combined with 20,000,000 NS-1 myeloma cells
and centrifuged at 400 g for five minutes. The 50% PEG 1500
solution at 37.degree. C. was transferred slowly over one minute to
the splenocyte-myeloma cell pellet with simultaneous cell
resuspension by use of a sterile Pasteur pipet. After one minute of
gentle stirring, ten ml of prewarmed, unsupplemented RPMI 1640 was
added over the ensuing two minutes. The cells were centrifuged at
400 g for five minutes, decanted, and resuspended using 50 ml of
RPMI 1640 supplemented with 20% FBS, penicillin-streptomicin,
L-glutamine, and HAT. 0.5 ml of HAT selection media with cells was
transferred to each well of 4 24-well tissue culture plates. Cells
were fed the next day with and equal volume of 2.times.HAT
selection media. 6 days later cells were fed again with 1.times.HAT
selection media, and supernatants were harvested from wells with
visible colonies of cells. Intermittent supernatant harvesting
continued for about two weeks as additional cell colonies became
visible. Supernatants were screened as described in Example 3
(Radioimmunoassays). Wells with supernatants positive in the
initial screen were rescreened. Those wells shown reproducibly to
bind heterodimeric hCG were expanded to 10 ml of culture and
frozen.
[0242] Limiting Dilution Subcloning. 96-well plates were prepared
with RPMI supplemented with 10% FBS, penicillin, streptomycin, and
L-glutamine. Hybridoma cells were counted then resuspended in 20
milliliters of media at 20, 10, and 2 cells/milliliter. Each cell
suspension was then plated to two 96-well plates (Fisher
Scientific, Ottawa, Ontario, Canada) at 100 microliters per well
using a multichannel pipettor (Fisher Scientific, Ottawa, Ontario,
Canada). Six days later cells were fed with the same media. Wells
with apparently clonal populations of cells were screened either by
radioimmunoassay as described or by use of an hCG enzyme-linked
immunosorbent assay (Rock, E. P., et al. Immunogenicity of a fusion
protein linking the beta subunit carboxyl terminal peptide (CTP) of
human chorionic gonadotropin to the B subunit of Escherichia coli
heat-labile enterotoxin (LTB).Vaccine. 14: 1560-1568. 1996).
Detection was via horseradish peroxidase-conjugated Rabbit
Anti-Goat IgG Heavy and Light Chain antibodies (Bethyl
Laboratories, Montgomery, Tex.), followed by Chemiluminescent
Peroxidase Substrate for ELISA (Sigma-Aldrich, St. Louis).
[0243] Isotype Determination Cryopreservation, and Mycoplasma
Testing. Isotype determination was performed for individual
monoclonal antibodies with the Immunotype Mouse monoclonal antibody
typing kit (Sigma Chemical Co., St. Louis, Mo.) by following the
manufacturer's instructions in an ELISA format with one microgram
of Protein G purified monoclonal antibody per well of a 96-well
plate as the solid phase. Monoclonal antibodies of known isotype
were used as controls. Freezing media for cryopreservation
contained 75% RPMI, 20% FBS, and 5% dimethyl sulfoxide (DMSO)
(Sigma-Aldrich, St. Louis, Mo.). For cryopreservation, cells were
spun at 4.degree. C., then resuspended slowly in freezing medium at
20,000,000 cells/milliliter and aliquoted to freezing vials. Vials
were frozen slowly in Styrofoam boxes to -70.degree. C. then
transferred one day later to liquid nitrogen. Mycoplasma testing
was performed by Bionique Testing Laboratories (Saranac Lake,
N.Y.).
[0244] Gene Sequencing. Cytoplasmic RNA was obtained from hybridoma
cells using a Cytoplasmic and Nuclear RNA Purification Kit from
Norgen Biotek (St Catherines, Ontario, Canada) via the
manufacturer's protocol. Full-length poly(A) RNA was then selected
and prepared for amplification using the Ambion FirstChoice
RLM-RACE Kit (Applied Biosystems, Foster City, Calif.) via the
manufacturer's protocol. 3' primers were obtained from Invitrogen
(Carlsbad, Calif.). Nested PCR was performed, and the inner
reaction was run on an agarose gel. A band of the correct predicted
fragment size was excised and extracted with a Gel Extraction Kit
(Qiagen, Valencia, Calif.) via the manufactuerer's protocol.
