U.S. patent application number 11/359185 was filed with the patent office on 2006-08-24 for extending time to disease progression or survival in cancer patients.
This patent application is currently assigned to Genentech, Inc.. Invention is credited to Mika K. Derynck, Stephen M. Kelsey.
Application Number | 20060188509 11/359185 |
Document ID | / |
Family ID | 36699124 |
Filed Date | 2006-08-24 |
United States Patent
Application |
20060188509 |
Kind Code |
A1 |
Derynck; Mika K. ; et
al. |
August 24, 2006 |
Extending time to disease progression or survival in cancer
patients
Abstract
The present application describes extending time to disease
progression or survival in a cancer patient, where the patient's
cancer displays HER activation, by treating the patient with a HER
dimerization inhibitor, such as pertuzumab.
Inventors: |
Derynck; Mika K.; (San
Mateo, CA) ; Kelsey; Stephen M.; (Montara,
CA) |
Correspondence
Address: |
GENENTECH, INC.
1 DNA WAY
SOUTH SAN FRANCISCO
CA
94080
US
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
36699124 |
Appl. No.: |
11/359185 |
Filed: |
February 21, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60655277 |
Feb 23, 2005 |
|
|
|
Current U.S.
Class: |
424/155.1 |
Current CPC
Class: |
A61P 13/08 20180101;
A61P 35/04 20180101; A61P 35/00 20180101; A61P 43/00 20180101; C07K
2317/21 20130101; A61K 31/7068 20130101; C07K 2317/24 20130101;
C07K 16/32 20130101; A61P 15/00 20180101; A61K 2039/505 20130101;
A61K 45/06 20130101; A61K 39/395 20130101; C07K 16/3069 20130101;
C07K 2317/56 20130101; A61P 1/04 20180101; A61P 11/00 20180101;
A61K 39/39558 20130101 |
Class at
Publication: |
424/155.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Claims
1. A method for extending time to disease progression (TTP) or
survival in a cancer patient comprising administering a HER
dimerization inhibitor to the patient in an amount which extends
TTP or survival in the patent, wherein the patient's cancer
displays HER activation.
2. The method of claim 1 wherein the HER dimerization inhibitor is
a HER2 dimerization inhibitor.
3. The method of claim 1 wherein the HER dimerization inhibitor
inhibits HER heterodimerization.
4. The method of claim 1 wherein the patient's cancer displays HER2
activation.
5. The method of claim 1 wherein the patient's cancer displays HER2
phosphorylation.
6. The method of claim 5 wherein the patient's cancer displays HER2
phosphorylation as evaluated in a phospho-ELISA assay.
7. The method of claim 1 wherein the patient's cancer displays HER2
activation as evaluated by gene expression profiling.
8. The method of claim 1 wherein the HER dimerization inhibitor is
an antibody.
9. The method of claim 8 wherein the antibody binds to a HER
receptor selected from the group consisting of EGFR, HER2, and
HER3.
10. The method of claim 9 wherein the antibody binds to HER2.
11. The method of claim 10 wherein the HER2 antibody binds to
Domain II of HER2 extracellular domain.
12. The method of claim 11 wherein the antibody binds to a junction
between domains I, II and III of HER2 extracellular domain.
13. The method of claim 12 wherein the HER2 antibody comprises the
variable light and variable heavy amino acid sequences in SEQ ID
NOS. 3 and 4, respectively.
14. The method of claim 13 wherein the HER2 antibody is
pertuzumab.
15. The method of claim 8 wherein the HER antibody is a naked
antibody.
16. The method of claim 8 wherein the HER antibody is an intact
antibody.
17. The method of claim 8 wherein the HER antibody is an antibody
fragment comprising an antigen binding region.
18. The method of claim 1 wherein the cancer is selected from the
group consisting of ovarian cancer, peritoneal cancer, fallopian
tube cancer, metastatic breast cancer (MBC), non-small cell lung
cancer (NSCLC), prostate cancer, and colorectal cancer.
19. The method of claim 18 wherein the cancer is ovarian,
peritoneal, or fallopian tube cancer.
20. The method of claim 18 wherein the cancer is advanced,
refractory or recurrent ovarian cancer.
21. The method of claim 1 wherein the HER dimerization inhibitor is
administered as a single anti-tumor agent.
22. The method of claim 1 comprising administering a second
therapeutic agent to the patient.
23. The method claim 22 wherein the second therapeutic agent is
selected from the group consisting of chemotherapeutic agent, 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.
24. The method of claim 23 wherein the second therapeutic agent is
a chemotherapeutic agent.
25. The method of claim 24 wherein the chemotherapeutic agent is an
antimetabolite chemotherapeutic agent.
26. The method of claim 25 wherein the antimetabolite
chemotherapeutic agent is gemcitabine.
27. The method of claim 22 wherein the second therapeutic agent is
trastuzumab, erlotinib, or bevacizumab.
28. The method of claim 1 wherein TTP is extended.
29. The method of claim 1 wherein survival is extended.
30. The method of claim 1 wherein administration of the HER
dimerization inhibitor extends TTP or survival at least about 20%
more than TTP or survival achieved by administering an approved
anti-tumor agent to the cancer patient.
31. A method for extending time to disease progression (TTP) or
survival in a patient with ovarian, peritoneal, or fallopian tube
cancer comprising administering pertuzumab to the patient in an
amount which extends TTP or survival in the patent, wherein the
patient's cancer displays HER2 activation.
32. The method of claim 31 wherein patient has ovarian cancer.
33. The method of claim 31 wherein the patient has advanced,
refractory or recurrent cancer.
34. The method of claim 31 wherein administration of pertuzumab to
the patient extends TTP or survival at least about 20% more than
TTP or survival achieved by administering topotecan or liposomal
doxorubicin to the patient.
35. The method of claim 31 further comprising administering a
chemotherapeutic agent to the patient.
36. The method of claim 35 wherein the chemotherapeutic agent is an
antimetabolite chemotherapeutic agent.
37. The method of claim 36 wherein the antimetabolite
chemotherapeutic agent is gemcitabine.
Description
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 60/655,277, filed 23 Feb. 2005, the entire
disclosure of which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention concerns extending time to disease
progression or survival in a cancer patient, where the patient's
cancer displays HER activation, by treating the patient with a HER
dimerization inhibitor, such as pertuzumab.
BACKGROUND OF THE INVENTION
HER Receptors and Antibodies Thereagainst
[0003] The HER family of receptor tyrosine kinases are important
mediators of cell growth, differentiation and survival. The
receptor family includes four distinct members including epidermal
growth factor receptor (EGFR, ErbB1, or HER1), HER2 (ErbB2 or
p185.sup.neu), HER3 (ErbB3) and HER4 (ErbB4 or tyro2).
[0004] EGFR, encoded by the erbB1 gene, has been causally
implicated in human malignancy. In particular, increased expression
of EGFR has been observed in breast, bladder, lung, head, neck and
stomach cancer as well as glioblastomas. Increased EGFR receptor
expression is often associated with increased production of the
EGFR ligand, transforming growth factor alpha (TGF-.alpha.), by the
same tumor cells resulting in receptor activation by an autocrine
stimulatory pathway. Baselga and Mendelsohn Pharmac. Ther.
64:127-154 (1994). Monoclonal antibodies directed against the EGFR
or its ligands, TGF-.alpha. and EGF, have been evaluated as
therapeutic agents in the treatment of such malignancies. See,
e.g., Baselga and Mendelsohn., supra; Masui et al. Cancer Research
44:1002-1007 (1984); and Wu et al. J. Clin. Invest. 95:1897-1905
(1995).
[0005] The second member of the HER family, p185.sup.neu, was
originally identified as the product of the transforming gene from
neuroblastomas of chemically treated rats. The activated form of
the neu proto-oncogene results from a point mutation (valine to
glutamic acid) in the transmembrane region of the encoded protein.
Amplification of the human homolog of neu is observed in breast and
ovarian cancers and correlates with a poor prognosis (Slamon et
al., Science, 235:177-182 (1987); Slamon et al., Science,
244:707-712 (1989); and U.S. Pat. No. 4,968,603). To date, no point
mutation analogous to that in the neu proto-oncogene has been
reported for human tumors. Overexpression of HER2 (frequently but
not uniformly due to gene amplification) has also been observed in
other carcinomas including carcinomas of the stomach, endometrium,
salivary gland, lung, kidney, colon, thyroid, pancreas and bladder.
See, among others, King et al., Science, 229:974 (1985); Yokota et
al., Lancet: 1:765-767 (1986); Fukushige et al., Mol Cell Biol.,
6:955-958 (1986); Guerin et al., Oncogene Res., 3:21-31 (1988);
Cohen et al., Oncogene, 4:81-88 (1989); Yonemura et al., Cancer
Res., 51:1034 (1991); Borst et al., Gynecol. Oncol., 38:364 (1990);
Weiner et al., Cancer Res., 50:421-425 (1990); Kern et al., Cancer
Res., 50:5184 (1990); Park et al., Cancer Res., 49:6605 (1989);
Zhau et al., Mol. Carcinog., 3:254-257 (1990); Aasland et al. Br.
J. Cancer 57:358-363 (1988); Williams et al. Pathobiology 59:46-52
(1991); and McCann et al., Cancer, 65:88-92 (1990). HER2 may be
overexpressed in prostate cancer (Gu et al. Cancer Lett. 99:185-9
(1996); Ross et al. Hum. Pathol. 28:827-33 (1997); Ross et al.
Cancer 79:2162-70 (1997); and Sadasivan et al. J. Urol. 150:126-31
(1993)).
[0006] 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, p185.sup.neu See, for example, Drebin et al.,
Cell 41:695-706 (1985); Myers et al., Meth. Enzym. 198:277-290
(1991); and WO94/22478. Drebin et al. Oncogene 2:273-277 (1988)
report that mixtures of antibodies reactive with two distinct
regions of p185.sup.neu result in synergistic anti-tumor effects on
neu-transformed NIH-3T3 cells implanted into nude mice. See also
U.S. Pat. No. 5,824,311 issued Oct. 20, 1998.
[0008] Hudziak et al., Mol. Cell. Biol. 9(3):1165-1172 (1989)
describe the generation of a panel of 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.
[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, US2003/0147884A 1, US2003/0170234A
1, US2005/0002928A 1, U.S. Pat. No. 6,573,043, US2003/0152987A1,
WO99/48527, US2002/0141993A1, WO01/00245, US2003/0086924,
US2004/0013667A1, WO00/69460, WO01/00238, WO/015730, U.S. Pat. No.
6,627,196B1, U.S. Pat. No. 6,632,979B1, WO01/00244,
US2002/0090662A1, WO01/89566, US2002/0064785, US2003/0134344, WO
04/24866, US2004/0082047, US2003/0175845A1, WO03/087131,
US2003/0228663, WO2004/008099A2, US2004/0106161, WO2004/048525,
US2004/0258685A1, U.S. Pat. No. 5,985,553, U.S. Pat. No. 5,747,261,
U.S. Pat. No. 4,935,341, U.S. Pat. No. 5,401,638, U.S. Pat. No.
5,604,107, WO 87/07646, WO 89/10412, WO 91/05264, EP 412,116 B1, EP
494,135 B1, U.S. Pat. No. 5,824,311, EP 444,181 B1, EP 1,006,194
A2, US 2002/0155527A1, WO 91/02062, U.S. Pat. No. 5,571,894, U.S.
Pat. No. 5,939,531, EP 502,812 B1, WO 93/03741, EP 554,441 B1, EP
656,367 A1, U.S. Pat. No. 5,288,477, U.S. Pat. No. 5,514,554, U.S.
Pat. No. 5,587,458, WO 93/12220, WO 93/16185, U.S. Pat. No.
5,877,305, WO 93/21319, WO 93/21232, U.S. Pat. No. 5,856,089, WO
94/22478, U.S. Pat. No. 5,910,486, U.S. Pat. No. 6,028,059, WO
96/07321, U.S. Pat. No. 5,804,396, U.S. Pat. No. 5,846,749, EP
711,565, WO 96/16673, U.S. Pat. No. 5,783,404, U.S. Pat. No.
5,977,322, U.S. Pat. No. 6,512,097, WO 97/00271, U.S. Pat. No.
6,270,765, U.S. Pat. No. 6,395,272, U.S. Pat. No. 5,837,243, WO
96/40789, U.S. Pat. No. 5,783,186, U.S. Pat. No. 6,458,356, WO
97/20858, WO 97/38731, U.S. Pat. No. 6,214,388, U.S. Pat. No.
5,925,519, WO 98/02463, U.S. Pat. No. 5,922,845, WO 98/18489, WO
98/33914, U.S. Pat. No. 5,994,071, WO 98/45479, U.S. Pat. No.
6,358,682 B1, US 2003/0059790, WO 99/55367, WO 01/20033, US
2002/0076695 A1, WO 00/78347, WO 01/09187, WO 01/21192, WO
01/32155, WO 01/53354, WO 01/56604, WO 01/76630, WO02/05791, WO
02/11677, U.S. Pat. No. 6,582,919, US2002/0192652A1, US
2003/0211530A1, WO 02/44413, US 2002/0142328, U.S. Pat. No.
6,602,670 B2, WO 02/45653, WO 02/055106, US 2003/0152572, US
2003/0165840, WO 02/087619, WO 03/006509, WO03/012072, WO
03/028638, US 2003/0068318, WO 03/041736, EP 1,357,132, US
2003/0202973, US 2004/0138160, U.S. Pat. No. 5,705,157, U.S. Pat.
No. 6,123,939, EP 616,812 B1, US 2003/0103973, US 2003/0108545,
U.S. Pat. No. 6,403,630 B1, WO 00/61145, WO 00/61185, U.S. Pat. No.
6,333,348 B1, WO 01/05425, WO 01/64246, US 2003/0022918, US
2002/0051785 A1, U.S. Pat. No. 6,767,541, WO 01/76586, US
2003/0144252, WO 01/87336, US 2002/0031515 A1, WO 01/87334, WO
02/05791, WO 02/09754, US 2003/0157097, US 2002/0076408, WO
02/055106, WO 02/070008, WO 02/089842 and WO 03/86467.
Diagnostics
[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, US2003/0152987, Cohen et al, concerning immunohistochemistry
(1HC) 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. US2004/013297A 1 (Bacus
et al.) concerns determining or predicting response to ABX0303 EGFR
antibody therapy. WO2004/000094 (Bacus et al.) is directed to
determining response to GW572016, a small molecule, EGFR-HER2
tyrosine kinase inhibitor. WO2004/063709, Amler et al., refers to
biomarkers and methods for determining sensitivity to EGFR
inhibitor, erlotinib HCl. US2004/0209290, Cobleigh et al., concerns
gene expression markers for breast cancer prognosis. Patients
treated with pertuzumab can be selected for therapy based on HER
activation or dimerization. Patent publications concerning
pertuzumab and selection of patients for therapy therewith include:
WO01/00245 (Adams et al.); US2003/0086924 (Sliwkowski, M.);
US2004/0013667A1 (Sliwkowski, M.); as well as WO2004/008099A2, and
US2004/0106161 (Bossenmaier et al.).
[0017] Cronin et al. Am. J. Path. 164(1): 35-42 (2004) describes
measurement of gene expression in archival paraffin-embedded
tissues. Ma et al. Cancer Cell 5:607-616 (2004) describes gene
profiling by gene oliogonucleotide microarray using isolated RNA
from tumor-tissue sections taken from archived primary
biopsies.
[0018] 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).
[0019] 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)).
[0020] 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)).
