U.S. patent application number 12/609737 was filed with the patent office on 2010-06-03 for methods of treatment using anti-erbb antibody-maytansinoid conjugates.
This patent application is currently assigned to IMMUNOGEN, INC.. Invention is credited to Walter Blattler, Sharon Erickson, Ralph Schwall, Mark Sliwkowski.
Application Number | 20100136033 12/609737 |
Document ID | / |
Family ID | 27392641 |
Filed Date | 2010-06-03 |
United States Patent
Application |
20100136033 |
Kind Code |
A1 |
Erickson; Sharon ; et
al. |
June 3, 2010 |
METHODS OF TREATMENT USING ANTI-ErbB ANTIBODY-MAYTANSINOID
CONJUGATES
Abstract
The application concerns methods of treatment using anti-ErbB
receptor antibody-maytansinoid conjugates, and articles of
manufacture suitable for use in such methods. In particular, the
invention concerns ErbB receptor-directed cancer therapies, using
anti-ErbB receptor antibody-maytansinoid conjugates.
Inventors: |
Erickson; Sharon;
(Hillsborough, CA) ; Schwall; Ralph; (Pacifica,
CA) ; Sliwkowski; Mark; (San Carlos, CA) ;
Blattler; Walter; (Brookline, MA) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W., SUITE 800
WASHINGTON
DC
20037
US
|
Assignee: |
IMMUNOGEN, INC.
Cambridge
MA
|
Family ID: |
27392641 |
Appl. No.: |
12/609737 |
Filed: |
October 30, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11949351 |
Dec 3, 2007 |
|
|
|
12609737 |
|
|
|
|
11488545 |
Jul 17, 2006 |
7575748 |
|
|
11949351 |
|
|
|
|
09811123 |
Mar 16, 2001 |
7097840 |
|
|
11488545 |
|
|
|
|
60238327 |
Oct 5, 2000 |
|
|
|
60327563 |
Jun 23, 2000 |
|
|
|
60189844 |
Mar 16, 2000 |
|
|
|
Current U.S.
Class: |
424/181.1 ;
530/391.9 |
Current CPC
Class: |
A61K 47/6871 20170801;
A01K 2207/15 20130101; A01K 2217/05 20130101; C12N 15/8509
20130101; A61K 9/0019 20130101; C07K 16/2863 20130101; C07K 16/32
20130101; A61K 39/39558 20130101; A01K 2217/00 20130101; C07K
16/3015 20130101; A61P 35/00 20180101; A01K 2227/105 20130101; A01K
67/0275 20130101; A01K 67/0271 20130101; A61K 47/6855 20170801;
A61K 2039/505 20130101; A01K 67/0278 20130101; A61K 47/6803
20170801; A01K 2217/052 20130101; C07K 2317/24 20130101; A01K
2267/0331 20130101 |
Class at
Publication: |
424/181.1 ;
530/391.9 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/00 20060101 C07K016/00; A61P 35/00 20060101
A61P035/00 |
Claims
1. A composition of matter comprising an anti-ErbB2 antibody
covalently linked to a maytansinoid by
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),
wherein the anti-ErbB2 antibody is a humanized or human
antibody.
2. The composition of claim 1, wherein the anti-ErbB2 antibody
binds essentially the same epitope as monoclonal antibody 4D5.
3. The composition of claim 1, wherein the anti-ErbB2 antibody is a
humanized form of murine monoclonal antibody 4D5.
4. The composition of claim 3, wherein the humanized antibody is
huMAb4D5-8 (HERCEPTIN.RTM.).
5. The composition of claim 1, wherein the composition has a growth
inhibitory effect on SK-BR3 cells in vitro.
6. The composition of claim 5, wherein the growth inhibitory effect
comprises a significant inhibition of the in vitro growth of SK-BR3
cells at concentrations greater than or equal to about 0.01
.mu.g/mL.
7. The composition of claim 1, wherein the composition has
anti-tumor activity in a mouse tumor model that overexpresses HER2
and does not respond to HERCEPTIN.RTM..
8. The composition of claim 1 wherein the anti-ErbB2 antibody is an
antigen binding fragment selected from the group consisting of a
Fab, Fab', F(ab').sub.2, F.sub.v fragment, diabody, linear
antibody, and single chain antibody.
9. The composition of claim 1, wherein the maytansinoid is selected
from the group consisting of maytansine, maytansinol and a
maytansinol ester.
10. The composition of claim 9, wherein the maytansinoid is DM1
having the structure ##STR00001##
11. The composition of claim 1 comprising from about 1 to about 10
maytansinoid molecules per antibody molecule.
12. The composition of claim 1 comprising from about 3 to about 5
maytansinoid molecules per antibody molecule.
13. A pharmaceutical composition comprising the composition of
claim 1 and a pharmaceutically acceptable carrier.
14. A composition comprising huMAb4D5-8 (HERCEPTIN.RTM.) conjugated
to DM1 by
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),
wherein DM1 has the structure ##STR00002##
15. The composition of claim 14 comprising from about 1 to about 10
maytansinoid molecules per antibody molecule.
16. The composition of claim 14 comprising from about 3 to about 5
maytansinoid molecules per antibody molecule.
17. A pharmaceutical composition comprising the composition of
claim 14 and a pharmaceutically acceptable carrier.
18. The composition of claim 1, wherein said SMCC linker is
covalently linked to a lysine residue of said anti-ErbB2
antibody.
19. The composition of claim 14, wherein the SMCC is covalently
linked to a lysine residue of huMAb4D5-8 (HERCEPTIN.RTM.), wherein
the lysine residue is selected from lysine 13 in the light chain,
lysine 32 in the heavy chain, lysine 26 in both Fab fragments, and
lysine 38 in the Fc fragment of huMAb4D5-8 (HERCEPTIN.RTM.).
20. An immunoconjugate comprising an anti-ErbB2 antibody conjugated
to a maytansinoid, wherein the antibody is huMAb4D5-8.
21. The immunoconjugate of claim 20, wherein the maytansinoid is
DM1 having the structure: ##STR00003## and wherein the antibody is
chemically linked to the maytansinoid via a disulfide or thioether
group at "R" shown in the structure.
22. The immunoconjugate of claim 20, wherein the immunoconjugate
comprises from 3 to 5 maytansinoid molecules per antibody
molecule.
23. The immunoconjugate of claim 20 or claim 21, wherein the
antibody and the maytansinoid are conjugated by a chemical linker
selected from N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),
N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) and
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
(SMCC).
24. A pharmaceutical composition comprising an immunoconjugate of
any of claims 20 to 22, and a pharmaceutically acceptable carrier.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S. Ser.
No. 11/488,545, filed Jul. 7, 2006, which is a continuation
application of U.S. Ser. No. 09/811,123, filed Mar. 16, 2001 which
application claims priority to U.S. Provisional Application Nos.
60/238,327, filed Oct. 5, 2000, 60/189,844 filed Mar. 16, 2000, and
60/327,563 filed Jun. 23, 2000 (converted from U.S. application
Ser. No. 09/602,530) under 35 USC .sctn.119(e).
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention concerns methods of treatment,
especially ErbB receptor-directed cancer therapies, using anti-ErbB
receptor antibody-maytansinoid conjugates, and articles of
manufacture suitable for use in such methods.
[0004] 2. Description of the Related Art
[0005] 1. Maytansine and Maytansinoids
[0006] Maytansine was first isolated from the east African shrub
Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it was
discovered that certain microbes also produce maytansinoids, such
as maytansinol and C-3 maytansinol esters (U.S. Pat. No.
4,151,042). Synthetic maytansinol and maytansinol analogues are
disclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;
4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;
4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;
4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663;
and 4,371,533, the disclosures of which are hereby expressly
incorporated by reference.
[0007] Maytansine and maytansinoids are highly cytotoxic but their
clinical use in cancer therapy has been greatly limited by their
severe systemic side-effects primarily attributed to their poor
selectivity for tumors. Clinical trials with maytansine had been
discontinued due to serious adverse effects on the central nervous
system and gastrointestinal system (Issel et al., Can. Trtmnt. Rev.
5:199-207 [1978]).
[0008] 2. The ErbB Family of Receptor Tyrosine Kinases and
Anti-ErbB Antibodies
[0009] Members of the ErbB family of receptor tyrosine kinases are
important mediators of cell growth, differentiation and survival.
The receptor family includes four distinct members, including
epidermal growth factor receptor (EGFR or ErbB1), HER2 (ErbB2 or
p185.sup.neu), HER3 (ErbB3) and HER4 (ErbB4 or tyro2).
[0010] 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 homologue
of neu is observed in breast and ovarian cancers and correlates
with a poor prognosis (Slamon et al., Science, 235:177-182 (1987);
Slamon et al., Science, 244:707-712 (1989); and U.S. Pat. No.
4,968,603). To date, no point mutation analogous to that in the neu
proto-oncogene has been reported for human tumors. Overexpression
of ErbB2 (frequently but not uniformly due to gene amplification)
has also been observed in other carcinomas including carcinomas of
the stomach, endometrium, salivary gland, lung, kidney, colon,
thyroid, pancreas and bladder. See, among others, King et al.,
Science, 229:974 (1985); Yokota et al., Lancet: 1:765-767 (1986);
Fukushigi et al., Mol Cell Biol., 6:955-958 (1986); Geurin et al.,
Oncogene Res., 3:21-31 (1988); Cohen et al., Oncogene, 4:81-88
(1989); Yonemura et al., Cancer Res., 51:1034 (1991); Borst et al.,
Gynecol. Oncol., 38:364 (1990); Weiner et al., Cancer Res.,
50:421-425 (1990); Kern et al., Cancer Res., 50:5184 (1990); Park
et al., Cancer Res., 49:6605 (1989); Zhau et al., Mol. Carcinog.,
3:354-357 (1990); Aasland et al. Br. J. Cancer 57:358-363 (1988);
Williams et al. Pathobiology 59:46-52 (1991); and McCann et al.,
Cancer, 65:88-92 (1990). ErbB2 may be overexpressed in prostate
cancer (Gu et al. Cancer Lett. 99:185-9 (1996); Ross et al. Hum.
Pathol. 28:827-33 (1997); Ross et al. Cancer 79:2162-70 (1997); and
Sadasivan et al. J. Urol. 150:126-31 (1993)).
[0011] A spliced form of erbB2 oncogen encoding a constitutively
tyrosine phosphorylated ErbB2 receptor is disclosed in PCT
publication WO 00/20579, published on Apr. 13, 2000. The erbB2
protein encoded by the splice variant has an in frame deletion of
16 amino acids (CVDLDDKGCPAEQRAS (SEQ ID NO: 11)), two of which are
conserved cysteine residues.
[0012] Antibodies directed against the rat p185.sup.neu and human
ErbB2 protein products have been described. Drebin and colleagues
have raised antibodies against the rat neu gene product,
p185.sup.neu. See, for example, Drebin et al., Cell 41:695-706
(1985); Myers et al., Meth. Enzym. 198:277-290 (1991); and
WO94/22478. Drebin et al. Oncogene 2:273-277 (1988) report that
mixtures of antibodies reactive with two distinct regions of
p185.sup.neu result in synergistic anti-tumor effects on
neu-transformed NIH-3T3 cells implanted into nude mice. See also
U.S. Pat. No. 5,824,311 issued Oct. 20, 1998.
[0013] Other anti-ErbB2 antibodies with various properties have
been described in Tagliabue et al. Int. J. Cancer 47:933-937
(1991); McKenzie et al. Oncogene 4:543-548 (1989); Maier et al.
Cancer Res. 51:5361-5369 (1991); Bacus et al. Molecular
Carcinogenesis 3:350-362 (1990); Stancovski et al. PNAS (USA)
88:8691-8695 (1991); Bacus et al. Cancer Research 52:2580-2589
(1992); Xu et al. Int. J. Cancer 53:401-408 (1993); WO94/00136;
Kasprzyk et al. Cancer Research 52:2771-2776 (1992); Hancock et al.
Cancer Res. 51:4575-4580 (1991); Shawver et al. Cancer Res.
54:1367-1373 (1994); Arteaga et al. Cancer Res. 54:3758-3765
(1994); Harwerth et al. J. Biol. Chem. 267:15160-15167 (1992); U.S.
Pat. No. 5,783,186; and Klapper et al. Oncogene 14:2099-2109
(1997).
[0014] Hudziak et al., Mol. Cell. Biol. 9(3): 1165-1172 (1989)
describe the generation of a panel of anti-ErbB2 antibodies which
were characterized using the human breast tumor cell line SK-BR-3.
Relative cell proliferation of the SK-BR-3 cells following exposure
to the antibodies was determined by crystal violet staining of the
monolayers after 72 hours. Using this assay, maximum inhibition was
obtained with the antibody called 4D5 which inhibited cellular
proliferation by 56%. Other antibodies in the panel reduced
cellular proliferation to a lesser extent in this assay. The
antibody 4D5 was further found to sensitize ErbB2-overexpressing
breast tumor cell lines to the cytotoxic effects of TNF-.alpha..
See also U.S. Pat. No. 5,677,171 issued Oct. 14, 1997. The
anti-ErbB2 antibodies discussed in Hudziak et al. are further
characterized in Fendly et al. Cancer Research 50:1550-1558 (1990);
Kotts et al. In Vitro 26(3):59A (1990); Sarup et al. Growth
Regulation 1:72-82 (1991); Shepard et al. J. Clin. Immunol.
