U.S. patent application number 14/063885 was filed with the patent office on 2014-03-27 for herceptin.rtm. adjuvant therapy.
This patent application is currently assigned to Genentech, Inc.. The applicant listed for this patent is Genentech, Inc.. Invention is credited to John L. Bryant.
Application Number | 20140086940 14/063885 |
Document ID | / |
Family ID | 37450489 |
Filed Date | 2014-03-27 |
United States Patent
Application |
20140086940 |
Kind Code |
A1 |
Bryant; John L. |
March 27, 2014 |
HERCEPTIN.RTM. ADJUVANT THERAPY
Abstract
The present application describes adjuvant therapy of
nonmetastatic breast cancer using HERCEPTIN.RTM..
Inventors: |
Bryant; John L.; (Allison
Road, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genentech, Inc. |
South San Francisco |
CA |
US |
|
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
37450489 |
Appl. No.: |
14/063885 |
Filed: |
October 25, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13205523 |
Aug 8, 2011 |
8597654 |
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14063885 |
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11400638 |
Apr 6, 2006 |
8591897 |
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13205523 |
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60681125 |
May 13, 2005 |
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Current U.S.
Class: |
424/174.1 |
Current CPC
Class: |
A61P 31/00 20180101;
A61K 2039/505 20130101; C07K 16/32 20130101; A61P 15/14 20180101;
A61P 43/00 20180101; Y02A 90/10 20180101; A61P 35/00 20180101; C07K
2317/24 20130101; A61P 15/00 20180101; A61K 39/39558 20130101; A61K
47/6803 20170801; G16H 20/10 20180101; A61K 39/39558 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
424/174.1 |
International
Class: |
A61K 47/48 20060101
A61K047/48 |
Claims
1. A method of adjuvant therapy comprising administering to a human
subject having HER2 positive cancer, following definitive surgery,
anthracycline cyclophosphamide (AC) based chemotherapy, followed by
administration of an effective amount of (a) an immunoconjugate
comprising an anti-ErbB2 antibody that binds to HER2 Domain IV
bound by trastuzumab (HERCEPTIN.RTM.), wherein the anti-ErbB2
antibody is conjugated to a maytansinoid, and (b) a second
anti-ErbB2 antibody, wherein the second anti-ErbB2 antibody is a
HER2 dimerization inhibitor.
2. The method of claim 1 wherein the anti-ErbB2 antibody conjugated
to said maytansinoid is huMAb4D5-8.
3. The method of claim 2 wherein the maytansinoid is DM1 having the
following structure ##STR00009## and wherein the anti-ErbB2
antibody is chemically linked to the maytansinoid via a disulfide
or thioether group at "R" shown in the structure.
4. The method of claim 3, wherein the immunoconjugate comprises
from 3 to 5 maytansinoid molecules per antibody molecule.
5. The method of any one of claims 1 to 3, wherein the anti-ErbB2
antibody and the maytansinoid are conjugated by a chemical linker
selected from N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),
N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) and
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
(SMCC).
6. The method of claim 5, wherein the anti-ErbB2 antibody and the
maytansinoid are conjugated by
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
(SMCC).
7. The method of any one of claims 1-4, wherein the second
anti-ErbB2 antibody is pertuzumab.
8. The method of claim 6, wherein the second anti-ErbB2 antibody is
pertuzumab.
9. The method of any one of claims 1-4, wherein the HER2 positive
cancer is HER2 positive breast cancer.
10. The method of claim 9, wherein the HER2 positive breast cancer
is nonmetastatic HER2 positive breast cancer.
11. The method of claim 7, wherein the HER2 positive cancer is HER2
positive breast cancer.
12. The method of claim 11, wherein the HER2 positive breast cancer
is nonmetastatic HER2 positive breast cancer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a non-provisional application 37 C.F.R.
.sctn.1.53(b), claiming priority under 37 C.F.R. .sctn.119(e) to
U.S. Provisional Patent Application Ser. No. 60/681,125 filed on
May 13, 2005, the entire disclosure of which is hereby expressly
incorporated by reference.
FIELD OF THE INVENTION
[0002] The present invention concerns adjuvant therapy of
nonmetastatic breast cancer using HERCEPTIN.RTM..
BACKGROUND OF THE INVENTION
HER Receptors and Antibodies Thereagainst
[0003] The HER 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, ErbB1, or HER1), HER2 (ErbB2 or
p185.sup.neu), HER3 (ErbB3) and HER4 (ErbB4 or tyro2).
[0004] 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).
[0005] The second member of the HER 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 HER2 (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., 3:254-257 (1990); Aasland et al., Br.
J. Cancer, 57:358-363 (1988); Williams et al., Pathobiology,
59:46-52 (1991); and McCann et al., Cancer, 65:88-92 (1990). HER2
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)).
[0006] HER2 amplification/overexpression is an early event in
breast cancer that is associated with aggressive disease and poor
prognosis. HER2 gene amplification is found in 20-25% of primary
breast tumors (Slamon et al., Science, 244:707-12 (1989); Owens et
al., Breast Cancer Res Treat, 76:S68 abstract 236 (2002)). HER2
positive disease correlates with decreased relapse-free and overall
survival (Slamon et al., Science, 235:177-82 (1987); Pauletti et
al., J Clin Oncol, 18:3651-64 (2000)). Amplification of the HER2
gene is associated with significantly reduced time to relapse and
poor survival in node-positive disease (Slamon et al. (1987);
Pauletti et al. (2000)) and poor outcome in node-negative disease
(Press et al., J Clin Oncol, 1997; 15:2894-904 (1997); Pauletti et
al. (2000)).
[0007] Antibodies directed against the rat p185.sup.neu and human
HER2 protein products have been described.
[0008] 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.
[0009] Hudziak et al., Mol. Cell. Biol., 9(3):1165-1172 (1989)
describe the generation of a panel of HER2 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 HER2-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 HER2
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); Disouza 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).
[0010] A recombinant humanized version of the murine HER2 antibody
4D5 (huMAb4D5-8, rhuMAb HER2, trastuzumab or HERCEPTIN.RTM., U.S.
Pat. No. 5,821,337) is clinically active in patients with
HER2-overexpressing metastatic breast cancers that have received
extensive prior anti-cancer therapy (Baselga et al., J. Clin.
14:737-744 (1996)). Trastuzumab 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
HER2 protein. Trastuzumab is indicated for weekly treatment of
patients both as first-line therapy in combination with paclitaxel
and as a single agent in second- and third-line therapy.
[0011] In clinical trials, HERCEPTIN.RTM. has shown a survival
benefit when used in combination with chemotherapy in metastatic
breast cancer patients. In December 2001, Genentech received FDA
approval to include data that showed a 24 percent increase in
median overall survival for women with HER2-positive metastatic
breast cancer treated initially with HERCEPTIN.RTM. and
chemotherapy compared to chemotherapy alone (median 25.1 months
compared to 20.3 months).
[0012] HERCEPTIN.RTM. has been used in combination with various
chemotherapeutic agents, including taxoids such as paclitaxel
(Slamon et al., N. Engl. J. of Med, 344:783-792 (2001);
Leyland-Jones et al., J. Clin. Oncol., 21(21):3965-3971 (2003)),
and docetaxel (Esteva et al., J. Clin. Oncol., 20(7):1800-1808
(2002); (Extra et al., Breast Cancer Res Treat, 82 (Suppl 1):217
(2003)); taxoids and platinum compounds (Pegram et al., J. Natl.
Cancer Inst., 96(10):759-69 (2004); Yardley et al., Breast Cancer
Res Treat, 76:S113 abstract 439 (2002)); platinum compound (such as
cisplatin or carboplatin) (Robert et al., Ann. Oncol., 15(suppl
3):39 (abstract 144P); (2004); Pegram et al., J Clin Oncol,
16:2659-71 (1998)); vincas such as vinorelbine (NAVELBINE.RTM.)
(Burstein et al., J. Clin. Oncol., 19(10); 2722-2730 (2001));
aromatase inhibitors such as letrozole and anastrazole (Jones, A.,
Annals of Oncology, 14:1697-1794 (2003); Wong et al., Breast Cancer
Res Treat, 82(Suppl 1):444 (2003)); anti-estrogen such as
fulvestrant (FASLODEX.RTM.) (Jones, A., supra); gemcitabine
(GEMZAR.RTM.) (Miller et al., Oncology, 15(2):38-40 (2001);
O'Shaughnessy et al., Breast Cancer Res Treat, 69:302 abstract 523
(2001)); liposomal doxorubicin (Theodoulou et al., Proc Am Soc Clin
Oncol, 21:216 abstract 216 (2002)); docetaxel/vinorelbine (with
G-CSF and quinolone prophylaxis) Limentani et al., Breast Cancer
Res Treat, 76:abstract 162 (2002)); epirubicin and cyclophosphomide
(Untch et al., Eur. J. Cancer, 40: 988-97 (2004b). See also Pegram
et al., J. Natl. Cancer. Inst., 96(10):739-49 (2004) for various
combination therapies including trastuzumab.
[0013] Other references describing Trastuzumab clinical trials
include Bendell et al., Cancer, 97:2972-7 (2003); Clayton et al.,
Brit. J. Cancer, 91:639-43 (2004); Seidman et al., J. Clin. Oncol.,
20:1215-21 (2002); and Ewer et al., Proc. Am. Soc. Clin. Oncol.,
(abstr. 489) (2002).
[0014] Other HER2 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).
[0015] Homology screening has resulted in the identification of two
other HER receptor family members; HER3 (U.S. Pat. Nos. 5,183,884
and 5,480,968 as well as Kraus et al., PNAS (USA), 86:9193-9197
(1989)) and HER4 (EP Patent Application 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.
[0016] The HER receptors are generally found in various
combinations in cells and heterodimerization is thought to increase
the diversity of cellular responses to a variety of HER 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 HER3 and
HER4. 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 HER ligands were
identified; neuregulin-2 (NRG-2) which is reported to bind either
1-IER3 or HER4 (Chang et al., Nature, 387 509-512 (1997); and
Carraway et al., Nature, 387:512-516 (1997)); neuregulin-3 which
binds HER4 (Zhang et al., PNAS (USA), 94(18):9562-7 (1997)); and
neuregulin-4 which binds HER4 (Harari et al., Oncogene, 18:2681-89
(1999)) HB-EGF, betacellulin and epiregulin also bind to HER4.
[0017] While EGF and TGF.alpha. do not bind HER2, EGF stimulates
EGFR and HER2 to form a heterodimer, which activates EGFR and
results in transphosphorylation of HER2 in the heterodimer.
Dimerization and/or transphosphorylation appears to activate the
HER2 tyrosine kinase. See Earp et al., supra. Likewise, when HER3
is co-expressed with HER2, an active signaling complex is formed
and antibodies directed against HER2 are capable of disrupting this
complex (Sliwkowski et al., J. Biol. Chem., 269(20):14661-14665
(1994)). Additionally, the affinity of HER3 for heregulin (HRG) is
increased to a higher affinity state when co-expressed with HER2.
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 HER2-HER3 protein complex. HER4, like HER3, forms an
active signaling complex with HER2 (Carraway and Cantley, Cell,
78:5-8 (1994)).
[0018] Patent publications related to HER antibodies include: U.S.
Pat. No. 5,677,171, U.S. Pat. No. 5,720,937, U.S. Pat. No.
5,720,954, U.S. Pat. No. 5,725,856, U.S. Pat. No. 5,770,195, U.S.
Pat. No. 5,772,997, U.S. Pat. No. 6,165,464, U.S. Pat. No.
6,387,371, U.S. Pat. No. 6,399,063, US2002/0192211A1, U.S. Pat. No.
6,015,567, U.S. Pat. No. 6,333,169, U.S. Pat. No. 4,968,603, U.S.
Pat. No. 5,821,337, U.S. Pat. No. 6,054,297, U.S. Pat. No.
6,407,213, U.S. Pat. No. 6,719,971, U.S. Pat. No. 6,800,738,
US2004/0236078A1, U.S. Pat. No. 5,648,237, U.S. Pat. No. 6,267,958,
U.S. Pat. No. 6,685,940, U.S. Pat. No. 6,821,515, WO98/17797, U.S.
Pat. No. 6,127,526, U.S. Pat. No. 6,333,398, U.S. Pat. No.
6,797,814, U.S. Pat. No. 6,339,142, U.S. Pat. No. 6,417,335, U.S.
Pat. No. 6,489,447, WO99/31140, US2003/0147884A1, US2003/0170234A1,
US2005/0002928A1, U.S. Pat. No. 6,573,043, US2003/0152987A1,
WO99/48527, US2002/0141993A1, WO01/00245, US2003/0086924,
US2004/0013667A1, WO00/69460, WO01/00238, WO01/15730, U.S. Pat. No.
6,627,196B1, U.S. Pat. No. 6,632,979B1, WO01/00244,
US2002/0090662A1, WO01/89566, US2002/0064785, US2003/0134344, WO
04/24866, US2004/0082047, US2003/0175845A1, WO03/087131,
US2003/0228663, WO2004/008099A2, US2004/0106161, WO2004/048525,
US2004/0258685A1, U.S. Pat. No. 5,985,553, U.S. Pat. No. 5,747,261,
U.S. Pat. No. 4,935,341, U.S. Pat. No. 5,401,638, U.S. Pat. No.
5,604,107, WO 87/07646, WO 89/10412, WO 91/05264, EP 412,116 B1, EP
494,135 B1, U.S. Pat. No. 5,824,311, EP 444,181 B1, EP 1,006,194
A2, US 2002/0155527A1, WO 91/02062, U.S. Pat. No. 5,571,894, U.S.
Pat. No. 5,939,531, EP 502,812 B1, WO 93/03741, EP 554,441 B1, EP
656,367 A1, U.S. Pat. No. 5,288,477, U.S. Pat. No. 5,514,554, U.S.
Pat. No. 5,587,458, WO 93/12220, WO 93/16185, U.S. Pat. No.
5,877,305, WO 93/21319, WO 93/21232, U.S. Pat. No. 5,856,089, WO
94/22478, U.S. Pat. No. 5,910,486, U.S. Pat. No. 6,028,059, WO
96/07321, U.S. Pat. No. 5,804,396, U.S. Pat. No. 5,846,749, EP
711,565, WO 96/16673, U.S. Pat. No. 5,783,404, U.S. Pat. No.
5,977,322, U.S. Pat. No. 6,512,097, WO 97/00271, U.S. Pat. No.
6,270,765, U.S. Pat. No. 6,395,272, U.S. Pat. No. 5,837,243, WO
96/40789, U.S. Pat. No. 5,783,186, U.S. Pat. No. 6,458,356, WO
97/20858, WO 97/38731, U.S. Pat. No. 6,214,388, U.S. Pat. No.
5,925,519, WO 98/02463, U.S. Pat. No. 5,922,845, WO 98/18489, WO
98/33914, U.S. Pat. No. 5,994,071, WO 98/45479, U.S. Pat. No.
6,358,682 B1, US 2003/0059790, WO 99/55367, WO 01/20033, US
2002/0076695 A1, WO 00/78347, WO 01/09187, WO 01/21192, WO
01/32155, WO 01/53354, WO 01/56604, WO 01/76630, WO02/05791, WO
02/11677, U.S. Pat. No. 6,582,919, US2002/0192652A1, US
2003/0211530A1, WO 02/44413, US 2002/0142328, U.S. Pat. No.
6,602,670 B2, WO 02/45653, WO 02/055106, US 2003/0152572, US
2003/0165840, WO 02/087619, WO 03/006509, WO03/012072, WO
03/028638, US 2003/0068318, WO 03/041736, EP 1,357,132, US
2003/0202973, US 2004/0138160, U.S. Pat. No. 5,705,157, U.S. Pat.
No. 6,123,939, EP 616,812 B1, US 2003/0103973, US 2003/0108545,
U.S. Pat. No. 6,403,630 B1, WO 00/61145, WO 00/61185, U.S. Pat. No.
6,333,348 B1, WO 01/05425, WO 01/64246, US 2003/0022918, US
2002/0051785 A1, U.S. Pat. No. 6,767,541, WO 01/76586, US
2003/0144252, WO 01/87336, US 2002/0031515 A1, WO 01/87334, WO
02/05791, WO 02/09754, US 2003/0157097, US 2002/0076408, WO
02/055106, WO 02/070008, WO 02/089842 and WO 03/86467.
[0019] Patients treated with the HER2 antibody trastuzumab may be
selected for therapy based on HER2 overexpression/amplification.
See, for example, WO99/31140 (Paton et al.), US2003/0170234A1
(Hellmann, S.), and US2003/0147884 (Paton et al.); as well as
WO01/89566, US2002/0064785, and US2003/0134344 (Mass et al.). See,
also, US2003/0152987, Cohen et al., concerning immunohistochemistry
(IHC) and fluorescence in situ hybridization (FISH) for detecting
HER2 overexpression and amplification.
[0020] WO2004/053497 and US2004/024815A1 (Bacus et al.), as well as
US 2003/0190689 (Crosby and Smith), refer to determining or
predicting response to trastuzumab therapy. US2004/013297A1 (Bacus
et al.) concerns determining or predicting response to ABX0303 EGFR
antibody therapy. WO2004/000094 (Bacus et al.) is directed to
determining response to GW572016, a small molecule, EGFR-HER2
tyrosine kinase inhibitor. WO2004/063709, Amler et al., refers to
biomarkers and methods for determining sensitivity to EGFR
inhibitor, erlotinib HCl. US2004/0209290, Cobleigh et al., concerns
gene expression markers for breast cancer prognosis.
[0021] Patients treated with pertuzumab can be selected for therapy
based on HER activation or dimerization. Patent publications
concerning pertuzumab and selection of patients for therapy
therewith include: WO01/00245 (Adams et al.); US2003/0086924
(Sliwkowski, M.); US2004/0013667A1 (Sliwkowski, M.); as well as
WO2004/008099A2, and US2004/0106161 (Bossenmaier et al.).