Purification from contaminating primers, nucleotides, DNA
polymerase, oil, and salts was performed using the GenElute PCR
Clean-Up Kit (Sigma-Aldrich, St. Louis, Mo.). DNA sequencing was
then performed on an Applied Biosystems sequencer (Foster City,
Calif.) via the manufacturer's protocol.
[0245] Monoclonal Antibody Production and Purification. Hybridoma
cells were grown in 250 ml RPMI with 10% FBS, penicillin,
streptomycin, and L-glutamine using Corning polystyrene roller
bottles revolving at two revolutions per minute (Fisher Scientific,
Ottawa, Ontario, Canada). As the culture reached saturation of
about 1,000,000 cells per milliliter, cells were removed by
centrifugation. Antibody was concentrated by ammonium sulfate
precipitation followed by resuspension and dialysis overnight at
4.degree. C. to 20 millimolar sodium phosphate, pH 7.0. Subsequent
antibody purification was performed using Hi-Trap Protein G columns
(GE Healthcare/Amersham Biosciences, Uppsala, Sweden) via the
manufacturers protocol. Elution from the column was with 0.1 molar
glycine-HCl, pH 2.7. Following elution, the eluate was subsequently
buffered with 1.0 molar Tris to pH 7.0, followed by dialysis
overnight at 4.degree. C. to phosphate-buffered saline (PBS) using
12,000 molecular weight cut-off tubing. Antibodies were stored at
4.degree. C. or frozen in either PBS or water with 50%
glycerol.
Example 5
Cancer Cell Proliferation and Xenografts
[0246] Effects on cancer cell proliferation were assessed as
follows. 96-well plates were seeded with 10.sup.3 cells per well in
culture media. Either 2 or 20 micrograms of purified monoclonal
antibody was added to each well. Murine monoclonal antibody,
muromonab-CD3 (OKT3; Orthoclone OKT3, Ortho Biotech Products, L.P.,
Bridgewater N.J.), was used as a negative control. Cells were
cultured for 72 hours prior to harvesting for measurement of cell
proliferation by two methods.
[0247] The MTS calorimetric assay measures cellular reductive
capacity of NADH and NADPH. These moieties are produced by
dehydrogenases in metabolically active cells and decrease with
declining cell viability. MTS reagent was added to 96-well cell
cultures as recommended by the manufacturer (CelTiter 96 AQueous
Non-Radioactive Cell Proliferation Assay; Promega, Madison Wis.),
and absorbance was read at 492 nm. Data are presented as mean %
inhibition [(1-(experimental absorbance-background
absorbance)/(absorbance of control cultures-background
absorbance)).times.100].+-.SD for triplicate determinations.
[0248] Intracellular ATP concentration was determined with the
ATPlite.TM. Luminescence ATP Detection Assay System (PerkinElmer
Life Sciences, Wellesley, Mass.) according to the manufacturer's
directions. The procedure is described in U.S. patent application
Ser. No. 09/806,165, which is incorporated as a reference herein.
Advantages of this assay include sensitivity, linearity, and
simplicity. In this test, ATP is converted to light by firefly
(Photinus pyralis) luciferase. The light generated can be
quantified as counts per second (cps) in a luminescence counter.
Care was taken to avoid contamination of reagents in this kit with
ATP present in the environment, e.g. on the hand. Gloves are worn
at all times during the procedure. 50 microliters of the
manufacturer's cell lysis solution is added to 100 microliters of
cell suspension in wells of a 96-well microplate. The plate is
incubated for five minutes on an orbital shaker operating at 700
revolutions per minute (rpm). 50 microliters of the manufacturer's
substrate solution is then added to wells containing cell lysates,
and the plate is again incubated for five minutes in an orbital
shaker at 700 rpm. The plate is dark adapted for at least ten
minutes, following which luminescence is read in either of two
PerkinElmer luminometers. In each experiment, only one type of
luminometer was used. Data in FIGS. 8 and 9 are presented as mean %
inhibition [(1-experimental cps/control cps).times.100%].+-.SD for
triplicate determinations.