11(3):117-127 (1991); Kumar et al. Mol. Cell. Biol. 11(2):979-986
(1991); Lewis et al. Cancer Immunol. Immunother. 37:255-263 (1993);
Pietras et al. Oncogene 9:1829-1838 (1994); Vitetta et al. Cancer
Research 54:5301-5309 (1994); Sliwkowski et al. J. Biol. Chem.
269(20):14661-14665 (1994); Scott et al. J. Biol. Chem. 266:14300-5
(1991); D' souza et al. Proc. Natl. Acad. Sci. 91:7202-7206 (1994);
Lewis et al. Cancer Research 56:1457-1465 (1996); and Schaefer et
al. Oncogene 15:1385-1394 (1997).
[0015] The murine monoclonal anti-HER2 antibody inhibits the growth
of breast cancer cell lines that overexpress HER2 at the 2+ and 3+
level, but has no activity on cells that express lower levels of
HER2 (Lewis et al., Cancer Immunol. Immunother. [1993]). Based on
this observation, antibody 4D5 was humanized (Carter et al., Proc.
Natl. Acad. Sci. USA 89: 4285-4289 [1992]). The humanized version
designated HERCEPTIN.RTM. (huMAb4D5-8, rhuMAb HER2, U.S. Pat. No.
5,821,337) was tested in breast cancer patients whose tumors
overexpress HER2 but who had progressed after conventional
chemotherapy (Baselga et al., J. Clin. Oncol. 14:737-744 [1996]);
Cobleigh et al., J. Clin. Oncol. 17: 2639-2648 [1999]). Most
patients in this trial expressed HER2 at the 3+ level, though a
fraction was 2+ tumors. Remarkably, HERCEPTIN.RTM. induced clinical
responses in 15% of patients (complete responses in 4% of patients,
and partial responses in 11%) and the median duration of those
responses was 9.1 months. HERCEPTIN.RTM. received marketing
approval from the Food and Drug Administration Sep. 25, 1998 for
the treatment of patients with metastatic breast cancer whose
tumors overexpress the ErbB2 protein.
[0016] Homology screening has resulted in the identification of two
other ErbB receptor family members; ErbB3 (U.S. Pat. Nos. 5,183,884
and 5,480,968 as well as Kraus et al. PNAS (USA) 86:9193-9197
(1989)) and ErbB4 (EP Pat Appln No 599,274; Plowman et al., Proc.
Natl. Acad. Sci. USA, 90:1746-1750 (1993); and Plowman et al.,
Nature, 366:473-475 (1993)). Both of these receptors display
increased expression on at least some breast cancer cell lines.
[0017] 3. Maytansinoid-Antibody Conjugates
[0018] In an attempt to improve their therapeutic index, maytansine
and maytansinoids have been conjugated to antibodies specifically
binding to tumor cell antigens. Immunoconjugates containing
maytansinoids are disclosed, for example, in U.S. Pat. Nos.
5,208,020; 5,416,064 and European Patent EP 0 425 235 B1, the
disclosures of which are hereby expressly incorporated by
reference. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623
(1996) described immunoconjugates comprising a maytansinoid
designated DM1 linked to the monoclonal antibody C242 directed
against human colorectal cancer. The conjugate was found to be
highly cytotoxic towards cultured colon cancer cells, and showed
antitumor activity in an in vivo tumor growth assay. Chari et al.
Cancer Research 52:127-131 (1992) describe immunoconjugates in
which a maytansinoid was conjugated via a disulfide linker to the
murine antibody A7 binding to an antigen on human colon cancer cell
lines, or to another murine monoclonal antibody TA.1 that binds the
HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansonoid
conjugate was tested in vitro on the human breast cancer cell line
SK-BR-3, which expresses 3.times.10.sup.5 HER-2 surface antigens
per cell. The drug conjugate achieved a degree of cytotoxicity
similar to the free maytansinoid drug, which could be increased by
increasing the number of maytansinoid molecules per antibody
molecule. The A7-maytansinoid conjugate showed low systemic
cytotoxicity in mice.
[0019] Although HERCEPTIN.RTM. is a breakthrough in treating
patients with ErbB2-overexpressing breast cancers that have
received extensive prior anti-cancer therapy, generally
approximately 85% of the patients in this population fail to
respond, or respond only poorly, to HERCEPTIN.RTM. treatment, and
in the clinical trial preceding marketing approval, the median time
to disease progression in all treated patients was only 3.1 months.
Therefore, there is a significant clinical need for developing
further HER2-directed cancer therapies for those patients with
HER2-overexpressing tumors or other diseases associated with HER2
expression that do not respond, or respond poorly, to
HERCEPTIN.RTM. treatment.
SUMMARY OF THE INVENTION
[0020] The present invention is based on the unexpected
experimental finding that HERCEPTIN.RTM.-maytansinoid conjugates
are highly effective in the treatment of HER2 (ErbB2)
overexpressing tumors that do not respond, or respond poorly, to
HERCEPTIN.RTM. therapy. The anti-ErbB2-maytansinoid conjugates of
the present invention are expected to have superior clinical
activity compared to treatment with HERCEPTIN.RTM. alone, including
a better objective response rate and/or longer duration of response
and/or increased survival.
[0021] In one aspect, the present invention concerns a method for
the treatment of a tumor in a mammal, wherein the tumor is
characterized by the overexpression of an ErbB receptor and does
not respond or responds poorly to treatment with a monoclonal
anti-ErbB antibody, comprising administering to the mammal a
therapeutically effective amount of a conjugate of the anti-ErbB
antibody with a maytansinoid.
[0022] In a preferred embodiment, the patient is human. In another
preferred embodiment, the ErbB receptor is (human) ErbB2 (HER2).
The method is not limited by the mechanism of action of the
anti-ErbB antibody used. Thus, the anti-ErbB antibody may, for
example, have growth inhibitory properties and/or may induce cell
death and/or apoptosis. In a particularly preferred embodiment, the
method concerns the treatment of cancer including, without
limitation, breast, ovarian, stomach, endometrial, salivary gland,
lung, kidney, colon, colorectal, thyroid, pancreatic, prostate and
bladder cancer. Preferably the cancer is breast cancer, in
particular, breast cancer which overexpresses ErbB2 at a 2+ level
or above, more preferably at a 3+ level. A preferred group of
antibodies has a biological characteristic of a 4D5 monoclonal
antibody, or binds essentially the same epitope as a 4D5 monoclonal
antibody, a humanized form of the murine monoclonal antibody 4D5
(ATCC CRL 10463) being particularly preferred.
[0023] The maytansinoid used in the conjugates of the present
invention may be maytansine or, preferably, maytansinol or a
maytansinol ester. The antibody and maytansinoid may be conjugated
by a bispecific chemical linker, such as
N-succinimidyl-4-(2-pyridylthio)propanoate (SPDP) or
N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP). The linking group
between the antibody and the maytansinoid may, for example, be a
disulfide, thioether, acid labile, photolabile, peptidase labile,
or esterase labile group.
[0024] In another aspect, the invention concerns an article of
manufacture comprising a container and a composition contained
therein, wherein the composition comprises an anti-ErbB
antibody-maytansinoid conjugate, and further comprising a package
insert or label indicating that the composition can be used to
treat cancer characterized by overexpression of an ErbB receptor,
preferably at a 2+ level or above.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 shows the heavy chain variable region sequence of a
humanized anti-HER2 antibody 2C4 (SEQ ID NO: 1) aligned with the
heavy chain variable region sequences of antibody 574 (SEQ ID NO:
2) and the human subgroup V.sub.HIII upon which the humanized
sequences are based (SEQ ID NO: 3).
[0026] FIG. 2 shows the light chain variable region sequence of a
humanized anti-HER2 antibody 2C4 (SEQ ID NO: 4) aligned with the
light chain variable region sequences of antibody 574 (SEQ ID NO:
5) and the sequence of the human subgroup upon which the humanized
sequences are based (SEQ ID NO: 6).
[0027] FIG. 3 shows the structure of the maytansinoid, designated
"DM1." In the structure of DM1, "R" can be occupied by a variety of
groups capable of forming a chemical bond with a selected linker.
Preferably, "R" is an SH group or a protected derivative thereof,
which forms an S--S bond with a linker, such as
N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP).
[0028] FIG. 4 illustrates the structure of a HERCEPTIN.RTM.-DM1
conjugate.
[0029] FIG. 5 is the elution profile of HERCEPTIN.RTM.-DM1
conjugate on a Sephacryl S300 gel filtration column.
[0030] FIG. 6 shows the anti-proliferative effect of HERCEPTIN.RTM.
and HERCEPTIN.RTM.-DM1 conjugate on SK-BR3 cells in vitro. As
control, the unrelated monoclonal antibody RITUXAN.RTM. or
RITUXAN.RTM.-DM1 conjugate was used.
[0031] FIG. 7A-GG shows the nucleotide sequence of a HER2 transgene
plasmid construct (SEQ ID NO: 7) directing the expression of native
human HER2 (ErbB2) in the mammary gland of a transgenic mouse. The
reverse strand is also depicted (SEQ ID NO: 10). The figure
includes the nucleotide sequence of HER2 (ErbB2) cDNA insert (SEQ
ID NO: 8) as well as the deduced amino acid sequence of HER2
(ErbB2) (SEQ ID NO: 9), including the signal sequence. Within SEQ
ID NO: 9, residues from about 22 to about 645, inclusive represent
the HER2 (ErbB2) extracellular domain.
[0032] FIGS. 8A and B show that the amount of HER2 extracellular
domain (ECD) shed into serum increases following transplant (FIG.
8A) and is proportional to the weight of the resulting tumor (FIG.
8B).
[0033] FIG. 9 illustrates the effect of HERCEPTIN.RTM.-DM1 on
HER2-transgenic tumors. Two mm.sup.3 pieces of MMTV-HER2-transgenic
tumors were transplanted into the mammary fat pad of FVB mice. When
tumors reached 250 mm.sup.3, groups of 8 mice were injected i.v. on
5 consecutive days with a HERCEPTIN.RTM.-DM1 conjugate. Two other
groups of mice were treated IP twice per week with 10 mg/kg of
either HERCEPTIN.RTM. or RITUXAN.RTM..
[0034] FIG. 10 shows that tumor cells originating from Founder 5
show binding to cy3-HERCEPTIN.RTM. and to an
anti-tyrosine-phosphorylated-HER2 antibody. Antibodies were
injected intravenously into transgenic mice and the next day tumors
were collected and sectioned. Antibody binding was visualized by
fluorescence microscopy.
[0035] FIG. 11 shows the effect of HERCEPTIN.RTM. and
HERCEPTIN.RTM.-DM1 conjugate on the growth of HER2 transgenic tumor
transplant. HERCEPTIN.RTM.-DM1 was administered once a week for 5
weeks at 300 .mu.g DM1/kg or 18 mg/kg of HERCEPTIN.RTM..
HERCEPTIN.RTM. or a control mAb (RITUXAN.RTM.) was administered
twice a week at 18 mg/kg.
[0036] FIG. 12 shows evaluation of doses and schedule of treatment
with HERCEPTIN.RTM.-DM1 conjugate in HER2 transgenic tumor
transplant in nude mice. HERCEPTIN.RTM.-DM1 conjugate was
administered either twice a week at various doses (300, 100, 30 or
10 .mu.g DM1/kg) or once a week at various doses (300 or 100 .mu.g
DM1/kg) for 5 weeks. HERCEPTIN.RTM. or a control mAb E25
(RITUXAN.RTM.) was administered twice a week at 18 mg/kg.
[0037] FIG. 13 shows the effect of different HERCEPTIN.RTM.-DM1
dosing regimens on HER2-transgenic tumors compared to matching
doses of RITUXAN.RTM.-DM1. Mice with 100 mm.sup.3 tumors were
injected i.v. with HERCEPTIN.RTM.-DM1 or RITUXAN.RTM.-DM1 at doses
of 100 or 300 .mu.g DM1/kg twice a week or 300 .mu.g DM1/kg once a
week. All animals received 5 doses.
[0038] FIG. 14 is a comparison of the most effective observed dose
of HERCEPTIN.RTM.-DM1 and RITUXAN.RTM.-DM1. A second dose of
HERCEPTIN.RTM.-DM1 following the beginning of tumor regrowth was
effective in shrinking the tumors again.
DETAILED DESCRIPTION OF THE INVENTION
1. Definitions
[0039] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Singleton et al., Dictionary of Microbiology and Molecular Biology
2nd ed., J. Wiley & Sons (New York, N.Y. 1994). One skilled in
the art will recognize many methods and materials similar or
equivalent to those described herein, which could be used in the
practice of the present invention. Indeed, the present invention is
in no way limited to the methods and materials described. For
purposes of the present invention, the following terms are defined
below.
[0040] An "ErbB receptor" or "ErbB" is a receptor protein tyrosine
kinase which belongs to the ErbB receptor family and includes ErbB1
(EGFR), ErbB2 (HER2), ErbB3 (HER3) and ErbB4 (HER4) receptors and
other members of this family to be identified in the future. The
definition specifically includes ErbB receptors encoded by spliced
forms of the corresponding erbB oncogens, including, without
limitation, the deletion variant of ErbB2 disclosed in PCT
publication No. WO 00/20579 (published on Apr. 13, 2000). The ErbB
receptor will generally comprise an extracellular domain, which may
bind an ErbB ligand; a lipophilic transmembrane domain; a conserved
intracellular tyrosine kinase domain; and a carboxyl-terminal
signaling domain harboring several tyrosine residues which can be
phosphorylated. The ErbB receptor may be a "native sequence" ErbB
receptor or a functional derivative, such as an "amino acid
sequence variant" thereof. Preferably the ErbB receptor is native
sequence human ErbB receptor.