[0022] Cronin et al., Am. J. Path., 164(1):35-42 (2004) describes
measurement of gene expression in archival paraffin-embedded
tissues. Ma et al., Cancer Cell, 5:607-616 (2004) describes gene
profiling by gene oliogonucleotide microarray using isolated RNA
from tumor-tissue sections taken from archived primary
biopsies.
Adjuvant Therapy
[0023] Adjuvant therapy, in the broadest sense, is treatment given
in addition to the primary therapy to kill any cancer cells that
may have spread, even if the spread cannot be detected by
radiologic or laboratory tests. Contemporary clinical trials have
evaluated the efficacy of chemotherapeutic agents for breast cancer
adjuvant therapy, namely BCIRG 001 (comparing paclitaxel,
doxorubicin, and cyclophosphomide (TAC) to fluorouracil,
doxorubicin, and cyclophosphomide FAC); CALGB 9741 (dose dense
tiral); and CALGC 9344 (anthracycline+cyclosphosphomide (AC)
compared to AC+paclitaxel (AC/T)).
[0024] In the BCRIG 001 trial, the disease free survival (DFS)
hazard ratio was 0.72 (p=0.0010), 5 year DFS for TAC was 75%, and
for FAC was 68%. Overal survival (OS) hazard ratio was 0.70
(p=0.0080), 5 year OS for TAC was 87%, and for FAC was 81%. For
HER2 positive (HER2+) subjects (n=328) in this trial, DFS hazard
ratio was 0.60 (p=0.0088).
[0025] CALGB 9741 was a dose dense trial comparing AC.times.4 to
T.times.4; sequential A.times.4 to T.times.4 to C.times.4; dose
dense sequential A.times.4 to T.times.4 to C.times.4; and dose
dense AC.times.4 to T.times.4 (A=anthracycline; C=cyclophosphomide;
T=paclitaxel). DFS hazard ratio (dose dense versus standard) was
0.74 (p=0.010); 4 year DFS was 82% versus 75%. OS hazard ratio
(dose dense versus standard) was 0.69 (p=0.013).
[0026] CALGB 9344 compared the efficacy of AC to AC/T. DFS hazard
ratio was 0.83 (p=0.002), with 5 year DFS of 65% for AC and 70% for
AC/T. OS hazard ratio was 0.82 (p=0.0064), with 5 year OS for AC of
77% and for AC/T of 80%.
[0027] According to the American Cancer Society, an estimated
211,000 women will be diagnosed with breast cancer and
approximately 40,000 women will die of the disease in the United
States in 2005. Breast cancer is the most common cause of cancer
among women in the United States and a woman is diagnosed with
breast cancer in the United States every three minutes. About 30%
of women diagnosed with breast cancer will have lymph node-positive
breast cancer.
[0028] Publications or seminars related to adjuvant therapy
include: Paik et al., J. Natl. Cancer Inst., 92(24):1991-1998
(2000); Paik et al., J. Natl. Cancer Inst., 94:852-854 (2002); Paik
et al. Successful quality assurance program for HER2 testing in the
NSABP Trial for Herceptin. San Antonio Breast Cancer Symposium,
2002; Roche P C et al., J. Natl. Cancer Inst., 94(11):855-7 (2002);
Albain et al., Proceedings of the American Society of Clinical
Oncology Thirty-Eighth Annual Meeting, May 18-21 2002, Orlando,
Fla., Abstract 143; The ATAC (Arimidex, Tamoxifen Alone or in
Combination) Trialists' Group, Lancet, 359:2131-39 (2002); Geyer et
al., 26th Annual San Antonio Breast Cancer Symposium (SABCS),
December 2003, Abstract 12; Perez et al., Proc. ASCO, 2005,
Abstract 556.
[0029] U.S. Patent Publication No. 2004/0014694 (published Jan. 22,
2004) describes a method of adjuvant therapy for the treatment of
early breast cancer, comprising administration of docetaxel,
doxorubicin and cyclophosphamide.
SUMMARY OF THE INVENTION
[0030] The invention herein concerns the results obtained in
clinical studies of the adjuvant use of HERCEPTIN.RTM. in human
subjects with nonmetastatic, high risk, breast cancer. The
efficacy, as evaluated by disease free survival (DFS) and overall
survival (OS) was remarkable, especially when compared to DFS and
OS data for chemotherapeutic agents recently tested in clinical
trials for use in the adjuvant setting. Surprisingly, subjects in
the clinical trials who received HERCEPTIN.RTM. in combination with
paclitaxel, following anthracycline (doxorubicin)/cyclophosphamide
(AC) chemotherapy, had a 52% decrease in disease recurrence (first
breast cancer event) compared to subjects treated with AC followed
by paclitaxel alone at 3 years. The difference was highly
significant.
[0031] The results were particularly impressive and surprising,
given that the subjects were HER2 positive, and therefore at high
risk for recurrence, since HER2 amplification or overexpression has
been linked with more aggressive disease and greater risk of
recurrence. In addition, aside from their HER2 positivity, the
subjects included in the trials were selected by criteria that
further increased their risk for recurring, including the number of
involved lymph nodes, size of the primary tumor, etc. The
significant improvement over chemotherapy alone, is particularly
unexpected in such subjects.
[0032] This invention constitutes a significant medical break
through providing for the more effective care of subjects with
nonmetastatic breast cancer.
[0033] In one aspect, the invention concerns a method of adjuvant
therapy comprising administering to a human subject with
nonmetastatic HER2 positive breast cancer, following definitive
surgery, an effective amount of an antibody which binds to
HER2Domain IV bound by trastuzumab (HERCEPTIN.RTM.) and at least
one chemotherapeutic agent, so as to extend disease free survival
(DFS) or overall survival (OS) in the subject, wherein the DFS or
the OS is evaluated about 2 to 5 years after initiation of
treatment.
[0034] In another aspect, the invention concerns a method of curing
nonmetastatic breast cancer in a population of human subjects with
nonmetastatic HER2 positive breast cancer comprising administering
an effective amount of trastuzumab (HERCEPTIN.RTM.) and taxoid to
the population of subjects following definitive surgery, and
evaluating the population of subjects after about four years to
confirm no disease recurrence has occurred in at least about 80% of
the population.
[0035] In yet another aspect, the invention concerns a method of
decreasing disease recurrence in a population of human subjects
with nonmetastatic HER2 positive breast cancer comprising
administering an effective amount of trastuzumab (HERCEPTIN.RTM.)
and taxoid to the subjects following definitive surgery, wherein
disease recurrence at about 3 years is decreased by at least about
50% compared to subjects treated with taxoid alone.
[0036] In a particular embodiment of these methods, the
administration of the antibody and chemotherapeutic agent decreases
disease cancer recurrence in a population of subjects by about 50%
compared to subjects treated with chemotherapy, such as
anthacycline/cyclophosphamide followed by paclitaxel, alone. In
another embodiment, the subject has a high risk of cancer
recurrence. In another embodiment, the population comprises 3000 or
more human subjects.
[0037] In a further aspect, the invention concerns a method of
adjuvant therapy comprising administering to a human subject with
nonmetastatic HER2 positive breast cancer, following definitive
surgery, an antibody which binds to HER2Domain IV bound by
trastuzumab (HERCEPTIN.RTM.) and at least one chemotherapeutic
agent, in an amount effective to extend disease free survival (DFS)
or overall survival (OS), relative to standard of care
chemotherapy, wherein the DFS or the OS is evaluated at least once
a year for at least about 3 years after initiation of treatment,
wherein DFS is extended if the patient remains alive, without
cancer recurrence for at least one year, and OS is extended if the
patient remains alive for at least one year, from initiation of
treatment.
[0038] In a still further aspect, the invention concerns a method
of instructing a human subject with non-metastatic HER2 positive
breast cancer identified as having a high risk of cancer recurrence
or low likelihood of survival following definitive surgery, and who
is being treated solely by standard of care chemotherapy to receive
treatment with an antibody which binds to HER2Domain IV bound by
trastuzumab (HERCEPTIN.RTM.) and at least one chemotherapeutic
agent.
[0039] In a different aspect, the invention concerns a promotional
method, comprising promoting, for the treatment of HER2 positive
nonmetastatic breast cancer in human subjects identified as being
at high risk of cancer recurrence or low likelihood of survival
following definitive surgery: (a) a chemotherapeutic agent in
combination with an antibody which binds to HER2Domain IV bound by
trastuzumab (HERCEPTIN.RTM.); or (b) an antibody which binds to
HER2Domain IV bound by trastuzumab (HERCEPTIN.RTM.) in combination
with a chemotherapeutic agent.
[0040] In yet another aspect, the invention concerns a business
method, comprising marketing a chemotherapeutic agent for treating
HER2 positive nonmetastatic breast cancer in human subjects
identified as being at high risk of cancer recurrence or low
likelihood of survival following definitive surgery in combination
with an antibody which binds to HER2Domain IV bound by trastuzumab
(HERCEPTIN.RTM.), so as to decrease the subjects' likelihood of
cancer recurrence or increase the subjects' likelihood of
survival.
[0041] In a further aspect, the invention concerns a business
method, comprising marketing an antibody which binds to HER2Domain
IV bound by trastuzumab (HERCEPTIN.RTM.) for treating HER2 positive
nonmetastatic breast cancer in human subjects identified as being
at high risk of cancer recurrence or low likelihood of survival
following definitive surgery in combination with a chemotherapeutic
agent, so as to decrease the subjects' likelihood of cancer
recurrence or increase the subjects' likelihood of survival.
[0042] The invention also concerns a method of adjuvant therapy
comprising administering to a human subject with nonmetastatic HER2
positive breast cancer, following definitive surgery, an antibody
which binds to HER2Domain IV bound by trastuzumab (HERCEPTIN.RTM.),
as a single agent, in an amount effective to extend disease free
survival (DFS) or overall survival (OS), wherein the DFS or the OS
is confirmed at least about one year after an initial
administration of the antibody.
[0043] In all aspects, a preferred antibody blocks binding of
trastuzumab (HERCEPTIN.RTM.) to HER2. More preferably, the antibody
comprises trastuzumab (HERCEPTIN.RTM.). The chemotherapeutic agent
can be selected, without limitation, from the group consisting of
taxoid, vinca, platinum compound, aromatase inhibitor,
anti-estrogen, etoposide, thiotepa, cyclophosphamide, methotrexate,
liposomal doxorubicin, pegylated liposomal doxorubicin,
capecitabine, and gemcitabine. In a preferred embodiment, the
chemotherapeutic agent is a taxoid, such as, for example,
paclitaxel or docetaxel, most preferably paclitaxel.
[0044] In all aspects, preferably the chemotherapeutic agent, such
a taxoid, and the antibody are administered concurrently.
[0045] In all aspects, the chemotherapeutic agent, such as taxoid,
and the antibody are preferably administered following other
standard chemotherapy, administered post-operation. In a preferred
embodiment, the standard chemotherapy is the administration of
anthracycline (doxorubicin) and cyclophosphamide.
[0046] In all aspects, the subject is preferably relatively young,
e.g., less than about 50 years, or less than about 45 years, or
less than about 40 years old.
[0047] In all aspects, the methods include treatment of subjects
having a tumor greater than 2 centimeters in diameter, and/or
subjects with lymph node-positive cancer (having 4-9, or 10 or more
involved lymph nodes), and/or estrogen receptor (ER) negative
subjects, and/or progesterone receptor (PG) negative subjects.
[0048] In all aspects, the antibody can, for example, be an intact,
naked antibody.
[0049] In a particular embodiment, DFS or OS is evaluated 5 years
after initiation of treatment.
[0050] In a further embodiment, administration of the antibody and
chemotherapeutic agent decreases disease recurrence in a population
of subjects by about 50% compared to subjects treated with the
chemotherapteutic agent, without the antibody.
BRIEF DESCRIPTION OF THE DRAWINGS
[0051] FIG. 1 provides a schematic of the HER2 protein structure,
and amino acid sequences for Domains I-IV (SEQ ID NOS: 1-4,
respectively) of the extracellular domain thereof.
[0052] FIGS. 2A and 2B show the amino acid sequences of trastuzumab
light chain (FIG. 2A; SEQ ID NO: 5) and heavy chain (FIG. 2B: SEQ
ID No: 6), respectively.
[0053] FIG. 3 depicts differences between functions of two
different HER2 antibodies; trastuzumab and pertuzumab.
[0054] FIG. 4A depicts the study design for the NSABP B-31 and
NCCTG N9831 (Intergroup) studies, respectively.
[0055] FIG. 4B depicts the study design used for the joint analysis
of the NSABP B31 and NCCTG N9831 (Intergroup) study results.
AC=anthracycline/cyclosphosphomide combination. Efficacy data in
Example 1 herein included from all subjects from NSABP B-31 but
excludes the patients from Intergroup who did not start
HERCEPTIN.RTM. simultaneously with TAXOL.RTM. (arm 2).
[0056] FIG. 5 depicts patient and tumor characteristics for the
AC.fwdarw.paclitaxel and AC.fwdarw.paclitaxel+trastuzumab arms of
the B-31 and N9831 studies. The results are grouped by the age of
patients, number of positive lymph nodes, hormone receptor status,
and tumor size.
[0057] FIG. 6 represents disease-free survival for the B31/N9831
studies
[0058] FIG. 7 is a Forest plot for disease-free survival, where
patients are grouped by age, hormone status, tumor size, and number
of positive nodes.
[0059] FIG. 8 shows disease-free survival for the AC.fwdarw.T and
AC.fwdarw.TH arms of the B-31 (left panel) and N9831 (right panel)
studies.
[0060] FIG. 9 shows time to distant recurrence for the AC.fwdarw.T
and AC.fwdarw.TH arms of the B31/N9831 studies.
[0061] FIG. 10 depicts hazard of distant recurrence for the
AC.fwdarw.T and AC.fwdarw.TH arms of the B31/N9831 studies.
[0062] FIG. 11 shows survival data for the AC.fwdarw.T and
AC.fwdarw.TH arms of the 831/N9831 studies.
[0063] FIG. 12 is a summary of efficacy endpoint analyses.
[0064] FIG. 13 presents the cumulative incidence of cardiac events
in the evaluable cohort (for NSAPB B-31 study only).
[0065] FIGS. 14A and 14B show the amino acid sequences of
pertuzumab light chain (FIG. 14A; SEQ ID NO: 7) and heavy chain
(FIG. 14B; SEQ ID NO: 8). CDRs are shown in bold. Calculated
molecular mass of the light chain and heavy chain are 23,526.22 Da
and 49,216.56 Da (cysteines in reduced form). The carbohydrate
moiety is attached to Asn 299 of the heavy chain.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions
[0066] "Adjuvant therapy" herein refers to therapy given after
definitive surgery, where no evidence of residual disease can be
detected, so as to reduce the risk of disease recurrence. The goal
of adjuvant therapy is to prevent recurrence of the cancer, and
therefore to reduce the chance of cancer-related death. Adjuvant
therapy herein specifically excludes neoadjuvant therapy, e.g.,
where the subject is treated with a chermotherapeutic agent and/or
HERCEPTIN.RTM., prior to definitive surgery.
[0067] "Definitive surgery" refers to complete removal of tumor and
surrounding tissue as well as any involved lymph nodes. Such
surgery includes lumpectomy, mastectomy, such as total mastectomy
plus axillary dissection, double mastectomy etc.
[0068] "Breast cancer" herein refers to cancer involving breast
cells or tissue.
[0069] "Metastatic" breast cancer refers to cancer which has spread
to parts of the body other than the breast and the regional lymph
nodes.
[0070] "Nonmetastatic" breast cancer is cancer which is confined to
the breast and/or regional lymph nodes.
[0071] "Survival" refers to the patient remaining alive, and
includes disease free survival (DFS) as well as overall survival
(OS).
[0072] "Disease free survival (DFS)" refers to the patient
remaining alive, without return of the cancer, for a defined period
of time such as about 1 year, about 2 years, about 3 years, about 4
years, about 5 years, about 10 years, etc., from initiation of
treatment or from initial diagnosis. In the studies underlying the
present invention, DFS was analyzed according to the
intent-to-treat principle, ie, patients were evaluated on the basis
of their assigned therapy. The events used in the analysis of DFS
included local, regional and distant recurrence of cancer,
occurrence of secondary cancer, death from any cause in patients
without a prior event (breast cancer recurrence or second primary
cancer).
[0073] "Overall survival" refers to the patient remaining alive for
a defined period of time, such as about 1 year, about 2 years,
about 3 years, about 4 years, about 5 years, about 10 years, etc.,
from initiation of treatment or from initial diagnosis. In the
studies underlying the present invention the event used for
survival analysis was death from any cause.
[0074] The term "effective amount" refers to an amount of a drug or
drug combination effective to treat cancer in the patient. The
effective amount of the drug may reduce the number of cancer cells;
reduce the tumor size; inhibit (i.e., slow to some extent and
preferably stop) cancer cell infiltration into peripheral organs;
inhibit (i.e., slow to some extent and preferably stop) tumor
metastasis; inhibit, to some extent, tumor growth; and/or relieve
to some extent one or more of the symptoms associated with the
cancer. To the extent the drug may prevent growth and/or kill
existing cancer cells, it may be cytostatic and/or cytotoxic. The
effective amount may improve disease free survival (DFS), improve
overall survival (OS), decrease likelihood of recurrence, extend
time to recurrence, extend time to distant recurrence (i.e.,
recurrence outside of the breast), cure cancer, improve symptoms of
breast cancer (e.g., as gauged using a breast cancer specific
survey), reduce contralateral breast cancer, reduce appearance of
second primary cancer, etc.
[0075] By "extending survival" is meant increasing DFS and/or OS in
a treated patient relative to an untreated patient (i.e., relative
to a patient not treated with the HER2 antibody, HERCEPTIN.RTM.),
or relative to a control treatment protocol, such as treatment only
with the chemotherapeutic agent, such as paclitaxel. Survival is
monitored for at least about six months, or at least about 1 year,
or at least about 2 years, or at least about 3 years, or at least
about 4 years, or at least about 5 years, or at least about 10
years, etc., following the initiation of treatment or following the
initial diagnosis.