SUMMARY OF THE INVENTION
[0021] The present invention provides the clinical data from human
cancer patients treated with a HER dimerization inhibitor,
pertuzumab. Patients were evaluated for HER activation, as
determined using a phospho-ELISA bioassay. Clinical benefit, as
measured by time to disease progression (TTP) and survival, was
observed in patients displaying HER activation.
[0022] Accordingly, the invention provides a method for extending
time to disease progression (TTP) or survival in a cancer patient
comprising administering a HER dimerization inhibitor to the
patient in an amount which extends TTP or survival in the patent,
wherein the patient's cancer displays HER activation.
[0023] The invention also concerns a method for extending time to
disease progression (TTP) or survival in a patient with ovarian,
peritoneal, or fallopian tube cancer comprising administering
pertuzumab to the patient in an amount which extends TTP or
survival in the patent, wherein the patient's cancer displays HER2
activation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 provides a schematic of the HER2 protein structure,
and amino acid sequences for Domains I-IV (SEQ ID NOS. 19-22,
respectively) of the extracellular domain thereof.
[0025] 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.
[0026] FIGS. 3A and 3B show the amino acid sequences of pertuzumab
light chain (FIG. 3A; SEQ ID NO. 13) and heavy chain (FIG. 3B; SEQ
ID NO. 14). 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.
[0027] FIG. 4 depicts, schematically, binding of 2C4 at the
heterodimeric binding site of HER2, thereby preventing
heterodimerization with activated EGFR or HER3.
[0028] FIG. 5 depicts coupling of HER2/HER3 to the MAPK and Akt
pathways.
[0029] FIG. 6 compares various activities of trastuzumab and
pertuzumab.
[0030] FIGS. 7A and 7B show the amino acid sequences of trastuzumab
light chain (FIG. 7A; SEQ ID No. 15) and heavy chain (FIG. 7B; SEQ
ID NO. 16), respectively.
[0031] FIGS. 8A and 8B depict a variant pertuzumab light chain
sequence (FIG. 8A; SEQ ID NO. 17) and a variant pertuzumab heavy
chain sequence (FIG. 8B; SEQ ID NO. 18), respectively.
[0032] FIG. 9 provides baseline demographics of patients treated in
Example 1.
[0033] FIG. 10 shows all grade 3-4 adverse events (irrespective of
relatedness to treatment).
[0034] FIG. 11 shows serious adverse events (irrespective or
relatedness to treatment).
[0035] FIG. 12 summarizes serious adverse events judged to be
related to study drug by investigators.
[0036] FIG. 13 provides information on selected adverse events.
[0037] FIG. 14 depicts cardiac serious adverse events and adverse
events requiring expedited reporting.
[0038] FIG. 15 summarizes efficacy results for the phase II study
of pertuzumab in Example 1.
[0039] FIG. 16 shows time to disease progression (TTP) efficacy for
evaluable ovarian cancer subjects treated with either a low dose
(420 mg) or high dose (1050 mg) of pertuzumab.
[0040] FIG. 17 shows overall survival efficacy for evaluable
ovarian cancer subjects treated with either low dose (420 mg) or
high dose (1050 mg) of pertuzumab. Historical median survival for
ovarian cancer subjects treated with topotecan was 43 weeks, and
for liposomal doxorubicin was 36 weeks.
[0041] FIG. 18 provides CA-125 responses for ovarian cancer
subjects treated with either 420 mg or 1050 mg of pertuzumab.
[0042] FIG. 19 provides phospho-HER2 (pHER2) status, as determined
by ELISA, for ovarian cancer subjects treated with 420 mg of
pertuzumab.
[0043] FIG. 20 provides clinical efficacy results by pHER2 status,
as determined by ELISA, for ovarian cancer subjects treated with
420 mg of pertuzumab.
[0044] FIG. 21 provides pHER2 status, as determined by ELISA, for
ovarian patients treated with 420 mg of pertuzumab showing evidence
of activity (partial response, PR, or stable disease, SD, for
greater than 18 weeks). BSLD refers to baseline sum of longest
diameter.
[0045] FIG. 22 shows TTP efficacy by pHER2 status. Ovarian cancer
subjects were treated with 420 mg of pertuzumab. Overall TTP was
6.6 weeks; TTP in pHER positive subjects was 20.9 weeks; TTP in
pHER2 negative subjects was 6.0 weeks; and TTP in subjects with
unknown pHER2 status was 9.1 weeks.
[0046] FIG. 23 depicts overall survival by pHER2 status. Ovarian
cancer subjects were treated with 420 mg of pertuzumab. Historical
median survival for ovarian cancer subjects treated with topotecan
was 43 weeks, and for liposomal doxorubicin was 36 weeks.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0047] I. Definitions
[0048] Herein "time to disease progression" or "TTP" refer to the
time, generally measured in weeks or months, from the time of
initial treatment (e.g. with a HER dimerization inhibitor, such as
pertuzumab), until the cancer progresses or worsens. Such
progression can be evaluated by the skilled clinician. In the case
of ovarian cancer, for instance, progression can be evaluated by
RECIST (see, for example, Therasse et al., J. Nat. Cancer Inst.
92(3): 205-216 (2000)).
[0049] By "extending TTP" is meant increasing the time to disease
progression in a treated patient relative to an untreated patient
(i.e. relative to a patient not treated with a HER dimerization
inhibitor, such as pertuzumab), or relative to a patient who does
not display HER activation, and/or relative to a patient treated
with an approved anti-tumor agent (such as topotecan or liposomal
doxorubicin, where the cancer is ovarian cancer).
[0050] "Survival" refers to the patient remaining alive, and
includes overall survival as well as progression free survival.
[0051] "Overall survival" refers to the patient remaining alive for
a defined period of time, such as I year, 5 years, etc from the
time of diagnosis or treatment.
[0052] "Progression free survival" refers to the patient remaining
alive, without the cancer progressing or getting worse.
[0053] By "extending survival" is meant increasing overall or
progression free survival in a treated patient relative to an
untreated patient (i.e. relative to a patient not treated with a
HER dimerization inhibitor, such as pertuzumab), or relative to a
patient who does not display HER activation, and/or relative to a
patient treated with an approved anti-tumor agent (such as
topotecan or liposomal doxorubicin, where the cancer is ovarian
cancer).
[0054] An "objective response" refers to a measurable response,
including complete response (CR) or partial response (PR).
[0055] 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.
[0056] "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.
[0057] 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.
[0058] The terms "ErbB1," "HER1", "epidermal growth factor
receptor" and "EGFR" are used interchangeably herein and refer to
EGFR as disclosed, for example, in Carpenter et al. Ann. Rev.
Biochem. 56:881-914 (1987), including naturally occurring mutant
forms thereof (e.g. a deletion mutant EGFR as in Humphrey et al.
PNAS (USA) 87:4207-4211 (1990)). erbB1 refers to the gene encoding
the EGFR protein product.
[0059] 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.
[0060] Herein, "HER2 extracellular domain" or "HER2 ECD" refers to
a domain of HER2 that is outside of a cell, either anchored to a
cell membrane, or in circulation, including fragments thereof. In
one embodiment, the extracellular domain of HER2 may comprise four
domains: "Domain I" (amino acid residues from about 1-195; SEQ ID
NO: 19), "Domain II" (amino acid residues from about 196-319; SEQ
ID NO:20), "Domain III" (amino acid residues from about 320-488:
SEQ ID NO:21), and "Domain IV" (amino acid residues from about
489-630; SEQ ID NO:22) (residue numbering without signal peptide).
See Garrett et al. Mol. Cell. 11: 495-505 (2003), Cho et al. Nature
421: 756-760 (2003), Franklin et al. Cancer Cell 5:317-328 (2004),
and Plowman et al. Proc. Natl. Acad. Sci. 90:1746-1750 (1993), as
well as FIG. 1 herein.
[0061] "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).
[0062] 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.
[0063] 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.
[0064] "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)).
[0065] 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.
[0066] 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.
[0067] A "HER inhibitor" is an agent which interferes with HER
activation or function. Examples of HER inhibitors include HER
antibodies (e.g. EGFR, HER2, HER3, or HER4 antibodies);
EGFR-targeted drugs; small molecule HER antagonists; HER tyrosine
kinase inhibitors; HER2 and EGFR dual tyrosine kinase inhibitors
such as lapatinib/GW572016; antisense molecules (see, for example,
WO2004/87207); and/or agents that bind to, or interfere with
function of, downstream signaling molecules, such as MAPK or Akt
(see FIG. 5). Preferably, the HER inhibitor is an antibody or small
molecule which binds to a HER receptor.
[0068] A "HER dimerization inhibitor" is an agent which inhibits
formation of a HER dimer or HER heterodimer. Preferably, the HER
dimerization inhibitor is an antibody, for example an antibody
which binds to HER2 at the heterodimeric binding site thereof. The
most preferred HER dimerization inhibitor herein is pertuzumab or
MAb 2C4. Binding of 2C4 to the heterodimeric binding site of HER2
is illustrated in FIG. 4. Other examples of HER dimerization
inhibitors 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;
antibodies which bind to HER4 and inhibit dimerization thereof with
one or more other HER receptors; peptide dimerization inhibitors
(U.S. Pat. No. 6,417,168); antisense dimerization inhibitors;
etc.
[0069] A "HER2 dimerization inhibitor" is an agent that inhibits
formation of a dimer or heterodimer comprising HER2.
[0070] 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. A HER2 antibody of particular interest herein is
pertuzumab. Another example of a HER2 antibody is trastuzumab.
Examples of EGFR antibodies include cetuximab and ABX0303.
[0071] "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, see, FIG. 5, for example.
[0072] "Phosphorylation" refers to the addition of one or more
phosphate group(s) to a protein, such as a HER receptor, or
substrate thereof.
[0073] 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. Binding of 2C4 to the
heterodimeric binding site of HER2 is illustrated in FIG. 4. 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.
[0074] An antibody which "blocks ligand activation of a HER
receptor more effectively than trastuzumab" is one which reduces or
eliminates HER ligand activation of HER receptor(s) or HER dimer(s)
more effectively (for example at least about 2-fold more
effectively) than trastuzumab. Preferably, such an antibody blocks
HER ligand activation of a HER receptor at least about as
effectively as murine monoclonal antibody 2C4 or a Fab fragment
thereof, or as pertuzumab or a Fab fragment thereof. One can
evaluate the ability of an antibody to block ligand activation of a
HER receptor by studying HER dimers directly, or by evaluating HER
activation, or downstream signaling, which results from HER
dimerization, and/or by evaluating the antibody-HER2 binding site,
etc. Assays for screening for antibodies with the ability to
inhibit ligand activation of a HER receptor more effectively than
trastuzumab are described in Agus et al. Cancer Cell 2: 127-137
(2002) and WO01/00245 (Adams et al.). By way of example only, one
may assay for: inhibition of HER dimer formation (see, e.g., FIG.
1A-B of Agus et al. Cancer Cell 2: 127-137 (2002); and WO01/00245);
reduction in HER ligand activation of cells which express HER
dimers (WO01/00245 and FIG. 2A-B of Agus et al. Cancer Cell 2:
127-137 (2002), for example); blocking of HER ligand binding to
cells which express HER dimers (WO01/00245, and FIG. 2E of Agus et
al. Cancer Cell 2: 127-137 (2002), for example); cell growth
inhibition of cancer cells (e.g. MCF7, MDA-MD-134, ZR-75-1,
MD-MB-175, T-47D cells) which express HER dimers in the presence
(or absence) of HER ligand (WO01/00245 and FIGS. 3A-D of Agus et
al. Cancer Cell 2: 127-137 (2002), for instance); inhibition of
downstream signaling (for instance, inhibition of HRG-dependent AKT
phosphorylation or inhibition of HRG- or TGF.alpha.-dependent MAPK
phosphorylation) (see, WO01/00245, and FIG. 2C-D of Agus et al.
Cancer Cell 2: 127-137 (2002), for example). One may also assess
whether the antibody inhibits HER dimerization by studying the
antibody-HER2 binding site, for instance, by evaluating a structure
or model, such as a crystal structure, of the antibody bound to
HER2 (See, for example, Franklin et al. Cancer Cell 5:317-328
(2004)).
[0075] 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. Franklin et al. Cancer Cell 5:317-328 (2004).
[0076] The HER2 antibody may "inhibit HRG-dependent AKT
phosphorylation" and/or inhibit "HRG- or TGF.alpha.-dependent MAPK
phosphorylation" more effectively (for instance at least 2-fold
more effectively) than trastuzumab (see Agus et al. Cancer Cell 2:
127-137 (2002) and WO01/00245, by way of example).
[0077] The HER2 antibody may be one which, like pertuzumab, does
"not inhibit HER2 ectodomain cleavage" (Molina et al. Cancer Res.
61:4744-4749(2001)). Trastuzumab, on the other hand, can inhibit
HER2 ectodomain cleavage.
[0078] A HER2 antibody that "binds to a heterodimeric binding site"
of HER2, binds to residues in domain II (and optionally also binds
to residues in other of the domains of the HER2 extracellular
domain, such as domains I and III), and can sterically hinder, at
least to some extent, formation of a HER2-EGFR, HER2-HER3, or
HER2-HER4 heterodimer. Franklin et al. Cancer Cell 5:317-328 (2004)
characterize the HER2-pertuzumab crystal structure, deposited with
the RCSB Protein Data Bank (ID Code IS78), illustrating an
exemplary antibody that binds to the heterodimeric binding site of
HER2.
[0079] An antibody that "binds to domain II" of HER2 binds to
residues in domain II and optionally residues in other domain(s) of
HER2, such as domains I and III. Preferably the antibody that binds
to domain II binds to the junction between domains I, II and III of
HER2.
[0080] Protein "expression" refers to conversion of the information
encoded in a gene into messenger RNA (mRNA) and then to the
protein.
[0081] 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.
[0082] 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.
[0083] "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).
[0084] The term "microarray" refers to an ordered arrangement of
hybridizable array elements, preferably polynucleotide probes, on a
substrate.
[0085] The term "polynucleotide," when used in singular or plural,
generally refers to any polyribonucleotide or
polydeoxribonucleotide, 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.
[0086] 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.
[0087] 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.
[0088] "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).
[0089] "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.
[0090] "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.
[0091] 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.
[0092] 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.
[0093] 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)).
[0094] The monoclonal antibodies herein specifically include
"chimeric" antibodies in which a portion of the heavy and/or light
chain is identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad.
Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest
herein include "primatized" antibodies comprising variable domain
antigen-binding sequences derived from a non-human primate (e.g.
Old World Monkey, Ape etc) and human constant region sequences, as
well as "humanized" antibodies.
[0095] "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.
[0096] 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).
[0097] 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.
[0098] For the purposes herein, "trastuzumab," "HERCEPTIN.RTM.,"
and "huMAb4D5-8" refer to an antibody comprising the light and
heavy chain amino acid sequences in SEQ ID NOS. 15 and 16,
respectively.
[0099] Herein, "pertuzumab" and "OMNITARG.TM." refer to an antibody
comprising the light and heavy chain amino acid sequences in SEQ ID
NOS. 13 and 14, respectively.
[0100] Differences between trastuzumab and pertuzumab functions are
illustrated in FIG. 6.
[0101] 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.
[0102] "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).
[0103] "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.
[0104] 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).
[0105] 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.
[0106] 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.
[0107] "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.
[0108] 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.
[0109] 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.
[0110] 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.
[0111] 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.
[0112] 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.
[0113] 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.
[0114] 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.