[0041] The terms "ErbB1", "epidermal growth factor receptor" and
"EGFR" are used interchangeably herein and refer to native sequence
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)), and its functional derivatives,
such as amino acid sequence variants. erbB1 refers to the gene
encoding the EGFR protein product.
[0042] The expressions "ErbB2" and "HER2" are used interchangeably
herein and refer to native sequence 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), and functional derivatives, such as amino acid
sequence variants thereof. The term erbB2 refers to the gene
encoding human HER2 and neu refers to the gene encoding rat
p185.sup.neu. Preferred HER2 is native sequence human HER2.
Examples of antibodies which bind HER2 include MAbs 4D5 (ATCC CRL
10463), 2C4 (ATCC HB-12697), 7F3 (ATCC HB-12216), and 7C2 (ATCC HB
12215) (see, U.S. Pat. No. 5,772,997; WO98/77797; and U.S. Pat. No.
5,840,525, expressly incorporated herein by reference). Humanized
anti-HER2 antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3,
huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8
(HERCEPTIN.RTM.) as described in Table 3 of U.S. Pat. No. 5,821,337
expressly incorporated herein by reference; humanized 520C9
(WO93/21319). Human anti-HER2 antibodies are described in U.S. Pat.
No. 5,772,997 issued Jun. 30, 1998 and WO 97/00271 published Jan.
3, 1997.
[0043] "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), and
functional derivatives, including amino acid sequence variants
thereof. Examples of antibodies which bind HER3 are described in
U.S. Pat. No. 5,968,511 (Akita and Sliwkowski), e.g. the 8B8
antibody (ATCC HB 12070) or a humanized variant thereof.
[0044] 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), and functional
derivatives, including amino acid sequence variants thereof such as
the HER4 isoforms disclosed in WO 99/19488.
[0045] A "native" or "native sequence" EGFR, HER2, HER3 or HER4
polypeptide may be isolated from nature, produced by techniques of
recombinant DNA technology, chemically synthesized, or produced by
any combinations of these or similar methods.
[0046] "Functional derivatives" include amino acid sequence
variants, and covalent derivatives of the native polypeptides as
long as they retain a qualitative biological activity of the
corresponding native polypeptide. Amino acid sequence variants
generally differ from a native sequence in the substitution,
deletion and/or insertion of one or more amino acids anywhere
within a native amino acid sequence. Deletional variants include
fragments of the native polypeptides, and variants having N- and/or
C-terminal truncations. Ordinarily, amino acid sequence variants
will possess at least about 70% homology, preferably at least about
80%, more preferably at least about 90% homology with a native
polypeptide.
[0047] "Homology" is defined as the percentage of residues in the
amino acid sequence variant that are identical after aligning the
sequences and introducing gaps, if necessary, to achieve the
maximum percent homology. Methods and computer programs for the
alignment are well known in the art. One such computer program is
"Align 2", authored by Genentech, Inc., which was filed with user
documentation in the United States Copyright Office, Washington,
D.C. 20559, on Dec. 10, 1991.
[0048] By "ErbB ligand" is meant a polypeptide which binds to
and/or activates an ErbB receptor. The ErbB ligand of particular
interest herein is a native sequence human ErbB ligand such as
Epidermal Growth Factor (EGF) (Savage et al., J. Biol. Chem.
247:7612-7621 (1972)); Transforming Growth Factor alpha (TGF-alpha)
(Marquardt et al., Science 223:1079-1082 (1984)); amphiregulin also
known as schwanoma or keratinocyte autocrine growth factor (Shoyab
et al. Science 243:1074-1076 (1989); Kimura et al. Nature
348:257-260 (1990); and Cook et al. Mol. Cell. Biol. 11:2547-2557
(1991)); betacellulin (Shing et al., Science 259:1604-1607 (1993);
and Sasada et al. Biochem. Biophys. Res. Commun. 190:1173 (1993));
heparin-binding epidermal growth factor (HB-EGF) (Higashiyama et
al., Science 251:936-939 (1991)); epiregulin (Toyoda et al., J.
Biol. Chem. 270:7495-7500 (1995); and Komurasaki et al. Oncogene
15:2841-2848 (1997)), a heregulin (see below); neuregulin-2 (NRG-2)
(Carraway et al., Nature 387:512-516 (1997)); neuregulin-3 (NRG-3)
(Zhang et al., Proc. Natl. Acad. Sci. 94:9562-9567 (1997)); or
cripto (CR-1) (Kannan et al. J. Biol. Chem. 272(6):3330-3335
(1997)). ErbB ligands which bind EGFR include EGF, TGF-alpha,
amphiregulin, betacellulin, HB-EGF and epiregulin. ErbB ligands
which bind HER3 include heregulins. ErbB ligands capable of binding
HER4 include betacellulin, epiregulin, HB-EGF, NRG-2, NRG-3 and
heregulins.
[0049] "Heregulin" (HRG) when used herein refers to a polypeptide
which activates the ErbB2-ErbB3 and ErbB2-ErbB4 protein complexes
(i.e. induces phosphorylation of tyrosine residues in the complex
upon binding thereto). Various heregulin polypeptides encompassed
by this term are disclosed in Holmes et al., Science 256:1205-1210
(1992); WO 92/20798; Wen et al., Mol. Cell. Biol. 14(3):1909-1919
(1994) and Marchionni et al., Nature 362:312-318 (1993), for
example. The term includes biologically active fragments and/or
variants of a naturally occurring HRG polypeptide, such as an
EGF-like domain fragment thereof (e.g. HRG.beta..sub.177-244).
[0050] An "ErbB hetero-oligomer" herein is a noncovalently
associated oligomer comprising at least two different ErbB
receptors. Such complexes may form when a cell expressing two or
more ErbB receptors is exposed to an ErbB 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. Examples of such ErbB hetero-oligomers include
EGFR-HER2, HER2-HER3 and HER3-HER4 complexes. Moreover, the ErbB
hetero-oligomer may comprise two or more HER2 receptors combined
with a different ErbB receptor, such as HER3, HER4 or EGFR. Other
proteins, such as a cytokine receptor subunit (e.g. gp130), may be
included in the hetero-oligomer.
[0051] In the context of HER2 variants, such as HER2 fragments, the
phrase "having the biological activity of a native human HER2" is
used to refer to the qualitative ability of such fragments to
induce tumor growth when overexpressed in an animal model
(transgenic or non-transgenic) of the present invention.
[0052] "Tumor", as used herein, refers to all neoplastic cell
growth and proliferation, whether malignant or benign, and all
pre-cancerous and cancerous cells and tissues.
[0053] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. Examples of cancer include, but are not
limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or
lymphoid malignancies. More particular examples of such cancers
include squamous cell cancer (e.g. epithelial squamous cell
cancer), lung cancer including small-cell lung cancer, non-small
cell lung cancer, adenocarcinoma of the lung and squamous carcinoma
of the lung, cancer of the peritoneum, hepatocellular cancer,
gastric or stomach cancer including gastrointestinal cancer,
pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer,
liver cancer, bladder cancer, hepatoma, breast cancer, colon
cancer, rectal cancer, colorectal cancer, endometrial or uterine
carcinoma, salivary gland carcinoma, kidney or renal cancer,
prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma,
anal carcinoma, penile carcinoma, as well as head and neck
cancer.
[0054] A cancer which "overexpresses" an ErbB receptor is one which
has significantly higher levels of an ErbB receptor, such as HER2,
at the cell surface thereof, compared to a noncancerous cell of the
same tissue type. Such overexpression may be caused by gene
amplification or by increased transcription or translation. ErbB
receptor overexpression may be determined in a diagnostic or
prognostic assay by evaluating increased levels of the ErbB protein
present on the surface of a cell (e.g. via an immunohistochemistry
assay; IHC). Alternatively, or additionally, one may measure levels
of ErbB-encoding nucleic acid in the cell, e.g. via fluorescent in
situ hybridization (FISH; see WO98/45479 published October, 1998),
Southern blotting, or polymerase chain reaction (PCR) techniques,
such as real time quantitative PCR (RT-PCR). One may also study
ErbB receptor overexpression by measuring shed antigen (e.g., ErbB
extracellular domain) in a biological fluid such as serum (see,
e.g., U.S. Pat. No. 4,933,294 issued Jun. 12, 1990; WO91/05264
published Apr. 18, 1991; U.S. Pat. No. 5,401,638 issued Mar. 28,
1995; and Sias et al. J. Immunol. Methods 132: 73-80 (1990)). Aside
from the above assays, various in vivo assays are available to the
skilled practitioner. For example, one may expose cells within the
body of the patient to an antibody which is optionally labeled with
a detectable label, e.g. a radioactive isotope, and binding of the
antibody to cells in the patient can be evaluated, e.g. by external
scanning for radioactivity or by analyzing a biopsy taken from a
patient previously exposed to the antibody.
[0055] The tumors overexpressing HER2 are 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. 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]).
[0056] Conversely, a cancer which is "not characterized by
overexpression of an ErbB receptor" is one which, in a diagnostic
assay, does not express higher than normal levels of ErbB receptor
compared to a noncancerous cell of the same tissue type.
[0057] A "hormone independent" cancer is one in which proliferation
thereof is not dependent on the presence of a hormone which binds
to a receptor expressed by cells in the cancer. Such cancers do not
undergo clinical regression upon administration of pharmacological
or surgical strategies that reduce the hormone concentration in or
near the tumor. Examples of hormone independent cancers include
androgen independent prostate cancer, estrogen independent breast
cancer, endometrial cancer and ovarian cancer. Such cancers may
begin as hormone dependent tumors and progress from a
hormone-sensitive stage to a hormone-refractory tumor following
anti-hormonal therapy.
[0058] The term "antibody" herein is used in the broadest sense and
specifically covers intact monoclonal antibodies, polyclonal
antibodies, multispecific antibodies (e.g. bispecific antibodies)
formed from at least two intact antibodies, and antibody fragments,
so long as they exhibit the desired biological activity.
[0059] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to polyclonal antibody
preparations which include different antibodies directed against
different determinants (epitopes), each monoclonal antibody is
directed against a single determinant on the antigen. In addition
to their specificity, the monoclonal antibodies are advantageous in
that they may be synthesized uncontaminated by other antibodies.
The modifier "monoclonal" indicates the character of the antibody
as being obtained from a substantially homogeneous population of
antibodies, and is not to be construed as requiring production of
the antibody by any particular method. For example, the monoclonal
antibodies to be used in accordance with the present invention may
be made by the hybridoma method first described by Kohler et al.,
Nature, 256:495 (1975), or may be made by recombinant DNA methods
(see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies"
may also be isolated from phage antibody libraries using the
techniques described in Clackson et al., Nature, 352:624-628 (1991)
and Marks et al., J. Mol. Biol., 222:581-597 (1991), for
example.
[0060] 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.
[0061] "Antibody fragments" comprise a portion of an intact
antibody, preferably comprising the antigen-binding or variable
region thereof. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2, and Fv fragments; diabodies; linear antibodies;
single-chain antibody molecules; and multispecific antibodies
formed from antibody fragment(s).
[0062] An "intact" antibody is one which comprises an
antigen-binding variable region as well as a light chain constant
domain (C.sub.L) and heavy chain constant domains, C.sub.H1,
C.sub.H2 and C.sub.H3. The constant domains may be native sequence
constant domains (e.g. human native sequence constant domains) or
amino acid sequence variant thereof. Preferably, the intact
antibody has one or more effector functions.
[0063] "Humanized" forms of non-human (e.g., rodent) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity, and capacity. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
[0064] Humanized anti-ErbB2 antibodies include huMAb4D5-1,
huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6,
huMAb4D5-7 and huMAb4D5-8 (HERCEPTIN.RTM.) as described in Table 3
of U.S. Pat. No. 5,821,337 expressly incorporated herein by
reference; humanized 520C9 (WO93/21319) and humanized 2C4
antibodies. The heavy chain and light chain of humanized antibody
2C4 are shown in FIGS. 1 and 2, respectively.
[0065] Antibody "effector functions" refer to those biological
activities attributable to the Fc region (a native sequence Fc
region or amino acid sequence variant Fc region) of an antibody.
Examples of antibody effector functions include C1q binding;
complement dependent cytotoxicity; Fc receptor binding;
antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis;
down regulation of cell surface receptors (e.g. B cell receptor;
BCR), etc.
[0066] 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.
[0067] "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).
[0068] "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.
[0069] 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 Fey RIII
subclasses, including allelic variants and alternatively spliced
forms of these receptors. Fc.gamma.RII receptors include
Fc.gamma.RIIA (an "activating receptor") and Fc.gamma.RIIB (an
"inhibiting receptor"), which have similar amino acid sequences
that differ primarily in the cytoplasmic domains thereof.
Activating receptor Fc.gamma.RIIA contains an immunoreceptor
tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
Inhibiting receptor Fc.gamma.RIIB contains an immunoreceptor
tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain.
(see review M. in Daedron, Annu. Rev. Immunol. 15:203-234 (1997)).
FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92
(1991); Capel et al., Immuno methods 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)).
[0070] "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.
[0071] "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.
[0072] The term "variable", as used in connection with antibodies,
refers to the fact that certain portions of the antibody 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).
[0073] 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.
[0074] 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.