[0076] "Hazard ratio" in survival analysis is a summary of the
difference between two survival curves, representing the reduction
in the risk of death on treatment compared to control, over a
period of follow-up. Hazard ratio is a statistical definition for
rates of events. For the purpose of the present invention, hazard
ratio is defined as representing the probability of an event in the
experimental arm divided by the probability of an event in the
control arm at any specific point in time.
[0077] The term "concurrently" is used herein to refer to
administration of two or more therapeutic agents, where at least
part of the administration overlaps in time. Accordingly,
concurrent administration includes a dosing regimen when the
administration of one or more agent(s) continues after
discontinuing the administration of one or more other agent(s).
[0078] For the methods of the present invention, the term
"instructing" a subject means providing directions for applicable
therapy, medication, treatment, treatment regimens, and the like,
by any means, but preferably in writing, such as in the form of
package inserts or other written promotional material.
[0079] For the methods of the present invention, the term
"promoting" means offering, advertising, selling, or describing a
particular drug, combination of drugs, or treatment modality, by
any means, including writing, such as in the form of package
inserts. Promoting herein refers to promotion of a therapeutic
agent, such as a HER2 antibody or chemotherapeutic agent, for an
indication, such as adjuvant breast cancer, where such promoting is
authorized by the Food and Drug Administration (FDA) as having been
demonstrated to be associated with statistically significant
therapeutic efficacy and acceptable safety in a population of
subjects.
[0080] The term "marketing" is used herein to describe the
promotion, selling or distribution of a product (e.g., drug).
Marketing specifically includes packaging, advertising, and any
business activity with the purpose of commercializing a
product.
[0081] A "subject" herein is a human subject.
[0082] A "population" of subjects refers to a group of subjects
with breast cancer, such as in a clinical trial, or as seen by
oncologists following FDA approval for a particular indication,
such as breast cancer adjuvant therapy. In one embodiment, the
population comprises at least 3000 subjects.
[0083] "Node-positive breast cancer" is breast cancer that has
spread to the regional lymph nodes (usually those under the arm).
Subjects with node-positive breast cancer herein included those
with 1-3 involved nodes; 4-9 involved nodes; and 10 or more
involved nodes. Subjects with 4 or more involved nodes are at
higher risk of recurrence than those with less or no involved
nodes.
[0084] "Cancer recurrence" herein refers to a return of cancer
following treatment, and includes return of cancer in the breast,
as well as distant recurrence, where the cancer returns outside of
the breast.
[0085] A subject at "high risk of cancer recurrence" is one who has
a greater chance of experiencing recurrence of cancer, for example,
relatively young subjects (e.g., less than about 50 years old),
those with positive lymph nodes, particularly 4 or more involved
lymph nodes (including 4-9 involved lymph nodes, and 10 or more
involved lymph nodes), those with tumors greater than 2 cm in
diameter, those with HER2-positive breast cancer, and those with
hormone receptor negative breast cancer (i.e., estrogen receptor
(ER) negative and progesterone receptor (PR) negative). A subject's
risk level can be determined by a skilled physician. Generally,
such high risk subjects will have lymph node involvement (for
example with 4 or more involved lymph nodes); however, subjects
without lymph node involvement are also high risk, for example if
their tumor is greater or equal to 2 cm.
[0086] "Estrogen receptor (ER) positive" cancer is cancer which
tests positive for expression of ER. Conversely, "ER negative"
cancer tests negative for such expression. Analysis of ER status
can be performed by any method known in the art. For the purpose of
the studies herein, ER-positive tumors are defined as 10 fmol/mg
cytosol protein by the Dextran-coated charcoal or sucrose-density
gradient method, or positive (using individual laboratory criteria)
by the enzyme immunoassay (EIA) method, or by immunocytochemical
assay.
[0087] "Progesterone receptor (PR) positive" cancer is cancer which
tests positive for expression of PR. Conversely, "PR negative"
cancer tests negative for such expression. Analysis of PR status
can be performed by any method known in the art. For the purpose of
the studies herein, acceptable methods include the Dextran-coated
charcoal or sucrose-density gradient methods, enzyme immunoassay
(EIA) techniques, and immunocytochemical assays.
[0088] Herein, "initiation of treatment" refers to the start of a
treatment regimen following surgical removal of the tumor. In one
embodiment, such may refer to administration of AC following
surgery. Alternatively, this can refer to an initial administration
of the HER2 antibody and/or chemotherapeutic agent.
[0089] By an "initial administration" of a HER2 antibody and
chemotherapeutic agent is meant a first dose of the HER2 antibody
or chemotherapeutic agent as part of a treatment schedule.
[0090] By "curing" cancer herein is meant the absence of cancer
recurrence at about 4 or about 5 years after beginning adjuvant
therapy.
[0091] A "HER receptor" is a receptor protein tyrosine kinase which
belongs to the HER receptor family and includes EGFR, HER2, HER3
and HER4 receptors. The HER receptor will generally comprise an
extracellular domain, which may bind an HER ligand and/or dimerize
with another HER receptor molecule; 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 HER receptor may be a
Anative sequence@ HER receptor or an Aamino acid sequence variant@
thereof. Preferably the HER receptor is native sequence human HER
receptor.
[0092] "HER activation" refers to activation, or phosphorylation,
of any one or more HER receptors. Generally, HER activation results
in signal transduction (e.g., that caused by an intracellular
kinase domain of a HER receptor phosphorylating tyrosine residues
in the HER receptor or a substrate polypeptide). HER activation may
be mediated by HER ligand binding to a HER dimer comprising the HER
receptor of interest. HER ligand binding to a HER dimer may
activate a kinase domain of one or more of the HER receptors in the
dimer and thereby results in phosphorylation of tyrosine residues
in one or more of the HER receptors and/or phosphorylation of
tyrosine residues in additional substrate polypeptides(s), such as
Akt or MAPK intracellular kinases.
[0093] 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 "AerbB2" refers to the gene encoding human ErbB2 and Aneu@
refers to the gene encoding rat p185.sup.neu. Preferred HER2 is
native sequence human HER2.
[0094] Herein, "HER2 extracellular domain" or "HER2ECD" refers to a
domain of HER2 that is outside of a cell, either anchored to a cell
membrane, or in circulation, including fragments thereof. In one
embodiment, the extracellular domain of HER2 may comprise four
domains: ADomain I@ (amino acid residues from about 1-195; SEQ ID
NO: 1), ADomain II@ (amino acid residues from about 196-319; SEQ ID
NO: 2), ADomain III@ (amino acid residues from about 320-488: SEQ
ID NO: 3), and ADomain IV@ (amino acid residues from about 489-630;
SEQ ID NO: 4) (residue numbering without signal peptide). See
Garrett et al., Mol. Cell., 11: 495-505 (2003), Cho et al., Nature,
421:756-760 (2003), Franklin et al., Cancer Cell, 5:317-328 (2004),
and Plowman et al., Proc. Natl. Acad. Sci., 90:1746-1750 (1993), as
well as FIG. 1 herein.
[0095] An antibody which "binds to HER2Domain IV bound by
trastuzumab (HERCEPTIN.RTM.)" binds to an epitope comprising or
including residues from about 489-630 (SEQ ID NO:4) of HER2 ECD.
The preferred such antibody is trastuzumab, or an affinity matured
variant thereof, and/or comprising a variant Fc region (for
instance with improved effector function).
[0096] An antibody which "blocks binding of trastuzumab
(HERCEPTIN.RTM.) to HER2" is one which can be demonstrated to block
trastuzumab's binding to HER2, or compete with trastuzumab for
binding to HER2. Such antibodies may be identified using
cross-blocking assays such as those described in Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and
David Lane (1988); or Fendly et al., Cancer Research, 50:1550-1558
(1990), for example.
[0097] The "trastuzumab (HERCEPTIN.RTM.) epitope" herein is the
region in the extracellular domain of HER2 to which the antibody
4D5 (ATCC CRL 10463) or trastuzumab bind. This epitope is close to
the transmembrane domain of HER2, and within Domain IV of HER2. To
screen for antibodies which bind to this epitope, a cross-blocking
assay such as that described in Antibodies, A Laboratory Manual,
Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988) or
Fendly et al., Cancer Research, 50:1550-1558 (1990), can be
performed. Alternatively, epitope mapping can be performed to
assess whether the antibody binds to the Trastuzumab epitope of
HER2 (e.g., any one or more residues in the region from about
residue 529 to about residue 625, inclusive of the HER2ECD, residue
numbering including signal peptide). One can also study the
antibody-HER2 structure (Franklin et al, Cancer Cell, 5:317-328
(2004)) to see what epitope of HER2 is bound by the antibody.
[0098] For the purposes herein, "trastuzumab," "HERCEPTIN.RTM." and
"huMAb4D5-8" refer to an antibody comprising the light and heavy
chain amino acid sequences in SEQ ID NOS: 5 and 6,
respectively.
[0099] For the purposes herein, a "HER2 positive" cancer or tumor
is one which expresses HER2 at a level which exceeds the level
found on normal breast cells or tissue. Such HER2 positivity may be
caused by HER2 gene amplification, and/or increased transcription
and/or translation. HER2 positive tumors can be identified in
various ways, for instance, by evaluating protein
expression/overexpression (e.g., using the DAKO HERCEPTEST.RTM.)
immunohistochemistry assay, by evaluating HER2 nucleic acid in the
cell (for example via fluorescent in situ hybridization (FISH), see
WO98/45479 published October, 1998, including as the Vysis
PATHVISION.RTM. FISH assay; southern blotting; or polymerase chain
reaction (PCR) techniques, including quantitative real time PCR
(qRT-PCR)), by measuring shed antigen (e.g., HER 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)), or by exposing 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.
Moreover, HER2 positive cancer or tumor samples can be identified
indirectly, for instance by evaluating downstream signaling
mediated through HER2 receptor, gene expression profiling etc.
[0100] The terms "ErbB1," "HER1", "epidermal growth factor
receptor" 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)). erbB1 refers to the gene encoding
the EGFR protein product.
[0101] "AErbB3" and "AHER3" 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).
[0102] The terms "ErbB4" and "AHER4" herein refer to the receptor
polypeptide as disclosed, for example, in EP Patent Application 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.
[0103] By "HER ligand" is meant a polypeptide which binds to and/or
activates a HER receptor. The HER ligand of particular interest
herein is a native sequence human HER 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));
and cripto (CR-1) (Kannan et al., J. Biol. Chem., 272(6):3330-3335
(1997)). HER ligands which bind EGFR include EGF, TGF-.alpha.,
amphiregulin, betacellulin, HB-EGF and epiregulin. HER ligands
which bind HER3 include heregulins. HER ligands capable of binding
HER4 include betacellulin, epiregulin, HB-EGF, NRG-2, NRG-3, NRG-4,
and heregulins.
[0104] "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)).
[0105] A "HER dimmer" herein is a noncovalently associated dimer
comprising at least two HER receptors. Such complexes may form when
a cell expressing two or more HER receptors is exposed to an HER
ligand and can be isolated by immunoprecipitation and analyzed by
SDS-PAGE as described in Sliwkowski et al., J. Biol. Chem.,
269(20):14661-14665 (1994), for example. Other proteins, such as a
cytokine receptor subunit (e.g., gp130) may be associated with the
dimer. Preferably, the HER dimer comprises HER2.
[0106] A "HER heterodimer" herein is a noncovalently associated
heterodimer comprising at least two different HER receptors, such
as EGFR-HER2, HER2-HER3 or HER2-HER4 heterodimers.
[0107] A "HER inhibitor" is an agent which interferes with HER
activation or function. Examples of HER inhibitors include HER
antibodies (e.g., EGFR, HER2, HER3, or HER4 antibodies);
EGFR-targeted drugs; small molecule HER antagonists; HER tyrosine
kinase inhibitors; HER2 and EGFR dual tyrosine kinase inhibitors
such as lapatinib/GW572016; antisense molecules (see, for example,
WO2004/87207); and/or agents that bind to, or interfere with
function of, downstream signaling molecules, such as MAPK or Akt.
Preferably, the HER inhibitor is an antibody or small molecule
which binds to a HER receptor.
[0108] A "HER2 heterodimerization inhibitor" is an agent which
inhibits formation of a heterodimer comprising HER2. Preferably,
the HER2 heterodimerization inhibitor is an antibody, for example
an antibody which binds to HER2 at the heterodimeric binding site
thereof. The most preferred HER2 heterodimerization inhibitor
herein is pertuzumab or MAb 2C4. Other examples of HER2
heterodimerization inhibitors include antibodies which bind to EGFR
and inhibit dimerization thereof with HER2 (for example EGFR
monoclonal antibody 806, MAb 806, which binds to activated or
"untethered" EGFR; see Johns et al., J. Biol. Chem.,
279(29):30375-30384 (2004)); antibodies which bind to HER3 and
inhibit dimerization thereof with HER2; antibodies which bind to
HER4 and inhibit dimerization thereof with HER2; peptide
dimerization inhibitors (U.S. Pat. No. 6,417,168); antisense
dimerization inhibitors; etc.
[0109] A HER2 antibody that Abinds to a heterodimeric binding site@
of HER2, binds to residues in domain II (and optionally also binds
to residues in other of the domains of the HER2 extracellular
domain, such as domains I and III), and can sterically hinder, at
least to some extent, formation of a HER2-EGFR, HER2-HER3, or
HER2-HER4 heterodimer. Franklin et al., Cancer Cell, 5:317-328
(2004) characterize the HER2-pertuzumab crystal structure,
deposited with the RCSB Protein Data Bank (ID Code IS78),
illustrating an exemplary antibody that binds to the heterodimeric
binding site of HER2.
[0110] Protein "expression" refers to conversion of the information
encoded in a gene into messenger RNA (mRNA) and then to the
protein.
[0111] Herein, a sample or cell that "expresses" a protein of
interest (such as HER2) is one in which mRNA encoding the protein,
or the protein, including fragments thereof, is determined to be
present in the sample or cell.
[0112] A "native sequence" polypeptide is one which has the same
amino acid sequence as a polypeptide (e.g., HER receptor or HER
ligand) derived from nature, including naturally occurring or
allelic variants. 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.
[0113] The term "antibody" herein is used in the broadest sense and
specifically covers monoclonal antibodies, polyclonal antibodies,
multispecific antibodies (e.g., bispecific antibodies), and
antibody fragments, so long as they exhibit the desired biological
activity.
[0114] The term "monoclonal antibody" as used herein refers to an
antibody from a population of substantially homogeneous antibodies,
i.e., the individual antibodies comprising the population are
identical and/or bind the same epitope(s), except for possible
variants that may arise during production of the monoclonal
antibody, such variants generally being present in minor amounts.
Such monoclonal antibody typically includes an antibody comprising
a polypeptide sequence that binds a target, wherein the
target-binding polypeptide sequence was obtained by a process that
includes the selection of a single target binding polypeptide
sequence from a plurality of polypeptide sequences. For example,
the selection process can be the selection of a unique clone from a
plurality of clones, such as a pool of hybridoma clones, phage
clones or recombinant DNA clones. It should be understood that the
selected target binding sequence can be further altered, for
example, to improve affinity for the target, to humanize the target
binding sequence, to improve its production in cell culture, to
reduce its immunogenicity in vivo, to create a multispecific
antibody, etc., and that an antibody comprising the altered target
binding sequence is also a monoclonal antibody of this invention.
In contrast to polyclonal antibody preparations which typically
include different antibodies directed against different
determinants (epitopes), each monoclonal antibody of a monoclonal
antibody preparation is directed against a single determinant on an
antigen. In addition to their specificity, the monoclonal antibody
preparations are advantageous in that they are typically
uncontaminated by other immunoglobulins. 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 a
variety of techniques, including, for example, the hybridoma method
(e.g., Kohler et al., Nature, 256:495 (1975); Harlow et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory
Press, 2nd ed. 1988); Hammerling et al., in Monoclonal Antibodies
and T-Cell Hybridomas 563-681, (Elsevier, N.Y., 1981)), recombinant
DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display
technologies (see, e.g., Clackson et al., Nature, 352:624-628
(1991); Marks et al., J. Mol. Biol., 222:581-597 (1991); Sidhu et
al., J. Mol. Biol., 338(2):299-310 (2004); Lee et al., J. Mol.
Biol., 340(5):1073-1093 (2004); Fellouse, Proc. Nat. Acad. Sci.
USA, 101(34):12467-12472 (2004); and Lee et al., J. Immunol.
Methods, 284(1-2):119-132 (2004), and technologies for producing
human or human-like antibodies in animals that have parts or all of
the human immunoglobulin loci or genes encoding human
immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096;
WO 1996/33735; WO 1991/10741; 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); U.S. Pat.
Nos. 5,545,806; 5,569,825; 5,591,669 (all of GenPharm); U.S. Pat.
No. 5,545,807; WO 1997/17852; U.S. Pat. Nos. 5,545,807; 5,545,806;
5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al.,
Bio/Technology, 10:779-783 (1992); Lonberg et al., Nature, 368:
856-859 (1994); Morrison, Nature, 368: 812-813 (1994); Fishwild et
al., Nature Biotechnology, 14:845-851 (1996); Neuberger, Nature
Biotechnology, 14:826 (1996); and Lonberg and Huszar, Intern. Rev.
Immunol., 13:65-93 (1995)).
[0115] 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 Aprimatized@ 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,
as well as "humanized" antibodies.
[0116] "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 2:593-596 (1992).
[0117] Humanized HER2 antibodies include huMAb4D5-1, huMAb4D5-2,
huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and
huMAb4D5-8 or trastuzumab (HERCEPTIN7) 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 such as
pertuzumab as described herein.
[0118] Herein, Apertuzumab@ and AOMNITARGJ@ refer to an antibody
comprising the light and heavy chain amino acid sequences in SEQ ID
NOS: 7 and 8, respectively.
[0119] An Aintact antibody@ herein is one which comprises two
antigen binding regions, and an Fc region. Preferably, the intact
antibody has a functional Fc region.
