U.S. patent application number 12/484440 was filed with the patent office on 2009-12-24 for treatment of metastatic breast cancer.
Invention is credited to Anne Blackwood Chirchir, Pam Klein, Virginia Paton.
Application Number | 20090317387 12/484440 |
Document ID | / |
Family ID | 40436247 |
Filed Date | 2009-12-24 |
United States Patent
Application |
20090317387 |
Kind Code |
A1 |
Paton; Virginia ; et
al. |
December 24, 2009 |
TREATMENT OF METASTATIC BREAST CANCER
Abstract
The present invention concerns treatment of previously untreated
HER2-positive metastatic breast cancer with a combination of a
growth inhibitory HER2 antibody, a HER2 dimerization inhibitor
antibody and a taxane. In particular, the invention concerns the
treatment of HER2-positive metastatic breast cancer in patients who
did not receive prior chemotherapy or biologic therapy with a HER2
antibody binding essentially to epitope 2C4, a HER2 antibody
binding essentially to epitope 4D5, and a taxane. The invention
further comprises extending survival of such patients by the
combination therapy of the present invention. In a preferred
embodiment, the treatment involves administration of trastuzumab,
pertuzumab and docetaxel.
Inventors: |
Paton; Virginia; (Piedmont,
CA) ; Chirchir; Anne Blackwood; (Emerald Hills,
CA) ; Klein; Pam; (San Mateo, CA) |
Correspondence
Address: |
GOODWIN PROCTER LLP
135 COMMONWEALTH DRIVE
MENLO PARK
CA
94025
US
|
Family ID: |
40436247 |
Appl. No.: |
12/484440 |
Filed: |
June 15, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61061962 |
Jun 16, 2008 |
|
|
|
Current U.S.
Class: |
424/133.1 ;
424/138.1 |
Current CPC
Class: |
C07K 2317/73 20130101;
C07K 2317/24 20130101; A61P 35/00 20180101; A61K 39/3955 20130101;
C07K 16/3015 20130101; C07K 16/2863 20130101; A61K 31/337 20130101;
C07K 16/32 20130101; A61K 39/39558 20130101; A61P 35/04 20180101;
C07K 2317/76 20130101; C07K 2317/21 20130101; A61K 45/06 20130101;
A61K 2039/507 20130101; A61K 39/39558 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/133.1 ;
424/138.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Claims
1. A method for the treatment of breast cancer, comprising
administering to a HER2 positive metastatic breast cancer patient
an effective amount of a growth inhibitory HER2 antibody, a HER2
dimerization inhibitor antibody, and a taxane, wherein the patient
did not receive prior chemotherapy or biologic therapy.
2. The method of claim 1 wherein the growth inhibitory HER2
antibody binds to an epitope within Domain IV (SEQ ID NO: 17) of
the HER2 amino acid sequence.
3. The method of claim 2 wherein the growth inhibitory HER2
antibody binds essentially to epitope 4D5 of HER2.
4. The method of claim 1 wherein the HER2 dimerization inhibitor
antibody binds HER2 at the junction of domains I, II and III (SEQ
ID NOs: 14, 15, and 16).
5. The method of claim 4 wherein the HER2 dimerization inhibitor
antibody binds essentially to epitope 2C4.
6. The method of claim 1 wherein the growth inhibitory and/or the
HER2 dimerization inhibitor antibody is an antibody fragment.
7. The method of claim 1 wherein the growth inhibitory and/or the
HER2 dimerization inhibitor antibody is chimeric, humanized, or
human.
8. The method of claim 1 wherein the growth inhibitory antibody is
trastuzumab, or a fragrement thereof, the HER2 dimerization
antibody is pertuzumab, or a fragment thereof, and the taxane is
docetaxel.
9. A method for the treatment of breast cancer, comprising
administering to a HER2 positive metastatic breast cancer patient
an effective amount of a first HER2 antibody binding essentially to
epitope 2C4, a second HER2 antibody binding essentially to epitope
4D5, and a taxane, wherein the patient did not receive prior
chemotherapy or biologic therapy.
10. The method of claim 9 wherein the patient is a human
patient.
11. The method of claim 10 wherein said first and second antibodies
are monoclonal antibodies.
12. The method of claim 10 wherein at least one of said first and
said second antibody is an antibody fragment.
13. The method of claim 11 wherein at least one of said first and
said second antibody is chimeric humanized, or human.
14. The method of claim 10 wherein said first antibody is
pertuzumab.
15. The method of claim 10 or claim 14 wherein said second antibody
is trastuzumab.
16. The method of claim 14 wherein said taxane is docetaxel.
17. The method of claim 15 wherein said taxane is docetaxel.
18. The method of claim 10 wherein said first and second antibodies
and said taxane are administered concurrently.
19. The method of claim 10 wherein said first and second antibodies
and said taxane are administered consecutively, in any order.
20. The method of claim 10 wherein administration of the first
antibody precedes administration of the second antibody and the
taxane.
21. The method of claim 16 wherein at least one of the pertuzumab
and the transtuzumab is a naked antibody.
22. The method of claim 16 wherein at least one of the pertuzumab
and the transtuzumab is an intact antibody.
23. The method of claim 16 wherein administration of the
pertuzumab, trastuzumab and docetaxel results in a synergistic
effect.
24. The method of claim 16 wherein administration of the
pertuzumab, trastuzumab and docetaxel extends survival of the human
patient relative to treatment in the absence of at least one of
pertuzumab, trastuzumab and docetaxel.
25. The method of claim 24 wherein progression free survival (PFS)
is extended.
26. The method of claim 24 wherein overall survival (OS) is
extended.
27. The method of claim 10 further comprising the administration of
a further therapeutic agent selected from the group consisting of
chemotherapeutic agent, a different HER antibody, antibody directed
against a tumor associated antigen, anti-hormonal compound,
cardioprotectant, cytokine, EGFR-targeted drug, anti-angiogenic
agent, tyrosine kinase inhibitor, COX inhibitor, non-steroidal
anti-inflammatory drug, farnesyl transferase inhibitor, antibody
that binds oncofetal protein CA 125, HER2 vaccine, HER targeting
therapy, Raf or ras inhibitor, liposomal doxorubicin, topotecan,
taxane, dual tyrosine kinase inhibitor, TLK286, EMD-7200, a
medicament that treats nausea, a medicament that prevents or treats
skin rash or standard acne therapy, a medicament that treats or
prevents diarrhea, a body temperature-reducing medicament, and a
hematopoietic growth factor.
28. A kit comprising a first HER2 antibody binding essentially to
epitope 2C4, a second HER2 antibody binding essentially to epitope
4D5, and a taxane, and a package insert or label with directions to
treat a HER2 positive metastatic breast cancer patient, who did not
receive prior chemotherapy or biologic therapy.
29. A method of promoting pertuzumab for the treatment of a HER2
positive metastatic breast cancer patient who did not receive prior
chemotherapy or biologic therapy, in combination with trastuzumab
and a taxane.
30. The method of claim 29 wherein the taxane is docetaxel.
31. A method of promoting trastuzumab for the treatment of a HER2
positive metastatic breast cancer patient who did not receive prior
chemotherapy or biologic therapy, in combination with pertuzumab
and a taxane.
32. The method of claim 31 wherein the taxane is docetaxel.
33. A method for promoting a taxane for the treatment of a HER2
positive metastatic breast cancer patient who did not receive prior
chemotherapy or biologic therapy, in combination with pertuzumab
and trastuzumab
34. The method of claim 33 wherein the taxane is docetaxel.
35. The method of any one of claims 29-34, wherein the promotion is
in the form of a written material.
36. The method of any one of claims 29-34, wherein the promotion is
in the form of a package insert.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of U.S.
Provisional Patent Application Ser. No. 61/061,962, filed Jun. 16,
2008, all of which is fully incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention concerns treatment of previously
untreated HER2-positive metastatic breast cancer with a combination
of a growth inhibitory HER2 antibody, a HER2 dimerization inhibitor
antibody and a taxane. In particular, the invention concerns the
treatment of HER2-positive metastatic breast cancer in patients who
did not receive prior chemotherapy or biologic therapy with a HER2
antibody binding essentially to epitope 2C4, a HER2 antibody
binding essentially to epitope 4D5, and a taxane. The invention
further comprises extending survival of such patients by the
combination therapy of the present invention. In a preferred
embodiment, the treatment involves administration of trastuzumab,
pertuzumab and docetaxel.
BACKGROUND OF THE INVENTION
HER Receptors and Antibodies Thereagainst
[0003] Members of 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 erbB 1 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] Antibodies directed against the rat p185.sup.neu and human
HER2 protein products have been described.
[0007] Drebin and colleagues have raised antibodies against the rat
neu gene product, p185neu See, for example, Drebin et al., Cell
41:695-706 (1985); Myers et al., Meth. Enzym. 198:277-290 (1991);
and WO94/22478. Drebin et al. Oncogene 2:273-277 (1988) report that
mixtures of antibodies reactive with two distinct regions of
p185.sup.neu result in synergistic anti-tumor effects on
neu-transformed NIH-3T3 cells implanted into nude mice. See also
U.S. Pat. No. 5,824,311 issued Oct. 20, 1998.
[0008] Hudziak et al., Mol. Cell. Biol. 9(3): 1165-1172 (1989)
describe the generation of a panel of 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); D'souza et al. Proc. Natl. Acad. Sci. 91:7202-7206 (1994);
Lewis et al. Cancer Research 56:1457-1465 (1996); and Schaefer et
al. Oncogene 15:1385-1394 (1997).
[0009] A recombinant humanized version of the murine 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.
Oncol. 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. While the administration of
trastuzumab has led to excellent results in the treatment of breast
cancer, recent data from a clinical trial of lapirinib appear to
suggest that even with administration of trastuzumab, HER2 plays an
active role in tumor biology (Geyer et al., N Engl J Med 2006;
355:2733-2743).
[0010] 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).
[0011] 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 Pat Appln No 599,274; Plowman et al., Proc.
Natl. Acad. Sci. USA, 90:1746-1750 (1993); and Plowman et al.,
Nature, 366:473-475 (1993)). Both of these receptors display
increased expression on at least some breast cancer cell lines.
[0012] The 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 HER3 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.
[0013] 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)).
[0014] 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,
US2004/0037823A1, US2005/0002928A1, U.S. Pat. No. 6,573,043, U.S.
Pat. No. 6,905,830, US2003/0152987A1, WO99/48527, US2002/0141993A1,
US2005/0244417A1, U.S. Pat. No. 6,949,245, US2003/0086924,
US2004/0013667A1, WO00/69460, US2003/0170235A1, U.S. Pat. No.
7,041,292, WO01/00238, US2006/0083739, WO01/15730, U.S. Pat. No.
6,627,196B1, U.S. Pat. No. 6,632,979B1, WO01/00244,
US2002/0001587A1, US2002/0090662A1, U.S. Pat. No. 6,984,494B2,
WO01/89566, US2002/0064785, US2003/0134344, WO 2005/099756,
US2006/0013819, WO2006/07398A1, US2006/0018899, WO 2006/33700,
US2006/0088523, US 2006/0034840, WO 04/24866, US2004/0082047,
US2003/0175845A1, WO03/087131, US2003/0228663, WO2004/008099A2,
US2004/0106161, WO2004/048525, US2004/0258685A1, WO 2005/16968,
US2005/0038231A1 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.
[0015] Patients treated with the HER2 antibody trastuzumab are
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, U.S. Pat. No. 6,573,043, U.S. Pat. No. 6,905,830, and
US2003/0152987, Cohen et al., concerning immunohistochemistry (IHC)
and fluorescence in situ hybridization (FISH) for detecting HER2
overexpression and amplification.
[0016] 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 and WO04/065583, Cobleigh
et al., concern gene expression markers for breast cancer
prognosis. See, also, WO03/078662 (Baker et al.), and WO03/040404
(Bevilacqua et al.). WO02/44413 (Danenberg, K.) refers to
determining EGFR and HER2 gene expression for determining a
chemotherapeutic regimen.
[0017] 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: U.S. Pat. No. 6,949,245, WO01/00245,
US2005/0208043, US2005/0238640, US2006/0034842, and US2006/0073143
(Adams et al.); US2003/0086924 (Sliwkowski, M.); US2004/0013667A1
(Sliwkowski, M.); as well as WO2004/008099A2, and US2004/0106161
(Bossenmaier et al.).
[0018] 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 oligonucleotide microarray using isolated RNA
from tumor-tissue sections taken from archived primary
biopsies.
[0019] Pertuzumab (also known as recombinant human monoclonal
antibody 2C4; OMNITARG.TM., Genentech, Inc, South San Francisco)
represents the first in a new class of agents known as HER
dimerization inhibitors (HDI) and functions to inhibit the ability
of HER2 to form active heterodimers with other HER receptors (such
as EGFR/HER1, HER3 and HER4) and is active irrespective of HER2
expression levels. See, for example, Harari and Yarden Oncogene
19:6102-14 (2000); Yarden and Sliwkowski. Nat. Rev Mol Cell Biol
2:127-37 (2001); Sliwkowski Nat Struct Biol 10:158-9 (2003); Cho et
al. Nature 421:756-60 (2003); and Malik et al. Pro Am Soc Cancer
Res 44:176-7 (2003).
[0020] Pertuzumab blockade of the formation of HER2-HER3
heterodimers in tumor cells has been demonstrated to inhibit
critical cell signaling, which results in reduced tumor
proliferation and survival (Agus et al. Cancer Cell 2:127-37
(2002)).
[0021] Pertuzumab has undergone testing as a single agent in the
clinic with a phase Ia trial in patients with advanced cancers and
phase II trials in patients with ovarian cancer and breast cancer
as well as lung and prostate cancer. In a Phase I study, patients
with incurable, locally advanced, recurrent or metastatic solid
tumors that had progressed during or after standard therapy were
treated with pertuzumab given intravenously every 3 weeks.
Pertuzumab was generally well tolerated. Tumor regression was
achieved in 3 of 20 patients evaluable for response. Two patients
had confirmed partial responses. Stable disease lasting for more
than 2.5 months was observed in 6 of 21 patients (Agus et al. Pro
Am Soc Clin Oncol 22:192 (2003)). At doses of 2.0-15 mg/kg, the
pharmacokinetics of pertuzumab was linear, and mean clearance
ranged from 2.69 to 3.74 mL/day/kg and the mean terminal
elimination half-life ranged from 15.3 to 27.6 days. Antibodies to
pertuzumab were not detected (Allison et al. Pro Am Soc Clin Oncol
22:197 (2003)).
[0022] US 2006/0034842 describes methods for treating
ErbB-expressing cancer with anti-ErbB2 antibody combinations. WO
08/031,531 describes the use of trastuzumab and pertuzumab in the
treatment of HER2-positive metastatic cancer, such as breast
cancer. Baselga et al., J Clin Oncol, 2007 ASCO Annual Meeting
Proceedings Part I, Col. 25, No. 18S (June 20 Supplement),
2007:1004 report the treatment of patients with pre-treated HER2
positive breast cancer, which has progressed during treatment with
trastuzumab, with a combination of trastuzumab and pertuzumab.
Portera et al., J Clin Oncol, 2007 ASCO Annual Meeting Proceedings
Part I. Vol. 25, No. 18S (June 20 Supplement), 2007:1028 evaluated
the efficacy and safety of trastuzumab+pertuzumab combination
therapy in HER2-positive breast cancer patients, who had
progressive disease on trastuzumab-based therapy. The authors
concluded that further evaluation of the efficacy of combination
treatment was required to define the overall risk and benefit of
this treatment regimen.
[0023] Pertuzumab has been evaluated in Phase II studies in
combination with trastuzumab in patients with HER2-positive
metastatic breast cancer who have previously received trastuzumab
for metastatic disease. One study, conducted by the National cancer
Institute (NCI), enrolled 11 patients with previously treated
HER2-positive metastatic breast cancer. Two out of the 11 patients
exhibited a partial response (PR) (Baselga et al., J Clin Oncol
2007 ASCO Annual Meeting Proceedings; 25:18 S (June 20 Supplement):
1004.
[0024] Breast cancer is the most common cancer in women, with a
global prevalence of more than 1 million patients and a mortality
rate of approximately 400,000 deaths per year (International Agency
for Research on Cancer; http://www-dep.iarc.fr; Globocan 2002).
While improved early detection and advances in systemic therapy for
early stage disease have resulted in a decline in breast cancer
mortality since 1989, metastatic breast cancer (MBC) remains
largely incurable with a median survival of approximately 24
months. Factors associated with poor survival include age.gtoreq.50
years, visceral disease, shorter disease-free interval (DFI),
aneuploid tumors, tumors with a high S-phase fraction, p53
accumulation, low bcl-2 expression, negative hormone receptor
status, and positive human epidermal growth factor receptor 2
(HER2) status (Chang J, et al., Cancer 2003; 97:545-53).
[0025] Although chemotherapy agents, such as anthracyclines,
taxanes, alkylating agents, and/or vinca alkaloids, used as single
agents, have produced important results in extending the survival
of patients with metastatic breast cancer, the rare complete
responses are short-lived, and usually the disease continues to
progress. (Chung C, Carlson R. The Oncologist 2003; 8:514-20;
Bernard-Marty C, et al., The Oncologist 2003; 9:617-32).
[0026] The HER2-antibody trastuzumab is approved for use as
monotherapy or in combination with chemotherapy in the metastatic
setting, and in combination with chemotherapy as adjuvant treatment
for HER2-positive breast cancer. The optimal management of
metastatic breast cancer now takes into account not only a
patient's general condition, medical history, tumor burden, and
receptor status, but also the HER2 status.
[0027] A randomized Phase II study evaluated trastuzumab and
docetaxel vs. docetaxel alone as a first-line treatment for
HER2-positive metastatic breast cancer (Marty et al., J Clin Oncol
2005; 23:4265-4274).
[0028] Improvement in survival is an important goal in the
treatment of patients diagnosed with HER2-positive metastatic
breast cancer. Despite advances in cancer therapy, there is
significant medical need for new treatment regimens in order to
achieve this goal.
SUMMARY OF THE INVENTION
[0029] The present invention provides clinical data from human
breast cancer patients treated with a combination of trastuzumab,
pertuzumab and docetaxel.
[0030] In one aspect, the invention concerns a method for the
treatment of breast cancer, comprising administering to a HER2
positive metastatic breast cancer patient an effective amount of a
growth inhibitory HER2 antibody, a HER2 dimerization inhibitor
antibody, and a taxane, wherein the patient did not receive prior
chemotherapy or biologic therapy.
[0031] In one embodiment, the growth inhibitory HER2 antibody binds
to an epitope within Domain IV (SEQ ID NO: 17) of the HER2 amino
acid sequence.
[0032] In another embodiment, the growth inhibitory HER2 antibody
binds essentially to epitope 4D5 of HER2.
[0033] In yet another embodiment, the HER2 dimerization inhibitor
antibody binds HER2 at the junction of domains I, II and III (SEQ
ID NOs: 14, 15, and 16).
[0034] In a further embodiment, the HER2 dimerization inhibitor
antibody binds essentially to epitope 2C4.
[0035] In a still further embodiment, the growth inhibitory and/or
the HER2 dimerization inhibitor antibody is an antibody
fragment.
[0036] In an additional embodiment, the growth inhibitory and/or
the HER2 dimerization inhibitor antibody is chimeric, humanized, or
human.
[0037] In a particular embodiment, the growth inhibitory antibody
is trastuzumab, or a fragrement thereof, the HER2 dimerization
antibody is pertuzumab, or a fragment thereof, and the taxane is
docetaxel.
[0038] In another aspect, the invention concerns a method for the
treatment of breast cancer, comprising administering to a HER2
positive metastatic breast cancer patient an effective amount of a
first HER2 antibody binding essentially to epitope 2C4, a second
HER2 antibody binding essentially to epitope 4D5, and a taxane,
wherein the patient did not receive prior chemotherapy or biologic
therapy.
[0039] In one embodiment, the patient is a human patient.
[0040] In another embodiment, the first and second antibodies are
monoclonal antibodies.
[0041] In yet another embodiment, at least one of the first and the
second antibodies is an antibody fragment.
[0042] In a different embodiment, at least one of the first and the
second antibodies is chimeric humanized, or human.
[0043] In a particular embodiment, the first antibody is
pertuzumab.
[0044] In another particular embodiment, the second antibody is
trastuzumab.
[0045] In yet another particular embodiment, the taxane is
docetaxel.
[0046] In a further embodiment, the first and second antibodies and
said taxane are administered concurrently.
[0047] In a still further embodiment, the first and second
antibodies and the taxane are administered consecutively, in any
order.
[0048] In another embodiment, administration of the first antibody
precedes administration of the second antibody and the taxane.
[0049] In yet another embodiment, at least one of the pertuzumab
and the transtuzumab is a naked antibody.
[0050] In a different embodiment, at least one of the pertuzumab
and the transtuzumab is an intact antibody.
[0051] In a further embodiment, administration of the pertuzumab,
trastuzumab and docetaxel results in a synergistic effect.
[0052] In a still further embodiment, administration of the
pertuzumab, trastuzumab and docetaxel extends survival of the human
patient relative to treatment in the absence of at least one of
pertuzumab, trastuzumab and docetaxel. In a particular embodiment,
progression free survival (PFS) or overall survival (OS) is
extended.
[0053] Although the methods of the present invention may be
performed in the absence of any other means of cancer therapy, e.g.
in the absence of a further therapeutic agent, including
chemotherapeutic agents and biologics, the methods may optionally
comprise the administration of a further therapeutic agent selected
from the group consisting of chemotherapeutic agent, a different
HER antibody, antibody directed against a tumor associated antigen,
anti-hormonal compound, cardioprotectant, cytokine, EGFR-targeted
drug, anti-angiogenic agent, tyrosine kinase inhibitor, COX
inhibitor, non-steroidal anti-inflammatory drug, farnesyl
transferase inhibitor, antibody that binds oncofetal protein CA
125, HER2 vaccine, HER targeting therapy, Raf or ras inhibitor,
liposomal doxorubicin, topotecan, taxane, dual tyrosine kinase
inhibitor, TLK286, EMD-7200, a medicament that treats nausea, a
medicament that prevents or treats skin rash or standard acne
therapy, a medicament that treats or prevents diarrhea, a body
temperature-reducing medicament, and a hematopoietic growth
factor.
[0054] In another aspect, the invention concerns a kit comprising a
first HER2 antibody binding essentially to epitope 2C4, a second
HER2 antibody binding essentially to epitope 4D5, and a taxane, and
a package insert or label with directions to treat a HER2 positive
metastatic breast cancer patient, who did not receive prior
chemotherapy or biologic therapy.