[0115] Depending on the amino acid sequence of the constant domain
of their heavy chains, intact antibodies can be assigned to
different "classes". There are five major classes of intact
antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may
be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2,
IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that
correspond to the different classes of antibodies are called
.alpha., .delta., .epsilon., .gamma., and .mu., respectively. The
subunit structures and three-dimensional configurations of
different classes of immunoglobulins are well known.
[0116] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC"
refer to a cell-mediated reaction in which nonspecific cytotoxic
cells that express Fc receptors (FcRs) (e.g. Natural Killer (NK)
cells, neutrophils, and macrophages) recognize bound antibody on a
target cell and subsequently cause lysis of the target cell. The
primary cells for mediating ADCC, NK cells, express Fc.gamma.RIII
only, whereas monocytes express Fc.gamma.RI, Fc.gamma.RII and
Fc.gamma.RIII. FcR expression on hematopoietic cells in summarized
is Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol
9:457-92 (1991). To assess ADCC activity of a molecule of interest,
an in vitro ADCC assay, such as that described in U.S. Pat. No.
5,500,362 or 5,821,337 may be performed. Useful effector cells for
such assays include peripheral blood mononuclear cells (PBMC) and
Natural Killer (NK) cells. Alternatively, or additionally, ADCC
activity of the molecule of interest may be assessed in vivo, e.g.,
in a animal model such as that disclosed in Clynes et al. PNAS
(USA) 95:652-656 (1998).
[0117] "Human effector cells" are leukocytes which express one or
more FcRs and perform effector functions. Preferably, the cells
express at least Fc.gamma.RIII and perform ADCC effector function.
Examples of human leukocytes which mediate ADCC include peripheral
blood mononuclear cells (PBMC), natural killer (NK) cells,
monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK
cells being preferred. The effector cells may be isolated from a
native source thereof, e.g. from blood or PBMCs as described
herein.
[0118] The terms "Fc receptor" or "FcR" are used to describe a
receptor that binds to the Fc region of an antibody. The preferred
FcR is a native sequence human FcR. Moreover, a preferred FcR is
one which binds an IgG antibody (a gamma receptor) and includes
receptors of the Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma. RIII
subclasses, including allelic variants and alternatively spliced
forms of these receptors. Fc.gamma.RII receptors include
Fc.gamma.RIIA (an "activating receptor") and Fc.gamma.RIIB (an
"inhibiting receptor"), which have similar amino acid sequences
that differ primarily in the cytoplasmic domains thereof.
Activating receptor Fc.gamma.RIIA contains an immunoreceptor
tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
Inhibiting receptor Fc.gamma.RIIB contains an immunoreceptor
tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain
(see review M. in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)).
FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92
(1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et
al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including
those to be identified in the future, are encompassed by the term
"FcR" herein. The term also includes the neonatal receptor, FcRn,
which is responsible for the transfer of maternal IgGs to the fetus
(Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J.
Immunol. 24:249 (1994)), and regulates homeostasis of
immunoglobulins.
[0119] "Complement dependent cytotoxicity" or "CDC" refers to the
ability of a molecule to lyse a target in the presence of
complement. The complement activation pathway is initiated by the
binding of the first component of the complement system (C1q) to a
molecule (e.g. an antibody) complexed with a cognate antigen. To
assess complement activation, a CDC assay, e.g. as described in
Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be
performed.
[0120] "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.
[0121] 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).
[0122] A "naked antibody" is an antibody that is not conjugated to
a heterologous molecule, such as a cytotoxic moiety or
radiolabel.
[0123] 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.
[0124] An "affinity matured" antibody is one with one or more
alterations in one or more hypervariable regions thereof which
result an improvement in the affinity of the antibody for antigen,
compared to a parent antibody which does not possess those
alteration(s). Preferred affinity matured antibodies will have
nanomolar or even picomolar affinities for the target antigen.
Affinity matured antibodies are produced by procedures known in the
art. Marks et al. Bio/Technology 10:779-783 (1992) describes
affinity maturation by V.sub.H and V.sub.L domain shuffling. Random
mutagenesis of CDR and/or framework residues is described by:
Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier
et al. Gene 169:147-155(1995); Yelton et al. J. Immunol.
155:1994-2004(1995); Jackson et al., J. Immunol. 154(7):3310-9
(1995); and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).
[0125] 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 in SEQ ID Nos. 3 and 4, and most preferably comprising
the light chain and heavy chain amino acid sequences in SEQ ID NOS.
13 and 14 (pertuzumab).
[0126] 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.
[0127] A "glycosylation variant" antibody herein is an antibody
with one or more carbohydrate moeities attached thereto which
differ from one or more carbohydate 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.
[0128] 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.
[0129] Unless indicated otherwise, a "G1 oligosaccharide structure"
herein includes G-1, G1-1, G1(1-6) and G1(1-3) structures.
[0130] 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.
[0131] 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.
[0132] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. Examples of cancer include, but are not
limited to, carcinoma, lymphoma, blastoma (including
medulloblastoma and retinoblastoma), sarcoma (including liposarcoma
and synovial cell sarcoma), neuroendocrine tumors (including
carcinoid tumors, gastrinoma, and islet cell cancer), mesothelioma,
schwannoma (including acoustic neuroma), meningioma,
adenocarcinoma, melanoma, and leukemia or lymphoid malignancies.
More particular examples of such cancers include squamous cell
cancer (e.g. epithelial squamous cell cancer), lung cancer
including small-cell lung cancer (SCLC), non-small cell lung cancer
(NSCLC), adenocarcinoma of the lung and squamous carcinoma of the
lung, cancer of the peritoneum, hepatocellular cancer, gastric or
stomach cancer including gastrointestinal cancer, pancreatic
cancer, glioblastoma, cervical cancer, ovarian cancer, liver
cancer, bladder cancer, hepatoma, breast cancer (including
metastatic breast cancer), colon cancer, rectal cancer, colorectal
cancer, endometrial or uterine carcinoma, salivary gland carcinoma,
kidney or renal cancer, prostate cancer, vulval cancer, thyroid
cancer, hepatic carcinoma, anal carcinoma, penile carcinoma,
testicular cancer, esophagael cancer, tumors of the biliary tract,
as well as head and neck cancer.
[0133] An "advanced" cancer is one which has spread outside the
site or organ of origin, either by local invasion or
metastasis.
[0134] 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.
[0135] A "recurrent" cancer is one which has regrown, either at the
initial site or at a distant site, after a response to initial
therapy.
[0136] 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.
[0137] A "tumor sample" herein is a sample derived from, or
comprising tumor cells from, a patient's tumor. Examples of tumor
samples herein include, but are not limited to, tumor biopsies,
circulating tumor cells, circulating plasma proteins, ascitic
fluid, primary cell cultures or cell lines derived from tumors or
exhibiting tumor-like properties, as well as preserved tumor
samples, such as formalin-fixed, paraffin-embedded tumor samples or
frozen tumor samples.
[0138] A "fixed" tumor sample is one which has been histologically
preserved using a fixative.
[0139] A "formalin-fixed" tumor sample is one which has been
preserved using formaldehyde as the fixative.
[0140] 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).
[0141] A "paraffin-embedded" tumor sample is one surrounded by a
purified mixture of solid hydrocarbons derived from petroleum.
[0142] Herein, a "frozen" tumor sample refers to a tumor sample
which is, or has been, frozen.
[0143] 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.
[0144] 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).
[0145] Herein, "gene expression profiling" refers to an evaluation
of expression of one or more genes as a surrogate for determining
HER phosphorylation directly.
[0146] 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.
[0147] 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.
[0148] 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.
[0149] 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.
[0150] 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.
[0151] 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.
[0152] By "standard of care" herein is intended the anti-tumor
agent or agents that are routinely used to treat a particular form
of cancer. For example, for platinum-resistant ovarian cancer, the
standard of care is topotecan or liposomal doxorubicin.
[0153] 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. (W B Saunders:
Philadelphia, 1995), especially p. 13.
[0154] 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/ml, where the
growth inhibition is determined six days after exposure of the
SK-BR-3 cells to the antibody (see U.S. Pat. No. 5,677,171 issued
Oct. 14, 1997). The SK-BR-3 cell growth inhibition assay is
described in more detail in that patent and hereinbelow. The
preferred growth inhibitory antibody is a humanized variant of
murine monoclonal antibody 4D5, e.g., trastuzumab.
[0155] 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.
[0156] 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 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 to the 2C4 epitope
of HER2. Epitope 2C4 comprises residues from Domain II 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. Franklin et al. Cancer Cell 5:317-328 (2004).
[0157] 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. To screen for antibodies which bind
to the 4D5 epitope, a routine cross-blocking assay such as that
described in Antibodies, A Laboratory Manual, Cold Spring Harbor
Laboratory, Ed Harlow and David Lane (1988), can be performed.
Alternatively, epitope mapping can be performed to assess whether
the antibody binds to the 4D5 epitope of HER2 (e.g. any one or more
residues in the region from about residue 529 to about residue 625,
inclusive of the HER2 ECD, residue numbering including signal
peptide).
[0158] The "epitope 7C2/7F3" is the region at the N terminus,
within Domain I, 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 to the 7C2/7F3 epitope, a
routine cross-blocking assay such as that described in Antibodies,
A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and
David Lane (1988), can be performed. Alternatively, epitope mapping
can be performed to establish whether the antibody binds to the
7C2/7F3 epitope on HER2 (e.g. any one or more of residues in the
region from about residue 22 to about residue 53 of the HER2 ECD,
residue numbering including signal peptide).
[0159] "Treatment" refers to both therapeutic treatment and
prophylactic or preventative measures. Those in need of treatment
include those already with cancer as well as those in which cancer
is to be prevented. Hence, the patient to be treated herein may
have been diagnosed as having cancer or may be predisposed or
susceptible to cancer.
[0160] The term "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).
[0161] 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.
[0162] 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, chlornaphazine,
cholophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranimnustine; 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.RTM.,
TDX); inhibitors of dihydropyrimidine dehydrogenase such as
eniluracil; aldophosphamide glycoside; aminolevulinic acid;
amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;
demecolcine; diaziquone; elfomithine; 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; PSKS polysaccharide complex (JHS Natural Products,
Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium;
tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine;
trichothecenes (especially T-2 toxin, verracurin A, roridin A and
anguidine); urethan; 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; difluorometlhylornithine (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.
[0163] 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. 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@), azocytosine, deoxycytosine, pyridmidene, fludarabine
(FLUDARA.RTM.), cladrabine, 2-deoxy-D-glucose etc. The preferred
antimetabolite chemotherapeutic agent is gemcitabine.
[0164] "Gemcitabine" or "2'-deoxy-2',2'-difluorocytidine
monohydrochloride (b-isomer)" is a nucleoside analogue that
exhibits antitumor activity. The empirical formula for gemcitabine
HCl is C9H11F2N3O4.HCl. Gemcitabine HCl is sold by Eli Lilly under
the trademark GEMZAR.RTM..
[0165] 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.
[0166] 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.
[0167] 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.
[0168] 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.
[0169] 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.).
[0170] 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.
[0171] 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.TM.; Astra Zeneca); CP-358774 or Erlotinib
(TARCEVA.TM.; Genentech/OSI); and AG1478, AG1571 (SU 5271; Sugen);
EMD-7200.
[0172] A "tyrosine kinase inhibitor" is a molecule which inhibits
tyrosine kinase activity of a tyrosine kinase such as a HER
receptor. Examples of such inhibitors include the EGFR-targeted
drugs noted in the preceding paragraph; small molecule HER2
tyrosine kinase inhibitor such as TAK165 available from Takeda;
CP-724,714, an oral selective inhibitor of the ErbB2 receptor
tyrosine kinase (Pfizer and OSI); dual-HER inhibitors such as
EKB-569 (available from Wyeth) which preferentially binds EGFR but
inhibits both HER2 and EGFR-overexpressing cells; 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).
[0173] 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.
[0174] 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).
[0175] 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.
[0176] II. Production of Antibodies
[0177] Since, in the preferred embodiment, the HER dimerization
inhibitor is an antibody, a description follows as to exemplary
techniques for the production of HER antibodies used in accordance
with the present invention. 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.
[0178] (i) Polyclonal Antibodies
[0179] 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.
[0180] 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.
[0181] (ii) Monoclonal antibodies
[0182] 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).
[0183] 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)).
[0184] 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.
[0185] 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)).
[0186] 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).
[0187] 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).
[0188] 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.
[0189] 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.
[0190] 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).
[0191] 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.
[0192] 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.
[0193] 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.
[0194] (iii) Humanized Antibodies
[0195] 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.
[0196] 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)).
[0197] 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.
[0198] WO01/00245 describes production of exemplary humanized HER2
antibodies which bind HER2 and block ligand activation of a HER
receptor. The humanized antibody of particular interest herein
blocks EGF, TGF-.alpha. and/or HRG mediated activation of MAPK
essentially as effectively as murine monoclonal antibody 2C4 (or a
Fab fragment thereof) and/or binds 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.
[0199] An exemplary humanized antibody of interest herein comprises
variable heavy domain complementarity determining residues
GFTFTDYTMX, where X is preferrably D or S (SEQ ID NO:7);
DVNPNSGGSIYNQRFKG (SEQ ID NO:8); and/or NLGPSFYFDY (SEQ ID NO:9),
optionally comprising amino acid modifications of those CDR
residues, e.g. where the modifications essentially maintain or
improve affinity of the antibody. For example, the antibody variant
of interest may have from about one to about seven or about five
amino acid substitutions in the above variable heavy CDR sequences.
Such antibody variants may be prepared by affinity maturation,
e.g., as described below. The most preferred humanized antibody
comprises the variable heavy domain amino acid sequence in SEQ ID
NO:4.
[0200] The humanized antibody may comprise variable light domain
complementarity determining residues KASQDVSIGVA (SEQ ID NO:10);
SASYX.sup.1X.sup.2X.sup.3, where X.sup.1 is preferably R or L,
X.sup.2 is preferably Y or E, and X.sup.3 is preferably T or S (SEQ
ID NO:11); and/or QQYYIYPYT (SEQ ID NO:12), e.g. in addition to
those variable heavy domain CDR residues in the preceding
paragraph. Such humanized antibodies optionally comprise amino acid
modifications of the above CDR residues, e.g. where the
modifications essentially maintain or improve affinity of the
antibody. For example, the antibody variant of interest may have
from about one to about seven or about five amino acid
substitutions in the above variable light CDR sequences. Such
antibody variants may be prepared by affinity maturation, e.g., as
described below. The most preferred humanized antibody comprises
the variable light domain amino acid sequence in SEQ ID NO:3.
[0201] The present application also contemplates affinity matured
antibodies which bind HER2 and block ligand activation of a HER
receptor. 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. 3 and 4, respectively (i.e.
comprising the VL and/or VH of pertuzumab). The affinity matured
antibody 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).
[0202] 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. The preferred
intact IgG1 antibody comprises the light chain sequence in SEQ ID
NO: 13 and the heavy chain sequence in SEQ ID NO: 14.
[0203] (iv) Human Antibodies
[0204] 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.
[0205] 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).
[0206] 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.
[0207] (v) Antibody Fragments
[0208] 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.
[0209] (vi) Bispecific Antibodies
[0210] 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).
[0211] 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 IDMI (Osidem). A bispecific HER2/Fca
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.
[0212] 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
(Milistein 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).
[0213] 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.
[0214] 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).
[0215] 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.
[0216] 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.
[0217] 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.
[0218] 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.
[0219] 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).
[0220] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al. J.
Immunol. 147: 60 (1991).
[0221] (vii) Other Amino Acid Sequence Modifications
[0222] 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.
[0223] 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.
[0224] 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.