[0075] An antibody "which binds" an antigen of interest, e.g. ErbB2
antigen, is one capable of binding that antigen with sufficient
affinity such that the antibody is useful as a diagnostic and/or
therapeutic agent in targeting a cell expressing the antigen and/or
for targeted delivery of a cytotoxic or other chemotherapeutic
agent, such as a maytansinoid. Where the antibody is one which
binds ErbB2, it will usually preferentially bind ErbB2 as opposed
to other ErbB receptors, and may be one which does not
significantly cross-react with other proteins such as EGFR, ErbB3
or ErbB4. In such embodiments, the extent of binding of the
antibody to these non-ErbB2 proteins (e.g., cell surface binding to
endogenous receptor) will be less than 10% as determined by
fluorescence activated cell sorting (FACS) analysis or
radioimmunoprecipitation (RIA). Sometimes, the anti-ErbB2 antibody
will not significantly cross-react with the rat neu protein, e.g.,
as described in Schecter et al. Nature 312:513 (1984) and Drebin et
al., Nature 312:545-548 (1984).
[0076] Unless indicated otherwise, the expressions "monoclonal
antibody 4D5", and "4D5 monoclonal antibody" refer to an antibody
that has antigen binding residues of, or derived from, the murine
4D5 antibody. For example, the monoclonal antibody 4D5 may be
murine monoclonal antibody 4D5 (ATCC CRL 10463) or a variant
thereof, such as humanized antibody 4D5, possessing antigen binding
amino acid residues of murine monoclonal antibody 4D5. Exemplary
humanized 4D5 antibodies include huMAb4D5-1, huMAb4D5-2,
huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and
huMAb4D5-8 (HERCEPTIN.RTM.) as in U.S. Pat. No. 5,821,337, with
huMAb4D5-8 (HERCEPTIN.RTM.) being a preferred humanized 4D5
antibody.
[0077] An antibody having a "biological characteristic" of a
designated antibody, such as the monoclonal antibody designated
4D5, is one which possesses one or more of the biological
characteristics of that antibody which distinguish it from other
antibodies that bind to the same antigen (e.g. ErbB2). For example,
an antibody with a biological characteristic of 4D5 may show growth
inhibitory effect on ErbB2 overexpressing cells in a manner that is
dependent on the ErbB2 expression level and/or bind the same
epitope in the extracellular domain of ErbB2 as that bound by 4D5
(e.g. which blocks binding of monoclonal antibody 4D5 to
ErbB2).
[0078] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell, especially
an ErbB expressing cancer cell either in vitro or in vivo. Thus,
the growth inhibitory agent may be one which significantly reduces
the percentage of ErbB expressing cells in S phase. Examples of
growth inhibitory agents include agents that block cell cycle
progression (at a place other than S phase), such as agents that
induce G1 arrest and M-phase arrest. Classical M-phase blockers
include the vincas (vincristine and vinblastine), taxanes, and topo
II inhibitors such as doxorubicin, epirubicin, daunorubicin,
etoposide, and bleomycin. Those agents that arrest G1 also spill
over into S-phase arrest, for example, DNA alkylating agents such
as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin,
methotrexate, 5-fluorouracil, and ara-C. Further information can be
found in The Molecular Basis of Cancer, Mendelsohn and Israel.
eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and
antineoplastic drugs" by Murakami et al. (WB Saunders:
Philadelphia, 1995), especially p. 13.
[0079] Examples of "growth inhibitory" antibodies are those which
bind to ErbB2 and inhibit the growth of cancer cells overexpressing
ErbB2. Preferred growth inhibitory anti-ErbB2 antibodies inhibit
growth of SK-BR-3 breast tumor cells in cell culture by greater
than 20%, and preferably greater than 50% (e.g. from about 50% to
about 100%) at an antibody concentration of about 0.5 to 30
.mu.g/ml, where the growth inhibition is determined six days after
exposure of the SK-BR-3 cells to the antibody (see U.S. Pat. No.
5,677,171 issued Oct. 14, 1997). The SK-BR-3 cell growth inhibition
assay is described in more detail in that patent and hereinbelow.
The preferred growth inhibitory antibody is monoclonal antibody
4D5, e.g., humanized 4D5.
[0080] A molecule (e.g. antibody) which "induces cell death" is one
which causes a viable cell to become nonviable. The cell is
generally one which expresses the ErbB2 receptor, especially where
the cell overexpresses the ErbB2 receptor. Preferably, the cell is
a cancer cell, e.g. a breast, ovarian, stomach, endometrial,
salivary gland, lung, kidney, colon, thyroid, pancreatic, prostate
or bladder cancer cell. In vitro, the cell may be a SK-BR-3, BT474,
Calu 3, MDA-MB-453, MDA-MB-361 or SKOV3 cell. Cell death in vitro
may be determined in the absence of complement and immune effector
cells to distinguish cell death induced by antibody-dependent
cell-mediated cytotoxicity (ADCC) or complement dependent
cytotoxicity (CDC). Thus, the assay for cell death may be performed
using heat inactivated serum (i.e. in the absence of complement)
and in the absence of immune effector cells. To determine whether
the molecule is able to induce cell death, loss of membrane
integrity as evaluated by uptake of propidium iodide (PI), trypan
blue (see Moore et al. Cytotechnology 17:1-11 (1995)) or 7AAD can
be assessed relative to untreated cells. Preferred cell
death-inducing antibodies are those which induce PI uptake in the
PI uptake assay in BT474 cells. Examples of antibodies which induce
cell death include anti-ErbB2 antibodies 7C2 and 7F3 (WO 98/17797,
expressly incorporated herein by reference), including humanized
and/or affinity matured variants thereof.
[0081] A molecule (e.g. antibody) which "induces apoptosis" is one
which induces programmed cell death as determined by binding of
annexin V, fragmentation of DNA, cell shrinkage, dilation of
endoplasmic reticulum, cell fragmentation, and/or formation of
membrane vesicles (called apoptotic bodies). The cell is usually
one which overexpresses the ErbB2 receptor. Preferably the cell is
a tumor cell, e.g. a breast, ovarian, stomach, endometrial,
salivary gland, lung, kidney, colon, thyroid, pancreatic, prostate
or bladder cancer 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 molecule 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. Sometimes the pro-apoptotic molecule will
be one which further blocks ErbB ligand activation of an ErbB
receptor. In other situations, the molecule is one which does not
significantly block ErbB ligand activation of an ErbB receptor.
Further, the molecule may induce apoptosis, without inducing a
large reduction in the percent of cells in S phase (e.g. one which
only induces about 0-10% reduction in the percent of these cells
relative to control). Examples of antibodies which induce apoptosis
include anti-ErbB2 antibodies 7C2 and 7F3 (WO 98/17797, expressly
incorporated herein by reference), including humanized and/or
affinity matured variants thereof.
[0082] An antibody which "blocks" ligand activation of an ErbB
receptor is one which reduces or prevents such activation as
hereinabove defined, wherein the antibody is able to block ligand
activation of the ErbB receptor substantially more effectively than
monoclonal antibody 4D5, e.g. about as effectively as monoclonal
antibodies 7F3 or 2C4 or Fab fragments thereof and preferably about
as effectively as monoclonal antibody 2C4 or a Fab fragment
thereof. For example, the antibody that blocks ligand activation of
an ErbB receptor may be one which is about 50-100% more effective
than 4D5 at blocking formation of an ErbB hetero-oligomer. Blocking
of ligand activation of an ErbB receptor can occur by any means,
e.g. by interfering with: ligand binding to an ErbB receptor, ErbB
complex formation, tyrosine kinase activity of an ErbB receptor in
an ErbB complex and/or phosphorylation of tyrosine kinase
residue(s) in or by an ErbB receptor. Examples of antibodies which
block ligand activation of an ErbB receptor include monoclonal
antibodies 2C4 and 7F3 (which block HRG activation of ErbB2/ErbB3
and ErbB2/ErbB4 hetero-oligomers; and EGF, TGF-.alpha.,
amphiregulin, HB-EGF and/or epiregulin activation of an EGFR/ErbB2
hetero-oligomer); and L26, L96 and L288 antibodies (Klapper et al.
Oncogene 14:2099-2109 (1997)), which block EGF and NDF binding to
T47D cells which express EGFR, ErbB2, ErbB3 and ErbB4. Humanized
and/or affinity matured variants these and other antibodies within
the definition are specifically included.
[0083] The term "epitope" is used to refer to binding sites for
(monoclonal or polyclonal) antibodies on protein antigens.
[0084] Antibodies that bind to a certain epitope are identified by
"epitope mapping." There are many methods known in the art for
mapping and characterizing the location of epitopes on proteins,
including solving the crystal structure of an antibody-antigen
complex, competition assays, gene fragment expression assays, and
synthetic peptide-based assays, as described, for example, in
Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., 1999. Competition assays are discussed below. According to
the gene fragment expression assays, the open reading frame
encoding the protein is fragmented either randomly or by specific
genetic constructions and the reactivity of the expressed fragments
of the protein with the antibody to be tested is determined. The
gene fragments may, for example, be produced by PCR and then
transcribed and translated into protein in vitro, in the presence
of radioactive amino acids. The binding of the antibody to the
radioactively labeled protein fragments is then determined by
immunoprecipitation and gel electrophoresis. Certain epitopes can
also be identified by using large libraries of random peptide
sequences displayed on the surface of phage particles (phage
libraries). Alternatively, a defined library of overlapping peptide
fragments can be tested for binding to the test antibody in simple
binding assays. The latter approach is suitable to define linear
epitopes of about 5 to 15 amino acids.
[0085] An antibody binds "essentially the same epitope" as a
reference antibody, when the two antibodies recognize identical or
sterically overlapping epitopes. The most widely used and rapid
methods for determining whether two epitopes bind to identical or
sterically overlapping epitopes are competition assays, which can
be configured in all number of different formats, using either
labeled antigen or labeled antibody. Usually, the antigen is
immobilized on a 96-well plate, and the ability of unlabeled
antibodies to block the binding of labeled antibodies is measured
using radioactive or enzyme labels.
[0086] The "epitope 4D5" is the region in the extracellular domain
of ErbB2 to which the antibody 4D5 (ATCC CRL 10463) binds. This
epitope is close to the transmembrane domain of ErbB2, and extends
from about residue 519 to about residue 625, inclusive within the
ErbB2 extracellular domain sequence included in SEQ ID NO: 3, FIG.
4. To screen for antibodies which bind to the 4D5 epitope, a
routine cross-blocking assay such as that described in Harlow and
Lane, supra, can be performed. Alternatively, epitope mapping can
be performed to assess whether the antibody binds to the 4D5
epitope of ErbB2 (e.g. any one or more residues in the region from
about residue 529 to about residue 625, inclusive in SEQ ID NO:
3).
[0087] The "epitope 31-14" is the region in the extracellular
domain of ErbB2 to which the antibody 3H4 binds. This epitope
includes residues from about 541 to about 599, inclusive, in the
amino acid sequence of ErbB2 extracellular domain (see FIG. 4 and
SEQ ID NO: 3).
[0088] The "epitope 7C2/7F3" is the region at the N terminus of the
extracellular domain of ErbB2 to which the 7C2 and/or 7F3
antibodies (each deposited with the ATCC, see below) bind. To
screen for antibodies which bind to the 7C2/7F3 epitope, a routine
cross-blocking assay such as that described in Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and
David Lane (1988), can be performed. Alternatively, epitope mapping
can be performed to establish whether the antibody binds to the
7C2/7F3 epitope on ErbB2 (e.g. any one or more of residues in the
region from about residue 22 to about residue 53 of ErbB2; see FIG.
4, and SEQ ID NO: 3).
[0089] A tumor which "does not respond, or responds poorly, to
treatment with a monoclonal anti-ErbB antibody" does not show
statistically significant improvement in response to anti-ErbB
antibody treatment when compared to no treatment or treatment with
placebo in a recognized animal model or a human clinical trial, or
which responds to initial treatment with anti-ErbB antibodies but
grows as treatment is continued. A particularly suitable animal
model for testing the efficacy of anti-ErbB antibodies is the
transgenic animal model disclosed herein, and illustrated in
Example 3.
[0090] The terms "treat" or "treatment" refer to both therapeutic
treatment and prophylactic or preventative measures, wherein the
object is to prevent or slow down (lessen) an undesired
physiological change or disorder, such as the development or spread
of cancer. For purposes of this invention, beneficial or desired
clinical results include, but are not limited to, alleviation of
symptoms, diminishment of extent of disease, stabilized (i.e., not
worsening) state of disease, delay or slowing of disease
progression, amelioration or palliation of the disease state, and
remission (whether partial or total), whether detectable or
undetectable. "Treatment" can also mean prolonging survival as
compared to expected survival if not receiving treatment. Those in
need of treatment include those already with the condition or
disorder as well as those prone to have the condition or disorder
or those in which the condition or disorder is to be prevented.
[0091] A "disorder" is any condition that would benefit from
treatment of the present invention. This includes chronic and acute
disorders or diseases including those pathological conditions which
predispose the mammal to the disorder in question. Non-limiting
examples of disorders to be treated herein include benign and
malignant tumors; leukemias and lymphoid malignancies, in
particular breast, ovarian, stomach, endometrial, salivary gland,
lung, kidney, colon, thyroid, pancreatic, prostate or bladder
cancer. A preferred disorder to be treated in accordance with the
present invention is malignant tumor, such as breast cancer, that
overexpresses an ErbB receptor (e.g. ErbB2 and/or EGFR), and does
not respond or responds poorly to treatment with antibody to the
receptor(s) that is/are overexpressed. A particularly preferred
disorder is an ErbB2-overexpressing breast cancer that does not
respond or responds poorly to HERCEPTIN.RTM. therapy.