[0055] In yet another aspect, the invention concerns a method of
promoting pertuzumab for the treatment of a HER2 positive
metastatic breast cancer patient who did not receive prior
chemotherapy or biologic therapy, in combination with trastuzumab
and a taxane. Just as before the taxane may, for example, be
docetaxel.
[0056] In a further aspect, the invention concerns a method of
promoting trastuzumab for the treatment of a HER2 positive
metastatic breast cancer patient who did not receive prior
chemotherapy or biologic therapy, in combination with pertuzumab
and a taxane, such as docetaxel.
[0057] In a still further aspect, the invention concerns a method
for promoting a taxane for the treatment of a HER2 positive
metastatic breast cancer patient who did not receive prior
chemotherapy or biologic therapy, in combination with pertuzumab
and trastuzumab, wherein the taxane may, for example, be docetaxel.
Without limitation, the promotion may be in the form of a written
material, or a package insert.
BRIEF DESCRIPTION OF THE DRAWINGS
[0058] FIG. 1 provides a schematic of the HER2 protein structure,
and amino acid sequences for Domains I-IV (SEQ ID Nos.14-17,
respectively) of the extracellular domain thereof.
[0059] FIGS. 2A and 2B depict alignments of the amino acid
sequences of the variable light (V.sub.L) (FIG. 2A) and variable
heavy (V.sub.H) (FIG. 2B) domains of murine monoclonal antibody 2C4
(SEQ ID Nos. 1 and 2, respectively); V.sub.L and V.sub.H domains of
variant 574/pertuzumab (SEQ ID Nos. 3 and 4, respectively), and
human V.sub.L and V.sub.H consensus frameworks (hum .kappa.1, light
kappa subgroup I; humIII, heavy subgroup III) (SEQ ID Nos. 5 and 6,
respectively). Asterisks identify differences between variable
domains of pertuzumab and murine monoclonal antibody 2C4 or between
variable domains of pertuzumab and the human framework.
Complementarity Determining Regions (CDRs) are in brackets.
[0060] FIGS. 3A and 3B show the amino acid sequences of pertuzumab
light chain (FIG. 3A; SEQ ID NO. 7) and heavy chain (FIG. 3B; 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.
[0061] FIGS. 4A and 4B show the amino acid sequences of trastuzumab
light chain (FIG. 4A; SEQ ID NO. 9) and heavy chain (FIG. 4B; SEQ
ID NO. 10), respectively.
[0062] FIGS. 5A and 5B depict a variant pertuzumab light chain
sequence (FIG. 5A; SEQ ID NO. 11) and a variant pertuzumab heavy
chain sequence (FIG. 5B; SEQ ID NO. 12), respectively.
[0063] FIG. 6. Serum Pertuzumab Concentrations (.mu.g/mL) for the
First 84 Days (through Study Day 85) for Studies TOC2689g and
BO16934.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions
[0064] The terms "biologic therapy" "immunotherapy" are used herein
interchangeably, and refer to cancer treatments utilizing the
body's immune system to fight cancer, regardless of their mechanism
of action. Biologic therapy specifically includes antibody
treatment.
[0065] Ther term "chemotherapy" as used herein refers to treatment
comprising the administration of a chemotherapeutic agent, as
defined hereinbelow.
[0066] "Survival" refers to the patient remaining alive, and
includes overall survival as well as progression free survival.
[0067] "Overall survival" or "OS" refers to the patient remaining
alive for a defined period of time, such as 1 year, 5 years, etc
from the time of diagnosis or treatment. For the purposes of the
clinical trial described in the examples, overall survival (OS) is
defined as the time from the date of randomization of patient
population to the date of death from any cause.
[0068] "Progression-free survival" or "PFS" refers to the patient
remaining alive, without the cancer progressing or getting worse.
For the purpose of the clinical trial described in the examples,
progression-free survival (PFS) is defined as the time from
randomization of study population to the first documented
progressive disease, or death from any cause, whichever occurs
first. Disease progression can be documented by any clinically
accepted methods, such as, for example, radiographical progressive
disease, as determined by Response Evaluation Criteria in Solid
Tumors (RECIST) (Therasse et al., J Natl Ca Inst 2000;
92(3):205-216), carcinomatous meningitis diagnosed by cytologic
evaluation of cerebral spinal fluid, and/or medical photography to
monitor chest wall recurrences of subcutaneous lesions.
[0069] By "extending survival" is meant increasing overall or
progression free survival in a patient treated in accordance with
the present invention relative to an untreated patient and/or
relative to a patient treated with one or more approved anti-tumor
agents, but not receiving treatment in accordance with the present
invention. In a particular example, "extending survival" means
extending progression-free survival (PFS) and/or overall survival
(OS) of breast cancer patients receiving the combination therapy of
the present invention (e.g. treatment with a combination of a HER2
antibody binding essentially to epitope 2C4, a HER2 antibody
binding essentially to epitope 4D5, and a taxane, e.g.
pertuzumab+trastuzumab+docetaxel) relative to patients treated with
a HER2 antibody binding essentially to epitope 4D5, and a taxane,
e.g. trastuzumab+docetaxel, in the absence of a HER2 antibody
binding essentially to epitope 2C4, i.e. pertuzumab.
[0070] Herein "time to disease progression" or "TTP" refer to the
time, generally measured in weeks or months, from the time of
initial treatment until the cancer progresses or worsens. Such
progression can be evaluated by the skilled clinician. Disease
progression can be evaluated and documented by any clinically
accepted methods, such as, for example, radiographical progressive
disease, as determined by Response Evaluation Criteria in Solid
Tumors (RECIST) (Therasse et al., J Natl Ca Inst 2000;
92(3):205-216), carcinomatous meningitis diagnosed by cytologic
evaluation of cerebral spinal fluid, and/or medical photography to
monitor chest wall recurrences of subcutaneous lesions.
[0071] By "extending TTP" is meant increasing the time to disease
progression in a patient treated in accordance with the present
invention relative to an untreated patient and/or relative to a
patient treated with one or more approved anti-tumor agents, but
not receiving treatment in accordance with the present invention.
In a particular example, "extending TTP" means extending time to
disease progression (TTP) of breast cancer patients receiving the
combination therapy of the present invention (treatment with a
combination of a HER2 antibody binding essentially to epitope 2C4,
a HER2 antibody binding essentially to epitope 4D5, and a taxane,
e.g. pertuzumab+trastuzumab+docetaxel) relative to patients treated
with a HER2 antibody binding essentially to epitope 4D5, and a
taxane, e.g. trastuzumab+docetaxel, in the absence of a HER2
antibody binding essentially to epitope 2C4, i.e. pertuzumab.
[0072] An "objective response" refers to a measurable response,
including complete response (CR) or partial response (PR).
[0073] By "complete response" or "CR" is intended the disappearance
of all signs of cancer in response to treatment. This does not
always mean the cancer has been cured.
[0074] "Partial response" or "PR" refers to a decrease in the size
of one or more tumors or lesions, or in the extent of cancer in the
body, in response to treatment.
[0075] 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
"native sequence" HER receptor or an "amino acid sequence variant"
thereof. Preferably the HER receptor is native sequence human HER
receptor.
[0076] The terms "ErbB 1," "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)). erbB 1 refers to the gene encoding
the EGFR protein product.
[0077] The expressions "ErbB2" and "HER2" are used interchangeably
herein and refer to human HER2 protein described, for example, in
Semba et al., PNAS (USA) 82:6497-6501 (1985) and Yamamoto et al.
Nature 319:230-234 (1986) (Genebank accession number X03363). The
term "erbB2" refers to the gene encoding human ErbB2 and "neu@
refers to the gene encoding rat p185.sup.neu. Preferred HER2 is
native sequence human HER2.
[0078] 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. The amino
acid sequence of HER2 is shown in FIG. 1. In one embodiment, the
extracellular domain of HER2 may comprise four domains: "Domain I"
(amino acid residues from about 1-195; SEQ ID NO:14), "Domain II"
(amino acid residues from about 196-319; SEQ ID NO:15), "Domain
III" (amino acid residues from about 320-488: SEQ ID NO:16), and
"Domain IV" (amino acid residues from about 489-630; SEQ ID NO: 17)
(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. 6
herein.
[0079] "ErbB3" and "HER3" refer to the receptor polypeptide as
disclosed, for example, in U.S. Pat. Nos. 5,183,884 and 5,480,968
as well as Kraus et al. PNAS (USA) 86:9193-9197 (1989).
[0080] The terms "ErbB4" and "HER4" herein refer to the receptor
polypeptide as disclosed, for example, in EP Pat Appln No 599,274;
Plowman et al., Proc. Natl. Acad. Sci. USA, 90:1746-1750 (1993);
and Plowman et al., Nature, 366:473-475 (1993), including isoforms
thereof, e.g., as disclosed in WO99/19488, published Apr. 22,
1999.
[0081] 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.
[0082] "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)).
[0083] A "HER dimer" 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.
[0084] 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.
[0085] A "HER antibody" is an antibody that binds to a HER
receptor. Optionally, the HER antibody further interferes with HER
activation or function. Preferably, the HER antibody binds to the
HER2 receptor. HER2 antibodies of interest herein are pertuzumab
and trastuzumab.
[0086] "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.
[0087] "Phosphorylation" refers to the addition of one or more
phosphate group(s) to a protein, such as a HER receptor, or
substrate thereof.
[0088] An antibody which "inhibits HER dimerization" is an antibody
which inhibits, or interferes with, formation of a HER dimer.
Preferably, such an antibody binds to HER2 at the heterodimeric
binding site thereof. The most preferred dimerization inhibiting
antibody herein is pertuzumab or MAb 2C4. Other examples of
antibodies which inhibit HER dimerization include antibodies which
bind to EGFR and inhibit dimerization thereof with one or more
other HER receptors (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 one or more
other HER receptors; and antibodies which bind to HER4 and inhibit
dimerization thereof with one or more other HER receptors.
[0089] A "HER2 dimerization inhibitor" is an agent that inhibits
formation of a dimer or heterodimer comprising HER2.
[0090] A "heterodimeric binding site" on HER2, refers to a region
in the extracellular domain of HER2 that contacts, or interfaces
with, a region in the extracellular domain of EGFR, HER3 or HER4
upon formation of a dimer therewith. The region is found in Domain
II of HER2 (SEQ ID NO: 15). Franklin et al. Cancer Cell 5:317-328
(2004).
[0091] A HER2 antibody that "binds to a heterodimeric binding site"
of HER2, binds to residues in Domain II (SEQ ID NO: 15) and
optionally also binds to residues in other of the domains of the
HER2 extracellular domain, such as domains I and III, SEQ ID NOs:
14 and 16), 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.
[0092] An antibody that "binds to domain II" of HER2 binds to
residues in domain II (SEQ ID NO: 15) and optionally residues in
other domain(s) of HER2, such as domains I and III (SEQ ID NOs: 14
and 16, respectively). Preferably the antibody that binds to domain
II binds to the junction between domains I, II and III of HER2.
[0093] Protein "expression" refers to conversion of the information
encoded in a gene into messenger RNA (mRNA) and then to the
protein.
[0094] Herein, a sample or cell that "expresses" a protein of
interest (such as a HER receptor or HER ligand) is one in which
mRNA encoding the protein, or the protein, including fragments
thereof, is determined to be present in the sample or cell.
[0095] The technique of "polymerase chain reaction" or "PCR" as
used herein generally refers to a procedure wherein minute amounts
of a specific piece of nucleic acid, RNA and/or DNA, are amplified
as described in U.S. Pat. No. 4,683,195 issued 28 Jul. 1987.
Generally, sequence information from the ends of the region of
interest or beyond needs to be available, such that oligonucleotide
primers can be designed; these primers will be identical or similar
in sequence to opposite strands of the template to be amplified.
The 5' terminal nucleotides of the two primers may coincide with
the ends of the amplified material. PCR can be used to amplify
specific RNA sequences, specific DNA sequences from total genomic
DNA, and cDNA transcribed from total cellular RNA, bacteriophage or
plasmid sequences, etc. See generally Mullis et al., Cold Spring
Harbor Symp. Quant. Biol., 51: 263 (1987); Erlich, ed., PCR
Technology, (Stockton Press, NY, 1989). As used herein, PCR is
considered to be one, but not the only, example of a nucleic acid
polymerase reaction method for amplifying a nucleic acid test
sample, comprising the use of a known nucleic acid (DNA or RNA) as
a primer and utilizes a nucleic acid polymerase to amplify or
generate a specific piece of nucleic acid or to amplify or generate
a specific piece of nucleic acid which is complementary to a
particular nucleic acid.
[0096] "Quantitative real time polymerase chain reaction" or
"qRT-PCR" refers to a form of PCR wherein the amount of PCR product
is measured at each step in a PCR reaction. This technique has been
described in various publications including Cronin et al., Am. J.
Pathol. 164(1):35-42 (2004); and Ma et al., Cancer Cell 5:607-616
(2004).
[0097] The term "microarray" refers to an ordered arrangement of
hybridizable array elements, preferably polynucleotide probes, on a
substrate.
[0098] The term "polynucleotide," when used in singular or plural,
generally refers to any polyribonucleotide or
polydeoxyribonucleotide, which may be unmodified RNA or DNA or
modified RNA or DNA. Thus, for instance, polynucleotides as defined
herein include, without limitation, single- and double-stranded
DNA, DNA including single- and double-stranded regions, single- and
double-stranded RNA, and RNA including single- and double-stranded
regions, hybrid molecules comprising DNA and RNA that may be
single-stranded or, more typically, double-stranded or include
single- and double-stranded regions. In addition, the term
"polynucleotide" as used herein refers to triple-stranded regions
comprising RNA or DNA or both RNA and DNA. The strands in such
regions may be from the same molecule or from different molecules.
The regions may include all of one or more of the molecules, but
more typically involve only a region of some of the molecules. One
of the molecules of a triple-helical region often is an
oligonucleotide. The term "polynucleotide" specifically includes
cDNAs. The term includes DNAs (including cDNAs) and RNAs that
contain one or more modified bases. Thus, DNAs or RNAs with
backbones modified for stability or for other reasons are
"polynucleotides" as that term is intended herein. Moreover, DNAs
or RNAs comprising unusual bases, such as inosine, or modified
bases, such as tritiated bases, are included within the term
"polynucleotides" as defined herein. In general, the term
"polynucleotide" embraces all chemically, enzymatically and/or
metabolically modified forms of unmodified polynucleotides, as well
as the chemical forms of DNA and RNA characteristic of viruses and
cells, including simple and complex cells.
[0099] The term "oligonucleotide" refers to a relatively short
polynucleotide, including, without limitation, single-stranded
deoxyribonucleotides, single- or double-stranded ribonucleotides,
RNA:DNA hybrids and double-stranded DNAs. Oligonucleotides, such as
single-stranded DNA probe oligonucleotides, are often synthesized
by chemical methods, for example using automated oligonucleotide
synthesizers that are commercially available. However,
oligonucleotides can be made by a variety of other methods,
including in vitro recombinant DNA-mediated techniques and by
expression of DNAs in cells and organisms.
[0100] The phrase "gene amplification" refers to a process by which
multiple copies of a gene or gene fragment are formed in a
particular cell or cell line. The duplicated region (a stretch of
amplified DNA) is often referred to as "amplicon." Usually, the
amount of the messenger RNA (mRNA) produced also increases in the
proportion of the number of copies made of the particular gene
expressed.
[0101] "Stringency" of hybridization reactions is readily
determinable by one of ordinary skill in the art, and generally is
an empirical calculation dependent upon probe length, washing
temperature, and salt concentration. In general, longer probes
require higher temperatures for proper annealing, while shorter
probes need lower temperatures. Hybridization generally depends on
the ability of denatured DNA to reanneal when complementary strands
are present in an environment below their melting temperature. The
higher the degree of desired homology between the probe and
hybridizable sequence, the higher the relative temperature which
can be used. As a result, it follows that higher relative
temperatures would tend to make the reaction conditions more
stringent, while lower temperatures less so. For additional details
and explanation of stringency of hybridization reactions, see
Ausubel et al., Current Protocols in Molecular Biology, Wiley
Interscience Publishers, (1995).
[0102] "Stringent conditions" or "high stringency conditions", as
defined herein, typically: (1) employ low ionic strength and high
temperature for washing, for example 0.015 M sodium chloride/0.0015
M sodium citrate/0.1% sodium dodecyl sulfate at 50.degree. C.; (2)
employ during hybridization a denaturing agent, such as formamide,
for example, 50% (v/v) formamide with 0.1% bovine serum
albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium
phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM
sodium citrate at 42.degree. C.; or (3) employ 50% formamide,
5.times.SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium
phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5.times.Denhardt's
solution, sonicated salmon sperm DNA (50 &gr; g/ml), 0.1% SDS,
and 10% dextran sulfate at 42.degree. C., with washes at 42.degree.
C. in 0.2.times.SSC (sodium chloride/sodium citrate) and 50%
formamide at 55.degree. C., followed by a high-stringency wash
consisting of 0.1.times.SSC containing EDTA at 55.degree. C.
[0103] "Moderately stringent conditions" may be identified as
described by Sambrook et al., Molecular Cloning: A Laboratory
Manual, New York: Cold Spring Harbor Press, 1989, and include the
use of washing solution and hybridization conditions (e.g.,
temperature, ionic strength and % SDS) less stringent that those
described above. An example of moderately stringent conditions is
overnight incubation at 37.degree. C. in a solution comprising: 20%
formamide, 5.times.SSC (150 mM NaCl, 15 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times.Denhardt's solution, 10%
dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA,
followed by washing the filters in 1.times.SSC at about
37-50.degree. C. The skilled artisan will recognize how to adjust
the temperature, ionic strength, etc. as necessary to accommodate
factors such as probe length and the like.
[0104] 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.
[0105] 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.
[0106] 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)).
[0107] 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.
[0108] "Humanized" forms of non-human (e.g., rodent) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity, and capacity. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
[0109] Humanized HER2 antibodies include huMAb4D5-1, huMAb4D5-2,
huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and
huMAb4D5-8 or trastuzumab (HERCEPTIN.RTM.) as described in Table 3
of U.S. Pat. No. 5,821,337 expressly incorporated herein by
reference; humanized 520C9 (WO93/21319); and humanized 2C4
antibodies such as pertuzumab as described herein.
[0110] 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. 9 and 10,
respectively.
[0111] Herein, "pertuzumab" and "OMNITARG.TM." refer to an antibody
comprising the light and heavy chain amino acid sequences in SEQ ID
NOs. 7 and 8, respectively.
[0112] An "intact antibody" herein is one which comprises two
antigen binding regions, and an Fc region. Preferably, the intact
antibody has a functional Fc region.
[0113] "Antibody fragments" comprise a portion of an intact
antibody, preferably comprising the antigen binding region thereof.
Examples of antibody fragments include Fab, Fab', F(ab').sub.2, and
Fv fragments; diabodies; linear antibodies; single-chain antibody
molecules; and multispecific antibodies formed from antibody
fragment(s).
[0114] "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.
[0115] 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).
[0116] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody which are responsible for
antigen-binding. The hypervariable region generally comprises amino
acid residues from a "complementarity determining region" or "CDR"
(e.g. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light
chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in
the heavy chain variable domain; Kabat et al., Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)) and/or those
residues from a "hypervariable loop" (e.g. residues 26-32 (L1),
50-52 (L2) and 91-96 (L3) in the light chain variable domain and
26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable
domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
"Framework Region" or "FR" residues are those variable domain
residues other than the hypervariable region residues as herein
defined.
[0117] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
yields an F(ab').sub.2 fragment that has two antigen-binding sites
and is still capable of cross-linking antigen.
[0118] "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.
[0119] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (CH1) of the heavy chain.
Fab=fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear at least one free thiol
group. F(ab').sub.2 antibody fragments originally were produced as
pairs of Fab' fragments which have hinge cysteines between them.
Other chemical couplings of antibody fragments are also known.
[0120] 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.
[0121] 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.
[0122] 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.
[0123] 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.
[0124] 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.
[0125] 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.
[0126] Depending on the amino acid sequence of the constant domain
of their heavy chains, intact antibodies can be assigned to
different "classes". There are five major classes of intact
antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may
be further divided into 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.
[0127] "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.
[0128] 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).
[0129] A "naked antibody" is an antibody that is not conjugated to
a heterologous molecule, such as a cytotoxic moiety or
radiolabel.
[0130] 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.
[0131] 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 VH and VL 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).
[0132] 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 to Domain II of HER2, antibody that inhibits
HER dimerization more effectively than trastuzumab, and/or an
antibody which binds to a heterodimeric binding site of HER2. The
preferred embodiment herein of the main species antibody is one
comprising the variable light and variable heavy amino acid
sequences of SEQ ID Nos. 3 and 4, and most preferably comprising
the light chain and heavy chain amino acid sequences in SEQ ID Nos.
7 and 8 (pertuzumab).
[0133] 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 an
amino-terminal leader extension (e.g. VHS-) on one or two light
chains thereof, 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. The antibody variant of particular interest herein is the
antibody comprising an amino-terminal leader extension on one or
two light chains thereof, optionally further comprising other amino
acid sequence and/or glycosylation differences relative to the main
species antibody.
[0134] 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.
[0135] 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). For
pertuzumab, G0 was the predominant oligosaccharide structure, with
other oligosaccharide structures such as G0-F, G-1, Man5, Man6,
G1-1, G1(1-6), G1(1-3) and G2 being found in lesser amounts in the
pertuzumab composition.
[0136] Unless indicated otherwise, a AG1 oligosaccharide structure@
herein includes G-1, G1-1, G1(1-6) and G1(1-3) structures.
[0137] An "amino-terminal leader extension" herein refers to one or
more amino acid residues of the amino-terminal leader sequence that
are present at the amino-terminus of any one or more heavy or light
chains of an antibody. An exemplary amino-terminal leader extension
comprises or consists of three amino acid residues, VHS, present on
one or both light chains of an antibody variant.
[0138] 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.
[0139] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. The cancer treated in accordance with
the present invention is any type of metastatic HER2 positive
breast cancer, including, without limitation, any histologically or
cytologically confirmed adenocaracinoma of the breast with locally
recurrent or metastatic disease (where the locally recurrent
disease is not amenable to resection with curative intent),
HER2-positive metastatic ductal carcinoma, HER2-positive metastatic
lobular carcinoma, specifically including both ER-positive and
ER-negative breast cancers, and may, but are not required to,
express other HER receptors, such as EGFR and/or HER3 and/or HER4
and/or one or more HER ligands.
[0140] An "advanced" cancer is one which has spread outside the
site or organ of origin, either by local invasion or
metastasis.