[0225] 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
[0226] 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)): [0227] (1) non-polar: Ala (A), Val
(V), Leu (L), Ile (1), Pro (P), Phe (F), Trp (W), Met (M) [0228]
(2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y),
Asn (N), Gin (O) [0229] (3) acidic: Asp (D), Glu (E) [0230] (4)
basic: Lys (K), Arg (R), His(H)
[0231] Alternatively, naturally occurring residues may be divided
into groups based on common side-chain properties: [0232] (1)
hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; [0233] (2)
neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; [0234] (3) acidic:
Asp, Glu; [0235] (4) basic: His, Lys, Arg; [0236] (5) residues that
influence chain orientation: Gly, Pro; [0237] (6) aromatic: Trp,
Tyr, Phe.
[0238] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0239] 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).
[0240] 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.
[0241] 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.
[0242] 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.
[0243] 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).
[0244] 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.
[0245] 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).
[0246] 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.
[0247] 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.
6,528,624B 1 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).
[0248] 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.
[0249] Antibodies with improved binding to the neonatal Fc receptor
(FcRn), and increased half-lives, are described in WO00/42072
(Presta, L.) and US2005/0014934A 1 (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).
[0250] Engineered antibodies with three or more (preferably four)
functional antigen binding sites are also contemplated (US Appln
No. US2002/0004587 A1, Miller et al.).
[0251] 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.
[0252] (viii) Screening for Antibodies with the Desired
Properties
[0253] Techniques for generating antibodies have been described
above. One may further select antibodies with certain biological
characteristics, as desired.
[0254] 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.
[0255] 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 WO01/00245. HER2 monoclonal antibodies may be added to each well
and incubated for 30 minutes. .sup.125I-labeled
rHRG.beta.1.sub.177-224 (25 pm) may then be added, and the
incubation may be continued for 4 to 16 hours. Dose response curves
may be prepared and an IC.sub.50 value may be calculated for the
antibody of interest. In one embodiment, the antibody which blocks
ligand activation of 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.
[0256] 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.
[0257] In one embodiment, one may screen for an antibody which
inhibits HRG stimulation of p180 tyrosine phosphorylation in MCF7
cells essentially as described in WO01/00245. 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 rHRGP1.sub.177-244 may be added to each well to a
final concentration of 0.2 nM, and the incubation may be continued
for 8 minutes. Media may be aspirated from each well, and reactions
may be stopped by the addition of 100 .mu.l of SDS sample buffer
(5% SDS, 25 mM DTT, and 25 mM Tris-HCl, pH 6.8). Each sample (25
.mu.l) may be electrophoresed on a 4-12% gradient gel (Novex) and
then electrophoretically transferred to polyvinylidene difluoride
membrane. Antiphosphotyrosine (at 1 .mu.g/ml) immunoblots may be
developed, and the intensity of the predominant reactive band at
M.sub.r.about.180,000 may be quantified by reflectance
densitometry. The antibody selected will preferably significantly
inhibit HRG stimulation of p 180 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.
[0258] 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.
[0259] In one embodiment, the HER2 antibody of interest may block
heregulin dependent association of HER2 with HER3 in both MCF7 and
SK-BR-3 cells as determined in a co-immunoprecipitation experiment
such as that described in WO01/00245 substantially more effectively
than monoclonal antibody 4D5, and preferably substantially more
effectively than monoclonal antibody 7F3.
[0260] 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/ml. To identify
such antibodies, the SK-BR-3 assay described in U.S. Pat. No.
5,677,171 can be performed. According to this assay, SK-BR-3 cells
are grown in a 1:1 mixture of F12 and DMEM medium supplemented with
10% fetal bovine serum, glutamine and penicillin streptomycin. The
SK-BR-3 cells are plated at 20,000 cells in a 35 mm cell culture
dish (2 mls/35 mm dish). 0.5 to 30 .mu.g/ml of the HER2 antibody is
added per dish. After six days, the number of cells, compared to
untreated cells are counted using an electronic COULTER.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.
[0261] 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.
[0262] 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.
[0263] (ix) Pertuzumab Compositions
[0264] 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 selected from SEQ ID NO. 13 and 17, and a heavy chain
amino acid sequence selected from SEQ ID NO. 14 and 18 (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.
[0265] 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.
[0266] 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.
[0267] (x) Immunoconjugates
[0268] 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).
[0269] Chemotherapeutic agents useful in the generation of such
immunoconjugates have been described above. Conjugates of an
antibody and one or more small molecule toxins, such as a
calicheamicin, a maytansine (U.S. Pat. No. 5,208,020), a
trichothene, and CC1065 are also contemplated herein.
[0270] 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.
[0271] 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.1, .alpha..sub.2.sup.1,
.alpha..sub.3.sup.1, N-acetyl-.gamma..sub.1.sup.1, 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.
[0272] 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.
[0273] 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).
[0274] 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, Re.sup.188,
Sm.sup.153, Bi.sup.212, P.sup.32 and radioactive isotopes of
Lu.
[0275] Conjugates of the antibody and cytotoxic agent may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al. Science 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026. The linker may be
a "cleavable linker" facilitating release of the cytotoxic drug in
the cell. For example, an acid-labile linker, peptidase-sensitive
linker, dimethyl linker or disulfide-containing linker (Chari et
al. Cancer Research 52: 127-131 (1992)) may be used.
[0276] Alternatively, a fusion protein comprising the antibody and
cytotoxic agent may be made, e.g. by recombinant techniques or
peptide synthesis.
[0277] 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).
[0278] 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.
[0279] 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).
[0280] III. Selecting Patients for Therapy
[0281] The patient herein is optionally subjected to a diagnostic
test prior to therapy. For example, the diagnostic test may
evaluate HER (e.g. HER2 or EGFR) expression (including
overexpression), amplification, and/or activation (including
phosphorylation or dimerization).
[0282] Generally, if a diagnostic test is performed, a sample may
be obtained from a patient in need of therapy. Where the subject
has cancer, the sample is generally a tumor sample. In the
preferred embodiment, the tumor sample is from an ovarian cancer,
peritoneal cancer, fallopian tube cancer, metastatic breast cancer
(MBC), non-small cell lung cancer (NSCLC), prostate cancer, or
colorectal cancer tumor sample.
[0283] The biological sample herein may be a fixed sample, e.g. a
formalin fixed, paraffin-embedded (FFPE) sample, or a frozen
sample.
[0284] According to one embodiment of the invention herein, the
patient selected for therapy has a tumor displaying HER (and
preferably HER2) activation. In one embodiment, the extent of HER
(or HER2) activation in cancer cells significantly exceeds the
level of activation of that receptor in non-cancerous cells of the
same tissue type. Such excessive activation may result from
overexpression of the HER receptor and/or greater than normal
levels of a HER ligand available for activating the HER receptor in
the cancer cells. Such excessive activation may cause and/or be
caused by the malignant state of a cancer cell. In some
embodiments, the cancer will be subjected to a diagnostic or
prognostic assay to determine whether amplification and/or
overexpression of a HER receptor is occurring which results in such
excessive activation of the HER receptor. Alternatively, or
additionally, the cancer may be subjected to a diagnostic or
prognostic assay to determine whether amplification and/or
overexpression a HER ligand is occurring in the cancer which
attributes to excessive activation of the receptor. In a subset of
such cancers, excessive activation of the receptor may result from
an autocrine stimulatory pathway. Various assays for determining
HER activation will be described in more detail below. The
preferred methods for determining HER activation are: detecting the
presense of HER dimers or heterodimers, evaluating HER or HER2
phosphorylation, and gene expression profiling.
[0285] (i) HER Dimers
[0286] Tumors samples can be assessed for the presence of HER
dimers, as indicating HER or HER2 activation. Any method known in
the art may be used to detect HER2 dimers, such as EGFR-HER2,
HER2-HER3, in tumors. Several preferred methods are described
below. These methods detect noncovalent protein-protein
interactions or otherwise indicate proximity between proteins of
interest.
[0287] Immunoaffinity-based methods, such as immunoprecipitation or
ELISA, may be used to detect HER dimers. In one embodiment, HER2
antibodies are used to immunoprecipitate complexes comprising HER2
from tumor cells, and the resulting immunoprecipitant is then
probed for the presence of EGFR or HER3 by immunoblotting. In
another embodiment, EGFR or HER3 antibodies may be used for the
immunoprecipitation step and the immunoprecipitant then probed with
HER2 antibodies. In a further embodiment, HER ligands specific to
EGFR, HER3, EGFR-HER2 complexes or HER2-HER3 complexes may be used
to precipitate complexes, which are then probed for the presence of
HER2. For example, ligands may be conjugated to avidin and
complexes purified on a biotin column.
[0288] In other embodiments, such as ELISA or antibody
"sandwich"-type assays, antibodies to HER2 are immobilized on a
solid support, contacted with tumor cells or tumor cell lysate,
washed, and then exposed to antibody against EGFR or HER3. Binding
of the latter antibody, which may be detected directly or by a
secondary antibody conjugated to a detectable label, indicates the
presence of heterodimers. In certain embodiments, EGFR or HER3
antibody is immobilized, and HER2 antibody is used for the
detection step. In other embodiments HER ligands may be used in
place of, or in combination with HER antibodies.
[0289] Chemical or UV cross-linking may also be used to covalently
join dimers on the surface of living cells. Examples of chemical
cross-linkers include dithiobis(succinimidyl)propionate (DSP) and
3,{acute over (3)}dithiobis(sulphosuccinimidyl)propionate (DTSSP).
In one embodiment, cell extracts from chemically cross-linked tumor
cells are analyzed by SDS-PAGE and immunoblotted with antibodies to
EGFR and/or HER3. A supershifted band of the appropriate molecular
weight most likely represents EGFR-HER2 or HER2-HER3 dimers, as
HER2 is the preferred dimerization partner for EGFR and HER3. This
result may be confirmed by subsequent immunoblotting with HER2
antibodies.
[0290] Fluorescence resonance energy transfer (FRET) may also be
used to detect EGFR-HER2 or HER2-HER3 dimers. FRET detects protein
conformational changes and protein-protein interactions in vivo and
in vitro based on the transfer of energy from a donor fluorophore
to an acceptor fluorophore. Selvin, Nat. Struct. Biol., 7:730-34
(2000). Energy transfer takes place only if the donor fluorophore
is in sufficient proximity to the acceptor fluorophore. In a
typical FRET experiment, two proteins or two sites on a single
protein are labeled with different fluorescent probes. One of the
probes, the donor probe, is excited to a higher energy state by
incident light of a specified wavelength. The donor probe then
transmits its energy to the second probe, the acceptor probe,
resulting in a reduction in the donor's fluorescence intensity and
an increase in the acceptor's fluorescence emission. To measure the
extent of energy transfer, the donor's intensity in a sample
labeled with donor and acceptor probes is compared with its
intensity in a sample labeled with donor probe only. Optionally,
acceptor intensity is compared in donor/acceptor and acceptor only
samples. Suitable probes are known in the art and include, for
example, membrane permeant dyes, such as fluorescein and rhodamine,
organic dyes, such as the cyanine dyes, and lanthanide atoms.
Methods and instrumentation for detecting and measuring energy
transfer are also known in the art.
[0291] FRET-based techniques suitable for detecting and measuring
protein-protein interactions in individual cells are also known in
the art. For example, donor photobleaching fluorescence resonance
energy transfer (pbFRET) microscopy and fluorescence lifetime
imaging microscopy (FLIM) may be used to detect the dimerization of
cell surface receptors. Gadella & Jovin, J. Cell Biol.,
129:1543-58 (1995). In one embodiment, pbFRET is used on cells
either "in suspension" or "in situ" to detect and measure the
formation of EGFR-HER2 or HER2-HER3 dimers, as described in Nagy et
al., Cytometry, 32:120-131 (1998). These techniques measure the
reduction in a donor's fluorescence lifetime due to energy
transfer. In a particular embodiment, a flow cytometric
Foerster-type FRET technique (FCET) may be used to investigate
EGFR-HER2 and HER2-HER3 dimerization, as described in Nagy et al.,
supra, and Brockhoff et al, Cytometry, 44:338-48 (2001).
[0292] FRET is preferably used in conjunction with standard
immunohistochemical labeling techniques. Kenworthy, Methods,
24:289-96 (2001). For example, antibodies conjugated to suitable
fluorescent dyes can be used as probes for labeling two different
proteins. If the proteins are within proximity of one another, the
fluorescent dyes act as donors and acceptors for FRET. Energy
transfer is detected by standard means. Energy transfer may be
detected by flow cytometric means or by digital microscopy systems,
such as confocal microscopy or wide-field fluorescence microscopy
coupled to a charge-coupled device (CCD) camera.
[0293] In one embodiment of the present invention, HER2 antibodies
and either EGFR or HER3 antibodies are directly labeled with two
different fluorophores, for example as described in Nagy et al,
supra. Tumor cells or tumor cell lysates are contacted with the
differentially labeled antibodies, which act as donors and
acceptors for FRET in the presence of EGFR-HER2 or HER2-HER3
dimers. Alternatively, unlabeled antibodies against HER2 and either
EGFR or HER3 are used along with differentially labeled secondary
antibodies that serve as donors and acceptors. See, for example,
Brockhoff et al., supra. Energy transfer is detected and the
presence of dimers is determined if the labels are found to be in
close proximity.
[0294] In other embodiments HER receptor ligands that are specific
for HER2 and either EGFR or HER3 are fluorescently labeled and used
for FRET studies.
[0295] In still other embodiments of the present invention, the
presence of dimers on the surface of tumor cells is demonstrated by
co-localization of HER2 with either EGFR or HER3 using standard
direct or indirect immunofluorescence techniques and confocal laser
scanning microscopy. Alternatively, laser scanning imaging (LSI) is
used to detect antibody binding and co-localization of HER2 with
either EGFR or HER3 in a high-throughput format, such as a
microwell plate, as described in Zuck et al, Proc. Natl. Acad. Sci.
USA, 96:11122-27 (1999).
[0296] In further embodiments, the presence of EGFR-HER2 and/or
HER2-HER3 dimers is determined by identifying enzymatic activity
that is dependent upon the proximity of the dimer components. A
HER2 antibody is conjugated with one enzyme and an EGFR or HER3
antibody is conjugated with a second enzyme. A first substrate for
the first enzyme is added and the reaction produces a second
substrate for the second enzyme. This leads to a reaction with
another molecule to produce a detectable compound, such as a dye.
The presence of another chemical breaks down the second substrate,
so that reaction with the second enzyme is prevented unless the
first and second enzymes, and thus the two antibodies, are in close
proximity. In a particular embodiment tumor cells or cell lysates
are contacted with a HER2 antibody that is conjugated with glucose
oxidase and a HER3 or EGFR antibody that is conjugated with horse
radish peroxidase. Glucose is added to the reaction, along with a
dye precursor, such as DAB, and catalase. The presence of dimers is
determined by the development of color upon staining for DAB.
[0297] Dimers may also be detected using methods based on the
eTag.TM. assay system (Aclara Bio Sciences, Mountain View, Calif.),
as described, for example, in U.S. Patent Application 2001/0049105,
published Dec. 6, 2001, both of which are expressly incorporated by
reference in their entirety. An eTag.TM., or "electrophoretic tag,"
comprises a detectable reporter moiety, such as a fluorescent
group. It may also comprise a "mobility modifier," which consists
essentially of a moiety having a unique electrophoretic mobility.