[0092] The term "therapeutically effective amount" refers to an
amount of a drug effective to treat a disease or disorder in a
mammal. In the case of cancer, the therapeutically 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. For cancer therapy, efficacy can,
for example, be measured by assessing the time to disease
progression (TTP) and/or determining the response rate (RR).
[0093] The term "objective response rate" refers to the number of
treated individuals that respond to treatment as determined by a
quantitative measure.
[0094] 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.
[0095] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include alkylating agents such as thiotepa and cyclosphosphamide
(CYTOXAN.TM.); alkyl sulfonates such as busulfan, improsulfan and
piposulfan; aziridines such as benzodopa, carboquone, meturedopa,
and uredopa; ethylenimines and methylamelamines including
altretamine, triethylenemelamine, trietylenephosphoramide,
triethylenethiophosphaoramide and trimethylolomelamine; nitrogen
mustards such as chlorambucil, chlornaphazine, cholophosphamide,
estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide
hydrochloride, melphalan, novembichin, phenesterine, prednimustine,
trofosfamide, uracil mustard; nitrosureas such as carmustine,
chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;
antibiotics such as aclacinomysins, actinomycin, authramycin,
azaserine, bleomycins, cactinomycin, calicheamicin, carabicin,
caminomycin, carzinophilin, chromomycins, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin,
epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins,
mycophenolic acid, nogalamycin, olivomycins, peplomycin,
potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,
streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;
anti-metabolites such as methotrexate and 5-fluorouracil (5-FU);
folic acid analogues such as denopterin, methotrexate, pteropterin,
trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine,
thiamiprine, thioguanine; pyrimidine analogs such as ancitabine,
azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,
doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as
calusterone, dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;
demecolcine; diaziquone; elformithine; elliptinium acetate;
etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine;
mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin;
phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide;
procarbazine; PSK7; razoxane; sizofuran; spirogermanium; tenuazonic
acid; triaziquone; 2,2',2'=-trichlorotriethylamine; urethan;
vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol;
pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide;
thiotepa; taxanes, e.g. paclitaxel (TAXOL.RTM., Bristol-Myers
Squibb Oncology, Princeton, N.J.) and doxetaxel (TAXOTERE.RTM.,
Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine;
6-thioguanine; mercaptopurine; methotrexate; platinum analogs such
as cisplatin and carboplatin; vinblastine; platinum; etoposide
(VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine;
vinorelbine; navelbine; novantrone; teniposide; daunomycin;
aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor
RFS 2000; difluoromethylornithine (DMFO); retinoic acid;
esperamicins; capecitabine; and pharmaceutically acceptable salts,
acids or derivatives of any of the above. Also included in this
definition are anti-hormonal agents that act to regulate or inhibit
hormone action on tumors such as anti-estrogens including for
example tamoxifen, raloxifene, aromatase inhibiting
4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene,
LY117018, onapristone, and toremifene (Fareston); and
anti-androgens such as flutamide, nilutamide, bicalutamide,
leuprolide, and goserelin; and pharmaceutically acceptable salts,
acids or derivatives of any of the above.
[0096] The term "prodrug" as used in this application refers to a
precursor or derivative form of a pharmaceutically active substance
that is less cytotoxic to tumor cells compared to the parent drug
and is capable of being enzymatically activated or converted into
the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer
Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382,
615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A
Chemical Approach to Targeted Drug Delivery," Directed Drug
Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press
(1985). The prodrugs of this invention include, but are not limited
to, phosphate-containing prodrugs, thiophosphate-containing
prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,
D-amino acid-modified prodrugs, glycosylated prodrugs,
.beta.-lactam-containing prodrugs, optionally substituted
phenoxyacetamide-containing prodrugs or optionally substituted
phenylacetamide-containing prodrugs, 5-fluorocytosine and other
5-fluorouridine prodrugs which can be converted into the more
active cytotoxic free drug. Examples of cytotoxic drugs that can be
derivatized into a prodrug form for use in this invention include,
but are not limited to, those chemotherapeutic agents described
above.
[0097] The term "nucleic acid" refers to polynucleotides such as
deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic
acid (RNA). The term also includes, as equivalents, analogs of
either DNA or RNA made from nucleotide analogs, and as applicable,
single (sense or antisense) and double-stranded polynucleotides. An
"isolated" nucleic acid molecule is a nucleic acid molecule that is
identified and separated from at least one contaminant nucleic acid
molecule with which it is ordinarily associated in the natural
source of the nucleic acid. An isolated nucleic acid molecule is
other than in the form or setting in which it is found in nature.
Isolated nucleic acid molecules therefore are distinguished from
the nucleic acid molecule as it exists in natural cells. However,
an isolated nucleic acid molecule includes a nucleic acid molecule
contained in cells that ordinarily express the antibody where, for
example, the nucleic acid molecule is in a chromosomal location
different from that of natural cells.
[0098] As used herein, the term "vector" refers to a nucleic acid
molecule capable of transporting another nucleic acid to which it
has been linked. The term "expression vector" includes plasmids,
cosmids or phages capable of synthesizing the subject HER2 protein
encoded by the respective recombinant gene carried by the vector.
Preferred vectors are those capable of autonomous replication
and/expression of nucleic acids to which they are linked. In the
present specification, "plasmid" and "vector" are used
interchangeably, as the plasmid is the most commonly used form of
vector.
[0099] As used herein, the terms "transcriptional regulatory
elements" and "transcriptional regulatory sequences" are used
interchangeably and refer to nucleic acid, e.g. DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to utilize promoters, enhancers,
splicing signals and polyadenylation signals. These terms are
intended to encompass all elements that promote or regulate
transcription, including promoters, core elements required for
basic interaction of RNA polymerase and transcription factors,
upstream elements, enhancers, and response elements (Lewin, "Genes
V" (Oxford University Press, Oxford) pages 847-873). Reference
herein to the transcriptional regulatory elements of a gene or
class of gene includes both all or an intact region of the
naturally occurring transcriptional regulatory elements and
modified forms of the transcriptional regulatory elements of the
gene or group of genes. Such modified forms include rearrangements
of the elements, deletions of some elements or extraneous
sequences, and insertion of heterologous elements. The modular
nature of transcriptional regulatory elements and the absence of
position-dependence of the function of some regulatory elements
such as enhancers make such modifications possible. Numerous
techniques are available for dissecting the regulatory elements of
genes to determine their location and function. Such information
can be used to direct modification of the elements, if desired. It
is preferred, however, that an intact region of the transcriptional
regulatory elements of a gene be used.
[0100] The term "tissue-specific promoter" means a nucleotide
sequence that serves as a promoter, i.e., regulates expression of a
selected DNA sequence operably linked to the promoter, and which
effects expression of the selected DNA sequence in specific cells
of a tissue, such as cells of a mammary gland. In an illustrative
embodiment, gene constructs utilizing mammary gland-specific
promoters can be used to preferentially direct expression of a HER2
protein or protein fragment in the mammary gland tissue.
[0101] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
[0102] The term "transfection" refers to the introduction of a
nucleic acid, e.g., an expression vector, into a recipient cell by
nucleic acid-mediated gene transfer. "Transformation", as used
herein, refers to a process in which a cell's genotype is changed
as a result of the cellular uptake of exogenous DNA or RNA, and,
for example, the transformed cell expresses a recombinant form of
HER2.
[0103] As used herein, the term "transgene" refers to a nucleic
acid sequence which is partly or entirely heterologous, i.e.,
foreign, to the transgenic animal or cell into which it is
introduced, or, is homologous to an endogenous gene of the
transgenic animal or cell into which it is introduced, but which is
designed to be inserted, or is inserted, into the animal's genome
in such a way as to alter the genome of the cell into which it is
inserted (e.g., it is inserted at a location which differs from
that of the natural gene or its insertion results in a knockout). A
transgene can be operably linked to one or more transcriptional
regulatory sequences and any other nucleic acid, such as introns,
that may be necessary for optimal expression of a selected nucleic
acid.
[0104] Accordingly, the term "transgene construct" refers to a
nucleic acid which includes a transgene, and (optionally) such
other nucleic acid sequences as transcriptionally regulatory
sequence, polyadenylation sites, replication origins, marker genes,
etc., which may be useful in the general manipulation of the
transgene for insertion in the genome of a host organism.
[0105] The term "transgenic" is used herein as an adjective to
describe the property, for example, of an animal or a construct, of
harboring a transgene. For instance, as used herein, a "transgenic
organism" is any animal, preferably a non-human mammal, in which
one or more of the cells of the animal contain heterologous nucleic
acid introduced by way of human intervention, such as by transgenic
techniques well known in the art. The nucleic acid is introduced
into the cell, directly or indirectly by introduction into a
precursor of the cell, by way of deliberate genetic manipulation,
such as by microinjection or by infection with a recombinant virus.
The term genetic manipulation does not include classical
cross-breeding, or in vitro fertilization, but rather is directed
to the introduction of a recombinant DNA molecule. This molecule
may be integrated within a chromosome, or it may be
extrachromosomally replicating DNA. In the typical transgenic
animals described herein, the transgene causes cells to express or
overexpress a recombinant form of the subject HER2 proteins. The
terms "founder line" and "founder animal" refer to those animals
that are the mature product of the embryos to which the transgene
was added, i.e., those animals that grew from the embryos into
which DNA was inserted, and that were implanted into one or more
surrogate hosts.
[0106] The terms "progeny" and "progeny of the transgenic animal"
refer to any and all offspring of every generation subsequent to
the originally transformed mammals. The term "non-human mammal"
refers to all members of the class Mammalia except humans. "Mammal"
refers to any animal classified as a mammal, including humans,
domestic and farm animals, and zoo, sports, or pet animals, such as
mouse, rat, rabbit, pig, sheep, goat, cattle and higher
primates.
[0107] As used herein, the expressions "cell," "cell line," and
"cell culture" are used interchangeably and all such designations
include progeny. Thus, the words "transformants" and "transformed
cells" include the primary subject cell and cultures derived
therefrom without regard for the number of transfers. It is also
understood that all progeny may not be precisely identical in DNA
content, due to deliberate or inadvertent mutations. Mutant progeny
that have the same function or biological activity as screened for
in the originally transformed cell are included. Where distinct
designations are intended, it will be clear from the context.
[0108] A "liposome" is a small vesicle composed of various types of
lipids, phospholipids and/or surfactant which is useful for
delivery of a drug (such as the anti-ErbB2 antibodies disclosed
herein and, optionally, a chemotherapeutic agent) to a mammal. The
components of the liposome are commonly arranged in a bilayer
formation, similar to the lipid arrangement of biological
membranes.
[0109] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, contraindications and/or warnings
concerning the use of such therapeutic products.
[0110] A "cardioprotectant" is a compound or composition which
prevents or reduces myocardial dysfunction (i.e. cardiomyopathy
and/or congestive heart failure) associated with administration of
a drug, such as an anti-ErbB antibody or its maytansinoid
conjugate, to a patient. The cardioprotectant may, for example,
block or reduce a free-radical-mediated cardiotoxic effect and/or
prevent or reduce oxidative-stress injury. Examples of
cardioprotectants encompassed by the present definition include the
iron-chelating agent dexrazoxane (ICRF-187) (Seifert et al. The
Annals of Pharmacotherapy 28:1063-1072 (1994)); a lipid-lowering
agent and/or anti-oxidant such as probucol (Singal et al. J. Mol.
Cell. Cardiol. 27:1055-1063 (1995)); amifostine (aminothiol
2-[(3-aminopropyl)amino]ethanethiol-dihydrogen phosphate ester,
also called WR-2721, and the dephosphorylated cellular uptake form
thereof called WR-1065) and
S-3-(3-methylaminopropylamino)propylphosphorothioic acid
(WR-151327), see Green et al. Cancer Research 54:738-741 (1994);
digoxin (Bristow, M. R. In: Bristow M R, ed. Drug-Induced Heart
Disease. New York: Elsevier 191-215 (1980)); beta-blockers such as
metoprolol (Hjalmarson et al. Drugs 47:Suppl 4:31-9 (1994); and
Shaddy et al. Am. Heart J. 129:197-9 (1995)); vitamin E; ascorbic
acid (vitamin C); free radical scavengers such as oleanolic acid,
ursolic acid and N-acetylcysteine (NAC); spin trapping compounds
such as alpha-phenyl-tert-butyl nitrone (PBN); (Paracchini et al.,
Anticancer Res. 13:1607-1612 (1993)); selenoorganic compounds such
as P251 (Elbesen); and the like.
2. Detailed Description
[0111] The present invention is based on results obtained in a
novel murine HER2-transgenic tumor model in which HERCEPTIN.RTM. or
the murine antibody 4D5 from which HERCEPTIN.RTM. was derived, had
little effect on tumor growth. Using this model to test the
efficacy of HERCEPTIN.RTM. and HERCEPTIN.RTM.-maytansinoid
conjugates, it was surprisingly found that while the transplanted
tumor obtained from such transgenic mice responded poorly to
HERCEPTIN.RTM. treatment, the HERCEPTIN.RTM.-maytansinoid
conjugates were highly efficacious.
[0112] Accordingly, the present invention is based on the use of
anti-ErbB antibody-maytansinoid conjugates in the treatment of ErbB
overexpressing tumors that do not respond well to anti-ErbB
antibody and/or maytansinoid treatment.
[0113] A. Production of Anti-ErbB Antibodies
[0114] A description follows as to exemplary techniques for the
production of the antibodies used in accordance with the present
invention. The production of antibodies will be illustrated with
reference to anti-ErbB2 antibodies but it will be apparent for
those skilled in the art that antibodies to other members of the
ErbB receptor family can be produced and modified in a similar
manner.