[0141] A "refractory" cancer is one which progresses even though an
anti-tumor agent, such as a chemotherapeutic agent, is being
administered to the cancer patient. An example of a refractory
cancer is one which is platinum refractory.
[0142] A "recurrent" cancer is one which has regrown, either at the
initial site or at a distant site, after a response to initial
therapy.
[0143] Herein, a "patient" is a human patient. The patient may be a
"cancer patient," i.e. one who is suffering or at risk for
suffering from one or more symptoms of cancer.
[0144] A "tumor sample" herein is a sample derived from, or
comprising tumor cells from, a patients tumor, including cancer, as
hereinabove defined. 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.
[0145] A "fixed" tumor sample is one which has been histologically
preserved using a fixative.
[0146] A "formalin-fixed" tumor sample is one which has been
preserved using formaldehyde as the fixative.
[0147] 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).
[0148] A "paraffin-embedded" tumor sample is one surrounded by a
purified mixture of solid hydrocarbons derived from petroleum.
[0149] Herein, a "frozen" tumor sample refers to a tumor sample
which is, or has been, frozen.
[0150] A cancer or biological sample which "displays HER
expression, amplification, or activation" is one which, in a
diagnostic test, expresses (including overexpresses) a HER
receptor, has amplified HER gene, and/or otherwise demonstrates
activation or phosphorylation of a HER receptor.
[0151] A cancer or biological sample which "displays HER
activation" is one which, in a diagnostic test, demonstrates
activation or phosphorylation of a HER receptor. Such activation
can be determined directly (e.g. by measuring HER phosphorylation
by ELISA) or indirectly (e.g. by gene expression profiling or by
detecting HER heterodimers, as described herein).
[0152] Herein, "gene expression profiling" refers to an evaluation
of expression of one or more genes as a surrogate for determining
HER phosphorylation directly.
[0153] 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.
[0154] A cancer cell with "HER receptor overexpression or
amplification" is one which has significantly higher levels of a
HER receptor protein or gene compared to a noncancerous cell of the
same tissue type. Such overexpression may be caused by gene
amplification or by increased transcription or translation. HER
receptor overexpression or amplification may be determined in a
diagnostic or prognostic assay by evaluating increased levels of
the HER protein present on the surface of a cell (e.g. via an
immunohistochemistry assay; IHC). Alternatively, or additionally,
one may measure levels of HER-encoding nucleic acid in the cell,
e.g. via fluorescent in situ hybridization (FISH; see WO98/45479
published October, 1998), southern blotting, or polymerase chain
reaction (PCR) techniques, such as quantitative real time PCR
(qRT-PCR). One may also study HER receptor overexpression or
amplification 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)). Aside from the above
assays, various in vivo assays are available to the skilled
practitioner. For example, one may expose cells within the body of
the patient to an antibody which is optionally labeled with a
detectable label, e.g. a radioactive isotope, and binding of the
antibody to cells in the patient can be evaluated, e.g. by external
scanning for radioactivity or by analyzing a biopsy taken from a
patient previously exposed to the antibody.
[0155] Conversely, a cancer which "does not overexpress or amplify
HER receptor" is one which does not have higher than normal levels
of HER receptor protein or gene compared to a noncancerous cell of
the same tissue type. Antibodies that inhibit HER dimerization,
such as pertuzumab, may be used to treat cancer which does not
overexpress or amplify HER2 receptor.
[0156] Herein, an "anti-tumor agent" refers to a drug used to treat
cancer. Non-limiting examples of anti-tumor agents herein include
chemotherapeutic agents, HER dimerization inhibitors, HER
antibodies, antibodies directed against tumor associated antigens,
anti-hormonal compounds, cytokines, EGFR-targeted drugs,
anti-angiogenic agents, tyrosine kinase inhibitors, growth
inhibitory agents and antibodies, cytotoxic agents, antibodies that
induce apoptosis, COX inhibitors, farnesyl transferase inhibitors,
antibodies that binds oncofetal protein CA 125, HER2 vaccines, Raf
or ras inhibitors, liposomal doxorubicin, topotecan, taxane, dual
tyrosine kinase inhibitors, TLK286, EMD-7200, pertuzumab,
trastuzumab, erlotinib, and bevacizumab.
[0157] An "approved anti-tumor agent" is a drug used to treat
cancer which has been accorded marketing approval by a regulatory
authority such as the Food and Drug Administration (FDA) or foreign
equivalent thereof.
[0158] Where a HER dimerization inhibitor is administered as a
"single anti-tumor agent" it is the only anti-tumor agent
administered to treat the cancer, i.e. it is not administered in
combination with another anti-tumor agent, such as
chemotherapy.
[0159] 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), taxanes, and topo II
inhibitors such as doxorubicin, epirubicin, daunorubicin,
etoposide, and bleomycin. Those agents that arrest G1 also spill
over into S-phase arrest, for example, DNA alkylating agents such
as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin,
methotrexate, 5-fluorouracil, and ara-C. Further information can be
found in The Molecular Basis of Cancer, Mendelsohn and Israel,
eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and
antineoplastic drugs" by Murakami et al. (WB Saunders:
Philadelphia, 1995), especially p. 13.
[0160] Examples of "growth 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/mil, 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.
[0161] 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-MB-453,
MDA-MB-361 or SKOV3 cell. Various methods are available for
evaluating the cellular events associated with apoptosis. For
example, phosphatidyl serine (PS) translocation can be measured by
annexin binding; DNA fragmentation can be evaluated through DNA
laddering; and nuclear/chromatin condensation along with DNA
fragmentation can be evaluated by any increase in hypodiploid
cells. Preferably, the antibody which induces apoptosis is one
which results in about 2 to 50 fold, preferably about 5 to 50 fold,
and most preferably about 10 to 50 fold, induction of annexin
binding relative to untreated cell in an annexin binding assay
using BT474 cells (see below). Examples of HER2 antibodies that
induce apoptosis are 7C2 and 7F3.
[0162] The "epitope 2C4" is the region in the extracellular domain
of HER2 to which the antibody 2C4 binds. In order to screen for
antibodies which bind essentially to the 2C4 epitope, a routine
cross-blocking assay such as that described in Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and
David Lane (1988), can be performed. Preferably the antibody blocks
2C4's binding to HER2 by about 50% or more. Alternatively, epitope
mapping can be performed to assess whether the antibody binds
essentially to the 2C4 epitope of HER2. Epitope 2C4 comprises
residues from Domain II (SEQ ID NO: 15) in the extracellular domain
of HER2. 2C4 and pertuzumab binds to the extracellular domain of
HER2 at the junction of domains I, II and III (SEQ ID NOs: 14, 15,
and 16, respectively). Franklin et al. Cancer Cell 5:317-328
(2004).
[0163] The "epitope 4D5" is the region in the extracellular domain
of HER2 to which the antibody 4D5 (ATCC CRL 10463) and trastuzumab
bind. This epitope is close to the transmembrane domain of HER2,
and within Domain IV of HER2 (SEQ ID NO: 17). To screen for
antibodies which bind essentially to the 4D5 epitope, a routine
cross-blocking assay such as that described in Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and
David Lane (1988), can be performed. Alternatively, epitope mapping
can be performed to assess whether the antibody binds essentially
to the 4D5 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).
[0164] The "epitope 7C2/7F3" is the region at the N terminus,
within Domain I (SEQ ID NO: 14), of the extracellular domain of
HER2 to which the 7C2 and/or 7F3 antibodies (each deposited with
the ATCC, see below) bind. To screen for antibodies which bind
essentially to the 7C2/7F3 epitope, a routine cross-blocking assay
such as that described in Antibodies, A Laboratory Manual, Cold
Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be
performed. Alternatively, epitope mapping can be performed to
establish whether the antibody binds essentially to the 7C2/7F3
epitope on HER2 (e.g. any one or more of residues in the region
from about residue 22 to about residue 53 of the HER2ECD, residue
numbering including signal peptide).
[0165] "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.
[0166] The term "effective amount" refers to an amount of a drug
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 extend
progression free survival (e.g. as measured by Response Evaluation
Criteria for Solid Tumors, RECIST, or CA-125 changes), result in an
objective response (including a partial response, PR, or complete
respose, CR), increase overall survival time, and/or improve one or
more symptoms of cancer (e.g. as assessed by FOSI).
[0167] 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.
[0168] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include alkylating agents such as thiotepa and CYTOXAN.RTM.
cyclosphosphamide; 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; TLK 286 (TELCYTA.TM.); 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, chlomaphazine,
cholophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranimnustine; bisphosphonates, such as
clodronate; antibiotics such as the enediyne antibiotics (e.g.,
calicheamicin, especially calicheamicin gamma1I and calicheamicin
omegaI1 (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186
(1994)) and anthracyclines such as annamycin, AD 32, alcarubicin,
daunorubicin, dexrazoxane, DX-52-1, epirubicin, GPX-100,
idarubicin, KRN5500, menogaril, dynemicin, including dynemicin A,
an esperamicin, neocarzinostatin chromophore and related
chromoprotein enediyne antiobiotic chromophores, aclacinomysins,
actinomycin, authramycin, azaserine, bleomycins, cactinomycin,
carabicin, carminomycin, carzinophilin, chromomycinis,
dactinomycin, detorubicin, 6-diazo-5-oxo-L-norleucine,
ADRIAMYCIN.RTM. doxorubicin (including morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal
doxorubicin, and deoxydoxorubicin), esorubicin, marcellomycin,
mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,
olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,
rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,
zinostatin, and zorubicin; folic acid analogues such as denopterin,
pteropterin, and trimetrexate; purine analogs such as fludarabine,
6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs
such as ancitabine, azacitidine, 6-azauridine, carmofur,
cytarabine, dideoxyuridine, doxifluridine, enocitabine, and
floxuridine; androgens such as calusterone, dromostanolone
propionate, epitiostanol, mepitiostane, and testolactone;
anti-adrenals such as aminoglutethimide, mitotane, and trilostane;
folic acid replenisher such as folinic acid (leucovorin);
aceglatone; anti-folate anti-neoplastic agents such as ALIMTA.RTM.,
LY231514 pemetrexed, dihydrofolate reductase inhibitors such as
methotrexate, anti-metabolites such as 5-fluorouracil (5-FU) and
its prodrugs such as UFT, S-1 and capecitabine, and thymidylate
synthase inhibitors and glycinamide ribonucleotide
formyltransferase inhibitors such as raltitrexed (TOMUDEX.TM.,
TDX); inhibitors of dihydropyrimidine dehydrogenase such as
eniluracil; aldophosphamide glycoside; aminolevulinic acid;
amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;
demecolcine; diaziquone; elformithine; elliptinium acetate; an
epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan;
lonidainine; maytansinoids such as maytansine and ansamitocins;
mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin;
phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide;
procarbazine; PSK7 polysaccharide complex (JHS Natural Products,
Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium;
tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine;
trichothecenes (especially T-2 toxin, verracurin A, roridin A and
anguidine); urethan; vindesine (ELDISINE.RTM., FILDESIN.RTM.);
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;
taxoids and taxanes, e.g., TAXOL.RTM. paclitaxel (Bristol-Myers
Squibb Oncology, Princeton, N.J.), ABRAXANE.TM. Cremophor-free,
albumin-engineered nanoparticle formulation of paclitaxel (American
Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE.RTM.
docetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;
gemcitabine (GEMZAR.RTM.); 6-thioguanine; mercaptopurine; platinum;
platinum analogs or platinum-based analogs such as cisplatin,
oxaliplatin and carboplatin; vinblastine (VELBAN.RTM.); etoposide
(VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN.RTM.);
vinca alkaloid; vinorelbine (NAVELBINE.RTM.); novantrone;
edatrexate; daunomycin; aminopterin; xeloda; ibandronate;
topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);
retinoids such as retinoic acid; 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
leucovorin.
[0169] Also included in this definition are anti-hormonal agents
that act to regulate or inhibit hormone action on tumors such as
anti-estrogens and selective estrogen receptor modulators (SERMs),
including, for example, tamoxifen (including NOLVADEX.RTM.
tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen,
trioxifene, keoxifene, LY117018, onapristone, and FARESTON.RTM.
toremifene; aromatase inhibitors that inhibit the enzyme aromatase,
which regulates estrogen production in the adrenal glands, such as,
for example, 4(5)-imidazoles, aminoglutethimide, MEGASE.RTM.
megestrol acetate, AROMASIN.RTM. exemestane, formestanie,
fadrozole, RIVISOR.RTM. vorozole, FEMARA.RTM. letrozole, and
ARIMIDEX.RTM. anastrozole; and anti-androgens such as flutamide,
nilutamide, bicalutamide, leuprolide, and goserelin; as well as
troxacitabine (a 1,3-dioxolane nucleoside cytosine analog);
antisense oligonucleotides, particularly those that inhibit
expression of genes in signaling pathways implicated in abherant
cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras,
and epidermal growth factor receptor (EGF-R); vaccines such as gene
therapy vaccines, for example, ALLOVECTIN.RTM. vaccine,
LEUVECTIN.RTM. vaccine, and VAXID.RTM. vaccine; PROLEUKIN.RTM.
rIL-2; LURTOTECAN.RTM. topoisomerase 1 inhibitor; ABARELIX.RTM.
rmRH; and pharmaceutically acceptable salts, acids or derivatives
of any of the above.
[0170] An "antimetabolite chemotherapeutic agent" is an agent which
is structurally similar to a metabolite, but can not be used by the
body in a productive manner. Many antimetabolite chemotherapeutic
agents interfere with the production of the nucleic acids, RNA and
DNA. Examples of antimetabolite chemotherapeutic agents include
gemcitabine (GEMZAR.RTM.), 5-fluorouracil (5-FU), capecitabine
(XELODA.TM.), 6-mercaptopurine, methotrexate, 6-thioguanine,
pemetrexed, raltitrexed, arabinosylcytosine ARA-C cytarabine
(CYTOSAR-U.RTM.), dacarbazine (DTIC-DOME.RTM.), azocytosine,
deoxycytosine, pyridmidene, fludarabine (FLUDARA.RTM.), cladrabine,
2-deoxy-D-glucose etc. The preferred antimetabolite
chemotherapeutic agent is gemcitabine.
[0171] "Gemcitabine" or A "2'-deoxy-2',2'-difluorocytidine
monohydrochloride (b-isomer)" is a nucleoside analogue that
exhibits antitumor activity. The empirical formula for gemcitabine
HCl is C9H11F2N3O4 A HCl. Gemcitabine HCl is sold by Eli Lilly
under the trademark GEMZAR.RTM..
[0172] A "platinum-based chemotherapeutic agent" comprises an
organic compound which contains platinum as an integral part of the
molecule. Examples of platinum-based chemotherapeutic agents
include carboplatin, cisplatin, and oxaliplatinum.
[0173] By "platinum-based chemotherapy" is intended therapy with
one or more platinum-based chemotherapeutic agents, optionally in
combination with one or more other chemotherapeutic agents.
[0174] By "chemotherapy-resistant" cancer is meant that the cancer
patient has progressed while receiving a chemotherapy regimen (i.e.
the patient is "chemotherapy refractory"), or the patient has
progressed within 12 months (for instance, within 6 months) after
completing a chemotherapy regimen.
[0175] By "platinum-resistant" cancer is meant that the cancer
patient has progressed while receiving platinum-based chemotherapy
(i.e. the patient is "platinum refractory"), or the patient has
progressed within 12 months (for instance, within 6 months) after
completing a platinum-based chemotherapy regimen.
[0176] An "anti-angiogenic agent" refers to a compound which
blocks, or interferes with to some degree, the development of blood
vessels. The anti-angiogenic factor may, for instance, be a small
molecule or antibody that binds to a growth factor or growth factor
receptor involved in promoting angiogenesis. The preferred
anti-angiogenic factor herein is an antibody that binds to vascular
endothelial growth factor (VEGF), such as bevacizumab
(AVASTIN.RTM.).
[0177] 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.
[0178] As used herein, the term "EGFR-targeted drug" refers to a
therapeutic agent that binds to EGFR and, optionally, inhibits EGFR
activation. Examples of such agents include antibodies and small
molecules that bind to EGFR. Examples of antibodies which bind to
EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507),
MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, U.S. Pat.
No. 4,943,533, Mendelsohn et al.) and variants thereof, such as
chimerized 225 (C225 or Cetuximab; ERBUTIX.RTM.) and reshaped human
225 (H225) (see, WO 96/40210, Imclone Systems Inc.); 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
(see WO98/50433, Abgenix); 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; 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).
Examples of small molecules that bind to EGFR include ZD1839 or
Gefitinib (IRESSA; Astra Zeneca); CP-358774 or Erlotinib
(TARCEVA.TM.; Genentech/OSI); and AG1478, AG1571 (SU 5271; Sugen);
EMD-7200.
[0179] 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 TAK 165 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; GW572016
(available from Glaxo) 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 (Gleevac.TM.) available from Glaxo; MAPK
extracellular regulated kinase I 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 Cyanimid); WO98/43960
(American Cyanamid); WO97/38983 (Warner Lambert); WO99/06378
(Warner Lambert); WO99/06396 (Warner Lambert); WO96/30347 (Pfizer,
Inc); WO96/33978 (Zeneca); WO96/3397 (Zeneca); and WO96/33980
(Zeneca).
[0180] A "fixed" or "flat" dose of a therapeutic agent herein
refers to a dose that is administered to a human patient without
regard for the weight (WT) or body surface area (BSA) of the
patient. The fixed or flat dose is therefore not provided as a
mg/kg dose or a mg/m.sup.2 dose, but rather as an absolute amount
of the therapeutic agent.
[0181] 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).
[0182] 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
[0183] The HER antigen to be used for production of antibodies may
be, e.g., a soluble form of the extracellular domain of a HER
receptor or a portion thereof, containing the desired epitope.
Alternatively, cells expressing HER 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 HER receptor useful for generating antibodies will be
apparent to those skilled in the art.
[0184] (i) Polyclonal Antibodies
[0185] 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.
[0186] 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.
[0187] (ii) Monoclonal Antibodies
[0188] 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).
[0189] 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)).
[0190] 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.
[0191] 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)).
[0192] 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).
[0193] 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).
[0194] 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.
[0195] 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.
[0196] 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).
[0197] 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.
[0198] 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.
[0199] 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.
[0200] (iii) Humanized Antibodies
[0201] 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.
[0202] 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)).
[0203] 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.
[0204] U.S. Pat. No. 6,949,245 describes production of exemplary
humanized HER2 antibodies which bind HER2 and block ligand
activation of a HER receptor. A humanized antibody used in the
methods of the present invention is rhuMAb 2C4 (pertuzumab), or an
antibody that binds essentially to the same epitope within the HER2
extracellular domain as pertuzumab. In other embodiments, one of
the antibodies used in the methods of the present invention blocks
EGF, TGF-.alpha. and/or HRG mediated activation of MAPK essentially
as effectively as murine monoclonal antibody 2C4 (or a Fab fragment
thereof) and/or binds HER2 essentially as effectively as murine
monoclonal antibody 2C4 (or a Fab fragment thereof). The humanized
antibody herein may, for example, comprise nonhuman hypervariable
region residues incorporated into a human variable heavy domain and
may further comprise a framework region (FR) substitution at a
position selected from the group consisting of 69H, 71H and 73H
utilizing the variable domain numbering system set forth in Kabat
et al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991). In one embodiment, the humanized antibody comprises FR
substitutions at two or all of positions 69H, 71H and 73H.
[0205] An exemplary humanized antibody of interest herein comprises
variable heavy domain complementarity determining residues
GFTFTDYTMX (SEQ ID NO: 18), where X is preferrably D or S;
DVNPNSGGSIYNQRFKG (SEQ ID NO: 19); and/or NLGPSFYFDY (SEQ ID
NO:20), optionally comprising amino acid modifications of those CDR
residues, e.g. where the modifications essentially maintain or
improve affinity of the antibody. For example, an antibody variant
for use in the methods of the present invention may have from about
one to about seven or about five amino acid substitutions in the
above variable heavy CDR sequences. Such antibody variants may be
prepared by affinity maturation, e.g., as described below.
[0206] The humanized antibody may comprise variable light domain
complementarity determining residues KASQDVSIGVA (SEQ ID NO:21);
SASYX.sup.1X.sup.2X.sup.3, where X.sup.1 is preferably R or L,
X.sup.2 is preferably Y or E, and X.sup.3 is preferably T or S (SEQ
ID NO:22); and/or QQYYIYPYT (SEQ ID NO:23), e.g. in addition to
those variable heavy domain CDR residues in the preceding
paragraph. Such humanized antibodies optionally comprise amino acid
modifications of the above CDR residues, e.g. where the
modifications essentially maintain or improve affinity of the
antibody. For example, the antibody variant of interest may have
from about one to about seven or about five amino acid
substitutions in the above variable light CDR sequences. Such
antibody variants may be prepared by affinity maturation, e.g., as
described below.
[0207] The present application also contemplates affinity matured
antibodies which bind HER2. The parent antibody may be a human
antibody or a humanized antibody, e.g., one comprising the variable
light and/or variable heavy sequences of SEQ ID Nos. 7 and 8,
respectively (i.e. comprising the VL and/or VH of pertuzumab). An
affinity matured variant of pertuzumab preferably binds to HER2
receptor with an affinity superior to that of murine 2C4 or
pertuzumab (e.g. from about two or about four fold, to about 100
fold or about 1000 fold improved affinity, e.g. as assessed using a
HER2-extracellular domain (ECD) ELISA). Exemplary variable heavy
CDR residues for substitution include H28, H30, H34, H35, H64, H96,
H99, or combinations of two or more (e.g. two, three, four, five,
six, or seven of these residues). Examples of variable light CDR
residues for alteration include L28, L50, L53, L56, L91, L92, L93,
L94, L96, L97 or combinations of two or more (e.g. two to three,
four, five or up to about ten of these residues).
[0208] 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 activity of
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 K 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.
[0209] 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.
[0210] (iv) Human Antibodies
[0211] As an alternative to humanization, human antibodies can be
generated. For example, it is now possible to produce transgenic
animals (e.g., mice) that are capable, upon immunization, of
producing a full repertoire of human antibodies in the absence of
endogenous immunoglobulin production. For example, it has been
described that the homozygous deletion of the antibody heavy-chain
joining region (J.sub.H) gene in chimeric and germ-line mutant mice
results in complete inhibition of endogenous antibody production.
Transfer of the human germ-line immunoglobulin gene array in such
germ-line mutant mice will result in the production of human
antibodies upon antigen challenge. See, e.g., Jakobovits et al.,
Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al.,
Nature, 362:255-258 (1993); 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.
[0212] 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).
[0213] 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.
[0214] (v) Antibody Fragments
[0215] 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 "linear antibody", e.g., as
described in U.S. Pat. No. 5,641,870 for example. Such linear
antibody fragments may be monospecific or bispecific.