[0249] Human-Mouse Tumor Xenografts. Seven-week-old NCR male
athymic nude homozygous (nu/nu) mice were purchased from Taconic
(Germantown, N.Y.). Four groups of ten mice per group were
inoculated subcutaneously on the flank in the mid-axillary line
with 2.times.10.sup.6 BXPC-3 cells on Day 1. A negative control
group subsequently received no treatment (NT). The docetaxel group
received intraperitoneal docetaxel at 30 milligrams/kilogram body
weight in three treatments at 1-week intervals starting on Day 10.
The 6F5 group received 100 micrograms of anti-beta-hCG L2 loop
monoclonal antibody 6F5 in six treatments at twice weekly intervals
starting on Day 6. A 6F5+docetaxel group received both docetaxel
and monoclonal antibody 6F5 at the above doses and intervals. Mice
were assessed twice weekly for bidimensional size of human tumor
xenografts by use of a digital microcaliper device. Tumor volume in
cubic millimeters was calculated for each mouse by use of the
formula (length.times.width.sup.2)/2. Data are expressed for groups
as the mean tumor volume+/-the standard error of the mean.
Example 6
LP-DT Conjugate Generates Monoclonal Antibodies Specific for the
Beta-hCG L2 Loop
[0250] Three female C57BL/6 mice were immunized with LP-DT
conjugate on four occasions at three week intervals prior to Fusion
1. Similar mice were immunized on three occasions at three week
intervals prior to Fusion 2. The mouse with the highest RIA titer
against hCG after each series of immunizations was sacrificed for
fusion with NS-1 cells and consequent hybridoma production. Fusion
1 yielded monoclonal antibodies 2B3.3E8 (3E8), 2B3.3F5 (3F5), and
2B3.6A11 (6A11). Fusion 2 yielded 2B2.6F5 (6F5). Specific binding
to .sup.125I hCG was verified after growth and purification of all
monoclonal antibodies. All four of these antibodies were found to
have an IgG1 isotype.
[0251] Cross-reactivity to LH was assessed by RIA via competition
experiments with unlabeled LH. Standard curves with unlabeled hCG
were constructed for each antibody and compared to curves generated
using LH. The relative amount of LH to generate an equivalent drop
in counts to that of the hCG standard curve was used to establish
the degree of cross-reactivity. A similar experiment was performed
with antibody 6F5 from Fusion 2. However, in the latter instance
culture supernatant of undocumented concentration was tested.
Results are shown in FIG. 6. Fusion 1 antibodies 3E8, 3F5, and 6A11
displayed 8.6%, 12%, and 55% cross-reactivity for LH, respectively.
Fusion 2 antibody 6F5 displayed 4.6% cross-reactivity.
[0252] These results demonstrate that immunization with the LP-DT
conjugate is able to lead to generation of monoclonal antibodies
with strong preferential binding to hCG over LH despite only a one
amino acid difference between these two proteins in the
surface-accessible residues of the beta-hCG L2 loop.
[0253] Monoclonal antibody 2B2.6F5 was grown in greater quantity
for a xenograft experiment. In one production run, 781 milliliters
of culture supernatant yielded 23 mg of antibody by Protein G
purification. In a second such production experiment with 2B2.6F5
after limiting dilution subcloning, 1.5 liters of culture yielded
48 milligrams of purified antibody.
Example 7
Anti-Beta-L2 Loop Antibodies Block Binding of hCG to LH/hCG
Receptor
[0254] Iodinated hCG (0.25 ng hCG) was incubated with approximately
0.2 milligrams of rat testicular extract in duplicate tubes. This
resulted in 28% of the labeled hCG being bound. This was then
considered to represent 100% binding to available receptors. FIG. 7
shows as Series 1 an unlabeled hCG standard curve constructed using
doses of 5, 10, 25, 50 and 100 ng hCG, all in duplicate tubes with
rat testicular extract and 0.25 ng .sup.125I hCG. As expected, this
curve demonstrates that unlabeled hCG competes with labeled hCG for
binding to the LH/hCG receptor. Ascending doses of monoclonal
antibody 3E8 were then tested for their ability to abrogate
inhibition of 0.25 ng .sup.125I hCG binding to the LH/hCG receptor
by 100 ng unlabeled hCG. 100, 200, 400 and 800 ng of monoclonal
antibody 3E8 resulted in 12%, 18%, 21% and 29% of control binding,
respectively. Reading from the standard curve, these effects imply
ng hCG binding inhibition of 9, 24, 30, and 50 ng, respectively.