[0120] "Antibody fragments" comprise a portion of an intact
antibody, preferably comprising the antigen binding region thereof.
Examples of antibody fragments include Fab, Fab', F(abs).sub.2, and
Fv fragments; diabodies; linear antibodies; single-chain antibody
molecules; and multispecific antibodies formed from antibody
fragment(s).
[0121] "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.
[0122] 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).
[0123] The term Ahypervariable 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 Acomplementarity determining region@ or ACDR@
(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 Ahypervariable 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.
[0124] 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 crosslinking antigen.
[0125] "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.
[0126] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (CHI) 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.
[0127] The "light chains" of antibodies from any vertebrate species
can be assigned to one of two clearly distinct types, called kappa
(.kappa.) and lambda (.lamda.), based on the amino acid sequences
of their constant domains.
[0128] The term "Fc region" herein is used to define a C-terminal
region of an immunoglobulin heavy chain, including native sequence
Fc regions and variant Fc regions. Although the boundaries of the
Fc region of an immunoglobulin heavy chain might vary, the human
IgG heavy chain Fc region is usually defined to stretch from an
amino acid residue at position Cys226, or from Pro230, to the
carboxyl-terminus thereof. The C-terminal lysine (residue 447
according to the EU numbering system) of the Fc region may be
removed, for example, during production or purification of the
antibody, or by recombinantly engineering the nucleic acid encoding
a heavy chain of the antibody. Accordingly, a composition of intact
antibodies may comprise antibody populations with all K447 residues
removed, antibody populations with no K447 residues removed, and
antibody populations having a mixture of antibodies with and
without the K447 residue.
[0129] Unless indicated otherwise, herein the numbering of the
residues in an immunoglobulin heavy chain is that of the EU index
as in Kabat et al., Sequences of Proteins of Immunological
Interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda, Md. (1991), expressly incorporated herein by
reference. The "EU index as in Kabat" refers to the residue
numbering of the human IgG1 EU antibody.
[0130] A "functional Fc region" possesses an "effector function" of
a native sequence Fc region. Exemplary "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. Such effector functions generally require
the Fc region to be combined with a binding domain (e.g., an
antibody variable domain) and can be assessed using various assays
as herein disclosed, for example.
[0131] A "native sequence Fc region" comprises an amino acid
sequence identical to the amino acid sequence of an Fc region found
in nature. Native sequence human Fc regions include a native
sequence human IgG1 Fc region (non-A and A allotypes); native
sequence human IgG2 Fc region; native sequence human IgG3 Fc
region; and native sequence human IgG4 Fc region as well as
naturally occurring variants thereof.
[0132] A "variant Fc region" comprises an amino acid sequence which
differs from that of a native sequence Fc region by virtue of at
least one amino acid modification, preferably one or more amino
acid substitution(s). Preferably, the variant Fc region has at
least one amino acid substitution compared to a native sequence Fc
region or to the Fc region of a parent polypeptide, e.g., from
about one to about ten amino acid substitutions, and preferably
from about one to about five amino acid substitutions in a native
sequence Fc region or in the Fc region of the parent polypeptide.
The variant Fc region herein will preferably possess at least about
80% homology with a native sequence Fc region and/or with an Fc
region of a parent polypeptide, and most preferably at least about
90% homology therewith, more preferably at least about 95% homology
therewith.
[0133] Depending on the amino acid sequence of the constant domain
of their heavy chains, intact antibodies can be assigned to
different Aclasses@. There are five major classes of intact
antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may
be further divided into Asubclasses@ (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.
[0134] AAntibody-dependent cell-mediated cytotoxicity@ and AADCC@
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 U.S. Pat. No. 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).
[0135] AHuman effector cells@ are leukocytes which express one or
more FcR5 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.
[0136] The terms "Fc receptor" or AFcR@ 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 Fey 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)), and regulates homeostasis
of immunoglobulins.
[0137] "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.
[0138] "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). HER2 antibody scFv fragments are described in WO93/16185;
U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458.
[0139] 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).
[0140] A Anaked antibody@ herein is an antibody that is not
conjugated to a cytotoxic moiety or radiolabel.
[0141] 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.
[0142] An "affinity matured" antibody is one with one or more
alterations in one or more hypervariable regions thereof which
result an improvement in the affinity of the antibody for antigen,
compared to a parent antibody which does not possess those
alteration(s). Preferred affinity matured antibodies will have
nanomolar or even picomolar affinities for the target antigen.
Affinity matured antibodies are produced by procedures known in the
art. Marks et al., Bio/Technology, 10:779-783 (1992) describes
affinity maturation by V.sub.H and V.sub.L domain shuffling. Random
mutagenesis of CDR and/or framework residues is described by:
Barbas et al., Proc Nat. Acad. Sci, USA, 91:3809-3813 (1994);
Schier et al., Gene, 169:147-155 (1995); Yelton et al., J.
Immunol., 155:1994-2004 (1995); Jackson et al., J. Immunol.,
154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol., 226:889-896
(1992).
[0143] The term "main species antibody" herein refers to the
antibody structure in a composition which is the quantitatively
predominant antibody molecule in the composition. In one
embodiment, the main species antibody is a HER2 antibody, such as
an antibody that binds Domain IV of HER2 ECD bound by trastuzumab
(HERCEPTIN.RTM.). The preferred embodiment herein of the main
species antibody is one comprising the light chain and heavy chain
amino acid sequences in SEQ ID Nos. 5 and 6 (trastuzumab).
[0144] An "amino acid sequence variant" antibody herein is an
antibody with an amino acid sequence which differs from a main
species antibody. Ordinarily, amino acid sequence variants will
possess at least about 70% homology with the main species antibody,
and preferably, they will be at least about 80%, more preferably at
least about 90% homologous with the main species antibody. The
amino acid sequence variants possess substitutions, deletions,
and/or additions at certain positions within or adjacent to the
amino acid sequence of the main species antibody. Examples of amino
acid sequence variants herein include an acidic variant (e.g.,
deamidated antibody variant), a basic variant, an antibody with a
C-terminal lysine residue on one or two heavy chains thereof, etc,
and includes combinations of variations to the amino acid sequences
of heavy and/or light chains.
[0145] A "glycosylation variant" antibody herein is an antibody
with one or more carbohydrate moeities attached thereto which
differ from one or more carbohydrate moieties attached to a main
species antibody. Examples of glycosylation variants herein include
antibody with a G1 or G2 oligosaccharide structure, instead a G0
oligosaccharide structure, attached to an Fc region thereof,
antibody with one or two carbohydrate moieties attached to one or
two light chains thereof, antibody with no carbohydrate attached to
one or two heavy chains of the antibody, etc., and combinations of
glycosylation alterations.
[0146] Where the antibody has an Fc region, an oligosaccharide
structure may be attached to one or two heavy chains of the
antibody, e.g., at residue 299 (298, Eu numbering of residues).
[0147] A "deamidated" antibody is one in which one or more
asparagine residues thereof has been derivitized, e.g., to an
aspartic acid, a succinimide, or an iso-aspartic acid.
[0148] A "tumor sample" herein is a sample derived from, or
comprising tumor cells from, a patient=s tumor. Examples of tumor
samples herein include, but are not limited to, tumor biopsies,
circulating tumor cells, circulating plasma proteins, ascitic
fluid, primary cell cultures or cell lines derived from tumors or
exhibiting tumor-like properties, as well as preserved tumor
samples, such as formalin-fixed, paraffin-embedded tumor samples or
frozen tumor samples.
[0149] A "fixed" tumor sample is one which has been histologically
preserved using a fixative.
[0150] A "formalin-fixed" tumor sample is one which has been
preserved using formaldehyde as the fixative.
[0151] An "embedded" tumor sample is one surrounded by a firm and
generally hard medium such as paraffin, wax, celloidin, or a resin.
Embedding makes possible the cutting of thin sections for
microscopic examination or for generation of tissue microarrays
(TMAs).
[0152] A "paraffin-embedded" tumor sample is one surrounded by a
purified mixture of solid hydrocarbons derived from petroleum.
[0153] Herein, a "frozen" tumor sample refers to a tumor sample
which is, or has been, frozen.
[0154] Herein, "gene expression profiling" refers to an evaluation
of expression of one or more genes as a surrogate for determining
HER2 receptor expression directly.
[0155] A "phospho-ELISA assay" herein is an assay in which
phosphorylation of one or more HER receptors, especially HER2, is
evaluated in an enzyme-linked immunosorbent assay (ELISA) using a
reagent, usually an antibody, to detect phosphorylated HER
receptor, substrate, or downstream signaling molecule. Preferably,
an antibody which detects phosphorylated HER2 is used. The assay
may be performed on cell lysates, preferably from fresh or frozen
biological samples.
[0156] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell, especially
a HER expressing cancer cell either in vitro or in vivo. Thus, the
growth inhibitory agent may be one which significantly reduces the
percentage of HER 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), taxoids, 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. (WB Saunders:
Philadelphia, 1995), especially p. 13.
[0157] Examples of Agrowth inhibitory@ antibodies are those which
bind to HER2 and inhibit the growth of cancer cells overexpressing
HER2. Preferred growth inhibitory HER2 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 a humanized variant of
murine monoclonal antibody 4D5, e.g., trastuzumab.
[0158] 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 HER2 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-M13-453,
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. Examples of HER2 antibodies that induce
apoptosis are 7C2 and 7F3. See, in particular, WO98/17797.
[0159] "Treatment" refers to both therapeutic treatment and
prophylactic or preventative measures. Those in need of treatment
include those already with cancer as well as those in which cancer
is to be prevented. Hence, the patient to be treated herein may
have been diagnosed as having cancer or may be predisposed or
susceptible to cancer.
[0160] 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.
[0161] 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,
triethiylenethiophosphoramide and trimethylolomelamine; acetogenins
(especially bullatacin and bullatacinone);
delta-9-tetrahydrocannabinol (dronabinol, MARINOL.RTM.);
beta-lapachone; lapachol; colchicines; betulinic acid; a
camptothecin (including the synthetic analogue topotecan
(HYCAMTIN.RTM.), CPT-11 (irinotecan, CAMPTOSAR.RTM.),
acetylcamptothecin, scopolectin, and 9-aminocamptothecin);
bryostatin; callystatin; CC-1065 (including its adozelesin,
carzelesin and bizelesin synthetic analogues); podophyllotoxin;
podophyllinic acid; teniposide; cryptophycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogues, KW-2189 and CB1-TM1);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlornaphazine,
cholophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranimnustine; antibiotics such as the
enediyne antibiotics (e.g., calicheamicin, especially calicheamicin
gammaII and calicheamicin omegaII (see, e.g., Agnew, Chem Intl. Ed.
Engl., 33:183-186 (1994)); dynemicin, including dynemicin A; an
esperamicin; as well as neocarzinostatin chromophore and related
chromoprotein enediyne antiobiotic chromophores), aclacinomysins,
actinomycin, authramycin, azaserine, bleomycins, cactinomycin,
carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin
(including ADRIAMYCIN.RTM., morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin
HCl liposome injection (DOXIL.RTM.), liposomal doxorubicin TLC D-99
(MYOCET.RTM.), peglylated liposomal doxorubicin (CAELYX.RTM.), and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate, gemcitabine (GEMZAR.RTM.), tegafur (UFTORAL.RTM.),
capecitabine (XELODA.RTM.), an epothilone, 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;
anti-adrenals such as aminoglutethimide, mitotane, trilostane;
folic acid replenisher such as frolinic acid; aceglatone;
aldophosphamide glycoside; aminolevulinic acid; eniluracil;
amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;
demecolcine; diaziquone; elformithine; elliptinium acetate;
etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine;
maytansinoids such as maytansine and ansamitocins; mitoguazone;
mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet;
pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK.RTM.
polysaccharide complex (JHS Natural Products, Eugene, Oreg.);
razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid;
triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes
(especially T-2 toxin, verracurin A, roridin A and anguidine);
urethan; dacarbazine; mannomustine; mitobronitol; mitolactol;
pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoid,
e.g., paclitaxel (TAXOL.RTM.), albumin-engineered nanoparticle
formulation of paclitaxel (ABRAXANE.TM.), and docetaxel
(TAXOTERE.RTM.); chloranbucil; 6-thioguanine; mercaptopurine;
methotrexate; platinum agents such as cisplatin, oxaliplatin, and
carboplatin; vincas, which prevent tubulin polymerization from
forming microtubules, including vinblastine (VELBAN.RTM.),
vincristine (ONCOVIN.RTM.), vindesine (ELDISINE.RTM.,
FILDESIN.RTM.), and vinorelbine (NAVELBINE.RTM.); etoposide
(VP-16); ifosfamide; mitoxantrone; leucovovin; novantrone;
edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase
inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such
as retinoic acid, including bexarotene (TARGRETIN.RTM.);
bisphosphonates such as clodronate (for example, BONEFOS.RTM. or
OSTAC.RTM.), etidronate (DIDROCAL.RTM.), NE-58095, zoledronic
acid/zoledronate (ZOMETA.RTM.), alendronate (FOSAMAX.RTM.),
pamidronate (AREDIA.RTM.), tiludronate (SKELID.RTM.), or
risedronate (ACTONEL.RTM.); troxacitabine (a 1,3-dioxolane
nucleoside cytosine analog); antisense oligonucleotides,
particularly those that inhibit expression of genes in signaling
pathways implicated in aberrant cell proliferation, such as, for
example, PKC-alpha, Raf, H-Ras, and epidermal growth factor
receptor (EGF-R); vaccines such as THERATOPE.RTM. vaccine and gene
therapy vaccines, for example, ALLOVECTIN.RTM. vaccine,
LEUVECTIN.RTM. vaccine, and VAXID.RTM. vaccine; topoisomerase 1
inhibitor (e.g., LURTOTECAN.RTM.); rmRH (e.g., ABARELIX.RTM.);
BAY439006 (sorafenib; Bayer); SU-11248 (Pfizer); perifosine, COX-2
inhibitor (e.g., celecoxib or etoricoxib), proteosome inhibitor
(e.g., PS341); bortezomib (VELCADE.RTM.); CCI-779; tipifarnib
(R11577); orafenib, ABT510; Bcl-2 inhibitor such as oblimersen
sodium (GENASENSE.RTM.); pixantrone; EGFR inhibitors (see
definition below); tyrosine kinase inhibitors (see definition
below); and pharmaceutically acceptable salts, acids or derivatives
of any of the above; as well as combinations of two or more of the
above such as CHOP, an abbreviation for a combined therapy of
cyclophosphamide, doxorubicin, vincristine, and prednisolone, and
FOLFOX, an abbreviation for a treatment regimen with oxaliplatin
(ELOXATIN.TM.) combined with 5-FU and leucovovin.
[0162] Herein, chemotherapeutic agents include "anti-hormonal
agents" or "endocrine therapeutics" which act to regulate, reduce,
block, or inhibit the effects of hormones that can promote the
growth of cancer. They may be hormones themselves, including, but
not limited to: anti-estrogens with mixed agonist/antagonist
profile, including, tamoxifen (NOLVADEX.RTM.), 4-hydroxytamoxifen,
toremifene (FARESTON.RTM.), idoxifene, droloxifene, raloxifene
(EVISTA.RTM.), trioxifene, keoxifene, and selective estrogen
receptor modulators (SERMs) such as SERM3; pure anti-estrogens
without agonist properties, such as fulvestrant (FASLODEX.RTM.),
and EM800 (such agents may block estrogen receptor (ER)
dimerization, inhibit DNA binding, increase ER turnover, and/or
suppress ER levels); aromatase inhibitors, including steroidal
aromatase inhibitors such as formestane and exemestane
(AROMASIN.RTM.), and nonsteroidal aromatase inhibitors such as
anastrazole (ARIMIDEX.RTM.), letrozole (FEMARA.RTM.) and
aminoglutethimide, and other aromatase inhibitors include vorozole
(RIVISOR.RTM.), megestrol acetate (MEGASE.RTM.), fadrozole, and
4(5)-imidazoles; lutenizing hormone-releaseing hormone agonists,
including leuprolide (LUPRON.RTM. and ELIGARD.RTM.), goserelin,
buserelin, and tripterelin; sex steroids, including progestines
such as megestrol acetate and medroxyprogesterone acetate,
estrogens such as diethylstilbestrol and premarin, and
androgens/retinoids such as fluoxymesterone, all transretionic acid
and fenretinide; onapristone; anti-progesterones; estrogen receptor
down-regulators (ERDs); anti-androgens such as flutamide,
nilutamide and bicalutamide; and pharmaceutically acceptable salts,
acids or derivatives of any of the above; as well as combinations
of two or more of the above.
[0163] Herein, a "taxoid" is a chemotherapeutic agent that
functions to inhibit microtubule depolymerization. Examples include
paclitaxel (TAXOL.RTM.), albumin-engineered nanoparticle
formulation of paclitaxel (ABRAXANE.TM.), and docetaxel
(TAXOTERE.RTM.). The preferred taxoid is paclitaxel.
[0164] As used herein, the term "EGFR inhibitor" refers to
compounds that bind to or otherwise interact directly with EGFR and
prevent or reduce its signaling activity, and is alternatively
referred to as an "EGFR antagonist." 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.); IMC-11F8, a fully human, EGFR-targeted
antibody (Imclone); 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 or Panitumumab (see WO98/50433,
Abgenix/Amgen); EMD 55900 (Stragliotto et al., Eur. J. Cancer,
32A:636-640 (1996)); EMD7200 (matuzumab) a humanized EGFR antibody
directed against EGFR that competes with both EGF and TGF-alpha for
EGFR binding (EMD/Merck); human EGFR antibody, HuMax-EGFR (GenMab);
fully human antibodies known as E1.1, E2.4, E2.5, E6.2, E6.4,
E2.11, E6. 3 and E7.6. 3 and described in U.S. Pat. No. 6,235,883;
MDX-447 (Medarex Inc); and mAb 806 or humanized mAb 806 (Johns et
al., J. Biol. Chem., 279(29):30375-30384 (2004)). The anti-EGFR
antibody may be conjugated with a cytotoxic agent, thus generating
an immunoconjugate (see, e.g., EP659,439A2, Merck Patent GmbH).
