U.S. patent application number 11/102120 was filed with the patent office on 2006-02-16 for erbb antagonists for pain therapy.
Invention is credited to David B. Agus.
Application Number | 20060034840 11/102120 |
Document ID | / |
Family ID | 35150505 |
Filed Date | 2006-02-16 |
United States Patent
Application |
20060034840 |
Kind Code |
A1 |
Agus; David B. |
February 16, 2006 |
ErbB antagonists for pain therapy
Abstract
The present application describes the use of ErbB antagonist,
especially ErbB2 antibodies such as rhuMAb 2C4, for treating
pain.
Inventors: |
Agus; David B.; (Beverly
Hills, CA) |
Correspondence
Address: |
HELLER EHRMAN LLP
275 MIDDLEFIELD ROAD
MENLO PARK
CA
94025-3506
US
|
Family ID: |
35150505 |
Appl. No.: |
11/102120 |
Filed: |
April 7, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60561076 |
Apr 8, 2004 |
|
|
|
Current U.S.
Class: |
424/143.1 |
Current CPC
Class: |
C07K 16/30 20130101;
C07K 16/2863 20130101; A61P 25/04 20180101; C07K 2317/76 20130101;
A61K 2039/505 20130101; C07K 16/32 20130101; C07K 2317/55 20130101;
C07K 2317/565 20130101; C07K 2317/73 20130101; A61P 35/00 20180101;
A61P 29/00 20180101; A61P 43/00 20180101; C07K 2317/24
20130101 |
Class at
Publication: |
424/143.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Claims
1. A method of treating pain in a patient comprising administering
an ErbB antagonist to the patient in a dose confirmed to reduce or
eliminate the pain or the analgesia requirement of the patient.
2. The method of claim 1 wherein pain is measured by a pain score
or quality of life score reflective of pain.
3. The method of claim 2 wherein pain is measured by the McGill
Pain Index on a six point scale of 0 to 5.
4. The method of claim 2 wherein pain is measured using a visual
analog scale of 0-100 reflective of the subjective feeling of pain
of the patient.
5. The method of claim 1 wherein analgesia requirement is measured
using an analgesia score.
6. The method of claim 5 wherein one dose of a non-steroidal
analgesic agent corresponds to an analgesia score of 1, and one 10
mg dose of morphine, or an equivalent dose of another opiate
analgesic agent corresponds to an analgesia score of 2.
7. The method of claim 1 wherein pain is monitored daily.
8. The method of claim 1 wherein analgesia requirement is monitored
daily.
9. The method of claim 1 wherein the antagonist is an antibody that
binds an ErbB.
10. The method of claim 9 wherein the antibody blocks ligand
activation of an ErbB.
11. The method of claim 9 wherein the antibody blocks formation of
an ErbB heterodimer.
12. The method of claim 9 wherein the antibody blocks binding of
monoclonal antibody 2C4 to ErbB2.
13. The method of claim 9 wherein the antibody has a biological
characteristic of monoclonal antibody 2C4.
14. The method of claim 9 wherein the antibody comprises monoclonal
antibody 2C4 or humanized 2C4.
15. The method of claim 9 wherein the antibody is an antibody
fragment.
16. The method of claim 15 wherein the antibody fragment is a Fab
fragment.
17. The method of claim 9 wherein the antibody is not conjugated
with a cytotoxic agent.
18. The method of claim 9 wherein the antibody is conjugated with a
cytotoxic agent.
19. The method of claim 15 wherein the antibody fragment is not
conjugated with a cytotoxic agent.
20. The method of claim 15 wherein the antibody fragment is
conjugated with a cytotoxic agent.
21. The method of claim 1 wherein the pain is chronic pain.
22. The method of claim 21 wherein the chronic pain is selected
from the group consisting of nociceptive pain, neuropathic pain and
psychogenic pain.
23. The method of claim 22 wherein the pain is nociceptive
pain.
24. The method of claim 1 wherein the pain is cancer related
pain.
25. The method of claim 24 wherein the cancer expresses an ErbB
receptor.
26. The method of claim 25 wherein the ErbB receptor is ErbB2 or
EGFR.
27. The method of claim 24 wherein the cancer is metastatic
cancer.
28. The method of claim 27 wherein the pain is associated with
cancer metastasis.
29. The method of claim 26 wherein the cancer is prostate
cancer.
30. The method of claim 29 wherein the metastasis is bone
metastasis.
31. The method of claim 24 wherein the cancer is selected from the
group consisting of breast cancer, ovarian cancer, prostate cancer,
pancreatic cancer, squamous cell cancer, lung cancer, cancer of the
peritoneum, hepatocellular cancer, gastric cancer, glioblastoma,
cervical cancer, liver cancer, bladder cancer, hepatoma, colon
cancer, rectal cancer, colorectal cancer, endometrial or uterine
carcinoma, salivary gland carcinoma, kidney cancer, vulval cancer,
thyroid cancer, hepatic carcinoma, anal carcinoma, penile
carcinoma, and head and neck cancer.
32. The method of claim 31 wherein the cancer is prostate
cancer.
33. The method of claim 32 wherein the cancer is androgen
independent prostate cancer.
34. The method of claim 24 wherein the cancer is not in remission
or continues to grow during treatment.
35. The method of claim 1 wherein the pain is non-cancer
related.
36. The method of claim 1 wherein the patient is not suffering from
malignancy.
37. A method for treating cancer-related pain in a patient
diagnosed with cancer, comprising administering an effective amount
of an ErbB antagonist to the patient, wherein the cancer is not in
remission or continues to grow during said treatment.
38. The method of claim 37 wherein the cancer is not in remission
during said treatment.
39. The method of claim 37 wherein the cancer continues to grow
during said treatment.
40. The method of claim 37 wherein the cancer is selected from the
group consisting of breast cancer, ovarian cancer, prostate cancer,
pancreatic cancer, squamous cell cancer, lung cancer, cancer of the
peritoneum, hepatocellular cancer, gastric cancer, glioblastoma,
cervical cancer, liver cancer, bladder cancer, hepatoma, colon
cancer, rectal cancer, colorectal cancer, endometrial or uterine
carcinoma, salivary gland carcinoma, kidney cancer, vulval cancer,
thyroid cancer, hepatic carcinoma, anal carcinoma, penile
carcinoma, and head and neck cancer.
41. The method of claim 37 wherein the cancer is metastatic
cancer.
42. The method of claim 41 wherein the metastasis is soft tissue
metastasis.
43. The method of claim 41 wherein the metastasis includes bone
metastasis.
44. The method of claim 40 wherein the cancer is prostate
cancer.
45. The method of claim 44 wherein the cancer is androgen
independent prostate cancer.
46. The method of claim 44 wherein the patient's PSA shows no
reduction during treatment.
47. The method of claim 44 wherein the patient's PSA becomes
elevated during treatment.
48. The method of claim 37 wherein the antagonist is an antibody
that binds an ErbB.
49. The method of claim 48 wherein the antibody blocks ligand
activation of an ErbB.
50. The method of claim 48 wherein the antibody blocks formation of
an ErbB heterodimer.
51. The method of claim 48 wherein the antibody blocks binding of
monoclonal antibody 2C4 to ErbB2.
52. The method of claim 48 wherein the antibody has a biological
characteristic of monoclonal antibody 2C4.
53. The method of claim 48 wherein the antibody comprises
monoclonal antibody 2C4 or humanized 2C4.
54. The method of claim 48 wherein the antibody is an antibody
fragment.
55. The method of claim 54 wherein the antibody fragment is a Fab
fragment.
56. The method of claim 48 wherein the antibody is not conjugated
with a cytotoxic agent.
57. The method of claim 48 wherein the antibody is conjugated with
a cytotoxic agent.
58. The method of claim 54 wherein the antibody fragment is not
conjugated with a cytotoxic agent.
59. The method of claim 54 wherein the antibody fragment is
conjugated with a cytotoxic agent.
60. A method for treating non-cancer related pain in a patient
comprising administering an effective amount of an ErbB antagonist
to the patient.
61. The method of claim 60 wherein the antagonist is an anti-ErbB
antibody.
62. The method of claim 61 wherein the antibody blocks ligand
activation of an ErbB.
63. The method of claim 61 wherein the antibody blocks formation of
an ErbB heterodimer.
64. The method of claim 61 wherein the antibody blocks binding of
monoclonal antibody 2C4 to ErbB2.
65. The method of claim 61 wherein the antibody has a biological
characteristic of monoclonal antibody 2C4.
66. The method of claim 61 wherein the antibody comprises
monoclonal antibody 2C4 or humanized 2C4.
67. The method of claim 61 wherein the antibody is an antibody
fragment.
68. The method of claim 67 wherein the antibody fragment is a Fab
fragment.
69. A kit comprising an effective amount of an ErbB antagonist, and
instructions to administer said antagonist for the treatment of
pain.
70. The kit of claim 69 wherein the antagonist is an antibody that
binds an ErbB.
71. The kit of claim 70 wherein the antibody blocks ligand
activation of an ErbB.
72. The kit of claim 70 wherein the antibody blocks formation of an
ErbB heterodimer.
73. The kit of claim 70 wherein the antibody blocks binding of
monoclonal antibody 2C4 to ErbB2.
74. The kit of claim 70 wherein the antibody has a biological
characteristic of monoclonal antibody 2C4.
75. The kit of claim 70 wherein the antibody comprises monoclonal
antibody 2C4 or humanized 2C4.
76. The kit of claim 70 wherein the antibody is an antibody
fragment.
77. The kit of claim 76 wherein the antibody fragment is a Fab
fragment.
78. The kit of claim 69 wherein the pain is cancer-related.
79. The kit of claim 69 wherein the pain is non-cancer related.
80. The kit of claim 69 wherein the pain is chronic pain.
Description
CROSS-REFERENCE TO RELATED APPLICATION(S)
[0001] The is a non-provisional application filed under 37 CFR
1.53(b), claiming priority under USC Section 119(e) to Provisional
Application Ser. No. 60/561,076, filed on Apr. 8, 2004.
FIELD OF THE INVENTION
[0002] The present invention concerns ErbB antagonists for treating
pain.
BACKGROUND OF THE INVENTION
[0003] Anti-ErbB Antibodies and their Use in Cancer Treatment
[0004] The ErbB family of receptor tyrosine kinases are important
mediators of cell growth, differentiation and survival. The
receptor family includes four distinct members including epidermal
growth factor receptor (EGFR or ErbB1), HER2 (ErbB2 or
p185.sup.neu), HER3 (ErbB3) and HER4 (ErbB4 or tyro2).
[0005] EGFR, encoded by the erbB1 gene, has been causally
implicated in human malignancy. In particular, increased expression
of EGFR has been observed in breast, bladder, lung, head, neck and
stomach cancer as well as glioblastomas. Increased EGFR receptor
expression is often associated with increased production of the
EGFR ligand, transforming growth factor alpha (TGF-.alpha.), by the
same tumor cells resulting in receptor activation by an autocrine
stimulatory pathway. Baselga and Mendelsohn Pharmac. Ther.
64:127-154 (1994). Monoclonal antibodies directed against the EGFR
or its ligands, TGF-.alpha. and EGF, have been evaluated as
therapeutic agents in the treatment of such malignancies. See,
e.g., Baselga and Mendelsohn., supra; Masui et al. Cancer Research
44:1002-1007 (1984); and Wu et al. J. Clin. Invest. 95:1897-1905
(1995).
[0006] The second member of the ErbB family, p185.sup.neu, was
originally identified as the product of the transforming gene from
neuroblastomas of chemically treated rats. The activated form of
the neu proto-oncogene results from a point mutation (valine to
glutamic acid) in the transmembrane region of the encoded protein.
Amplification of the human homolog of neu is observed in breast and
ovarian cancers and correlates with a poor prognosis (Slamon et
al., Science, 235:177-182 (1987); Slamon et al., Science,
244:707-712 (1989); and U.S. Pat. No. 4,968,603). To date, no point
mutation analogous to that in the neu proto-oncogene has been
reported for human tumors. Overexpression of ErbB2 (frequently but
not uniformly due to gene amplification) has also been observed in
other carcinomas including carcinomas of the stomach, endometrium,
salivary gland, lung, kidney, colon, thyroid, pancreas and bladder.
See, among others, King et al., Science, 229:974 (1985); Yokota et
al., Lancet: 1:765-767 (1986); Fukushige et al., Mol Cell Biol.,
6:955-958 (1986); Guerin et al., Oncogene Res., 3:21-31 (1988);
Cohen et al., Oncogene, 4:81-88 (1989); Yonemura et al., Cancer
Res., 51:1034 (1991); Borst et al., Gynecol. Oncol., 38:364 (1990);
Weiner et al., Cancer Res., 50:421-425 (1990); Kern et al., Cancer
Res., 50:5184 (1990); Park et al., Cancer Res., 49:6605 (1989);
Zhau et al., Mol. Carcinog., 3:354-357(1990); Aasland et al. Br. J.
Cancer 57:358-363 (1988); Williams et al. Pathiobiology 59:46-52
(1991); and McCann et al., Cancer, 65:88-92 (1990). ErbB2 may be
overexpressed in prostate cancer (Gu et al. Cancer Lett. 99:185-9
(1996); Ross et al. Hum. Pathol. 28:827-33 (1997); Ross et al.
Cancer 79:2162-70 (1997); and Sadasivan et al. J. Urol. 150:126-31
(1993)).
[0007] Antibodies directed against the rat p185.sup.neu and human
ErbB2 protein products have been described. Drebin and colleagues
have raised antibodies against the rat neu gene product,
p185.sup.neu See, for example, Drebin et al., Cell 41:695-706
(1985); Myers et al., Meth. Enzym. 198:277-290 (1991); and
WO94/22478. Drebin et al. Oncogene 2:273-277 (1988) report that
mixtures of antibodies reactive with two distinct regions of
p185.sup.neu result in synergistic anti-tumor effects on
neu-transformed NIH-3T3 cells implanted into nude mice. See also
U.S. Pat. No. 5,824,311 issued Oct. 20, 1998.
[0008] Hudziak et al., Mol. Cell. Biol. 9(3):1165-1172 (1989)
describe the generation of a panel of anti-ErbB2 antibodies which
were characterized using the human breast tumor cell line SK-BR-3.
Relative cell proliferation of the SK-BR-3 cells following exposure
to the antibodies was determined by crystal violet staining of the
monolayers after 72 hours. Using this assay, maximum inhibition was
obtained with the antibody called 4D5 which inhibited cellular
proliferation by 56%. Other antibodies in the panel reduced
cellular proliferation to a lesser extent in this assay. The
antibody 4D5 was further found to sensitize ErbB2-overexpressing
breast tumor cell lines to the cytotoxic effects of TNF-.alpha..
See also U.S. Pat. No. 5,677,171 issued Oct. 14, 1997. The
anti-ErbB2 antibodies discussed in Hudziak et al. are further
characterized in Fendly et al. Cancer Research 50:1550-1558 (1990);
Kotts et al. In Vitro 26(3):59A (1990); Sarup et al. Growth
Regulation 1:72-82 (1991); Shepard et al. J. Clin. Immunol. 11(3):
117-127 (1991); Kumar et al. Mol. Cell. Biol. 11(2):979-986 (1991);
Lewis et al. Cancer Immunol. Immunother. 37:255-263 (1993); Pietras
et al. Oncogene 9:1829-1838(1994); Vitetta et al. Cancer Research
54:5301-5309 (1994); Sliwkowski et al. J. Biol. Chem.
269(20):14661-14665 (1994); Scott et al. J. Biol. Chem. 266:14300-5
(1991); D'souza et al. Proc. Natl. Acad. Sci. 91:7202-7206 (1994);
Lewis et al. Cancer Research 56:1457-1465(1996); and Schaefer et
al. Oncogene 15:1385-1394 (1997).
[0009] A recombinant humanized version of the murine anti-ErbB2
antibody 4D5 (huMAb4D5-8, rhuMAb HER2 or HERCEPTIN.RTM.; U.S. Pat.
No. 5,821,337) is clinically active in patients with
ErbB2-overexpressing metastatic breast cancers that have received
extensive prior anti-cancer therapy (Baselga et al., J. Clin.
Oncol. 14:737-744 (1996)). HERCEPTIN.RTM. received marketing
approval from the Food and Drug Administration Sep. 25, 1998 for
the treatment of patients with metastatic breast cancer whose
tumors overexpress the ErbB2 protein.
[0010] Other anti-ErbB2 antibodies with various properties have
been described in Tagliabue et al. Int. J. Cancer 47:933-937
(1991); McKenzie et al. Oncogene 4:543-548 (1989); Maier et al.
Cancer Res. 51:5361-5369 (1991); Bacus et al. Molecular
Carcinogenesis 3:350-362 (1990); Stancovski et al. PNAS (USA)
88:8691-8695 (1991); Bacus et al. Cancer Research 52:2580-2589
(1992); Xu et al. Int. J. Cancer 53:401-408 (1993); WO94/00136;
Kasprzyk et al. Cancer Research 52:2771-2776 (1992); Hancock et al.
Cancer Res. 51:4575-4580 (1991); Shawver et al. Cancer Res.
54:1367-1373 (1994); Arteaga et al. Cancer Res. 54:3758-3765
(1994); Harwerth et al. J. Biol. Chem. 267:15160-15167 (1992); U.S.
Pat. No. 5,783,186; and Klapper et al. Oncogene 14:2099-2109
(1997).
[0011] Homology screening has resulted in the identification of two
other ErbB receptor family members; ErbB3 (U.S. Pat. Nos. 5,183,884
and 5,480,968 as well as Kraus et al. PNAS (USA) 86:9193-9197
(1989)) and ErbB4 (EP Pat Appln No 599,274; Plowman et al., Proc.
Natl. Acad. Sci. USA, 90:1746-1750 (1993); and Plowman et al.,
Nature, 366:473-475 (1993)). Both of these receptors display
increased expression on at least some breast cancer cell lines.
[0012] The ErbB receptors are generally found in various
combinations in cells and heterodimerization is thought to increase
the diversity of cellular responses to a variety of ErbB ligands
(Earp et al. Breast Cancer Research and Treatment 35: 115-132
(1995)). EGFR is bound by six different ligands; epidermal growth
factor (EGF), transforming growth factor alpha (TGF-.alpha.),
amphiregulin, heparin binding epidermal growth factor (HB-EGF),
betacellulin and epiregulin (Groenen et al. Growth Factors
11:235-257 (1994)). A family of heregulin proteins resulting from
alternative splicing of a single gene are ligands for ErbB3 and
ErbB4. The heregulin family includes alpha, beta and gamma
heregulins (Holmes et al., Science, 256:1205-1210 (1992); U.S. Pat.
No. 5,641,869; and Schaefer et al. Oncogene 15:1385-1394 (1997));
neu differentiation factors (NDFs), glial growth factors (GGFs);
acetylcholine receptor inducing activity (ARIA); and sensory and
motor neuron derived factor (SMDF). For a review, see Groenen et
al. Growth Factors 11:235-257 (1994); Lemke, G. Molec. & Cell.
Neurosci. 7:247-262 (1996) and Lee et al. Pharm. Rev. 47:51-85
(1995). Recently three additional ErbB ligands were identified;
neuregulin-2 (NRG-2) which is reported to bind either ErbB3 or
ErbB4 (Chang et al. Nature 387 509-512 (1997); and Carraway et al
Nature 387:512-516 (1997)); neuregulin-3 which binds ErbB4 (Zhang
et al. PNAS (USA) 94(18):9562-7 (1997)); and neuregulin-4 which
binds ErbB4 (Harari et al. Oncogene 18:2681-89 (1999)) HB-EGF,
betacellulin and epiregulin also bind to ErbB4.
[0013] While EGF and TGF.alpha. do not bind ErbB2, EGF stimulates
EGFR and ErbB2 to form a heterodimer, which activates EGFR and
results in transphosphorylation of ErbB2 in the heterodimer.
Dimerization and/or transphosphorylation appears to activate the
ErbB2 tyrosine kinase. See Earp et al., supra. Likewise, when ErbB3
is co-expressed with ErbB2, an active signaling complex is formed
and antibodies directed against ErbB2 are capable of disrupting
this complex (Sliwkowski et al., J. Biol. Chem.,
269(20):14661-14665 (1994)). Additionally, the affinity of ErbB3
for heregulin (HRG) is increased to a higher affinity state when
co-expressed with ErbB2. See also, Levi et al., Journal of
Neuroscience 15: 1329-1340 (1995); Morrissey et al., Proc. Natl.
Acad. Sci. USA 92: 1431-1435 (1995); and Lewis et al., Cancer Res.,
56:1457-1465 (1996) with respect to the ErbB2-ErbB3 protein
complex. ErbB4, like ErbB3, forms an active signaling complex with
ErbB2 (Carraway and Cantley, Cell 78:5-8 (1994)).
[0014] Pain Management
[0015] Chronic pain is a common symptom of a variety of diseases
and pathologic conditions, and includes nociceptive pain (pain
caused by an injury to body tissues), neuropathic pain (pain caused
by abnormalities in the nerves, spinal cord, or brain), and
psychogenic pain (entirely or mostly related to a psychological
disorder). Nociceptive pain includes somatic pain, which arises
from bone, joint, muscle, skin, or connective tissue, and visceral
pain, which arises from visceral organs, such as the
gastrointestinal tract and the pancreas.
[0016] Mild to moderate pain is typically treated by nonsteroidal
antiinflammatory drugs (NSAIDs), such as acetaminophen, ibuprofen,
aspirin, ketorolac, etodolac, and the like. Treatment of more
severe chronic pain may include opiate and NSAID combinations, such
as aspirin and oxycodone (Percodan), acetaminophen and hydrocodone
(Vicodin and Lortab).
[0017] Pain is also a frequent symptom of advanced cancer. For
example, about 60% of patients with hormone-refractory prostate
cancer suffer significant pain. Typically the pain results directly
from the cancer (including cancer metastasis), although sometimes
it can also be associated with the cancer treatment itself. For
example, chronic pain may develop if there has been nerve damage
during surgical removal of cancer. Chemotherapy can also cause pain
in several ways. Some chemotherapy drugs, referred to as vesicants,
can harm tissues if they leak out of the vein. In some instances,
chemotherapy causes sores in the mouth (stomatitis) or lining of
the intestines (mucositis). Peripheral neuropathy can occur with
certain chemotherapy drugs when they are administered long-term in
high doses. Radiation treatment can also cause pain because it can
affect normal cells that surround the cancerous tumor being
treated.
[0018] At present, cancer-related pain is usually managed by opiate
analgesics, such as morphine or heroin with the goal to relieve the
patient's pain by adjusting the opiate dosage to maintain a pain
score of 3 or less on a 10-point visual analog scale. This
treatment, however, is not optimal. Common side effects include
drowsiness and constipation. In addition, patients often experience
tolerance and develop a physical dependency on opiate analgesics,
which reduces the effectiveness of the pain treatment and raises
serious issues of drug dependency. When an opioid is discontinued,
withdrawal symptoms may appear, the character and severity of which
are dependent upon such factors as the particular opioid being
withdrawn, the daily dose of the opioid that is being withdrawn,
the duration of opioid treatment, and the condition of the
drug-dependent individual. Withdrawal itself is associated with
symptoms including severe pain. Often the only effective treatment
for cancer-related pain is successful eradication of the tumor.
[0019] Severe, persisting pain is debilitating for patients and
their caregivers, and is often under-treated due to fear of opioid
addition by both patients and medical professionals. Since current
therapies are unsatisfactory, it is important to develop further
treatment modalities for the management of chronic pain, including
cancer-related pain, that are more effective and are devoid of the
undesired side-effects and risks associated with current treatment
approaches.
SUMMARY OF THE INVENTION
[0020] This invention is based, at least in part, on the surprising
observation that patients with prostate cancer treated with an ErbB
antagonist, namely rhuMAb 2C4, experienced diminished pain or
showed reduced analgesia requirement, even where the tumor was
progressing. This indicates that rhuMAb 2C4 has analgesic
properties.
[0021] In one aspect, the invention concern a method of treating
pain in a patient comprising administering an effective dose of an
ErbB antagonist to the patient.
[0022] In another aspect, the invention concerns a method of
treating pain in a patient comprising administering an ErbB
antagonist to the patient in a dose confirmed to reduce or
eliminate the pain or the analgesia requirement of the patient.
[0023] In one embodiment, the pain is measured by a pain score or
quality of life score reflective of pain. Pain may, for example, be
measured by the McGill Pain Index on a six point scale of 0 to 5.
Pain may alternatively be measured by using a visual analog scale
of 0-100 reflective of the subjective feeling of pain of the
patient. The analgesia requirement may be measured using an
analgesia score. In a particular embodiment, one dose of a
non-steroidal analgesic agent corresponds to an analgesia score of
1, and one 10 mg dose of morphine, or an equivalent dose of another
opiate analgesic agent corresponds to an analgesia score of 2.
[0024] In another embodiment, pain is monitored daily.
[0025] In yet another embodiment, analgesia requirement is
monitored daily.