This is shown in FIG. 7 as Series 2. Thus on average monoclonal
antibody 3E8 neutralized 88 micrograms of hCG per milligram of
antibody. These results demonstrate that a monoclonal antibody
specific for the beta-hCG L2 loop demonstrates dose dependent
inhibition of hCG binding to the LH/hCG receptor.
Example 8
Effects of Anti-Beta-hCG Antibodies on the Growth of Human Tumor
Cells
[0255] The ability of three anti-beta-hCG antibodies targeting the
L2 long loop to modulate the growth of cancer cell lines in vitro
was examined. Monoclonal antibodies 3F5, 3E8, 6A11 were generated
as described in examples above. Each has both specificity for the
L2 long loop of beta-hCG and an IgG1 isotype. A negative control
monoclonal antibody was also tested. The negative control was
murine monoclonal antibody muromonab-CD3 (OKT3; Orthoclone OKT3;
Ortho Biotech Products, L.P., Bridgewater, N.J.). These four
antibodies were each tested for in vitro activity against the
following three cancer cell lines. BXPC-3 pancreatic cancer cells
produce beta-hCG but do not express the LH/hCG receptor. MCF-7
breast cancer cells produce relatively little beta-hCG but do
express the LH/hCG receptor. HeLa cervical cancer cells produce
alpha-hCG and express the LH/hCG receptor but not produce
beta-hCG.
[0256] 1.0.times.10.sup.3 cells were incubated in triplicate with
either 2 or 20 .mu.g of purified monoclonal antibody for 72 hours
prior to cell proliferation assays. Two cell proliferation assays
with employed. These were based on either cellular reductive
capacity (MTS) or intracellular ATP concentration (ATPlite), as
described in examples above.
[0257] As shown in FIG. 8, each of the anti-beta-hCG monoclonal
antibodies inhibited in vitro proliferation of BXPC-3 pancreatic
carcinoma cells, known to produce and secrete beta hCG. Inhibition
of proliferation was dose dependent with each antibody. In each
instance the effect on intracellular ATP concentrations (ATPlite)
was greater than the effect on cellular reductive capacity by MTS.
By contrast none of the three monoclonal antibodies had any
discernible effect on proliferation of either MCF-7 or HeLa cells.
As expected, OKT3 had no inhibitory effect on any of the three cell
lines (data not shown). Thus anti-beta-hCG antibodies that target
the L2 long loop are able as single agents to inhibit proliferation
of cells that express beta-hCG but not cells that don't express
beta-hCG.
Example 9
Anti-Beta-hCG L2 Loop Antibodies Synergize with Cytotoxic
Chemotherapy
[0258] Ability of an anti-beta-hCG antibody targeting the L2 long
loop to synergize with cytotoxic chemotherapy in modulating growth
of cancer cell lines in vitro was also examined. Monoclonal
antibody 6F5 was generated as described in examples above. This
antibody binds with specificity for the L2 long loop of beta-hCG
and also has an IgG1 isotype. A negative control monoclonal
antibody was also tested. The negative control was murine
monoclonal antibody muromonab-CD3 (OKT3; Orthoclone OKT3; Ortho
Biotech Products, L.P., Bridgewater, N.J.). OKT3 is specific for
the CD3 antigen found on human T lymphocytes and does not bind to
the epithelial cancer cells described below. Monoclonal antibodies
6F5 and OKT3 were tested in combination with two cytotoxic
chemotherapy drugs approved by the U.S. Food and Drug
Administration (FDA) for treatment of pancreatic and/or breast
cancer. Gemcitabine (Gemzar.RTM., Eli Lilly and Company,
Indianapolis, Ind.), an antimetabolite, is a fluorine-substituted
deoxycitidine analog that received FDA approval for treatment of
both advanced pancreatic and metastatic breast cancer. Docetaxel
(Taxotere.RTM., Sanofi-Aventis, Bridgewater, N.J.) targets
microtubules and is a semi-synthetic taxane that received FDA
approval for treatment of breast cancer after failure of prior
chemotherapy. BXPC-3 pancreatic cancer cells produce beta-hCG but
do not express the LH/hCG receptor. MCF-7 breast cancer cells
produce relatively little beta-hCG but do express the LH/hCG
receptor.