EGFR antagonists include small molecules such as compounds
described in U.S. Pat. Nos. 5,616,582, 5,457,105, 5,475,001,
5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620,
6,596,726, 6,713,484, 5,770,599, 6,140,332, 5,866,572, 6,399,602,
6,344,459, 6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008,
and 5,747,498, as well as the following PCT publications:
WO98/14451, WO98/50038, WO99/09016, and WO99/24037. Particular
small molecule EGFR antagonists include OSI-774 (CP-358774,
erlotinib, TARCEVA.RTM. Genentech/OSI Pharmaceuticals); PD 183805
(CI-1033, 2-propenamide,
N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quin-
azolinyl]-, dihydrochloride, Pfizer Inc.); ZD1839, gefitinib
(IRESSAJ)
4-(3'-Chloro-4'-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoli-
ne, AstraZeneca); ZM 105180
((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zeneca); BIBX-1382
(N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4--
d]pyrimidine-2,8-diamine, Boehringer Ingelheim); PKI-166
((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol)-
;
(R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimi-
dine); CL-387785
(N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide); EKB-569
(N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(-
dimethylamino)-2-butenamide) (Wyeth); AG1478 (Sugen); AG1571 (SU
5271; Sugen); dual EGFR/HER2 tyrosine kinase inhibitors such as
lapatinib (GW 572016 or N-[3-chloro-4-[(3
fluorophenyl)methoxy]phenyl]6[5[[[2-methylsulfonyl)ethyl]amino]methyl]-2--
furanyl]-4-quinazolinamine; Glaxo-SmithKline).
[0165] A "tyrosine kinase inhibitor" is a molecule which inhibits
tyrosine kinase activity of a tyrosine kinase such as a HER
receptor. Examples of such inhibitors include the EGFR-targeted
drugs noted in the preceding paragraph; small molecule HER2
tyrosine kinase inhibitor such as TAK165 available from Takeda;
CP-724,714, an oral selective inhibitor of the ErbB2 receptor
tyrosine kinase (Pfizer and OSI); dual-HER inhibitors such as
EKB-569 (available from Wyeth) which preferentially binds EGFR but
inhibits both HER2 and EGFR-overexpressing cells; lapatinib
(GW572016; available from Glaxo-SmithKline) an oral HER2 and EGFR
tyrosine kinase inhibitor; PKI-166 (available from Novartis);
pan-HER inhibitors such as canertinib (CI-1033; Pharmacia); Raf-1
inhibitors such as antisense agent ISIS-5132 available from ISIS
Pharmaceuticals which inhibits Raf-1 signaling; non-HER targeted TK
inhibitors such as Imatinib mesylate (GLEEVACJ) available from
Glaxo; MAPK extracellular regulated kinase 1 inhibitor CI-1040
(available from Pharmacia); quinazolines, such as PD
153035,4-(3-chloroanilino) quinazoline; pyridopyrimidines;
pyrimidopyrimidines; pyrrolopyrimidines, such as CGP 59326, CGP
60261 and CGP 62706; pyrazolopyrimidines,
4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidines; curcumin (diferuloyl
methane, 4,5-bis(4-fluoroanilino)phthalimide); tyrphostines
containing nitrothiophene moieties; PD-0183805 (Warner-Lamber);
antisense molecules (e.g., those that bind to HER-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-HER 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); Semaxinib (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 Cyanamid); 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).
[0166] Herein, "standard of care" chemotherapy refers to the
chemotherapeutic agents routinely used to treat a particular
cancer. For example, for operable breast cancer, including node
positive breast cancer, the standard of care adjuvant therapy can
be anthracycline/cyclophosphamide (AC) chemotherapy,
cyclophosphamide, methotrexate, fluorouracil (CMF) chemotherapy,
fluorouracil, anthracycline and cyclophosphamide (FAC)
chemotherapy, or AC followed by paclitaxel (T) (AC.fwdarw.T). For
the patients described in the examples herein, "standard of care"
has been AC.fwdarw.T treatment.
[0167] Where an anti-cancer agent, such as HERCEPTIN.RTM., is
administered as a "single agent" it is the only agent administered
to the subject, during a treatment regimen, to treat the cancer,
i.e., the agent is not provided in combination with other
anti-cancer agents. However, such treatment includes the
administration of other anti-cancer agents substantially prior to,
or following, administration of the anti-cancer agent.
[0168] An Aanti-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 bevacizumab
(AVASTIN.RTM.).
[0169] 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.
[0170] A "loading" dose herein generally comprises an initial dose
of a therapeutic agent administered to a patient, and is followed
by one or more maintenance dose(s) thereof. Generally, a single
loading dose is administered, but multiple loading doses are
contemplated herein. Usually, the amount of loading dose(s)
administered exceeds the amount of the maintenance dose(s)
administered and/or the loading dose(s) are administered more
frequently than the maintenance dose(s), so as to achieve the
desired steady-state concentration of the therapeutic agent earlier
than can be achieved with the maintenance dose(s).
[0171] A "maintenance" dose herein refers to one or more doses of a
therapeutic agent administered to the patient over a treatment
period. Usually, the maintenance doses are administered at spaced
treatment intervals, such as approximately every week,
approximately every 2 weeks, approximately every 3 weeks, or
approximately every 4 weeks.
II. Production of Antibodies
[0172] A description follows as to exemplary techniques for the
production of HER2 antibodies used in accordance with the present
invention. The HER2 antigen to be used for production of antibodies
may be, e.g., a soluble form of the extracellular domain of a HER2
receptor or a portion thereof, containing the desired epitope.
Alternatively, cells expressing HER2 at their cell surface (e.g.,
NIH-3T3 cells transformed to overexpress HER2; 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 HER2 useful for generating antibodies will be apparent to
those skilled in the art.
[0173] (i) Polyclonal Antibodies
[0174] 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.
[0175] 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.
[0176] (ii) Monoclonal Antibodies
[0177] Various methods for making monoclonal antibodies herein are
available in the art. For example, the monoclonal antibodies may be
made using the hybridoma method first described by Kohler et al.,
Nature, 256:495 (1975), by recombinant DNA methods (U.S. Pat. No.
4,816,567).
[0178] 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)).
[0179] 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.
[0180] 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)).
[0181] 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).
[0182] 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).
[0183] 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.
[0184] 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.
[0185] 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).
[0186] 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.
[0187] 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.
[0188] 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.
[0189] (iii) Humanized Antibodies
[0190] 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.
[0191] 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)).
[0192] 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.
[0193] 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.
[0194] Humanization of murine 4D5 antibody to generate humanized
variants thereof, including Trastuzumab, is described in U.S. Pat.
Nos. 5,821,337, 6,054,297, 6,407,213, 6,639,055, 6,719,971, and
6,800,738, as well as Carter et al. PNAS (USA), 89:4285-4289
(1992). HuMAb4D5-8 (trastuzumab) bound HER2 antigen 3-fold more
tightly than the mouse 4D5 antibody, and had secondary immune
function (ADCC) which allowed for directed cytotoxic activityof the
humanized antibody in the presence of human effector cells.
HuMAb4D5-8 comprised variable light (V.sub.L) CDR residues
incorporated in a V.sub.L kappa subgroup I consensuse framework,
and variable heavy (V.sub.H) CDR residues incorporated into a
V.sub.H subgroup III consensus framework. The antibody further
comprised framework region (FR) substitutions as positions: 71, 73,
78, and 93 of the V.sub.H (Kabat numbering of FR residues; and a FR
substitution at position 66 of the V.sub.L (Kabat numbering of FR
residues). Trastuzumab comprises non-A allotype human gamma 1 Fc
region.
[0195] (iv) Human Antibodies
[0196] 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. LISA, 90:2551 (1993); Jakobovits et al.,
Nature, 362:255-258 (1993); Bruggermann et al., Year in Immuno.,
7:33 (1993); and U.S. Pat. Nos. 5,591,669, 5,589,369 and 5,545,807.
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.
[0197] 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).
[0198] Human HER2 antibodies are described in U.S. Pat. No.
5,772,997 issued Jun. 30, 1998 and WO 97/00271 published Jan. 3,
1997.
[0199] (v) Antibody Fragments
[0200] Various techniques have been developed for the production of
antibody fragments comprising one or more antigen binding regions.
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 Alinear antibody@, e.g., as
described in U.S. Pat. No. 5,641,870 for example. Such linear
antibody fragments may be monospecific or bispecific.
[0201] (vi) Bispecific Antibodies
[0202] 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
HER2 protein. Other such antibodies may combine a HER2 binding site
with binding site(s) for EGFR, HER3 and/or HER4. Alternatively, a
HER2 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 HER2-expressing
cell. Bispecific antibodies may also be used to localize cytotoxic
agents to cells which express HER2. These antibodies possess a
HER2-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).
[0203] WO 96/16673 describes a bispecific HER2/Fc.gamma.RIII
antibody and U.S. Pat. No. 5,837,234 discloses a bispecific
HER2/Fc.gamma.RI antibody IDM 1 (Osidem). A bispecific
HER2/Fc.alpha. antibody is shown in WO98/02463. U.S. Pat. No.
5,821,337 teaches a bispecific HER2/CD3 antibody. MDX-210 is a
bispecific HER2-Fc.gamma.RIII Ab.
[0204] 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
(Millstein 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).
[0205] 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.
[0206] 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).
[0207] 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.
[0208] 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 HIV 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.
[0209] 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.
[0210] 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
HER2 receptor and normal human T-cells, as well as trigger the
lytic activity of human cytotoxic lymphocytes against human breast
tumor targets.
[0211] 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).
[0212] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al.,
J. Immunol., 147:60 (1991).
[0213] (vii) Other Amino Acid Sequence Modifications
[0214] Amino acid sequence modification(s) of the 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 antibody are
prepared by introducing appropriate nucleotide changes into the
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
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
antibody, such as changing the number or position of glycosylation
sites.
[0215] A useful method for identification of certain residues or
regions of the 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
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 antibody
variants are screened for the desired activity.
[0216] 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 antibody, with an
N-terminal methionyl residue or the antibody fused to a cytotoxic
polypeptide. Other insertional variants of the antibody molecule
include the fusion to the N- or C-terminus of the antibody to an
enzyme (e.g., for ADEPT) or a polypeptide which increases the serum
half-life of the antibody.
[0217] Another type of variant is an amino acid substitution
variant. These variants have at least one amino acid residue in the
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; Leu Phe; Norleucine Leu (L) Norleucine; Ile; Val; Ile
Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu
Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)
Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe;
Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Norleucine
[0218] 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. Amino acids may be grouped
according to similarities in the properties of their side chains
(in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth
Publishers, New York (1975)): [0219] (1) non-polar: Ala (A), Val
(V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M) [0220]
(2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y),
Asn (N), Gln (O) [0221] (3) acidic: Asp (D), Glu (E) [0222] (4)
basic: Lys (K), Arg (R), His (H)
[0223] Alternatively, naturally occurring residues may be divided
into groups based on common side-chain properties: [0224] (1)
hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; [0225] (2)
neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; [0226] (3) acidic:
Asp, Glu; [0227] (4) basic: His, Lys, Arg; [0228] (5) residues that
influence chain orientation: Gly, Pro; [0229] (6) aromatic: Trp,
Tyr, Phe.
[0230] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0231] Any cysteine residue not involved in maintaining the proper
conformation of the 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).
[0232] 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 HER2. 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.
[0233] Exemplary trastuzumab variants herein include those
described in US2003/0228663A1 (Lowman et al.), including
substitutions of one or more of the following V.sub.L positions:
Q27, D28, N30, T31, A32, Y49, F53, Y55, R66, H91, Y92, and/or T94;
and/or substitutions of one or more of V.sub.H positions: W95, D98,
F100, Y100a, and/or Y102.
[0234] 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.
[0235] 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.
[0236] 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).
[0237] Where the antibody comprises an Fc region, the carbohydrate
attached thereto may be altered. For example, antibodies with a
mature carbohydrate structure that lacks fucose attached to an Fc
region of the antibody are described in US Patent Application No.
US 2003/0157108 A1, Presta, L. See also US 2004/0093621 A1 (Kyowa
Hakko Kogyo Co., Ltd). Antibodies with a bisecting
N-acetylglucosamine (GlcNAc) in the carbohydrate attached to an Fc
region of the antibody are referenced in WO03/011878, Jean-Mairet
et al. and U.S. Pat. No. 6,602,684, Umana et al. Antibodies with at
least one galactose residue in the oligosaccharide attached to an
Fc region of the antibody are reported in WO97/30087, Patel et al.
See, also, WO98/58964 (Raju, S.) and WO99/22764 (Raju, S.)
concerning antibodies with altered carbohydrate attached to the Fc
region thereof.
[0238] 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 cyotoxicity (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 anti-tumor 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).
[0239] WO00/42072 (Presta, L.) describes antibodies with improved
ADCC function in the presence of human effector cells, where the
antibodies comprise amino acid substitutions in the Fc region
thereof. Preferably, the antibody with improved ADCC comprises
substitutions at positions 298, 333, and/or 334 of the Fc region
(Eu numbering of residues). Preferably the altered Fc region is a
human IgG1 Fc region comprising or consisting of substitutions at
one, two or three of these positions. Such substitutions are
optionally combined with substitution(s) which increase Clq binding
and/or CDC.
[0240] Antibodies with altered Clq binding and/or complement
dependent cytotoxicity (CDC) are described in WO99/51642, U.S. Pat.
No. 6,194,551B1, U.S. Pat. No. 6,242,195B1, U.S. Pat. No.
6,528,624B1 and U.S. Pat. No. 6,538,124 (Idusogie et al.). The
antibodies comprise an amino acid substitution at one or more of
amino acid positions 270, 322, 326, 327, 329, 313, 333 and/or 334
of the Fc region thereof (Eu numbering of residues).
[0241] 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.
[0242] Antibodies with improved binding to the neonatal Fc receptor
(FcRn), and increased half-lives, are described in WO00/42072
(Presta, L.) and US2005/0014934A1 (Hinton et al.). These antibodies
comprise an Fc region with one or more substitutions therein which
improve binding of the Fc region to FcRn. For example, the Fc
region may have substitutions at one or more of positions 238, 250,
256, 265, 272, 286, 303, 305, 307, 311, 312, 314, 317, 340, 356,
360, 362, 376, 378, 380, 382, 413, 424, 428 or 434 (Eu numbering of
residues). The preferred Fc region-comprising antibody variant with
improved FcRn binding comprises amino acid substitutions at one,
two or three of positions 307, 380 and 434 of the Fc region thereof
(Eu numbering of residues).
[0243] Engineered antibodies with three or more (preferably four)
functional antigen binding sites are also contemplated (US Appin
No. US2002/0004587 A1, Miller et al.).
[0244] Nucleic acid molecules encoding amino acid sequence variants
of the 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 antibody.
[0245] (viii) Screening for Antibodies with the Desired
Properties
[0246] Techniques for generating antibodies have been described
above. One may further select antibodies with certain biological
characteristics, as desired.
[0247] To identify a HER2 antibody which binds to HER2 Domain IV
bound by trastuzumab (HERCEPTIN.RTM.), one can evaluate the ability
to bind to the isolated Domain IV peptide, Domain IV as present in
HER2 ECD; or as it exists in the intact HER2 receptor (where the
ECD or receptor can be isolated or present on the surface of a
cell), etc. Optionally, one may evaluate whether the HER2 antibody
of interest binds to the Trastuzumab or 4D5 epitope, or blocks or
competes with binding of Trastuzumab or 4D5 to HER2; such
antibodies would necessarily be considered to bind to HER2 Domain
IV bound by trastuzumab (HERCEPTIN.RTM.). To screen for antibodies
which bind to an epitope on HER2 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 to assess whether
the antibody blocks binding of an antibody, such as trastuzumab or
4D5 to HER2. See, also, Fendly et al., Cancer Research,
50:1550-1558 (1990), where cross-blocking studies were done on HER2
antibodies by direct fluorescence on intact HER2 positive cells.
HER2 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 the studies in
Fendly et al. 3H4 and 4D5 bound to the same epitope. Alternatively,
or additionally, epitope mapping can be performed by methods known
in the art and/or one can study the antibody-HER2 structure
(Franklin et al., Cancer Cell, 5:317-328 (2004)) to see what domain
or epitope of HER2 is/are bound by the antibody.
[0248] Trastuzumab has been shown in both in vitro assays and in
animals, to inhibit the proliferation of human tumor cells that
overexpress HER2. Hudziak et al., Mol. Cell. Biol., 9:1165-1172
(1989); U.S. Pat. No. 5,677,171; Lewis et al., Cancer Immunol.
Immunother, 37:255-263 (1993); Pietras et al., Oncogene, 1998;
17:2235-49 (1998); and Baselga et al., Cancer Res., 58:2825-2831
(1998). HERCEPTIN.RTM. has both cytostatic and cytotoxic effects on
HER2-positive tumor cell lines (Lewis et al., (1993)).
[0249] In order to select another growth inhibitory HER2 antibody
with this property, those in vitro or in vivo assays can be used to
screen HER2 antibodies for growth inhibition biological activity.
In particular, to identify growth inhibitory HER2 antibodies, one
may screen for antibodies which inhibit the growth of cancer cells
which overexpress HER2 in vitro. 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
HER2 antibody is added per dish. After six days, the number of
cells, compared to untreated cells are counted using an electronic
COULTER.RTM. 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. See U.S. Pat. No. 5,677,171 for
assays for screening for growth inhibitory antibodies, such as 4D5
and 3E8.
[0250] In order to select HER2 antibodies that inhibit growth of
HER2 positive tumors in vivo, xenograft studies, such as those in
Pietras et al. (1998) and Baselga et al. (1998), can be used to
screen HER2 antibodies for this property.