These moieties allow for separation and detection of the eTag.TM.
from a complex mixture under defined electrophoretic conditions,
such as capillary electrophoresis (CE). The portion of the eTag.TM.
containing the reporter moiety and, optionally, the mobility
modifier is linked to a first target binding moiety by a cleavable
linking group to produce a first binding compound. The first target
binding moiety specifically recognizes a particular first target,
such as a nucleic acid or protein. The first target binding moiety
is not limited in any way, and may be for example, a polynucleotide
or a polypeptide. Preferably, the first target binding moiety is an
antibody or antibody fragment. Alternatively, the first target
binding moiety may be a HER receptor ligand or binding-competent
fragment thereof.
[0298] The linking group preferably comprises a cleavable moiety,
such as an enzyme substrate, or any chemical bond that may be
cleaved under defined conditions. When the first target binding
moiety binds to its target, the cleaving agent is introduced and/or
activated, and the linking group is cleaved, thus releasing the
portion of the eTag.TM. containing the reporter moiety and mobility
modifier. Thus, the presence of a "free" eTag.TM. indicates the
binding of the target binding moiety to its target.
[0299] Preferably, a second binding compound comprises the cleaving
agent and a second target binding moiety that specifically
recognizes a second target. The second target binding moiety is
also not limited in any way and may be, for example, an antibody or
antibody fragment or a HER receptor ligand or binding competent
ligand fragment. The cleaving agent is such that it will only
cleave the linking group in the first binding compound if the first
binding compound and the second binding compound are in close
proximity.
[0300] In an embodiment of the present invention, a first binding
compound comprises an eTag.TM. in which an antibody to HER2 serves
as the first target binding moiety. A second binding compound
comprises an antibody to EGFR or HER3 joined to a cleaving agent
capable of cleaving the linking group of the eTag.TM.. Preferably
the cleaving agent must be activated in order to be able to cleave
the linking group. Tumor cells or tumor cell lysates are contacted
with the eTag.TM., which binds to HER2, and with the modified EGFR
or HER3 antibody, which binds to EGFR or HER3 on the cell surface.
Unbound binding compound is preferable removed, and the cleaving
agent is activated, if necessary. If EGFR-HER2 or HER2-HER3 dimers
are present, the cleaving agent will cleave the linking group and
release the eTag.TM. due to the proximity of the cleaving agent to
the linking group. Free eTag.TM. may then be detected by any method
known in the art, such as capillary electrophoresis.
[0301] In one embodiment, the cleaving agent is an activatable
chemical species that acts on the linking group. For example, the
cleaving agent may be activated by exposing the sample to
light.
[0302] In another embodiment, the eTag.TM. is constructed using an
antibody to EGFR or HER3 as the first target binding moiety, and
the second binding compound is constructed from an antibody to
HER2.
[0303] In yet another embodiment, the HER dimer is detected using
an antibody or other reagent which specifically or preferentially
binds to the dimer as compared to binding thereof to either HER
receptor in the dimer.
[0304] (ii) HER2 Phosphorylation
[0305] Phosphorylation of HER receptor may be assessed by
immunoprecipitation of one or more HER receptors, such as HER2
receptor, and analysis of phosphorylated tyrosine residue(s) in the
immunoprecipitated receptor(s). For example, positivity is
determined by the presence of a phospho-HER2 band on the gel, using
an anti-phosphotyrosine antibody to detect phosphorylated tyrosine
residue(s) in the immunoprecipitated HER receptor(s).
Anti-phosphotyrosine antibodies are commercially available from
PanVera (Madison, Wis.), a subsidiary of Invitrogen, Chemicon
International Inc. (Temecula, Calif.), or Upstate Biotechnology
(Lake Placid, N.Y.). Negativity is determined by the absence of the
band. Various assay formats for detecting phosphorylated proteins
are contemplated including Western blot analysis,
immunohistochemistry, ELISA, etc.
[0306] In one embodiment, phosphorylation of HER2 (HER2) receptor
is assessed by immunohistochemistry using a phospho-specific HER2
antibody (clone PN2A; Thor et al., J. Clin. Oncol, 18(18):3230-3239
(2000)).
[0307] Other methods for detecting phosphorylation of HER
receptor(s) include, but are not limited to, KIRA ELISA (U.S. Pat.
Nos. 5,766,863; 5,891,650; 5,914,237; 6,025,145; and 6,287,784),
mass spectrometry (comparing size of phosphorylated and
non-phosphorylated HER2), and e-tag proximity assay with both a HER
(e.g. HER2) antibody and phospho-specific or phospho-tyrosine
specific antibody (e.g., using the eTag.TM.assay kit available from
Aclara BioSciences (Mountain View, Calif.). Details of the eTag
assay are described hereinabove.
[0308] One may also use phospho-specific antibodies in cellular
array to detect phosphorylation status in a cellular sample of
signal transduction protein (US2003/0190689).
[0309] Example 2 below describes a preferred method for determining
HER2 phosphorylation by phospho-HER2 ELISA.
[0310] (iii) Gene Expression Profiling
[0311] In one embodiment, gene expression profiling can serve as a
surrogate for measuring HER phosphorylation directly. This is
particularly useful where the sample is a fixed sample (e.g.
parrafin-embedded, formalin fixed tumor sample) where HER
phosphorylation may be difficult to reliably quantify. For example,
expression of two or more HER receptors and one or more HER ligand
in a sample is evaluated, wherein expression of the two or more HER
receptors and one or more HER ligand indicates positive HER
activation in the sample. Alternatively or additionally, expression
of betacellulin and/or amphiregulin in the sample can be measured,
wherein betacellulin and/or amphiregulin expression indicates
positive HER activation in the sample.
[0312] According to a preferred embodiment of gene expression
profiling for evaluating HER2 activation, a sample from the patient
is tested for expression of two or more HER receptors (preferably
selected from EGFR, HER2, and HER3) and one or more HER ligands
(preferably selected from betacellulin, amphiregulin, epiregulin,
and TGF-.alpha., most preferably betacellulin or amphiregulin). For
example, the two or more HER receptors may be EGFR and HER2, or
HER2 and HER3, and the one or more HER ligands may be betacellulin
or amphiregulin. Preferably, expression of HER2 and EGFR or HER3,
as well as betacellulin or amphiregulin is determined. The sample
may be tested for expression of betacellulin or amphiregulin alone,
or in combination with testing for expression of two or more HER
receptors. Positive expression of the identified gene(s) indicates
the patient is a candidate for therapy with a HER dimerization
inhibitor, such as pertuzumab. Moreover, positive expression of the
gene(s) indicates the patient is more likely to respond favorably
to therapy with the HER dimerization inhibitor than a patient who
does not have such positive expression.
[0313] Various methods for determining expression of mRNA or
protein include, but are not limited to, gene expression profiling,
polymerase chain reaction (PCR) including quantitative real time
PCR (qRT-PCR), microarray analysis, serial analysis of gene
expression (SAGE), MassARRAY, Gene Expression Analysis by Massively
Parallel Signature Sequencing (MPSS), proteomics,
immunohistochemistry (IHC), etc. Preferably mRNA is quantified.
Such mRNA analysis is preferably performed using the technique of
polymerase chain reaction (PCR), or by microarray analysis. Where
PCR is employed, a preferred form of PCR is quantitative real time
PCR (qRT-PCR). In one embodiment, expression of one or more of the
above noted genes is deemed positive expression if it is at the
median or above, e.g. compared to other samples of the same
tumor-type. The median expression level can be determined
essentially contemporaneously with measuring gene expression, or
may have been determined previously.
[0314] The steps of a representative protocol for profiling gene
expression using fixed, paraffin-embedded tissues as the RNA
source, including mRNA isolation, purification, primer extension
and amplification are given in various published journal articles
(for example: Godfrey et al. J. Molec. Diagnostics 2: 84-91 (2000);
Specht et al., Am. J. Pathol. 158: 419-29 (2001)). Briefly, a
representative process starts with cutting about 10 microgram thick
sections of paraffin-embedded tumor tissue samples. The RNA is then
extracted, and protein and DNA are removed. After analysis of the
RNA concentration, RNA repair and/or amplification steps may be
included, if necessary, and RNA is reverse transcribed using gene
specific promoters followed by PCR. Finally, the data are analyzed
to identify the best treatment option(s) available to the patient
on the basis of the characteristic gene expression pattern
identified in the tumor sample examined.
[0315] Example 3 herein describes preferred methods for determining
HER2 activation by gene expression profiling.
[0316] (iv) HER Expression and Amplification
[0317] To determine HER expression or amplification in the cancer,
various diagnostic/prognostic assays are available. In one
embodiment, HER overexpression may be analyzed by IHC, e.g. using
the HERCEPTEST.RTM. (Dako). Parrafin embedded tissue sections from
a tumor biopsy may be subjected to the IHC assay and accorded a
HER2 protein staining intensity criteria as follows:
Score 0 no staining is observed or membrane staining is observed in
less than 10% of tumor cells.
Score 1+ a faint/barely perceptible membrane staining is detected
in more than 10% of the tumor cells. The cells are only stained in
part of their membrane.
Score 2+ a weak to moderate complete membrane staining is observed
in more than 10% of the tumor cells.
Score 3+ a moderate to strong complete membrane staining is
observed in more than 10% of the tumor cells.
[0318] Those tumors with 0 or I+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.
[0319] 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:
0=0-10,000 copies/cell,
1+=at least about 200,000 copies/cell,
2+=at least about 500,000 copies/cell,
3+=at least about 2,000,000 copies/cell.
[0320] 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)).
[0321] Alternatively, or additionally, FISH assays such as the
INFORM.TM. (sold by Ventana, Ariz.) or PATHVISION.TM. (Vysis, Ill.)
may be carried out on formalin-fixed, paraffin-embedded tumor
tissue to determine the extent (if any) of HER2 amplification in
the tumor.
[0322] In one embodiment, the cancer will be one which expresses
(and may overexpress) EGFR, such expression may be evaluated as for
the methods for evaluating HER2 expression as noted above.
[0323] HER receptor or HER ligand overexpression or amplification
may also be evaluated using an in vivo diagnostic assay, e.g. by
administering a molecule (such as an antibody) which binds the
molecule to be detected and is tagged with a detectable label (e.g.
a radioactive isotope) and externally scanning the patient for
localization of the label.
[0324] IV. Pharmaceutical Formulations
[0325] Therapeutic formulations of the HER dimerization inhibitors
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
octadecyidimethylbenzyl 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.
[0326] 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.
[0327] 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.
[0328] 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).
[0329] 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.
[0330] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0331] V. Treatment with HER Dimerization Inhibitors
[0332] The invention herein provides a method for extending TTP or
survival in a cancer patient, whose cancer displays HER activation,
comprising administering a HER dimerization inhibitor to the
patient in an amount which extends the patient's TTP or survival.
Preferably, the HER dimerization inhibitor is a HER2 dimerization
inhibitor and/or inhibits HER heterodimerization.
[0333] In one embodiment, the patient's cancer displays HER2
activation, including HER2 phosphorylation. Preferably, HER2
phosphorylation is evaluated using a phospho-ELISA assay.
Alternatively, HER2 activation can be evaluated by gene expression
profiling or by detecting HER dimers or heterodimers.
[0334] Examples of various cancers that can be treated with a HER
dimerization inhibitor are listed in the definition section above.
Preferred cancer indications include ovarian cancer; peritoneal
cancer; fallopian tube cancer; breast cancer, including metastatic
breast cancer (MBC); lung cancer, including non-small cell lung
cancer (NSCLC); prostate cancer; and colorectal cancer. In one
embodiment, the cancer which is treated is advanced, refractory,
recurrent, chemotherapy-resistant, and/or platinum-resistant
cancer.
[0335] Therapy with the HER dimerization inhibitor extends TTP
and/or survival. In one embodiment, therapy with the HER
dimerization inhibitor extends TTP or survival at least about 20%
more than TTP or survival achieved by administering an approved
anti-tumor agent, or standard of care, for the cancer being
treated.
[0336] In the preferred embodiment, the invention provides a method
for extending time to disease progression (TTP) or survival in a
patient with ovarian, peritoneal, or fallopian tube cancer, whose
cancer displays HER2 activation, comprising administering
pertuzumab to the patient in an amount which extends the patient's
TTP or survival. The patient may have advanced, refractory,
recurrent, chemotherapy-resistant, and/or platinum-resistant
ovarian, peritoneal or fallopian tube cancer. Administration of
pertuzumab to the patient may, for example, extend TTP or survival
at least about 20% more than TTP or survival achieved by
administering topotecan or liposomal doxorubicin to such a
patient.
[0337] The HER dimerization inhibitor is administered to a human
patient in accord with known methods, such as intravenous
administration, e.g., as a bolus or by continuous infusion over a
period of time, by intramuscular, intraperitoneal,
intracerobrospinal, subcutaneous, intra-articular, intrasynovial,
intrathecal, oral, topical, or inhalation routes. Intravenous
administration of the antibody is preferred.
[0338] For the prevention or treatment of cancer, the dose of HER
dimerization inhibitor will depend on the type of cancer to be
treated, as defined above, the severity and course of the cancer,
whether the antibody is administered for preventive or therapeutic
purposes, previous therapy, the patient's clinical history and
response to the antibody, and the discretion of the attending
physician.
[0339] In one embodiment, a fixed dose of HER dimerization
inhibitor is administered. The fixed dose may suitably be
administered to the patient at one time or over a series of
treatments. Where a fixed dose is administered, preferably it is in
the range from about 20 mg to about 2000 mg of the HER dimerization
inhibitor. For example, the fixed dose may be approximately 420 mg,
approximately 525 mg, approximately 840 mg, or approximately 1050
mg of the HER dimerization inhibitor, such as pertuzumab.
[0340] Where a series of doses are administered, these may, for
example, be administered approximately every week, approximately
every 2 weeks, approximately every 3 weeks, or approximately every
4 weeks, but preferably approximately every 3 weeks. The fixed
doses may, for example, continue to be administered until disease
progression, adverse event, or other time as determined by the
physician. For example, from about two, three, or four, up to about
17 or more fixed doses may be administered.
[0341] In one embodiment, one or more loading dose(s) of the
antibody are administered, followed by one or more maintenance
dose(s) of the antibody. In another embodiment, a plurality of the
same dose are administered to the patient.
[0342] 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, up to 17 or
more doses.
[0343] According to another preferred embodiment of the invention,
one or more fixed dose(s) of approximately 1050 mg of the HER
dimerization inhibitor (e.g. pertzumab) are administered, for
example every 3 weeks. According to this embodiment, one, two or
more of the fixed doses are administered, e.g. for up to one year
(17 cycles), and longer as desired.
[0344] In another embodiment, a fixed dose of approximately 1050 mg
of the HER dimerization inhibitor (e.g. pertuzumab) is administered
as a loading dose, followed by one or more maintenance dose(s) of
approximately 525 mg. About one, two or more maintenance doses may
be administered to the patient every 3 weeks according to this
embodiment.
[0345] While the HER dimerization inhibitor may be administered as
a single anti-tumor agent, the patient is optionally treated with a
combination of the HER dimerization inhibitor, and one or more
chemotherapeutic agent(s). Preferably at least one of the
chemotherapeutic agents is an antimetabolite chemotherapeutic agent
such as gemcitabine. The combined administration includes
coadministration or concurrent administration, using separate
formulations or a single pharmaceutical formulation, and
consecutive administration in either order, wherein preferably
there is a time period while both (or all) active agents
simultaneously exert their biological activities. Thus, the
antimetabolite chemotherapeutic agent may be administered prior to,
or following, administration of the HER dimerization inhibitor. In
this embodiment, the timing between at least one administration of
the antimetabolite chemotherapeutic agent and at least one
administration of the HER dimerization inhibitor is preferably
approximately 1 month or less, and most preferably approximately 2
weeks or less. Alternatively, the antimetabolite chemotherapeutic
agent and the HER dimerization inhibitor are administered
concurrently to the patient, in a single formulation or separate
formulations. Treatment with the combination of the
chemotherapeutic agent (e.g. antimetabolite chemotherapeutic agent
such as gemcitabine) and the HER dimerization inhibitor (e.g.
pertuzumab) may result in a synergistic, or greater than additive,
therapeutic benefit to the patient.