[0259] 1.0.times.10.sup.3 cells were incubated in triplicate with
20 .mu.g of purified 6F5 or OKT3 for 24 hours prior to addition of
gemcitabine or docetaxel. Final docetaxel concentrations were 0, 1,
3, 10, 30, and 100 nanograms per milliliter. Final gemcitabine
concentrations were 0, 5, 10, 20, 40, 80, and 160 nanomolar. After
48 hours of culture in the presence of cytotoxic chemotherapy, cell
proliferation was assessed by an assay of intracellular ATP
concentration (ATPlite), as described in examples above. Data shown
in FIG. 9 are presented as mean percent inhibition of total
intracellular ATP for triplicate assays. The data show that an
anti-beta-hCG monoclonal antibody which targets the L2 long loop
synergizes with cytotoxic chemotherapy in decreasing cell
proliferation of cancer cells that are known to produce and secrete
beta-hCG.
[0260] Pre-treatment with monoclonal antibody 6F5 increased
anti-proliferative activity of docetaxel against BXPC-3 cells
(p=0.00001, paired t test). Anti-proliferative activity of the
antimetabolite gemcitabine was not significantly enhanced. As
expected, monoclonal antibody 6F5 increased anti-proliferative
activity of neither cytotoxic agent against MCF-7 cells, which do
not produce beta-hCG. Taken together, these results indicate that
an anti-beta-hCG monoclonal antibody targeting the L2 long loop
synergizes with microtubule-targeting, cytotoxic chemotherapy
(docetaxel) in diminishing cell proliferation of human cancer cells
that produce beta-hCG.
Example 10
Anti-Beta-hCG L2 Loop Antibodies Inhibit Human-Mouse Tumor
Xenografts
[0261] Ability of an anti-hCG antibody targeting the L2 long loop
to modulate growth of cancer cells in vivo was also examined.
Monoclonal antibody 6F5 was generated as described in examples
above. Monoclonal antibody 6F5 was tested in combination with
docetaxel (Taxotere.RTM., Sanofi-Aventis, Bridgewater, N.J.), a
semi-synthetic taxane that targets microtubules. BXPC-3 pancreatic
cancer cells produce beta-hCG but do not express the LH/hCG
receptor. Seven-week-old NCR female athymic nude homozygous (nu/nu)
mice (Taconic, Germantown, N.Y.) were inoculated with BXPC-3 human
pancreatic carcinoma xenografts. Four experimental groups of ten
mice per group were studied. Groups include animals not treated
(NT) and animals treated with docetaxel, anti-beta-hCG monoclonal
antibody 6F5 alone, or antibody 6F5 plus docetaxel.
[0262] FIG. 10 shows that an anti-beta-hCG monoclonal antibody
which targets the L2 long loop is able to diminish tumor growth in
vivo, both by itself and in synergy with cytotoxic
chemotherapy.
[0263] 2.times.10.sup.6 cultured BXPC-3 tumor cells were inoculated
into the flank in the mid-axillary line of each mouse on Day 1.
Animals receiving antibody were given 100 micrograms purified 6F5
via the intraperitoneal route on each of Days 6, 9, 13, 16, 20, and
23. This is twice weekly for three weeks starting on Day 6. Animals
receiving chemotherapy were given 30 mg/kg docetaxel via the
intraperitoneal route on each of Days 10, 17, and 24. This is
weekly for three weeks starting on Day 10. Tumors of all mice were
measured bidimensionally with calipers every two to three days.
Tumor volume was calculated using the formula
(length.times.width.sup.2)/2. Data shown in FIG. 10 are expressed
as mean tumor volume.+-.standard error of the mean (SEM, N=10).