[0251] Trastuzumab is a mediator of antibody-dependent cellular
cytotoxicity (ADCC). Hotaling et al., Proc. Am. Assoc. Cancer Res.,
37:471 (1996), Abstract 3215; Pegram et al., Proc. Am. Assoc Cancer
Res, 38:602 (1997), Abstract 4044; U.S. Pat. Nos. 5,821,337,
6,054,297, 6,407,213, 6,639,055, 6,719,971, and 6,800,738; Carter
et al., PNAS (USA), 89:4285-4289 (1992); and Clynes et al., Nature
Medicine, 6:443-6 (2000). Other HER2 antibodies which mediate ADCC
can be identified using various assays, including those described
in these references.
[0252] Trastuzumab has also been reported to inhibit HER2
ectodomain cleavage (Molina et al., Cancer Res., 61:4744-4749
(2001)), and other HER2 antibodies with this function can be
identified using the methodology used by Molina et al., for
example.
[0253] HERCEPTIN.RTM. has also been reported to induce
normalization and regression of tumor vasculature in HER2 positive
human breast tumors by modulating the effects of angiogenic factors
(Izumi et al., Nature, 416:279-80 (2002)). Other HER2 antibodies
with this property can be identified using the experiments
described in Izumi et al.
[0254] (ix) HERCEPTIN.RTM. Compositions
[0255] The HERCEPTIN.RTM. composition generally comprises a mixture
of a main species antibody (comprising light and heavy chain
sequences of SEQ ID NOS: 5 and 6, respectively), and variant forms
thereof, in particular acidic variants (including deamidated
variants). Preferably, the amount of such acidic variants in the
composition is less than about 25%. See, U.S. Pat. No. 6,339,142.
See, also, Harris et al., J. Chromatography, B 752:233-245 (2001)
concerning forms of trastuzumab resolvable by cation-exchange
chromatography, including Peak A (Asn30 deamidated to Asp in both
light chains); Peak B (Asn55 deamidated to isoAsp in one heavy
chain); Peak 1 (Asn30 deamidated to Asp in one light chain); Peak 2
(Asn30 deamidated to Asp in one light chain, and Asp102 isomerized
to isoAsp in one heavy chain); Peak 3 (main peak form, or main
species antibody); Peak 4 (Asp102 isomerized to isoAsp in one heavy
chain); and Peak C (Asp102 succinimide (Asu) in one heavy chain).
Such variant forms and compositions are included in the invention
herein.
[0256] (x) Immunoconjugates
[0257] In another aspect, the invention provides immunoconjugates,
or antibody-drug conjugates (ADC), comprising an antibody
conjugated to a cytotoxic agent such as a chemotherapeutic agent, a
drug, a growth inhibitory agent, a toxin (e.g., an enzymatically
active toxin of bacterial, fungal, plant, or animal origin, or
fragments thereof), or a radioactive isotope (i.e., a
radioconjugate).
[0258] The use of antibody-drug conjugates for the local delivery
of cytotoxic or cytostatic agents, i.e., drugs to kill or inhibit
tumor cells in the treatment of cancer (Syrigos and Epenetos,
Anticancer Research, 19:605-614 (1999); Niculescu-Duvaz and
Springer Adv. Drug Del. Rev., 26:151-172 (1997); U.S. Pat. No.
4,975,278) allows targeted delivery of the drug moiety to tumors,
and intracellular accumulation therein, where systemic
administration of these unconjugated drug agents may result in
unacceptable levels of toxicity to normal cells as well as the
tumor cells sought to be eliminated. Maximal efficacy with minimal
toxicity is sought thereby. Both polyclonal antibodies and
monoclonal antibodies have been reported as useful in these
strategies (Rowland et al., Cancer Immunol. Immunother., 21:183-87
(1986)). Drugs used in these methods include daunomycin,
doxorubicin, methotrexate, and vindesine. Toxins used in
antibody-toxin conjugates include bacterial toxins such as
diphtheria toxin, plant toxins such as ricin, small molecule toxins
such as geldanamycin (Mandler et al., Jour. of the Nat. Cancer
Inst., 92(19):1573-1581 (2000); Mandler et al., Bioorganic &
Med. Chem. Letters, 10:1025-1028 (2000); Mandler et al.,
Bioconjugate Chem., 13:786-791 (2002)), maytansinoids (EP 1391213;
Liu et al., Proc. Natl. Acad. Sci. USA, 93:8618-8623 (1996)), and
calicheamicin (Lode et al., Cancer Res., 58:2928 (1998); Hinman et
al., Cancer Res., 53:3336-3342 (1993)). The toxins may affect their
cytotoxic and cytostatic effects by mechanisms including tubulin
binding, DNA binding, or topoisomerase inhibition. Some cytotoxic
drugs tend to be inactive or less active when conjugated to large
antibodies or protein receptor ligands.
[0259] Chemotherapeutic agents useful in the generation of
immunoconjugates are described above. Enzymatically active toxins
and fragments thereof that 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, e.g., WO 93/21232
published Oct. 28, 1993.
[0260] Conjugates of the antibody and cytotoxic agent are made
using a variety of bifunctional protein-coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 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.
[0261] Conjugates of an antibody and one or more small molecule
toxins, such as a calicheamicin, maytansinoids, dolastatins,
auristatins, a trichothecene, and CC1065, and the derivatives of
these toxins that have toxin activity, are also contemplated
herein.
[0262] In some embodiments, the immunoconjugate comprises an
antibody (full length or fragments) of the invention conjugated to
one or more maytansinoid molecules.
[0263] Maytansinoids are mitototic inhibitors which act by
inhibiting tubulin polymerization. Maytansine was first isolated
from the east African shrub Maytenus serrata (U.S. Pat. No.
3,896,111). Subsequently, it was discovered that certain microbes
also produce maytansinoids, such as maytansinol and C-3 maytansinol
esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and
derivatives and analogues thereof are disclosed, for example, in
U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608;
4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428;
4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650;
4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533.
[0264] Maytansinoid drug moieties are attractive drug moieties in
antibody drug conjugates because they are: (i) relatively
accessible to prepare by fermentation or chemical modification,
derivatization of fermentation products, (ii) amenable to
derivatization with functional groups suitable for conjugation
through the non-disulfide linkers to antibodies, (iii) stable in
plasma, and (iv) effective against a variety of tumor cell
lines.
[0265] Exemplary embodiments of maytansinoid drug moieities
include: DM1; DM3; and DM4, having the structures:
##STR00001##
[0266] wherein, the wavy line indicates the covalent attachment of
the sulfur atom of the drug to a linker (L) of an antibody drug
conjugate. HERCEPTIN.RTM. (trastuzumab) linked by SMCC to DM1 has
been reported (WO 2005/037992).
[0267] Other exemplary maytansinoid antibody drug conjugates have
the following structures and abbreviations, (wherein Ab is antibody
and p is 1 to about 8):
##STR00002##
[0268] Exemplary antibody drug conjugates where DMI is linked
through a BMPEO linker to a thiol group of the antibody have the
structure and abbreviation:
##STR00003##
[0269] where Ab is antibody; n is 0, 1, or 2; and p is 1, 2, 3, or
4.
[0270] Immunoconjugates containing maytansinoids, methods of making
same, and their therapeutic use are disclosed, for example, in U.S.
Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1,
the disclosures of which are hereby expressly incorporated by
reference. Liu et al., Proc. Natl. Acad. Sci. USA, 93:8618-8623
(1996) described immunoconjugates comprising a maytansinoid
designated DM1 linked to the monoclonal antibody C242 directed
against human colorectal cancer. The conjugate was found to be
highly cytotoxic towards cultured colon cancer cells, and showed
antitumor activity in an in vivo tumor growth assay. Chari et al.,
Cancer Research, 52:127-131 (1992) describe immunoconjugates in
which a maytansinoid was conjugated via a disulfide linker to the
murine antibody A7 binding to an antigen on human colon cancer cell
lines, or to another murine monoclonal antibody TA.1 that binds
HER2. Antibody-maytansinoid conjugates are prepared by chemically
linking an antibody to a maytansinoid molecule without
significantly diminishing the biological activity of either the
antibody or the maytansinoid molecule. See, e.g., U.S. Pat. No.
5,208,020. An average of 3-4 maytansinoid molecules conjugated per
antibody molecule has shown efficacy in enhancing cytotoxicity of
target cells without negatively affecting the function or
solubility of the antibody, although even one molecule of
toxin/antibody would be expected to enhance cytotoxicity over the
use of naked antibody. Maytansinoids are well known in the art and
can be synthesized by known techniques or isolated from natural
sources. Suitable maytansinoids are disclosed, for example, in U.S.
Pat. No. 5,208,020 and in the other patents and nonpatent
publications referred to hereinabove. Preferred maytansinoids are
maytansinol and maytansinol analogues modified in the aromatic ring
or at other positions of the maytansinol molecule, such as various
maytansinol esters.
[0271] There are many linking groups known in the art for making
antibody-maytansinoid conjugates, including, for example, those
disclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1,
Chari et al., Cancer Research, 52:127-131 (1992).
Antibody-maytansinoid conjugates comprising the linker component
SMCC may also be prepared. The linking groups include disulfide
groups, thioether groups, acid labile groups, photolabile groups,
peptidase labile groups, or esterase labile groups, as disclosed in
the above-identified patents, disulfide and thioether groups being
preferred. Additional linking groups are described and exemplified
herein.
[0272] Conjugates of the antibody and maytansinoid may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
Particularly, preferred coupling agents include
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlsson et
al., Biochem. J., 173:723-737 (1978)) and
N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for a
disulfide linkage.
[0273] The linker may be attached to the maytansinoid molecule at
various positions, depending on the type of the link. For example,
an ester linkage may be formed by reaction with a hydroxyl group
using conventional coupling techniques. The reaction may occur at
the C-3 position having a hydroxyl group, the C-14 position
modified with hydroxymethyl, the C-15 position modified with a
hydroxyl group, and the C-20 position having a hydroxyl group. In a
preferred embodiment, the linkage is formed at the C-3 position of
maytansinol or a maytansinol analogue.
[0274] In some embodiments, the immunoconjugate comprises an
antibody of the invention conjugated to dolastatins or dolostatin
peptidic analogs and derivatives, the auristatins (U.S. Pat. Nos.
5,635,483 and 5,780,588). Dolastatins and auristatins have been
shown to interfere with microtubule dynamics, GTP hydrolysis, and
nuclear and cellular division (Woyke et al., Antimicrob. Agents and
Chemother., 45(12):3580-3584 (2001)) and have anticancer (U.S. Pat.
No. 5,663,149) and antifungal activity (Pettit et al., Antimicrob.
Agents Chemother., 42:2961-2965 (1998)). The dolastatin or
auristatin drug moiety may be attached to the antibody through the
N (amino) terminus or the C (carboxyl) terminus of the peptidic
drug moiety (WO 02/088172).
[0275] Exemplary auristatin embodiments include the N-terminus
linked monomethylauristatin drug moieties DE and DF, disclosed in
"Senter et al., Proceedings of the American Association for Cancer
Research, Volume 45, Abstract Number 623, presented Mar. 28,
2004.
[0276] An exemplary auristatin embodiment is MMAE (wherein the wavy
line indicates the covalent attachment to a linker (L) of an
antibody drug conjugate).
##STR00004##
[0277] Another exemplary auristatin embodiment is MMAF (wherein the
wavy line indicates the covalent attachment to a linker (L) of an
antibody drug conjugate):
##STR00005##
[0278] Additional exemplary embodiments comprising MMAE or MMAF and
various linker components (described further herein) have the
following structures and abbreviations (wherein Ab means antibody
and p is 1 to about 8):
##STR00006##
[0279] Typically, peptide-based drug moieties can be prepared by
forming a peptide bond between two or more amino acids and/or
peptide fragments. Such peptide bonds can be prepared, for example,
according to the liquid phase synthesis method (see E. Schroder and
K. Lake, "The Peptides", volume 1, pp 76-136, Academic Press
(1965)) that is well known in the field of peptide chemistry. The
auristatin/dolastatin drug moieties may be prepared according to
the methods of: U.S. Pat. No. 5,635,483; U.S. Pat. No. 5,780,588;
Pettit et al., J. Am. Chem. Soc., 111:5463-5465 (1989); and Pettit
et al., Anti-Cancer Drug Design, 13:243-277 (1998).
[0280] In other embodiments, the immunoconjugate comprises an
antibody of the invention conjugated to one or more calicheamicin
molecules. The calicheamicin family of antibiotics are capable of
producing double-stranded DNA breaks at sub-picomolar
concentrations. For the preparation of conjugates of the
calicheamicin family, see U.S. Pat. Nos. 5,712,374, 5,714,586,
5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296
(all assigned to American Cyanamid Company). 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 (Hinman et
al., Cancer Research, 53:3336-3342 (1993), Lode et al., Cancer
Research, 58:2925-2928 (1998) and the aforementioned U.S. patents
to American Cyanamid). Another anti-tumor drug that the antibody
can be conjugated is QFA which is an antifolate. Both calicheamicin
and QFA have intracellular sites of action and do not readily cross
the plasma membrane. Therefore, cellular uptake of these agents
through antibody mediated internalization greatly enhances their
cytotoxic effects.
[0281] Other antitumor agents that can be conjugated to the
antibodies of the invention include BCNU, streptozoicin,
vincristine and 5-fluorouracil, the family of agents known
collectively LL-E33288 complex described in U.S. Pat. Nos.
5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No.
5,877,296).
[0282] 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.
[0283] 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).
[0284] For selective destruction of the tumor, the antibody may
comprise a highly radioactive atom. A variety of radioactive
isotopes are available for the production of radioconjugated
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,
Pb.sup.212 and radioactive isotopes of Lu. When the conjugate is
used for detection, it may comprise a radioactive atom for
scintigraphic studies, for example tc.sup.99m or I.sup.123, or a
spin label for nuclear magnetic resonance (NMR) imaging (also known
as magnetic resonance imaging, mri), such as iodine-123 again,
iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15,
oxygen-17, gadolinium, manganese or iron.
[0285] The radio- or other labels may be incorporated in the
conjugate in known ways. For example, the peptide may be
biosynthesized or may be synthesized by chemical amino acid
synthesis using suitable amino acid precursors involving, for
example, fluorine-19 in place of hydrogen. Labels such as
tc.sup.99m or I.sup.123, Re.sup.186, Re.sup.188 and In.sup.111 can
be attached via a cysteine residue in the peptide. Yttrium-90 can
be attached via a lysine residue. The method disclosed in Fraker et
al., Biochem. Biophys. Res. Commun., 80:49-57 (1978) can be used to
incorporate iodine-123.
[0286] Conjugates of the antibody and cytotoxic agent may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 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, photolabile linker, dimethyl linker or disulfide-containing
linker (Chari et al., Cancer Research, 52:127-131 (1992); U.S. Pat.
No. 5,208,020) may be used.
[0287] The compounds of the invention expressly contemplate, but
are not limited to, ADC prepared with cross-linker reagents: BMPS,
EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB,
SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB,
sulfo-SMCC, and sulfo-SMPB, and SVSB
(succinimidyl-(4-vinylsulfone)benzoate) which are commercially
available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill.,
U.S.A). See pages 467-498, 2003-2004 Applications Handbook and
Catalog.
[0288] In the antibody drug conjugates (ADC) of the invention, an
antibody (Ab) is conjugated to one or more drug moieties (D), e.g.,
about 1 to about 20 drug moieties per antibody, through a linker
(L). The ADC of Formula I may be prepared by several routes,
employing organic chemistry reactions, conditions, and reagents
known to those skilled in the art, including: (1) reaction of a
nucleophilic group of an antibody with a bivalent linker reagent,
to form Ab-L, via a covalent bond, followed by reaction with a drug
moiety D; and (2) reaction of a nucleophilic group of a drug moiety
with a bivalent linker reagent, to form D-L, via a covalent bond,
followed by reaction with the nucleophilic group of an antibody.
Additional methods for preparing ADC are described herein.
Ab-(L-D).sub.p 1
[0289] The linker may be composed of one or more linker components.
Exemplary linker components include 6-maleimidocaproyl ("MC"),
maleimidopropanoyl ("MP"), valine-citrulline ("val-cit"),
alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl
("PAB"), N-Succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"),
N-Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate
("SMCC"), and N-Succinimidyl (4-iodo-acetyl)aminobenzoate ("SIAB").
Additional linker components are known in the art and some are
described herein.
[0290] In some embodiments, the linker may comprise amino acid
residues. Exemplary amino acid linker components include a
dipeptide, a tripeptide, a tetrapeptide or a pentapeptide.
Exemplary dipeptides include: valine-citrulline (vc or val-cit),
alanine-phenylalanine (af or ala-phe). Exemplary tripeptides
include: glycine-valine-citrulline (gly-val-cit) and
glycine-glycine-glycine (gly-gly-gly). Amino acid residues which
comprise an amino acid linker component include those occurring
naturally, as well as minor amino acids and non-naturally occurring
amino acid analogs, such as citrulline. Amino acid linker
components can be designed and optimized in their selectivity for
enzymatic cleavage by a particular enzymes, for example, a
tumor-associated protease, cathepsin B, C and D, or a plasmin
protease.
[0291] Exemplary linker component structures are shown below
(wherein the wavy line indicates sites of covalent attachment to
other components of the ADC):
##STR00007##
[0292] Additional exemplary linker components and abbreviations
include (wherein the antibody (Ab) and linker are depicted, and p
is 1 to about 8):
##STR00008##
[0293] Nucleophilic groups on antibodies include, but are not
limited to: (i) N-terminal amine groups, (ii) side chain amine
groups, e.g., lysine, (iii) side chain thiol groups, e.g.,
cysteine, and (iv) sugar hydroxyl or amino groups where the
antibody is glycosylated. Amine, thiol, and hydroxyl groups are
nucleophilic and capable of reacting to form covalent bonds with
electrophilic groups on linker moieties and linker reagents
including: (i) active esters such as NHS esters, HOBt esters,
haloformates, and acid halides; (ii) alkyl and benzyl halides such
as haloacetamides; (iii) aldehydes, ketones, carboxyl, and
maleimide groups. Certain antibodies have reducible interchain
disulfides, i.e., cysteine bridges. Antibodies may be made reactive
for conjugation with linker reagents by treatment with a reducing
agent such as DTT (dithiothreitol). Each cysteine bridge will thus
form, theoretically, two reactive thiol nucleophiles. Additional
nucleophilic groups can be introduced into antibodies through the
reaction of lysines with 2-iminothiolane (Traut's reagent)
resulting in conversion of an amine into a thiol. Reactive thiol
groups may be introduced into the antibody (or fragment thereof) by
introducing one, two, three, four, or more cysteine residues (e.g.,
preparing mutant antibodies comprising one or more non-native
cysteine amino acid residues).