[0026] In another aspect, the invention cocnerns a method for
treating cancer-related pain in a patient diagnosed with cancer,
comprising administering an effective amount of an ErbB antagonist
to the patient, wherein the cancer is not in remission or continues
to grow during said treatment.
[0027] In a specific embodiment of the method, the cancer is not in
remission during said treatment, or continues to group during
treatment.
[0028] The cancer can be any kind of cancer, including, for
example, breast cancer, ovarian cancer, prostate cancer, pancreatic
cancer, squamous cell cancer, lung cancer, cancer of the
peritoneum, hepatocellular cancer, gastric cancer, glioblastoma,
cervical cancer, liver cancer, bladder cancer, hepatoma, colon
cancer, rectal cancer, colorectal cancer, endometrial or uterine
carcinoma, salivary gland carcinoma, kidney cancer, vulval cancer,
thyroid cancer, hepatic carcinoma, anal carcinoma, penile
carcinoma, and head and neck cancer.
[0029] In a particular embodiment, the cancer is metastatic
cancer.
[0030] In another embodiment, the metastasis is soft tissue
metastasis.
[0031] In yet another embodiment, the metastasis includes bone
metastasis.
[0032] In a preferred embodiment, the cancer is prostate cancer,
specifically including androgen independent prostate cancer.
[0033] In another embodiment, the cancer is prostate cancer, and
the patient's PSA shows no reduction during treatment, or becomes
elevated during treatment.
[0034] In a further aspect, the invention concerns a method for
treating non-cancer related pain in a patient comprising
administering an effective amount of an ErbB antagonist to the
patient.
[0035] In a still further aspect, the invention concerns a kit
comprising an effective amount of an ErbB antagonist, and
instructions to administer said antagonist for the treatment of
pain.
[0036] In all aspects, the ErbB antagonist preferably is an
antibody. The antibody can, for example, be a monoclonal antibody
that binds an ErbB. In another embodiment, the antibody blocks
ligand activation of an ErbB. In yet another embodiment, the
antibody blocks formation of an ErbB heterodimer. In a preferred
embodiment, the antibody blocks binding of monoclonal antibody 2C4
to ErbB2. In another preferred embodiment, the antibody has a
biological characteristic of monoclonal antibody 2C4. In a further
preferred embodiment, the antibody comprises monoclonal antibody
2C4 or humanized 2C4.
[0037] The antibody may be an antibody fragment, such as, for
example, a Fab fragment and may, or may not be conjugated with a
cytotoxic agent.
[0038] The pain treated in accordance with the present invention
can be acute pain or chronic pain, such as, without limitation,
nociceptive pain, neuropathic pain and psychogenic pain, and can be
cancer related or not associated with cancer. When the pain is
cancer related the cancer may, but does not have to, express an
ErbB receptor, such as, ErbB2 and/or EGFR. In a specific
embodiment, the cancer is metastatic cancer, where the metastasis
can be soft tissue and/or bone metastasis.
[0039] In another embodiment, the cancer is selected from the group
consisting of breast cancer, ovarian cancer, prostate cancer,
pancreatic cancer, squamous cell cancer, lung cancer, cancer of the
peritoneum, hepatocellular cancer, gastric cancer, glioblastoma,
cervical cancer, liver cancer, bladder cancer, hepatoma, colon
cancer, rectal cancer, colorectal cancer, endometrial or uterine
carcinoma, salivary gland carcinoma, kidney cancer, vulval cancer,
thyroid cancer, hepatic carcinoma, anal carcinoma, penile
carcinoma, and head and neck cancer.
[0040] In a particular embodiment, the cancer is prostate cancer,
such as, for example, androgen independent prostate cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIGS. 1A and 1B depict epitope mapping of residues 22-645
within the extracellular domain (ECD) of ErbB2 (amino acid
sequence, including signal sequence, shown in FIG. 1A; SEQ ID
NO:13) as determined by truncation mutant analysis and
site-directed mutagenesis (Nakamura et al. J. of Virology
67(10):6179-6191 (1993); and Renz et al. J. Cell Biol. 125(6):
1395-1406 (1994)). The various ErbB2-ECD truncations or point
mutations were prepared from cDNA using polymerase chain reaction
technology. The ErbB2 mutants were expressed as gD fusion proteins
in a mammalian expression plasmid. This expression plasmid uses the
cytomegalovirus promoter/enhancer with SV40 termination and
polyadenylation signals located downstream of the inserted cDNA.
Plasmid DNA was transfected into 293 cells. One day following
transfection, the cells were metabolically labeled overnight in
methionine and cysteine-free, low glucose DMEM containing 1%
dialyzed fetal bovine serum and 25 .mu.Ci each of .sup.35S
methionine and .sup.35S cysteine. Supernatants were harvested and
either the anti-ErbB2 monoclonal antibodies or control antibodies
were added to the supernatant and incubated 2-4 hours at 4.degree.
C. The complexes were precipitated, applied to a 10-20% Tricine SDS
gradient gel and electrophoresed at 100 V. The gel was
electroblotted onto a membrane and analyzed by autoradiography. As
shown in FIG. 1B, the anti-ErbB2 antibodies 7C2, 7F3, 2C4, 7D3,
3E8, 4D5, 2H11 and 3H4 bind various ErbB2 ECD epitopes.
[0042] FIGS. 2A and 2B show the effect of anti-ErbB2 monoclonal
antibodies 2C4 and 7F3 on rHRG.beta.1 activation of MCF7 cells.
FIG. 2A shows dose-response curves for 2C4 or 7F3 inhibition of HRG
stimulation of tyrosine phosphorylation. FIG. 2B shows
dose-response curves for the inhibition of .sup.125I-labeled
rHRG.beta.1.sub.177-244 binding to MCF7 cells by 2C4 or 7F3.
[0043] FIG. 3 depicts inhibition of specific .sup.125I-labeled
rHRG.beta.1.sub.177-244 binding to a panel of human tumor cell
lines by the anti-ErbB2 monoclonal antibodies 2C4 or 7F3.
Monoclonal antibody-controls are isotype-matched murine monoclonal
antibodies that do not block rHRG binding. Nonspecific
.sup.125I-labeled rHRG.beta.1.sub.177-244 binding was determined
from parallel incubations performed in the presence of 100 nM
rHRG.beta.1. Values for nonspecific .sup.125I-labeled
rHRG.beta.1.sub.177-244 binding were less than 1% of the total for
all the cell lines tested.
[0044] FIGS. 4A and 4B show the effect of monoclonal antibodies 2C4
and 4D5 on proliferation of MDA-MB-175 (FIG. 4A) and SK-BR-3 (FIG.
4B) cells. MDA-MB-175 and SK-BR-3 cells were seeded in 96 well
plates and allowed to adhere for 2 hours. Experiment was carried
out in medium containing 1% serum. Anti-ErbB2 antibodies or medium
alone were added and the cells were incubated for 2 hours at
37.degree. C. Subsequently rHRG.beta.1 (1 nM) or medium alone were
added and the cells were incubated for 4 days. Monolayers were
washed and stained/fixed with 0.5% crystal violet. To determine
cell proliferation the absorbance was measured at 540 nm.
[0045] FIGS. 5A and 5B show the effect of monoclonal antibody 2C4,
HERCEPTIN.RTM. antibody or an anti-EGFR antibody on heregulin (HRG)
dependent association of ErbB2 with ErbB3 in MCF7 cells expressing
low/normal levels of ErbB2 (FIG. 5A) and SK-BR-3 cells expressing
high levels of ErbB2 (FIG. 5B); see Example 2 below.
[0046] FIGS. 6A and 6B compare the activities of intact murine
monoclonal antibody 2C4 (mu 2C4) and a chimeric 2C4 Fab fragment.
FIG. 6A shows inhibition of .sup.125I-HRG binding to MCF7 cells by
chimeric 2C4 Fab or intact murine monoclonal antibody 2C4. MCF7
cells were seeded in 24-well plates (1.times.10.sup.5 cells/well)
and grown to about 85% confluency for two days. Binding experiments
were conducted as described in Lewis et al. Cancer Research
56:1457-1465 (1996). FIG. 6B depicts inhibition of rHRG.beta.1
activation of p180 tyrosine phosphorylation in MCF7 cells performed
as described in Lewis et al. Cancer Research 56:1457-1465
(1996).
[0047] FIGS. 7A and 7B depict alignments of the amino acid
sequences of the variable light (V.sub.L) (FIG. 7A) and variable
heavy (V.sub.H) (FIG. 7B) domains of murine monoclonal antibody 2C4
(SEQ ID Nos. 1 and 2, respectively); V.sub.L and V.sub.H domains of
humanized 2C4 version 574 (SEQ ID Nos. 3 and 4, respectively), and
human V.sub.L and V.sub.H consensus frameworks (hum .kappa.1, light
kappa subgroup I; humIII, heavy subgroup III) (SEQ ID Nos. 5 and 6,
respectively). Asterisks identify differences between humanized 2C4
version 574 and murine monoclonal antibody 2C4 or between humanized
2C4 version 574 and the human framework. Complementarity
Determining Regions (CDRs) are in brackets.
[0048] FIGS. 8A to C show binding of chimeric Fab 2C4 (Fab.v1) and
several humanized 2C4 variants to ErbB2 extracellular domain (ECD)
as determined by ELISA in Example 3.
[0049] FIG. 9 is a ribbon diagram of the V.sub.L and V.sub.H
domains of monoclonal antibody 2C4 with white CDR backbone labeled
(L1, L2, L3, H1, H2, H3). V.sub.H side chains evaluated by
mutagenesis during humanization (see Example 3, Table 2) are also
shown.
[0050] FIG. 10 depicts the effect of monoclonal antibody 2C4 or
HERCEPTIN.RTM. on EGF, TGF-.alpha., or HRG-mediated activation of
mitogen-activated protein kinase (MAPK).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0051] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Singleton et al., Dictionary of Microbiology and Molecular Biology
2nd ed., J. Wiley & Sons (New York, N.Y. 1994) provides one
skilled in the art with a general guide to many of the terms used
in the present disclosure.
I. Definitions
[0052] The term "pain" is used herein in the broadest sense and
refers to all types of pain, including acute and chronic pain, such
as nociceptive pain, e.g. somatic pain and visceral pain;
neuropathic pain, e.g. centrally generated pain and peripherally
generated pain; and psychogenic pain. The term preferably refers to
chronic pain, most preferably nociceptive pain, including somatic
pain and visceral pain, which can be cancer related, not associated
with cancer, or only partially associated with cancer.
[0053] The term "nociceptive pain" is used to include all pain
caused by injury to body tissues, including, without limitation, by
a cut, bruise, bone fracture, crush injury, burn, and the like.
This type of pain is typically aching, sharp, or throbbing. Pain
receptors for tissue injury (nociceptor) are located mostly in the
skin or in the internal organs.
[0054] The term "somatic pain" is used to refer to pain arising
from bone, joint, muscle, skin, or connective tissue. This type of
pain is typically aching or throbbing in quality and is well
localized.
[0055] The term "visceral pain" is used herein to refer to pain
arising from visceral organs, such as the gastrointestinal tract
and pancreas. Visceral pain includes aching and fairly well
localized pain caused by tumor involvement of the organ capsule.
Another type of visceral pain, which is typically caused by
obstruction of hollow viscus, is characterized by intermittent
cramping and poorly localized pain.
[0056] The term "neuropathic pain" is used herein to refer to pain
originating from abnormal processing of sensory input by the
peripheral or central nervous system.
[0057] The term "analgesia" is used to refer to the absence of pain
in response to a stimulus that would be normally painful and to a
treatment with an analgesic agent. The term "analgesic agent" and
grammatical equivalents thereof refer to agents that are capable of
invoking analgesia.
[0058] The term "analgesia requirement" of a patient is used herein
to refer to the need of administering an analgesic agent (other
than an ErbB antagonist) to a patient in order to manage the
patient's pain.
[0059] "Treatment" refers to both therapeutic treatment and
prophylactic or preventative measures. Those in need of treatment
include those already experiencing pain as well as those in which
pain is to be prevented.
[0060] The term "effective amount" refers to an amount of the ErbB
antagonist effective to reduce pain, at least to some extent, or to
reduce or eliminate the analgesia requirement while maintaining the
same or reduced pain score or subjective feeling of pain as
experienced under analgesia, prior to the administration of the
ErbB antagonist. Pain and reduction in pain can be evaluated by
using any of the pain score systems well known in the art of pain
management and/or a Quality of Life Score system for pain.
Preferably, pain is measured based on the McGill Pain Index using a
6 point scale (0-5), where 0=no pain, 1=mild pain, 2=discomforting
pain, 3=distressing pain, 4=horrible pain, 5=excruciating pain.
Quality of Life Score for pain can be determined using a visual
analog scale of 0-100.
[0061] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. Examples of cancer include, but are not
limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or
lymphoid malignancies. More particular examples of such cancers
include squamous cell cancer (e.g. epithelial squamous cell
cancer), lung cancer including small-cell lung cancer, non-small
cell lung cancer, adenocarcinoma of the lung and squamous carcinoma
of the lung, cancer of the peritoneum, hepatocellular cancer,
gastric or stomach cancer including gastrointestinal cancer,
pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer,
liver cancer, bladder cancer, hepatoma, breast cancer, colon
cancer, rectal cancer, colorectal cancer, endometrial or uterine
carcinoma, salivary gland carcinoma, kidney or renal cancer,
prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma,
anal carcinoma, penile carcinoma, as well as head and neck
cancer.
[0062] The term "PSA" is used herein to refer to the level of a
prostate-specific antigen in the blood produced by the prostate, as
determined by the prostate-specific antigen test. The amount of
this antigen increases if the prostate is cancerous, and typically
continues to increase as the cancer progresses.
[0063] An "ErbB" is a receptor protein tyrosine kinase which
belongs to the ErbB receptor family and includes EGFR, ErbB2, ErbB3
and ErbB4 receptors and other members of this family to be
identified in the future. The ErbB will generally comprise an
extracellular domain, which may bind an ErbB ligand; a lipophilic
transmembrane domain; a conserved intracellular tyrosine kinase
domain; and a carboxyl-terminal signaling domain harboring several
tyrosine residues which can be phosphorylated. The ErbB may be a
"native sequence" ErbB or an "amino acid sequence variant" thereof.
Preferably the ErbB is native sequence human ErbB.
[0064] The terms "ErbB1", "epidermal growth factor receptor,"
"HER1," and "EGFR" are used interchangeably herein and refer to
EGFR as disclosed, for example, in Carpenter et al. Ann. Rev.
Biochem. 56:881-914 (1987), including naturally occurring mutant
forms thereof (e.g. a deletion mutant EGFR as in Humphrey et al.
PNAS (USA) 87:4207-4211 (1990); type II EGFR mutant (U.S. Pat. No.
6,455,498) etc). erbB1 refers to the gene encoding the EGFR protein
product.
[0065] The expressions "ErbB2" and "HER2" are used interchangeably
herein and refer to human HER2 protein described, for example, in
Semba et al., PNAS (USA) 82:6497-6501 (1985) and Yamamoto et al.
Nature 319:230-234 (1986) (Genebank accession number X03363). The
term "erbB2" refers to the gene encoding human ErbB2 and "neu"
refers to the gene encoding rat p185.sup.neu. Preferred ErbB2 is
native sequence human ErbB2.
[0066] "ErbB3" and "HER3" refer to the receptor polypeptide as
disclosed, for example, in U.S. Pat. Nos. 5,183,884 and 5,480,968
as well as Kraus et al. PNAS (USA) 86:9193-9197 (1989).
[0067] The terms "ErbB4" and "HER4" herein refer to the receptor
polypeptide as disclosed, for example, in EP Pat Appln No 599,274;
Plowman et al., Proc. Natl. Acad. Sci. USA, 90:1746-1750 (1993);
and Plowman et al., Nature, 366:473-475 (1993), including isoforms
thereof, e.g., as disclosed in WO99/19488, published Apr. 22,
1999.
[0068] By "ErbB ligand" is meant a polypeptide which binds to
and/or activates an ErbB. The term includes membrane-bound
precursor forms of the ErbB ligand, as well as proteolytically
processed soluble forms of the ErbB ligand. The ErbB ligand of
particular interest herein is a native sequence human ErbB ligand
such as epidermal growth factor (EGF) (Savage et al., J. Biol.
Chem. 247:7612-7621 (1972)); transforming growth factor alpha
(TGF-.alpha.) (Marquardt et al., Science 223:1079-1082 (1984));
amphiregulin also known as schwanoma or keratinocyte autocrine
growth factor (Shoyab et al. Science 243:1074-1076 (1989); Kimura
et al. Nature 348:257-260 (1990); and Cook et al. Mol. Cell. Biol.
11:2547-2557 (1991)); betacellulin (Shing et al., Science
259:1604-1607 (1993); and Sasada et al. Biochem. Biophys. Res.
Commun. 190:1173 (1993)); heparin-binding epidermal growth factor
(HB-EGF) (Higashiyama et al., Science 251:936-939 (1991));
epiregulin (Toyoda et al., J. Biol. Chem. 270:7495-7500 (1995); and
Komurasaki et al. Oncogene 15:2841-2848 (1997)); a heregulin (see
below); neuregulin-2 (NRG-2) (Carraway et al., Nature 387:512-516
(1997)); neuregulin-3 (NRG-3) (Zhang et al., Proc. Natl. Acad. Sci.
94:9562-9567 (1997)); neuregulin-4 (NRG-4) (Harari et al. Oncogene
18:2681-89 (1999)) or cripto (CR-1) (Kannan et al. J. Biol. Chem.
272(6):3330-3335 (1997)). ErbB ligands which bind EGFR include EGF,
TGF-.alpha., amphiregulin, betacellulin, HB-EGF and epiregulin.
ErbB ligands which bind ErbB3 include heregulins. ErbB ligands
capable of binding ErbB4 include betacellulin, epiregulin, HB-EGF,
NRG-2, NRG-3, NRG-4 and heregulins.
[0069] "Heregulin" (HRG) when used herein refers to a polypeptide
encoded by the heregulin gene product as disclosed in U.S. Pat. No.
5,641,869 or Marchionni et al., Nature, 362:312-318 (1993).
Examples of heregulins include heregulin-.alpha.,
heregulin-.beta.1, heregulin-.beta.2 and heregulin-.beta.3 (Holmes
et al., Science, 256:1205-1210 (1992); and U.S. Pat. No.
5,641,869); neu differentiation factor (NDF) (Peles et al. Cell 69:
205-216 (1992)); acetylcholine receptor-inducing activity (ARIA)
(Falls et al. Cell 72:801-815 (1993)); glial growth factors (GGFs)
(Marchionni et al., Nature, 362:312-318 (1993)); sensory and motor
neuron derived factor (SMDF) (Ho et al. J. Biol. Chem.
270:14523-14532 (1995)); .gamma.-heregulin (Schaefer et al.
Oncogene 15:1385-1394 (1997)). The term includes biologically
active fragments and/or amino acid sequence variants of a native
sequence HRG polypeptide, such as an EGF-like domain fragment
thereof (e.g. HRG.beta.1.sub.177-244).
[0070] An "ErbB hetero-oligomer" herein is a noncovalently
associated oligomer comprising at least two different ErbBs. Such
complexes may form when a cell expressing two or more ErbBs is
exposed to an ErbB ligand (Sliwkowski et al., J. Biol. Chem.,
269(20):14661-14665 (1994)). Examples of such ErbB hetero-oligomers
include EGFR-ErbB2, ErbB2-ErbB3 and ErbB3-ErbB4 complexes.
Moreover, the ErbB hetero-oligomer may comprise two or more ErbB2
receptors combined with a different ErbB, such as ErbB3, ErbB4 or
EGFR. Other proteins, such as a cytokine receptor subunit (e.g.
gp130) may be included in the hetero-oligomer. The patient herein
may have been subjected to an assay to determine whether ErbB
heterodimers, especially an EGFR-ErbB2 and/or ErbB2-ErbB3
heterodimer are present in cells of the patient, e.g. in diseased
tissue therefrom.
[0071] By "ligand activation of an ErbB" is meant signal
transduction (e.g. that caused by an intracellular kinase domain of
an ErbB phosphorylating tyrosine residues in the ErbB or a
substrate polypeptide) mediated by ErbB ligand binding to a ErbB
hetero-oligomer comprising the ErbB of interest. Generally, this
will involve binding of an ErbB ligand to an ErbB hetero-oligomer
which activates a kinase domain of one or more of the ErbBs in the
hetero-oligomer and thereby results in phosphorylation of tyrosine
residues in one or more of the ErbBs and/or phosphorylation of
tyrosine residues in additional substrate polypeptides(s). ErbB
activation can be quantified using various tyrosine phosphorylation
assays.
[0072] An "ErbB antagonist" is a molecule which blocks (reduces or
prevents) a biological activity of one or more ErbB(s). Preferably,
the antagonist blocks (reduces or prevents) ligand activation of an
ErbB. Generally, the antagonist will be an antibody, small peptide
or non-peptide (organic) molecule, antisense molecule,
oligonucleotide decoy, and the like, which inhibits a biological
activity of an ErbB receptor. Thus, for example, an antagonist may
bind to or otherwise associate with an ErbB and reduce tyrosine
kinase activation thereof. ErbB antagonists also include molecules
that bind to or associate with ErbB ligands or other members of the
ErbB signaling pathway, thereby inhibiting ErbB biological
activity. The preferred ErbB antagonist is an antibody that binds
ErbB2, or EGFR, or a hetero-oligomer (e.g. a heterodimer)
comprising ErbB2 and/or EGFR, and blocks ligand activation of an
ErbB. The most preferred antagonist is rhuMAb 2C4 or a molecule
having a biological characteristic of rhuMAb 2C4. By way of
example, the antagonist may also be an EGFR-targeted drug and/or a
tyrosine kinase inhibitor.
[0073] As used herein, the term "EGFR-targeted drug" refers to a
therapeutic agent that binds to EGFR and, optionally, inhibits EGFR
activation. Examples of such agents include antibodies and small
molecules that bind to EGFR. Examples of antibodies which bind to
EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507),
MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, U.S. Pat.
No. 4,943,533, Mendelsohn et al.) and variants thereof, such as
chimerized 225 (C225 or Cetuximab; ERBUTIX.RTM.) and reshaped human
225 (H225) (see, WO 96/40210, Imclone Systems Inc.); antibodies
that bind type II mutant EGFR (U.S. Pat. No. 5,212,290); humanized
and chimeric antibodies that bind EGFR as described in U.S. Pat.
No. 5,891,996; and human antibodies that bind EGFR, such as ABX-EGF
(see WO98/50433, Abgenix). The anti-EGFR antibody may be conjugated
with a cytotoxic agent, thus generating an immunoconjugate (see,
e.g., EP659,439A2, Merck Patent GmbH). Examples of small molecules
that bind to EGFR include ZD1839 or Gefitinib (IRESSA.RTM.; Astra
Zeneca), CP-358,774 (TARCEVA.RTM.; Genentech/OSI) and AG1478,
AG1571 (SU 5271; Sugen).
[0074] A "tyrosine kinase inhibitor" is a molecule which inhibits
to some extent tyrosine kinase activity of a tyrosine kinase such
as an ErbB. Examples of such inhibitors include the EGFR-targeted
drugs noted in the preceding paragraph as well as quinazolines such
as PD 153035, 4-(3-chloroanilino) quinazoline, pyridopyrimidines,
pyrimidopyrimidines, pyrrolopyrimidines, such as CGP 59326, CGP
60261 and CGP 62706, and pyrazolopyrimidines,
4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidines, curcumin (diferuloyl
methane, 4,5-bis (4-fluoroanilino)phthalimide), tryphostines
containing nitrothiophene moieties; PD-0183805 (Warner-Lamber);
antisense molecules (e.g. those that bind to ErbB-encoding nucleic
acid); quinoxalines (U.S. Pat. No. 5,804,396); tryphostins (U.S.
Pat. No. 5,804,396); ZD6474 (Astra Zeneca); PTK-787
(Novartis/Schering AG); pan-ErbB inhibitors such as CI-1033
(Pfizer); Affinitac (ISIS 3521; Isis/Lilly); Imatinib mesylate
(Gleevac; Novartis); PKI 166 (Novartis); GW2016 (Glaxo SmithKline);
CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxanib (Sugen); ZD6474
(AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11 (Imclone);
or as described in any of the following patent publications: U.S.
Pat. No. 5,804,396; WO99/09016 (American Cyanimid); WO98/43960
(American Cyanamid); WO97/38983 (Warner Lambert); WO99/06378
(Warner Lambert); WO99/06396 (Warner Lambert); WO96/30347 (Pfizer,
Inc); WO96/33978 (Zeneca); WO96/3397 (Zeneca); and WO96/33980
(Zeneca).
[0075] A "native sequence" polypeptide is one which has the same
amino acid sequence as a polypeptide (e.g., ErbB or ErbB ligand)
derived from nature. Such native sequence polypeptides can be
isolated from nature or can be produced by recombinant or synthetic
means. Thus, a native sequence polypeptide can have the amino acid
sequence of naturally occurring human polypeptide, murine
polypeptide, or polypeptide from any other mammalian species.