[0264] Treatment of mice with the anti-beta-hCG monoclonal antibody
6F5 alone resulted in significant anti-tumor activity in vivo
relative to the NT negative control group. (p=0.000001, paired t
test). Furthermore, treatment of mice bearing xenografts with
antibody 6F5 also improved the anti-tumor activity of docetaxel in
vivo relative to docetaxel alone (p=0.00001, paired t test). These
results confirm and extend those obtained in vitro. We have shown
that a monoclonal antibody directed against the L2 long loop of
beta-hCG by itself generates significant anti-tumor activity in
vivo against human pancreatic cancer. This anti-tumor activity is
coincident with ability of the antibody to block binding of
beta-hCG to a cognate receptor on the surface of cancer cells.
Furthermore, the monoclonal antibody described synergizes with a
separate agent, docetaxel. Docetaxel acts by a distinct mechanism,
interference with microtubule function, and is known to be
effective for the treatment of cancer. Thus the monoclonal antibody
described offers the prospect for generation of a non-toxic cancer
therapy that will supplement those already of demonstrable
efficacy.
Example 11
DNA and Amino Acid Sequences of Beta-hCG L2 Loop Monoclonal
Antibody Heavy Chain
[0265] FIG. 11 shows DNA and amino acid sequences of mAb 2B2.6F5
heavy chain DNA and amino acid sequence, mAb 2B3.3E8 heavy chain
DNA and amino acid sequence, and mAb 2B3.3F5 heavy chain DNA and
amino acid sequence. The open reading frame is highlighted. mAb
2B3.3E8 heavy chain amino acid sequence, and mAb 2B3.3F5 heavy
chain amino acid sequence show an identical heavy chain
sequence.
[0266] When ranges are used herein for physical properties, such as
molecular weight, or chemical properties, such as chemical
formulae, all combinations and subcombinations of ranges and
specific embodiments therein are intended to be included.
[0267] The disclosures of each patent, patent application and
publication cited or described in this document are hereby
incorporated herein by reference in their entirety.
[0268] Those skilled in the art will appreciate that numerous
changes and modifications can be made to the embodiments of the
invention and that such changes and modifications can be made
without departing from the spirit of the invention. It is,
therefore, intended that the appended claims cover all such
equivalent variations as fall within the true spirit and scope of
the invention.
Sequence CWU 1
1
10 1 458 DNA Homo sapiens 1 tgtccatgtc ctctccacag acactgaaca
cactgactct aaccatgaga tggagctgga 60 tctttctctt cctcctgtca
ggaactgcag gtgtccactc tgaggtccac ctgcaacagt 120 ctggacctgt
gctggtgaag cctggggctt cagtgaagat gtcctgtaag gcttctggat 180
acacattcac tgactactat atgacctggg tgaagcagag ccatgaaaag agccttgagt
240 ggattggaat tattgatcct tataacggtg atactagcta caaccagaag
ttcatgggca 300 aggccacatt gactgttgac atgtcctcca gcacagccta
catggagctc aacagcctga 360 catctgacga ctctgcagtc tattactgtg
caagagacat tgactactgg ggccgcggca 420 ccactctcac cgtctcccca
gctagcacaa caccccca 458 2 151 PRT Homo sapiens 2 Met Ser Ser Pro
Gln Thr Leu Asn Thr Leu Thr Leu Thr Met Arg Trp 1 5 10 15 Ser Trp
Ile Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly Val His Ser 20 25 30
Glu Val His Leu Gln Gln Ser Gly Pro Val Leu Val Lys Pro Gly Ala 35