[0346] An antimetabolite chemotherapeutic agent, if administered,
is usually administered at dosages known therefor, or optionally
lowered due to combined action of the drugs or negative side
effects attributable to administration of the antimetabolite
chemotherapeutic agent. Preparation and dosing schedules for such
chemotherapeutic agents may be used according to manufacturers'
instructions or as determined empirically by the skilled
practitioner. Where the antimetabolite chemotherapeutic agent is
gemcitabine, preferably, it is administered at a dose between about
600 mg/m.sup.2 to 1250 mg/m.sup.2 (for example approximately 1000
mg/m.sup.2), for instance, on days 1 and 8 of a 3-week cycle.
[0347] Aside from the HER dimerization inhibitor and antimetabolite
chemotherapeutic agent, other therapeutic regimens may be combined
therewith. For example, a second (third, fourth, etc)
chemotherapeutic agent(s) may be administered, wherein the second
chemotherapeutic agent is either another, different antimetabolite
chemotherapeutic agent, or a chemotherapeutic agent that is not an
antimetabolite. For example, the second chemotherapeutic agent may
be a taxane (such as paclitaxel or docetaxel), capecitabine, or
platinum-based chemotherapeutic agent (such as carboplatin,
cisplatin, or oxaliplatin), anthracycline (such as doxorubicin,
including, liposomal doxorubicin), topotecan, pemetrexed, vinca
alkaloid (such as vinorelbine), and TLK 286. "Cocktails" of
different chemotherapeutic agents may be administered.
[0348] Other therapeutic agents that may be combined with the HER
dimerization inhibitor include any one or more of: a second,
different HER dimerization inhibitor (for example, a growth
inhibitory HER2 antibody such as trastuzumab, or a HER2 antibody
which induces apoptosis of a HER2-overexpressing cell, such as 7C2,
7F3 or humanized variants thereof); an antibody directed against a
different tumor associated antigen, such as EGFR, HER3, HER4;
anti-hormonal compound, e.g., an anti-estrogen compound such as
tamoxifen, or an aromatase inhibitor; a cardioprotectant (to
prevent or reduce any myocardial dysfunction associated with the
therapy); a cytokine; an EGFR-targeted drug (such as TARCEVA.RTM.,
IRESSA.RTM. or cetuximab); an anti-angiogenic agent (especially
bevacizumab sold by Genentech under the trademark AVAST.TM.); a
tyrosine kinase inhibitor; a COX inhibitor (for instance a COX-1 or
COX-2 inhibitor); non-steroidal anti-inflammatory drug, celecoxib
(CELEBREX.RTM.); farnesyl transferase inhibitor (for example,
Tipifarnib/ZARNESTRA.RTM. R115777 available from Johnson and
Johnson or Lonafarnib SCH66336 available from Schering-Plough);
antibody that binds oncofetal protein CA 125 such as Oregovomab
(MoAb B43.13); HER2 vaccine (such as HER2 AutoVac vaccine from
Pharmexia, or APC8024 protein vaccine from Dendreon, or HER2
peptide vaccine from GSK/Corixa); another HER targeting therapy
(e.g. trastuzumab, cetuximab, ABX-EGF, EMD7200, gefitinib,
erlotinib, CP724714, C11033, GW572016, IMC-11F8, TAK165, etc); Raf
and/or ras inhibitor (see, for example, WO 2003/86467); doxorubicin
HCl liposome injection (DOXIL.RTM.); topoisomerase I inhibitor such
as topotecan; taxane; HER2 and EGFR dual tyrosine kinase inhibitor
such as lapatinib/GW572016; TLK286 (TELCYTA.RTM.); EMD-7200; a
medicament that treats nausea such as a serotonin antagonist,
steroid, or benzodiazepine; a medicament that prevents or treats
skin rash or standard acne therapies, including topical or oral
antibiotic; a medicament that treats or prevents diarrhea; a body
temperature-reducing medicament such as acetaminophen,
diphenhydramine, or meperidine; hematopoietic growth factor,
etc.
[0349] Suitable dosages for any of the above coadministered agents
are those presently used and may be lowered due to the combined
action (synergy) of the agent and HER dimerization inhibitor.
[0350] In addition to the above therapeutic regimes, the patient
may be subjected to surgical removal of cancer cells and/or
radiation therapy.
[0351] Where the inhibitor is an antibody, preferably the
administered antibody is a naked antibody. 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.
[0352] The present application contemplates administration of the
HER dimerization inhibitor by gene therapy. See, for example,
WO96/07321 published Mar. 14, 1996 concerning the use of gene
therapy to generate intracellular antibodies.
[0353] There are two major approaches to getting the nucleic acid
(optionally contained in a vector) into the patient's cells; in
vivo and ex vivo. For in vivo delivery the nucleic acid is injected
directly into the patient, usually at the site where the antibody
is required. For ex vivo treatment, the patient's cells are
removed, the nucleic acid is introduced into these isolated cells
and the modified cells are administered to the patient either
directly or, for example, encapsulated within porous membranes
which are implanted into the patient (see, e.g. U.S. Pat. Nos.
4,892,538 and 5,283,187). There are a variety of techniques
available for introducing nucleic acids into viable cells. The
techniques vary depending upon whether the nucleic acid is
transferred into cultured cells in vitro, or in vivo in the cells
of the intended host. Techniques suitable for the transfer of
nucleic acid into mammalian cells in vitro include the use of
liposomes, electroporation, microinjection, cell fusion,
DEAE-dextran, the calcium phosphate precipitation method, etc. A
commonly used vector for ex vivo delivery of the gene is a
retrovirus.
[0354] The currently preferred in vivo nucleic acid transfer
techniques include transfection with viral vectors (such as
adenovirus, Herpes simplex I virus, or adeno-associated virus) and
lipid-based systems (useful lipids for lipid-mediated transfer of
the gene are DOTMA, DOPE and DC-Chol, for example). In some
situations it is desirable to provide the nucleic acid source with
an agent that targets the target cells, such as an antibody
specific for a cell surface membrane protein or the target cell, a
ligand for a receptor on the target cell, etc. Where liposomes are
employed, proteins which bind to a cell surface membrane protein
associated with endocytosis may be used for targeting and/or to
facilitate uptake, e.g. capsid proteins or fragments thereof tropic
for a particular cell type, antibodies for proteins which undergo
internalization in cycling, and proteins that target intracellular
localization and enhance intracellular half-life. The technique of
receptor-mediated endocytosis is described, for example, by Wu et
al., J. Biol. Chem. 262:4429-4432 (1987); and Wagner et al., Proc.
Natl. Acad. Sci. USA 87:3410-3414 (1990). For review of the
currently known gene marking and gene therapy protocols see
Anderson et al., Science 256:808-813 (1992). See also WO 93/25673
and the references cited therein.
[0355] VI. Deposit of Materials
[0356] 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
[0357] Further details of the invention are illustrated by the
following non-limiting Examples. The disclosures of all citations
in the specification are expressly incorporated herein by
reference.
EXAMPLE 1
Clinical Activity of Pertuzumab in Advanced, Refractory or
Recurrent Ovarian Cancer and the Role of HER2 Activation Status
[0358] This example concerns a single arm, open label, multicenter
phase II clinical trial of ovarian cancer patients. Patients with
advanced, refractory or recurrent ovarian cancer were treated with
pertuzumab, a humanized HER2 antibody. Pertuzumab represents a new
class of targeted agents called HER dimerization inhibitors (HDIs)
that inhibit dimerization of HER2 with EGFR, HER3 and HER4, and
inhibit signaling through MAP and P13 kinase.
[0359] 65 patients with relapsed ovarian cancer were enrolled with
61 receiving therapy with "low dose" single agent pertuzumab;
pertuzumab was administered intravenously (IV) with a loading of
840 mg followed by 420 mg every 3 weeks.
[0360] A second cohort of patients was treated with "high dose"
pertuzumab; 1050 mg every 3 weeks, administered as a single agent.
In this cohort, 64 subjects were enrolled, with 62 subjects being
treated.
[0361] Tumor assessments were obtained after 2, 4, 6, 8, 12 and 16
cycles.
[0362] Response Rate (RR) by RECIST was the primary endpoint. Fresh
tumor biopsies were mandatory in order to assay for HER2
phosphorylation (pHER2) status using a pHER2 enzyme-linked
immunosorbent assay as described in Example 2 below. pHER2 for
cohort 1 subjects was assessed. Safety and tolerability were
additionally evaluated.
[0363] Secondary endpoints were TTP, duration of response, duration
of survival, pharmacokinetics (PK), and FOSI (cohort 2).
Results
[0364] Baseline demographics of the patients are provided in FIG.
9. Median age was 57 years (range 35-83) and median ECOG PS was 2.
The median number of prior chemotherapy regimens was 5.
[0365] FIGS. 10-14 depict any adverse events in the treated
patients. Pertuzumab was well tolerated. Diarrhea (grade 1-3) was
experienced by 61% of patients. 5% of patients had a drop in
ejection fraction to less than 50%.
[0366] Efficacy results are summarized in FIG. 15. 4% of patients
had a partial response (PR). 39% of patients had stable disease
(SD). As shown in FIG. 16, median TTP for patients treated with 420
mg pertuzumab was 7 weeks, and, for patients treated with 1050 mg
pertuzumab was 6.6 weeks. FIG. 17 provides overall survival for
patients treated with low dose or high dose pertuzumab. Median
survival was 40 weeks. CA-125 responses are provided in FIG. 18.
Pertuzumab was efficacious in reducing CA-125 levels. Such
reduction is an indication of therapeutic effectiveness in ovarian
cancer.
[0367] HER2 activation status of patients in cohort 1, treated with
420 mg of pertuzumab, was evaluated. The results are shown in FIGS.
19-23. Approximately 30% of ovarian cancer subjects were pHER2
positive (greater than 30% of tumor, ELISA performed as described
in Example 2). Of the subjects evaluable for efficacy and pHER2
data, 26% were pHER2 positive. See FIG. 19.
[0368] The median TTP for pHER2+patients was 21 weeks, compared to
6 weeks in pHER2-patients, and 9 weeks in patients with unknown
pHER2 status (FIGS. 20 and 22).
[0369] Fourteen of 61 patients in cohort 1 showed evidence of
pertuzumab activity. The only patient with a partial response (PR)
was phospho-HER2 positive. See FIG. 21.
[0370] Overall survival of patients was also evaluated. As shown in
FIG. 23, overall survival of pHER2 positive patients treated with
pertuzumab appears superior to survival achieved with topotecan
(median survival 43 weeks) or liposomal doxorubicin (36 weeks).
Conclusions
[0371] As a single agent, pertuzumab is well tolerated. Pertuzumab
toxicity and efficacy do not appear to be dose-related. Pertuzumab
has activity in advanced, refractory or recurrent ovarian cancer.
Subjects with positive pHER2 status displayed enhanced TTP and
survival efficacy compared to subjects with negative pHER2 status.
Efficacy, as measured by TTP or survival, of pertuzumab in patients
displaying HER2 activation appeared superior to that achieved using
topotecan or liposomal doxorubicin, agents presently used to treat
patients with advanced, refractory or recurrent ovarian cancer.
EXAMPLE 2
Phospho-HER2 ELISA for Determining HER2 Activation
[0372] Example 1 above describes the clinical trial which evaluated
the efficacy of pertuzumab in subjects with advanced, refractory or
recurrent ovarian cancer. This example describes development of the
assay used to determine HER2 activation in the patients treated in
Example 1.
[0373] The phospho-HER2 ELISA was developed to measure the
concentration of HER2-associated tyrosine phosphorylation
(HER2/pTyr) in human ovarian tumor tissue lysates. The assay
utilizes COSTAR.TM. 96-well, half-area, microtiter plates because
of limited sample volume. The coat antibody is an affinity purified
goat anti-HER2 ECD and the secondary antibody is a biotinylated
murine monoclonal (clone 4G10) specific for phosphotyrosine. The
reference standard is a SK-BR-3 cell lysate with an assay range of
132 U/mL. One unit equals the amount of phosphorylated tyrosine
measured in a SK-BR-3 cell lysate containing 277 pg total HER2 as
determined by the Total HER2 ELISA (Total HER2 ELISA). The ELISA
uses AMDEX.TM. streptavidin-HRP for detection and TMB as the
substrate.
Materials
[0374] 1. Standard Material, SK-BR-3 Cell lysate 1,056 U/mL
HER2/pTyr [0375] 2. Control Source, SK-BR-3 Cell lysate 1,056 U/mL
[0376] 3. Coat antibody, goat anti-HER2 ECD 9.6 mg/mL [0377] 4.
Secondary antibody, biotinylated murine anti-phosphotyrosine, clone
4G10, 971 .mu.g/mL (Upstate Biotech Cat #16-103) [0378] 5.
AMDEX.TM. Streptavidin conjugated to HRP (SA-HRP) (Amersham
Biosciences Catalog No. RPN4401) [0379] 6. Substrate, Tetramethyl
Benzidine (TMB) Peroxidase Substrate (Kirkegaard & Perry Labs
[KPL] Catalog No. 50-76-01) [0380] 7. Coat Buffer, 0.05 M sodium
carbonate buffer, pH 9.6 [0381] 8. Assay Diluent, PBS/0.5%
BSA/0.05% Polysorbate 20/0.05% PROCLIN 300.TM., pH 7.4 [0382] 9.
Lysis Buffer: Base Lysis Buffer (50 mM Tris-HCl/150 mM NaCl/5 mM
EDTA/1% TRITON X-100.TM.)/1:10 Protease Inhibitor Cocktail/1:100
Phosphatase Inhibitor Cocktail I/1:100 Phosphatase Inhibitor
Cocktail II/50 mM sodium fluoride/2 mM sodium ortho-vanadate, pH
8.1 [0383] 10. Sample, Standard, and Control Diluent: Lysis Buffer
[0384] 11. MDA-468 (ATCC# HTB-132). HER2 Expression level: None.
Tissue: human mammary gland, breast, adenocarcinoma [0385] 12.
MCF-7 (ATCC# HTB-22). HER2 Expression level: 0 (normal expression
levels of HER2). Tissue: human mammary gland; breast; epithelial;
metastatic site: pleural effusion adenocarcinoma [0386] 13. SK-BR-3
(ATCC# HTB-30, Manassas, Va.). HER2 Expression level: 3 (high level
HER2 overexpression). Tissue: human mammary gland; breast;
metastatic site: pleural effusion adenocarcinoma [0387] 14. BT-474
(ATCC# HTB-20, Manassas, Va.). HER2 Expression level: 3 (high level
HER2 overexpression) Tissue: human mammary gland; breast; duct;
ductal carcinoma [0388] 15. BT-474 Tumor Lysates. Mice were
inoculated with BT-474. After 2 weeks tumors were harvested.
Harvest tumors were homogenized to produce tumor lysates
Preparation of Materials
[0389] Standard Material/Stock: The phospho HER2 ELISA Standard
Stock is neat Standard Material. The Standard Material was prepared
by collecting lysates from three 245.times.245 mm cell culture
trays containing SK-BR-3 (SKBR3) cells, which were 80% to 90%
confluent. Cell lysates clarified by centrifugation and the
supernatant was collected. The supernatant is used as the Standard
Material.