[0216] (vi) Bispecific Antibodies
[0217] 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).
[0218] 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 IDM1 (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.
[0219] 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).
[0220] 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.
[0221] 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).
[0222] 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.
[0223] 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.
[0224] 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.
[0225] 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.
[0226] 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).
[0227] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al. J.
Immunol. 147: 60 (1991).
[0228] (vii) Other Amino Acid Sequence Modifications
[0229] 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.
[0230] 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.
[0231] 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.
[0232] 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
[0233] 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)):
[0234] (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P),
Phe (F), Trp (W), Met (M)
[0235] (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr
(Y), Asn (N), Gln (Q)
[0236] (3) acidic: Asp (D), Glu (E)
[0237] (4) basic: Lys (K), Arg (R), His (H)
[0238] Alternatively, naturally occurring residues may be divided
into groups based on common side-chain properties:
[0239] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
[0240] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0241] (3) acidic: Asp, Glu;
[0242] (4) basic: H is, Lys, Arg;
[0243] (5) residues that influence chain orientation: Gly, Pro;
[0244] (6) aromatic: Trp, Tyr, Phe.
[0245] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0246] 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).
[0247] 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.
[0248] 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.
[0249] 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.
[0250] 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).
[0251] 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 Pat Appl 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.
[0252] 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).
[0253] 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 C1q binding
and/or CDC.
[0254] Antibodies with altered C1q 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).
[0255] 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.
[0256] Antibodies with improved binding to the neonatal Fc receptor
(FcRn), and increased half-lives, are described in WO0/42072
(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).
[0257] Engineered antibodies with three or more (preferably four)
functional antigen binding sites are also contemplated (US Appln
No. US2002/0004587 A1, Miller et al.).
[0258] 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.
[0259] (viii) Screening for Antibodies with the Desired
Properties
[0260] Techniques for generating antibodies have been described
above. One may further select antibodies with certain biological
characteristics, as desired.
[0261] To identify an antibody which blocks ligand activation of a
HER receptor, the ability of the antibody to block HER ligand
binding to cells expressing the HER receptor (e.g. in conjugation
with another HER receptor with which the HER receptor of interest
forms a HER hetero-oligomer) may be determined. For example, cells
naturally expressing, or transfected to express, HER receptors of
the HER hetero-oligomer may be incubated with the antibody and then
exposed to labeled HER ligand. The ability of the antibody to block
ligand binding to the HER receptor in the HER hetero-oligomer may
then be evaluated.
[0262] For example, inhibition of HRG binding to MCF7 breast tumor
cell lines by HER2 antibodies may be performed using monolayer MCF7
cultures on ice in a 24-well-plate format essentially as described
in U.S. Pat. No. 6,949,245. HER2 monoclonal antibodies may be added
to each well and incubated for 30 minutes. 125I-labeled
rHRG.beta.1177-224 (25 pm) may then be added, and the incubation
may be continued for 4 to 16 hours. Dose response curves may be
prepared and an IC50 value may be calculated for the antibody of
interest. In one embodiment, the antibody which blocks ligand
activation of a HER receptor will have an IC.sub.50 for inhibiting
HRG binding to MCF7 cells in this assay of about 50 nM or less,
more preferably 10 nM or less. Where the antibody is an antibody
fragment such as a Fab fragment, the IC.sub.50 for inhibiting HRG
binding to MCF7 cells in this assay may, for example, be about 100
nM or less, more preferably 50 nM or less.
[0263] Alternatively, or additionally, the ability of an antibody
to block HER ligand-stimulated tyrosine phosphorylation of a HER
receptor present in a HER hetero-oligomer may be assessed. For
example, cells endogenously expressing the HER receptors or
transfected to expressed them may be incubated with the antibody
and then assayed for HER ligand-dependent tyrosine phosphorylation
activity using an anti-phosphotyrosine monoclonal (which is
optionally conjugated with a detectable label). The kinase receptor
activation assay described in U.S. Pat. No. 5,766,863 is also
available for determining HER receptor activation and blocking of
that activity by an antibody.
[0264] In one embodiment, one may screen for an antibody which
inhibits HRG stimulation of p180 tyrosine phosphorylation in MCF7
cells essentially as described in U.S. Pat. No. 6,949,245. For
example, the MCF7 cells may be plated in 24-well plates and
monoclonal antibodies to HER2 may be added to each well and
incubated for 30 minutes at room temperature; then
rHRGP.beta.1.sub.177-244 may be added to each well to a final
concentration of 0.2 nM, and the incubation may be continued for 8
minutes. Media may be aspirated from each well, and reactions may
be stopped by the addition of 100 .mu.l of SDS sample buffer (5%
SDS, 25 mM DTT, and 25 mM Tris-HCl, pH 6.8). Each sample (25 .mu.l)
may be electrophoresed on a 4-12% gradient gel (Novex) and then
electrophoretically transferred to polyvinylidene difluoride
membrane. Antiphosphotyrosine (at 1 .mu.g/mil) immunoblots may be
developed, and the intensity of the predominant reactive band at
M.sub.r-180,000 may be quantified by reflectance densitometry. The
antibody selected will preferably significantly inhibit HRG
stimulation of p180 tyrosine phosphorylation to about 0-35% of
control in this assay. A dose-response curve for inhibition of HRG
stimulation of p180 tyrosine phosphorylation as determined by
reflectance densitometry may be prepared and an IC.sub.50 for the
antibody of interest may be calculated. In one embodiment, the
antibody which blocks ligand activation of a HER receptor will have
an IC.sub.50 for inhibiting HRG stimulation of p180 tyrosine
phosphorylation in this assay of about 50 nM or less, more
preferably 10 nM or less. Where the antibody is an antibody
fragment such as a Fab fragment, the IC.sub.50 for inhibiting HRG
stimulation of p180 tyrosine phosphorylation in this assay may, for
example, be about 100 nM or less, more preferably 50 nM or
less.
[0265] One may also assess the growth inhibitory effects of the
antibody on MDA-MB-175 cells, e.g, essentially as described in
Schaefer et al. Oncogene 15:1385-1394 (1997). According to this
assay, MDA-MB-175 cells may be treated with a HER2 monoclonal
antibody (10 .mu.g/mL) for 4 days and stained with crystal violet.
Incubation with a HER2 antibody may show a growth inhibitory effect
on this cell line similar to that displayed by monoclonal antibody
2C4. In a further embodiment, exogenous HRG will not significantly
reverse this inhibition. Preferably, the antibody will be able to
inhibit cell proliferation of MDA-MB-175 cells to a greater extent
than monoclonal antibody 4D5 (and optionally to a greater extent
than monoclonal antibody 7F3), both in the presence and absence of
exogenous HRG.
[0266] To identify growth inhibitory HER2 antibodies, one may
screen for antibodies which inhibit the growth of cancer cells
which overexpress HER2. 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/mil. 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.TM. 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.
[0267] In order to select for antibodies which induce apoptosis, an
annexin binding assay using BT474 cells is available. The BT474
cells are cultured and seeded in dishes as discussed in the
preceding paragraph. The medium is then removed and replaced with
fresh medium alone or medium containing 10 .mu.g/ml of the
monoclonal antibody. Following a three day incubation period,
monolayers are washed with PBS and detached by trypsinization.
Cells are then centrifuged, resuspended in Ca.sup.2+ binding buffer
and aliquoted into tubes as discussed above for the cell death
assay. Tubes then receive labeled annexin (e.g. annexin V-FTIC) (1
.mu.g/ml). Samples may be analyzed using a FACSCAN.TM. flow
cytometer and FACSCONVERT.TM. CellQuest software (Becton
Dickinson). Those antibodies which induce statistically significant
levels of annexin binding relative to control are selected as
apoptosis-inducing antibodies. In addition to the annexin binding
assay, a DNA staining assay using BT474 cells is available. In
order to perform this assay, BT474 cells which have been treated
with the antibody of interest as described in the preceding two
paragraphs are incubated with 9 .mu.g/ml HOECHST 33342.TM. for 2 hr
at 37.degree. C., then analyzed on an EPICS ELITE.TM. flow
cytometer (Coulter Corporation) using MODFIT LT.TM. software
(Verity Software House). Antibodies which induce a change in the
percentage of apoptotic cells which is 2 fold or greater (and
preferably 3 fold or greater) than untreated cells (up to 100%
apoptotic cells) may be selected as pro-apoptotic antibodies using
this assay. See WO98/17797 for assays for screening for antibodies
which induce apoptosis, such as 7C2 and 7F3.
[0268] 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 cross-blocks binding of an
antibody, such as 2C4 or pertuzumab, to HER2. 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(s) of HER2
is/are bound by the antibody.
[0269] (ix) Pertuzumab Compositions
[0270] In one embodiment of a HER2 antibody composition, the
composition comprises a mixture of a main species pertuzumab
antibody and one or more variants thereof. The preferred embodiment
herein of a pertuzumab main species antibody is one comprising the
variable light and variable heavy amino acid sequences in SEQ ID
Nos. 3 and 4, and most preferably comprising a light chain amino
acid sequence of SEQ ID No. 7, and a heavy chain amino acid
sequence of SEQ ID No. 8 (including deamidated and/or oxidized
variants of those sequences). In one embodiment, the composition
comprises a mixture of the main species pertuzumab antibody and an
amino acid sequence variant thereof comprising an amino-terminal
leader extension. Preferably, the amino-terminal leader extension
is on a light chain of the antibody variant (e.g. on one or two
light chains of the antibody variant). The main species HER2
antibody or the antibody variant may be an full length antibody or
antibody fragment (e.g. Fab of F(ab=)2 fragments), but preferably
both are full length antibodies. The antibody variant herein may
comprise an amino-terminal leader extension on any one or more of
the heavy or light chains thereof. Preferably, the amino-terminal
leader extension is on one or two light chains of the antibody. The
amino-terminal leader extension preferably comprises or consists of
VHS-. Presence of the amino-terminal leader extension in the
composition can be detected by various analytical techniques
including, but not limited to, N-terminal sequence analysis, assay
for charge heterogeneity (for instance, cation exchange
chromatography or capillary zone electrophoresis), mass
spectrometry, etc. The amount of the antibody variant in the
composition generally ranges from an amount that constitutes the
detection limit of any assay (preferably N-terminal sequence
analysis) used to detect the variant to an amount less than the
amount of the main species antibody. Generally, about 20% or less
(e.g. from about 1% to about 15%, for instance from 5% to about
15%) of the antibody molecules in the composition comprise an
amino-terminal leader extension. Such percentage amounts are
preferably determined using quantitative N-terminal sequence
analysis or cation exchange analysis (preferably using a
high-resolution, weak cation-exchange column, such as a PROPAC
WCX-10.TM. cation exchange column). Aside from the amino-terminal
leader extension variant, further amino acid sequence alterations
of the main species antibody and/or variant are contemplated,
including but not limited to an antibody comprising a C-terminal
lysine residue on one or both heavy chains thereof, a deamidated
antibody variant, etc.
[0271] Moreover, the main species antibody or variant may further
comprise glycosylation variations, non-limiting examples of which
include antibody comprising a G1 or G2 oligosaccharide structure
attached to the Fc region thereof, antibody comprising a
carbohydrate moiety attached to a light chain thereof (e.g. one or
two carbohydrate moieties, such as glucose or galactose, attached
to one or two light chains of the antibody, for instance attached
to one or more lysine residues), antibody comprising one or two
non-glycosylated heavy chains, or antibody comprising a sialidated
oligosaccharide attached to one or two heavy chains thereof
etc.
[0272] The composition may be recovered from a genetically
engineered cell line, e.g. a Chinese Hamster Ovary (CHO) cell line
expressing the HER2 antibody, or may be prepared by peptide
synthesis.
[0273] (x) Trastuzumab Compositions
[0274] The trastuzumab composition generally comprises a mixture of
a main species antibody (comprising light and heavy chain sequences
of SEQ ID NOS: 9 and 10, 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.
[0275] (xi) Immunoconjugates
[0276] The invention also pertains to immunoconjugates comprising
an antibody conjugated to a cytotoxic agent such as a
chemotherapeutic agent, toxin (e.g. a small molecule toxin or an
enzymatically active toxin of bacterial, fungal, plant or animal
origin, including fragments and/or variants thereof), or a
radioactive isotope (i.e., a radioconjugate).
[0277] Chemotherapeutic agents useful in the generation of such
immunoconjugates have been described above. Conjugates of an
antibody and one or more small molecule toxins, such as a
calicheamicin, a maytansine (U.S. Pat. No. 5,208,020), a
trichothene, and CC 1065 are also contemplated herein.
[0278] In one preferred embodiment of the invention, the antibody
is conjugated to one or more maytansine molecules (e.g. about 1 to
about 10 maytansine molecules per antibody molecule). Maytansine
may, for example, be converted to May-SS-Me which may be reduced to
May-SH3 and reacted with modified antibody (Chari et al. Cancer
Research 52: 127-131 (1992)) to generate a maytansinoid-antibody
immunoconjugate.
[0279] Another immunoconjugate of interest comprises an antibody
conjugated to one or more calicheamicin molecules. The
calicheamicin family of antibiotics are capable of producing
double-stranded DNA breaks at sub-picomolar concentrations.
Structural analogues of calicheamicin which may be used include,
but are not limited to, .gamma..sub.1.sup.I, .alpha..sub.2.sup.I,
.alpha..sub.3.sup.I, N-acetyl-.gamma..sub.1.sup.I, PSAG and
.theta..sup.I.sub.1 (Hinman et al. Cancer Research 53: 3336-3342
(1993) and Lode et al. Cancer Research 58: 2925-2928 (1998)). See,
also, U.S. Pat. Nos. 5,714,586; 5,712,374; 5,264,586; and 5,773,001
expressly incorporated herein by reference.
[0280] 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.
[0281] 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).
[0282] A variety of radioactive isotopes are available for the
production of radioconjugated HER2 antibodies. Examples include
At.sup.211, I.sup.131, I.sup.125, Y.sup.90, Re.sup.186, R.sup.188,
Sm.sup.153, Bi.sup.212, P.sup.32 and radioactive isotopes of
Lu.
[0283] Conjugates of the antibody and cytotoxic agent may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al. Science 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026. The linker may be
a Acleavable linker@ facilitating release of the cytotoxic drug in
the cell. For example, an acid-labile linker, peptidase-sensitive
linker, dimethyl linker or disulfide-containing linker (Chari et
al. Cancer Research 52: 127-131 (1992)) may be used.
[0284] Alternatively, a fusion protein comprising the antibody and
cytotoxic agent may be made, e.g. by recombinant techniques or
peptide synthesis.
[0285] Other immunoconjugates are contemplated herein. For example,
the antibody may be linked to one of a variety of nonproteinaceous
polymers, e.g., polyethylene glycol, polypropylene glycol,
polyoxyalkylenes, or copolymers of polyethylene glycol and
polypropylene glycol. The antibody also may be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization (for example, hydroxymethylcellulose
or gelatin-microcapsules and poly(methylmethacylate) microcapsules,
respectively), in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules), or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A.,
Ed., (1980).
[0286] 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.
[0287] 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).
[0288] (xii) Docetaxel
[0289] Docetaxel is an anti-neoplastic agent that binds to free
tubulin and promotes the assembly of tubulin into stable
microtubules while simultaneously inhibiting their assembly. This
leads to the production of microtubule bundles without normal
function and to the stabilization of microtubules, blocking cells
in the M-phase of the cell cycle and leading to cell death.
III. Selecting Patients for Therapy
[0290] The present invention concerns the treatment of patients who
have HER2-positive metastatic breast cancer and have not received
prior chemotherapy or biologic therapy (including approved or
investigational tyrosine kinase/HER inhibitors or vaccines) for
their metastatic disease. Patient could have received one prior
hormonal treatment for metastatic breast cancer. Patients may have
received systemic breast cancer treatment in the neo-adjuvant or
adjuvant setting, provided that the patient has experienced a
disease-free interval (DFI) of .gtoreq.12 months from completion of
adjuvant systemic treatment (excluding hormonal therapy) to
metastatic diagnosis. Patients may have received trastuzumab and/or
a taxane during the neo-adjuvant or adjuvant treatment.
[0291] Detection of HER2 protein overexpression is important for
selection of patients for treatment in accordance with the present
invention. Several FDA-approved commercial assays are available to
identify breast cancer patients whose cancer overexpresses HER2.
These methods include HERCEPTEST.TM. (Dako) and PATHWAY.RTM.
HER-2/neu (immunohistochemistry (IHC) assays) and PathVysion.RTM.
and HER2FISH pharmDx.TM. (FISH assays). Users should refer to the
package inserts of specific assay kits for information on the
validation and performance of each assay.
[0292] For example, HER2 overexpression may be analyzed byIHC, e.g.
using the HERCEPTEST.RTM. (Dako). Paraffin embedded tissue sections
from a tumor biopsy may be subjected to the IHC assay and accorded
a HER2 protein staining intensity criteria as follows:
[0293] Score 0 no staining is observed or membrane staining is
observed in less than 10% of tumor cells.
[0294] 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.
[0295] Score 2+ a weak to moderate complete membrane staining is
observed in more than 10% of the tumor cells.
[0296] Score 3+ a moderate to strong complete membrane staining is
observed in more than 10% of the tumor cells.
[0297] 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.
[0298] 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:
[0299] 0=0-10,000 copies/cell,
[0300] 1+=at least about 200,000 copies/cell,
[0301] 2+=at least about 500,000 copies/cell,
[0302] 3+=at least about 2,000,000 copies/cell.
[0303] 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)).
[0304] The presence of HER2 protein overexpression and gene
amplification are highly correlated, therefore, alternatively, or
additionally, the use of FISH assays to detect gene amplification
may also be employed for selection of patients appropriate for
treatment in accordance with the present invention. FISH assays
such as the INFORM.TM. (sold by Ventana, Arizona) or
PathVysion.RTM. (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.
[0305] Most commonly, HER2-positive status is confirmed using
archival paraffin-embedded tumor tissue, using any of the foregoing
methods.
[0306] Preferably, HER2-positive patients having a 3+IHC score or a
.gtoreq.2.0 FISH amplification ratio are selected for treatment in
accordance with the present invention.
IV. Pharmaceutical Formulations
[0307] 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 Appln 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
TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG). Lyophilized
antibody formulations are described in WO 97/04801, expressly
incorporated herein by reference.
[0308] 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. (trastuzumab)
formulation is a sterile, white to pale yellow preservative-free
lyophilized powder for intravenous (IV) administration, comprising
440 mg trastuzumab, 400 mg .alpha.alpha.,.alpha.-trehalose
dihydrate, 9.9 mg L-histidine-HCl, 6.4 mg L-histidine, and 1.8 mg
polysorbate 20, USP. Reconstitution 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. For further details, see
the trastuzumab prescribing information.
[0309] 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.
[0310] The formulation of the placebo used in the clinical trials
described in the Examples is equivalent to pertuzumab, without the
active agent.
[0311] 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 HER dimerization inhibitor are described in the Method
Section below. Such molecules are suitably present in combination
in amounts that are effective for the purpose intended.
[0312] 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).
[0313] 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.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid.
[0314] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
V. Treatment Methods
[0315] The treatment methods of the present invention comprise,
consist essentially of, or consist of the administration of a
growth inhibitory HER2 antibody, a HER2 dimerization inhibitor
antibody and a taxane. In a particular embodiment, the treatment
methods of the present invention comprise, consist essentially of,
or consist of the administration of an antibody binding essentially
to epitope 2C4, a HER2 antibody binding essentially to epitope 4D5,
and a taxane to HER2 positive metastatic breast cancer patients as
hereinabove defined, who did not receive prior chemotherapy or
biologic therapy for their metastatic disease. In a preferred
embodiment, the treatment comprises, consists essentially of or
consists of treatment with pertuzumab+trastuzumab+docetaxel. The
treatment methods herein may result in a synergistic, or greater
than additive, therapeutic benefit to the patient.
[0316] Therapy in accordance with the present invention extends
progression-free survival (PFS) and/or overall survival (OS) of the
patient treatment. In one embodiment, the treatment extends PFS or
OS at least about 5%, or at least about 10%, or at least about 15%
or at least about 20%, or at least about 25% more than PFS or OS
achieved by administering trastuzumab+docetaxel to the metastatic
breast cancer patient to be treated.
[0317] Antibodies binding essentially to epitope 2C4 specifically
include, without limitation, rhuMAb 2C4 (pertuzumab). Antibodies
binding essentially to epitope 4D5 specifically include, without
limitation, huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4,
huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8
(trastuzumab).
[0318] The antibodies and taxane, such as pertuzumab, trastuzumab,
and docetaxel are 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.
For antibodies, Intravenous administration is preferred.
[0319] According to one preferred embodiment of the invention, a
fixed dose of HER dimerization inhibitor (e.g. pertuzumab) of
approximately 840 mg (loading dose) is administered, followed by
one or more doses of approximately 420 mg (maintenance dose(s)) of
the antibody. The maintenance doses are preferably administered
about every 3 weeks, for a total of at least two doses, until
radiographic or clinical progressive disease, or unmanageable
toxicity, preferably up to 17 or more doses.
[0320] The growth inhibitory HER2 antibody preferably is
trastuzumab, which typically is administered as an intravenous
loading dose of about 8 mg/kg, followed by the administration of 6
mg/kg doses in subsequent cyclies. Trastuzumab is typically
administered every 3 weeks until radiographic or clinical
progressive disease or unmanageable toxicity, preferably up to 17
or more doses.
[0321] The taxane preferably is docetaxel, which is typically
administered as an IV dose of 75 mg/m.sup.2 every 3 weeks for at
least 6 cycles until radiographic or clinical progressive disease
or unmanageable toxicity.
[0322] The HER2 antibodies preferably are administered as naked
antibodies. However, the inhibitor administered may be conjugated
with a cytotoxic agent. Preferably, the conjugated inhibitor 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.
[0323] In one embodiment, treatment starts with the administration
of pertuzumab, or HER2 dimerization inhibitor antibody, followed by
administration of trastuzumab or another growth inhibitory HER2
antibody and a taxane, e.g. docetaxel, on the following day. In
another embodiment, treatment starts with trastuzumab, or another
growth inhibitory HER2 antibody, followed by the administration of
pertuzumab, or another HER2 dimerization inhibitor antibody, and a
taxane, e.g. docetaxel. In yet another embodiment, all three agents
are administered on the same day, in any order.
[0324] The dosages and treatment protocols described herein are for
information purposes only, and can be altered by a skilled
physician considering factors specific to the patient and cancer to
be treated, such as the patient's age, weight, overall physical
condition, treatment history, the severity and type of the breast
cancer to be treated, the extent and nature of the metastasis, and
the like.