40 45 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Tyr 50 55 60 Tyr Met Thr Trp Val Lys Gln Ser His Glu Lys Ser Leu
Glu Trp Ile 65 70 75 80 Gly Ile Ile Asp Pro Tyr Asn Gly Asp Thr Ser
Tyr Asn Gln Lys Phe 85 90 95 Met Gly Lys Ala Thr Leu Thr Val Asp
Met Ser Ser Ser Thr Ala Tyr 100 105 110 Met Glu Leu Asn Ser Leu Thr
Ser Asp Asp Ser Ala Val Tyr Tyr Cys 115 120 125 Ala Arg Asp Ile Asp
Tyr Trp Gly Arg Gly Thr Thr Leu Thr Val Ser 130 135 140 Pro Ala Ser
Thr Thr Pro Pro 145 150 3 457 DNA Homo sapiens 3 agttgtagtt
accatagtag catcttgcac agaaatatgt agccgtgtcc tcatttttga 60
ggttgttgat ctgcaaatag gcagtgctgg cagaggtttc caaagagaag gcaaaccgtc
120 ccttgaagtc atcagcatat gttggcactc cagagtaggt gtttatccag
cccatccact 180 ttaaaccctt tcctggagcc tgtttcaccc agctcattcc
ataggttgtg aaggtatacc 240 cagaagcctt gcaggagatc ttgactgtct
ctccaggctt cttcagctca ggtccagact 300 gtaccaactg gatctgtgct
tgggcacttt gggcagctgc catcaggaat agcaagttcc 360 acagccaacc
catgatgtct aagacttggg ctcagtggtg ccttaagact aactggtcac 420
tccctttttc atcaaagcca gcaaacgcag tgttcgg 457 4 124 PRT Homo sapiens
4 Met Gly Trp Leu Trp Asn Leu Leu Phe Leu Met Ala Ala Ala Gln Ser 1
5 10 15 Ala Gln Ala Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys
Lys 20 25 30 Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly
Tyr Thr Phe 35 40 45 Thr Thr Tyr Gly Met Ser Trp Val Lys Gln Ala
Pro Gly Lys Gly Leu 50 55 60 Lys Trp Met Gly Trp Ile Asn Thr Tyr
Ser Gly Val Pro Thr Tyr Ala 65 70 75 80 Asp Asp Phe Lys Gly Arg Phe
Ala Phe Ser Leu Glu Thr Ser Ala Ser 85 90 95 Thr Ala Tyr Leu Gln
Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr 100 105 110 Tyr Phe Cys
Ala Arg Cys Tyr Tyr Gly Asn Tyr Asn 115 120 5 407 DNA Homo sapiens
5 ttgtagttac catagtagca tcttgcacag aaatatgtag ccgtgtcctc atttttgagg
60 ttgttgatct gcaaataggc agtgctggca gaggtttcca aagagaaggc
aaaccgtccc 120 ttgaagtcat cagcatatgt tggcactcca gagtaggtgt
ttatccagcc catccacttt 180 aaaccctttc ctggagcctg tttcacccag
ctcattccat aggttgtgaa ggtataccca 240 gaagccttgc aggagatctt
gactgtctct ccaggcttct tcagctcagg tccagactgt 300 accaactgga
tctgtgcttg ggcactttgg gcagctgcca tcaggaatag caagttccac 360
agccaaccca tgatgtctaa aacttgggct cagtggtgcc ttaaaac 407 6 123 PRT
Homo sapiens 6 Met Gly Trp Leu Trp Asn Leu Leu Phe Leu Met Ala Ala
Ala Gln Ser 1 5 10 15 Ala Gln Ala Gln Ile Gln Leu Val Gln Ser Gly
Pro Glu Leu Lys Lys 20 25 30 Pro Gly Glu Thr Val Lys Ile Ser Cys
Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Thr Tyr Gly Met Ser Trp
Val Lys Gln Ala Pro Gly Lys Gly Leu 50 55 60 Lys Trp Met Gly Trp
Ile Asn Thr Tyr Ser Gly Val Pro Thr Tyr Ala 65 70 75 80 Asp Asp Phe
Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser 85 90 95 Thr
Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr 100 105
110 Tyr Phe Cys Ala Arg Cys Tyr Tyr Gly Asn Tyr 115 120 7 20 PRT
Homo sapiens 7 Cys Pro Thr Met Thr Arg Val Leu Gln Gly Val Leu Pro
Ala Leu Pro 1 5 10 15 Gln Val Val Cys 20 8 20 PRT Homo sapiens 8
Cys Pro Thr Met Met Arg Val Leu Gln Ala Val Leu Pro Pro Leu Pro 1 5
10 15 Gln Val Val Cys 20 9 20 PRT Homo sapiens 9 Cys Tyr Thr Arg
Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro Lys Ile 1 5 10 15 Gln Lys
Thr Cys 20 10 27 PRT Artificial Sequence Synthetic construct
MISC_FEATURE (9)..(9) Xaa = hydroxyproline 10 Ala Pro Pro Pro Pro
Pro Pro Cys Xaa Thr Met Thr Arg Val Leu Gln 1 5 10 15 Gly Val Leu
Pro Ala Leu Pro Gln Val Val Cys 20 25
* * * * *