[0390] The Standard Material was assigned a concentration of 1,056
U/mL so that the lowest calibrator in the assay reporting range
would be 1 U/mL. One unit is defined as the amount of
phosphorylated tyrosine measured in a SK-BR-3 cell lysate
containing 277 pg total HER2 as determined by the Total HER2
ELISA.
[0391] Cell lysate controls: Cell lysate controls were prepared
from the Standard Material. Standard Material was diluted in Lysis
Buffer to obtain HER2/pTyr levels that represent the low, middle,
and high ranges of the assay standard curve.
[0392] Tissue lysate controls: Tissue lysate controls were prepared
from the BT474 tumor lysates. BT474 tumor lysates were diluted in
Lysis Buffer to obtain HER2/pTyr levels that represent the high
range of the assay standard curve.
[0393] Coat source: Goat anti-HER2 ECD Stock I was prepared by
diluting the source material (9.6 mg/mL) to 100 .mu.g/mL in
PBS.
[0394] Biotinylated conjugate: The biotinylated murine
anti-phosphotyrosine antibody (1 .mu.g/mL) was purchased from
Upstate Biotech. The antibody is a biotinylated, protein
A-purified, monoclonal IgG2b-kappa raised against phosphotyramine
coupled to KLH. The biotinylated monoclonal antiphosphotyrosine
antibody (clone 4G10, Cat #05-321) is specific for phosphotyrosine
and does not cross-react with phosphoserine or
phosphothreonine.
[0395] Specificity of goat anti-HER2 ECD: HER2 receptor activation
initiates when receptor dimerization occurs with other family
members. Unless signaling is strictly due to HER2 homodimerization,
EGFR, HER3, and/or HER4 must be expressed within the active tumor.
Each of these receptors may be present in ovarian tissue lysate
samples and could interfere in accurately measuring HER2-associated
tyrosine phosphorylation (HER2/pTyr) if these receptors cross-react
to the coat antibody.
[0396] The specificity of the goat anti-HER2 ECD antibody was
determined by surface plasmon resonance analysis (BIACORE
3000.RTM., BIACORE.RTM. International AB, Neuchatel, Switzerland).
The goat anti-HER2 ECD antibody was immobilized onto a CM5 sensor
chip using amine coupling chemistry. The sensor chip was blocked
with 1 M ethanolamine-HCl, pH 8.5, and conditioned with 10 mM HCl.
Specificity was determined by injecting soluble recombinant EGFR
(sEGFR) (Research Diagnostics, Inc., Flanders, N.J.) and
recombinant human HER2 ECD/human IgG1 Fc fusion proteins over the
immobilized goat anti-HER2 antibody. The fusion proteins consisted
of the ECD of HER2, HER3, or HER4 fused to the carboxy-terminal
6.times. histidine-tagged Fc region of human IgG1 via a peptide
linker (R&D Systems, Minneapolis, Minn.).
[0397] Reference subtracted relative responses for sEGFR, HER3-Fc,
and HER4-Fc were -41 RU, 0.3 RU, and 2.5 RU, respectively. The
negative relative response obtained for sEGFR was due to refractive
index changes between the mobile phase (HBS-EP) and the sample
excipient. The relative response for HER2-Fc was 374 RU.
Methods
[0398] The phospho HER2 ELISA utilizes COSTAR.TM. half-area (A/2)
plates coated with goat anti-HER2 ECD at 4 .mu.g/mL in 0.5 M sodium
carbonate buffer, pH 9.6, and incubated 18-72 hours at 2.degree.
C.-8.degree. C. The wells are blocked with approximately 150
.mu.L/well assay diluent for 1-2 hours and then 50 .mu.L/well of
standards, controls, and samples are added. The minimum dilution
for ovarian tumor tissue lysates is 1/40 in Lysis Buffer. The
standards, controls, and samples are incubated 2 hours at ambient
temperature with agitation. The wells are washed with PBS/0.05
TWEEN 20.TM. and 250 ng/mL of biotinylated anti-phosphotyrosine is
added. After 2 hours, the wells are washed and AMDEX.TM.
streptavidin-HRP is added. AMDEX.TM. streptavidin-HRP (SA-HRP) is a
polymeric conjugate with multiple enzyme labels linked to the
streptavidin. After 15 minutes the wells are washed and a
tetramethyl-benzidine substrate (TMB) is added and allowed to
develop for 15 minutes before being stopped with 1 M phosphoric
acid. The absorbance is measured using a SPECTRAMAX.TM. plate
reader (Molecular Devices Corp., Sunnyvale, Calif.) with a 450 nm
filter and a 650 nm reference filter. The sample concentrations are
calculated relative to a nonlinear, four-parameter logistic fit of
a seven-point standard curve (Marquardt, D. J. Soc. Indust. Appl.
Math. 431-441 (1963)). The assay range of the ELISA is 1 to 32
U/mL. The units are arbitrary units, where 1 U equals the amount of
phosphorylated tyrosine measured in a SK-BR-3 cell lysate
containing 277 pg total HER2. The lower limit of the assay was set
to 1 U/mL and was defined by the lower limit of detection.
[0399] Precision of the phospho HER2 ELISA was re-evaluated after
the Standard Material concentration was re-assigned. Intra- and
inter-assay precision were evaluated by determining the coefficient
of variation (CV) of HER2/pTyr in a SKBR3 cell lysate at three
different levels. The SKBR3 cell lysate was diluted to obtain HER2
levels that represent the low, middle, and high ranges of the assay
standard curve. After the Standard Material concentration was
re-assigned the High Control was not within the high range of the
assay standard curve. Therefore, a BT474 tissue lysate control was
diluted to fall within the high range. The lysates, which were run
as assay controls, were analyzed in duplicate over 5 days. The
control data were imported into STATVIEW for ANOVA.TM. analysis to
determine the intra- and inter-assay standard deviation.
[0400] The CVs were calculated as follows: 100.times.(Standard
Deviation)/(mean control value)
[0401] The intra-assay precision CVs were 4%, 4%, 3%, and 11% for
the BT474, High, Mid, and Low controls, respectively. The
inter-assay precision CVs were 5%, 6%, 5%, and 14% for the BT474,
High, Mid, and Low controls, respectively.
[0402] During the development of the phospho HER2 ELISA, a SKBR3
cell lysate was diluted to 22.9, 6.39, and 1.71 U/mL in neat MDA468
cell lysate. The samples were diluted in Lysis buffer containing
SKBR3 HER2/pTyr to maintain a constant level of HER2/pTyr
throughout the entire dilution series while matrix effects are
diluted out. The dilution series was analyzed in the Phospho HER2
ELISA and compared to SKBR3 HER2/pTyr without MDA468 to determine
recovery. Percent recovery was calculated as follows: 100 .times.
SKBR .times. .times. 3 .times. .times. HER .times. .times. 2
.times. / .times. pTyr .times. .times. diluted .times. .times. in
.times. .times. MDA .times. .times. 468 SKBR .times. .times. 3
.times. .times. HER .times. .times. 2 .times. / .times. pTyr
.times. .times. diluted .times. .times. in .times. .times. Lysis
.times. .times. Buffer ##EQU1##
[0403] The results for SKBR3 HER2/pTyr recovery at the three levels
in the presence of MDA468 cell lysate, revealed the matrix
significantly enhances recovery in sample dilutions between neat
and 1/16 at the 1.71 U/mL level, with HER2/pTyr recoveries between
120% and 127%. Matrix interference was not observed at any other
level. Recovery between 80% and 120% is demonstrated in each level
starting at a sample dilution of 1/16 in Lysis Buffer.
[0404] Ovarian and BT474 tumor lysates were serially diluted
two-fold in Lysis Buffer and analyzed in the Phospho HER2 ELISA.
The starting dilution for the BT474 samples, which were analyzed
during development, was 1/20. The ovarian lysates were analyzed
after the Standard Material concentration was re-assigned. The
starting dilution for the ovarian lysates varied according to
expected HER2/pTyr concentrations.
[0405] The percent difference for the dilution series, which is an
indicator of sample dilution linearity, was calculated as follows:
100 .times. ( greatest .times. .times. Corrected .times. .times.
Result .times. .times. value - lowest .times. .times. Corrected
.times. .times. Result .times. .times. value ) ( average .times.
.times. of .times. .times. greatest .times. .times. and .times.
.times. lowest .times. .times. Corrected .times. .times. Result
.times. .times. values ) ##EQU2##
[0406] Percent differences for ovarian tissue lysate HF8198 were
calculated starting at a dilution of either 1/80, 1/160, or 1/320.
Percent differences for ovarian tissue lysate HF7945 were
calculated starting at a dilution of either 1/320, 1/640, or
1/1280. Percent differences for the remaining ovarian tissue
lysates were calculated starting at a dilution of either 1/20, or
1/40, to determine the minimum dilution as well as assess the
linearity of dilution.
[0407] The differences for the BT474 dilution series ranged from
-9% to 12%. The differences for HF7930 and HF7934 dilution series
starting at a 1/10 dilution were 43% and 38%, respectively. The
differences for HF8197 were 2%, 8%, and 5% for dilution series
starting at 1/320, 1/160, and 1/1280, respectively. The differences
for HF8198 were 72%, 34%, and 3%, for dilution series starting at
1/80, 1/160, and 1/320, respectively.
[0408] Eighteen different ovarian tissue lysates were analyzed in
the phospho HER2 ELISA after the Standard Material concentration
was changed. Samples were diluted two-fold starting from 1/20 to
1/160. One sample was LTR at a 1/20 dilution; 10 samples were LTR
at a 1/40 dilution. Seven samples had measurable levels of
HER2/pTyr at 1/20 and 1/40 dilutions and one sample had measurable
levels of HER2/pTyr up to a 1/80 dilution. The differences for
samples that had measurable levels of HER2/pTyr at 1/20 and 1/40
dilutions ranged from 16% to 34%, with three of seven samples with
differences less than 20%. The one sample that had measurable
levels of HER2/pTyr up to a 1/80 dilution, sample HF7931, had
differences of 16% and 4% for dilution series starting at 1/20 and
1/40, respectively.
[0409] Pertuzimab and trastuzumab were analyzed in the phospho HER2
ELISA to determine if these therapeutics interfere. The antibodies
were diluted to concentrations ranging from 1.5 to 10,000 ng/mL in
heregulin stimulated MCF7 (MCF7+) cell lysates containing 98.48
U/mL HER2/pTyr.
[0410] During development, cell and tissue lysates were subjected
to four cycles of freezing and thawing to determine the effects of
temperature cycling. Frozen SKBR3 cell lysate and BT474 tumor
lysates were thawed at ambient temperature. From each lysate 10
.mu.L were removed and diluted in Assay Diluent. The remaining
lysates were flash frozen in a mixture of dry ice and methanol and
thawed again. Test samples were once again removed and diluted in
Assay Diluent (first freeze/thaw cycle, 1 x). The flash freeze,
thaw and sample collection procedure was repeated twice to obtain
samples from the second and third freeze/thaw cycles (2.times. and
3.times., respectively).
[0411] Diluted samples were assayed in the phospho HER2 ELISA to
determine HER2/pTyr recovery with respect to the "fresh" sample.
The "fresh" sample is the sample taken from the initial
thawing.
[0412] Recovery of SKBR3 HER2/pTyr for 1.times., 2.times., and
3.times. samples were 104%, 109%, and 113%, respectively. Recovery
of BT474 HER2/pTyr in sample 314A were 99%, 103%, and 99%, for
1.times., 2.times., and 3.times. samples, respectively. Recovery of
BT474 HER2/pTyr in sample 365 were 111%, 96%, and 99%, for
1.times., 2.times., and 3.times. samples, respectively.
[0413] The Lower Limit of Quantitation (LLOQ) was set as the
average concentration of the low control, 1.35 U/mL. Because the
low control is included within each experiment, it is a reliable
indicator of the lower limit to which samples can be accurately
measured. Therefore, the minimum quantifiable concentration in the
phospho HER2 ELISA is the LLOQ multiplied by the minimum sample
dilution ( 1/40), or 54 U/mL.
Conclusions
[0414] A sensitive and accurate ELISA was developed to measure
HER2-associated tyrosine phosphorylation (HER2/pTyr) in tumor
tissue lysates. The phospho HER2 ELISA demonstrated sensitivity
down to 1.35 U/mL with a minimum quantifiable concentration of 54
U/mL, where 1 U is equal to the amount of phosphorylated tyrosine
measured in a SK-BR-3 cell lysate containing 277 pg total HER2. The
phospho-HER2 ELISA demonstrated good precision at four levels. The
intra-assay precision CVs were 4%, 3%, 3%, and 11%, for the BT474
tissue lysate control and the High, Mid, and Low SKBR3 cell lysate
controls, respectively. The inter-assay precision CVs were 5%, 6%,
5%, and 14%, for the BT474 tissue lysate control and the High, Mid,
and Low SKBR3 cell lysate controls, respectively.
[0415] The phospho HER2 ELISA demonstrated good recovery of
HER2/pTyr in the presence MDA468 cell lysate. Starting at a 1/16
dilution, recoveries ranged from 88% to 120%. The ELISA
demonstrated high specificity as EGFR, HER3-IgG Fc, and HER4-IgG Fc
do not cross-react with the assay coat.
[0416] Human ovarian tumor and BT474 mouse xenograft tumor tissue
lysates were used to analyze linearity of dilution and minimum
sample dilution. The differences of dilution corrected values for
the BT474 tumor lysates ranged from -9% to 12%. Out of the seven
ovarian lysates that had measurable levels of HER2/pTyr at 1/20 and
1/40 dilutions, only three had differences less than 20%, while six
out of seven had differences less than or equal to 23%. The one
sample that had measurable levels of HER2/pTyr up to a 1/80
dilution, sample HF7931, had a difference of 4% for dilution series
starting at 1/40. All of the above samples did not meet the
.ltoreq.20% criteria at a minimum dilution of 1/20, therefore, the
minimum sample dilution will be 1/40.
[0417] The BT474 tissue lysates and human ovarian tissue lysate
samples HF8197 and HF8198 had high measurable levels of HER2/pTyr
and required dilutions between 1/80 to 1/320 to fall within the
quantitative range of the assay. The BT474 samples and sample
HF8197, which had the highest measured HER2/pTyr concentration
within the human ovarian tumor tissue subset, diluted linearly
throughout the entire assay range. In contrast, sample HF8198
diluted nonlinearly as the corrected for dilution HER2/pTyr
concentrations monotonically increased throughout the assay range
and appear to plateau at a 1/320 dilution.
[0418] SKBR3 cell lysates subjected to three freeze/thaw cycles
demonstrated very good recovery. HER2/pTyr recovery ranged from
104% to 113% with respect to the same freshly thawed sample.
[0419] Two BT474 tumor lysates were also subjected to three
freeze/thaw cycles. BT474 phospho HER2 recovery ranged from 99% to
103%. HER2/pTyr recovery from BT474 ranged from 96% to 111%.
Therefore, temperature cycling does not appear to effect phospho
HER2 activity.
[0420] The phospho HER2 ELISA does not demonstrate any interference
from either pertuzimab or trastuzumab.
EXAMPLE 3
Gene Expression Profiling for Determining HER2 Activation
[0421] This example shows how HER2 activation can be evaluated by
determining gene expression profiles as an alternative to
determining HER2 phosphorylation directly. This profiling may be
done on fresh, frozen, or formalin-fixed, paraffin-embedded ovarian
tumor specimens, but preferably the latter.
[0422] Ovarian cancer specimens treated with pertuzumab were
profiled for gene expression using AFFYMETRIX.RTM. microarray
analysis performed according to the manufacturer's instructions.