[0294] Antibody drug conjugates of the invention may also be
produced by modification of the antibody to introduce electrophilic
moieties, which can react with nucleophilic subsituents on the
linker reagent or drug. The sugars of glycosylated antibodies may
be oxidized, e.g., with periodate oxidizing reagents, to form
aldehyde or ketone groups which may react with the amine group of
linker reagents or drug moieties. The resulting imine Schiff base
groups may form a stable linkage, or may be reduced, e.g., by
borohydride reagents to form stable amine linkages. In one
embodiment, reaction of the carbohydrate portion of a glycosylated
antibody with either glactose oxidase or sodium meta-periodate may
yield carbonyl (aldehyde and ketone) groups in the protein that can
react with appropriate groups on the drug (Hermanson, Bioconjugate
Techniques). In another embodiment, proteins containing N-terminal
serine or threonine residues can react with sodium meta-periodate,
resulting in production of an aldehyde in place of the first amino
acid (Geoghegan & Stroh, Bioconjugate Chem. 3:138-146 (1992);
U.S. Pat. No. 5,362,852). Such aldehyde can be reacted with a drug
moiety or linker nucleophile.
[0295] Likewise, nucleophilic groups on a drug moiety include, but
are not limited to: amine, thiol, hydroxyl, hydrazide, oxime,
hydrazine, thiosemicarbazone, hydrazine carboxylate, and
arylhydrazide groups capable of reacting to form covalent bonds
with electrophilic groups on linker moieties and linker reagents
including: (i) active esters such as NHS esters, HOBt esters,
haloformates, and acid halides; (ii) alkyl and benzyl halides such
as haloacetamides; (iii) aldehydes, ketones, carboxyl, and
maleimide groups.
[0296] Alternatively, a fusion protein comprising the antibody and
cytotoxic agent may be made, e.g., by recombinant techniques or
peptide synthesis. The length of DNA may comprise respective
regions encoding the two portions of the conjugate either adjacent
one another or separated by a region encoding a linker peptide
which does not destroy the desired properties of the conjugate.
[0297] In yet another embodiment, the antibody may be conjugated to
a "receptor" (such streptavidin) for utilization in tumor
pre-targeting 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).
[0298] Other immunoconjugates are contemplated herein. For example,
the antibody or antibody fragment 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).
[0299] The 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.
[0300] 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).
III. Selecting Patients for Therapy
[0301] The patient herein is generally subjected to a diagnostic
test prior to therapy so as to identify HER2 positive subjects. For
example, the diagnostic test may evaluate HER2 expression
(including overexpression), amplification, and/or activation
(including phosphorylation or dimerization).
[0302] Generally, if a diagnostic test is performed, a sample may
be obtained from a patient in need of therapy. Where the subject
has cancer, the sample is generally a tumor sample. In the
preferred embodiment, the tumor sample is from a breast cancer
biopsy. The biological sample herein may be a fixed sample, e.g., a
formalin fixed, paraffin-embedded (FFPE) sample, or a frozen
sample.
[0303] To determine HER2 expression or amplification in the cancer,
various diagnostic/prognostic assays are available. In one
embodiment, HER2 overexpression may be analyzed by IHC, e.g., using
the HERCEPTEST.RTM. (Dako). Parrafin embedded tissue sections from
a tumor biopsy may be subjected to the IHC assay and accorded a
HER2 protein staining intensity criteria as follows:
Score 0 no staining is observed or membrane staining is observed in
less than 10% of tumor cells. Score 1+ a faint/barely perceptible
membrane staining is detected in more than 10% of the tumor cells.
The cells are only stained in part of their membrane. Score 2+ a
weak to moderate complete membrane staining is observed in more
than 10% of the tumor cells. Score 3+ a moderate to strong complete
membrane staining is observed in more than 10% of the tumor
cells.
[0304] Those tumors with 0 or 1+ scores for HER2 overexpression
assessment may be characterized as not overexpressing HER2, whereas
those tumors with 2+ or 3+ scores may be characterized as
overexpressing HER2.
[0305] Tumors overexpressing HER2 may be rated by
immunohistochemical scores corresponding to the number of copies of
HER2 molecules expressed per cell, and can been determined
biochemically: [0306] 0=0-10,000 copies/cell, [0307] 1+=at least
about 200,000 copies/cell, [0308] 2+=at least about 500,000
copies/cell, [0309] 3+=at least about 2,000,000 copies/cell.
[0310] Overexpression of HER2 at the 3+ level, which leads to
ligand-independent activation of the tyrosine kinase (Hudziak et
al., Proc. Natl. Acad. Sci. USA, 84:7159-7163 (1987)), occurs in
approximately 30% of breast cancers, and in these patients,
relapse-free survival and overall survival are diminished (Slamon
et al., Science, 244:707-712 (1989); Slamon et al., Science,
235:177-182 (1987)).
[0311] Alternatively, or additionally, FISH assays such as the
INFORM.TM. (sold by Ventana, Arizona) or PATHVISION.TM. (Vysis,
Illinois) may be carried out on formalin-fixed, paraffin-embedded
tumor tissue to determine the extent (if any) of HER2 amplification
in the tumor.
[0312] HER2 positivity may also be evaluated using an in vivo
diagnostic assay, e.g. by administering a molecule (such as an
antibody) which binds the molecule to be detected and is tagged
with a detectable label (e.g., a radioactive isotope) and
externally scanning the patient for localization of the label.
[0313] Other methods for identifying HER2 positive tumors are
contemplated herein, including but not limited to measuring shed
antigen, and detecting HER2 positive tumors indirectly, such as by
evaluating downstream signaling mediated through HER2 receptor,
gene expression profiling, etc.
[0314] Preferably, subjects are selected which have a HER2 positive
tumor or sample which overexpresses HER2 as evaluated by
immunohistochemistry (IHC) and/or has amplified HER2 gene as
evaluated by FISH.
IV. Pharmaceutical Formulations
[0315] Therapeutic formulations of the HER2 antibodies used in
accordance with the present invention are prepared for storage by
mixing an antibody having the desired degree of purity with
optional pharmaceutically acceptable carriers, excipients or
stabilizers (Remington's Pharmaceutical Sciences 16th edition,
Osol, A. Ed. (1980)), generally in the form of lyophilized
formulations or aqueous solutions. Antibody crystals are also
contemplated (see US Pat Appin 2002/0136719). 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 TWEENJ,
PLURONICSJ or polyethylene glycol (PEG).
[0316] Lyophilized antibody formulations are described in U.S. Pat.
Nos. 6,267,958, 6,685,940 and 6,821,515, expressly incorporated
herein by reference. The preferred HERCEPTIN.RTM. formulation is a
sterile, white to pale yellow preservative-free lyophilized powder
for intravenous (IV) administration, comprising 440 mg trastuzumab,
400 mg .alpha., .alpha.-trehalose dihyrate, 9.9 mg L-histidine-HCl,
6.4 mg L-histidine, and 1.8 mg polysorbate 20, USP. Reconsitution
of 20 mL of bacteriostatic water for injection (BWFI), containing
1.1% benzyl alcohol as a preservative, yields a multi-dose solution
containing 21 mg/mL trastuzumab, at pH of approximately 6.0.
[0317] The preferred pertuzumab formulation for therapeutic use
comprises 30 mg/mL pertuzumab in 20 mM histidine acetate, 120 mM
sucrose, 0.02% polysorbate 20, at pH 6.0. An alternate pertuzumab
formulation comprises 25 mg/mL pertuzumab, 10 mM histidine-HCl
buffer, 240 mM sucrose, 0.02% polysorbate 20, pH 6.0.
[0318] 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. Various drugs which can be combined
with the HER2 antibody are described in the Adjuvant Therapy
section below. Such molecules are suitably present in combination
in amounts that are effective for the purpose intended.
[0319] 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
16th edition, Osol, A. Ed. (1980).
[0320] 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.
[0321] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
V. Adjuvant Therapy
[0322] The present invention provides a method of adjuvant therapy
comprising administering to a human subject with nonmetastatic HER2
positive breast cancer, following definitive surgery, an antibody
which binds to HER2 Domain IV bound by trastuzumab
(HERCEPTIN.RTM.), in an amount effective to extend disease free
survival (DFS) or overall survival (OS), wherein the DFS or the OS
is evaluated about 2 to 5 years after an initial administration of
the antibody. Preferably the subject's DFS or OS is evaluated about
3-5 years, about 4-5 years, or at least about 4, or at least about
5 years after initiation of treatment or after initial diagnosis.
Preferably, the antibody is trastuzumab (HERCEPTIN.RTM.).
[0323] The subject treated herein is generally at high risk of
recurrence. Where the subject's tumor is HER2 positive, this is
known to be more aggressive, and linked to a higher likelihood of
recurrence. In addition, the subject may be at increased risk due
to younger age (for instance, where the subject is less than about
50 years old); may have had a large primary tumor (for example a
tumor greater than 2 centimeters in diameter); may be lymph
node-positive (for example, having 4 or more involved lymph nodes,
including 4-9 involved lymph nodes, and 10 or more involved lymph
nodes); may be estrogen receptor (ER) negative; and/or may be
progesterone receptor (PG) negative.
[0324] The HER2 antibody is administered to a human patient in
accord with known methods, such as intravenous administration,
e.g., as a bolus or by continuous infusion over a period of time,
by intramuscular, intraperitoneal, intracerobrospinal,
subcutaneous, intra-articular, intrasynovial, intrathecal, oral,
topical, or inhalation routes. Intravenous administration of the
antibody is preferred.
[0325] Preferred dosages for the HER2 antibody are in the range
from about 1 mg/kg to about 20 mg/kg, most preferably from about 2
mg/kg to about 12 mg/kg. Preferred dosage regimens for trastuzumab
include 4 mg/kg trastuzumab administered as a 90-minute infusion,
followed by a weekly maintenance dose of 2 mg/kg trastuzumab which
can be administed as a 30-minute infusion if the initial loading
dose is well tolerated. Other dosage regimens for trastuzumab are
however contemplated, including less than weekly dosing, for
example administration every 3 weeks, for example at a dose of 6
mg/kg, 8 mg/kg or 12 mg/kg; and including an initial dose of 8
mg/kg, followed by 6 mg/kg every three weeks (see, U.S. Pat. No.
6,627,196 B1, Baughman et al.; Leyland-Jones et al., J. Clin.
Oncol., 21:3965-71 (2003)). The number of doses of Trastuzumab
administered may be at least 20 or more, preferably at least 50,
for example 52 doses (where the antibody is administered every
week). Where less frequent dosing of Trastuzumab is used, such as
every 3 week dosing, fewer doses may be administered. Generally,
the subject will receive Trastuzumab for at least about 1 year, and
the subject's progress will be followed after that time.
[0326] While the HER2 antibody may be administered as single agent,
the patient is preferably treated with a combination of the HER2
antibody, and one or more chemotherapeutic agent(s). Preferably at
least one of the chemotherapeutic agents is a taxoid. The combined
administration includes coadministration or concurrent
administration, 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. Thus, the chemotherapeutic agent may be administered
prior to, or following, administration of the HER2 antibody. In
this embodiment, the timing between at least one administration of
the chemotherapeutic agent and at least one administration of the
HER2 antibody is preferably approximately 1 month or less, and most
preferably approximately 2 weeks or less. Alternatively, the
chemotherapeutic agent and the HER2 antibody are administered
concurrently to the patient, in a single formulation or separate
formulations. Treatment with the combination of the
chemotherapeutic agent (e.g., taxoid) and the HER2 antibody (e.g.,
trastuzumab) may result in a synergistic, or greater than additive,
therapeutic benefit to the patient.
[0327] The chemotherapeutic agent, if administered, is usually
administered at dosages known therefor, or optionally lowered due
to combined action of the drugs or negative side effects
attributable to administration of the antimetabolite
chemotherapeutic agent. Preparation and dosing schedules for such
chemotherapeutic agents may be used according to manufacturers'
instructions or as determined empirically by the skilled
practitioner. Where the chemotherapeutic agent is paclitaxel,
preferably, it is administered every week (e.g., at 80 mg/m.sup.2)
or every 3 weeks (for example at 175 mg/m.sup.2 or 135 mg/m.sup.2).
Suitable docetaxel dosages include 60 mg/m.sup.2, 70 mg/m.sup.2, 75
mg/m.sup.2, 100 mg/m.sup.2 (every 3 weeks); or 35 mg/m.sup.2 or 40
mg/m.sup.2 (every week).
[0328] Various chemotherapeutic agents that can be combined are
disclosed above. Preferred chemotherapeutic agents to be combined
with the HER2 antibody are selected from the group consisting of a
taxoid (including docetaxel and paclitaxel), vinca (such as
vinorelbine or vinblastine), platinum compound (such as carboplatin
or cisplatin), aromatase inhibitor (such as letrozole, anastrazole,
or exemestane), anti-estrogen (e.g., fulvestrant or tamoxifen),
etoposide, thiotepa, cyclophosphamide, methotrexate, liposomal
doxorubicin, pegylated liposomal doxorubicin, capecitabine,
gemcitabine, COX-2 inhibitor (for instance, celecoxib), or
proteosome inhibitor (e.g., PS342).
[0329] Most preferably, the HER2 antibody is combined with a
taxoid, such as paclitaxel or docetaxel, optionally in combination
with at least one other chemotherapeutic agent, such as a platinum
compound (for example carboplatin or cisplatin).
[0330] Where an anthracycline (e.g., doxorubicin or epirubicin) is
administered to the subject, preferably this is given prior to
and/or following administration of the HER2 antibody, such as in
the protocols disclosed in Example 1 below where an
anthracycline/cyclophosphomide combination was administered to the
subject following surgery, but prior to administration of the HER2
antibody and taxoid. However, a modified anthracycline, such as
liposomal doxorubicin (TLC D-99; (MYOCET.RTM.), pegylated liposomal
doxorubicin (CAELYX.RTM.), or epirubicin, with reduced cardiac
toxicity, may be combined with the HER2 antibody.
[0331] Administration of the antibody and chemotherapy can decrease
disease recurrence (cancer recurrence in the breast and/or distant
recurrence), in a population of subjects by about 50% at 3 years
(where "about 50%" herein, includes a range from about 45% to about
70%), for example decreases recurrence in the breast by about 52%
at 3 years, and/or decreases distant recurrence by about 53% at 3
years, compared to subjects treated with chemotherapy (e.g.,
taxoid, such as paclitaxel) alone.
[0332] The invention herein provides a method of curing
nonmetastatic breast cancer in a population of human subjects with
nonmetastatic HER2 positive breast cancer comprising administering
an effective amount of trastuzumab (HERCEPTIN.RTM.) and a taxoid to
the subjects following definitive surgery, and evaluating the
subjects after four (or more) years to confirm no disease
recurrence has occurred in at least about 80% (preferably at least
about 85%) of the subjects. The population may comprise 3000 or
more human subjects.
[0333] The invention further concerns a method of decreasing
disease recurrence in a population of human subjects with
nonmetastatic HER2 positive breast cancer comprising administering
an effective amount of trastuzumab (HERCEPTIN.RTM.) and a taxoid to
the subjects following definitive surgery, wherein disease
recurrence is decreased by at least about 50% at 3 years compared
to subjects treated with taxoid alone.
[0334] Aside from the HER2 antibody and the chemotherapeutic agent,
other therapeutic regimens may be combined therewith. For example,
a second (third, fourth, etc) chemotherapeutic agent(s) may be
administered, wherein the second chemotherapeutic agent is either
another, different taxoid chemotherapeutic agent, or a
chemotherapeutic agent that is not a taxoid. For example, the
second chemotherapeutic agent may be a taxoid (such as paclitaxel
or docetaxel), a vinca (such as vinorelbine), a platinum compound
(such as cisplatin or carboplatin), an anti-hormonal agent (such as
an aromatase inhibitor or antiestrogen), gemcitabine, capecitabine,
etc. Exemplary combinations include taxoid/platinum compound,
gemcitabine/taxoid, gemcitabine/vinorelbine, vinorelbine/taxoid,
capecitabine/taxoid, etc. "Cocktails" of different chemotherapeutic
agents may be administered. Exemplary chemotherapy cocktails
include: TAC (TAXOTERE.RTM., ADRIAMYCIN.RTM., cyclophosphamide);
CEF (cyclophosphamide administered orally, epirubicin, 5-FU); CMF
(cyclophosphamide, methotrexate, 5-FU); dose dense ACT
(ADRIAMYCIN.RTM. administered every 2 weeks with cytokine support,
G-CSF, cyclophosphamide, TAXOTERE.RTM.), AC (ADRIAMYCIN.RTM.,
cyclophosphamide); FEC (5-FU, epirubicin, cyclophosphamide, all
drugs administered intravenously); FAC (5-FU, ADRIAMYCIN.RTM.,
cyclophosphamide).
[0335] The preferred treatment regimen herein comprises
lumpectomy/mastectomy and axilliary dissection with pathologically
involved lymph nodes, followed by anthracycline+cyclosphosphomide
(AC), for example for 4 cycles, then administration of a taxoid
with HERCEPTIN.RTM. for about one year.