[0115] The ErbB2 antigen to be used for production of antibodies
may be, e.g., a soluble form of the extracellular domain of ErbB2
or a portion thereof, containing the desired epitope.
Alternatively, cells expressing ErbB2 at their cell surface (e.g.
NIH-3T3 cells transformed to overexpress ErbB2; or a carcinoma cell
line such as SK-BR-3 cells, see Stancovski et al. PNAS (USA)
88:8691-8695 (1991)) can be used to generate antibodies. Other
forms of ErbB2 useful for generating antibodies will be apparent to
those skilled in the art.
[0116] (i) Polyclonal Antibodies
[0117] 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.
[0118] 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.
[0119] (ii) Monoclonal Antibodies
[0120] Monoclonal antibodies are obtained from a population of
substantially homogeneous antibodies, i.e., the individual
antibodies comprising the population are identical except for
possible naturally occurring mutations that may be present in minor
amounts. Thus, the modifier "monoclonal" indicates the character of
the antibody as not being a mixture of discrete antibodies.
[0121] For example, the monoclonal antibodies may be made using the
hybridoma method first described by Kohler et al., Nature, 256:495
(1975), or may be made by recombinant DNA methods (U.S. Pat. No.
4,816,567).
[0122] 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)).
[0123] 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.
[0124] 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)).
[0125] 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).
[0126] 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).
[0127] 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.
[0128] 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.
[0129] 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).
[0130] 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.
[0131] 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.
[0132] 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.
[0133] (iii) Humanized Antibodies
[0134] 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.
[0135] 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)).
[0136] 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.
[0137] Example 1 below describes production of an exemplary
humanized anti-ErbB2 antibody. 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.
[0138] Various forms of the humanized antibody are contemplated.
For example, the humanized antibody may be an antibody fragment,
such as a Fab. Alternatively, the humanized antibody may be an
intact antibody, such as an intact IgG1 antibody.
[0139] (iv) Human Antibodies
[0140] 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.
[0141] 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.
[0142] 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).
[0143] Human anti-ErbB2 antibodies are described in U.S. Pat. No.
5,772,997 issued Jun. 30, 1998 and WO 97/00271 published Jan. 3,
1997.
[0144] (v) Antibody Fragments
[0145] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., Journal of Biochemical and Biophysical Methods 24:107-117
(1992); and Brennan et al., Science, 229:81 (1985)). However, these
fragments can now be produced directly by recombinant host cells.
For example, the antibody fragments can be isolated from the
antibody phage libraries discussed above. Alternatively, Fab'-SH
fragments can be directly recovered from E. coli and chemically
coupled to form F(ab').sub.2 fragments (Carter et al.,
Bio/Technology 10:163-167 (1992)). According to another approach,
F(ab').sub.2 fragments can be isolated directly from recombinant
host cell culture. Other techniques for the production of antibody
fragments will be apparent to the skilled practitioner. In other
embodiments, the antibody of choice is a single chain Fv fragment
(scFv). See WO 93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No.
5,587,458. The antibody fragment may also be a "linear antibody",
e.g., as described in U.S. Pat. No. 5,641,870 for example. Such
linear antibody fragments may be monospecific or bispecific.
[0146] (vi) Bispecific Antibodies
[0147] Bispecific antibodies are antibodies that have binding
specificities for at least two different epitopes. Exemplary
bispecific antibodies may bind to two different epitopes of the
ErbB2 protein. Other such antibodies may combine an ErbB2 binding
site with binding site(s) for EGFR, ErbB3 and/or ErbB4.
Alternatively, an anti-ErbB2 arm may be combined with an arm which
binds to a triggering molecule on a leukocyte such as a T-cell
receptor molecule (e.g. CD2 or CD3), or Fc receptors for IgG
(Fc.gamma.R), such as Fc.gamma.RI (CD64), Fc.gamma.RII (CD32) and
Fc.gamma.RIII (CD16) so as to focus cellular defense mechanisms to
the ErbB2-expressing cell. Bispecific antibodies may also be used
to localize cytotoxic agents to cells which express ErbB2. WO
96/16673 describes a bispecific anti-ErbB2/anti-Fc.gamma.RIII
antibody and U.S. Pat. No. 5,837,234 discloses a bispecific
anti-ErbB2/anti-Fc.gamma.RI antibody. A bispecific
anti-ErbB2/Fc.alpha. antibody is shown in WO98/02463. U.S. Pat. No.
5,821,337 teaches a bispecific anti-ErbB2/anti-CD3 antibody.
[0148] Methods for making bispecific antibodies are known in the
art. Traditional production of full length bispecific antibodies is
based on the coexpression of two immunoglobulin heavy chain-light
chain pairs, where the two chains have different specificities
(Millstein et al., Nature, 305:537-539 (1983)). Because of the
random assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a potential mixture of 10 different
antibody molecules, of which only one has the correct bispecific
structure. Purification of the correct molecule, which is usually
done by affinity chromatography steps, is rather cumbersome, and
the product yields are low. Similar procedures are disclosed in WO
93/08829, and in Traunecker et al., EMBO J., 10:3655-3659
(1991).
[0149] 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.
[0150] 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).
[0151] 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.
[0152] 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.
[0153] Recent progress has facilitated the direct recovery of
Fab'-SH fragments from E. coli, which can be chemically coupled to
form bispecific antibodies. Shalaby et al., J. Exp. Med., 175:
217-225 (1992) describe the production of a fully humanized
bispecific antibody F(ab').sub.2 molecule. Each Fab' fragment was
separately secreted from E. coli and subjected to directed chemical
coupling in vitro to form the bispecific antibody. The bispecific
antibody thus formed was able to bind to cells overexpressing the
ErbB2 receptor and normal human T cells, as well as trigger the
lytic activity of human cytotoxic lymphocytes against human breast
tumor targets.
[0154] 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).
[0155] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al. J.
Immunol. 147: 60 (1991).
[0156] (vii) Other Amino Acid Sequence Modifications
[0157] Amino acid sequence modification(s) of the anti-ErbB2
antibodies described herein are contemplated. For example, it may
be desirable to improve the binding affinity and/or other
biological properties of the antibody. Amino acid sequence variants
of the anti-ErbB2 antibody are prepared by introducing appropriate
nucleotide changes into the anti-ErbB2 antibody nucleic acid, or by
peptide synthesis. Such modifications include, for example,
deletions from, and/or insertions into and/or substitutions of,
residues within the amino acid sequences of the anti-ErbB2
antibody. Any combination of deletion, insertion, and substitution
is made to arrive at the final construct, provided that the final
construct possesses the desired characteristics. The amino acid
changes also may alter post-translational processes of the
anti-ErbB2 antibody, such as changing the number or position of
glycosylation sites.
[0158] A useful method for identification of certain residues or
regions of the anti-ErbB2 antibody that are preferred locations for
mutagenesis is called "alanine scanning mutagenesis" as described
by Cunningham and Wells Science, 244:1081-1085 (1989). Here, a
residue or group of target residues are identified (e.g., charged
residues such as arg, asp, his, lys, and glu) and replaced by a
neutral or negatively charged amino acid (most preferably alanine
or polyalanine) to affect the interaction of the amino acids with
ErbB2 antigen. Those amino acid locations demonstrating functional
sensitivity to the substitutions then are refined by introducing
further or other variants at, or for, the sites of substitution.
Thus, while the site for introducing an amino acid sequence
variation is predetermined, the nature of the mutation per se need
not be predetermined. For example, to analyze the performance of a
mutation at a given site, ala scanning or random mutagenesis is
conducted at the target codon or region and the expressed
anti-ErbB2 antibody variants are screened for the desired
activity.
[0159] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an anti-ErbB2 antibody with
an N-terminal methionyl residue or the antibody fused to a
cytotoxic polypeptide. Other insertional variants of the anti-ErbB2
antibody molecule include the fusion to the N- or C-terminus of the
anti-ErbB2 antibody to an enzyme (e.g. for ADEPT) or a polypeptide
which increases the serum half-life of the antibody.
[0160] Another type of variant is an amino acid substitution
variant. These variants have at least one amino acid residue in the
anti-ErbB2 antibody molecule replaced by a different residue. The
sites of greatest interest for substitutional mutagenesis include
the hypervariable regions, but FR alterations are also
contemplated. Conservative substitutions are shown in Table 1 under
the heading of "preferred substitutions". If such substitutions
result in a change in biological activity, then more substantial
changes, denominated "exemplary substitutions" in Table 1, or as
further described below in reference to amino acid classes, may be
introduced and the products screened.
TABLE-US-00001 TABLE 1 Exemplary Preferred Original 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) leu; val; ile; ala; tyr tyr Pro (P) ala ala Ser (S) thr thr
Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe
Val (V) ile; leu; met; phe; leu ala; norleucine
[0161] Substantial modifications in the biological properties of
the antibody are accomplished by selecting substitutions that
differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. Naturally occurring residues are
divided into groups based on common side-chain properties:
[0162] (1) hydrophobic: norleucine, met, ala, val, leu, ile;
[0163] (2) neutral hydrophilic: cys, ser, thr;
[0164] (3) acidic: asp, glu;
[0165] (4) basic: asn, gln, his, lys, arg;
[0166] (5) residues that influence chain orientation: gly, pro;
and
[0167] (6) aromatic: trp, tyr, phe.
[0168] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0169] Any cysteine residue not involved in maintaining the proper
conformation of the anti-ErbB2 antibody also may be substituted,
generally with serine, to improve the oxidative stability of the
molecule and prevent aberrant crosslinking. Conversely, cysteine
bond(s) may be added to the antibody to improve its stability
(particularly where the antibody is an antibody fragment such as an
Fv fragment).
[0170] A particularly preferred type of substitutional variant
involves substituting one or more hypervariable region residues of
a parent antibody (e.g. a humanized or human antibody). Generally,
the resulting variant(s) selected for further development will have
improved biological properties relative to the parent antibody from
which they are generated. A convenient way for generating such
substitutional variants involves affinity maturation using phage
display. Briefly, several hypervariable region sites (e.g. 6-7
sites) are mutated to generate all possible amino substitutions at
each site. The antibody variants thus generated are displayed in a
monovalent fashion from filamentous phage particles as fusions to
the gene III product of M13 packaged within each particle. The
phage-displayed variants are then screened for their biological
activity (e.g. binding affinity) as herein disclosed. In order to
identify candidate hypervariable region sites for modification,
alanine scanning mutagenesis can be performed to identify
hypervariable region residues contributing significantly to antigen
binding. Alternatively, or additionally, it may be beneficial to
analyze a crystal structure of the antigen-antibody complex to
identify contact points between the antibody and human ErbB2. Such
contact residues and neighboring residues are candidates for
substitution according to the techniques elaborated herein. Once
such variants are generated, the panel of variants is subjected to
screening as described herein and antibodies with superior
properties in one or more relevant assays may be selected for
further development.
[0171] 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.
[0172] 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).
[0173] 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.
[0174] (viii) Glycosylation Variants
[0175] Antibodies are glycosylated at conserved positions in their
constant regions (Jefferis and Lund, Chem. Immunol. 65:111-128
[1997]; Wright and Morrison, TibTECH 15:26-32 [1997]). The
oligosaccharide side chains of the immunoglobulins affect the
protein's function (Boyd et al., Mol. Immunol. 32:1311-1318 [1996];
Wittwe and Howard, Biochem. 29:4175-4180 [1990]), and the
intramolecular interaction between portions of the glycoprotein
which can affect the conformation and presented three-dimensional
surface of the glycoprotein (Hefferis and Lund, supra; Wyss and
Wagner, Current Opin. Biotech. 7:409-416 [1996]). Oligosaccharides
may also serve to target a given glycoprotein to certain molecules
based upon specific recognition structures. For example, it has
been reported that in agalactosylated IgG, the oligosaccharide
moiety `flips` out of the inter-CH2 space and terminal
N-acetylglucosamine residues become available to bind mannose
binding protein (Malhotra et al., Nature Med. 1:237-243 [1995]).
Removal by glycopeptidase of the oligosaccharides from CAMPATH-1H
(a recombinant humanized murine monoclonal IgG1 antibody which
recognizes the CDw52 antigen of human lymphocytes) produced in
Chinese Hamster Ovary (CHO) cells resulted in a complete reduction
in complement mediated lysis (CMCL) (Boyd et al., Mol. Immunol.
32:1311-1318 [1996]), while selective removal of sialic acid
residues using neuraminidase resulted in no loss of DMCL.
Glycosylation of antibodies has also been reported to affect
antibody-dependent cellular cytotoxicity (ADCC). In particular, CHO
cells with tetracycline-regulated expression of
.beta.(1,4)--N-acetylglucosaminyltransferase III (GnTIII), a
glycosyltransferase catalyzing formation of bisecting GlcNAc, was
reported to have improved ADCC activity (Umana et al., Mature
Biotech. 17:176-180 [1999]).
[0176] 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.
[0177] Glycosylation variants of antibodies are variants in which
the glycosylation pattern of an antibody is altered. By altering is
meant deleting one or more carbohydrate moieties found in the
antibody, adding one or more carbohydrate moieties to the antibody,
changing the composition of glycosylation (glycosylation pattern),
the extent of glycosylation, etc.
[0178] 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). Similarly, removal of
glycosylation sites can be accomplished by amino acid alteration
within the native glycosylation sites of the antibody.