[0076] The term "amino acid sequence variant" refers to
polypeptides having amino acid sequences that differ to some extent
from a native sequence polypeptide. Ordinarily, amino acid sequence
variants will possess at least about 70% homology with at least one
receptor binding domain of a native ErbB ligand or with at least
one ligand binding domain of a native ErbB, and preferably, they
will be at least about 80%, more preferably at least about 90%
homologous with such receptor or ligand binding domains. The amino
acid sequence variants possess substitutions, deletions, and/or
insertions at certain positions within the amino acid sequence of
the native amino acid sequence.
[0077] "Homology" is defined as the percentage of residues in the
amino acid sequence variant that are identical after aligning the
sequences and introducing gaps, if necessary, to achieve the
maximum percent homology. Methods and computer programs for the
alignment are well known in the art. One such computer program is
"Align 2", authored by Genentech, Inc., which was filed with user
documentation in the United States Copyright Office, Washington,
D.C. 20559, on Dec. 10, 1991.
[0078] The term "antibody" herein is used in the broadest sense and
specifically covers intact monoclonal antibodies, polyclonal
antibodies, multispecific antibodies (e.g. bispecific antibodies)
formed from at least two intact antibodies, and antibody fragments,
so long as they exhibit the desired biological activity.
[0079] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for variants that may arise during
production of the antibody. Monoclonal antibodies are highly
specific, being directed against a single antigenic site.
Furthermore, in contrast to polyclonal antibody preparations which
include different antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed
against a single determinant on the antigen. In addition to their
specificity, the monoclonal antibodies are advantageous in that
they may be synthesized uncontaminated by other antibodies. The
modifier "monoclonal" indicates the character of the antibody as
being obtained from a substantially homogeneous population of
antibodies, and is not to be construed as requiring production of
the antibody by any particular method. For example, the monoclonal
antibodies to be used in accordance with the present invention may
be made by the hybridoma method first described by Kohler et al.,
Nature, 256:495 (1975), or may be made by recombinant DNA methods
(see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies"
may also be isolated from phage antibody libraries using the
techniques described in Clackson et al., Nature, 352:624-628 (1991)
and Marks et al., J. Mol. Biol., 222:581-597 (1991), for
example.
[0080] The monoclonal antibodies herein specifically include
"chimeric" antibodies in which a portion of the heavy and/or light
chain is identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad.
Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest
herein include "primatized" antibodies comprising variable domain
antigen-binding sequences derived from a non-human primate (e.g.
Old World Monkey, Ape etc) and human constant region sequences.
[0081] "Antibody fragments" comprise a portion of an intact
antibody, preferably comprising the antigen-binding or variable
region thereof. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2, and Fv fragments; diabodies; linear antibodies;
single-chain antibody molecules; and multispecific antibodies
formed from antibody fragment(s).
[0082] An "intact" antibody is one which comprises an
antigen-binding variable region as well as a light chain constant
domain (CL) and heavy chain constant domains, C.sub.H1, C.sub.H2
and C.sub.H3. The constant domains may be native sequence constant
domains (e.g. human native sequence constant domains) or amino acid
sequence variant thereof. Preferably, the intact antibody has one
or more effector functions.
[0083] Antibody "effector functions" refer to those biological
activities attributable to the Fc region (a native sequence Fc
region or amino acid sequence variant Fc region) of an antibody.
Examples of antibody effector functions include C1q binding;
complement dependent cytotoxicity; Fc receptor binding;
antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis;
down regulation of cell surface receptors (e.g. B cell receptor;
BCR), etc.
[0084] Depending on the amino acid sequence of the constant domain
of their heavy chains, intact antibodies can be assigned to
different "classes". There are five major classes of intact
antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may
be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2,
IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that
correspond to the different classes of antibodies are called
.alpha., .delta., .epsilon., .gamma., and .mu., respectively. The
subunit structures and three-dimensional configurations of
different classes of immunoglobulins are well known.
[0085] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC"
refer to a cell-mediated reaction in which nonspecific cytotoxic
cells that express Fc receptors (FcRs) (e.g. Natural Killer (NK)
cells, neutrophils, and macrophages) recognize bound antibody on a
target cell and subsequently cause lysis of the target cell. The
primary cells for mediating ADCC, NK cells, express Fc.gamma.RIII
only, whereas monocytes express Fc.gamma.RI, Fc.gamma.RII and
Fc.gamma.RIII. FcR expression on hematopoietic cells in summarized
is Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol
9:457-92 (1991). To assess ADCC activity of a molecule of interest,
an in vitro ADCC assay, such as that described in U.S. Pat. No.
5,500,362 or 5,821,337 may be performed. Useful effector cells for
such assays include peripheral blood mononuclear cells (PBMC) and
Natural Killer (NK) cells. Alternatively, or additionally, ADCC
activity of the molecule of interest may be assessed in vivo, e.g.,
in a animal model such as that disclosed in Clynes et al. PNAS
(USA) 95:652-656 (1998).
[0086] "Human effector cells" are leukocytes which express one or
more FcRs and perform effector functions. Preferably, the cells
express at least Fc.gamma.RIII and perform ADCC effector function.
Examples of human leukocytes which mediate ADCC include peripheral
blood mononuclear cells (PBMC), natural killer (NK) cells,
monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK
cells being preferred. The effector cells may be isolated from a
native source thereof, e.g. from blood or PBMCs as described
herein.
[0087] The terms "Fc receptor" or "FcR" are used to describe a
receptor that binds to the Fc region of an antibody. The preferred
FcR is a native sequence human FcR. Moreover, a preferred FcR is
one which binds an IgG antibody (a gamma receptor) and includes
receptors of the Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII
subclasses, including allelic variants and alternatively spliced
forms of these receptors. Fc.gamma.RII receptors include
Fc.gamma.RIIA (an "activating receptor") and Fc.gamma.RIIB (an
"inhibiting receptor"), which have similar amino acid sequences
that differ primarily in the cytoplasmic domains thereof.
Activating receptor Fc.gamma.RIIA contains an immunoreceptor
tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
Inhibiting receptor Fc.gamma.RIIB contains an immunoreceptor
tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain.
(see review M. in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)).
FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92
(1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et
al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including
those to be identified in the future, are encompassed by the term
"FcR" herein. The term also includes the neonatal receptor, FcRn,
which is responsible for the transfer of maternal IgGs to the fetus
(Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J.
Immunol. 24:249 (1994)).
[0088] "Complement dependent cytotoxicity" or "CDC" refers to the
ability of a molecule to lyse a target in the presence of
complement. The complement activation pathway is initiated by the
binding of the first component of the complement system (C1q) to a
molecule (e.g. an antibody) complexed with a cognate antigen. To
assess complement activation, a CDC assay, e.g. as described in
Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be
performed.
[0089] "Native antibodies" are usually heterotetrameric
glycoproteins of about 150,000 daltons, composed of two identical
light (L) chains and two identical heavy (H) chains. Each light
chain is linked to a heavy chain by one covalent disulfide bond,
while the number of disulfide linkages varies among the heavy
chains of different immunoglobulin isotypes. Each heavy and light
chain also has regularly spaced intrachain disulfide bridges. Each
heavy chain has at one end a variable domain (V.sub.H) followed by
a number of constant domains. Each light chain has a variable
domain at one end (V.sub.L) and a constant domain at its other end.
The constant domain of the light chain is aligned with the first
constant domain of the heavy chain, and the light-chain variable
domain is aligned with the variable domain of the heavy chain.
Particular amino acid residues are believed to form an interface
between the light chain and heavy chain variable domains.
[0090] The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed throughout the variable
domains of antibodies. It is concentrated in three segments called
hypervariable regions both in the light chain and the heavy chain
variable domains. The more highly conserved portions of variable
domains are called the framework regions (FRs). The variable
domains of native heavy and light chains each comprise four FRs,
largely adopting a .beta.-sheet configuration, connected by three
hypervariable regions, which form loops connecting, and in some
cases forming part of, the .beta.-sheet structure. The
hypervariable regions in each chain are held together in close
proximity by the FRs and, with the hypervariable regions from the
other chain, contribute to the formation of the antigen-binding
site of antibodies (see Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)). The constant domains
are not involved directly in binding an antibody to an antigen, but
exhibit various effector functions, such as participation of the
antibody in antibody dependent cellular cytotoxicity (ADCC).
[0091] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody which are responsible for
antigen-binding. The hypervariable region generally comprises amino
acid residues from a "complementarity determining region" or "CDR"
(e.g. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light
chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in
the heavy chain variable domain; Kabat et al., Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)) and/or those
residues from a "hypervariable loop" (e.g. residues 26-32 (L1),
50-52 (L2) and 91-96 (L3) in the light chain variable domain and
26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable
domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
"Framework Region" or "FR" residues are those variable domain
residues other than the hypervariable region residues as herein
defined.
[0092] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
yields an F(ab').sub.2 fragment that has two antigen-binding sites
and is still capable of cross-linking antigen.
[0093] "Fv" is the minimum antibody fragment which contains a
complete antigen-recognition and antigen-binding site. This region
consists of a dimer of one heavy chain and one light chain variable
domain in tight, non-covalent association. It is in this
configuration that the three hypervariable regions of each variable
domain interact to define an antigen-binding site on the surface of
the V.sub.H-V.sub.L dimer. Collectively, the six hypervariable
regions confer antigen-binding specificity to the antibody.
However, even a single variable domain (or half of an Fv comprising
only three hypervariable regions specific for an antigen) has the
ability to recognize and bind antigen, although at a lower affinity
than the entire binding site.
[0094] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (CH1) of the heavy chain.
Fab' fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear at least one free thiol
group. F(ab').sub.2 antibody fragments originally were produced as
pairs of Fab' fragments which have hinge cysteines between them.
Other chemical couplings of antibody fragments are also known.
[0095] The "light chains" of antibodies from any vertebrate species
can be assigned to one of two clearly distinct types, called kappa
(K) and lambda (X), based on the amino acid sequences of their
constant domains.
[0096] "Single-chain Fv" or "scFv" antibody fragments comprise the
V.sub.H and V.sub.L domains of antibody, wherein these domains are
present in a single polypeptide chain. Preferably, the Fv
polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the scFv to form the
desired structure for antigen binding. For a review of scFv see
Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315
(1994). Anti-ErbB2 antibody scFv fragments are described in
WO93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No.
5,587,458.
[0097] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a variable
heavy domain (V.sub.H) connected to a variable light domain
(V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L). By using
a linker that is too short to allow pairing between the two domains
on the same chain, the domains are forced to pair with the
complementary domains of another chain and create two
antigen-binding sites. Diabodies are described more fully in, for
example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.
Acad. Sci. USA, 90:6444-6448 (1993).
[0098] "Humanized" forms of non-human (e.g., rodent) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity, and capacity. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
[0099] Humanized anti-ErbB2 antibodies include huMAb4D5-1,
huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6,
huMAb4D5-7 and huMAb4D5-8 (HERCEPTIN.RTM.) as described in Table 3
of U.S. Pat. No. 5,821,337 expressly incorporated herein by
reference; humanized 520C9 (WO93/21319) and humanized 2C4
antibodies as described hereinbelow.
[0100] An "isolated" antibody is one which has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with diagnostic or therapeutic uses
for the antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred
embodiments, the antibody will be purified (1) to greater than 95%
by weight of antibody as determined by the Lowry method, and most
preferably more than 99% by weight, (2) to a degree sufficient to
obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, preferably, silver stain. Isolated antibody
includes the antibody in situ within recombinant cells since at
least one component of the antibody's natural environment will not
be present. Ordinarily, however, isolated antibody will be prepared
by at least one purification step.
[0101] An antibody "which binds" an antigen of interest, e.g. ErbB2
antigen, is one capable of binding that antigen with sufficient
affinity such that the antibody is useful as a therapeutic agent in
targeting a cell expressing the antigen. Where the antibody is one
which binds ErbB2, it will usually preferentially bind ErbB2 as
opposed to other ErbBs, and may be one which does not significantly
cross-react with other proteins such as EGFR, ErbB3 or ErbB4. In
such embodiments, the extent of binding of the antibody to these
non-ErbB2 proteins (e.g., cell surface binding to endogenous
receptor) will be less than 10% as determined by fluorescence
activated cell sorting (FACS) analysis or radioimmunoprecipitation
(RIA). Sometimes, the anti-ErbB2 antibody will not significantly
cross-react with the rat neu protein, e.g., as described in
Schecter et al. Nature 312:513 (1984) and Drebin et al., Nature
312:545-548 (1984).
[0102] An antibody which "blocks" ligand activation of an ErbB is
one which reduces or prevents such activation as hereinabove
defined, wherein the antibody is able to block ligand activation of
the ErbB substantially more effectively than monoclonal antibody
4D5, e.g. about as effectively as monoclonal antibodies 7F3 or 2C4
or Fab fragments thereof and preferably about as effectively as
monoclonal antibody 2C4 or a Fab fragment thereof. For example, the
antibody that blocks ligand activation of an ErbB may be one which
is about 50-100% more effective than 4D5 at blocking formation of
an ErbB hetero-oligomer. Blocking of ligand activation of an ErbB
can occur by any means, e.g. by interfering with: ligand binding to
an ErbB, ErbB complex formation, tyrosine kinase activity of an
ErbB in an ErbB complex and/or phosphorylation of tyrosine kinase
residue(s) in or by an ErbB. Examples of antibodies which block
ligand activation of an ErbB include monoclonal antibodies 2C4 and
7F3 (which block HRG activation of ErbB2/ErbB3 and ErbB2/ErbB4
hetero-oligomers; and EGF, TGF-.alpha., amphiregulin, HB-EGF and/or
epiregulin activation of an EGFR/ErbB2 hetero-oligomer); and L26,
L96 and L288 antibodies (Klapper et al. Oncogene 14:2099-2109
(1997)), which block EGF and NDF binding to T47D cells which
express EGFR, ErbB2, ErbB3 and ErbB4.
[0103] An antibody having a "biological characteristic" of a
designated antibody, such as the monoclonal antibody designated
2C4, is one which possesses one or more of the biological
characteristics of that antibody which distinguish it from other
antibodies that bind to the same antigen (e.g. ErbB2). For example,
an antibody with a biological characteristic of 2C4 may block HRG
activation of an ErbB hetero-oligomer comprising ErbB2 and ErbB3 or
ErbB4; block EGF, TGF-.alpha., HB-EGF, epiregulin and/or
amphiregulin activation of an ErbB comprising EGFR and ErbB2; block
EGF, TGF-.alpha. and/or HRG mediated activation of MAPK; and/or
bind the same epitope in the extracellular domain of ErbB2 as that
bound by 2C4 (e.g. which blocks binding of monoclonal antibody 2C4
to ErbB2).
[0104] Unless indicated otherwise, the expression "monoclonal
antibody 2C4" refers to an antibody that has antigen binding
residues of, or derived from, the murine 2C4 antibody of the
Examples below. For example, the monoclonal antibody 2C4 may be
murine monoclonal antibody 2C4 or a variant thereof, such as
humanized antibody 2C4, possessing antigen binding amino acid
residues of murine monoclonal antibody 2C4. Examples of humanized
2C4 antibodies are provided in Example 3 below. Unless indicated
otherwise, the expression "rhuMAb 2C4" when used herein refers to
an antibody comprising the variable light (V.sub.L) and variable
heavy (V.sub.H) sequences of SEQ ID Nos. 3 and 4, respectively,
fused to human light and heavy IgG1 (non-A allotype) constant
region sequences optionally expressed by a Chinese Hamster Ovary
(CHO) cell.
[0105] Unless indicated otherwise, the term "monoclonal antibody
4D5" refers to an antibody that has antigen binding residues of, or
derived from, the murine 4D5 antibody (ATCC CRL 10463). For
example, the monoclonal antibody 4D5 may be murine monoclonal
antibody 4D5 or a variant thereof, such as a humanized 4D5,
possessing antigen binding residues of murine monoclonal antibody
4D5. Exemplary humanized 4D5 antibodies include huMAb4D5-1,
huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6,
huMAb4D5-7 and huMAb4D5-8 (HERCEPTIN.RTM.) as in U.S. Pat. No.
5,821,337, with huMAb4D5-8 (HERCEPTIN.RTM.) being a preferred
humanized 4D5 antibody.
[0106] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell, especially
an ErbB expressing cell either in vitro or in vivo. Thus, the
growth inhibitory agent may be one which significantly reduces the
percentage of ErbB expressing cells in S phase. Examples of growth
inhibitory agents include agents that block cell cycle progression
(at a place other than S phase), such as agents that induce G1
arrest and M-phase arrest. Classical M-phase blockers include the
vincas (vincristine and vinblastine), taxanes, and topo II
inhibitors such as doxorubicin, epirubicin, daunorubicin,
etoposide, and bleomycin. Those agents that arrest G1 also spill
over into S-phase arrest, for example, DNA alkylating agents such
as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin,
methotrexate, 5-fluorouracil, and ara-C. Further information can be
found in The Molecular Basis of Cancer, Mendelsohn and Israel,
eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and
antineoplastic drugs" by Murakami et al. (W B Saunders:
Philadelphia, 1995), especially p. 13.
[0107] Examples of "growth inhibitory" antibodies are those which
bind to ErbB2 and inhibit the growth of cells overexpressing ErbB2.
Preferred growth inhibitory anti-ErbB2 antibodies inhibit growth of
SK-BR-3 breast tumor cells in cell culture by greater than 20%, and
preferably greater than 50% (e.g. from about 50% to about 100%) at
an antibody concentration of about 0.5 to 30 .mu.g/ml, where the
growth inhibition is determined six days after exposure of the
SK-BR-3 cells to the antibody (see U.S. Pat. No. 5,677,171 issued
Oct. 14, 1997). The SK-BR-3 cell growth inhibition assay is
described in more detail in that patent and hereinbelow. The
preferred growth inhibitory antibody is monoclonal antibody 4D5,
e.g., humanized 4D5.
[0108] An antibody which "induces cell death" is one which causes a
viable cell to become nonviable. The cell is generally one which
expresses the ErbB2 receptor, especially where the cell
overexpresses the ErbB2 receptor. Preferably, the cell is a cancer
cell, e.g. a breast, ovarian, stomach, endometrial, salivary gland,
lung, kidney, colon, thyroid, pancreatic or bladder cell. In vitro,
the cell may be a SK-BR-3, BT474, Calu 3, MDA-MB-453, MDA-MB-361 or
SKOV3 cell. Cell death in vitro may be determined in the absence of
complement and immune effector cells to distinguish cell death
induced by antibody-dependent cell-mediated cytotoxicity (ADCC) or
complement dependent cytotoxicity (CDC). Thus, the assay for cell
death may be performed using heat inactivated serum (i.e. in the
absence of complement) and in the absence of immune effector cells.
To determine whether the antibody is able to induce cell death,
loss of membrane integrity as evaluated by uptake of propidium
iodide (PI), trypan blue (see Moore et al. Cytotechnology 17:1-11
(1995)) or 7AAD can be assessed relative to untreated cells.
Preferred cell death-inducing antibodies are those which induce PI
uptake in the PI uptake assay in BT474 cells (see below).
[0109] An antibody which "induces apoptosis" is one which induces
programmed cell death as determined by binding of annexin V,
fragmentation of DNA, cell shrinkage, dilation of endoplasmic
reticulum, cell fragmentation, and/or formation of membrane
vesicles (called apoptotic bodies). The cell is usually one which
overexpresses the ErbB2 receptor. Preferably the cell is a tumor
cell, e.g. a breast, ovarian, stomach, endometrial, salivary gland,
lung, kidney, colon, thyroid, pancreatic or bladder cell. In vitro,
the cell may be a SK-BR-3, BT474, Calu 3 cell, MDA-MB-453,
MDA-MB-361 or SKOV3 cell. Various methods are available for
evaluating the cellular events associated with apoptosis. For
example, phosphatidyl serine (PS) translocation can be measured by
annexin binding; DNA fragmentation can be evaluated through DNA
laddering; and nuclear/chromatin condensation along with DNA
fragmentation can be evaluated by any increase in hypodiploid
cells. Preferably, the antibody which induces apoptosis is one
which results in about 2 to 50 fold, preferably about 5 to 50 fold,
and most preferably about 10 to 50 fold, induction of annexin
binding relative to untreated cell in an annexin binding assay
using BT474 cells (see below). Sometimes the pro-apoptotic antibody
will be one which further blocks ErbB ligand activation of an ErbB
(e.g. 7F3 antibody); i.e. the antibody shares a biological
characteristic with monoclonal antibody 2C4. In other situations,
the antibody is one which does not significantly block ErbB ligand
activation of an ErbB (e.g. 7C2). Further, the antibody may be one
like 7C2 which, while inducing apoptosis, does not induce a large
reduction in the percent of cells in S phase (e.g. one which only
induces about 0-10% reduction in the percent of these cells
relative to control).
[0110] The "epitope 2C4" is the region in the extracellular domain
of ErbB2 to which the antibody 2C4 binds. In order to screen for
antibodies which bind to the 2C4 epitope, a routine cross-blocking
assay such as that described in Antibodies, A Laboratory Manual,
Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can
be performed. Alternatively, epitope mapping can be performed to
assess whether the antibody binds to the 2C4 epitope of ErbB2 (e.g.
any one or more residues in the region from about residue 22 to
about residue 584 of ErbB2, inclusive; see FIGS. 1A-B).
[0111] The "epitope 4D5" is the region in the extracellular domain
of ErbB2 to which the antibody 4D5 (ATCC CRL 10463) binds. This
epitope is close to the transmembrane domain of ErbB2. To screen
for antibodies which bind to the 4D5 epitope, a routine
cross-blocking assay such as that described in Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and
David Lane (1988), can be performed. Alternatively, epitope mapping
can be performed to assess whether the antibody binds to the 4D5
epitope of ErbB2 (e.g. any one or more residues in the region from
about residue 529 to about residue 625, inclusive; see FIGS.
1A-B).
[0112] The "epitope 3H4" is the region in the extracellular domain
of ErbB2 to which the antibody 3H4 binds. This epitope includes
residues from about 541 to about 599, inclusive, in the amino acid
sequence of ErbB2 extracellular domain; see FIGS. 1A-B.
[0113] The "epitope 7C2/7F3" is the region at the N terminus of the
extracellular domain of ErbB2 to which the 7C2 and/or 7F3
antibodies (each deposited with the ATCC, see below) bind. To
screen for antibodies which bind to the 7C2/7F3 epitope, a routine
cross-blocking assay such as that described in Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and
David Lane (1988), can be performed. Alternatively, epitope mapping
can be performed to establish whether the antibody binds to the
7C2/7F3 epitope on ErbB2 (e.g. any one or more of residues in the
region from about residue 22 to about residue 53 of ErbB2; see
FIGS. 1A-B).
[0114] An "ErbB-expressing cell" is one which has ErbB protein
present at its cell surface, such that an anti-ErbB2 antibody can
bind thereto.
[0115] A cell "characterized by excessive activation" of an ErbB is
one in which the extent of ErbB activation therein significantly
exceeds the level of activation of that receptor in a normal cell
of the same tissue type. Such excessive activation may result from
overexpression or amplification of the ErbB and/or greater than
normal levels of an ErbB ligand available for activating the ErbB
in the cell. Such excessive activation may cause and/or be caused
by a diseased state of the cell. In some embodiments, a sample from
the patient will be subjected to a diagnostic or prognostic assay
to determine whether amplification and/or overexpression of an ErbB
is occurring which results in such excessive activation of the
ErbB. Alternatively, or additionally, a sample from the patient may
be subjected to a diagnostic or prognostic assay to determine
whether amplification, overexpression and/or increased proteolytic
processing of an ErbB ligand is occurring in the patient which
attributes to excessive activation of the receptor. Sometimes,
excessive activation of the receptor may result from an autocrine
stimulatory pathway.
[0116] In an "autocrine" stimulatory pathway, self stimulation
occurs by virtue of the cell producing both an ErbB ligand and its
cognate ErbB. For example, the cell may express or overexpress EGFR
and also express or overexpress an EGFR ligand (e.g. EGF,
TGF-.alpha., or HB-EGF). In another embodiment, the cell may
express or overexpress ErbB2 and also express or overexpress a
heregulin (e.g. .gamma.-HRG).
[0117] A cell which "overexpresses" an ErbB is one which has
significantly higher levels of an ErbB, such as ErbB2, at the cell
surface thereof, compared to a normal cell of the same tissue type.