The microarray expression data was analyzed to identify gene
patterns which would be associated with HER2 phosphorylation
status. Remarkably, a pattern emerged where tumors with relatively
high levels of expression of EGFR, HER2, HER3, and the HER ligand
betacelullin were also positive for HER2 phosphorylation. The
correlation was positive in six of the six HER2 phosphorylation
positive cases, and none of the HER2 phosphorylation negative cases
were predicted positive using microarray expression data as the
basis for the algorithm.
[0423] In a second analysis, prediction of HER2 phosphorylation
status was achieved by using a single gene only, namely
betacellulin. All six HER2 phosphorylation positive tumors had a
betacellulin expression above the median, again using microarray
expression data.
[0424] A second method for quantifying gene expression,
quantitative real time polymerase chain reaction (qRT-PCR), was
used to validate, and was compared with, the microarray data.
qRT-PCT would be a preferred method for measuring gene expression
in the typical patient sample available in a clinical setting.
Diagnostic technology platforms are already established for this
method. qRT-PCR was performed as described in Cronin et al., Am. J.
Pathol. 164(1):35-42 (2004); and Ma et al., Cancer Cell 5:607-616
(2004). RNA was extracted from frozen ovarian tumors using
commercially available reagents from Qiagen, Valencia, Calif.
Primers and probes for TAQMAN.TM. qRT-PCR analysis were designed to
give amplicon lengths of about 100 bases or less. Transcripts were
quantitated by qRT-PCR using a TAQMAN.TM. instrument (Applied
BioSystems), with expression levels of the test genes normalized to
those of the reference genes. The "house keeping" gene GUS was
selected as the control gene because of its low variance and high
expression.
[0425] Based on the experiments noted above an algorithm was
developed based on gene expression profiling date of tumors with
known HER2 phosphorylation status by ELISA. A tumor is deemed
positive for a gene expression profile associated with HER2
phosphorylation that has betacellulin or amphiregulin and HER2
expression at the median or above and/or EGFR and/or HER3
expression at the median or above. Alternatively, expression of
betacellulin or amphiregulin alone can be measured by qRT-PCR to
identify tumors with predicted phosphorylation of HER2.
Sequence CWU 1
1
22 1 107 PRT Mus musculus 1 Asp Thr Val Met Thr Gln Ser His Lys Ile
Met Ser Thr Ser Val 1 5 10 15 Gly Asp Arg Val Ser Ile Thr Cys Lys
Ala Ser Gln Asp Val Ser 20 25 30 Ile Gly Val Ala Trp Tyr Gln Gln
Arg Pro Gly Gln Ser Pro Lys 35 40 45 Leu Leu Ile Tyr Ser Ala Ser
Tyr Arg Tyr Thr Gly Val Pro Asp 50 55 60 Arg Phe Thr Gly Ser Gly
Ser Gly Thr Asp Phe Thr Phe Thr Ile 65 70 75 Ser Ser Val Gln Ala
Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 80 85 90 Tyr Tyr Ile Tyr
Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu 95 100 105 Ile Lys 2
119 PRT Mus musculus 2 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
Val Lys Pro Gly 1 5 10 15 Thr Ser Val Lys Ile Ser Cys Lys Ala Ser
Gly Phe Thr Phe Thr 20 25 30 Asp Tyr Thr Met Asp Trp Val Lys Gln
Ser His Gly Lys Ser Leu 35 40 45 Glu Trp Ile Gly Asp Val Asn Pro
Asn Ser Gly Gly Ser Ile Tyr 50 55 60 Asn Gln Arg Phe Lys Gly Lys
Ala Ser Leu Thr Val Asp Arg Ser 65 70 75 Ser Arg Ile Val Tyr Met
Glu Leu Arg Ser Leu Thr Phe Glu Asp 80 85 90 Thr Ala Val Tyr Tyr
Cys Ala Arg Asn Leu Gly Pro Ser Phe Tyr 95 100 105 Phe Asp Tyr Trp
Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 110 115 3 107 PRT
Artificial Sequence sequence is synthesized 3 Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val
Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser 20 25 30 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 50 55 60 Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85
90 Tyr Tyr Ile Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu 95
100 105 Ile Lys 4 119 PRT Artificial Sequence sequence is
synthesized 4 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Thr 20 25 30 Asp Tyr Thr Met Asp Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu 35 40 45 Glu Trp Val Ala Asp Val Asn Pro Asn Ser
Gly Gly Ser Ile Tyr 50 55 60 Asn Gln Arg Phe Lys Gly Arg Phe Thr
Leu Ser Val Asp Arg Ser 65 70 75 Lys Asn Thr Leu Tyr Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp 80 85 90 Thr Ala Val Tyr Tyr Cys Ala
Arg Asn Leu Gly Pro Ser Phe Tyr 95 100 105 Phe Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 110 115 5 107 PRT Artificial
Sequence sequence is synthesized 5 Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Ser Ile Ser 20 25 30 Asn Tyr Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45 Leu Leu Ile Tyr Ala
Ala Ser Ser Leu Glu Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90 Tyr Asn
Ser Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105 Ile
Lys 6 119 PRT Artificial Sequence sequence is synthesized 6 Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30
Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40
45 Glu Trp Val Ala Val Ile Ser Gly Asp Gly Gly Ser Thr Tyr Tyr 50
55 60 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
65 70 75 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp 80 85 90 Thr Ala Val Tyr Tyr Cys Ala Arg Gly Arg Val Gly Tyr
Ser Leu 95 100 105 Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 110 115 7 10 PRT Artificial sequence Sequence is
synthesized. 7 Gly Phe Thr Phe Thr Asp Tyr Thr Met Xaa 5 10 8 17
PRT Artificial sequence Sequence is synthesized. 8 Asp Val Asn Pro
Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe 1 5 10 15 Lys Gly 9 10
PRT Artificial sequence Sequence is synthesized. 9 Asn Leu Gly Pro
Ser Phe Tyr Phe Asp Tyr 5 10 10 11 PRT Artificial sequence Sequence
is synthesized. 10 Lys Ala Ser Gln Asp Val Ser Ile Gly Val Ala 5 10
11 7 PRT Artificial sequence sequence is synthesized 11 Ser Ala Ser
Tyr Xaa Xaa Xaa 5 12 9 PRT Artificial sequence Sequence is
synthesized. 12 Gln Gln Tyr Tyr Ile Tyr Pro Tyr Thr 5 13 214 PRT
Artificial sequence Sequence is synthesized. 13 Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val
Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser 20 25 30 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 50 55 60 Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85
90 Tyr Tyr Ile Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu 95
100 105 Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
110 115 120 Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys
Leu 125 130 135 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp
Lys Val 140 145 150 Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser
Val Thr Glu 155 160 165 Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
Ser Thr Leu Thr 170 175 180 Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr Ala Cys Glu 185 190 195 Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser Phe Asn 200 205 210 Arg Gly Glu Cys 14 448 PRT
Artificial sequence Sequence is synthesized. 14 Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr 20 25 30 Asp Tyr Thr
Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45 Glu Trp
Val Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr 50 55 60 Asn
Gln Arg Phe Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser 65 70 75
Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85
90 Thr Ala Val Tyr Tyr Cys Ala Arg Asn Leu Gly Pro Ser Phe Tyr 95
100 105 Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala
110 115 120 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
Lys 125 130 135 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp 140 145 150 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
Gly Ala Leu 155 160 165 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly 170 175 180 Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu 185 190 195 Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn 200 205 210 Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys Asp Lys Thr 215 220 225 His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro 230 235 240 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 260 265 270 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 305 310 315 Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 320 325 330
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 335 340
345 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 350
355 360 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
365 370 375 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu 380 385 390 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser 395 400 405 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln 410 415 420 Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His 425 430 435 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly 440 445 15 214 PRT Artificial sequence Sequence is
synthesized. 15 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val 1 5 10 15 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Val Asn 20 25 30 Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys 35 40 45 Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr
Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Arg Ser Gly Thr
Asp Phe Thr Leu Thr Ile 65 70 75 Ser Ser Leu Gln Pro Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln 80 85 90 His Tyr Thr Thr Pro Pro Thr
Phe Gly Gln Gly Thr Lys Val Glu 95 100 105 Ile Lys Arg Thr Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro 110 115 120 Ser Asp Glu Gln Leu
Lys Ser Gly Thr Ala Ser Val Val Cys Leu 125 130 135 Leu Asn Asn Phe
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val 140 145 150 Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu 155 160 165 Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr 170 175 180 Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu 185 190 195
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn 200 205
210 Arg Gly Glu Cys 16 449 PRT Artificial sequence sequence is
synthesized 16 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Asn Ile Lys 20 25 30 Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu 35 40 45 Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn
Gly Tyr Thr Arg Tyr 50 55 60 Ala Asp Ser Val Lys Gly Arg Phe Thr
Ile Ser Ala Asp Thr Ser 65 70 75 Lys Asn Thr Ala Tyr Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp 80 85 90 Thr Ala Val Tyr Tyr Cys Ser
Arg Trp Gly Gly Asp Gly Phe Tyr 95 100 105 Ala Met Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 110 115 120 Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 125 130 135 Lys Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 140 145 150 Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 155 160 165 Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 170 175 180 Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 185 190 195
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 200 205
210 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 215
220 225 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn 290 295 300 Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp 305 310 315 Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala 320 325 330 Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln 335 340 345 Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu 350 355 360 Met Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe 365 370 375 Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 380 385 390 Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 395 400 405 Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 410 415 420 Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 425 430 435 His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 440 445 17 217
PRT Artificial sequence Sequence is synthesized. 17 Val His Ser Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 1 5 10 15 Ala Ser Val
Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln 20 25 30 Asp Val
Ser Ile Gly Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala
Pro Lys Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly 50 55 60
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 65 70
75 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr 80
85 90 Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr Thr Phe Gly Gln Gly Thr
95 100 105 Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe
Ile 110 115 120 Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala
Ser Val 125 130
135 Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln 140
145 150 Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser
155 160 165 Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
Ser 170 175 180 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr 185 190 195 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys 200 205 210 Ser Phe Asn Arg Gly Glu Cys 215 18 449 PRT
Artificial sequence Sequence is synthesized. 18 Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr 20 25 30 Asp Tyr Thr
Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45 Glu Trp
Val Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr 50 55 60 Asn
Gln Arg Phe Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser 65 70 75
Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85
90 Thr Ala Val Tyr Tyr Cys Ala Arg Asn Leu Gly Pro Ser Phe Tyr 95
100 105 Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala
110 115 120 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
Lys 125 130 135 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp 140 145 150 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
Gly Ala Leu 155 160 165 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly 170 175 180 Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu 185 190 195 Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn 200 205 210 Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys Asp Lys Thr 215 220 225 His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro 230 235 240 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 260 265 270 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 305 310 315 Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 320 325 330
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 335 340
345 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 350
355 360 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
365 370 375 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu 380 385 390 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser 395 400 405 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln 410 415 420 Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His 425 430 435 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 440 445 19 195 PRT Homo sapiens 19 Thr Gln Val Cys
Thr Gly Thr Asp Met Lys Leu Arg Leu Pro Ala 1 5 10 15 Ser Pro Glu
Thr His Leu Asp Met Leu Arg His Leu Tyr Gln Gly 20 25 30 Cys Gln
Val Val Gln Gly Asn Leu Glu Leu Thr Tyr Leu Pro Thr 35 40 45 Asn
Ala Ser Leu Ser Phe Leu Gln Asp Ile Gln Glu Val Gln Gly 50 55 60
Tyr Val Leu Ile Ala His Asn Gln Val Arg Gln Val Pro Leu Gln 65 70
75 Arg Leu Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr 80
85 90 Ala Leu Ala Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr
95 100 105 Pro Val Thr Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln
Leu 110 115 120 Arg Ser Leu Thr Glu Ile Leu Lys Gly Gly Val Leu Ile
Gln Arg 125 130 135 Asn Pro Gln Leu Cys Tyr Gln Asp Thr Ile Leu Trp
Lys Asp Ile 140 145 150 Phe His Lys Asn Asn Gln Leu Ala Leu Thr Leu
Ile Asp Thr Asn 155 160 165 Arg Ser Arg Ala Cys His Pro Cys Ser Pro
Met Cys Lys Gly Ser 170 175 180 Arg Cys Trp Gly Glu Ser Ser Glu Asp
Cys Gln Ser Leu Thr Arg 185 190 195 20 124 PRT Homo sapiens 20 Thr
Val Cys Ala Gly Gly Cys Ala Arg Cys Lys Gly Pro Leu Pro 1 5 10 15
Thr Asp Cys Cys His Glu Gln Cys Ala Ala Gly Cys Thr Gly Pro 20 25
30 Lys His Ser Asp Cys Leu Ala Cys Leu His Phe Asn His Ser Gly 35
40 45 Ile Cys Glu Leu His Cys Pro Ala Leu Val Thr Tyr Asn Thr Asp
50 55 60 Thr Phe Glu Ser Met Pro Asn Pro Glu Gly Arg Tyr Thr Phe
Gly 65 70 75 Ala Ser Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu Ser
Thr Asp 80 85 90 Val Gly Ser Cys Thr Leu Val Cys Pro Leu His Asn
Gln Glu Val 95 100 105 Thr Ala Glu Asp Gly Thr Gln Arg Cys Glu Lys
Cys Ser Lys Pro 110 115 120 Cys Ala Arg Val 21 169 PRT Homo sapiens
21 Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu Val Arg Ala Val 1 5
10 15 Thr Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys Lys Ile Phe
20 25 30 Gly Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro
Ala 35 40 45 Ser Asn Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln Val
Phe Glu 50 55 60 Thr Leu Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser
Ala Trp Pro 65 70 75 Asp Ser Leu Pro Asp Leu Ser Val Phe Gln Asn
Leu Gln Val Ile 80 85 90 Arg Gly Arg Ile Leu His Asn Gly Ala Tyr
Ser Leu Thr Leu Gln 95 100 105 Gly Leu Gly Ile Ser Trp Leu Gly Leu
Arg Ser Leu Arg Glu Leu 110 115 120 Gly Ser Gly Leu Ala Leu Ile His
His Asn Thr His Leu Cys Phe 125 130 135 Val His Thr Val Pro Trp Asp
Gln Leu Phe Arg Asn Pro His Gln 140 145 150 Ala Leu Leu His Thr Ala
Asn Arg Pro Glu Asp Glu Cys Val Gly 155 160 165 Glu Gly Leu Ala 22
142 PRT Homo sapiens 22 Cys His Gln Leu Cys Ala Arg Gly His Cys Trp
Gly Pro Gly Pro 1 5 10 15 Thr Gln Cys Val Asn Cys Ser Gln Phe Leu
Arg Gly Gln Glu Cys 20 25 30 Val Glu Glu Cys Arg Val Leu Gln Gly
Leu Pro Arg Glu Tyr Val 35 40 45 Asn Ala Arg His Cys Leu Pro Cys
His Pro Glu Cys Gln Pro Gln 50 55 60 Asn Gly Ser Val Thr Cys Phe
Gly Pro Glu Ala Asp Gln Cys Val 65 70 75 Ala Cys Ala His Tyr Lys
Asp Pro Pro Phe Cys Val Ala Arg Cys 80 85 90 Pro Ser Gly Val Lys
Pro Asp Leu Ser Tyr Met Pro Ile Trp Lys 95 100 105 Phe Pro Asp Glu
Glu Gly Ala Cys Gln Pro Cys Pro Ile Asn Cys 110 115 120 Thr His Ser
Cys Val Asp Leu Asp Asp Lys Gly Cys Pro Ala Glu 125 130 135 Gln Arg
Ala Ser Pro Leu Thr 140
* * * * *