VI. Deposit of Materials
[0325] 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
[0326] These deposits were made under the provisions of the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedure and the
Regulations thereunder (Budapest Treaty). This assures maintenance
of a viable culture of the deposit for 30 years from the date of
deposit. The deposits will be made available by ATCC under the
terms of the Budapest Treaty, and subject to an agreement between
Genentech, Inc. and ATCC, which assures that all restrictions
imposed by the depositor on the availability to the public of the
deposited material will be irrevocably removed upon the granting of
the pertinent U.S. patent, assures permanent and unrestricted
availability of the progeny of the culture of the deposit to the
public upon issuance of the pertinent U.S. patent or upon laying
open to the public of any U.S. or foreign patent application,
whichever comes first, and assures availability of the progeny to
one determined by the U.S. Commissioner of Patents and Trademarks
to be entitled thereto according to 35 USC .sctn. 122 and the
Commissioner's rules pursuant thereto (including 37 CFR .sctn. 1.14
with particular reference to 886 OG 638).
[0327] 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.
GLOSSARY OF ABBREVIATIONS
[0328] FACT-B Functional Assessment of Cancer Therapy-Breast
[0329] FFPE Formalin-fixed paraffin-embedded
[0330] FISH Fluorescence in situ hybridization
[0331] GGT Gamma-glutamyl transferase
[0332] ICH International Conference on harmonization
[0333] IHC Immunohistochemistry
[0334] ITT Intent to treat
[0335] IV Intravenous
[0336] JVP Jugular Venous Pressure
[0337] LDH Lactate dehydrogenase
[0338] LLN Lower limit of normal
[0339] MBC Metastatic breast cancer
[0340] MRI Magnetic resonance imaging
[0341] NCI-CTC National Cancer Institute Common Toxicity
Criteria
[0342] NCI-CTCAE National Cancer Institute Common Terminology
Criteria for Adverse Events
[0343] ORR Objective response rate
[0344] OS Overall survival
[0345] PD Progressive disease
[0346] PFS Progression-free survival
[0347] PK Pharmacokinetic
[0348] PR Partial response
[0349] PS Performance status
[0350] aRTT Activated partial thromboplastin time
[0351] RECIST Response Evaluation Criteria in Solid Tumors
[0352] SAE Serious adverse event
[0353] SD Stable disease
[0354] TOI-PFB Trial Outcome Index-Physician Function Breast
[0355] ULN Upper limit of normal
Example 1
Phase III Clinical Study to Evaluate the Efficacy and Safety of
Pertuzumab+Trastuzumab+Docetaxel Treatment of Previously Untreated
Metastatic Breast Cancer
[0356] Primary Objectives
[0357] The primary objective of this study is to compare
progression-free survival (PFS) based on tumor assessments by an
independent review facility (IRF) between patients in two treatment
arms:
[0358] placebo+trastuzumab+docetaxel vs.
pertuzumab+trastuzumab+docetaxel.
[0359] Secondary Objectives [0360] To compare overall survival (OS)
between the two arms [0361] To compare PFS between the two
treatment arms based upon investigator assessment of progression
[0362] To compare the overall objective response rate between the
two treatment arms [0363] To compare the duration of objective
response between the two treatment arms [0364] To compare the
safety profile between the two treatment arms [0365] To compare
time to symptom progression, as assessed by the FACT Trial Outcome
Index-Physical Functional Breast (TOI-PFB) [0366] To evaluate if
biomarkers from tumor tissues or blood samples (e.g., HER3
expression, Fc.gamma., and serum ECD/HER2 and/or HER ligands
concentrations) correlate with clinical outcomes.
[0367] Target Population
[0368] The study enrolls 800 patients from approximately 250 sites
worldwide. The study population is patients with HER2-positive
metastatic breast cancer (MBC) who have not previously been treated
with chemotherapy and/or biologic therapy for their MBC. Patients
with Stage IV disease at initial disease presentation as well as
those who have progressed following either neo-adjuvant or adjuvant
therapy with a disease-free interval of at least 12 months are
included, and they may have received trastuzumab and/or taxanes in
the adjuvant setting.
[0369] Investigational Drug
[0370] The investigational drug is pertuzumab in combination with
trastuzumab and docetaxel, compared to the administration of
placebo in combination with trastuzumab and docetaxel.
[0371] Blinded Pertuzumab/Placebo
[0372] Pertuzumab/placebo are administered as an IV loading dose of
840 mg for Cycle I, and 420 mg for subsequent cyclies.
Pertuzumab/placebo are administered every 3 weeks until
investigator-assessed radiographic or clinical progressive disease,
or unmanageable toxicity. Administration may be delayed to assess
or treat adverse events such as cardiac adverse events or
mylosuppression. No dose reduction is allowed.
[0373] If the patient misses a dose of pertuzumab/placebo for 1
cycle (i.e., the 2 sequential administration times are 6 weeks or
more apart), a re-loading dose of pertuzumab/placebo (840 mg)
should be given. If re-loading is required for a given cycle, the 3
study therapies should be given on the same schedule as Cycle 1,
i.e., pertuzumab/placebo on Day 1, and trastuzumab and docetaxel on
Day 2. Subsequent maintenance pertuzumab doses of 420 mg are then
given every 3 weeks, starting 3 weeks later.
[0374] Because the pertuzumab/placebo formulation does not contain
a preservative, the vial seal may only be punctured once. Any
remaining solution should be discarded.
[0375] The indicated volume of pertuzumab/placebo solution should
be withdrawn from the vials and added to a 250-cc IV bag of 0.9%
sodium chloride injection.
[0376] Trastuzumab
[0377] Trastuzumab is administered as an IV loading does of 8 mg/kg
for Cycle 1, and 6 mg/kg for subsequent cyclies. The dose of
trastuzumab does not need to be recalculated unless the body weight
has changed by more than .+-.10% from baseline.
[0378] Trastuzumab is administered every 3 weeks until
investigator-assessed radiographic or clinical progressive disease,
or unmanageable toxicity. Administration may be delayed to assessed
or treat adverse events such as cardiac adverse events or
myelosuppression. No dose reduction is allowed.
[0379] If the patient misses a dose of trastuzumab for 1 cycle
(i.e., the 2 sequential administration times are 6 weeks or more
apart), a re-loading dose of pertuzumab/placebo (8 mg/kg) should be
given. If re-loading is required for a given cycle, the 3 study
therapies should be given on the same schedule as Cycle 1, i.e.,
pertuzumab/placebo on Day 1, and trastuzumab and docetaxel on Day
2. Subsequent maintenance trastuzumab doses of 6 mg/kg are then
given every 3 weeks, starting 3 weeks later.
[0380] For administration, each vial of trastuzumab 150 mg is
reconstituted with 7.2 mL of Sterile Water for Injection (SWFI).
This formulation does not contain a preservative and is suitable
for single use only.
[0381] Each vial of trastuzumab 440 mg is reconstituted with 20 mL
of either SWFI or Bacteriostatic Water for Injection (BWFI), USP,
1.1% benzyl alcohol preserved, as supplied. If the trastuzumab is
reconstituted with SWFI, it is suitable for single use only.
[0382] The reconstituted solution contains 21 mg/mL of trastuzumab,
at a pH of approximately 6.0, and the appropriate calculated volume
will be added in to 250 mL of 0.9 Sodium Chloride Injection. The
appropriate volume is calculated (in mL) using the following
formula:
Body Weight (in kg).times.Dose (8 mg/kg for loading or 6 mg/kg for
maintenance)/21 mg/mL (concentration of reconstituted
solution).
[0383] Docetaxel
[0384] Docetaxel is administered as an IV dose of 75 mg/m.sup.2
every 3 weeks for at least 6 cycles until investigator-assessed
radiographic or clinical progressive disease or unmanageable
toxicity. At the discretion of the treating physician, the
docetaxel dose is increased to 100 mg/m.sup.2 for patients who
tolerate at least 1 cycle without any of the following toxicitiesL
febrile neurtopenia, Grade 4 neutropenia for >5 days or
ANC<100 .mu.L for more than 1 day, or other non-hematological
toxicities of Grade>2 (NCI/CTCAE, Version 3). For further
details, refer to docetaxel Package Insert.
[0385] Treatment Schedule
[0386] For the first cycle of treatment, blinded pertuzumab/placebo
is given of Day 1 over 60 minutes followed by a 60-minute
observation period. Trastuzumab and docetaxel is administered on
Day 2 of Cycle I using the labeled guidelines for
administration.
[0387] If the administrations of all three agents are well
tolerated in Cycle 1, they may be given sequentially on Day 1 in
subsequent cycles thereafter. If the subject cannot tolerate all
three drugs given on the same day, the Cycle 1 dosing schedule
(pertuzumab/placebo on Day 1, trastuzumab and docetaxel on Day 2)
is followed.
[0388] If one or both of the monoclonal antibody study drugs needs
to be permanently discontinued or is held for more than two cycles,
the subject is taken off the study treatment. However, if docetaxel
needs to be permanently discontinued for reasons related to
toxicity, the subject can continue on monoclonal antibody study
drugs.
[0389] Inclusion Criteria
[0390] Disease-Specific Inclusion Criteria
[0391] 1. Histologically or cytologically confirmed adenocarcinoma
of the breast with locally recurrent or metastatic disease, and
candidate for chemotherapy. Patients with measurable and
nonmeasurable lesion are eligible.
[0392] Locally recurrent disease must not be amenable to resection
with curative intent.
[0393] Note: Patients with de-novo Stage IV disease are
eligible.
[0394] 2. HER2-positive (defined as 3+ IHC or FISH amplification
ratio.gtoreq.2.0) MBC confirmed by a Sponsor-designated central
laboratory. It is strongly recommended that a formalin-fixed
paraffin-embedded (FFPE) tissue block from the primary tumor be
submitted for central laboratory confirmation of HER2 eligibility;
however, if that is not possible, 25 unstained and freshly cut
slides will be submitted. (Tissue will subsequently be used for
assessment of biomarkers.)
[0395] General Inclusion Criteria:
[0396] 3. Age.gtoreq.18 years
[0397] 4. Left Ventricular Ejection Fraction (LVEF).gtoreq.50% at
baseline (within 42 days of randomization) as determined by either
ECHO or MUGA (ECHO is the preferred method. If the patient is
randomized, the same method of LVEF assessment, ECHO or MUGA, must
be used throughout the study, and to the extent possible, be
obtained at the same institution). All pre-study LVEF values during
and post-trastuzumab adjuvant treatment for patients who received
such adjuvant therapy prior to enrollment into the study will be
collected.
[0398] 5. Eastern Cooperative Oncology Group (ECOG) performance
status (PS) 0 or 1
[0399] 6. For women of childbearing potential, agreement to use an
effective form of contraception (patient and/or partner, e.g.,
surgical sterilization, a reliable barrier method) and to continue
its use for the duration of study treatment and for 6 months after
the last dose of study treatment.
[0400] 7. Signed, written informed consent (approved by the
Institutional Review Board or Independent Ethics Committee)
obtained prior to any study procedure.
[0401] Cancer-Related Exclusion Criteria
[0402] 1. History of anticancer therapy for MBC (with the exception
of one prior hormonal regimen for MBC). This includes any EGFR or
anti-HER2 agents or vaccines, cytotoxic chemotherapy, or more than
one prior hormonal regimen for MBC.
[0403] 2. History of approved or investigative tyrosine kinase/HER
inhibitors for breast cancer in any treatment setting, except
trastuzumab used in the neoadjuvant or adjuvant setting
[0404] 3. History of systemic breast cancer treatment in the
neo-adjuvant or adjuvant setting with a disease-free interval from
completion of the systemic treatment (excluding hormonal therapy)
to metastatic diagnosis of <12 months.
[0405] 4. History of persistent Grade.gtoreq.2 hematologic toxicity
resulting from previous adjuvant therapy.
[0406] 5. Current peripheral neuropathy of NCI-CTCAE, Version 3.0,
Grade.gtoreq.3 at randomization.
[0407] 6. History of other malignancy within the last 5 years,
except for carcinoma in situ of the cervix or basal cell
carcinoma.
[0408] 7. Current clinical or radiographic evidence of central
nervous system (CNS) metastases. CT or MRI scan of the brain is
mandatory (within 28 days of randomization) in cases of clinical
suspicion of brain metastases.
[0409] 8. History of exposure to the following cumulative doses of
anthracyclines: [0410] doxorubicin or liposomal doxorubicin>360
mg/m2 [0411] epirubicin>720 mg/m2 [0412] mitoxantrone>120
mg/m2 and idarubicin>90 mg/m2 [0413] Other (e.g., liposomal
doxorubicin or other anthracycline>the equivalent of 360 mg/m2
of doxorubicin) [0414] If more than 1 anthracycline has been used,
then the cumulative dose must not exceed the equivalent of 360
mg/m2 of doxorubicin.
[0415] Hematological, Biochemical, and Organ Function
[0416] 9. Current uncontrolled hypertension (systolic>150 mmHg
and/or diastolic>100 mmHg) or unstable angina
[0417] 10. History of CHF of any New York Heart Association (NYHA)
criteria, or serious cardiac arrhythmia requiring treatment
(exception, atrial fibrillation, paroxysmal supraventricular
tachycardia)
[0418] 11. History of myocardial infarction within 6 months of
randomization
[0419] 12. History of LVEF decline to below 50% during or after
prior trastuzumab neo-adjuvant or adjuvant therapy
[0420] 13. Current dyspnea at rest due to complications of advanced
malignancy, or other diseases that require continuous oxygen
therapy.
[0421] General Exclusion Criteria
[0422] 14. Inadequate organ function, evidenced by the following
laboratory results within 28 days prior to randomization: [0423]
Absolute neutrophil count<1,500 cells/mm3 [0424] Platelet
count<100,000 cells/mm3 [0425] Hemoglobin<9 g/dL [0426] Total
bilirubin>upper limit of normal (ULN) (unless the patient has
documented Gilbert's syndrome) [0427] AST (SGOT) and ALT
(SGPT)>2.5.times.ULN [0428] AST (SGOT) or ALT
(SGPT)>1.5.times.ULN with concurrent serum alkaline
phosphatase>2.5.times.ULN (unless bone metastases are present)
[0429] Serum creatinine>2.0 mg/dL or 177 .mu.mol/L [0430]
International normalized ratio (INR) and activated partial
thromboplastin time (aPTT)>1.5.times.ULN (unless on therapeutic
coagulation)
[0431] 15. Current severe, uncontrolled systemic disease (e.g.,
clinically significant cardiovascular, pulmonary, or metabolic
disease; wound healing disorders; ulcers; or bone fractures)
[0432] 16. Major surgical procedure or significant traumatic injury
within 28 days prior to study treatment start or anticipation of
the need for major surgery during the course of study treatment
[0433] 17. Pregnant or lactating women
[0434] 18. History of receiving any investigational treatment
within 28 days of randomization
[0435] 19. Current known infection with HIV, HBV, or HCV
[0436] 20. Receipt of IV antibiotics for infection within 14 days
of randomization
[0437] 21. Current chronic daily treatment with corticosteroids
(dose of >10 mg/day methylprednisolone equivalent) (excluding
inhaled steroids)
[0438] 22. Known hypersensitivity to any of the study drugs
[0439] 23. Assessed by the investigator to be unable or unwilling
to comply with the requirements of the protocol.
[0440] Assessments
[0441] Efficacy
[0442] The primary endpoint is PFS based on IRF evaluations. PFS is
defined as the time from randomization to the first documented
radiographical progressive disease, as determined by the IRF using
current RECIST (Therasse et al. 2000), or death from any cause,
whichever occurs first.
[0443] Carcinomatous meningitis diagnosed by cytologic evaluation
of cerebral spinal fluid will also define progressive disease.
Medical photography will also be allowed to monitor chest wall
recurrences of subcutaneous lesions.
[0444] Overall survival is the key secondary endpoint, and is
defined as the time from the date of randomization to the date of
death from any cause.
[0445] Safety [0446] Safety outcome measures are as follows: [0447]
Incidence of Symptomatic left ventricular systolic dysfunction
[Congestive Heart Failure (CHF)] and asymptomatic left ventricular
ejection fraction (LVEF) events [0448] LVEF measurements over the
course of the study [0449] Incidence and severity of adverse events
(AEs) and serious adverse events (SAEs) [0450] Laboratory test
abnormalities
[0451] Pharmacokinetics/OT (Substudy)
[0452] A subset of principal investigators and patients
participates in a pharmacokinetic, drug-drug interaction, and QTc
interval substudy as detailed in a separate protocol (see Example
2). Separate IRB/IEC approval and Informed Consent Form will be
required for participation in the substudy.
[0453] Quality of Life/Pharmacoeconomics
[0454] Patient-Reported Outcomes Assessments: This study uses the
Functional Assessment of Cancer Therapy-Breast (FACT-B), Version 4.
The FACT-B has a 28-item generic score for all patients, plus nine
items specific to breast cancer. Patients rate all items on a
five-point scale ranging from "not at all" to "very much." The
FACT-B provides supplemental domain valuative ratings or utility
weights, thus providing an estimate of the relative importance of
each quality of life domain to an individual patient. The FACT-B
provides a total QoL score as well as information about physical
well-being, social/family well-being, functional well-being, and
disease-specific concerns. The FACT-B has been used extensively and
has demonstrated reliability, validity, and sensitivity to change
over time. Only female patients on this study will be asked to
complete the FACT-B questionnaire.
[0455] Pharmacoeconomic Assessments
[0456] An economic assessment comparing various costs between the
two treatment arms is conducted by evaluating hospitalizations
while on study treatment. The number of hospital visits, number of
days admitted, and type of visits (emergency room vs. inpatient
care) will be collected. This information will be collected from
information submitted on AE and SAE electronic case report forms
(eCRFs).
[0457] Sample Collection
[0458] Archival tumor samples from the primary tumor (or metastatic
sites, if the primary tumor is not available) are submitted from
all subjects during screening and submitted to a central pathology
laboratory for assessment of HER2 status via IHC and FISH for study
eligibility, as well as for the assessment of tumor tissue
biomarkers for pertuzumab/trastuzumab response prediction. Tumor
tissue samples are submitted in the form of either paraffin blocks
or unstained, freshly cut slides containing formalin-fixed tumor
tissue. Because uncontrolled oxidation processes on the slides may
affect slides, tumor tissue blocks are preferred. However, if a
tumor block is not available, 25 unstained freshly cut slides of 4
.mu.m are submitted (the number of slides submitted may be reduced
pending on the regulatory and or IEC requirements of some
counties). The slides must be sent to the central lab within 2 days
of being cut. From submitted tumor blocks, at the central
laboratory a maximum of 15 slides will be cut and 2 cores will be
removed in order to construct tissue microarrays (TMAs) for later
analysis. The remaining part of the tumor block will be returned to
the institution. HER2 testing will be prioritized and the tissue
will subsequently be used for assessment of biomarkers.
[0459] For the assessment of tumor tissue biomarkers, a variety of
analysis methodologies may be used, including but not limited to,
qRT-PCR, IHC, in-situ hybridization, and gene expression profiling.
At the end of the collection process, the most suitable analytical
methodologies will be selected and employed.
[0460] Tissue Microarray (TMA) Construction
[0461] The tumor blocks are also used to set up a TMA: a maximum of
2 tissue cores of 1.5 mm each are taken out using a puncher and
then rearranged as an array into a block of wax. A single array may
include tissue cores from different patients. This process protects
the tissue against oxidation and allows for long-term storage and
later analysis.
[0462] For later analysis, tissue sections can be generated using
the latter tissue microarray. This technology allows a high
throughput (many patient samples on one glass slide) analysis of
biomarkers.
[0463] DNA/RNA Extraction
[0464] The submitted tumor blocks are used to generate sections on
glass slides for the extraction of tumor DNA and RNA for later
analysis. These slides are prepared in a central lab to ensure the
same quality for all samples and in later studies. Note that as
tumorigenesis is a multiple-step process linked to somatic events,
DNA analysis will focus on sporadic mutations specifically found in
tumor tissue but not inherited changes found in the whole body. For
this purpose, some sections containing tumor will be taken from the
block and used for the extraction process. The tumor tissue samples
will be stored at the study Sponsors' facility or a contract
laboratory facility for up to 7 years after database closure, at
which time the samples will be destroyed.
[0465] Metastatic Tumor Tissue Samples for Biomarker Analysis
(Optional)
[0466] If a biopsy of the patient's metastatic tumor tissue is
available, it is submitted from consenting patients at baseline
(after the patient has been determined to be eligible for the
study, but before the first administration of study medication) for
the assessment of tumor tissue biomarkers for
pertuzumab/trastuzumab response prediction.
[0467] Serum Samples for ECD/HER2 and HER Ligands Analysis
[0468] For assessment of serum biomarkers that may indicate
response to pertuzumab and trastuzumab, serum samples (from an
approximately 5 mL blood draw) are collected at baseline (after the
patient has been determined to be eligible for the study but before
the first administration of study medication) and during the study
at the time of each tumor assessment. Biomarker assessments with
these samples will include levels of ECD/HER2, selected HER
ligands, and/or markers thought to be important for HER family
signaling or response to HER inhibitors and HER activation.
Leftovers of samples may be used for re-testing or developing and
validating existing and/or new diagnostic tests related to
pertuzumab or trastuzumab, or both.
[0469] Whole Blood Sample for Fc.gamma. Polymorphism Analysis
(Clinical Genotyping)
[0470] A whole blood sample (3 mL) for assessment of Fc.gamma.
polymorphism is collected from patients at baseline (after the
patient has been determined to be eligible for the study but before
the first administration of study medication). An analysis of
Fc.gamma.-receptor polymorphism is correlated with clinical outcome
in order to further evaluate the mechanism of action of both
trastuzumab and pertuzumab. Mandatory blood collection for
polymorphic analysis will be pending on the regulatory and or IEC
requirements of the individual countries.
[0471] Serum and Plasma for Biomarker Sample Repository (BSR)
Research (Optional)
[0472] Blood samples for extraction of serum and plasma samples
(approximately 5 mL per sample) for biomarker discovery,
validation, and application will be collected from consenting
patients. These samples are collected at baseline (after the
patient has been determined to be eligible for the study but before
the first administration of study medication) and during the study
every 9 weeks at the time of every tumor assessment until
IRF-determined progressive disease. If IRF-determined PD occurs
prior to post-treatment Week 18, BSR samples will continue to be
collected every 9 weeks until posttreatment Week 18.