Such overexpression may be caused by gene amplification or by
increased transcription or translation. ErbB overexpression may be
determined in a diagnostic or prognostic assay by evaluating
increased levels of the ErbB protein present on the surface of a
cell (e.g. via an immunohistochemistry assay; IHC, immunoenzyme,
Western blot, ligand binding, kinase activity). Alternatively, or
additionally, one may measure levels of ErbB-encoding nucleic acid
in the cell, e.g. via fluorescent in situ hybridization (FISH; see
WO98/45479 published October, 1998), southern blotting, or
polymerase chain reaction (PCR) techniques, such as real time
quantitative PCR (RT-PCR). One may also study ErbB overexpression
by measuring shed antigen (e.g., ErbB extracellular domain) in a
biological fluid such as serum (see, e.g., U.S. Pat. No. 4,933,294
issued Jun. 12, 1990; WO91/05264 published Apr. 18, 1991; U.S. Pat.
No. 5,401,638 issued Mar. 28, 1995; and Sias et al. J. Immunol.
Methods 132: 73-80 (1990)). Aside from the above assays, various in
vivo assays are available to the skilled practitioner. For example,
one may expose cells within the body of the patient to an antibody
which is optionally labeled with a detectable label, e.g. a
radioactive isotope, and binding of the antibody to cells in the
patient can be evaluated, e.g. by external scanning for
radioactivity or by analyzing a biopsy taken from a patient
previously exposed to the antibody.
[0118] Conversely, a cell which is "not characterized by
overexpression of an ErbB" is one which, in a diagnostic assay,
does not express higher than normal levels of ErbB compared to a
normal cell of the same tissue type.
[0119] A cell which "overexpresses" an ErbB ligand is one which
produces significantly higher levels of that ligand compared to a
normal cell of the same tissue type. Such overexpression may be
caused by gene amplification or by increased transcription or
translation. Overexpression of the ErbB ligand may be determined
diagnostically by evaluating levels of the ligand (or nucleic acid
encoding it) in the patient, e.g. in a biopsy or by various
diagnostic assays such as the IHC, immunoenzyme, Western blot,
ligand binding, FISH, southern blotting, PCR or in vivo assays
described above.
[0120] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g. At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, p.sup.32
and radioactive isotopes of Lu), chemotherapeutic agents, and
toxins such as small molecule toxins or enzymatically active toxins
of bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof.
[0121] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include alkylating agents such as thiotepa and cyclosphosphamide
(CYTOXAN.RTM.); alkyl sulfonates such as busulfan, improsulfan and
piposulfan; aziridines such as benzodopa, carboquone, meturedopa,
and uredopa; ethylenimines and methylamelamines including
altretamine, triethylenemelamine, trietylenephosphoramide,
triethylenethiophosphaoramide and trimethylolomelamine; nitrogen
mustards such as chlorambucil, chlomaphazine, cholophosphamide,
estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide
hydrochloride, melphalan, novembichin, phenesterine, prednimustine,
trofosfamide, uracil mustard; nitrosureas such as carmustine,
chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;
antibiotics such as aclacinomysins, actinomycin, authramycin,
azaserine, bleomycins, cactinomycin, calicheamicin, carabicin,
carminomycin, carzinophilin, chromomycins, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin,
epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins,
mycophenolic acid, nogalamycin, olivomycins, peplomycin,
potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,
streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;
anti-metabolites such as methotrexate and 5-fluorouracil (5-FU);
folic acid analogues such as denopterin, methotrexate, pteropterin,
trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine,
thiamiprine, thioguanine; pyrimidine analogs such as ancitabine,
azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,
doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as
calusterone, dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
amusacrine; bestrabucil; bisantrene; edatraxate; defofamine;
demecolcine; diaziquone; elfomithine; elliptinium acetate;
etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine;
mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin;
phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide;
procarbazine; PSK.RTM.; razoxane; sizofiran; spirogermanium;
tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine;
urethan; vindesine; dacarbazine; mannomustine; mitobronitol;
mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");
cyclophosphamide; thiotepa; taxanes, e.g. paclitaxel (TAXOL.RTM.,
Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel
(TAXOTERE.RTM., Rhone-Poulenc Rorer, Antony, France); chlorambucil;
gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum
analogs such as cisplatin and carboplatin; vinblastine; platinum;
etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;
vincristine; vinorelbine; navelbine; novantrone; teniposide;
daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase
inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid;
esperamicins; capecitabine; and pharmaceutically acceptable salts,
acids or derivatives of any of the above. Also included in this
definition are anti-hormonal agents that act to regulate or inhibit
hormone action on cells such as anti-estrogens including for
example tamoxifen, raloxifene, aromatase inhibiting
4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene,
LY117018, onapristone, and toremifene (Fareston); and
anti-androgens such as flutamide, nilutamide, bicalutamide,
leuprolide, and goserelin; and pharmaceutically acceptable salts,
acids or derivatives of any of the above.
[0122] An "anti-angiogenic agent" refers to a compound which
blocks, or interferes with to some degree, the development of blood
vessels. The anti-angiogenic factor may, for instance, be a small
molecule or antibody that binds to a growth factor or growth factor
receptor involved in promoting angiogenesis. The preferred
anti-angiogenic factor herein is an antibody that binds to Vascular
Endothelial Growth Factor (VEGF), such as the recombinant humanized
anti-VEGF antibody AVASTIN.RTM. (Genentech).
[0123] The term "cytokine" is a generic term for proteins released
by one cell population which act on another cell as intercellular
mediators. Examples of such cytokines are lymphokines, monokines,
and traditional polypeptide hormones. Included among the cytokines
are growth hormone such as human growth hormone, N-methionyl human
growth hormone, and bovine growth hormone; parathyroid hormone;
thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein
hormones such as follicle stimulating hormone (FSH), thyroid
stimulating hormone (TSH), and luteinizing hormone (LH); hepatic
growth factor; fibroblast growth factor; prolactin; placental
lactogen; tumor necrosis factor-.alpha. and -.beta.;
mullerian-inhibiting substance; mouse gonadotropin-associated
peptide; inhibin; activin; vascular endothelial growth factor;
integrin; thrombopoietin (TPO); nerve growth factors such as
NGF-.beta.; platelet-growth factor; transforming growth factors
(TGFs) such as TGF-.alpha. and TGF-.beta.; insulin-like growth
factor-I and -II; erythropoietin (EPO); osteoinductive factors;
interferons such as interferon-.alpha., -.beta., and -.gamma.,
colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);
granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);
interleukins (ILs) such as IL-1, IL-1.alpha., IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; a tumor necrosis
factor such as TNF-.alpha. or TNF-.beta.; and other polypeptide
factors including LIF and kit ligand (KL). As used herein, the term
cytokine includes proteins from natural sources or from recombinant
cell culture and biologically active equivalents of the native
sequence cytokines.
[0124] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, contraindications and/or warnings
concerning the use of such therapeutic products.
[0125] A "cardioprotectant" is a compound or composition which
prevents or reduces myocardial dysfunction (i.e. cardiomyopathy
and/or congestive heart failure) associated with administration of
a drug, such as an anthracycline antibiotic and/or an anti-ErbB2
antibody, to a patient. The cardioprotectant may, for example,
block or reduce a free-radical-mediated cardiotoxic effect and/or
prevent or reduce oxidative-stress injury. Examples of
cardioprotectants encompassed by the present definition include the
iron-chelating agent dexrazoxane (ICRF-187) (Seifert et al. The
Annals of Pharmacotherapy 28:1063-1072 (1994)); a lipid-lowering
agent and/or anti-oxidant such as probucol (Singal et al. J. Mol.
Cell Cardiol. 27:1055-1063 (1995)); amifostine (aminothiol
2-[(3-aminopropyl)amino] ethanethiol-dihydrogen phosphate ester,
also called WR-2721, and the dephosphorylated cellular uptake form
thereof called WR-1065) and
S-3-(3-methylaminopropylamino)propylphosphorothioic acid
(WR-151327), see Green et al. Cancer Research 54:738-741 (1994);
digoxin (Bristow, M. R. In: Bristow M R, ed. Drug-Induced Heart
Disease. New York: Elsevier 191-215 (1980)); beta-blockers such as
metoprolol (Hjalmarson et al. Drugs 47:Suppl 4:31-9 (1994); and
Shaddy et al. Am. Heart J. 129:197-9 (1995)); vitamin E; ascorbic
acid (vitamin C); free radical scavengers such as oleanolic acid,
ursolic acid and N-acetylcysteine (NAC); spin trapping compounds
such as alpha-phenyl-tert-butyl nitrone (PBN); (Paracchini et al.,
Anticancer Res. 13:1607-1612 (1993)); selenoorganic compounds such
as P251 (Elbesen); and the like.
II. Detailed Description
[0126] The ErbB (HER) family of transmembrane tyrosine kinase
receptors is composed of four members, ErbB1 (HER1, EGFR); ErbB2
(HER2 or p185.sup.neu), ErbB3 (HER3) and ErbB4 (HER4 or tyro2).
[0127] ErbB2/HER2/p185.sup.neu and ErbB1/EGFR are significantly
over-expressed in most epithelial malignancies, including breast
cancer, head and neck cancer, stomach cancer, prostate cancer,
ovarian cancer, pancreatic cancer, lung cancer bladder, as well as
glioblastomas. HER2 amplifies the signal provided by other
receptors of the HER family by forming heterodimers. The essential
role of HER2 in the HER signaling network led to the development of
anti-HER2 monoclonal antibodies (MAbs) for cancer therapy. In
particular, a humanized anti-ErbB2 MAb (transtuzumab,
Herceptin.RTM., Genentech, Inc.) is widely used for the treatment
of women with HER2 overexpressing breast cancers. Trastuzumab
induces HER2 receptor downmodulation and, as a result, inhibits
critical signalling pathways (i.e. ras-Raf-MAPK and P13K/Akt) and
blocks cell cycle progression by inducing the formation of p27/Cdk2
complexes. Trastuzumab also inhibits HER2 cleavage, preceding
antibody-induced receptor downmodulation, which effect might
contribute to its antitumor activity in some cancers. In vivo,
trastuzumab inhibits angiogenesis and induces antibody-dependent
cellular cytotoxicity. HER2 is known to form heterodimers with HER1
(EGFR), HER3 or HER4. A humanized monoclonal antibody, called 2C4,
is in clinical development for cancer treatment. 2C4 binds to a
different epitope of HER2 ectodomain than trastuzumab and
sterically hinders HER2 recruitment in heterodimers with other HER
receptors. This results in the inhibition of signalling by
HER2-based heterodimers both in cells with low and high HER2
expression. In vitro and in vivo antitumor activity has been
reported in a range of breast and prostate tumor models.
Small-molecule anti-HER2/neu peptidomimetics (AHNP) have also been
developed and tested for efficacy in cancer therapy (Zhang et al.,
Drug News Perspect. 13(6):325-9 (2000).
[0128] The present invention is, at least partially, based on the
unexpected finding that ErbB antagonists exhibit analgesic effects
and are, therefore, useful in the management of pain, including
chronic pain, such as cancer-related pain. In particular, prostate
cancer patients treated with the humanized anti-ErbB2 antibody 2C4
recorded less pain and reduced analgesia even when their PSA value
was not reduced. Similarly, the analgesic effect of the humanized
2C4 antibody was observed during the treatment of liposarcoma.
[0129] Therefore, in its broadest aspect, the invention relates to
pain management using ErbB antagonists. In particular, the
invention concerns the management of pain, including acute and
chronic pain, either cancer related or not associated with cancer,
with ErbB antagonists. The invention concerns the treatment of any
type of pain, including, without limitation, nociceptive pain,
somatic pain, visceral pain, neuropathic pain, centrally generated
pain (including deafferentiation pain and sympathetically
maintained pain), and peripherally generated pain (including
painful polyneuropathies and painful mononeuropethies).
[0130] ErbB Antagonists
[0131] The ErbB antagonists of the present invention are molecules
that block (reduce or prevent) a biological activity of one or more
ErbB(s). Preferably, the antagonist blocks (reduces or prevents)
ligand activation of an ErbB. In a particular embodiment, the ErbB
antagonists of the present invention inhibit a biological activity
mediated by an ErbB2 (HER2) and/or ErbB1 (EGFR) receptor or a
receptor complex comprising such receptor(s).
[0132] The ErbB antagonists herein include, without limitation,
polypeptides (including antibodies and antibody fragments),
peptides, peptide mimetics, non-peptide small organic molecules,
antisense molecules, and oligonucleotide decoy molecules.
[0133] Small organic molecules that compete with ATP binding to the
kinase pocket of EGFR are reviewed, for example, by Arteaga C L.,
Exp. Cell. Res. 284(1):122-30 (2003). A small molecule EGFR
inhibitor, Iressa.TM. (ZD1839, gefinitib, Aztra-Zeneca) is
described, for example, in Kris M et al. JAMA, 290(16):2149-2158
(2003). Further small molecule EGFR antagonists include CP-358,774
(TARCEVA.RTM.; Genentech/OSI) and AG1478, AG1571 (SU 5271;
Sugen).
[0134] Anti-EGFR antibodies are also known in the art and include,
for example, Erbitux.RTM. (IMC-C225, cetuximab, ImClone) a chimeric
anti-EGFR MAb, and reshaped human 225 (H225) (see, WO 96/40210,
ImClone Systems Inc.). Further examples of antibodies which bind to
EGFR include MAb 579 (ATCC CRL HB. 8506), MAb 455 (ATCC CRL
HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see,
U.S. Pat. No. 4,943,533, Mendelsohn et al.) and variants thereof,
humanized and chimeric antibodies that bind EGFR as described in
U.S. Pat. No. 5,891,996, and human antibodies that bind EGFR, such
as ABX-EGF (see WO98/50433, Abgenix). The anti-EGFR antibody may be
conjugated with a cytotoxic agent, thus generating an
immunoconjugate (see, e.g., EP659,439A2, Merck Patent GmbH).
[0135] CP-654577 (Pfizer) a selective inhibitor of ErbB2 relative
to EGFR tyrosine kinase, is disclosed, for example, in Barbacci et
al., Cancer Res. 63(15):4450-9 (2003). A small molecule form of
anti-ErbB2 peptidomimetic is described by Zhang et al., Drug Nes
Perspect 13(6):325-9 (2000).
[0136] Identification of ErbB Antagonists
[0137] New agents that target and inhibit a biological activity of
ErbB receptors, such as EGFR and/or ErbB2, can be identified by
methods known in the art.
[0138] In general, the first step in identifying new ErbB
antagonists is in vitro screening followed by in vivo assays in an
apporpriate animal model and ultimately human clinical trials.
[0139] To identify compounds that bind to an ErbB receptor or
receptor complex, receptor-binding tests can be performed using
ErbB receptors or receptor complexes isolated from their respective
native sources, or produced by recombinant DNA technology and/or
chemical synthesis. The binding affinity of the candidate compounds
can be tested by direct binding or by indirect, e.g. competitive,
binding. In competitive binding experiments, the concentration of a
compound necessary to displace 50% of another compound bound to the
receptor (IC.sub.50) is usually used as a measure of binding
affinity.
[0140] In another method, in order to identify novel ErbB
antagonists, DNA encoding the sequence encoding the target ErbB
receptor (e.g. ErbB2 or EGFR) is cloned into an expression vector
containing a selectable marker. The vector is used to transfect
recombinant host cells. Following several rounds of selection
stable lines which express the ErbB receptor are identified. New
ErbB antagonists can then be identified by virtue of their ability
to compete effectively with a known inhibitor of the target ErbB
receptor. Binding coefficients can be determined by any known
manner, e.g. by Scatchard analysis.
[0141] Binding experiments can also be performed using cells or
cell lines known to express the target ErbB receptor.
[0142] To identify a candidate molecule which blocks ligand
activation of an ErbB, the ability of the molecule to block ErbB
ligand binding to cells expressing the ErbB (e.g. in conjugation
with another ErbB with which the ErbB of interest forms an ErbB
hetero-oligomer) may be determined. For example, cells naturally
expressing, or transfected to express, ErbBs of the ErbB
hetero-oligomer may be incubated with the candidate molecule and
then exposed to labeled ErbB ligand. The ability of the candidate
molecule to block ligand binding to the ErbB in the ErbB
hetero-oligomer may then be evaluated.
[0143] Alternatively, or additionally, the ability of a candidate
molecule to block ErbB ligand-stimulated tyrosine phosphorylation
of an ErbB present in an ErbB hetero-oligomer may be assessed. For
example, cells endogenously expressing the ErbBs or transfected to
expressed them may be incubated with the candidate molecule and
then assayed for ErbB ligand-dependent tyrosine phosphorylation
activity using an anti-phosphotyrosine monoclonal (which is
optionally conjugated with a detectable label). The kinase receptor
activation assay described in U.S. Pat. No. 5,766,863 is also
available for determining ErbB activation and blocking of that
activity by an antagonist.
[0144] One may also assess the growth inhibitory effects of a
candidate molecule on MDA-MB-175 cells, e.g, essentially as
described in Schaefer et al. Oncogene 15:1385-1394 (1997).
According to this assay, MDA-MB-175 cells may treated with a
candidate antagonist and stained with crystal violet. Incubation
with a candidate antagonist may show a growth inhibitory effect on
this cell line similar to that displayed by monoclonal antibody
2C4. In a further embodiment, exogenous HRG will not significantly
reverse this inhibition. Preferably, the antagonist will be able to
inhibit cell proliferation of MDA-MB-175 cells to a greater extent
than monoclonal antibody 4D5 (and optionally to a greater extent
than monoclonal antibody 7F3), both in the presence and absence of
exogenous HRG.
[0145] In one embodiment, the antagonist may block heregulin
dependent association of ErbB2 with ErbB3 in both MCF7 and SK-BR-3
cells as determined in a co-immunoprecipitation experiment such as
that described in Example 2 substantially more effectively than
monoclonal antibody 4D5, and preferably substantially more
effectively than monoclonal antibody 7F3.
[0146] To identify antagonists with growth inhibitory properties,
one may screen for antibodies which inhibit the growth of cancer
cells which overexpress ErbB2.
[0147] To select for antagonists which induce cell death, loss of
membrane integrity as indicated by, e.g., PI, trypan blue or 7AAD
uptake may be assessed relative to control. The preferred assay is
the PI uptake assay using BT474 cells. Details of this assay will
be provided hereinafter, with particular reference to
antibodies.
[0148] In order to select for antagonists which induce apoptosis,
for example an annexin binding assay using BT474 cells or a DNA
staining assay using BT 474 cells can be used, These assays will be
described in detail hereinbelow, with reference to antibodies with
apoptotic properties.
[0149] According to a further approach, ErbB2 and EGFR antagonists
are identified by correlating EGFR, TGF-.alpha. (a ligand for
EGFR), and ErbB2 mRNA expression levels with the results of
cytotoxicity assays of the 49000 compounds in the National Cancer
Institute (NCI) drug screen database (Wosikowski et al., J. Natl.
Cancer Inst. 89(20): 1505-15 (1997)).
[0150] After determining the inhibition of a target biological
activity in an in vitro assay, the ability of a candidate
antagonist to control pain can be tested in in vivo models of
pain.
[0151] In a preferred embodiment, the analgesic activity of a
candidate ErbB antagonist is tested in an animal model used in pain
research.
[0152] Animal Models of Pain
[0153] (i) Noxious Pain
[0154] A suitable animal model of noxious pain is the "tail flick"
test (Bass and Wanderbrook, J. Am. Pharm. Assoc. Sci. Ed. 41:569-70
(1952)) This method is based on measuring pain sensitivity in mice
or rats as they respond to the application of heat to a small area
of their tails with or without administration of a candidate ErbB
antagonist. The test is based on measuring the withdrawal time of
the tail following application of radiant heat, and can be
performed by using commercially available equipment, such as, for
example, the Tail Flick Analgesia Meter (SDI, San Diego,
Calif.).
[0155] Another animal model of noxious pain is the "hot plate
paw-licking" test. In this test, animals (rats or mice) are
individually placed on a hot plate. The latency to first sign of
hind paw licking is taken or jump response is taken as an index of
nociceptive threshold. The effect of drug administration on this
threshold is considered to be an index of analgesic response.
(O'Callaghan and Holzman, J. Pharmaceol. Exp. Ther. 192:497-505
(1975)). The route of drug administration may differ but typically
is subcutaneous.
[0156] The paw-pressure, or mechanical hyperalgesia, test uses a
pressure of increasing intensity applied to a punctiform area on
the hindpaw or, less commonly, on the tail. In practice, the paw or
tail is placed between a plane surface and a blunt, plastic-coated
point mounted on top of a system of cogwheels, with a cursor that
can be displaced along the length of a graduated beam (Green et
al., Br. J. Pharmacol. 6:572-85 (1957) for an automated readout.
The application of increasing pressure is interrupted when the
animal removes its tail, an action that is read out as force in
grams for the threshold of response.
[0157] Chemical stimuli can also be used to model noxious pain in
animals. Typically the chemical agents are administered
intradermally or intraperitoneally. For example, in the formalin
(paw) test usually a 0.5 to 15% (generally about 3.5%) solution of
formalin is injected into the dorsal or plantar surface of the rat
fore- or hindpaw, and produces a painful response of increasing and
decreasing intensity for about 60 minutes. Typical responses
include paw lifting, licking, nibbling or shaking, which can be
monitored in the absence and presence of a test substance
(Lariviere and Melzack, Pain 66:271-7 (1996)).
[0158] (ii) Visceral Pain
[0159] Visceral pain is typically tested by intraperitoneal
injections of irritants (Writhing Test), such as, for example,
acetic acid/ethacrinic acid or phenylbenzoquinone. The agents
provoke a stereotypical response in rodents, charcterized by
abdominal contractions, whole body movements, contortions of the
abdominal muscles, reduced motor activity and incoordination. These
responses are considered indicative of visceral pain associated
with visceral chemoreceptors. The writhing test has been
successfully used to predict effective analgesic doses for
treatment of humans (Dubinsky et al., Agents Actions 20:50-60
(1987)).
[0160] (iii) Inflammatory Pain
[0161] Inflammatory pain is typically modeled using
p-benzoquinone-induced Writhing Test or the carrageean-induced hind
paw edema model (DiRosa et al., J. Pathol. 101:15-29 (1971)).
[0162] In another animal model of inflammatory pain, intradermal
injection of capsaicin is used to neurogenic inflammation and
hyperalgesia. Originally, response was observed and quantitated by
visual inspectsion However, recent developments in thermography and
laser-Doppler Flowmetry have proved to be invaluable tools in
quantitating neurogenic inflammation and inflammatory responses.
For further details see, e.g. Sumikura et al., Pain 105(285-291
(2003).
[0163] (iv) Licking, Scratching and Biting Responses
[0164] Such responses can be induced, for example, by TNF-.alpha.
(typically 100 pg), IFN-.gamma. (typically 100 pg), or IL-1.beta.
(typically 100 pg) injected intratracheally (i.t.), especially at
higher dose (3 g/kg). In addition, similar responses can be
elicited by i.t. injections of glutamate (20 .mu.g),
N-methyl-D-aspartic acid (50 ng),
.alpha.-amino-3-hydrocy-5-methylisoxazole-4-proprionic acid (13 ng)
or kainic acid, or by administration fo substrance P or
capsaicin.
[0165] (v) Neuropathic Pain
[0166] Chronic constriction injury (CCI) of the sciatic nerve in
rats induces persistent mechanical hyperalgesia and allodyinia, and
is a widely used model of neuropathic pain (Bennett and Xie, Pain
33:87-107 (1988)).
[0167] (vi) Cancer Pain
[0168] Candidate agents for the treatment of cancer pain can be
tested in the above-listed animals models of acute and chronic pain
or inflammatory pain. In addition, there are several animal models
available that have been specifically designed to test cancer
pain.
[0169] For example, a candidate ErbB antagonist can be tested in a
murine model of bone cancer pain, as described in Menendez et al.,
Brain Res. 969(102): 102-9 (2003). This test investigated the
reactivity to noxious heat of C3G/HeJ mice receiving an intratibial
(i.t.) injetion of NCTC 2472 cells. NCTC2472 cells are able to
induce osteosarcoma, and break through bone into soft tissues about
two weeks after inoculation, producing a macroscopical increase of
the limb size from the fourth week. Thermal reactivity is
diminished during the first two weeks after cell implantation, and
this hyperalgesia is reversed by the administration of naloxone (10
mg/kg). According to the authors, during the fourth and fifth weeks
after NCTC 2472 cell implantation, an increased nociceptive heat
reactivity, instead of hypoalgesia, was observed. This hyperalgesia
was prevented by the systemic administration of morphine (15
mg/kg). This animal model of bone cancer pain can be used to
identify and study new agents, such as ErbB antagonists for the
management of bone cancer pain, and potentially cancer pain in
general.
[0170] Similarly, the effect of candidate ErbB antagonists on
cancer pain can be studied in tumor implanted mice, as described by
Wacnik et al.; Pain 101 (1-2):175-86 (2003).
[0171] A mouse model of neuropathic cancer pain has been developed
by Shimoyama et al., Pain 99(1-2): 167-74 (2002). In this model,
Meth A sarcoma cells were inoculated to the immediate proximity of
the scietic nerve in BALB/c mice. The growing tumor gradually
compresses the nerve, thereby causing nerve injury. Time courses of
thermal hyperalgesia and mechanical sensitivity to von Frey hairs
are determined and signs of spontaneous pain are evaluated.