[0179] The amino acid sequence is usually altered by altering the
underlying nucleic acid sequence. Nucleic acid molecules encoding
amino acid sequence variants of the anti-ErbB2 antibody are
prepared by a variety of methods known in the art. These methods
include, but are not limited to, isolation from a natural source
(in the case of naturally occurring amino acid sequence variants)
or preparation by oligonucleotide-mediated (or site-directed)
mutagenesis, PCR mutagenesis, and cassette mutagenesis of an
earlier prepared variant or a non-variant version of the anti-ErbB2
antibody.
[0180] The glycosylation (including glycosylation pattern) of
antibodies may also be altered without altering the amino acid
sequence or the underlying nucleotide sequence. Glycosylation
largely depends on the host cell used to express the antibody.
Since the cell type used for expression of recombinant
glycoproteins, e.g. antibodies, as potential therapeutics is rarely
the native cell, significant variations in the glycosylation
pattern of the antibodies can be expected (see, e.g. Hse et al., J.
Biol. Chem. 272:9062-9070 [1997]). In addition to the choice of
host cells, factors which affect glycosylation during recombinant
production of antibodies include growth mode, media formulation,
culture density, oxygenation, pH, purification schemes and the
like. Various methods have been proposed to alter the glycosylation
pattern achieved in a particular host organism including
introducing or overexpressing certain enzymes involved in
oligosaccharide production (U.S. Pat. Nos. 5,047,335; 5,510,261 and
5,278,299). Glycosylation, or certain types of glycosylation, can
be enzymatically removed from the glycoprotein, for example using
endoglycosidase H (Endo H). In addition, the recombinant host cell
can be genetically engineered, e.g. make defective in processing
certain types of polysaccharides. These and similar techniques are
well known in the art.
[0181] The glycosylation structure of antibodies can be readily
analyzed by conventional techniques of carbohydrate analysis,
including lectin chromatography, NMR, Mass spectrometry, HPLC, GPC,
monosaccharide compositional analysis, sequential enzymatic
digestion, and HPAEC-PAD, which uses high pH anion exchange
chromatography to separate oligosaccharides based on charge.
Methods for releasing oligosaccharides for analytical purposes are
also known, and include, without limitation, enzymatic treatment
(commonly performed using peptide-N-glycosidase
F/endo-.beta.-galactosidase), elimination using harsh alkaline
environment to release mainly O-linked structures, and chemical
methods using anhydrous hydrazine to release both N- and O-linked
oligosaccharides.
[0182] (viii) Screening for Antibodies with the Desired
Properties
[0183] Techniques for generating antibodies have been described
above. One may further select antibodies with certain biological
characteristics, as desired.
[0184] For example, to identify growth inhibitory anti-ErbB2
antibodies, one may screen for antibodies which inhibit the growth
of cancer cells which overexpress ErbB2. In one embodiment, the
growth inhibitory antibody of choice is able to inhibit growth of
SK-BR-3 cells in cell culture by about 20-100% and preferably by
about 50-100% at an antibody concentration of about 0.5 to 30
.mu.g/ml. To identify such antibodies, the SK-BR-3 assay described
in U.S. Pat. No. 5,677,171 can be performed. According to this
assay, SK-BR-3 cells are grown in a 1:1 mixture of F12 and DMEM
medium supplemented with 10% fetal bovine serum, glutamine and
penicillin streptomycin. The SK-BR-3 cells are plated at 20,000
cells in a 35 mm cell culture dish (2 mls/35 mm dish). 0.5 to 30
.mu.g/ml of the anti-ErbB2 antibody is added per dish. After six
days, the number of cells, compared to untreated cells are counted
using an electronic COULTER.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.
[0185] To select for antibodies which induce cell death, loss of
membrane integrity as indicated by, e.g., PI, trypan blue or 7AAD
uptake may be assessed relative to control. The preferred assay is
the PI uptake assay using BT474 cells. According to this assay,
BT474 cells (which can be obtained from the American Type Culture
Collection (Rockville, Md.)) are cultured in Dulbecco's Modified
Eagle Medium (D-MEM):Ham's F-12 (50:50) supplemented with 10%
heat-inactivated FBS (Hyclone) and 2 mM L-glutamine. (Thus, the
assay is performed in the absence of complement and immune effector
cells). The BT474 cells are seeded at a density of 3.times.10.sup.6
per dish in 100.times.20 mm dishes and allowed to attach overnight.
The medium is then removed and replaced with fresh medium alone or
medium containing 10 .mu.g/ml of the appropriate monoclonal
antibody. The cells are incubated for a 3 day time period.
Following each treatment, monolayers are washed with PBS and
detached by trypsinization. Cells are then centrifuged at 1200 rpm
for 5 minutes at 4.degree. C., the pellet resuspended in 3 ml ice
cold Ca.sup.2+ binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl,
2.5 mM CaCl.sub.2) and aliquoted into 35 mm strainer-capped
12.times.75 tubes (1 ml per tube, 3 tubes per treatment group) for
removal of cell clumps. Tubes then receive PI (10 .mu.m/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 cell
death as determined by PI uptake may be selected as cell
death-inducing antibodies.
[0186] 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.
[0187] 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.
[0188] To identify an antibody which blocks ligand activation of an
ErbB receptor, the ability of the antibody to block ErbB ligand
binding to cells expressing the ErbB receptor (e.g. in conjugation
with another ErbB receptor with which the ErbB receptor of interest
forms an ErbB hetero-oligomer) may be determined. For example,
cells naturally expressing, or transfected to express, ErbB
receptors of the ErbB hetero-oligomer may be incubated with the
antibody and then exposed to labeled ErbB ligand. The ability of
the anti-ErbB2 antibody to block ligand binding to the ErbB
receptor in the ErbB hetero-oligomer may then be evaluated.
[0189] For example, inhibition of HRG binding to MCF7 breast tumor
cell lines by anti-ErbB2 antibodies may be performed using
monolayer MCF7 cultures on ice in a 24-well-plate format
essentially as described in Example 1 below. Anti-ErbB2 monoclonal
antibodies may be added to each well and incubated for 30 minutes.
.sup.125I-labeled rHRG.beta.1.sub.177-224 (25 pm) may then be
added, and the incubation may be continued for 4 to 16 hours. Dose
response curves may be prepared and an IC.sub.50 value may be
calculated for the antibody of interest. In one embodiment, the
antibody which blocks ligand activation of an ErbB 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.
[0190] Alternatively, or additionally, the ability of the
anti-ErbB2 antibody to block ErbB ligand-stimulated tyrosine
phosphorylation of an ErbB receptor present in an ErbB
hetero-oligomer may be assessed. For example, cells endogenously
expressing the ErbB receptors or transfected to expressed them may
be incubated with the antibody and then assayed for ErbB
ligand-dependent tyrosine phosphorylation activity using an
anti-phosphotyrosine monoclonal (which is optionally conjugated
with a detectable label). The kinase receptor activation assay
described in U.S. Pat. No. 5,766,863 is also available for
determining ErbB receptor activation and blocking of that activity
by an antibody.
[0191] In one embodiment, one may screen for an antibody which
inhibits HRG stimulation of p180 tyrosine phosphorylation in MCF7
cells. For example, the MCF7 cells may be plated in 24-well plates
and monoclonal antibodies to ErbB2 may be added to each well and
incubated for 30 minutes at room temperature; then
rHRG.beta.1.sub.177-224 may be added to each well to a final
concentration of 0.2 nM, and the incubation may be continued for 8
minutes. Media may be aspirated from each well, and reactions may
be stopped by the addition of 100 .mu.l of SDS sample buffer (5%
SDS, 25 mM DTT, and 25 mM Tris-HCl, pH 6.8). Each sample (25 .mu.l)
may be electrophoresed on a 4-12% gradient gel (Novex) and then
electrophoretically transferred to polyvinylidene difluoride
membrane. Antiphosphotyrosine (at 1 .mu.g/ml) immunoblots may be
developed, and the intensity of the predominant reactive band at
M.sub.r.about.180,000 may be quantified by reflectance
densitometry. The antibody selected will preferably significantly
inhibit HRG stimulation of p180 tyrosine phosphorylation to about
0-35% of control in this assay. A dose-response curve for
inhibition of HRG stimulation of p180 tyrosine phosphorylation as
determined by reflectance densitometry may be prepared and an
IC.sub.50 for the antibody of interest may be calculated. In one
embodiment, the antibody which blocks ligand activation of an ErbB
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.
[0192] One may also assess the growth inhibitory effects of the
antibody on MDA-MB-175 cells, e.g., essentially as described in
Schaefer et al. Oncogene 15:1385-1394 (1997). According to this
assay, MDA-MB-175 cells may treated with an anti-ErbB2 monoclonal
antibody (10 .mu.g/mL) for 4 days and stained with crystal violet.
Incubation with an anti-ErbB2 antibody may show a growth inhibitory
effect on this cell line similar to that displayed by monoclonal
antibody 2C4. In a further embodiment, exogenous HRG will not
significantly reverse this inhibition. Preferably, the antibody
will be able to inhibit cell proliferation of MDA-MB-175 cells to a
greater extent than monoclonal antibody 4D5 (and optionally to a
greater extent than monoclonal antibody 7F3), both in the presence
and absence of exogenous HRG.
[0193] In one embodiment, the anti-ErbB2 antibody of interest may
block heregulin dependent association of ErbB2 with ErbB3 in both
MCF7 and SK-BR-3 cells as determined in a co-immunoprecipitation
experiment substantially more effectively than monoclonal antibody
4D5, and preferably substantially more effectively than monoclonal
antibody 7F3.
[0194] To screen for antibodies which bind to an epitope on ErbB2
bound by an antibody of interest, a routine cross-blocking assay
such as that described in Antibodies, A Laboratory Manual, Cold
Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be
performed. Alternatively, or additionally, epitope mapping can be
performed by methods known in the art (see, e.g. FIGS. 1A and 1B
herein).
[0195] The results obtained in the cell-based assays described
above can then be followed by testing in animal, e.g. murine,
models, and human clinical trials. In particular, the inability or
limited ability of an antibody to treat ErbB2 overexpressing tumors
can be demonstrated in the transgenic mouse model disclosed in the
present application as described in the Examples below.
[0196] B. Anti-ErbB Antibody-Maytansinoid Conjugates
(Immunoconjugates)
[0197] Anti-ErbB antibody-maytansinoid conjugates are prepared by
chemically linking an anti-ErbB antibody to a maytansinoid molecule
without significantly diminishing the biological activity of either
the antibody or the maytansinoid molecule. Maytansinoids are well
known in the art and can be synthesized by known techniques or
isolated from natural sources. Suitable maytansinoids are
disclosed, for example, in U.S. Pat. No. 5,208,020 and in the other
patents and nonpatent publications referred to hereinabove.
Preferred maytansinoids are maytansinol and maytansinol analogues
modified in the aromatic ring or at other positions of the
maytansinol molecule, such as various maytansinol esters.
[0198] There are many linking groups known in the art for making
antibody-maytansinoid conjugates, including, for example, those
disclosed in U.S. Pat. No. 5,208,020 (e.g., at column 7, lines
55-67 and at column 8, lines 1-14), or EP Patent 0 425 235 B1, and
Chari et al. Cancer Research 52: 127-131 (1992). The linking groups
include disulfide groups, thioether groups, acid labile groups,
photolabile groups, peptidase labile groups, or esterase labile
groups, as disclosed in the above-identified patents, disulfide and
thioether groups being preferred. For example, for a compound as
illustrated in FIG. 3, "R" may be SH or may be SSR.sub.1, where
R.sub.1 represents methyl, linear alkyl, branched alkyl, cyclic
alkyl, simple or substituted aryl or heterocyclic.
[0199] In addition, there are many possible sites within the
antibody molecule for linking maytansinoid to the antibody. For
example, in one embodiment HERCEPTIN.RTM. can be linked to the
maytansinoid at lysine 13 in the light chain, at lysine 32 in the
heavy chain, at lysine 26 in both Fab fragments and at lysine 38 in
the Fc fragment.
[0200] Conjugates of the antibody and maytansinoid may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,
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 toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
Particularly preferred coupling agents include
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlsson et
al., Biochem. J. 173:723-737 [1978]) and
N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for a
disulfide linkage.
[0201] The linker may be attached to the maytansinoid molecule at
various positions, depending on the type of the link. For example,
an ester linkage may be formed by reaction with a hydroxyl group
using conventional coupling techniques. The reaction may occur at
the C-3 position having a hydroxyl group, the C-14 position
modified with hydroxymethyl, the C-15 position modified with a
hydroxyl group, and the C-20 position having a hydroxyl group. In a
preferred embodiment, the linkage is formed at the C-3 position of
maytansinol or a maytansinol analogue.
[0202] C. Pharmaceutical Formulations
[0203] Therapeutic formulations of the antibody-maytansinoid
conjugates 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)), in the form of
lyophilized formulations or aqueous solutions. Acceptable carriers,
excipients, or stabilizers are nontoxic to recipients at the
dosages and concentrations employed, and include buffers such as
phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptides;
proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such
as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA;
sugars such as sucrose, mannitol, trehalose or sorbitol;
salt-forming counter-ions such as sodium; metal complexes (e.g.
Zn-protein complexes); and/or non-ionic surfactants such as
TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG). Preferred
lyophilized anti-ErbB2 antibody formulations are described in WO
97/04801, expressly incorporated herein by reference.