[0473] The collected BSR samples will be stored with the study
Sponsor's facility or a contract laboratory facility for up to 15
years after the end of the associated study (database closure), at
which time the samples will be destroyed. These samples will be
used only for research purposes to identify dynamic biomarkers that
may be predictive of response to pertuzumab and trastuzumab
treatment (in terms of dose, safety, tolerability, and efficacy)
and will help to better understand the pathogenesis, course, and
outcome of breast cancer and related diseases and adverse
events.
[0474] The collected blood samples may be used to develop and
validate diagnostic assays and allow the generation of
statistically meaningful biomarker data related to HER2-positive
breast cancer disease or response to pertuzumab and/or trastuzumab.
Since the identification of new markers that correlate with disease
activity and the efficacy or safety of treatment is rapidly
developing, the definitive list of analyses remains to be
determined.
[0475] Study Duration
[0476] Patients remain in the treatment phase of the study until
investigator-assessed radiographic or clinical progressive disease,
unmanageable toxicity, or study termination by the Sponsors.
Patients will not receive open-label pertuzumab after
discontinuation from study treatment. After discontinuation of
study treatment, tumor assessments will continue until IRF-assessed
progression. In addition, patients will be followed for survival
until death, loss to follow-up, withdrawal of consent, or study
termination by the Sponsors. Tumor assessments will be conducted
every 9 weeks from the date of randomization. Delays in treatment
administration will not impact the timing of the tumor assessments.
If a tumor assessment must be performed early/late, subsequent
assessments will be conducted according to the original schedule of
every 9 weeks from the date of randomization. Tumor assessments
must be conducted until IRF-determined progressive disease (PD),
even if treatment has been discontinued due to an
investigator-determined PD or unacceptable toxicity.
[0477] After termination of study treatment, patients will continue
be followed for survival until death, loss to follow-up, or study
termination.
[0478] Sample Size
[0479] A sample size of 800 patients is needed to provide 80% power
to detect a 33% improvement in OS (HR=0.75) at the two-sided
significance level of 5%. Since both PFS and OS analyses are
event-driven, and to avoid prolonged waiting period after final PFS
analysis for OS data to reach the required number of events, the
trial is designed to enroll sufficient number of patients such that
approximately 50% of the required deaths will have been observed at
the time of the final PFS analysis.
[0480] Assuming that the median OS in the control arm is 36 months
and OS is exponentially distributed, one interim analysis at 50% of
total requiredvdeaths, and a Lan-DeMets alpha-spending function
with the O'Brien-Fleming stopping boundary, approximately 385
deaths will be required. In addition, assuming that the accrual
rate is approximately 40 patients per month after a 9-month ramp-up
period, v800 patients will need to be enrolled and followed for an
additional 29.5 months to obtain 385 deaths. The enrollment period
is estimated to be 26.5 months, and 50% of the required deaths will
be reached at around 33.5 months.
[0481] Assuming that PFS is exponentially distributed with a median
of 10.5 months in the control arm, it is estimated that 381
IRF-assessed PFS events, corresponding to approximately 448
investigator-assessed events, will have occurred when 50% of the
required deaths (193 deaths) is reached. Final primary analysis of
PFS will be performed after 381 IRF-assessed PFS events have
occurred.
[0482] Statistical Methods
[0483] Efficacy Analyses
[0484] Analyses of PFS, OS, and time to symptom progression will be
based on the intent-to-treat (ITT) population, defined as patients
who have been randomized. For objective response, only patients
with measurable disease at baseline will be included in the
analysis. For duration of response, only responders will be
included in the analysis. All efficacy analyses will be based on
the treatment arm to which patients were randomized.
[0485] Analysis of Primary Variable
[0486] The primary endpoint is PFS based on IRF assessments. For
patients who discontinue study treatment due to reasons other than
death or IRF-assessed progression, every effort will be made to
continue tumor assessments until IRF-determined progressive disease
or patient death. Data for patients who do not have documented
progressive disease or who have not died within 18 weeks of the
last tumor assessment will be censored at the time of the last
IRF-evaluable tumor assessment (or, if no tumor assessments are
performed after the baseline visit, at the time of randomization
plus 1 day).
[0487] For patients whose IRF-determined progression event is not
available, surrogating death at any time as a progressive event can
artificially prolong the PFS time because of a much longer life
expectancy in this patient population compared with PFS. Therefore,
only deaths within 18 weeks of the last tumor assessments will be
included as an event in the primary analysis. However, a
sensitivity analysis will be performed including all deaths as an
event.
[0488] The log-rank test, stratified by prior treatment status (de
novo and prior adjuvant or neo-adjuvant therapy) and region
(Europe, North America, South America, and Asia), will be used to
compare PFS between the two treatment arms. The unstratified
log-rank test results will also be provided as a sensitivity
analysis. The Kaplan-Meier approach will be used to estimate median
PFS for each treatment arm. The Cox proportional hazard model,
stratified by prior treatment status and region, will be used to
estimate the HR between the two treatment arms (i.e., the magnitude
of treatment effect) and its 95% confidence interval (CI).
[0489] The aforementioned analyses will be performed in demographic
subgroups as appropriate. For example analysis may be performed in
patient subgroups based on racial origin provided there is a
reasonable sample size in the subgroups of interest.
[0490] Secondary Variables
[0491] Overall survival. Patients who are alive or lost to
follow-up at the time of the analysis will be censored at the last
known alive date. Patients with no post-baseline information will
be censored at the time of randomization plus 1 day. Analysis
methods are the same as those described for the primary endpoint.
To minimize the chance of a biased OS estimate resulting from
scheduled survival follow-up every 18 weeks, immediately prior to
the data cutoff for the final PFS analysis and final OS analysis,
the investigative sites will contact every patient that is alive to
confirm current survival status. (The study Sponsors will notify
all investigators of the timing of this survival data sweep.)
[0492] PFS based on investigator assessments. Data for patients who
do not have documented progressive disease or who have not died
within 18 weeks of the last tumor assessment will be censored at
the time of the last investigator tumor assessment (or, if no tumor
assessments are performed after the baseline visit, at the time of
randomization plus 1 day). Analysis methods are the same as those
described for the primary endpoint.
[0493] Objective response. Only patients with measurable disease at
baseline will be included in the analysis of the objective
response. Patients without a post-baseline tumor assessment will be
considered to be non-responders. Analysis of objective response
will be based on IRF assessments.
[0494] An estimate of the objective response rate and its 95% CI
will be calculated for each treatment arm. The difference in
objective response rate will also be provided with 95% CIs. The
Mantel-Haenszel .chi.2 test stratified by prior treatment status
and region will be used to compare the objective response rate
between the two treatment arms. An unadjusted Fisher's exact test
result will also be provided as a sensitivity analysis.
[0495] Duration of objective response. Only patients with an
objective response will be included in the analysis of duration of
objective response. The method for handling censoring is the same
as that described for the primary endpoint. Analysis of duration of
objective response will be based on IRF assessments.
[0496] Median duration of objective response for each arm will be
estimated using the Kaplan-Meier approach. The hazard ratio between
the two arms will also be estimated using Cox regression.
[0497] Time to symptom progression. A decrease of five points in
TOI-PFB is considered symptom progression. Data for patients who do
not have an observed symptom progression will be censored at the
last observed TOI-PFB assessment date. If baseline TOI-PFB
assessment is unavailable, or if there is no post-baseline TOI-PFB
assessment performed, data will be censored at the time of
randomization plus 1 day. Analysis methods are the same as those
described for the primary endpoint.
[0498] Biomarker analyses. To evaluate the effect of molecular
markers on efficacy outcome, efficacy outcomes will be summarized
for all patients, and by treatment arm, within each subgroup
determined by exploratory markers. Markers to be considered include
the status of HER receptors, HER ligands, Fc-.gamma., shed antigens
(e.g., ECD/HER2), and other markers relevant for the HER family
pathway. Special emphasis will be put on markers that have shown
association with clinical outcome in patients treated with
pertuzumab in previous studies:
[0499] qRT-PCR markers: tumor gene expression profiles associated
with HER2 activation
[0500] Baseline serum markers: levels of ECD/HER2 and HER
ligands
[0501] Efficacy outcomes considered for this analysis will include
PFS, objective response rate, and OS. The PFS and objective
response will be based on the IRF assessments.
[0502] The biomarker analyses at the time of protocol development
do not take the form of testing fixed hypotheses involving specific
cutoffs or other pre-specified prediction rules. It is planned for
the Statistical Analysis Plan (to be generated prior to unblinding
of this trial) to use all available scientific evidence from
independent studies or publications to specify testable prediction
rules. In addition, this plan will specify in due detail how
data-adaptive prediction rules will be derived (e.g., systematic
cutoff search) and how the inherent multiplicity/bias will be
corrected in order to prevent biased conclusions.
[0503] The difference in treatment benefit across biomarker
statuses defined by a suitable prediction rule will be evaluated by
testing the interaction effect of treatment and the prediction
status using Cox regression for PFS and OS, and using logistic
regression for response rate. These models involving an interaction
term will also be used to estimate the conditional efficacy
outcomes, conditional on biomarker prediction status or treatment
arm, including and excluding the stratification factors into the
model.
[0504] Clinical covariates can be of prognostic value and could
interact with treatment benefit and with biomarker status.
Candidates here are baseline variables of prognostic value
describing tumour properties and morbidity status or common lab
values. Biomarker prediction will be checked involving relevant
clinical covariates, which could be part of the biomarker
prediction function, if necessary.
[0505] Safety Analyses The safety of pertuzumab in combination with
trastuzumab and chemotherapy will be assessed through summaries of
AEs, cardiac-specific AEs, LVEF measurements, and laboratory test
results. Patients who receive any amount of study treatment will be
included in safety analyses. Safety results will be summarized by
the treatment patients actually receive.
Example 2
Pharmacokinetic, Drug-Drug Interaction, and QTc Interval
Substudy
[0506] This substudy has two main goals: (1) to describe the
potential effects of pertuzumab on the QTc interval, and (2) to
evaluate the pharmacokinetic profile of pertuzumab in the presence
of trastuzumab and docetaxel and to describe any drug-drug
interactions that might be observed when all three drugs are
co-administered.
[0507] OTC Prolongation
[0508] Drug-induced prolongation of the QT/corrected QT (QTc)
interval resulting in increased susceptibility to cardiac
arrhythmia is a recognized complication of many drugs across a wide
therapeutic spectrum (Morissette et al. Can J Cardiol 2005;
21:857-64). Prolongation of the QT/QTc interval, which is usually
asymptomatic, may be manifested by syncope resulting from cardiac
arrhythmias such as torsades de pointes (TdP), ventricular
arrhythmia, and sudden cardiac death (Morganroth rnst Schering Res
Found Workshop 2007; 59:171-84).
[0509] Measurement of QT is made by an electrocardiogram (ECG) and
is a surrogate for ventricular repolarization. The QT interval is
defined as the time from the beginning of the QRS complex to the
end of the T-wave. Because the QT interval is inversely related to
the heart rate, the following formulae are commonly used to correct
the QT interval (Strevel et al. J Clin Oncol 2007; 25:3362-71).
[0510] Fridericia's Correction (QTcF): QTcF=QT/RR.sup.0.33 [0511]
Bazett's Correction (QTcB): QTcB=QT/RR.sup.0.5
[0512] QTc is considered prolonged when it is greater than 450
milliseconds (ms) in duration. The QT/QTc interval can be affected
by the location of the ECG lead, gender, time of day, drug therapy,
or congenital conditions. Several pre-disposing factors may
influence drug-induced arrhythmia secondary to prolonged QT/QTc,
including electrolyte imbalance, bradycardia, toxins,
cerebrovascular disease, inhibition of cytochrome p450, and
inhibition of p-glycoprotein (Kannankeril and Roden Curr Opin
Cardiol 2007; 22:39-43, Morissette et al 2005, supra).
[0513] The common mechanism of drug-induced QT/QTc interval
prolongation is the direct blockade of specific potassium channels,
encoded by the human ether-a-go-go (hERG)-related gene, that
regulate cardiac repolarization or disrupt hERG channel protein
trafficking, or both. Drugs have been classified by their
propensity to prolong the QTc interval; the classification provided
in Table 2 is commonly used (Woosley http//www.arizonacert.org
(updated as of Mar. 1, 2006). To date, the drugs known to block
potassium channels are small molecules, such as antiarrhythmics,
some antibiotics, antiemetics, antihistamines, antipsychotics,
antidepressants, bronchodilators, and some central nervous system
(CNS) stimulants (Morissette et al. 2005m supra, Woosley 2006,
supra). The binding site for potassium channel blockade is located
on an intracellular domain, a site that is difficult for large
molecules (i.e., monoclonal antibodies) to access.
[0514] Classes of molecularly-targeted oncology therapeutic agents
associated with effects on the QT interval have been identified,
including farnesyl protein transferase inhibitors, arsenic, Scr/Abl
kinase inhibitors, multi-targeted tyrosine kinase inhibitors,
histone deacetylase inhibitors, vascular disruption agents, and
protein kinase C inhibitors. A direct mechanism common to these
agents in association with QT effects has not been described
(Streval et al. J Clin Oncol 2007; 25:3362-71).
TABLE-US-00003 TABLE 2 Classification of Drugs by Propensity to
Prolong QTc Interval Drug List 1 Generally accepted by authorities
to have a risk of causing torsades de pointes Drug List 2 Drugs
that in some reports may be associated with torsades de pointes but
at this time lack substantial evidence for causing torsades de
pointes Drug List 3 Drugs to be avoided for use in patients with
diagnosed or suspected congenital long QT syndrome. Drugs on Lists
1, 2, and 4 should also be avoided by patients with QT syndrome
Drug List 4 Drugs that, in some reports, have been weakly
associated with torsades de pointes, and/or QT prolongation but
that are unlikely to be a risk for torsades de pointes when used in
the usual recommended dosages and in patients with out other risk
factors (e.g., concomitant QT prolonging drugs, bradycardia,
electrolyte disturbances, congenital long QT syndrome, concomitant
drugs that inhibit metabolism) Females > Males Substantial
evidence indicates a greater risk (usually > 2 fold) of torsades
de pointes in women
[0515] Pertuzumab Mechanism of Action and Nonclinical
Experience
[0516] As described earlier, pertuzumab is a humanized monoclonal
antibody based on human IgG1 (.kappa.) framework sequences and
consists of two heavy chains (449 residues) and two light chains
(214 residues). Like trastuzumab (Herceptin.RTM.), pertuzumab is
produced in Chinese hamster ovary (CHO) cells and is directed
against HER2. However, it differs from trastuzumab in the
epitope-binding regions of the light chain (12 amino acid
differences) and heavy chain (29 amino acid differences). As a
result, pertuzumab binds to a different epitope on HER2.
[0517] Pertuzumab acts by blocking the association of HER2 with
other HER family members, including HER1 (EGFR), HER3, and HER4. As
a result, it inhibits ligand-initiated intracellular signaling
through two major signal pathways, MAP kinase and PI3 kinase.
Inhibition of these signaling pathways can result in growth arrest
and apoptosis, respectively (Hanahan and Weinberg Cell 2000;
100:57-70). Nonclinical data have demonstrated that overexpression
of HER2 is not required for the anti-tumor activity of
pertuzumab.
[0518] Proarrhythmias secondary to abnormal ventricular
repolarization and QT prolongation have been of concern in drug
development. Two International Conference on Harmonization (ICH)
guidelines for nonclinical (S7B) and clinical (E14) testing were
recently developed (International Conference on Harmonization of
Technical Requirements for Registration of Pharmaceuticals for
Human Use 2005). Based on these guidelines, the effect of
pertuzumab on QTc in patients with breast cancer will be
investigated in this substudy.
[0519] In order to fully characterize any potential effects of
pertuzumab on the heart, additional cardiac endpoints have been
included in two nonclinical multi-dose toxicology studies performed
to support the clinical development of pertuzumab. In a 7-week
intravenous toxicity and toxicokinetic study in cynomolgus monkeys
with a 4-week recovery period (Study 00-377-1821), telemetry was
measured in two animals per sex in the control and high-dose (150
mg/kg/dose) groups to collect electrocardiographic endpoints. At
two timepoints before the initiation of treatment, and on Days 1
and 28, telemetered animals had systolic, diastolic, and mean
arterial blood pressure, heart rate, PR interval, QRS, QT, and Lead
II ECG data recorded. Four 1-minute tracings were collected for
each animal from 2 to 22 hours after dosing and interpreted by a
board-certified veterinary cardiologist. In addition to the routine
serum chemistry parameters evaluated, serum was also analyzed for
creatinine kinase isozymes and Troponin T on Study Days 2/3 and
44/45 for all animals. In a 26-week intravenous toxicity and
toxicokinetic study with pertuzumab in cynomolgus monkeys with an
8-week recovery period (Study 01-458-1821), ECG (standard surface
leads) and blood pressure measurements were recorded on all
animals. Recordings were taken at two timepoints before the
initiation of treatment and once during Weeks 4, 16, and 26 on
anesthetized animals and were interpreted by a board-certified
veterinary cardiologist. In addition to the routine serum chemistry
parameters evaluated, serum was also analyzed for Troponin T on Day
1 predose and on Days 114, 184, and 239 (recovery) for all animals.
Results from both multidose toxicity studies concluded that there
was no evidence of cardiac injury caused by pertuzumab treatment,
as determined by histopathology, lack of increases in relevant
serum chemistry parameters (Troponin T and creatine kinase
isozymes), and normal ECGs, blood pressures, and heart rates.
[0520] In clinical trials as of November 2006, there has been no
association noted of an increase of TdP in pertuzumab-treated
patients.
[0521] Pertuzumab Pharmacokinetics
[0522] Summary
[0523] Pharmacokinetic (PK) results observed in previous pertuzumab
studies showed no change in clearance at doses from 2.0 to 15.0
mg/kg (140 mg-1050 mg for a 70 kg patient). A two-compartment model
adequately described the concentration-time data, with a systemic
serum clearance of approximately 0.24 L/day and a terminal
half-life of approximately 17 days for a typical patient. Based on
these data, a dosing interval of 3 weeks is recommended in clinical
studies. In Phase II studies, a loading dose of 840 mg (followed by
420 mg every 3 weeks), led to the attainment of steady-state trough
(C.sub.min) and peak (C.sub.max) concentrations by the second cycle
and achieved a PK target of 20 .mu.g/mL (based on pre-clinical
tumor xenograft models). Population PK modeling of data from Phase
Ia and Phase II studies support the continued use of fixed,
non-weight-based dosing in female patients. There was no evidence
that pertuzumab impacted the PK of co-administered chemotherapeutic
agents (docetaxel and capecitabine in Phase Ib studies and
gemcitabine in a Phase II study).
[0524] Pharmacokinetics in Single-Agent Studies
[0525] In Phase II single-agent studies (Studies TOC2689g and
B016934), concentration-time data show that the loading dose 840 mg
(followed by 420 mg every 3 weeks) resulted in the achievement of
steady-state C.sub.min and C.sub.max by the second cycle, and also
achieved serum pertuzumab target concentrations>20 .mu.g/mL in
most patients. Mean serum C.sub.min and C.sub.max for the first two
cycles of treatment in ovarian and metastatic breast cancer (MBC)
patients are presented in Table 3. FIG. 6 shows the
concentration-time profiles for the first 84 days.
TABLE-US-00004 TABLE 3 Mean (.+-.SD) Peak and Trough Serum
Concentrations at the End of the First and Second Treatment Cycles
(Studies TOC2689g and BO16934) Cycle 1 Cycle 2 C.sub.min C.sub.max
C.sub.min C.sub.max Study Dose .mu.g/mL .mu.g/mL .mu.g/mL .mu.g/mL
TOC2689g 420 mg 64.6 (.+-.20.9) 231.7 (.+-.50.6) 66.5 (.+-.38.0)
237.6 (.+-.55.0) Ovarian (n = 61) cancer 1050 mg 90.1 (.+-.68.1)
390.9 (.+-.114.7) 94.7 (.+-.47.3) 357.2 (.+-.112.4) (n = 62)
BO16934 420 mg 56.5 (.+-.21.2) 202.9 (.+-.78.4) 62.6 (.+-.21.1)
181.2 (.+-.63.0) Metastatic (n = 40) breast cancer 1050 mg 75.9
(.+-.34.5) 425.6 (.+-.166.9) 136.6 (.+-.53.5) 542.9 (.+-.173.7) (n
= 35)
[0526] PK parameters were estimated using a two-compartment model
(see Table 4). The mean systemic clearance for these patients
(0.225-0.285 L/day), and the mean volume of distribution of the
central compartment (2.70-3.11 L; i.e., approximately the serum
volume), were similar to that observed in the Phase Ia study (Study
TOC2297g). The mean initial half-life and the mean terminal
half-life were also within the ranges observed across 2-15 mg/kg
dose groups in the Phase Ia study.
TABLE-US-00005 TABLE 4 Estimates.sup.a of Selected Pertuzumab
Pharmacokinetic Parameters following Intravenous Infusion (Mean
.+-. SD).sup.b CL V.sub.c V.sub.ss t.sub.1/2 initial t.sub.1/2
terminal Study Dose Group (L/day) (L) (L) (days) (days) TOC2689g
420 mg 0.244 .+-. 0.095 2.70 .+-. 0.39 4.73 .+-. 0.78 1.37 .+-.
0.12 16.2 .+-. 4.7 (Ovarian (n = 61) cancer) 1050 mg 0.285 .+-.
0.119 3.11 .+-. 0.72 5.39 .+-. 1.31 1.34 .+-. 0.11 15.8 .+-. 5.2 (n
= 62) BO16934 420 mg 0.255 .+-. 0.096 2.98 .+-. 0.67 5.12 .+-. 0.98
1.36 .+-. 0.13 16.3 .+-. 3.5 (Metastatic (n = 40) breast 1050 mg
0.225 .+-. 0.121 2.95 .+-. 0.81 5.11 .+-. 1.12 1.39 .+-. 0.15 20.5
.+-. 8.1 cancer) (n = 35) CL = systemic clearance; V.sub.c= =
volume of central compartment; V.sub.ss = steady-state volume of
distribution; t.sub.1/2 initial = initial distribution half-life;
t.sub.1/2 terminal = terminal half-life .sup.aPharmacokinetic
parameters estimated by post-hoc analysis from 2-compartment
population pharmacokinetic model. .sup.bPharmacokinetics not
performed for Study TOC2572g.
[0527] Pharmacokinetics in Combination Therapy Studies
[0528] Analyses of the PK data from Phase Ib studies of
capecitabine (Study B017003) and docetaxel (Study B017021) indicate
that pertuzumab does not alter the PK of these two cytotoxic
agents. In both studies, the PK parameters for pertuzumab were
similar to the PK parameters obtained in single-agent pertuzumab
studies.