[0172] An example of using mutant mice for pain threshold research
is described by Guilherme et al., Nature Neurosci. 6:221-222
(2003).
[0173] For a general overview of common animal models for pain see,
e.g. Eaton, M., J. of Rehabilitation Research and Development 40(4)
Suppl. 1:41-54 (2003).
[0174] Human Clinical Trials
[0175] Pain measurement is essential for assessing analgesia. In
human clinical trials, the pain experienced by an individual
patient is assessed by using one or more of the known measures of
pain, including a Visual Analog Scale (VAS), descriptive scale,
numeric scale, Health Assessment Questionare (HAQ) pain index, and
the like. A workshop sponsored by the Initiative on Methods,
Measurement, and Pain Assessment in Clinical Trials (IMMPACT)
described six consensus domains recommended to be included in all
chronic pain trials (Turk et al., Pain, 106(3):337-45 (2003)).
[0176] Most frequently, pain is assessed using a VAS recorded on
various scales, such as a scale of 0 to 100, or 0 to 10, where the
lowest score represents no pain, and the highest score represents
the worst possible pain. Since it has been reported that patients
have difficulty discriminating 100 levels of pain, the most
frequently used VAS system operates on a scale of 0 to 10 (Miller,
G A, Psychological Review 63:81-97 (1956).
[0177] The descriptive scale typically includes the following
descriptions: no pain, mild pain, moderate pain, severe pain, very
severe pain, and worst possible pain.
[0178] In a preferred embodiment, the ErbB antagonists of the
present invention are anti-ErbB2 antibodies.
Production of Anti-ErbB2 Antibodies
[0179] A description follows as to exemplary techniques for the
production of the antibodies used in accordance with the present
invention. The ErbB2 antigen to be used for production of
antibodies may be, e.g., a soluble form of the extracellular domain
of ErbB2 or a portion thereof, containing the desired epitope.
Alternatively, cells expressing ErbB2 at their cell surface (e.g.
NIH-3T3 cells transformed to overexpress ErbB2; or a carcinoma cell
line such as SK-BR-3 cells, see Stancovski et al. PNAS (USA)
88:8691-8695 (1991)) can be used to generate antibodies. Other
forms of ErbB2 useful for generating antibodies will be apparent to
those skilled in the art.
[0180] (i) Polyclonal Antibodies
[0181] Polyclonal antibodies are preferably raised in animals by
multiple subcutaneous (sc) or intraperitoneal (ip) injections of
the relevant antigen and an adjuvant. It may be useful to conjugate
the relevant antigen to a protein that is immunogenic in the
species to be immunized, e.g., keyhole limpet hemocyanin, serum
albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a
bifunctional or derivatizing agent, for example, maleimidobenzoyl
sulfosuccinimide ester (conjugation through cysteine residues),
N-hydroxysuccinimide (through lysine residues), glutaraldehyde,
succinic anhydride, SOCl.sub.2, or R.sup.1N.dbd.C.dbd.NR, where R
and R.sup.1 are different alkyl groups.
[0182] Animals are immunized against the antigen, immunogenic
conjugates, or derivatives by combining, e.g., 100 .mu.g or 5 .mu.g
of the protein or conjugate (for rabbits or mice, respectively)
with 3 volumes of Freund's complete adjuvant and injecting the
solution intradermally at multiple sites. One month later the
animals are boosted with 1/5 to 1/10 the original amount of peptide
or conjugate in Freund's complete adjuvant by subcutaneous
injection at multiple sites. Seven to 14 days later the animals are
bled and the serum is assayed for antibody titer. Animals are
boosted until the titer plateaus. Preferably, the animal is boosted
with the conjugate of the same antigen, but conjugated to a
different protein and/or through a different cross-linking reagent.
Conjugates also can be made in recombinant cell culture as protein
fusions. Also, aggregating agents such as alum are suitably used to
enhance the immune response.
[0183] (ii) Monoclonal Antibodies
[0184] Monoclonal antibodies are obtained from a population of
substantially homogeneous antibodies, i.e., the individual
antibodies comprising the population are identical except for
variants that may arise during the production of the antibody.
Thus, the modifier "monoclonal" indicates the character of the
antibody as not being a mixture of discrete antibodies.
[0185] For example, the monoclonal antibodies may be made using the
hybridoma method first described by Kohler et al., Nature, 256:495
(1975), or may be made by recombinant DNA methods (U.S. Pat. No.
4,816,567).
[0186] In the hybridoma method, a mouse or other appropriate host
animal, such as a hamster, is immunized as hereinabove described to
elicit lymphocytes that produce or are capable of producing
antibodies that will specifically bind to the protein used for
immunization. Alternatively, lymphocytes may be immunized in vitro.
Lymphocytes then are fused with myeloma cells using a suitable
fusing agent, such as polyethylene glycol, to form a hybridoma cell
(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103
(Academic Press, 1986)).
[0187] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused,
parental myeloma cells. For example, if the parental myeloma cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically
will include hypoxanthine, aminopterin, and thymidine (HAT medium),
which substances prevent the growth of HGPRT-deficient cells.
[0188] Preferred myeloma cells are those that fuse efficiently,
support stable high-level production of antibody by the selected
antibody-producing cells, and are sensitive to a medium such as HAT
medium. Among these, preferred myeloma cell lines are murine
myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse
tumors available from the Salk Institute Cell Distribution Center,
San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from
the American Type Culture Collection, Rockville, Md. USA. Human
myeloma and mouse-human heteromyeloma cell lines also have been
described for the production of human monoclonal antibodies
(Kozbor, J. Immunol., 133:3001 (1984); and Brodeur et al.,
Monoclonal Antibody Production Techniques and Applications, pp.
51-63 (Marcel Dekker, Inc., New York, 1987)).
[0189] Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed against
the antigen. Preferably, the binding specificity of monoclonal
antibodies produced by hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay
(ELISA).
[0190] The binding affinity of the monoclonal antibody can, for
example, be determined by the Scatchard analysis of Munson et al.,
Anal. Biochem., 107:220 (1980).
[0191] After hybridoma cells are identified that produce antibodies
of the desired specificity, affinity, and/or activity, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, Monoclonal Antibodies: Principles and
Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture
media for this purpose include, for example, D-MEM or RPMI-1640
medium. In addition, the hybridoma cells may be grown in vivo as
ascites tumors in an animal.
[0192] The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional antibody purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0193] DNA encoding the monoclonal antibodies is readily isolated
and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of murine antibodies).
The hybridoma cells serve as a preferred source of such DNA. Once
isolated, the DNA may be placed into expression vectors, which are
then transfected into host cells such as E. coli cells, simian COS
cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do
not otherwise produce antibody protein, to obtain the synthesis of
monoclonal antibodies in the recombinant host cells. Review
articles on recombinant expression in bacteria of DNA encoding the
antibody include Skerra et al., Curr. Opinion in Immunol.,
5:256-262 (1993) and Pluckthun, Immunol. Revs., 130:151-188
(1992).
[0194] In a further embodiment, monoclonal antibodies or antibody
fragments can be isolated from antibody phage libraries generated
using the techniques described in McCafferty et al., Nature,
348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and
Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the
isolation of murine and human antibodies, respectively, using phage
libraries. Subsequent publications describe the production of high
affinity (nM range) human antibodies by chain shuffling (Marks et
al., Bio/Technology, 10:779-783 (1992)), as well as combinatorial
infection and in vivo recombination as a strategy for constructing
very large phage libraries (Waterhouse et al., Nuc. Acids. Res.,
21:2265-2266 (1993)). Thus, these techniques are viable
alternatives to traditional monoclonal antibody hybridoma
techniques for isolation of monoclonal antibodies.
[0195] The DNA also may be modified, for example, by substituting
the coding sequence for human heavy chain and light chain constant
domains in place of the homologous murine sequences (U.S. Pat. No.
4,816,567; and Morrison, et al., Proc. Natl. Acad. Sci. USA,
81:6851 (1984)), or by covalently joining to the immunoglobulin
coding sequence all or part of the coding sequence for a
non-immunoglobulin polypeptide.
[0196] Typically such non-immunoglobulin polypeptides are
substituted for the constant domains of an antibody, or they are
substituted for the variable domains of one antigen-combining site
of an antibody to create a chimeric bivalent antibody comprising
one antigen-combining site having specificity for an antigen and
another antigen-combining site having specificity for a different
antigen.
[0197] (iii) Humanized Antibodies
[0198] Methods for humanizing non-human antibodies have been
described in the art. Preferably, a humanized antibody has one or
more amino acid residues introduced into it from a source which is
non-human. These non-human amino acid residues are often referred
to as "import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers (Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327
(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by
substituting hypervariable region sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some hypervariable region residues and possibly
some FR residues are substituted by residues from analogous sites
in rodent antibodies.
[0199] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity. According to the so-called "best-fit" method,
the sequence of the variable domain of a rodent antibody is
screened against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the
rodent is then accepted as the human framework region (FR) for the
humanized antibody (Sims et al., J. Immunol., 151:2296 (1993);
Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses
a particular framework region derived from the consensus sequence
of all human antibodies of a particular subgroup of light or heavy
chains. The same framework may be used for several different
humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA,
89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).
[0200] It is further important that antibodies be humanized with
retention of high affinity for the antigen and other favorable
biological properties. To achieve this goal, according to a
preferred method, humanized antibodies are prepared by a process of
analysis of the parental sequences and various conceptual humanized
products using three-dimensional models of the parental and
humanized sequences. Three-dimensional immunoglobulin models are
commonly available and are familiar to those skilled in the art.
Computer programs are available which illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
hypervariable region residues are directly and most substantially
involved in influencing antigen binding.
[0201] Example 3 below describes production of exemplary humanized
anti-ErbB2 antibodies which bind ErbB2 and block ligand activation
of an ErbB. The humanized antibody of particular interest herein
blocks EGF, TGF-.alpha. and/or HRG mediated activation of MAPK
essentially as effectively as murine monoclonal antibody 2C4 (or a
Fab fragment thereof) and/or binds ErbB2 essentially as effectively
as murine monoclonal antibody 2C4 (or a Fab fragment thereof). The
humanized antibody herein may, for example, comprise nonhuman
hypervariable region residues incorporated into a human variable
heavy domain and may further comprise a framework region (FR)
substitution at a position selected from the group consisting of
69H, 71H and 73H utilizing the variable domain numbering system set
forth in Kabat et al., Sequences of Proteins of Immunological
Interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda, Md. (1991). In one embodiment, the humanized
antibody comprises FR substitutions at two or all of positions 69H,
71H and 73H.
[0202] An exemplary humanized antibody of interest herein comprises
variable heavy domain complementarity determining residues
GFTFTDYTMX, where X is preferrably D or S (SEQ ID NO:7);
DVNPNSGGSIYNQRFKG (SEQ ID NO:8); and/or NLGPSFYFDY (SEQ ID NO:9),
optionally comprising amino acid modifications of those CDR
residues, e.g. where the modifications essentially maintain or
improve affinity of the antibody. For example, the antibody variant
of interest may have from about one to about seven or about five
amino acid substitutions in the above variable heavy CDR sequences.
Such antibody variants may be prepared by affinity maturation,
e.g., as described below. The most preferred humanized antibody
comprises the variable heavy domain amino acid sequence in SEQ ID
NO:4.
[0203] The humanized antibody may comprise variable light domain
complementarity determining residues KASQDVSIGVA (SEQ ID NO:10);
SASYX.sup.1X.sup.2X.sup.3, where X.sup.1 is preferably R or L,
X.sup.2 is preferably Y or E, and X.sup.3 is preferably T or S (SEQ
ID NO:11); and/or QQYYIYPYT (SEQ ID NO:12), e.g. in addition to
those variable heavy domain CDR residues in the preceding
paragraph. Such humanized antibodies optionally comprise amino acid
modifications of the above CDR residues, e.g. where the
modifications essentially maintain or improve affinity of the
antibody. For example, the antibody variant of interest may have
from about one to about seven or about five amino acid
substitutions in the above variable light CDR sequences. Such
antibody variants may be prepared by affinity maturation, e.g., as
described below. The most preferred humanized antibody comprises
the variable light domain amino acid sequence in SEQ ID NO:3.
[0204] The present application also contemplates affinity matured
antibodies which bind ErbB2 and block ligand activation of an ErbB.
The parent antibody may be a human antibody or a humanized
antibody, e.g., one comprising the variable light and/or heavy
sequences of SEQ ID Nos. 3 and 4, respectively (i.e. variant 574).
The affinity matured antibody preferably binds to ErbB2 receptor
with an affinity superior to that of murine 2C4 or variant 574
(e.g. from about two or about four fold, to about 100 fold or about
1000 fold improved affinity, e.g. as assessed using a
ErbB2-extracellular domain (ECD) ELISA). Exemplary variable heavy
CDR residues for substitution include H28, H30, H34, H35, H64, H96,
H99, or combinations of two or more (e.g. two, three, four, five,
six, or seven of these residues). Examples of variable light CDR
residues for alteration include L28, L50, L53, L56, L91, L92, L93,
L94, L96, L97 or combinations of two or more (e.g. two to three,
four, five or up to about ten of these residues).
[0205] Various forms of the humanized antibody or affinity matured
antibody are contemplated. For example, the humanized antibody or
affinity matured antibody may be an antibody fragment, such as a
Fab, which is optionally conjugated with one or more cytotoxic
agent(s) in order to generate an immunoconjugate. Alternatively,
the humanized antibody or affinity matured antibody may be an
intact antibody, such as an intact IgG1 antibody.
[0206] (iv) Human Antibodies
[0207] As an alternative to humanization, human antibodies can be
generated. For example, it is now possible to produce transgenic
animals (e.g., mice) that are capable, upon immunization, of
producing a full repertoire of human antibodies in the absence of
endogenous immunoglobulin production. For example, it has been
described that the homozygous deletion of the antibody heavy-chain
joining region (J.sub.H) gene in chimeric and germ-line mutant mice
results in complete inhibition of endogenous antibody production.
Transfer of the human germ-line immunoglobulin gene array in such
germ-line mutant mice will result in the production of human
antibodies upon antigen challenge. See, e.g., Jakobovits et al.,
Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al.,
Nature, 362:255-258 (1993); Bruggemann et al., Year in Immuno.,
7:33 (1993); and U.S. Pat. Nos. 5,591,669, 5,589,369 and
5,545,807.
[0208] Alternatively, phage display technology (McCafferty et al.,
Nature 348:552-553 (1990)) can be used to produce human antibodies
and antibody fragments in vitro, from immunoglobulin variable (V)
domain gene repertoires from unimmunized donors. According to this
technique, antibody V domain genes are cloned in-frame into either
a major or minor coat protein gene of a filamentous bacteriophage,
such as M13 or fd, and displayed as functional antibody fragments
on the surface of the phage particle. Because the filamentous
particle contains a single-stranded DNA copy of the phage genome,
selections based on the functional properties of the antibody also
result in selection of the gene encoding the antibody exhibiting
those properties. Thus, the phage mimics some of the properties of
the B-cell. Phage display can be performed in a variety of formats;
for their review see, e.g., Johnson, Kevin S. and Chiswell, David
J., Current Opinion in Structural Biology 3:564-571 (1993). Several
sources of V-gene segments can be used for phage display. Clackson
et al., Nature, 352:624-628 (1991) isolated a diverse array of
anti-oxazolone antibodies from a small random combinatorial library
of V genes derived from the spleens of immunized mice. A repertoire
of V genes from unimmunized human donors can be constructed and
antibodies to a diverse array of antigens (including self-antigens)
can be isolated essentially following the techniques described by
Marks et al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al.,
EMBO J. 12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and
5,573,905.
[0209] As discussed above, human antibodies may also be generated
by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and
5,229,275).
[0210] Human anti-ErbB2 antibodies are described in U.S. Pat. No.
5,772,997 issued Jun. 30, 1998; WO 97/00271 published Jan. 3, 1997;
and WO01/09187 published Feb. 8, 2001 (Medarex).
[0211] (v) Antibody Fragments
[0212] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., Journal of Biochemical and Biophysical Methods 24:107-117
(1992); and Brennan et al., Science, 229:81 (1985)). However, these
fragments can now be produced directly by recombinant host cells.
For example, the antibody fragments can be isolated from the
antibody phage libraries discussed above. Alternatively, Fab'-SH
fragments can be directly recovered from E. coli and chemically
coupled to form F(ab').sub.2 fragments (Carter et al.,
Bio/Technology 10:163-167 (1992)). According to another approach,
F(ab').sub.2 fragments can be isolated directly from recombinant
host cell culture. Other techniques for the production of antibody
fragments will be apparent to the skilled practitioner. In other
embodiments, the antibody of choice is a single chain Fv fragment
(scFv). See WO 93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No.
5,587,458. The antibody fragment may also be a "linear antibody",
e.g., as described in U.S. Pat. No. 5,641,870 for example. Such
linear antibody fragments may be monospecific or bispecific. Single
chain intracellular antibodies (sFv) that bind ErbB2 are described
in WO01/56604 and U.S. Pat. No. 6,028,059.
[0213] (vi) Bispecific Antibodies
[0214] Bispecific antibodies are antibodies that have binding
specificities for at least two different epitopes. Exemplary
bispecific antibodies may bind to two different epitopes of the
ErbB2 protein. Other such antibodies may combine an ErbB2 binding
site with binding site(s) for EGFR, ErbB3 and/or ErbB4.
Alternatively, an anti-ErbB2 arm may be combined with an arm which
binds to a triggering molecule on a leukocyte such as a T-cell
receptor molecule (e.g. CD2 or CD3), or Fc receptors for IgG
(Fc.gamma.R), such as Fc.gamma.RI (CD64), Fc.gamma.RII (CD32) and
Fc.gamma.RIII (CD16) so as to focus cellular defense mechanisms to
the ErbB2-expressing cell. Bispecific antibodies may also be used
to localize cytotoxic agents to cells which express ErbB2. These
antibodies possess an ErbB2-binding arm and an arm which binds the
cytotoxic agent (e.g saporin, anti-interferon-.alpha., vinca
alkaloid, ricin A chain, methotrexate or radioactive isotope
hapten). Bispecific antibodies can be prepared as full length
antibodies or antibody fragments (e.g. F(ab').sub.2 bispecific
antibodies).
[0215] WO 96/16673 describes a bispecific
anti-ErbB2/anti-Fc.gamma.RIII antibody and U.S. Pat. No. 5,837,234
discloses a bispecific anti-ErbB2/anti-Fc.gamma.RI antibody. A
bispecific anti-ErbB2/Fc.alpha. antibody is shown in WO98/02463.
U.S. Pat. No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3
antibody.
[0216] Methods for making bispecific antibodies are known in the
art. Traditional production of full length bispecific antibodies is
based on the coexpression of two immunoglobulin heavy chain-light
chain pairs, where the two chains have different specificities
(Milstein et al., Nature, 305:537-539 (1983)). Because of the
random assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a potential mixture of 10 different
antibody molecules, of which only one has the correct bispecific
structure. Purification of the correct molecule, which is usually
done by affinity chromatography steps, is rather cumbersome, and
the product yields are low. Similar procedures are disclosed in WO
93/08829, and in Traunecker et al., EMBO J, 10:3655-3659
(1991).
[0217] According to a different approach, antibody variable domains
with the desired binding specificities (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences. The
fusion preferably is with an immunoglobulin heavy chain constant
domain, comprising at least part of the hinge, CH2, and CH3
regions. It is preferred to have the first heavy-chain constant
region (CH1) containing the site necessary for light chain binding,
present in at least one of the fusions. DNAs encoding the
immunoglobulin heavy chain fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression
vectors, and are co-transfected into a suitable host organism. This
provides for great flexibility in adjusting the mutual proportions
of the three polypeptide fragments in embodiments when unequal
ratios of the three polypeptide chains used in the construction
provide the optimum yields. It is, however, possible to insert the
coding sequences for two or all three polypeptide chains in one
expression vector when the expression of at least two polypeptide
chains in equal ratios results in high yields or when the ratios
are of no particular significance.
[0218] In a preferred embodiment of this approach, the bispecific
antibodies are composed of a hybrid immunoglobulin heavy chain with
a first binding specificity in one arm, and a hybrid immunoglobulin
heavy chain-light chain pair (providing a second binding
specificity) in the other arm. It was found that this asymmetric
structure facilitates the separation of the desired bispecific
compound from unwanted immunoglobulin chain combinations, as the
presence of an immunoglobulin light chain in only one half of the
bispecific molecule provides for a facile way of separation. This
approach is disclosed in WO 94/04690. For further details of
generating bispecific antibodies see, for example, Suresh et al.,
Methods in Enzymology, 121:210 (1986).
[0219] According to another approach described in U.S. Pat. No.
5,731,168, the interface between a pair of antibody molecules can
be engineered to maximize the percentage of heterodimers which are
recovered from recombinant cell culture. The preferred interface
comprises at least a part of the C.sub.H3 domain of an antibody
constant domain. In this method, one or more small amino acid side
chains from the interface of the first antibody molecule are
replaced with larger side chains (e.g. tyrosine or tryptophan).
Compensatory "cavities" of identical or similar size to the large
side chain(s) are created on the interface of the second antibody
molecule by replacing large amino acid side chains with smaller
ones (e.g. alanine or threonine). This provides a mechanism for
increasing the yield of the heterodimer over other unwanted
end-products such as homodimers.
[0220] Bispecific antibodies include cross-linked or
"heteroconjugate" antibodies. For example, one of the antibodies in
the heteroconjugate can be coupled to avidin, the other to biotin.
Such antibodies have, for example, been proposed to target immune
system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for
treatment of HUV infection (WO 91/00360, WO 92/200373, and EP
03089). Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents are well known
in the art, and are disclosed in U.S. Pat. No. 4,676,980, along
with a number of cross-linking techniques.
[0221] Techniques for generating bispecific antibodies from
antibody fragments have also been described in the literature. For
example, bispecific antibodies can be prepared using chemical
linkage. Brennan et al., Science, 229: 81 (1985) describe a
procedure wherein intact antibodies are proteolytically cleaved to
generate F(ab').sub.2 fragments. These fragments are reduced in the
presence of the dithiol complexing agent sodium arsenite to
stabilize vicinal dithiols and prevent intermolecular disulfide
formation. The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the
other Fab'-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced can be used as agents for the
selective immobilization of enzymes.
[0222] Recent progress has facilitated the direct recovery of
Fab'-SH fragments from E. coli, which can be chemically coupled to
form bispecific antibodies. Shalaby et al., J. Exp. Med., 175:
217-225 (1992) describe the production of a fully humanized
bispecific antibody F(ab').sub.2 molecule. Each Fab' fragment was
separately secreted from E. coli and subjected to directed chemical
coupling in vitro to form the bispecific antibody. The bispecific
antibody thus formed was able to bind to cells overexpressing the
ErbB2 receptor and normal human T cells, as well as trigger the
lytic activity of human cytotoxic lymphocytes against human breast
tumor targets.
[0223] Various techniques for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. Kostelny et al., J. Immunol.,
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos
and Jun proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This method can also be utilized for the
production of antibody homodimers. The "diabody" technology
described by Hollinger et al., Proc. Natl. Acad. Sci. USA,
90:6444-6448 (1993) has provided an alternative mechanism for
making bispecific antibody fragments. The fragments comprise a
heavy-chain variable domain (V.sub.H) connected to a light-chain
variable domain (V.sub.L) by a linker which is too short to allow
pairing between the two domains on the same chain. Accordingly, the
V.sub.H and V.sub.L domains of one fragment are forced to pair with
the complementary V.sub.L and V.sub.H domains of another fragment,
thereby forming two antigen-binding sites. Another strategy for
making bispecific antibody fragments by the use of single-chain Fv
(sFv) dimers has also been reported. See Gruber et al., J.
Immunol., 152:5368 (1994).
[0224] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al. J.
Immunol. 147: 60 (1991). U.S. Pat. No. 6,270,765B1 published Aug.
7, 2001 describes trispecific antibodies that bind ErbB2, EGFR and
FcR.
[0225] (vii) Other Amino Acid Sequence Modifications
[0226] Amino acid sequence modification(s) of the anti-ErbB2
antibodies described herein are contemplated. For example, it may
be desirable to improve the binding affinity and/or other
biological properties of the antibody. Amino acid sequence variants
of the anti-ErbB2 antibody are prepared by introducing appropriate
nucleotide changes into the anti-ErbB2 antibody nucleic acid, or by
peptide synthesis. Such modifications include, for example,
deletions from, and/or insertions into and/or substitutions of,
residues within the amino acid sequences of the anti-ErbB2
antibody. Any combination of deletion, insertion, and substitution
is made to arrive at the final construct, provided that the final
construct possesses the desired characteristics. The amino acid
changes also may alter post-translational processes of the
anti-ErbB2 antibody, such as changing the number or position of
glycosylation sites.