[0336] Other therapeutic agents that may be combined with the HER2
antibody include any one or more of: a second, different HER2
antibody (for example, a HER2 heterodimerization inhibitor such as
pertuzumab, or a HER2 antibody which induces apoptosis of a
HER2-overexpressing cell, such as 7C2, 7F3 or humanized variants
thereof); an antibody directed against a different tumor associated
antigen, such as EGFR, HER3, HER4; anti-hormonal compound or
endocrine therapeutic, e.g., an anti-estrogen compound such as
tamoxifen, or an aromatase inhibitor; a cardioprotectant (to
prevent or reduce any myocardial dysfunction associated with the
therapy); a cytokine; an EGFR inhibitor (such as TARCEVA.RTM.,
IRESSA.RTM. or cetuximab); an anti-angiogenic agent (especially
bevacizumab sold by Genentech under the trademark AVASTIN.RTM.); a
tyrosine kinase inhibitor; a COX inhibitor (for instance a COX-1 or
COX-2 inhibitor); non-steroidal anti-inflammatory drug, celecoxib
(CELEBREX.RTM.); farnesyl transferase inhibitor (for example,
Tipifarnib/ZARNESTRA.TM. R115777 available from Johnson and Johnson
or Lonafarnib SCH66336 available from Schering-Plough); HER2
vaccine (such as HER2 AutoVac vaccine from Pharmexia, or APC8024
protein vaccine from Dendreon, or HER2 peptide vaccine from
GSK/Corixa); another HER targeting therapy (e.g. trastuzumab,
cetuximab, ABX-EGF, EMD7200, gefitinib, erlotinib, CP724714,
CI1033, GW572016, IMC-11F8, TAM 65, etc.); Raf and/or ras inhibitor
(see, for example, WO 2003/86467); doxorubicin HCl liposome
injection (DOXIL.RTM.); topoisomerase I inhibitor such as
topotecan; taxoid; HER2 and EGFR dual tyrosine kinase inhibitor
such as lapatinib/GW572016; TLK286 (TELCYTA.RTM.); EMD-7200; AB1007
(Factor XII heavy chain antibody, B7C9); everolimis
(CERTICAN.RTM.); sirolimus (rapamycin, RAPAMUNE.RTM.); a body
temperature-reducing medicament such as acetaminophen,
diphenhydramine, or meperidine; hematopoietic growth factor,
etc.
[0337] 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 HER2 antibody.
[0338] In addition to the above therapeutic regimes, the patient
may be subjected to radiation therapy.
[0339] Preferably the administered HER2 antibody is an intact,
naked antibody. However, the HER2 antibody may be conjugated with a
cytotoxic agent. Preferably, the conjugated antibody and/or antigen
to which it is bound is/are internalized by the cell, resulting in
increased therapeutic efficacy of the conjugate in killing the
cancer cell to which it binds. In a preferred embodiment, the
cytotoxic agent targets or interferes with nucleic acid in the
cancer cell. Examples of such cytotoxic agents include
maytansinoids, calicheamicins, ribonucleases and DNA
endonucleases.
VI. Deposit of Materials
[0340] The following hybridoma cell lines have been deposited with
the American Type Culture Collection, 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
[0341] 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
[0342] This example concerns a joint interm analysis of results
obtained in human breast cancer subjects treated in National
Surgical Adjuvant Breast and Bowel Project (NSABP B-31) and the
North Central Cancer Treatment Group (NCCTG) Intergroup N9831
breast cancer clinical trials. The NCCTG study enrolled its first
patient in June 2000 and has enrolled 3,406 patients to date; the
NSABP study began enrollment in March 2000 and has enrolled 2,085
patients to date. The interim analysis of this example was based on
information from 3,300 patients. These trials evaluated the
efficacy of trastuzumab (HERCEPTIN.RTM.) as adjuvant therapy for
high risk operable breast cancer.
Study Design
[0343] The design of the NSABP B-31 and NCCTG N9831 studies is
depicted in FIG. 4A.
[0344] In the NSABP B-31 trial, subjects were treated with
anthracycline (60 mg/m.sup.2) plus cyclophosphamide (600
mg/m.sup.2), every 3 weeks, for four cycles (q 3 wk.times.4) then
received either: paclitaxel (TAXOL.RTM.) (175 mg/m.sup.2), every 3
weeks, for 4 cycles (q 3 wk.times.4) (Arm 1), or paclitaxel (175
mg/m.sup.2) every 3 weeks, for 4 cycles and trastuzumab (4 mg/kg/wk
loading dose (LD) for 4 weeks), followed by 2 mg/kg/wk maintenance
dose for 51 weeks (Arm 2).
[0345] In the NCCTG N9831 trial, which is an amended version of the
NSABP B-31 trial, the following treatment protocol was used:
[0346] Arm A: anthracycline (60 mg/m.sup.2) plus cyclophosphamide
(600 mg/m.sup.2), every 3 weeks, for four cycles (q 3 wk.times.4)
followed by paclitaxel (80 mg/m.sup.2/wk) for 12 weeks.
[0347] Arm B: anthracycline (60 mg/m.sup.2) plus cyclophosphamide
(600 mg/m.sup.2), every 3 weeks, for four cycles (q 3 wk.times.4),
followed by paclitaxel (80 mg/m.sup.2/wk) for 12 weeks, followed by
trastuzumab (4 mg/kg/wk loading dose (LD) for 4 weeks and 2
mg/kg/wk maintenance dose for 51 weeks).
[0348] Arm C: anthracycline (60 mg/m.sup.2) plus cyclophosphamide
(600 mg/m.sup.2), every 3 weeks, for four cycles (q 3 wk.times.4),
followed by paclitaxel (80 mg/m.sup.2/wk) for 12 weeks and
trastuzumab (4 mg/kg/wk loading dose (LD) for 4 weeks and 2
mg/kg/wk maintenance dose for 51 weeks).
[0349] The arms used in the joint analysis of the two study designs
are depicted in FIG. 4B.
[0350] HERCEPTIN.RTM. is a sterile, white to pale yellow
preservative-free lyophilized powder for intravenous (IV)
administration. The nominal content of each HERCEPTIN.RTM. vial is
440 mg trastuzumab, 400 mg .alpha., .alpha.-trehalose dihydrate,
9.9 mg L-histidine-HCl, 6.4 mg L-histidine, and 1.8 mg polysorbate
20, USP. Reconsitution of 20 mL of the supplied bacteriostatic
water for injection (BWFI), containing 1.1% benzyl alcohol as a
preservative, yields a multi-dose solution containing 21 mg/mL
trastuzumab, at pH of approximately 6.0. HERCEPTIN.RTM. was
administered as a loading dose of 4 mg/kg, following by maintenance
dose of 2 mg/kg every week.
[0351] To qualify for these trials, patients were required to have
invasive breast cancer, resected by either lumpectomy, or total
mastectomy, plus axillary dissection, with pathologically involved
axillary nodes. The protocol, as amended in N9831 allowed the
enlistment of high risk node negative patients. Patients were not
allowed to have locally advanced or distant disease, had normal
hematologic, hepatic, and renal function, received no prior
anthracycline or taxanes therapy, and had no significant
sensory/motor neuropathy. The patients were HER2 positive by FISH
or +++ by immunohistochemistry (IHC) verified centrally (N9831) or
by approved reference lab (B-31).
[0352] The patient and tumor characteristics for the subjects in
these studies are shown in FIG. 5. The joint analysis population
represents a very high risk group for recurrence and death compared
to more typical subjects included in adjuvant clinical trials. In
particular, the subjects: are younger (median age=48); have larger
tumors (60% greater than 2 cm); have more involved lymph nodes
(14-15% had more than 10 involved lymph nodes); and all were HER2
positive (HER2+). Outcomes of the treated population were expected
to be very poor with currently available chemotherapy.
Results
[0353] The primary endpoint of these trials was disease free
survival (DFS), analyzed according to the intent-to-treat
principle, ie, patients were evaluated on the basis of their
assigned therapy. Secondary endpoints were overall survival (OS)
and Time to 1st Distant Recurrence. Definite analysis was scheduled
after 710 DFS events. The first interm analysis was scheduled after
355 DFS events, then every 6 months thereafter; a total of 395
events on both trials are reported herein. The trials were to be
stopped only if equivalence was rejected at p=0.0005
(2p=0.001).
[0354] The DFS for the combined B31 and N9831 study results is
shown in FIG. 6.
[0355] FIG. 7 presents the DFS data for various patient groups,
classified based upon age, hormone receptor status, tumor size, and
number of positive nodes, relative to all data (from both studies),
and expressed as a hazard ratio. The individual results for the two
studies (N9831 and B-31, respectively) are shown at the bottom of
the plot.
[0356] FIG. 8 shows the DFS results for the N9831 and B-31 trials
individually.
[0357] Time to First Distant Recurrence for the combined results of
N9831 and B-31 is shown in FIG. 9.
[0358] FIG. 10 depicts the Hazard of Distant Recurrence for
randomized trials B31/N9831, for patients treated with
anthracycline and cyclophosphamide (AC), followed by paclitaxel (T)
compared with patients treated with anthracycline and
cyclophosphamide (AC), followed by paclitaxel and trastuzumab (TH)
treatment. The Figure illustrates the dramatic decrease in the
Hazard of Distant Recurrence in the group receiving TH
treatment.
[0359] Similarly, the survival data set forth in FIG. 11 show that
the survial of patients in the TH-treated group significantly
exceeded the survival of patients in the T-treated group. At 3
years from randomization, the AC+TH group was 94% vs. 92% of the
AC+T group. The difference was even greater at 4 years: 91% in the
AC+TH group vs. 87% in the AC+T group.
[0360] Efficacy endpoint analyses for the two studies are
summarized in FIG. 12.
[0361] The cumulative incidence of cardiac events in the evaluable
cohort is depicted and summarized in FIG. 13.
CONCLUSIONS
[0362] For node positive HER2 positive breast cancer, trastuzumab
given concurrently with paclitaxel following AC chemotherapy,
reduced the risk of a first breast cancer event at 3 years by
52%.
[0363] The relative risk reduction benefit was present and of
similar magnitude in all subsets of patients analyzed.
[0364] The addition of trastuzumab reduced the probability of
distant recurrence by 53% at 3 years, and the hazard of developing
distant metastases appeared to decrease over time.
[0365] Results at a median follow-up at 2 years show a
statistically significant survival advantage with a relative risk
reduction of 33%.
Sequence CWU 1
1
81195PRTHomo sapiens 1Thr Gln Val Cys Thr Gly Thr Asp Met Lys Leu
Arg Leu Pro Ala 1 5 10 15Ser Pro Glu Thr His Leu Asp Met Leu Arg
His Leu Tyr Gln Gly 20 25 30Cys Gln Val Val Gln Gly Asn Leu Glu Leu
Thr Tyr Leu Pro Thr 35 40 45Asn Ala Ser Leu Ser Phe Leu Gln Asp Ile
Gln Glu Val Gln Gly 50 55 60Tyr Val Leu Ile Ala His Asn Gln Val Arg
Gln Val Pro Leu Gln 65 70 75Arg Leu Arg Ile Val Arg Gly Thr Gln Leu
Phe Glu Asp Asn Tyr 80 85 90Ala Leu Ala Val Leu Asp Asn Gly Asp Pro
Leu Asn Asn Thr Thr 95 100 105Pro Val Thr Gly Ala Ser Pro Gly Gly
Leu Arg Glu Leu Gln Leu 110 115 120Arg Ser Leu Thr Glu Ile Leu Lys
Gly Gly Val Leu Ile Gln Arg 125 130 135Asn Pro Gln Leu Cys Tyr Gln
Asp Thr Ile Leu Trp Lys Asp Ile 140 145 150Phe His Lys Asn Asn Gln
Leu Ala Leu Thr Leu Ile Asp Thr Asn 155 160 165Arg Ser Arg Ala Cys
His Pro Cys Ser Pro Met Cys Lys Gly Ser 170 175 180Arg Cys Trp Gly
Glu Ser Ser Glu Asp Cys Gln Ser Leu Thr Arg 185 190 1952124PRTHomo
sapiens 2Thr Val Cys Ala Gly Gly Cys Ala Arg Cys Lys Gly Pro Leu
Pro 1 5 10 15Thr Asp Cys Cys His Glu Gln Cys Ala Ala Gly Cys Thr
Gly Pro 20 25 30Lys His Ser Asp Cys Leu Ala Cys Leu His Phe Asn His
Ser Gly 35 40 45Ile Cys Glu Leu His Cys Pro Ala Leu Val Thr Tyr Asn
Thr Asp 50 55 60Thr Phe Glu Ser Met Pro Asn Pro Glu Gly Arg Tyr Thr
Phe Gly 65 70 75Ala Ser Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu Ser
Thr Asp 80 85 90Val Gly Ser Cys Thr Leu Val Cys Pro Leu His Asn Gln
Glu Val 95 100 105Thr Ala Glu Asp Gly Thr Gln Arg Cys Glu Lys Cys
Ser Lys Pro 110 115 120Cys Ala Arg Val3169PRTHomo sapiens 3Cys Tyr
Gly Leu Gly Met Glu His Leu Arg Glu Val Arg Ala Val 1 5 10 15Thr
Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys Lys Ile Phe 20 25 30Gly
Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro Ala 35 40 45Ser
Asn Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe Glu 50 55 60Thr
Leu Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro 65 70 75Asp
Ser Leu Pro Asp Leu Ser Val Phe Gln Asn Leu Gln Val Ile 80 85 90Arg
Gly Arg Ile Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln 95 100
105Gly Leu Gly Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu Leu 110
115 120Gly Ser Gly Leu Ala Leu Ile His His Asn Thr His Leu Cys Phe
125 130 135Val His Thr Val Pro Trp Asp Gln Leu Phe Arg Asn Pro His
Gln 140 145 150Ala Leu Leu His Thr Ala Asn Arg Pro Glu Asp Glu Cys
Val Gly 155 160 165Glu Gly Leu Ala4142PRTHomo sapiens 4Cys His Gln
Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro 1 5 10 15Thr Gln
Cys Val Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys 20 25 30Val Glu
Glu Cys Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val 35 40 45Asn Ala
Arg His Cys Leu Pro Cys His Pro Glu Cys Gln Pro Gln 50 55 60Asn Gly
Ser Val Thr Cys Phe Gly Pro Glu Ala Asp Gln Cys Val 65 70 75Ala Cys
Ala His Tyr Lys Asp Pro Pro Phe Cys Val Ala Arg Cys 80 85 90Pro Ser
Gly Val Lys Pro Asp Leu Ser Tyr Met Pro Ile Trp Lys 95 100 105Phe
Pro Asp Glu Glu Gly Ala Cys Gln Pro Cys Pro Ile Asn Cys 110 115
120Thr His Ser Cys Val Asp Leu Asp Asp Lys Gly Cys Pro Ala Glu 125
130 135Gln Arg Ala Ser Pro Leu Thr 1405214PRTArtificial
sequencesequence is synthesized 5Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val 1 5 10 15Gly Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Asp Val Asn 20 25 30Thr Ala Val Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu Ile Tyr Ser Ala Ser
Phe Leu Tyr Ser Gly Val Pro Ser 50 55 60Arg Phe Ser Gly Ser Arg Ser
Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser Leu Gln Pro Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90His Tyr Thr Thr Pro Pro Thr
Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile Lys Arg Thr Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro 110 115 120Ser Asp Glu Gln Leu
Lys Ser Gly Thr Ala Ser Val Val Cys Leu 125 130 135Leu Asn Asn Phe
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val 140 145 150Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu 155 160 165Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr 170 175 180Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu 185 190
195Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn 200
205 210Arg Gly Glu Cys6449PRTArtificial sequencesequence is
synthesized 6Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly 1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Asn Ile Lys 20 25 30Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu 35 40 45Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr
Thr Arg Tyr 50 55 60Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala
Asp Thr Ser 65 70 75Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp 80 85 90Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp
Gly Phe Tyr 95 100 105Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 110 115 120Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser 125 130 135Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys 140 145 150Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala 155 160 165Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser 170 175 180Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser 185 190 195Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro Ser 200 205 210Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 215 220 225Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 230 235 240Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265
270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275
280 285Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
290 295 300Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp 305 310 315Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala 320 325 330Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln 335 340 345Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu 350 355 360Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe 365 370 375Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro 380 385 390Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly 395 400 405Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp 410 415 420Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu 425 430 435His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly 440 4457214PRTArtificial
sequencesequence is synthesized 7Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val 1 5 10 15Gly Asp Arg Val Thr Ile Thr
Cys Lys Ala Ser Gln Asp Val Ser 20 25 30Ile Gly Val Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu Ile Tyr Ser Ala Ser
Tyr Arg Tyr Thr Gly Val Pro Ser 50 55 60Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser Leu Gln Pro Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90Tyr Tyr Ile Tyr Pro Tyr Thr
Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile Lys Arg Thr Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro 110 115 120Ser Asp Glu Gln Leu
Lys Ser Gly Thr Ala Ser Val Val Cys Leu 125 130 135Leu Asn Asn Phe
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val 140 145 150Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu 155 160 165Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr 170 175 180Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu 185 190
195Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn 200
205 210Arg Gly Glu Cys8448PRTArtificial sequencesequence is
synthesized 8Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly 1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Thr 20 25 30Asp Tyr Thr Met Asp Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu 35 40 45Glu Trp Val Ala Asp Val Asn Pro Asn Ser Gly Gly
Ser Ile Tyr 50 55 60Asn Gln Arg Phe Lys Gly Arg Phe Thr Leu Ser Val
Asp Arg Ser 65 70 75Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp 80 85 90Thr Ala Val Tyr Tyr Cys Ala Arg Asn Leu Gly Pro
Ser Phe Tyr 95 100 105Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser Ala 110 115 120Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys 125 130 135Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp 140 145 150Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu 155 160 165Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly 170 175 180Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu 185 190 195Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn 200 205 210Thr Lys Val Asp
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 215 220 225His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 230 235 240Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265
270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275
280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
290 295 300Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp 305 310 315Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu 320 325 330Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro 335 340 345Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Glu Glu Met 350 355 360Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 365 370 375Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu 380 385 390Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser 395 400 405Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln 410 415 420Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His 425 430 435Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly 440 445
* * * * *