[0529] Preliminary PK analysis from a Phase II study to evaluate
the efficacy of pertuzumab in combination with gemcitabine in
patients with platinum-resistant ovarian cancer (Study TOC3258g)
indicates that pertuzumab does not alter the PK of gemcitabine or
its major metabolite, dFdU. In addition, the serum concentrations
of pertuzumab were similar to the concentrations in the
single-agent Phase II studies in ovarian cancer and MBC (Studies
TOC2689g and B016934)
[0530] Pertuzumab Dose Regimen
[0531] A dosing regimen of pertuzumab administered every 3 weeks to
patients in Phase II studies (Studies TOC2689g and B016934) using a
fixed 840 mg loading dose (equivalent to 12 mg/kg for a 70 kg
patient) for treatment Cycle 1 and a fixed 420 mg maintenance dose
(equivalent to 6 mg/kg for a 70 kg patient) for subsequent
treatment cycles resulted in steady-state serum C.sub.min of
approximately 60 .mu.g/mL by the second treatment cycle. In
nonclinical dose-response xenograft studies using nude mice
implanted with non-small cell lung cancer (NSCLC) and breast cancer
tumors (low and high HER2 expression levels), >80% suppression
of tumor growth was achieved when steady-state trough
concentrations of pertuzumab reached 5-25 .mu.g/mL. Thus, the
steady-state C.sub.min that were observed in patients in the Phase
II studies are in excess of concentrations shown to be efficacious
in animal tumor models, and therefore expected to result in a
biologic effect.
[0532] A preliminary population PK analysis of the Phase Ia (Study
TOC2297g) and Phase Ia studies (Studies TOC2689g and B016934),
comprising a total of 153 patients (weight range: 45.0-150.6 kg)
and 1458 concentration-timepoints, showed that the population
variability of steady-state trough concentration and exposure were
similar with fixed-dosing, body surface area-based dosing, and
weight-based dosing. Therefore, a dose based on body-surface area
or weight was not superior to a fixed dose. These data support the
continued use of a fixed dose of pertuzumab in female patients with
MBC and ovarian cancer.
[0533] The dependence of pertuzumab serum clearance on body weight
for both female and male patients will be evaluated further using
all available clinical PK data from the pertuzumab studies.
[0534] Objectives
[0535] Pharmacokinetic Objectives
[0536] The PK objectives of this substudy are the following: [0537]
To characterize the pharmacokinetics of pertuzumab in patients with
HER2-positive MBC, and to compare these data with PK data from
other clinical studies. [0538] To characterize the potential of a
drug-drug interaction of pertuzumab on the pharmacokinetics of
docetaxel (in the presence of trastuzumab), and on the
pharmacokinetics of trastuzumab (in the presence of docetaxel).
[0539] Electrocardiogram Objectives
[0540] The ECG objectives are exploratory and may include the
following: [0541] To describe the effect of pertuzumab on the
change from baseline in QTc interval as calculated using
Fridericia's correction (QTcF) [0542] To describe the effect of
pertuzumab on the change from baseline in QTc interval as
calculated using Bazett's correction (QTcB) [0543] To describe the
proportion of patients with QTc interval prolongation and change
from baseline in QTc interval, calculated using both the
Fridericia's and Bazett's corrections [0544] To describe the effect
of pertuzumab on the following ECG parameters: heart rate, QT
interval, PR interval, and QRS duration
[0545] Study Design
[0546] Description of the Study
[0547] This is a supplemental study to Study TOC4129g/WO20698 that
is designed to evaluate the effect of pertuzumab on QTc interval,
further evaluate the pharmacokinetics of pertuzumab, and
characterize the drug-drug interaction of pertuzumab on docetaxel
pharmacokinetics (in the presence of trastuzumab), and on
trastuzumab pharmacokinetics (in the presence of docetaxel).
[0548] A subset of investigative sites participating in Study
TOC4129g/WO20698 will participate in this substudy. Patients at
these sites who have consented to participate in and have been
determined to be eligible for enrollment into Study
TOC4129g/WO20698 will be invited to participate in this substudy.
Participation in this substudy is optional; therefore, informed
consent for this substudy will be obtained separately from the
consent to participate in Study TOC4129g/WO20698. Refusal to
participate in this substudy will not affect a patient's
eligibility a patient's eligibility for Study TOC4129g/WO20698.
Fifty evaluable patients (25 per treatment arm) will be enrolled to
this substudy.
[0549] Each enrolled patient will receive treatment as specified in
TOC4129g/WO20698. Day 1 of TOC4129g/WO20698 will correspond to Day
1 of this substudy.
[0550] All patients participating in this substudy will have
alpha-1-acid glycoprotein tested at baseline by a local laboratory,
in addition to the standard hematology and serum chemistry tests in
Study TOC4129g/WO20698.
[0551] Triplicate 12-lead ECG measurements will be taken during the
pre-treatment baseline period from Day -7 to Day -1 (i.e., within 7
days prior to Cycle 1 Day 1), at Cycle 1 Day 1, at Cycle 1 Day 3,
coincident with the 23-hour docetaxel PK sample, and at Cycle 3 Day
1 (corresponding to pertuzumab/placebo steady-state C.sub.min and
C.sub.max) On Day 1 of Cycle 1 and Cycle 3, triplicate 12-lead ECG
measurements will be taken at the following timepoints: -30 and -15
minutes pre-pertuzumab/placebo infusion (any premedication that is
required before the pertuzumab/placebo infusion must be given
between these two pre-dose timepoints), immediately
post-pertuzumab/placebo infusion, and 60-75 minutes
post-pertuzumab/placebo infusion. ECG results will be sent to a
central core cardiology laboratory for the determination of the
QT/QTc interval, which will be used as the data for this
substudy.
[0552] To minimize variations due to circadian rhythms, the Cycle 1
and Cycle 3 pertuzumab/placebo infusions should be administered at
the same time of day, and the baseline (Day -7 to Day -1) ECG
readings must be taken at the same corresponding time of day as the
Cycle 1 and Cycle 3 ECG measurements. The severity of QTc
prolongation will be graded according to the National Cancer
Institute Common Toxicity Criteria for Adverse Events (NCI-CTCAE),
Version 3.0. A treatment algorithm is provided to guide study
treatment decisions based upon the observed QT/QTc interval at each
timepoint. If at any time during this substudy the QTc interval
exceeds 500 ms or a high degree of artifact is present on the ECG,
cardiac consultation with the ECG core laboratory is available.
[0553] Blood samples for pertuzumab PK evaluation will be drawn
before and after the pertuzumab/placebo infusions at Cycles 1, 3,
6, 9, 12, 15, 18, with an additional sample drawn at the Treatment
Discontinuation Visit (28-42 days after the last dose of study
treatment). At Cycles 1 and 3, the post-pertuzumab PK samples will
be drawn 60-75 minutes after the end of the pertuzumab/placebo
infusion to correspond with the ECGs performed on those days.
[0554] Blood samples for trastuzumab PK evaluation will be
collected at Cycles 1 and 3, before and after the trastuzumab
infusions.
[0555] Blood samples for the docetaxel PK evaluation will be
collected at Cycle 1 at the following timepoints after the
initiation of docetaxel infusion (Timepoint 0): 0.5 hour (during
infusion), 1.0 hour (at the end of infusion, EOI), 1.25 hours (15
minutes after the EOI), 2 hours (1 hour after EOI), 4 hours (3
hours after EOI), 6 hours (5 hours after EOI), 8 hours (7 hours
after EOI), and 24 hours (Cycle 1 Day 3, 23 hours after EOI).
[0556] Rationale for Study Design
[0557] Rationale for QTc Study Design
[0558] The study will evaluate the effect of pertuzumab on the QTc
interval in patients with HER2-positive MBC. Ordinarily, a thorough
QTc study is performed in healthy volunteers when feasible. Because
the QTc is to be evaluated in a cancer population with multiple
confounders (baseline illness, baseline medications, including
antiemetics, antibiotics, and other supportive care medications),
this substudy has been designed to describe the change in QTc
interval from baseline to steady-state in pertuzumab-treated and
placebo control patients. If patients require antiemetics or other
premedications prior to the infusion of pertuzumab/placebo, they
must be given between the two pre-dose ECG measurements in an
attempt to control for concomitant medication effects on the QT/QTc
interval. ICH guidance recommends that studies should characterize
the effect of a drug on the QT/QTc and perform ECG recordings at
timepoints around the C.sub.max.
[0559] As stated in the ICH E14 guidance, Bazett's correction
generally over-corrects at elevated heart rates and under-corrects
at heart rates below 60 beats per minute (bpm). Fridericia's
correction has been chosen as the primary correction because it
accounts for the effect of altered heart rates on QT interval.
[0560] Pertuzumab PK samples will be drawn at the time of the
Cycles 1 and 3 ECG readings when therapeutic serum concentrations
of pertuzumab are expected to be achieved. Pertuzumab exposure will
be correlated with QTc. Cycle 3 Day 1 (assuming 21-day cycles) was
chosen for the measurement of QTc at steady-state concentration
based on the Phase II studies, during which a loading dose of 840
mg (followed by 420 mg every 3 weeks) resulted in the achievement
of steady-state C.sub.min and C.sub.max by the second cycle.
Therefore, the majority of the population should be at steady state
by Cycle 3.
[0561] Although a positive-control comparison drug (e.g.,
moxifloxacin) is recommended by ICH guidelines to validate assay
sensitivity, a positive control drug will not be administered to
patients in this substudy as it is felt that the use of a
positive-control medication would not be ethical in a metastatic
cancer patient population. Furthermore, patients have baseline
variability secondary to medications already being
administered.
[0562] Per ICH recommendations, rates of selected adverse events,
if observed (TdP; sudden death; ventricular tachycardia;
ventricular fibrillation and flutter; syncope; and seizures) will
be compared between the pertuzumab-treated and control patients, as
part of data collected for Study TOC4129g/WO20698. Additionally,
the incidence of QTc interval prolongation and the change from
baseline in QTc interval will be summarized.
[0563] Rationale for Pharmacokinetic Sampling
[0564] The proposed sampling scheme for pertuzumab concentration
assessments in this substudy should allow for the adequate
characterization of the pharmacokinetics of pertuzumab. The
pertuzumab concentration results will be compared with available
data from other pertuzumab clinical studies. In addition, the
pertuzumab concentration data will be used for population PK
modeling to generate PK parameter estimates. These data may also
contribute to a future population PK analysis to investigate the
effect of physiologic and disease-related covariates on PK
parameters.
[0565] Study TOC4129g/WO20698 proposes to combine pertuzumab with
trastuzumab and docetaxel. Based on the clearance mechanisms for
pertuzumab, there is no expectation that pertuzumab will alter the
pharmacokinetics of docetaxel. However, the concentrations of
docetaxel will be measured to assess a potential PK-related
interaction between docetaxel and pertuzumab (in the presence of
trastuzumab). In addition, concentrations of trastuzumab will be
measured to assess a potential PK-related interaction between
trastuzumab and pertuzumab (in the presence of docetaxel).
[0566] Dose-ranging pharmacokinetics will not be performed, and a
supratherapeutic dose will not be administered in Study
TOC4129g/WO20698.
[0567] Outcome Measures
[0568] Pharmacokinetic Outcome Measures: The PK outcome measures
are the following: [0569] Observed minimum and maximum pertuzumab
serum concentrations (C.sub.min and C.sub.max), and PK parameter
estimates (CL, AUC, Vd, t.sub.1/2) [0570] Minimum and maximum
trastuzumab serum concentrations (C.sub.min and C.sub.max) [0571]
Area-under-the-curve (AUC) for docetaxel plasma concentrations
[0572] Electrocardiogram Outcome Measures: The ECG outcome measures
are the following: [0573] Time-matched baseline-adjusted
placebo-corrected QTcF [0574] Time-matched baseline-adjusted
placebo-corrected QTcB [0575] Proportion of patients at each
timepoint whose ECG recordings meet the following criteria:
[0576] New incidence of absolute QTc interval prolongation (based
on Fridericia's correction) of >450 ms, >470 ms, and >500
ms
[0577] The following changes from baseline in QTc interval (based
on Fridericia's correction): QTc increases >30 ms, QTc increases
>60 ms Change from baseline in heart rate of .gtoreq.25%,
resulting in a final heart rate<50 beats per minute (bpm) or
>120 bpm
[0578] New incidence of abnormal U waves
[0579] New incidence of abnormal T waves
[0580] New incidence of abnormal ECG morphology [0581] The
time-matched baseline-adjusted placebo-corrected differences in the
following ECG parameters: heart rate, QT, PR interval, and QRS
duration.
[0582] Safety Plan
[0583] Clinically significant ECG changes detected during this
substudy will be reported and managed according to the safety
reporting and monitoring requirements of Study TOC4129g/WO20698.
The degree of QTc prolongation will be graded according to the
NCI-CTCAE, Version 3.0. A treatment algorithm is provided to guide
study treatment decisions in the event of QT/QTc prolongation
during the study, A central ECG core laboratory will be available
to evaluate any cases of QT/QTc prolongation,
[0584] Control Group
[0585] Because Study TOC4129g/WO20698 is a randomized,
double-blind, placebo-controlled study, the control group will
consist of patients randomized to receive placebo instead of
pertuzumab.
[0586] Minimization of Bias
[0587] For purposes of the main study, unblinding procedures will
be performed according to the TOC4129g/WO20698 protocol. For this
substudy, patient treatment (pertuzumab vs. control) will be
determined by the analysis of PK serum samples for the presence or
absence of pertuzumab; therefore, to maintain blinding of the main
study, Sponsor personnel involved in the analysis of pertuzumab PK
samples and analysis of this substudy will not be involved with any
of the operational or analysis aspects of the Study
TOC4129g/WO20698. All study personnel involved in the main
TOC4129g/WO20698 study will remain blinded (e.g., site personnel,
investigators, patients, statisticians, etc.).
[0588] Centralized ECG readers will be blinded to patient treatment
and ECG timepoint.
[0589] Patients
[0590] Patient Selection
[0591] Patients who have consented to participate in Study
TOC4129g/WO20698 at a subset of investigative sites will be
eligible for enrollment into this substudy.
[0592] Inclusion Criteria: Patients must meet the following
criteria to be eligible for substudy entry: [0593] Enrollment in
Study TOC4129g/WO20698 [0594] Signed Informed Consent Form for this
substudy [0595] Exclusion Criteria Patients who meet any of the
following criteria will be excluded from substudy entry: [0596]
Implantable pacemaker or automatic implantable cardioverter
defibrillator (AICD) [0597] Congenital long QT syndrome [0598]
Family history of long QT syndrome [0599] Baseline QTc>450 ms as
assessed locally at each study site [0600] Patients currently
requiring regular use of medications that are known to prolong QTc
interval or induce TdP (see Appendix B) [0601] Clinically
significant bradycardia (defined as <50 bpm) at baseline [0602]
Evidence of heart block [0603] Hypokalemia, hypomagnesemia, and
hypocalcemia that cannot be corrected with electrolyte
supplement
[0604] Method of Treatment Assignment and Blinding
[0605] Treatment assignment will be in accordance with the protocol
described in Example 1.
[0606] Unblinding of study treatment will be in accordance with the
procedures specified in Example 1. Centralized ECG readers will
remain blinded to patient treatment and ECG timepoints.
[0607] Study Treatment
[0608] Study treatment will be as specified in Example 1.
[0609] Concomitant and Excluded Therapies
[0610] Clinical judgment should be applied when determining
treatment options and supportive care treatment for each
patient.
[0611] Other concomitant and excluded medications will be as
directed in Example 1.
[0612] Study Assessments
[0613] Study treatment infusions, ECG measurements, and blood draws
should be consistently administered, recorded, and collected at the
same time of day, between 9:00 AM-12:00 PM and >1 hour
postprandial, in order to minimize variations due to circadian
rhythms.
[0614] Screening and Pretreatment Assessments
[0615] Informed consent must be obtained before study-specific
evaluations are performed. The informed consent process should be
documented in the patient's medical chart.
[0616] The following substudy evaluations and procedures will be
performed during the baseline period of the study described in
Example 1: [0617] Written informed consent [0618] Review of
inclusion and exclusion criteria [0619] Serum chemistry to evaluate
electrolyte values [0620] Collection of blood sample for
alpha-1-acid glycoprotein test and analysis by a local laboratory,
as an addition to the standard hematology and chemistry testing in
the study described in Example 1.
[0621] ECG Measurements
[0622] Serum potassium, magnesium and calcium levels must be within
normal limits before performing ECGs, as determined by local
laboratory testing performed as specified in the main protocol
TOC4129g/WO20698. Patients may receive electrolyte supplement per
institutional standard practice to bring electrolyte levels within
normal limits prior to performing the ECGs; retesting of potassium,
magnesium, and calcium levels should be performed according to
institutional standard practice.
[0623] Triplicate 12-lead ECG readings will be taken during the
baseline period (Day -7 to Day -1; i.e., within 7 days prior to
Cycle 1 Day 1) at the same time of day at which ECG measurements
will be performed at Cycles 1 and 3.
[0624] To minimize postural variability, it is important that
patients are resting and in a supine position for at least 10
minutes prior to each ECG collection. Blood draws and other
procedures should be avoided during the period immediately before
ECG measurement, and activity should be controlled as much as
possible in order to minimize variability due to the effects of
physiologic stress. Meals should be standardized as much as
possible between patients, to avoid postprandial effects. If
possible, ECGs should be collected on the same type of machine for
each site involved in the study, and the same machine should be
used for all ECGs for a specific patient. Detailed instructions on
ECG acquisitions are provided in the central ECG core laboratory
manual.
[0625] Triplicate runs of 12-lead ECG measurements must be obtained
at each assessment timepoint, and should be collected over a period
of 2 minutes (e.g., a single ECG each minute).
[0626] Assessments During Treatment
[0627] All visits and assessments during treatment are to be
performed on the days indicated. Per the protocol described in
Example 1, a cycle is 21 days in length.
[0628] ECG Measurements During Cycle 1 Day 1, Cycle 1 Day 3, and
Cycle 3 Day 1
[0629] The 12-lead ECGs (triplicate runs) will be performed before
collecting the corresponding PK samples. All ECGs for a patient
should be obtained on the same machine.
[0630] Serum potassium, magnesium and calcium levels must be within
normal limits before performing ECGs, as determined by local
laboratory testing performed as specified in the main protocol
TOC4129g/WO20698. Patients may receive electrolyte supplement per
institutional standard practice to bring electrolyte levels within
normal limits prior to performing the ECGs; retesting of potassium,
magnesium, and calcium levels should be performed according to
institutional standard practice.
[0631] Triplicate 12-lead ECG readings will be taken during Cycle 1
Day 1 and Cycle 3 Day 1 at the same time as the baseline ECG
measurements, at the following times of day: 30 minutes and 15
minutes (.+-.5 minutes) prior to pertuzumab/placebo infusion
[0632] Any premedications that are required for pertuzumab/placebo
infusions must be given between the two pre-infusion ECG
measurements [0633] 0-15 minutes post-pertuzumab/placebo infusion
[0634] 60-75 minutes post-pertuzumab/placebo infusion
[0635] Triplicate 12-lead ECG readings will be also taken during
Cycle 1 Day 3, post-docetaxel infusion and coincident with the
23-hour PK sample.
[0636] Pharmacokinetic Blood Samples
[0637] Unless otherwise specified, blood samples for PK evaluations
should be drawn at the following timepoints: [0638] Pre-dose:
within 15 minutes before the infusion [0639] Post-dose: within 15
minutes after the end of infusion
[0640] Approximately 5 mL of blood will be drawn at each PK
timepoint.
[0641] Pertuzumab Pharmacokinetics
[0642] Blood samples for pertuzumab PK evaluation will be drawn
pre- and post-pertuzumab/placebo infusion at the following cycles:
Cycles 1, 3, 6, 9, 12, 15, and 18. An additional sample will be
drawn at the Treatment Discontinuation Visit (28-42 days after the
last dose of study treatment). [0643] At Cycles 1 and 3, the
post-pertuzumab PK sample will be drawn 60-75 minutes after the end
of the pertuzumab/placebo infusion (prior to administration of
trastuzumab). [0644] At Cycles 1 and 3, the pre-pertuzumab and
60-75 minutes post-pertuzumab PK samples must be collected after
the corresponding ECGs are performed at those timepoints.
[0645] Trastuzumab Pharmacokinetics
[0646] Blood samples for trastuzumab PK evaluation will be drawn
pre- and post-trastuzumab infusion at Cycles 1 and 3.
[0647] At Cycle 3, the trastuzumab dose should be delayed until
after the 60-75 minute post-pertuzumab ECG assessments and PK
sample collection have been completed.
[0648] Docetaxel Pharmacokinetics
[0649] Blood samples for docetaxel PK evaluation will be collected
at Cycle 1 at the following timepoints after the initiation of the
docetaxel infusion (Timepoint 0): [0650] Cycle 1 Day 2
[0651] 0.5 hour (during infusion)
[0652] 1.0 hours (EOI)
[0653] 1.25 hours (15 minutes after EOI)
[0654] 2 hours (1 hour after EOI)
[0655] 4 hours (3 hours after EOI)
[0656] 6 hours (5 hours after EOI)
[0657] 8 hours (7 hours after EOI) [0658] Cycle 1: Day 3
[0659] 24 hours (Cycle 1: Day 3, 23 hours after EOI)
[0660] Adverse Events
[0661] Adverse events will be collected and followed according to
the requirements of the protocol of the study described in Example
1.
[0662] Assay Methods
[0663] Docetaxel Pharmacokinetic Assay
[0664] Plasma samples will be analyzed for docetaxel concentrations
using a high-performance liquid chromatography (HPLC) method (or
equivalent), and the plasma concentrations will be quantified by
comparing the results against known standards. The lower limit of
quantification (LLOQ) for docetaxel in human plasma will be
determined according to validated assay methods established by the
laboratory contracted to perform the analyses.
[0665] Pertuzumab Pharmacokinetic Assay
[0666] Serum samples will be assayed for pertuzumab concentrations
using an ELISA that is currently being developed. The minimum
quantifiable concentration (MQC) for pertuzumab in human serum
measured by this assay is to be determined.
[0667] Trastuzumab Pharmacokinetic Assay
[0668] Serum samples collected at baseline will be assayed for
trastuzumab concentrations using a receptor-binding ELISA. This
assay uses immobilized antigen HER2-ECD to capture trastuzumab from
serum samples. The MQC for trastuzumab in human serum measured by
this assay is 156 ng/mL.
[0669] Serum samples collected after pertuzumab administration will
be assayed for trastuzumab concentrations using an ELISA that is
currently being developed. The MQC for pertuzumab in human serum
measured by this assay is to be determined.