[0227] A useful method for identification of certain residues or
regions of the anti-ErbB2 antibody that are preferred locations for
mutagenesis is called "alanine scanning mutagenesis" as described
by Cunningham and Wells Science, 244:1081-1085 (1989). Here, a
residue or group of target residues are identified (e.g., charged
residues such as arg, asp, his, lys, and glu) and replaced by a
neutral or negatively charged amino acid (most preferably alanine
or polyalanine) to affect the interaction of the amino acids with
ErbB2 antigen. Those amino acid locations demonstrating functional
sensitivity to the substitutions then are refined by introducing
further or other variants at, or for, the sites of substitution.
Thus, while the site for introducing an amino acid sequence
variation is predetermined, the nature of the mutation per se need
not be predetermined. For example, to analyze the performance of a
mutation at a given site, ala scanning or random mutagenesis is
conducted at the target codon or region and the expressed
anti-ErbB2 antibody variants are screened for the desired
activity.
[0228] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an anti-ErbB2 antibody with
an N-terminal methionyl residue or the antibody fused to a
cytotoxic polypeptide. Other insertional variants of the anti-ErbB2
antibody molecule include the fusion to the - or C-terminus of the
anti-ErbB2 antibody to an enzyme (e.g. for ADEPT) or a polypeptide
which increases the serum half-life of the antibody.
[0229] Another type of variant is an amino acid substitution
variant. These variants have at least one amino acid residue in the
anti-ErbB2 antibody molecule replaced by a different residue. The
sites of greatest interest for substitutional mutagenesis include
the hypervariable regions, but FR alterations are also
contemplated. Conservative substitutions are shown in Table 1 under
the heading of "preferred substitutions". If such substitutions
result in a change in biological activity, then more substantial
changes, denominated "exemplary substitutions" in Table 1, or as
further described below in reference to amino acid classes, may be
introduced and the products screened. TABLE-US-00001 TABLE 1
Original Exemplary Preferred Residue Substitutions Substitutions
Ala (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln;
his; asp, lys; arg gln Asp (D) glu; asn glu Cys (C) ser; ala ser
Gln (Q) asn; glu asn Glu (E) asp; gln asp Gly (G) Ala ala His (H)
asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe; norleucine
leu Leu (L) norleucine; ile; val; met; ala; phe ile Lys (K) arg;
gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala;
tyr tyr Pro (P) Ala ala Ser (S) Thr thr Thr (T) Ser ser Trp (W)
tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met;
phe; ala; norleucine leu
[0230] Substantial modifications in the biological properties of
the antibody are accomplished by selecting substitutions that
differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. Naturally occurring residues are
divided into groups based on common side-chain properties: [0231]
(1) hydrophobic: norleucine, met, ala, val, leu, ile; [0232] (2)
neutral hydrophilic: cys, ser, thr; [0233] (3) acidic: asp, glu;
[0234] (4) basic: asn, gin, his, lys, arg; [0235] (5) residues that
influence chain orientation: gly, pro; and [0236] (6) aromatic:
trp, tyr, phe.
[0237] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0238] Any cysteine residue not involved in maintaining the proper
conformation of the anti-ErbB2 antibody also may be substituted,
generally with serine, to improve the oxidative stability of the
molecule and prevent aberrant crosslinking. Conversely, cysteine
bond(s) may be added to the antibody to improve its stability
(particularly where the antibody is an antibody fragment such as an
Fv fragment).
[0239] A particularly preferred type of substitutional variant
involves substituting one or more hypervariable region residues of
a parent antibody (e.g. a humanized or human antibody). Generally,
the resulting variant(s) selected for further development will have
improved biological properties relative to the parent antibody from
which they are generated. A convenient way for generating such
substitutional variants involves affinity maturation using phage
display. Briefly, several hypervariable region sites (e.g. 6-7
sites) are mutated to generate all possible amino substitutions at
each site. The antibody variants thus generated are displayed in a
monovalent fashion from filamentous phage particles as fusions to
the gene III product of M13 packaged within each particle. The
phage-displayed variants are then screened for their biological
activity (e.g. binding affinity) as herein disclosed. In order to
identify candidate hypervariable region sites for modification,
alanine scanning mutagenesis can be performed to identify
hypervariable region residues contributing significantly to antigen
binding. Alternatively, or additionally, it may be beneficial to
analyze a crystal structure of the antigen-antibody complex to
identify contact points between the antibody and human ErbB2. Such
contact residues and neighboring residues are candidates for
substitution according to the techniques elaborated herein. Once
such variants are generated, the panel of variants is subjected to
screening as described herein and antibodies with superior
properties in one or more relevant assays may be selected for
further development.
[0240] Another type of amino acid variant of the antibody alters
the original glycosylation pattern of the antibody. By altering is
meant deleting one or more carbohydrate moieties found in the
antibody, and/or adding one or more glycosylation sites that are
not present in the antibody.
[0241] Glycosylation of antibodies is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tripeptide
sequences asparagine-X-serine and asparagine-X-threonine, where X
is any amino acid except proline, are the recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine
side chain. Thus, the presence of either of these tripeptide
sequences in a polypeptide creates a potential glycosylation site.
O-linked glycosylation refers to the attachment of one of the
sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino
acid, most commonly serine or threonine, although 5-hydroxyproline
or 5-hydroxylysine may also be used.
[0242] Addition of glycosylation sites to the antibody is
conveniently accomplished by altering the amino acid sequence such
that it contains one or more of the above-described tripeptide
sequences (for N-linked glycosylation sites). The alteration may
also be made by the addition of, or substitution by, one or more
serine or threonine residues to the sequence of the original
antibody (for O-linked glycosylation sites).
[0243] Nucleic acid molecules encoding amino acid sequence variants
of the anti-ErbB2 antibody are prepared by a variety of methods
known in the art. These methods include, but are not limited to,
isolation from a natural source (in the case of naturally occurring
amino acid sequence variants) or preparation by
oligonucleotide-mediated (or site-directed) mutagenesis, PCR
mutagenesis, and cassette mutagenesis of an earlier prepared
variant or a non-variant version of the anti-ErbB2 antibody.
[0244] It may be desirable to modify the antibody of the invention
with respect to effector function, e.g. so as to enhance
antigen-dependent cell-mediated cytotoxicity (ADCC) and/or
complement dependent cytotoxicity (CDC) of the antibody. This may
be achieved by introducing one or more amino acid substitutions in
an Fc region of the antibody. Alternatively or additionally,
cysteine residue(s) may be introduced in the Fc region, thereby
allowing interchain disulfide bond formation in this region. The
homodimeric antibody thus generated may have improved
internalization capability and/or increased complement-mediated
cell killing and antibody-dependent cellular cytotoxicity (ADCC).
See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B.
J. Immunol. 148:2918-2922 (1992). Homodimeric antibodies with
enhanced activity may also be prepared using heterobifunctional
cross-linkers as described in Wolff et al. Cancer Research
53:2560-2565 (1993). Alternatively, an antibody can be engineered
which has dual Fc regions and may thereby have enhanced complement
lysis and ADCC capabilities. See Stevenson et al. Anti-Cancer Drug
Design 3:219-230 (1989).
[0245] Antibody variants with altered Fc region sequence and
improved or diminished C1q binding are described in WO99/51642.
Antibody variants with altered Fc region sequences and altered FcR
binding function are described in WO00/42072.
[0246] To increase the serum half life of the antibody, one may
incorporate a salvage receptor binding epitope into the antibody
(especially an antibody fragment) as described in U.S. Pat. No.
5,739,277, for example. As used herein, the term "salvage receptor
binding epitope" refers to an epitope of the Fc region of an IgG
molecule (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, or IgG.sub.4) that
is responsible for increasing the in vivo serum half-life of the
IgG molecule.
[0247] (viii) Screening for Antibodies with the Desired
Properties
[0248] Techniques for generating antibodies have been described
above. One may further select antibodies with certain biological
characteristics, as desired.
[0249] To identify an antibody which blocks ligand activation of an
ErbB, the ability of the antibody to block ErbB ligand binding to
cells expressing the ErbB (e.g. in conjugation with another ErbB
with which the ErbB of interest forms an ErbB hetero-oligomer) may
be determined. For example, cells naturally expressing, or
transfected to express, ErbBs of the ErbB hetero-oligomer may be
incubated with the antibody and then exposed to labeled ErbB
ligand. The ability of the anti-ErbB2 antibody to block ligand
binding to the ErbB in the ErbB hetero-oligomer may then be
evaluated.
[0250] For example, inhibition of HRG binding to MCF7 breast tumor
cell lines by anti-ErbB2 antibodies may be performed using
monolayer MCF7 cultures on ice in a 24-well-plate format
essentially as described in Example 1 below. Anti-ErbB2 monoclonal
antibodies may be added to each well and incubated for 30 minutes.
.sup.125I-labeled rHRG.beta.1.sub.177-224 (25 pm) may then be
added, and the incubation may be continued for 4 to 16 hours. Dose
response curves may be prepared and an IC.sub.50 value may be
calculated for the antibody of interest. In one embodiment, the
antibody which blocks ligand activation of an ErbB will have an
IC.sub.50 for inhibiting HRG binding to MCF7 cells in this assay of
about 50nM or less, more preferably 10 nM or less. Where the
antibody is an antibody fragment such as a Fab fragment, the
IC.sub.50 for inhibiting HRG binding to MCF7 cells in this assay
may, for example, be about 100 nM or less, more preferably 50 nM or
less. Other, non-antibody, ErbB antagonist candidates can be tested
in a similar manner.
[0251] Alternatively, or additionally, the ability of the
anti-ErbB2 antibody to block ErbB ligand-stimulated tyrosine
phosphorylation of an ErbB present in an ErbB hetero-oligomer may
be assessed. For example, cells endogenously expressing the ErbBs
or transfected to expressed them may be incubated with the antibody
and then assayed for ErbB ligand-dependent tyrosine phosphorylation
activity using an anti-phosphotyrosine monoclonal (which is
optionally conjugated with a detectable label). The kinase receptor
activation assay described in U.S. Pat. No. 5,766,863 is also
available for determining ErbB activation and blocking of that
activity by an antibody.
[0252] In one embodiment, one may screen for an antibody which
inhibits HRG stimulation of p180 tyrosine phosphorylation in MCF7
cells essentially as described in Example 1 below. For example, the
MCF7 cells may be plated in 24-well plates and monoclonal
antibodies to ErbB2 may be added to each well and incubated for 30
minutes at room temperature; then rHRG.beta.1.sub.177-244 may be
added to each well to a final concentration of 0.2 nM, and the
incubation may be continued for 8 minutes. Media may be aspirated
from each well, and reactions may be stopped by the addition of 100
.mu.l of SDS sample buffer (5% SDS, 25 mM DTT, and 25 mM Tris-HCl,
pH 6.8). Each sample (25 .mu.l) may be electrophoresed on a 4-12%
gradient gel (Novex) and then electrophoretically transferred to
polyvinylidene difluoride membrane. Antiphosphotyrosine (at 1
.mu.g/ml) immunoblots may be developed, and the intensity of the
predominant reactive band at M.sub.r.about.180,000 may be
quantified by reflectance densitometry. The antibody selected will
preferably significantly inhibit HRG stimulation of p180 tyrosine
phosphorylation to about 0-35% of control in this assay. A
dose-response curve for inhibition of HRG stimulation of p180
tyrosine phosphorylation as determined by reflectance densitometry
may be prepared and an IC.sub.50 for the antibody of interest may
be calculated. In one embodiment, the antibody which blocks ligand
activation of an ErbB will have an IC.sub.50 for inhibiting HRG
stimulation of p180 tyrosine phosphorylation in this assay of about
50 nM or less, more preferably 10 nM or less. Where the antibody is
an antibody fragment such as a Fab fragment, the IC.sub.50 for
inhibiting HRG stimulation of p180 tyrosine phosphorylation in this
assay may, for example, be about 100nM or less, more preferably 50
nM or less.
[0253] One may also assess the growth inhibitory effects of the
antibody on MDA-MB-175 cells, e.g, essentially as described in
Schaefer et al. Oncogene 15:1385-1394 (1997). According to this
assay, MDA-MB-175 cells may treated with an anti-ErbB2 monoclonal
antibody (10 .mu.g/mL) for 4 days and stained with crystal violet.
Incubation with an anti-ErbB2 antibody may show a growth inhibitory
effect on this cell line similar to that displayed by monoclonal
antibody 2C4. In a further embodiment, exogenous HRG will not
significantly reverse this inhibition. Preferably, the antibody
will be able to inhibit cell proliferation of MDA-MB-175 cells to a
greater extent than monoclonal antibody 4D5 (and optionally to a
greater extent than monoclonal antibody 7F3), both in the presence
and absence of exogenous HRG.
[0254] In one embodiment, the anti-ErbB2 antibody of interest may
block heregulin dependent association of ErbB2 with ErbB3 in both
MCF7 and SK-BR-3 cells as determined in a co-immunoprecipitation
experiment such as that described in Example 2 substantially more
effectively than monoclonal antibody 4D5, and preferably
substantially more effectively than monoclonal antibody 7F3.
[0255] To identify growth inhibitory anti-ErbB2 antibodies, one may
screen for antibodies which inhibit the growth of cancer cells
which overexpress ErbB2. In one embodiment, the growth inhibitory
antibody of choice is able to inhibit growth of SK-BR-3 cells in
cell culture by about 20-100% and preferably by about 50-100% at an
antibody concentration of about 0.5 to 30 .mu.g/ml. To identify
such antibodies, the SK-BR-3 assay described in U.S. Pat. No.
5,677,171 can be performed. According to this assay, SK-BR-3 cells
are grown in a 1:1 mixture of F12 and DMEM medium supplemented with
10% fetal bovine serum, glutamine and penicillin streptomycin. The
SK-BR-3 cells are plated at 20,000 cells in a 35 mm cell culture
dish (2 mls/35 mm dish). 0.5 to 30 .mu.g/ml of the anti-ErbB2
antibody is added per dish. After six days, the number of cells,
compared to untreated cells are counted using an electronic
COULTER.quadrature. cell counter. Those antibodies which inhibit
growth of the SK-BR-3 cells by about 20-100% or about 50-100% may
be selected as growth inhibitory antibodies.
[0256] To select for antibodies which induce cell death, loss of
membrane integrity as indicated by, e.g., PI, trypan blue or 7AAD
uptake may be assessed relative to control. The preferred assay is
the PI uptake assay using BT474 cells. According to this assay,
BT474 cells (which can be obtained from the American Type Culture
Collection (Rockville, Md.)) are cultured in Dulbecco's Modified
Eagle Medium (D-MEM):Ham's F-12(50:50) supplemented with 10%
heat-inactivated FBS (Hyclone) and 2 mM L-glutamine. (Thus, the
assay is performed in the absence of complement and immune effector
cells). The BT474 cells are seeded at a density of 3.times.10.sup.6
per dish in 100.times.20 mm dishes and allowed to attach overnight.
The medium is then removed and replaced with fresh medium alone or
medium containing 10 .mu.g/ml of the appropriate monoclonal
antibody. The cells are incubated for a 3 day time period.
Following each treatment, monolayers are washed with PBS and
detached by trypsinization. Cells are then centrifuged at 1200 rpm
for 5 minutes at 4.degree. C., the pellet resuspended in 3 ml ice
cold Ca.sup.2+ binding buffer (10 mM Hepes, pH 7.4,140 mM NaCl, 2.5
mM CaCl.sub.2) and aliquoted into 35 mm strainer-capped 12.times.75
tubes (1 ml per tube, 3 tubes per treatment group) for removal of
cell clumps. Tubes then receive PI (10 .mu.g/ml). Samples may be
analyzed using a FACSCAN.RTM. flow cytometer and FACSCONVERT.RTM.
CellQuest software (Becton Dickinson). Those antibodies which
induce statistically significant levels of cell death as determined
by PI uptake may be selected as cell death-inducing antibodies.
[0257] In order to select for antibodies which induce apoptosis, an
annexin binding assay using BT474 cells is available. The BT474
cells are cultured and seeded in dishes as discussed in the
preceding paragraph. The medium is then removed and replaced with
fresh medium alone or medium containing 10 .mu.g/ml of the
monoclonal antibody. Following a three day incubation period,
monolayers are washed with PBS and detached by trypsinization.
Cells are then centrifuged, resuspended in Ca.sup.2+ binding buffer
and aliquoted into tubes as discussed above for the cell death
assay. Tubes then receive labeled annexin (e.g. annexin V-FTIC) (1
.mu.g/ml). Samples may be analyzed using a FACSCAN.RTM. flow
cytometer and FACSCONVERT.RTM. CellQuest software (Becton
Dickinson). Those antibodies which induce statistically significant
levels of annexin binding relative to control are selected as
apoptosis-inducing antibodies.
[0258] In addition to the annexin binding assay, a DNA staining
assay using BT474 cells is available. In order to perform this
assay, BT474 cells which have been treated with the antibody of
interest as described in the preceding two paragraphs are incubated
with 9 .mu.g/ml HOECHST 33342.RTM. for 2 hr at 37.degree. C., then
analyzed on an EPICS ELITE.RTM. flow cytometer (Coulter
Corporation) using MODFIT LT.RTM. software (Verity Software House).
Antibodies which induce a change in the percentage of apoptotic
cells which is 2 fold or greater (and preferably 3 fold or greater)
than untreated cells (up to 100% apoptotic cells) may be selected
as pro-apoptotic antibodies using this assay.
[0259] To screen for antibodies which bind to an epitope on ErbB2
bound by an antibody of interest, a routine cross-blocking assay
such as that described in Antibodies, A Laboratory Manual, Cold
Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be
performed. Alternatively, or additionally, epitope mapping can be
performed by methods known in the art (see, e.g. FIGS. 1A and 1B
herein).
[0260] The antibodies identified as antagonists can then be tested
in any of the animal models of pain discussed above.
[0261] (ix) Immunoconjugates
[0262] The ErbB antagonist antibodies of the invention can also be
in the form of immunoconjugates comprising an antibody conjugated
to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g.
a small molecule toxin or an enzymatically active toxin of
bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof), or a radioactive isotope (i.e., a
radioconjugate).
[0263] Chemotherapeutic agents useful in the generation of such
immunoconjugates have been described above. Conjugates of an
antibody and one or more small molecule toxins, such as a
calicheamicin, a maytansine (U.S. Pat. No. 5,208,020), a
trichothene, and CC1065 are also contemplated herein.
[0264] In one preferred embodiment of the invention, the antibody
is conjugated to one or more maytansine molecules (e.g. about 1 to
about 10 maytansine molecules per antibody molecule). Maytansine
may, for example, be converted to May-SS-Me which may be reduced to
May-SH3 and reacted with modified antibody (Chari et al. Cancer
Research 52: 127-131 (1992)) to generate a maytansinoid-antibody
immunoconjugate.
[0265] Another immunoconjugate of interest comprises an anti-ErbB2
antibody conjugated to one or more calicheamicin molecules. The
calicheamicin family of antibiotics are capable of producing
double-stranded DNA breaks at sub-picomolar concentrations.
Structural analogues of calicheamicin which may be used include,
but are not limited to,
.gamma..sub.1.sup.1,.alpha..sub.2.sup.1,.alpha..sub.3.sup.1,N-acetyl--
.gamma..sub.1.sup.1, PSAG and .theta..sup.1.sub.1 (Hinman et al.
Cancer Research 53: 3336-3342 (1993) and Lode et al. Cancer
Research 58: 2925-2928 (1998)). See, also, U.S. Pat. Nos.
5,714,586; 5,712,374; 5,264,586; and 5,773,001 expressly
incorporated herein by reference.
[0266] Enzymatically active toxins and fragments thereof which can
be used include diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor,
curcin, crotin, sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin and the
tricothecenes. See, for example, WO 93/21232 published Oct. 28,
1993.
[0267] The present invention further contemplates an
immunoconjugate formed between an antibody and a compound with
nucleolytic activity (e.g. a ribonuclease or a DNA endonuclease
such as a deoxyribonuclease; DNase).
[0268] A variety of radioactive isotopes are available for the
production of radioconjugated anti-ErbB2 antibodies. Examples
include At.sup.211, I.sup.131, I.sup.125, Y.sup.90, Re.sup.186,
Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32 and radioactive
isotopes of Lu.
[0269] Conjugates of the antibody and cytotoxic agent may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al. Science 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026. The linker may be
a "cleavable linker" facilitating release of the cytotoxic drug in
the cell. For example, an acid-labile linker, peptidase-sensitive
linker, dimethyl linker or disulfide-containing linker (Chari et
al. Cancer Research 52: 127-131 (1992)) may be used.
[0270] Alternatively, a fusion protein comprising the anti-ErbB2
antibody and cytotoxic agent may be made, e.g. by recombinant
techniques or peptide synthesis. Immunoconjugates of anti-ErbB2
antibody fused to a chemokine (e.g. RANTES) are described in
WO98/33914).
[0271] In yet another embodiment, the antibody may be conjugated to
a "receptor" (such streptavidin) for utilization in pretargeting
wherein the antibody-receptor conjugate is administered to the
patient, followed by removal of unbound conjugate from the
circulation using a clearing agent and then administration of a
"ligand" (e.g. avidin) which is conjugated to a cytotoxic agent
(e.g. a radionucleotide).
[0272] (x) Antibody Dependent Enzyme Mediated Prodrug Therapy
(ADEPT)
[0273] The antibodies of the present invention may also be used in
ADEPT by conjugating the antibody to a prodrug-activating enzyme
which converts a prodrug (e.g. a peptidyl chemotherapeutic agent,
see WO81/01145) to an active drug. See, for example, WO 88/07378
and U.S. Pat. No. 4,975,278.
[0274] The enzyme component of the immunoconjugate useful for ADEPT
includes any enzyme capable of acting on a prodrug in such a way so
as to covert it into its more active, cytotoxic form.
[0275] Enzymes that are useful in the method of this invention
include, but are not limited to, alkaline phosphatase useful for
converting phosphate-containing prodrugs into free drugs;
arylsulfatase useful for converting sulfate-containing prodrugs
into free drugs; cytosine deaminase useful for converting non-toxic
5-fluorocytosine into the active drug, 5-fluorouracil; proteases,
such as serratia protease, thermolysin, subtilisin,
carboxypeptidases and cathepsins (such as cathepsins B and L), that
are useful for converting peptide-containing prodrugs into free
drugs; D-alanylcarboxypeptidases, useful for converting prodrugs
that contain D-amino acid substituents; carbohydrate-cleaving
enzymes such as .beta.-galactosidase and neuramimidase useful for
converting glycosylated prodrugs into free drugs; .beta.-lactamase
useful for converting drugs derivatized with .beta.-lactams into
free drugs; and penicillin amidases, such as penicillin V amidase
or penicillin G amidase, useful for converting drugs derivatized at
their amine nitrogens with phenoxyacetyl or phenylacetyl groups,
respectively, into free drugs. Alternatively, antibodies with
enzymatic activity, also known in the art as "abzymes", can be used
to convert the prodrugs of the invention into free active drugs
(see, e.g., Massey, Nature 328: 457-458 (1987)). Antibody-abzyme
conjugates can be prepared as described herein for delivery of the
abzyme to a cell population.
[0276] The enzymes of this invention can be covalently bound to the
anti-ErbB2 antibodies by techniques well known in the art such as
the use of the heterobifunctional crosslinking reagents discussed
above. Alternatively, fusion proteins comprising at least the
antigen binding region of an antibody of the invention linked to at
least a functionally active portion of an enzyme of the invention
can be constructed using recombinant DNA techniques well known in
the art (see, e.g., Neuberger et al., Nature, 312: 604-608
(1984).
[0277] (xi) Other Antibody Modifications
[0278] Other modifications of the antibody are contemplated herein.
For example, the antibody may be linked to one of a variety of
nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene
glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and
polypropylene glycol. The antibody also may be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization (for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively), in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules), or in macroemulsions. Such
techniques are disclosed in Remington's Pharmaceutical Sciences,
16th edition, Oslo, A., Ed., (1980).
[0279] The anti-ErbB2 antibodies disclosed herein may also be
formulated as immunoliposomes. Liposomes containing the antibody
are prepared by methods known in the art, such as described in
Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang
et al., Proc. Natl. Acad. Sci. USA, 77:4030 (1980); U.S. Pat. Nos.
4,485,045 and 4,544,545; and WO97/38731 published Oct. 23, 1997.
Liposomes with enhanced circulation time are disclosed in U.S. Pat.
No. 5,013,556.
[0280] Particularly useful liposomes can be generated by the
reverse phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes as described in Martin et al. J.
Biol. Chem. 257: 286-288 (1982) via a disulfide interchange
reaction. A chemotherapeutic agent is optionally contained within
the liposome. See Gabizon et al. J. National Cancer Inst.
81(19)1484 (1989).
[0281] Pharmaceutical Formulations
[0282] Therapeutic formulations of the ErbB antagonists of the
present invention can be prepared as pharmaceutical formulations,
using standard ingredients and techniques as described, for
example, in Remington's Pharmaceutical Sciences 16th edition, Osol,
A. Ed. (1980). Thus, anti-ErbB antibodies are prepared for storage
by mixing an antagonist having the desired degree of purity with
optional pharmaceutically acceptable carriers, excipients or
stabilizers, in the form of lyophilized formulations or aqueous
solutions. Acceptable carriers, excipients, or stabilizers are
nontoxic to recipients at the dosages and concentrations employed,
and include buffers such as phosphate, citrate, and other organic
acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g. Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.RTM., PLURONICS.RTM. or
polyethylene glycol (PEG). Preferred lyophilized anti-ErbB2
antibody formulations are described in WO 97/04801, expressly
incorporated herein by reference.
[0283] The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
preferably those with complementary activities that do not
adversely affect each other. For example, it may be desirable to
further provide antibodies which bind to EGFR, ErbB2 (e.g. an
antibody which binds a different epitope on ErbB2), ErbB3, ErbB4,
or vascular endothelial factor (VEGF) in the one formulation.
Alternatively, or additionally, the composition may further
comprise a chemotherapeutic agent, cytotoxic agent, cytokine,
growth inhibitory agent, anti-hormonal agent, EGFR-targeted drug,
tyrosine kinase inhibitor, immunosuppressive agent, anti-angiogenic
agent, and/or cardioprotectant. Such molecules are suitably present
in combination in amounts that are effective for the purpose
intended.
[0284] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington's Pharmaceutical Sciences,
supra.
[0285] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.RTM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid.
[0286] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0287] If the ErbB antagonist is a small organic molecule, it is
preferably formulated in a form suitable for oral administration,
although liquid preparations for injection or infusion
administration are also suitable.
[0288] Pain Treatment with the ErbB Antagonist
[0289] The ErbB antagonists can be administered to human patients
in accord with known methods. Thus, for example, anti-ErbB (e.g.
anti-ErbB2) antibodies can be administered intravenously, e.g., as
a bolus or by continuous infusion over a period of time, or by
intramuscular, intraperitoneal, intracerobrospinal, subcutaneous,
intra-articular, intrasynovial, intrathecal, oral, topical, or
inhalation routes. Intravenous or subcutaneous administration of
the antibodies is preferred.
[0290] Optimal dosage can be determined based upon dosing
experiments in relevant animal models and fine tuned in human
clinical trials. Typically, an effective dose will reduce the
patient's pain score by at least 1, preferably by at least 2, more
preferably by at least 3 grades, and preferably results in a pain
score (on a 0 to 10 scale) of no more than 5, more preferably no
more than 4, even more preferably no more than 3, most preferably
no more than 2. The effective dose will also depend on the nature
and severity of the initial pain.
[0291] The analgesic activity of a candidate ErbB antagonist is
generally tested in a double-blind, randomized, placebo-controlled
clinical trial. For general overview of the design of randomized
controlled trials see, e.g. Jadad A R, Haynes R B. Med Decis Making
1998; 18:2-9.
[0292] Other therapeutic regimens may be combined with the
administration of the ErbB antagonists, such as anti-ErbB2
antibodies. The combined administration includes coadministration,
using separate formulations or a single pharmaceutical formulation,
and consecutive administration in either order, wherein preferably
there is a time period while both (or all) active agents
simultaneously exert their biological activities.
[0293] In one preferred embodiment, the patient is treated with two
different anti-ErbB2 antibodies. For example, the patient may be
treated with a first anti-ErbB2 antibody which blocks ligand
activation of an ErbB or an antibody having a biological
characteristic of monoclonal antibody 2C4 as well as a second
anti-ErbB2 antibody which is growth inhibitory (e.g.
HERCEPTIN.RTM.) or an anti-ErbB2 antibody which induces apoptosis
of an ErbB2-overexpressing cell (e.g. 7C2, 7F3 or humanized
variants thereof). Preferably such combined therapy results in a
synergistic therapeutic effect. One may, for instance, treat the
patient with HERCEPTIN.RTM. and thereafter treat with rhuMAb 2C4,
e.g. where the patient does not respond to HERCEPTIN.RTM. therapy.
In another embodiment, the patient may first be treated with rhuMAb
2C4 and then receive HERCEPTIN.RTM. therapy. In yet a further
embodiment, the patient may be treated with both rhuMAb 2C4 and
HERCEPTIN.RTM. simultaneously.
[0294] It may also be desirable to combine administration of the
anti-ErbB2 antibody or antibodies, with administration of an
antibody directed against EGFR, ErbB3, ErbB4, or vascular
endothelial growth factor (VEGF).
[0295] In one embodiment, the treatment of the present invention
involves the combined administration of an anti-ErbB2 antibody (or
antibodies) and one or more chemotherapeutic agents, cytotoxic
agents, or growth inhibitory agents, including coadministration of
cocktails of different chemotherapeutic agents. Preparation and
dosing schedules for such chemotherapeutic agents may be used
according to manufacturers' instructions or as determined
empirically by the skilled practitioner. Preparation and dosing
schedules for such chemotherapy are also described in Chemotherapy
Service Ed., M. C. Perry, Williams & Wilkins, Baltimore, Md.
(1992).
[0296] The antibody may be combined with an anti-hormonal compound;
e.g., an anti-estrogen compound such as tamoxifen; an
anti-progesterone such as onapristone (see, EP 616 812); or an
anti-androgen such as flutamide, in dosages known for such
molecules.
[0297] Sometimes, it may be beneficial to also coadminister a
cardioprotectant (to prevent or reduce myocardial dysfunction
associated with the therapy) or one or more cytokines to the
patient. One may also coadminister an anti-angiogenic agent. In
addition to the above therapeutic regimes, the patient may be
subjected to surgery, radiation therapy, or phototherapy.
[0298] The anti-ErbB2 antibodies herein may also be combined with
an EGFR-targeted drug, tyrosine kinase inhibitor and/or
immunosuppressive agent, such as those discussed above in the
definitions section resulting in a complementary, and potentially
synergistic, therapeutic effect.
[0299] Suitable dosages for any of the above coadministered agents
are those presently used and may be lowered due to the combined
action (synergy) of the agent and anti-ErbB2 antibody.
[0300] As discussed above, for the prevention or treatment of pain,
the appropriate dosage of antibody will depend on the type of and
severity of pain to be treated, whether the antibody is
administered for preventive or therapeutic purposes, previous
therapy, the patient's clinical history and response to the
antibody, and the discretion of the attending physician. The
antibody is suitably administered to the patient at one time or
over a series of treatments. Depending on the type and severity of
the disease, about 1 .mu.g/kg to 15 mg/kg (e.g. preferably about
0.1 or 0.5 to about 20 or about 30 mg/kg) of antibody is an initial
candidate dosage for administration to the patient, whether, for
example, by one or more separate administrations, or by continuous
infusion. A typical daily dosage might range from about 1 .mu.g/kg
to 100 mg/kg or more, depending on the factors mentioned above. For
repeated administrations over several days or longer, depending on
the condition, the treatment is sustained until a desired
suppression of disease symptoms occurs. The preferred dosage of the
antibody will be in the range from about 0.5 mg/kg to about 30
mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0
mg/kg, 6 mg/kg, 8 mg/kg, 10 mg/kg or 15 mg/kg (or any combination
thereof) may be administered to the patient. Such doses may be
administered intermittently, e.g. every week, every three weeks,
monthly or less frequently, for instance every 3 or 4 months (e.g.
such that the patient receives from about two to about twenty, e.g.
about six doses of the anti-ErbB2 antibody). An initial higher
loading dose, followed by one or more lower doses may be
administered. An exemplary dosing regimen comprises administering
an initial loading dose of about 4 mg/kg, followed by a weekly
maintenance dose of about 2 mg/kg of the anti-ErbB2 antibody.
However, other dosage regimens may be useful. The progress of this
therapy is easily monitored by conventional techniques and
assays.
[0301] While the antibody administered is preferably "naked" or
"not conjugated with a cytotoxic agent," in certain embodiments, an
immunoconjugate comprising the anti-ErbB (e.g. anti-ErbB2) antibody
conjugated with a cytotoxic agent is administered to the patient.
Preferably, the immunoconjugate and/or ErbB2 protein to which it is
bound is/are internalized by the cell, resulting in increased
therapeutic efficacy of the immunoconjugate. In a preferred
embodiment, the cytotoxic agent targets or interferes with nucleic
acid in a cell. Examples of such cytotoxic agents include
maytansinoids, calicheamicins, ribonucleases and DNA
endonucleases.
[0302] Aside from administration of the antibody protein to the
patient, the present application contemplates administration of the
antibody by gene therapy. Such administration of nucleic acid
encoding the antibody is encompassed by the expression
"administering a therapeutically effective amount of an antibody".
See, for example, WO96/07321 published Mar. 14, 1996 and WO01/56604
published Aug. 9, 2001 concerning antibodies to HER2 administered
by gene therapy.
[0303] There are two major approaches to getting the nucleic acid
(optionally contained in a vector) into the patient's cells; in
vivo and ex vivo. For in vivo delivery the nucleic acid is injected
directly into the patient, usually at the site where the antibody
is required. For ex vivo treatment, the patient's cells are
removed, the nucleic acid is introduced into these isolated cells
and the modified cells are administered to the patient either
directly or, for example, encapsulated within porous membranes
which are implanted into the patient (see, e.g. U.S. Pat. Nos.
4,892,538 and 5,283,187). There are a variety of techniques
available for introducing nucleic acids into viable cells. The
techniques vary depending upon whether the nucleic acid is
transferred into cultured cells in vitro, or in vivo in the cells
of the intended host. Techniques suitable for the transfer of
nucleic acid into mammalian cells in vitro include the use of
liposomes, electroporation, microinjection, cell fusion,
DEAE-dextran, the calcium phosphate precipitation method, etc. A
commonly used vector for ex vivo delivery of the gene is a
retrovirus.
[0304] The currently preferred in vivo nucleic acid transfer
techniques include transfection with viral vectors (such as
adenovirus, Herpes simplex I virus, or adeno-associated virus) and
lipid-based systems (useful lipids for lipid-mediated transfer of
the gene are DOTMA, DOPE and DC-Chol, for example). In some
situations it is desirable to provide the nucleic acid source with
an agent that targets the target cells, such as an antibody
specific for a cell surface membrane protein or the target cell, a
ligand for a receptor on the target cell, etc. Where liposomes are
employed, proteins which bind to a cell surface membrane protein
associated with endocytosis may be used for targeting and/or to
facilitate uptake, e.g. capsid proteins or fragments thereof tropic
for a particular cell type, antibodies for proteins which undergo
internalization in cycling, and proteins that target intracellular
localization and enhance intracellular half-life. The technique of
receptor-mediated endocytosis is described, for example, by Wu et
al., J. Biol. Chem. 262:4429-4432 (1987); and Wagner et al., Proc.
Natl. Acad. Sci. USA 87:3410-3414 (1990). For review of the
currently known gene marking and gene therapy protocols see
Anderson et al., Science 256:808-813 (1992). See also WO 93/25673
and the references cited therein.
[0305] Deposit of Materials
[0306] The following hybridoma cell lines have been deposited with
the American Type Culture Collection (ATCC), 10801 University
Boulevard, Manassas, Va. 20110-2209, USA (ATCC): TABLE-US-00002
Antibody Designation ATCC No. Deposit Date 7C2 ATCC HB-12215 Oct.
17, 1996 7F3 ATCC HB-12216 Oct. 17, 1996 4D5 ATCC CRL 10463 May 24,
1990 2C4 ATCC HB-12697 Apr. 8, 1999
[0307] Further details of the invention are illustrated by the
following non-limiting Examples. The disclosures of all citations
in the specification are expressly incorporated herein by
reference.
EXAMPLE 1
Production and Characterization of Monoclonal Antibody 2C4
[0308] The murine monoclonal antibodies 2C4, 7F3 and 4D5 which
specifically bind the extracellular domain of ErbB2 were produced
as described in Fendly et al., Cancer Research 50:1550-1558 (1990).
Briefly, NIH 3T3/HER2-3.sub.400 cells (expressing approximately
1.times.10.sup.5 ErbB2 molecules/cell) produced as described in
Hudziak et al Proc. Natl. Acad. Sci. (USA) 84:7158-7163 (1987) were
harvested with phosphate buffered saline (PBS) containing 25 mM
EDTA and used to immunize BALB/c mice. The mice were given
injections i.p. of 10.sup.7 cells in 0.5 ml PBS on weeks 0, 2, 5
and 7. The mice with antisera that immunoprecipitated
.sup.32P-labeled ErbB2 were given i.p. injections of a wheat germ
agglutinin-Sepharose (WGA) purified ErbB2 membrane extract on weeks
9 and 13. This was followed by an i.v. injection of 0.1 ml of the
ErbB2 preparation and the splenocytes were fused with mouse myeloma
line X63-Ag8.653.
[0309] Hybridoma supernatants were screened for ErbB2-binding by
ELISA and radioimmunoprecipitation.
[0310] The ErbB2 epitopes bound by monoclonal antibodies 4D5, 7F3
and 2C4 were determined by competitive binding analysis (Fendly et
al. Cancer Research 50:1550-1558 (1990)). Cross-blocking studies
were done on antibodies by direct fluorescence on intact cells
using the PANDEX.RTM. Screen Machine to quantitate fluorescence.
Each monoclonal antibody was conjugated with fluorescein
isothiocyanate (FITC), using established procedures (Wofsy et al.
Selected Methods in Cellular Immunology, p. 287, Mishel and Schiigi
(eds.) San Francisco: W.J. Freeman Co. (1980)). Confluent
monolayers of NIH 3T3/HER2-3.sub.400 cells were trypsinized, washed
once, and resuspended at 1.75.times.10.sup.6 cell/ml in cold PBS
containing 0.5% bovine serum albumin (BSA) and 0.1% NaN.sub.3. A
final concentration of 1% latex particles (IDC, Portland, Oreg.)
was added to reduce clogging of the PANDEX.RTM. plate membranes.
Cells in suspension, 20 .mu.l, and 20 .mu.l of purified monoclonal
antibodies (100 .mu.g/ml to 0.1 .mu.g/ml) were added to the
PANDEX.RTM. plate wells and incubated on ice for 30 minutes. A
predetermined dilution of FITC-labeled monoclonal antibodies in 20
.mu.l was added to each well, incubated for 30 minutes, washed, and
the fluorescence was quantitated by the PANDEX.RTM.. Monoclonal
antibodies were considered to share an epitope if each blocked
binding of the other by 50% or greater in comparison to an
irrelevant monoclonal antibody control. In this experiment,
monoclonal antibodies 4D5, 7F3 and 2C4 were assigned epitopes I,
G/F and F, respectively.
[0311] The growth inhibitory characteristics of monoclonal
antibodies 2C4, 7F3 and 4D5 were evaluated using the breast tumor
cell line, SK-BR-3 (see Hudziak et al. Molec. Cell. Biol.
9(3):1165-1172 (1989)). Briefly, SK-BR-3 cells were detached by
using 0.25% (vol/vol) trypsin and suspended in complete medium at a
density of 4.times.10.sup.5 cells per ml. Aliquots of 100 .mu.l
(4.times.10.sup.4 cells) were plated into 96-well microdilution
plates, the cells were allowed to adhere, and 100 .mu.l of media
alone or media containing monoclonal antibody (final concentration
5 .mu.g/ml) was then added. After 72 hours, plates were washed
twice with PBS (pH 7.5), stained with crystal violet (0.5% in
methanol), and analyzed for relative cell proliferation as
described in Sugarman et al. Science 230:943-945 (1985). Monoclonal
antibodies 2C4 and 7F3 inhibited SK- BR-3 relative cell
proliferation by about 20% and about 38%, respectively, compared to
about 56% inhibition achieved with monoclonal antibody 4D5.
[0312] Monoclonal antibodies 2C4, 4D5 and 7F3 were evaluated for
their ability to inhibit HRG-stimulated tyrosine phosphorylation of
proteins in the M.sub.r 180,000 range from whole-cell lysates of
MCF7 cells (Lewis et al. Cancer Research 56:1457-1465 (1996)). MCF7
cells are reported to express all known ErbBs, but at relatively
low levels. Since ErbB2, ErbB3, and ErbB4 have nearly identical
molecular sizes, it is not possible to discern which protein is
becoming tyrosine phosphorylated when whole-cell lysates are
evaluated by Western blot analysis.
[0313] However, these cells are ideal for HRG tyrosine
phosphorylation assays because under the assay conditions used, in
the absence of exogenously added HRG, they exhibit low to
undetectable levels of tyrosine phosphorylation proteins in the
M.sub.r 180,000 range.
[0314] MCF7 cells were plated in 24-well plates and monoclonal
antibodies to ErbB2 were added to each well and incubated for 30
minutes at room temperature; then rHRG.beta.1.sub.177-244 was added
to each well to a final concentration of 0.2 nM, and the incubation
was continued for 8 minutes. Media was carefully aspirated from
each well, and reactions were stopped by the addition of 100 .mu.l
of SDS sample buffer (5% SDS, 25 mM DTT, and 25 mM Tris-HCl, pH
6.8). Each sample (25 .mu.l) was electrophoresed on a 4-12%
gradient gel (Novex) and then electrophoretically transferred to
polyvinylidene difluoride membrane. Antiphosphotyrosine (4G10, from
UBI, used at 1 .mu.g/ml) immunoblots were developed, and the
intensity of the predominant reactive band at M.sub.r.about.180,000
was quantified by reflectance densitometry, as described previously
(Holmes et al. Science 256:1205-1210 (1992); Sliwkowski et al. J.
Biol. Chem. 269:14661-14665 (1994)).
[0315] Monoclonal antibodies 2C4, 7F3, and 4D5, significantly
inhibited the generation of a HRG-induced tyrosine phosphorylation
signal at M.sub.r 180,000. In the absence of HRG, none of these
antibodies were able to stimulate tyrosine phosphorylation of
proteins in the M.sub.r 180,000 range. Also, these antibodies do
not cross-react with EGFR (Fendly et al. Cancer Research
50:1550-1558 (1990)), ErbB3, or ErbB4. Antibodies 2C4 and 7F3
significantly inhibited HRG stimulation of p180 tyrosine
phosphorylation to <25% of control. Monoclonal antibody 4D5 was
able to block HRG stimulation of tyrosine phosphorylation by
.about.50%. FIG. 2A shows dose-response curves for 2C4 or 7F3
inhibition of HRG stimulation of p180 tyrosine phosphorylation as
determined by reflectance densitometry. Evaluation of these
inhibition curves using a 4-parameter fit yielded an IC.sub.50 of
2.8.+-.0.7 nM and 29.0.+-.4.1 nM for 2C4 and 7F3, respectively.
[0316] Inhibition of HRG binding to MCF7 breast tumor cell lines by
anti-ErbB2 antibodies was performed with monolayer cultures on ice
in a 24-well-plate format (Lewis et al. Cancer Research
56:1457-1465 (1996)). Anti-ErbB2 monoclonal antibodies were added
to each well and incubated for 30 minutes .sup.125I-labeled
rHRG.beta.1.sub.177-224 (25 pm) was added, and the incubation was
continued for 4 to 16 hours. FIG. 2B provides dose-response curves
for 2C4 or 7F3 inhibition of HRG binding to MCF7 cells. Varying
concentrations of 2C4 or 7F3 were incubated with MCF7 cells in the
presence of .sup.125I-labeled rHRG.beta.1, and the inhibition
curves are shown in FIG. 2B. Analysis of these data yielded an
IC.sub.50 of 2.4.+-.0.3 nM and 19.0.+-.7.3 nM for 2C4 and 7F3,
respectively. A maximum inhibition of .about.74% for 2C4 and 7F3
were in agreement with the tyrosine phosphorylation data.
[0317] To determine whether the effect of the anti-ErbB2 antibodies
observed on MCF7 cells was a general phenomenon, human tumor cell
lines were incubated with 2C4 or 7F3 and the degree of specific
I-labeled rHRG.beta.1 binding was determined (Lewis et al. Cancer
Research 56:1457 1465 (1996)). The results from this study are
shown in FIG. 3. Binding of .sup.125I-labeled rHRG.beta.1 could be
significantly inhibited by either 2C4 or 7F3 in all cell lines,
with the exception of the breast cancer cell line MDA-MB-468, which
has been reported to express little or no ErbB2. The remaining cell
lines are reported to express ErbB2, with the level of ErbB2
expression varying widely among these cell lines. In fact, the
range of ErbB2 expression in the cell lines tested varies by more
than 2 orders of magnitude. For example, BT-20, MCF7, and Caov3
express .about.10.sup.4 ErbB2 receptors/cell, whereas BT-474 and
SK-BR-3 express .about.10.sup.6 ErbB2 receptors/cell. Given the
wide range of ErbB2 expression in these cells and the data above,
it was concluded that the interaction between ErbB2 and ErbB3 or
ErbB4, was itself a high-affinity interaction that takes place on
the surface of the plasma membrane.
[0318] The growth inhibitory effects of monoclonal antibodies 2C4
and 4D5 on MDA-MB-175 and SK-BR-3 cells in the presence or absence
of exogenous rHRG.beta.1 was assessed (Schaefer et al. Oncogene
15:1385-1394 (1997)). ErbB2 levels in MDA-MB-175 cells are 4-6
times higher than the level found in normal breast epithelial cells
and the ErbB2-ErbB4 receptor is constitutively tyrosine
phosphorylated in MDA-MB-175 cells. MDA-MB-175 cells were treated
with an anti-ErbB2 monoclonal antibodies 2C4 and 4D5 (10 .mu.g/mL)
for 4 days. In a crystal violet staining assay, incubation with 2C4
showed a strong growth inhibitory effect on this cell line (FIG.
4A). Exogenous HRG did not significantly reverse this inhibition.
On the other hand 2C4 revealed no inhibitory effect on the ErbB2
overexpressing cell line SK-BR-3 (FIG. 4B). Monoclonal antibody 2C4
was able to inhibit cell proliferation of MDA-MB-175 cells to a
greater extent than monoclonal antibody 4D5, both in the presence
and absence of exogenous HRG. Inhibition of cell proliferation by
4D5 is dependent on the ErbB2 expression level (Lewis et al. Cancer
Immunol. Immunother. 37:255-263 (1993)). A maximum inhibition of
66% in SK-BR-3 cells could be detected (FIG. 4B). However this
effect could be overcome by exogenous HRG.
EXAMPLE 2
HRG Dependent Association of ErbB2 with ErbB3 is Blocked by
Monoclonal Antibody 2C4
[0319] The ability of ErbB3 to associate with ErbB2 was tested in a
co-immunoprecipitation experiment. 1.0.times.10.sup.6 MCF7 or
SK-BR-3 cells were seeded in six well tissue culture plates in
50:50 DMEM/Ham's F12 medium containing 10% fetal bovine serum (FBS)
and 10 mM HEPES, pH 7.2 (growth medium), and allowed to attach
overnight. The cells were starved for two hours in growth medium
without serum prior to beginning the experiment.
[0320] The cells were washed briefly with phosphate buffered saline
(PBS) and then incubated with either 100 nM of the indicated
antibody diluted in 0.2% w/v bovine serum albumin (BSA), RPMI
medium, with 10 mM HEPES, pH 7.2 (binding buffer), or with binding
buffer alone (control). After one hour at room temperature, HRG was
added to a final concentration of 5 nM to half the wells (+). A
similar volume of binding buffer was added to the other wells (-).
The incubation was continued for approximately 10 minutes.
[0321] Supernatants were removed by aspiration and the cells were
lysed in RPMI, 10 mM HEPES, pH 7.2, 1.0% v/v TRITON X-100.RTM.,
1.0% w/v CHAPS (lysis buffer), containing 0.2 mM PMSF, 10 .mu.g/ml
leupeptin, and 10 TU/ml aprotinin. The lysates were cleared of
insoluble material by centrifugation.
[0322] ErbB2 was immunoprecipitated using a monoclonal antibody
covalently coupled to an affinity gel (Affi-Prep 10, Bio-Rad). This
antibody (Ab-3, Oncogene Sciences) recognizes a cytoplasmic domain
epitope. Immunoprecipitation was performed by adding 10 .mu.l of
gel slurry containing approximately 8.5 .mu.g of immobilized
antibody to each lysate, and the samples were allowed to mix at
room temperature for two hours. The gels were then collected by
centrifugation. The gels were washed batchwise three times with
lysis buffer to remove unbound material. SDS sample buffer was then
added and the samples were heated briefly in a boiling water
bath.
[0323] Supernatants were run on 4-12% polyacrylamide gels and
electroblotted onto nitrocellulose membranes. The presence of ErbB3
was assessed by probing the blots with a polyclonal antibody
against a cytoplasmic domain epitope thereof (c-17, Santa Cruz
Biotech). The blots were visualized using a chemiluminescent
substrate (ECL, Amersham).