[0670] Patient Discontinuation
[0671] Patients may voluntarily withdraw or be discontinued from
this substudy at any time. Patients who withdraw from this substudy
may continue participation in Study TOC4129g/WO20698. Reasons for
patient discontinuation from the substudy include, but are not
limited to, the following: [0672] Voluntary withdrawal of consent
[0673] Non-compliance [0674] Investigator determination that it is
not in the patient's best interest to continue (e.g., illness or
condition that requires the use of prohibited drugs or treatment)
[0675] Patient withdrawal from Protocol TOC4129g/WO20698
[0676] The primary reason for early discontinuation must be
recorded on the appropriate electronic case report form (eCRF).
[0677] Statistical Methods
[0678] Due to the small sample size, the emphasis of all analyses
will be on estimations. No formal statistical hypothesis testing is
planned.
[0679] Analysis of the Conduct of the Study
[0680] Enrollment and discontinuations from this substudy will be
summarized.
[0681] Analysis of Treatment Group Comparability
[0682] Demographic and baseline characteristics, such as age, sex,
and race, will be summarized using means, standard deviations,
medians, ranges (for continuous variables), and frequencies and
percentages (for categorical variables). Summaries will be
presented by study treatment patients actually received.
[0683] Pharmacokinetic Analyses
[0684] Population modeling will be used to derive post-hoc PK
parameter estimates (CL, AUC, V.sub.d, and t.sub.1/2) for
pertuzumab, and will be summarized for the treatment cohort.
Observed C.sub.max and C.sub.min for pertuzumab and trastuzumab
will be summarized for each specified PK sampling timepoint.
Descriptive statistics will include means, medians, ranges, and
standard deviations, as appropriate. Pertuzumab PK parameters and
serum concentration-time data will be compared with available data
from other pertuzumab clinical studies.
[0685] PK samples for docetaxel will be obtained on Days 2 and 3 of
Cycle 1 only. Plasma concentrations and the AUC for docetaxel will
be summarized by treatment arm using descriptive statistics as
described above. The geometric mean ratio for AUC between the
experimental and control arms will be computed and the
corresponding 90% confidence intervals will be provided.
[0686] Electrocardiogram Analyses
[0687] The ECG-evaluable analysis population will comprise all
patients who receive any study drug (as per Protocol
TOC4129g/WO20698) and who have ECG data available for baseline, the
pre-pertuzumab/placebo timepoint on Cycle 1 Day 1, and at least one
timepoint following pertuzumab/placebo administration at Cycle 1 or
Cycle 3. The average of the triplicate ECG readings for each
timepoint will be utilized in the analyses.
[0688] Descriptive statistics will be used for absolute QTcF value
and change from baseline in QTcF for each timepoint. The difference
in mean baseline-adjusted QTcF between the two treatment arms
(ddQTcF) will be provided as well as the two-sided 90% confidence
interval.
[0689] The time-matched, baseline-adjusted, and placebo-corrected
QTcB, HR, PR, and QRS will be summarized in a similar fashion.
[0690] The number and percentage of patients with ECG recordings
meeting the criteria as described in Section 3.2.2 will be
tabulated for each treatment arm and each post-baseline time point
as appropriate.
[0691] Missing Data
[0692] No missing ECG data will be imputed. As long as one of the
triplicate ECGs is interpretable at each timepoint, a QTc will be
calculated. If patients do not have corresponding ECGs at baseline
and post-baseline timepoint of interest, they will not be included
in the analysis for that timepoint.
[0693] Determination of Sample Size
[0694] At least 50 ECG-evaluable patients will be enrolled to this
substudy. The sample size for this substudy has been primarily
chosen to provide an estimate of key PK and ECG parameters. No
formal statistical hypothesis testing is planned. Assuming an equal
rate of participation between treatment arms (25 patients per
treatment arm) and an estimated standard deviation of 20 ms, the
two-sided 90% confidence interval for the baseline-adjusted
difference in QTcF between treatment arms (ddQTcF) will be within
10 ms of the observed difference.
[0695] It is expected that at least 40 patients enrolled to this
substudy will be PK-evaluable. A PK-evaluable patient is defined as
a patient who has had complete PK samples collected at Cycle 1 and
Cycle 3. With a sample size of 40 evaluable patients and an
inter-patient coefficient of variation in AUC of 30%, the 90%
confidence interval for the ratio of the geometric mean docetaxel
concentrations between treatment arms will be (86%, 117%) if the
observed geometric mean AUCs for both treatment arms are
identical.
[0696] An ECG-evaluable patient is defined as a patient who has an
interpretable baseline ECG as well as an interpretable ECG at Cycle
3 Day 1 immediately post-pertuzumab/placebo infusion (steady-state
C.sub.max). With this sample size of 40 evaluable patients and an
estimated standard deviation of 20 ms, a 95% confidence interval
for the difference between treatment arms in mean change in QTcF
from baseline to Cycle 3 will be .+-.12.4 ms from the observed mean
change.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 23 <210> SEQ ID NO 1 <211> LENGTH: 107 <212>
TYPE: PRT <213> ORGANISM: Mus sp. <400> SEQUENCE: 1 Asp
Thr Val Met Thr Gln Ser His Lys Ile Met Ser Thr Ser Val Gly 1 5 10
15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly
20 25 30 Val Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Lys Leu
Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp
Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr
Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Tyr Cys
Gln Gln Tyr Tyr Ile Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys 100 105 <210> SEQ ID NO 2 <211>
LENGTH: 119 <212> TYPE: PRT <213> ORGANISM: Mus sp.
<400> SEQUENCE: 2 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
Val Lys Pro Gly Thr 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser
Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Thr Met Asp Trp Val Lys Gln
Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asp Val Asn Pro
Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe 50 55 60 Lys Gly Lys
Ala Ser Leu Thr Val Asp Arg Ser Ser Arg Ile Val Tyr 65 70 75 80 Met
Glu Leu Arg Ser Leu Thr Phe Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> SEQ ID NO 3
<211> LENGTH: 107 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 3 Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Lys Ala Ser Gln Asp Val Ser Ile Gly 20 25 30 Val Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser
Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65
70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr
Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105 <210> SEQ ID NO 4 <211> LENGTH: 119 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <400> SEQUENCE: 4 Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25
30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln
Arg Phe 50 55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Leu Gly Pro Ser Phe
Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser
Ser 115 <210> SEQ ID NO 5 <211> LENGTH: 107 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
5 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn
Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Glu Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Tyr Asn Ser Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105 <210> SEQ ID NO 6 <211>
LENGTH: 119 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 6 Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile
Ser Gly Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Gly Arg Val Gly Tyr Ser Leu Tyr Asp Tyr Trp
Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210>
SEQ ID NO 7 <211> LENGTH: 214 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 7 Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly 20 25 30 Val
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr
Ile Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170
175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> SEQ ID
NO 8 <211> LENGTH: 448 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (65)..(65) <223> OTHER INFORMATION: Any
amino acid <400> SEQUENCE: 8 Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Thr Met Asp Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asp
Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe 50 55 60
Xaa Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr
Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185
190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310
315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435
440 445 <210> SEQ ID NO 9 <211> LENGTH: 214 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <400> SEQUENCE: 9 Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25
30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155
160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210>
SEQ ID NO 10 <211> LENGTH: 449 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 10 Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr
Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr
Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala
Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170
175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295
300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420
425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro 435 440 445 Gly <210> SEQ ID NO 11 <211> LENGTH:
217 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 11
Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 1 5
10 15 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp
Val 20 25 30 Ser Ile Gly Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys 35 40 45 Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr
Gly Val Pro Ser Arg 50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser 65 70 75 80 Leu Gln Pro Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile 85 90 95 Tyr Pro Tyr Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135
140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly
Glu Cys 210 215 <210> SEQ ID NO 12 <211> LENGTH: 449
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 12
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp
Tyr 20 25 30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile
Tyr Asn Gln Arg Phe 50 55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp
Arg Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Leu Gly
Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135
140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260
265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385
390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> SEQ ID NO 13
<400> SEQUENCE: 13 000 <210> SEQ ID NO 14 <211>
LENGTH: 195 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 14 Thr Gln Val Cys Thr Gly Thr Asp
Met Lys Leu Arg Leu Pro Ala Ser 1 5 10 15 Pro Glu Thr His Leu Asp
Met Leu Arg His Leu Tyr Gln Gly Cys Gln 20 25 30 Val Val Gln Gly
Asn Leu Glu Leu Thr Tyr Leu Pro Thr Asn Ala Ser 35 40 45 Leu Ser
Phe Leu Gln Asp Ile Gln Glu Val Gln Gly Tyr Val Leu Ile 50 55 60
Ala His Asn Gln Val Arg Gln Val Pro Leu Gln Arg Leu Arg Ile Val 65
70 75 80 Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr Ala Leu Ala Val
Leu Asp 85 90 95 Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro Val Thr
Gly Ala Ser Pro 100 105 110 Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser
Leu Thr Glu Ile Leu Lys 115 120 125 Gly Gly Val Leu Ile Gln Arg Asn
Pro Gln Leu Cys Tyr Gln Asp Thr 130 135 140 Ile Leu Trp Lys Asp Ile
Phe His Lys Asn Asn Gln Leu Ala Leu Thr 145 150 155 160 Leu Ile Asp
Thr Asn Arg Ser Arg Ala Cys His Pro Cys Ser Pro Met 165 170 175 Cys
Lys Gly Ser Arg Cys Trp Gly Glu Ser Ser Glu Asp Cys Gln Ser 180 185
190 Leu Thr Arg 195 <210> SEQ ID NO 15 <211> LENGTH:
124 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 15 Thr Val Cys Ala Gly Gly Cys Ala Arg Cys
Lys Gly Pro Leu Pro Thr 1 5 10 15 Asp Cys Cys His Glu Gln Cys Ala
Ala Gly Cys Thr Gly Pro Lys His 20 25 30 Ser Asp Cys Leu Ala Cys
Leu His Phe Asn His Ser Gly Ile Cys Glu 35 40 45 Leu His Cys Pro
Ala Leu Val Thr Tyr Asn Thr Asp Thr Phe Glu Ser 50 55 60 Met Pro
Asn Pro Glu Gly Arg Tyr Thr Phe Gly Ala Ser Cys Val Thr 65 70 75 80
Ala Cys Pro Tyr Asn Tyr Leu Ser Thr Asp Val Gly Ser Cys Thr Leu 85
90 95 Val Cys Pro Leu His Asn Gln Glu Val Thr Ala Glu Asp Gly Thr
Gln 100 105 110 Arg Cys Glu Lys Cys Ser Lys Pro Cys Ala Arg Val 115
120 <210> SEQ ID NO 16 <211> LENGTH: 169 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
16 Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu Val Arg Ala Val Thr
1 5 10 15 Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys Lys Ile Phe
Gly Ser 20 25 30 Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro
Ala Ser Asn Thr 35 40 45 Ala Pro Leu Gln Pro Glu Gln Leu Gln Val
Phe Glu Thr Leu Glu Glu 50 55 60 Ile Thr Gly Tyr Leu Tyr Ile Ser
Ala Trp Pro Asp Ser Leu Pro Asp 65 70 75 80 Leu Ser Val Phe Gln Asn
Leu Gln Val Ile Arg Gly Arg Ile Leu His 85 90 95 Asn Gly Ala Tyr
Ser Leu Thr Leu Gln Gly Leu Gly Ile Ser Trp Leu 100 105 110 Gly Leu
Arg Ser Leu Arg Glu Leu Gly Ser Gly Leu Ala Leu Ile His 115 120 125
His Asn Thr His Leu Cys Phe Val His Thr Val Pro Trp Asp Gln Leu 130
135 140 Phe Arg Asn Pro His Gln Ala Leu Leu His Thr Ala Asn Arg Pro
Glu 145 150 155 160 Asp Glu Cys Val Gly Glu Gly Leu Ala 165
<210> SEQ ID NO 17 <211> LENGTH: 142 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 17 Cys
His Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr 1 5 10
15 Gln Cys Val Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu
20 25 30 Glu Cys Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn
Ala Arg 35 40 45 His Cys Leu Pro Cys His Pro Glu Cys Gln Pro Gln
Asn Gly Ser Val 50 55 60 Thr Cys Phe Gly Pro Glu Ala Asp Gln Cys
Val Ala Cys Ala His Tyr 65 70 75 80 Lys Asp Pro Pro Phe Cys Val Ala
Arg Cys Pro Ser Gly Val Lys Pro 85 90 95 Asp Leu Ser Tyr Met Pro
Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala 100 105 110 Cys Gln Pro Cys
Pro Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp 115 120 125 Asp Lys
Gly Cys Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr 130 135 140
<210> SEQ ID NO 18 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (10)..(10) <223> OTHER
INFORMATION: Ser or Asp <400> SEQUENCE: 18 Gly Phe Thr Phe
Thr Asp Tyr Thr Met Xaa 1 5 10 <210> SEQ ID NO 19 <211>
LENGTH: 17 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic peptide <400>
SEQUENCE: 19 Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln
Arg Phe Lys 1 5 10 15 Gly <210> SEQ ID NO 20 <211>
LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic peptide <400>
SEQUENCE: 20 Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr 1 5 10
<210> SEQ ID NO 21 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 21 Lys Ala Ser Gln Asp Val
Ser Ile Gly Val Ala 1 5 10 <210> SEQ ID NO 22 <211>
LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic peptide <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(5)..(5) <223> OTHER INFORMATION: Arg or Leu <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(6)..(6) <223> OTHER INFORMATION: Tyr or Glu <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(7..(7) <223> OTHER INFORMATION: Thr or Ser <400>
SEQUENCE: 22 Ser Ala Ser Tyr Xaa Xaa Xaa 1 5 <210> SEQ ID NO
23 <211> LENGTH: 9 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <400> SEQUENCE: 23 Gln Gln Tyr Tyr Ile Tyr Pro Tyr
Thr 1 5
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 23 <210>
SEQ ID NO 1 <211> LENGTH: 107 <212> TYPE: PRT
<213> ORGANISM: Mus sp. <400> SEQUENCE: 1 Asp Thr Val
Met Thr Gln Ser His Lys Ile Met Ser Thr Ser Val Gly 1 5 10 15 Asp
Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly 20 25
30 Val Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe
Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser
Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln
Tyr Tyr Ile Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu
Glu Ile Lys 100 105 <210> SEQ ID NO 2 <211> LENGTH: 119
<212> TYPE: PRT <213> ORGANISM: Mus sp. <400>
SEQUENCE: 2 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro
Gly Thr 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Thr
Phe Thr Asp Tyr 20 25 30 Thr Met Asp Trp Val Lys Gln Ser His Gly
Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asp Val Asn Pro Asn Ser Gly
Gly Ser Ile Tyr Asn Gln Arg Phe 50 55 60 Lys Gly Lys Ala Ser Leu
Thr Val Asp Arg Ser Ser Arg Ile Val Tyr 65 70 75 80 Met Glu Leu Arg
Ser Leu Thr Phe Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110
Thr Thr Leu Thr Val Ser Ser 115 <210> SEQ ID NO 3 <211>
LENGTH: 107 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic polypeptide
<400> SEQUENCE: 3 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala
Ser Gln Asp Val Ser Ile Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr
Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210>
SEQ ID NO 4 <211> LENGTH: 119 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 4 Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Thr
Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe
50 55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe
Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
<210> SEQ ID NO 5 <211> LENGTH: 107 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 5 Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn Tyr
20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Glu Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Tyr Asn Ser Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys 100 105 <210> SEQ ID NO 6 <211>
LENGTH: 119 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 6 Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile
Ser Gly Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Gly Arg Val Gly Tyr Ser Leu Tyr Asp Tyr Trp
Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210>
SEQ ID NO 7 <211> LENGTH: 214 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 7 Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly 20 25 30 Val
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr
Ile Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170
175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
<210> SEQ ID NO 8 <211> LENGTH: 448 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (65)..(65) <223> OTHER
INFORMATION: Any amino acid <400> SEQUENCE: 8 Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30
Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg
Phe 50 55 60 Xaa Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn
Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Leu Gly Pro Ser Phe Tyr
Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165
170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290
295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410
415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly 435 440 445 <210> SEQ ID NO 9 <211> LENGTH: 214
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 9
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5
10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr
Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
<210> SEQ ID NO 10 <211> LENGTH: 449 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 10 Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr
Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr
Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala
Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170
175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295
300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420
425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro 435 440 445 Gly
<210> SEQ ID NO 11 <211> LENGTH: 217 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 11 Val His Ser Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 1 5 10 15 Ser Val Gly
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val 20 25 30 Ser
Ile Gly Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40
45 Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg
50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser 65 70 75 80 Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Tyr Tyr Ile 85 90 95 Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170
175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
<210> SEQ ID NO 12 <211> LENGTH: 449 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 12 Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Thr
Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe
50 55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe
Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295
300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420
425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly 435 440 445 Lys <210> SEQ ID NO 13 <400> SEQUENCE:
13 000 <210> SEQ ID NO 14 <211> LENGTH: 195 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
14 Thr Gln Val Cys Thr Gly Thr Asp Met Lys Leu Arg Leu Pro Ala Ser
1 5 10 15 Pro Glu Thr His Leu Asp Met Leu Arg His Leu Tyr Gln Gly
Cys Gln 20 25 30 Val Val Gln Gly Asn Leu Glu Leu Thr Tyr Leu Pro
Thr Asn Ala Ser 35 40 45 Leu Ser Phe Leu Gln Asp Ile Gln Glu Val
Gln Gly Tyr Val Leu Ile 50 55 60 Ala His Asn Gln Val Arg Gln Val
Pro Leu Gln Arg Leu Arg Ile Val 65 70 75 80 Arg Gly Thr Gln Leu Phe
Glu Asp Asn Tyr Ala Leu Ala Val Leu Asp 85 90 95 Asn Gly Asp Pro
Leu Asn Asn Thr Thr Pro Val Thr Gly Ala Ser Pro 100 105 110 Gly Gly
Leu Arg Glu Leu Gln Leu Arg Ser Leu Thr Glu Ile Leu Lys 115 120 125
Gly Gly Val Leu Ile Gln Arg Asn Pro Gln Leu Cys Tyr Gln Asp Thr 130
135 140 Ile Leu Trp Lys Asp Ile Phe His Lys Asn Asn Gln Leu Ala Leu
Thr 145 150 155 160 Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys His Pro
Cys Ser Pro Met 165 170 175 Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser
Ser Glu Asp Cys Gln Ser 180 185 190 Leu Thr Arg 195 <210> SEQ
ID NO 15 <211> LENGTH: 124 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 15 Thr Val Cys Ala Gly
Gly Cys Ala Arg Cys Lys Gly Pro Leu Pro Thr 1 5 10 15 Asp Cys Cys
His Glu Gln Cys Ala Ala Gly Cys Thr Gly Pro Lys His 20 25 30 Ser
Asp Cys Leu Ala Cys Leu His Phe Asn His Ser Gly Ile Cys Glu 35 40
45 Leu His Cys Pro Ala Leu Val Thr Tyr Asn Thr Asp Thr Phe Glu Ser
50 55 60 Met Pro Asn Pro Glu Gly Arg Tyr Thr Phe Gly Ala Ser Cys
Val Thr 65 70 75 80 Ala Cys Pro Tyr Asn Tyr Leu Ser Thr Asp Val Gly
Ser Cys Thr Leu 85 90 95 Val Cys Pro Leu His Asn Gln Glu Val Thr
Ala Glu Asp Gly Thr Gln 100 105 110 Arg Cys Glu Lys Cys Ser Lys Pro
Cys Ala Arg Val 115 120 <210> SEQ ID NO 16 <211>
LENGTH: 169 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 16 Cys Tyr Gly Leu Gly Met Glu His
Leu Arg Glu Val Arg Ala Val Thr 1 5 10 15 Ser Ala Asn Ile Gln Glu
Phe Ala Gly Cys Lys Lys Ile Phe Gly Ser 20 25 30
Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser Asn Thr 35
40 45 Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe Glu Thr Leu Glu
Glu 50 55 60 Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro Asp Ser
Leu Pro Asp 65 70 75 80 Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg
Gly Arg Ile Leu His 85 90 95 Asn Gly Ala Tyr Ser Leu Thr Leu Gln
Gly Leu Gly Ile Ser Trp Leu 100 105 110 Gly Leu Arg Ser Leu Arg Glu
Leu Gly Ser Gly Leu Ala Leu Ile His 115 120 125 His Asn Thr His Leu
Cys Phe Val His Thr Val Pro Trp Asp Gln Leu 130 135 140 Phe Arg Asn
Pro His Gln Ala Leu Leu His Thr Ala Asn Arg Pro Glu 145 150 155 160
Asp Glu Cys Val Gly Glu Gly Leu Ala 165 <210> SEQ ID NO 17
<211> LENGTH: 142 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 17 Cys His Gln Leu Cys Ala Arg
Gly His Cys Trp Gly Pro Gly Pro Thr 1 5 10 15 Gln Cys Val Asn Cys
Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu 20 25 30 Glu Cys Arg
Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg 35 40 45 His
Cys Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val 50 55
60 Thr Cys Phe Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr
65 70 75 80 Lys Asp Pro Pro Phe Cys Val Ala Arg Cys Pro Ser Gly Val
Lys Pro 85 90 95 Asp Leu Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp
Glu Glu Gly Ala 100 105 110 Cys Gln Pro Cys Pro Ile Asn Cys Thr His
Ser Cys Val Asp Leu Asp 115 120 125 Asp Lys Gly Cys Pro Ala Glu Gln
Arg Ala Ser Pro Leu Thr 130 135 140 <210> SEQ ID NO 18
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (10)..(10) <223> OTHER INFORMATION: Ser or Asp
<400> SEQUENCE: 18 Gly Phe Thr Phe Thr Asp Tyr Thr Met Xaa 1
5 10 <210> SEQ ID NO 19 <211> LENGTH: 17 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic peptide <400> SEQUENCE: 19 Asp Val Asn
Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe Lys 1 5 10 15 Gly
<210> SEQ ID NO 20 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 20 Asn Leu Gly Pro Ser Phe
Tyr Phe Asp Tyr 1 5 10 <210> SEQ ID NO 21 <211> LENGTH:
11 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <400> SEQUENCE: 21 Lys
Ala Ser Gln Asp Val Ser Ile Gly Val Ala 1 5 10 <210> SEQ ID
NO 22 <211> LENGTH: 7 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (5)..(5) <223> OTHER INFORMATION: Arg
or Leu <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (6)..(6) <223> OTHER INFORMATION: Tyr
or Glu <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (7..(7) <223> OTHER INFORMATION: Thr or
Ser <400> SEQUENCE: 22 Ser Ala Ser Tyr Xaa Xaa Xaa 1 5
<210> SEQ ID NO 23 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <400> SEQUENCE: 23 Gln Gln Tyr Tyr Ile Tyr
Pro Tyr Thr 1 5
* * * * *
References