[0324] As shown in the control lanes of FIGS. 5A and 5B, for MCF7
and SK-BR-3 cells, respectively, ErbB3 was present in an ErbB2
immunoprecipitate only when the cells were stimulated with HRG. If
the cells were first incubated with monoclonal antibody 2C4, the
ErbB3 signal was abolished in MCF7 cells (FIG. 5A, lane 2C4+) or
substantially reduced in SK-BR-3 cells (FIG. 5B, lane 2C4+). As
shown in FIGS. 5A-B, monoclonal antibody 2C4 blocks heregulin
dependent association of ErbB3 with ErbB2 in both MCF7 and SK-BR-3
cells substantially more effectively than HERCEPTIN.RTM..
Preincubation with HERCEPTIN.RTM. decreased the ErbB3 signal in
MCF7 lysates but had little or no effect on the amount of ErbB3
co-precipitated from SK-BR-3 lysates. Preincubation with an
antibody against the EGF receptor (Ab-1, Oncogene Sciences) had no
effect on the ability of ErbB3 to co-immunoprecipitate with ErbB2
in either cell line.
EXAMPLE 3
Humanized 2C4 Antibodies
[0325] The variable domains of murine monoclonal antibody 2C4 were
first cloned into a vector which allows production of a mouse/human
chimeric Fab fragment. Total RNA was isolated from the hybridoma
cells using a Stratagene RNA extraction kit following
manufacturer's protocols. The variable domains were amplified by
RT-PCR, gel purified, and inserted into a derivative of a
pUC119-based plasmid containing a human kappa constant domain and
human C.sub.H1 domain as previously described (Carter et al. PNAS
(USA) 89:4285 (1992); and U.S. Pat. No. 5,821,337). The resultant
plasmid was transformed into E. coli strain 16C9 for expression of
the Fab fragment. Growth of cultures, induction of protein
expression, and purification of Fab fragment were as previously
described (Werther et al. J. Immunol. 157:4986-4995 (1996); Presta
et al. Cancer Research 57: 4593-4599 (1997)).
[0326] Purified chimeric 2C4 Fab fragment was compared to the
murine parent antibody 2C4 with respect to its ability to inhibit
.sup.125I-HRG binding to MCF7 cells and inhibit rHRG activation of
p180 tyrosine phosphorylation in MCF7 cells. As shown in FIG. 6A,
the chimeric 2C4 Fab fragment is very effective in disrupting the
formation of the high affinity ErbB2-ErbB3 binding site on the
human breast cancer cell line, MCF7. The relative IC.sub.50 value
calculated for intact murine 2C4 is 4.0.+-.0.4 nM, whereas the
value for the Fab fragment is 7.7.+-.1.1 nM. As illustrated in FIG.
6B, the monovalent chimeric 2C4 Fab fragment is very effective in
disrupting HRG-dependent ErbB2-ErbB3 activation. The IC.sub.50
value calculated for intact murine monoclonal antibody 2C4 is
6.0.+-.2 nM, whereas the value for the Fab fragment is 15.0.+-.2
nM.
[0327] DNA sequencing of the chimeric clone allowed identification
of the CDR residues (Kabat et al., Sequences of Proteins of
Immunological Interest, 5.sup.th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)) (FIGS. 7A and
B). Using oligonucleotide site-directed mutagenesis, all six of
these CDR regions were introduced into a complete human framework
(V.sub.L kappa subgroup I and V.sub.H subgroup III) contained on
plasmid VX4 as previously described (Presta et al., Cancer Research
57: 4593-4599 (1997)). Protein from the resultant "CDR-swap" was
expressed and purified as above. Binding studies were performed to
compare the two versions. Briefly, a NUNC MAXISORP.RTM. plate was
coated with 1 microgram per ml of ErbB2 extracellular domain (ECD;
produced as described in WO 90/14357) in 50 mM carbonate buffer, pH
9.6, overnight at 4.degree. C., and then blocked with ELISA diluent
(0.5% BSA, 0.05% polysorbate 20, PBS) at room temperature for 1
hour. Serial dilutions of samples in ELISA diluent were incubated
on the plates for 2 hours. After washing, bound Fab fragment was
detected with biotinylated murine anti-human kappa antibody (ICN
634771) followed by streptavidin-conjugated horseradish peroxidase
(Sigma) and using 3,3',5,5'-tetramethyl benzidine (Kirkegaard &
Perry Laboratories, Gaithersburg, Md.) as substrate. Absorbance was
read at 450 nm. As shown in FIG. 8A, all binding was lost on
construction of the CDR-swap human Fab fragment.
[0328] To restore binding of the humanized Fab, mutants were
constructed using DNA from the CDR-swap as template. Using a
computer generated model (FIG. 9), these mutations were designed to
change human framework region residues to their murine counterparts
at positions where the change might affect CDR conformations or the
antibody-antigen interface. Mutants are shown in Table 2.
TABLE-US-00003 TABLE 2 Designation of Humanized 2C4 FR Mutations
Mutant no. Framework region (FR) substitutions 560 ArgH71Val 561
AspH73Arg 562 ArgH71Val, AspH73Arg 568 ArgH71Val, AspH73Arg,
AlaH49Gly 569 ArgH71Val, AspH73Arg, PheH67Ala 570 ArgH71Val,
AspH73Arg, AsnH76Arg 571 ArgH71Val, AspH73Arg, LeuH78Val 574
ArgH71Val, AspH73Arg, IleH69Leu 56869 ArgH71Val, AspH73Arg,
AlaH49Gly, PheH67Ala
[0329] Binding curves for the various mutants are shown in FIGS.
8A-C. Humanized Fab version 574, with the changes ArgH71Val,
AspH73Arg and IleH69Leu, appears to have binding restored to that
of the original chimeric 2C4 Fab fragment. Additional FR and/or CDR
residues, such as L2, L54, L55, L56, H35 and/or H48, may be
modified (e.g. substituted as follows--IleL2Thr; ArgL54Leu;
TyrL55Glu; ThrL56Ser; AspH35Ser; and ValH48Ile) in order to further
refine or enhance binding of the humanized antibody. Alternatively,
or additionally, the humanized antibody may be affinity matured
(see above) in order to further improve or refine its affinity
and/or other biological activities.
[0330] Humanized 2C4 version 574 was affinity matured using a
phage-display method. Briefly, humanized 2C4.574 Fab was cloned
into a phage display vector as a geneIII fusion. When phage
particles are induced by infection with M13KO7 helper phage, this
fusion allows the Fab to be displayed on the N-terminus of the
phage tail-fiber protein, geneIII (Baca et al. J Biol Chem.
272:10678 (1997)).
[0331] Individual libraries were constructed for each of the 6 CDRs
identified above. In these libraries, the amino acids in the CDRs
which were identified using a computer generated model (FIG. 9) as
being potentially significant in binding to ErbB2 were randomized
using oligos containing "NNS" as their codons. The libraries were
then panned against ErbB2 ECD coated on NUNC MAXISORP.TM. plates
with 3% dry milk in PBS with 0.2% TWEEN 20.RTM. (MPBST) used in
place of all blocking solutions. In order to select for phage with
affinities higher than that of 2C4.574, in panning rounds 3, 4, and
5, soluble ErbB2 ECD or soluble Fab 2C4.574 was added during the
wash steps as competitor. Wash times were extended to 1 hour at
room temperature.
[0332] After 5 rounds of panning, individual clones were again
analyzed by phage-ELISA. Individual clones were grown in Costar
96-well U-bottomed tissue culture plates, and phage were induced by
addition of helper phage. After overnight growth, E. coli cells
were pelleted, and the phage-containing supernates were transfered
to 96-well plates where the phage were blocked with MPBST for 1 hr
at room temperature. NUNC MAXISORP.TM. plates coated with ErbB2 ECD
were also blocked with MPBST as above. Blocked phage were incubated
on the plates for 2 hours. After washing, bound phage were detected
using horseradish-peroxidase-conjugated anti-M13 monoclonal
antibody (Amersham Pharmacia Biotech, Inc. 27-9421-01) diluted
1:5000 in MPBST, followed by 3,3',5,5',-tetramethyl benzidine as
substrate. Absorbance was read at 450 nm.
[0333] The 48 clones from each library which gave the highest
signals were DNA sequenced. Those clones whose sequences occurred
the most frequently were subcloned into the vector described above
which allows expression of soluble Fabs. These Fabs were induced,
proteins purified and the purified Fabs were analyzed for binding
by ELISA as described above and the binding was compared to that of
the starting humanized 2C4.574 version.
[0334] After interesting mutations in individual CDRs were
identified, additional mutants which were various combinations of
these were constructed and tested as above. Mutants which gave
improved binding relative to 574 are described in Table 3.
TABLE-US-00004 TABLE 3 Designation of mutants derived from affinity
maturation of 2C4.574 Mutant/ Mutant Name Change from 574 574*
H3.A1 serH99trp, metH34leu 0.380 L2.F5 serL50trp, tyrL53gly,
metH34leu 0.087 H1.3.B3 thrH28gln, thrH30ser, metH34leu 0.572 L3.G6
tyrL92pro, ileL93lys, metH34leu 0.569 L3.G11 tyrL92ser, ileL93arg,
tyrL94gly, metH34leu 0.561 L3.29 tyrL92phe, tyrL96asn, metH34leu
0.552 L3.36 tyrL92phe, tyrL94leu, tyrL96pro, metH34leu 0.215 654
serL50trp, metH34leu 0.176 655 metH34ser 0.542 659 serL50trp,
metH34ser 0.076 L2.F5.H3.A1 serL50trp, tyrL53gly, metH34leu,
serH99trp 0.175 L3G6.H3.A1 tyrL92pro, ileL93lys, metH34leu,
serH99trp 0.218 H1.3.B3.H3.A1 thrH28gln, thrH30ser, metH34leu,
serH99trp 0.306 L3.G11.H3.A1 tyrL92ser, ileL93arg, tyrL94gly,
metH34leu, 0.248 serH99trp 654.H3.A1 serL50trp, metH34leu,
serH99trp 0.133 654.L3.G6 serL50trp, metH34leu, tyrL92pro,
ileL93lys 0.213 654.L3.29 serL50trp, metH34leu, tyrL92phe,
tyrL96asn 0.236 654.L3.36 serL50trp, metH35leu, tyrL92phe,
tyrL94leu, 0.141 tyrL96pro *Ratio of the amount of mutant needed to
give the mid-OD of the standard curve to the amount of 574 needed
to give the mid-OD of the standard curve in an Erb2-ECD ELISA. A
number less than 1.0 indicates that the mutant binds Erb2 better
than 574 binds.
[0335] The following mutants have also been constructed, and are
currently under evaluation: TABLE-US-00005 659.L3.G6 serL50trp,
metH34ser, tyrL92pro, ileL93lys 659.L3.G11 serL50trp, metH34ser,
tyrL92ser, ileL93arg, tyrL94gly 659.L3.29 serL50trp, metH34ser,
tyrL92phe, tyrL96asn 659.L3.36 serL50trp, metH34ser, tyrL92phe,
tyrL94leu, tyrL96pro L2F5.L3G6 serL50trp, tyrL53gly, metH34leu,
tyrL92pro, ileL93lys L2F5.L3G11 serL50trp, tyrL53gly, metH34leu,
tyrL92ser, ileL93arg, tyrL94gly L2F5.L29 serL50trp, tyrL53gly,
metH34leu, tyrL92phe, tyrL96asn L2F5.L36 serL50trp, tyrL53gly,
metH34leu, tyrL92phe, tyrL94leu, tyrL96pro L2F5.L3G6.655 serL50trp,
tyrL53gly, metH35ser, tyrL92pro, ileL93lys L2F5.L3G11.655
serL50trp, tyrL53gly, metH34ser, tyrL92ser, ileL93arg, tyrL94gly
L2F5.L29.655 serL50trp, tyrL53gly, metH34ser, tyrL92phe, tyrL96asn
L2F5.L36.655 serL50trp, tyrL53gly, metH34ser, tyrL92phe, tyrL94leu,
tyrL96pro.
[0336] The following mutants, suggested by a homology scan, are
currently being constructed: TABLE-US-00006 678 thrH30ala 679
thrH30ser 680 lysH64arg 681 leuH96val 682 thrL97ala 683 thrL97ser
684 tyrL96phe 685 tyrL96ala 686 tyrL91phe 687 thrL56ala 688
glnL28ala 689 glnL28glu
[0337] The preferred amino acid at H34 would be methionine. A
change to leucine might be made if there were found to be oxidation
at this position.
[0338] AsnH52 and asnH53 were found to be strongly preferred for
binding. Changing these residues to alanine or aspartic acid
dramatically decreased binding.
[0339] An intact antibody comprising the variable light and heavy
domains of humanized version 574 with a human IgG1 heavy chain
constant region has been prepared (see U.S. Pat. No. 5,821,337).
The intact antibody is produced by Chinese Hamster Ovary (CHO)
cells. That molecule is designated rhuMAb 2C4 herein.
EXAMPLE 4
Monoclonal Antibody 2C4 Blocks EGF, TGF-.alpha. or HRG Mediated
Activation of MAPK
[0340] Many growth factor receptors signal through the
mitogen-activated protein kinase (MAPK) pathway. These dual
specificity kinases are one of the key endpoints in signal
transduction pathways that ultimately triggers cancer cells to
divide. The ability of monoclonal antibody 2C4 or HERCEPTIN.RTM. to
inhibit EGF, TGF-.alpha. or HRG activation of MAPK was assessed in
the following way.
[0341] MCF7 cells (10.sup.5 cells/well) were plated in serum
containing media in 12-well cell culture plates. The next day, the
cell media was removed and fresh media containing 0.1% serum was
added to each well. This procedure was then repeated the following
day and prior to assay the media was replaced with serum-free
binding buffer (Jones et al. J. Biol. Chem. 273:11667-74 (1998);
and Schaefer et al. J. Biol. Chem. 274:859-66 (1999)). Cells were
allowed to equilibrate to room temperature and then incubated for
30 minutes with 0.5 mL of 200 nM HERCEPTIN.RTM. or monoclonal
antibody 2C4. Cells were then treated with 1 nM EGF, 1 nM
TGF-.alpha. or 0.2 nM HRG for 15 minutes. The reaction was stopped
by aspirating the cell medium and then adding 0.2 mL SDS-PAGE
sample buffer containing 1% DTT. MAPK activation was assessed by
Western blotting using an anti-active MAPK antibody (Promega) as
described previously (Jones et al. J. Biol. Chem. 273:11667-74
(1998)).
[0342] As shown in FIG. 10, monoclonal antibody 2C4 significantly
blocks EGF, TGF-.alpha. and HRG mediated activation of MAPK to a
greater extent than HERCEPTIN.RTM.. These data suggest that
monoclonal antibody 2C4 binds to a surface of ErbB2 that is used
for its association with either EGFR or ErbB3 and thus prevents the
formation of the signaling receptor complex.
[0343] Monoclonal antibody 2C4 was also shown to inhibit heregulin
(HRG)-dependent Akt activation. Activation of the PI3 kinase signal
transduction pathway is important for cell survival (Carraway et
al. J. Biol. Chem. 270: 7111-6 (1995)). In tumor cells, PI3 kinase
activation may play a role in the invasive phenotype (Tan et al.
Cancer Research. 59: 1620-1625, (1999)). The survival pathway is
primarily mediated by the serine/threonine kinase AKT (Bos et al.
Trends Biochem Sci. 20: 441-442 (1995). Complexes formed between
ErbB2 and either ErbB3 or EGFR can initiate these pathways in
response to heregulin or EGF, respectively (Olayioye et al. Mol.
& Cell. Biol. 18: 5042-51 (1998); Karunagaran et al., EMBO
Journal. 15: 254-264 (1996); and Krymskaya et al. Am. J. Physiol.
276: L246-55 (1999)). Incubation of MCF7 breast cancer cells with
2C4 inhibits heregulin-mediated AKT activation. Moreover, the basal
level of AKT activation present in the absence of heregulin
addition is further reduced by the addition of 2C4. These data
suggest that 2C4 may inhibit ErbB ligand-activation of PI3 kinase
and that this inhibition may lead to apoptosis. The increased
sensitivity to apoptosis may manifest in a greater sensitivity of
tumor cells to the toxic effects of chemotherapy.
[0344] Thus, monoclonal antibody 2C4 inhibits ligand initiated ErbB
signaling through two major signal transduction pathways--MAP
Kinase (a major proliferative pathway) and P13 kinase (a major
survival/anti-apoptotic pathway).
EXAMPLE 5
rhuMAb 2C4 for Treating Pain Cancer-Associated Pain
[0345] A 43 years old male patient diagnosed with lipsarcoma and
receiving chemotherapy for two years was hospitalized with a deep
wound in the left leg, requiring opiate analgesia prior to packing.
The size of the tumor lesion was 6.6 cm. After two cycles of
treatment with monoclonal antibody 2C4 (10 mg/kg), the patient
could be wound packed without the administration of any additional
analgesic agent. The tumor itself did not respond to 2C4 antibody
treatment, but 2C4 exhibited analgesic activity.
EXAMPLE 6
rhuMAb 2C4 for Treating Pain Associated with Androgen-Independent
Prostate Cancer
[0346] Patients diagnosed with hormone-resistant
(androgen-independent) prostate cancer with bony metastasis, which
had progressed after taxane-based chemotherapy, were treated with
rhuMAb 2C4. All patients recorded a Present Pain Intensity Score of
1 or more on a 6 point scale (McGill Pain Index) prior to starting
rhuMAb 2C4 administration, where [0347] 0=no pain [0348] 1=mild
pain [0349] 2=discomforting pain [0350] 3=distressing pain [0351]
4=horrible pain [0352] 5=excrutiating pain.
[0353] The analgesia intake of the patients was measured using a
daily diary. One dose of a non-steroidal analgesic agent was
assigned a score of 1, one 10-mg dose of morphine or an equivalent
dose of another opiate analgesic was assigned a score of 2. [0354]
rhuMAb 2C4 was administered starting with a 840 mg loading bolus
dose followed by a 420 mg intravenous (i.v.) dose every three
weeks.
[0355] One patient has a fall in pain score from 3 to 1, with
modest reduction in analgesia requirement. This effect lasted for
24 weeks. The patient showed a reduction in the rate of rise of
PSA, but no reduction in absolute value.
[0356] One patient had a stable pain score of 2, but stopped all
analgesia. The effect is ongoing at 24 weeks, despite progression
of PSA.
[0357] One patient showed an initial improvement in pain (score was
reduced from 2 to 1) with more than 50% reduction in analgesia
requirement. The treatment had no effect on the PSA of the patient.
The effect on pain lasted for 6 weeks, then the disease progressed
and pain increased.
[0358] The data show that rhuMAb 2C4 was able to reduce and
stabilize the pain of patients even when prostate cancer was not in
remission or continued to progress.
Sequence CWU 1
1
13 1 107 PRT Artificial Sequence Murine monoclonal antibody 2C4
(variable light) 1 Asp Thr Val Met Thr Gln Ser His Lys Ile Met Ser
Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser
Gln Asp Val Ser Ile Gly 20 25 30 Val Ala Trp Tyr Gln Gln Arg Pro
Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg
Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly
Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp
Leu Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr 85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 2 119 PRT
Artificial Sequence Murine monoclonal antibody 2C4 (variable heavy)
2 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Thr 1
5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asp
Tyr 20 25 30 Thr Met Asp Trp Val Lys Gln Ser His Gly Lys Ser Leu
Glu Trp Ile 35 40 45 Gly Asp Val Asn Pro Asn Ser Gly Gly Ser Ile
Tyr Asn Gln Arg Phe 50 55 60 Lys Gly Lys Ala Ser Leu Thr Val Asp
Arg Ser Ser Arg Ile Val Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr
Phe Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Leu Gly
Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu
Thr Val Ser Ser 115 3 107 PRT Artificial Sequence Humanized
monoclonal antibody 2C4 version 574 (variable light) 3 Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly 20 25
30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Tyr Tyr Ile Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys 100 105 4 119 PRT Artificial Sequence Humanized
monoclonal antibody 2C4 version 574 (variable heavy) 4 Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25
30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln
Arg Phe 50 55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Leu Gly Pro Ser Phe
Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser
Ser 115 5 107 PRT Artificial Sequence Consensus human framework for
the humanized antibody hum kI (variable light) 5 Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn Tyr 20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Ala Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
Asn Ser Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105 6 119 PRT Artificial Sequence Consensus human
framework for the humanized antibody hum III (variable heavy) 6 Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ala Val Ile Ser Gly Asp Gly Gly Ser Thr Tyr Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Arg Val Gly
Tyr Ser Leu Tyr Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr
Val Ser Ser 115 7 10 PRT Artificial Sequence humanized anitbody
(variable heavy) 7 Gly Phe Thr Phe Thr Asp Tyr Thr Met Xaa 1 5 10 8
17 PRT Artificial Sequence humanized anitbody (variable heavy) 8
Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe Lys 1 5
10 15 Gly 9 10 PRT Artificial Sequence humanized anitbody (variable
heavy) 9 Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr 1 5 10 10 11 PRT
Artificial Sequence humanized anitbody (variable light) 10 Lys Ala
Ser Gln Asp Val Ser Ile Gly Val Ala 1 5 10 11 7 PRT Artificial
Sequence humanized anitbody (variable light) 11 Ser Ala Ser Tyr Xaa
Xaa Xaa 1 5 12 9 PRT Artificial Sequence humanized anitbody
(variable light) 12 Gln Gln Tyr Tyr Ile Tyr Pro Tyr Thr 1 5 13 525
PRT homo sapiens 13 Met Glu Leu Ala Ala Leu Cys Arg Trp Gly Leu Leu
Leu Ala Leu Leu 1 5 10 15 Pro Pro Gly Ala Ala Ser Thr Gln Val Cys
Thr Gly Thr Asp Met Lys 20 25 30 Leu Arg Leu Pro Ala Ser Pro Glu
Thr His Leu Asp Met Leu Arg His 35 40 45 Leu Tyr Gln Gly Cys Gln
Val Val Gln Gly Asn Leu Glu Leu Thr Tyr 50 55 60 Leu Pro Thr Asn
Ala Ser Leu Ser Phe Leu Gln Asp Ile Gln Glu Val 65 70 75 80 Ile Val
Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr Ala Leu Ala Val 85 90 95
Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro Val Thr Gly Ala 100
105 110 Ser Pro Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser Leu Thr Glu
Ile 115 120 125 Leu Lys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln Leu
Cys Tyr Gln 130 135 140 Asp Thr Ile Leu Trp Lys Asp Ile Phe His Lys
Asn Asn Gln Leu Ala 145 150 155 160 Gly Ser Arg Cys Trp Gly Glu Ser
Ser Glu Asp Cys Gln Ser Leu Thr 165 170 175 Arg Thr Val Cys Ala Gly
Gly Cys Ala Arg Cys Lys Gly Pro Leu Pro 180 185 190 Thr Asp Cys Cys
His Glu Gln Cys Ala Ala Gly Cys Thr Gly Pro Lys 195 200 205 His Ser
Asp Cys Leu Ala Cys Leu His Phe Asn His Ser Gly Ile Cys 210 215 220
Glu Leu His Cys Pro Ala Leu Val Thr Tyr Asn Thr Asp Thr Phe Glu 225
230 235 240 Tyr Asn Tyr Leu Ser Thr Asp Val Gly Ser Cys Thr Leu Val
Cys Pro 245 250 255 Leu His Asn Gln Glu Val Thr Ala Glu Asp Gly Thr
Gln Arg Cys Glu 260 265 270 Lys Cys Ser Lys Pro Cys Ala Arg Val Cys
Tyr Gly Leu Gly Met Glu 275 280 285 His Leu Arg Glu Val Arg Ala Val
Thr Ser Ala Asn Ile Gln Glu Phe 290 295 300 Ala Gly Cys Lys Lys Ile
Phe Gly Ser Leu Ala Phe Leu Pro Glu Ser 305 310 315 320 Glu Thr Leu
Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro 325 330 335 Asp
Ser Leu Pro Asp Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg 340 345
350 Gly Arg Ile Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu
355 360 365 Gly Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu Leu Gly
Ser Gly 370 375 380 Leu Ala Leu Ile His His Asn Thr His Leu Cys Phe
Val His Thr Val 385 390 395 400 Glu Asp Glu Cys Val Gly Glu Gly Leu
Ala Cys His Gln Leu Cys Ala 405 410 415 Arg Gly His Cys Trp Gly Pro
Gly Pro Thr Gln Cys Val Asn Cys Ser 420 425 430 Gln Phe Leu Arg Gly
Gln Glu Cys Val Glu Glu Cys Arg Val Leu Gln 435 440 445 Gly Leu Pro
Arg Glu Tyr Val Asn Ala Arg His Cys Leu Pro Cys His 450 455 460 Pro
Glu Cys Gln Pro Gln Asn Gly Ser Val Thr Cys Phe Gly Pro Glu 465 470
475 480 Pro Ser Gly Val Lys Pro Asp Leu Ser Tyr Met Pro Ile Trp Lys
Phe 485 490 495 Pro Asp Glu Glu Gly Ala Cys Gln Pro Cys Pro Ile Asn
Cys Thr His 500 505 510 Ser Cys Val Asp Leu Asp Asp Lys Gly Cys Pro
Ala Glu 515 520 525
* * * * *