[0204] The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
preferably those with complementary activities that do not
adversely affect each other. For example, it may be desirable to
further provide antibodies or antibody-maytansinoid conjugates
which bind to EGFR, ErbB2 (e.g. an antibody which binds a different
epitope on ErbB2), ErbB3, ErbB4, or vascular endothelial factor
(VEGF) in the one formulation. Alternatively, or additionally, the
composition may further comprise a chemotherapeutic agent,
cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonal
agent, and/or cardioprotectant. Such molecules are suitably present
in combination in amounts that are effective for the purpose
intended.
[0205] 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).
[0206] 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.
[0207] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0208] In one embodiment, the formulation comprises 5 mg/ml
HERCEPTIN.RTM.-DM1, 100 mg/ml sucrose, 0.1% polysorbate 20 and 10
mM sodium succinate at pH 5.0.
[0209] D. Treatment with the Anti-ErbB2 Antibody-Maytansinoid
Conjugates
[0210] It is contemplated that, according to the present invention,
the anti-ErbB2 antibody-maytansinoid conjugates may be used to
treat various diseases or disorders. Exemplary conditions or
disorders include benign or malignant tumors; leukemias and
lymphoid malignancies; other disorders such as neuronal, glial,
astrocytal, hypothalamic, glandular, macrophagal, epithelial,
stromal, blastocoelic, inflammatory, angiogenic and immunologic
disorders.
[0211] Generally, the disease or disorder to be treated is cancer.
Examples of cancer to be treated herein include, but are not
limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or
lymphoid malignancies. More particular examples of such cancers
include squamous cell cancer (e.g. epithelial squamous cell
cancer), lung cancer including small-cell lung cancer, non-small
cell lung cancer, adenocarcinoma of the lung and squamous carcinoma
of the lung, cancer of the peritoneum, hepatocellular cancer,
gastric or stomach cancer including gastrointestinal cancer,
pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer,
liver cancer, bladder cancer, hepatoma, breast cancer, colon
cancer, rectal cancer, colorectal cancer, endometrial or uterine
carcinoma, salivary gland carcinoma, kidney or renal cancer,
prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma,
anal carcinoma, penile carcinoma, as well as head and neck
cancer.
[0212] The cancer will comprise ErbB-expressing cells, such that an
anti-ErbB antibody herein is able to bind to the cancer, and will
be typically characterized by overexpression of the ErbB receptor.
In a preferred embodiment, the cancer comprises ErbB2-expressing
cells, even more preferably, cells which are characterized by
overexpression of the ErbB2 receptor. To determine ErbB, e.g. ErbB2
expression in the cancer, various diagnostic/prognostic assays are
available. In one embodiment, ErbB2 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 ErbB2 protein staining intensity criteria as
follows:
Score 0
[0213] no staining is observed or membrane staining is observed in
less than 10% of tumor cells.
Score 1+
[0214] 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+
[0215] a weak to moderate complete membrane staining is observed in
more than 10% of the tumor cells.
Score 3+
[0216] a moderate to strong complete membrane staining is observed
in more than 10% of the tumor cells.
[0217] Those tumors with 0 or 1+ scores for ErbB2 overexpression
assessment may be characterized as not overexpressing ErbB2,
whereas those tumors with 2+ or 3+ scores may be characterized as
overexpressing ErbB2.
[0218] Alternatively, or additionally, fluorescence in situ
hybridization (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 ErbB2 overexpression in the tumor. In comparison
with IHC assay, the FISH assay, which measures her2 gene
amplification, seems to correlate better with response of patients
to treatment with HERCEPTIN.RTM., and is currently considered to be
the preferred assay to identify patients likely to benefit from
HERCEPTIN.RTM. treatment or treatment with the immunoconjugates of
the present invention.
[0219] In one embodiment, the cancer will be one which expresses
(and may overexpress) EGFR. Examples of cancers which may
express/overexpress EGFR include squamous cell cancer (e.g.
epithelial squamous cell cancer), lung cancer including small-cell
lung cancer, non-small cell lung cancer, adenocarcinoma of the lung
and squamous carcinoma of the lung, cancer of the peritoneum,
hepatocellular cancer, gastric or stomach cancer including
gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical
cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,
breast cancer, colon cancer, rectal cancer, colorectal cancer,
endometrial or uterine carcinoma, salivary gland carcinoma, kidney
or renal cancer, prostate cancer, vulval cancer, thyroid cancer,
hepatic carcinoma, anal carcinoma, penile carcinoma, as well as
head and neck cancer.
[0220] Preferably, the immunoconjugates of the present invention
and/or ErbB, e.g. ErbB2 or EGFR protein to which they are bound are
internalized by the cell, resulting in increased therapeutic
efficacy of the immunoconjugate in killing the cancer cell to which
they bind. In a preferred embodiment, the cytotoxic agent
(maytansinoid) targets or interferes with nucleic acid in the
cancer cell.
[0221] The treatment of the present invention targets ErbB
overexpressing tumors that do not respond, or respond poorly, to
treatment with an unconjugated anti-ErbB antibody. Such patients
might have received prior treatment with an anti-ErbB antibody not
conjugated to a maytansinoid moiety, where the prior treatment
either did not result in significant improvement, or resulted in
transient response. Prior treatment of any particular patient with
an unconjugated anti-ErbB antibody is, however, not a prerequisite
of identifying patients who are candidates for treatment in
accordance with the present invention. An ordinary skilled
physician can readily identify patients who are expected to benefit
from treatment with the immunoconjugates of the present invention
based on publicly available clinical data and his or her own
experience. Treatment of mammals, and in particular human patients,
with or without prior treatment with an (unconjugated) anti-ErbB
antibody is specifically within the scope of the present
invention.
[0222] The anti-ErbB antibody-maytansinoid conjugates are
administered to a mammal, preferably 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 or subcutaneous administration of
the antibody is preferred.
[0223] Other therapeutic regimens may be combined with the
administration of the anti-ErbB antibody-maytansinoid conjugates.
The combined administration includes coadministration, using
separate formulations or a single pharmaceutical formulation, and
consecutive administration in either order, wherein preferably
there is a time period while both (or all) active agents
simultaneously exert their biological activities.
[0224] In one preferred embodiment, the patient is treated with two
or more different anti-ErbB antibodies, at least one of which is in
the form of a maytansinoid conjugate. For example, the patient may
be treated with a first anti-ErbB2 antibody-maytansinoid conjugate
in which the antibody is growth inhibitory (e.g. HERCEPTIN.RTM.),
and a second anti-ErbB2 antibody or antibody-immunoconjugate, e.g.
an antibody-maytansinoid conjugate which blocks ligand activation
of an ErbB receptor (e.g. 2C4 or a humanized and/or affinity
matured variant thereof) or induces apoptosis of an
ErbB2-overexpressing cell (e.g. 7C2, 7F3 or humanized variants
thereof). In another embodiment, the treatment involves the
administration of antibodies that specifically bind two or more
different ErbB receptors, such as, for example, ErbB2 and EGFR
receptors, where at least one of the anti-ErbB antibodies is
administered as a maytansinoid conjugate. Preferably such combined
therapy results in a synergistic therapeutic effect.
[0225] It may also be desirable to combine administration of the
anti-ErbB antibody-maytansinoid conjugates, with administration of
an antibody directed against another tumor-associated antigen,
which is not member of the ErbB family of receptors. The other
antibody in this case may, for example, bind to vascular
endothelial growth factor (VEGF), and may be in the form of a
maytansinoid conjugate, or another immunoconjugate.
[0226] In one embodiment, the treatment of the present invention
involves the combined administration of an anti-ErbB2
antibody-maytansinoid conjugate (or conjugates) and one or more
chemotherapeutic agents or growth inhibitory agents, including
coadministration of cocktails of different chemotherapeutic agents.
Preferred chemotherapeutic agents include taxanes (such as
paclitaxel and doxetaxel) and/or anthracycline antibiotics.
Preparation and dosing schedules for such chemotherapeutic agents
may be used according to manufacturers' instructions or as
determined empirically by the skilled practitioner. Preparation and
dosing schedules for such chemotherapy are also described in
Chemotherapy Service Ed., M. C. Perry, Williams & Wilkins,
Baltimore, Md. (1992).
[0227] In a preferred embodiment, the treatment is initiated with
an anti-ErbB antibody-maytansinoid conjugate, followed by
maintenance treatment with an unconjugated or `naked` anti-ErbB
antibody. This strategy may eliminate or reduce tumor cells
resistant to the naked antibody in the initial round because of the
ability of the antibody-DM1 conjugate to effectively kill such
tumor cells.
[0228] The antibody-maytansinoid conjugates may be combined with an
anti-hormonal compound; e.g., an anti-estrogen compound such as
tamoxifen; an anti-progesterone such as onapristone (see, EP 616
812); or an anti-androgen such as flutamide, in dosages known for
such molecules. Where the cancer to be treated is hormone
independent cancer, the patient may previously have been subjected
to anti-hormonal therapy and, after the cancer becomes hormone
independent, the anti-ErbB2 antibody (and optionally other agents
as described herein) may be administered to the patient.
[0229] Sometimes, it may be beneficial to also coadminister a
cardioprotectant (to prevent or reduce myocardial dysfunction
associated with the therapy) or one or more cytokines to the
patient. In addition to the above therapeutic regimes, the patient
may be subjected to surgical removal of cancer cells and/or
radiation therapy.
[0230] Suitable dosages for any of the above coadministered agents
are those presently used and may be lowered due to the combined
action (synergy) of the agent and anti-ErbB2 antibody.
[0231] For the prevention or treatment of disease, the appropriate
dosage of antibody-maytansinoid conjugates will depend on the type
of disease to be treated, as defined above, the severity and course
of the disease, whether the antibody is administered for preventive
or therapeutic purposes, previous therapy, the patient's clinical
history and response to the antibody, and the discretion of the
attending physician. The antibody-maytansinoid conjugate is
suitably administered to the patient at one time or over a series
of treatments. Depending on the type and severity of the disease,
about 1 .mu.g/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of
antibody-maytansinoid conjugate is an initial candidate dosage for
administration to the patient, whether, for example, by one or more
separate administrations, or by continuous infusion. A typical
daily dosage might range from about 1 .mu.g/kg to 100 mg/kg or
more, depending on the factors mentioned above. For repeated
administrations over several days or longer, depending on the
condition, the treatment is sustained until a desired suppression
of disease symptoms occurs. A preferred dosing regimen comprises
administering an initial loading dose of about 4 mg/kg, followed by
a weekly maintenance dose of about 2 mg/kg of the anti-ErbB2
antibody-maytansinoid conjugate. However, other dosage regimens may
be useful. The progress of this therapy is easily monitored by
conventional techniques and assays.
[0232] Based on the data disclosed herein, it is anticipated that
one useful dosing protocol may entail approximately weekly (or less
frequent) administration of the anti-ErbB antibody-maytansinoid
conjugate where each dose of the conjugate is about 0.2-10 mg/kg,
preferably about 1-3 mg/kg of the conjugate (e.g. where there are 1
to about 10, preferably about 3-4, maytansinoid molecules
conjugated to each antibody molecule). From about 2-10, preferably
about 4-6, dosages of the conjugate may be administered to the
patient approximately every week.
[0233] In a preferred embodiment, the patients are treated
initially with anti-ErbB-maytansinoid conjugate followed by therapy
with unconjugated anti-ErbB antibody. Preferably, the anti-ErbB
antibody in the conjugate and the unconjugated antibody are the
same antibody. For example, treatment could be initiated with
weekly injections of HERCEPTIN.RTM.-DM1 at about 0.5-5 mg/kg,
preferably at about 1-3 mg/kg for 4-6 weeks, with the option of
repeating this treatment. Patients can then be rolled over to
conventional HERCEPTIN.RTM. therapy, which typically consists of
treatment with a 4 mg/kg initial dose of HERCEPTIN.RTM., followed
by weekly treatment with a maintenance dose of 2 mg/kg. However,
the 4 mg/kg initial dose may be omitted, with therapy going
straight to the 2 mg/kg maintenance dose.
[0234] E. Articles of Manufacture
[0235] In another embodiment of the invention, an article of
manufacture containing materials useful for the treatment of the
disorders described above is provided. The article of manufacture
comprises a container and a label or package insert on or
associated with the container. Suitable containers include, for
example, bottles, vials, syringes, etc. The containers may be
formed from a variety of materials such as glass or plastic. The
container holds a composition which is effective for treating the
condition and may have a sterile access port (for example the
container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). At least one
active agent in the composition is an anti-ErbB2
antibody-maytansinoid conjugate. In one embodiment the container is
a 10 cc vial containing 10 mL of a solution comprising
HERCEPTIN.RTM.-DM1.
[0236] The label or package insert indicates that the composition
is used for treating the condition of choice, such as cancer. In
one embodiment, the label or package inserts indicates that the
composition comprising the antibody which binds ErbB2 can be used
to treat cancer which expresses an ErbB receptor selected from the
group consisting of epidermal growth factor receptor (EGFR), ErbB2,
ErbB3 and ErbB4, preferably EGFR. In addition, the label or package
insert may indicate that the patient to be treated is one having
cancer characterized by excessive activation of an ErbB receptor
selected from EGFR, ErbB2, ErbB3 or ErbB4. For example, the cancer
may be one which overexpresses one of these receptors and/or which
overexpresses an ErbB ligand (such as TGF-.alpha.). The label or
package insert may also indicate that the composition can be used
to treat cancer, wherein the cancer is not characterized by
overexpression of the ErbB2 receptor. For example, whereas the
present package insert for HERCEPTIN.RTM. indicates that the
antibody is used to treat patients with metastatic breast cancer
whose tumors overexpress the ErbB2 protein, the package insert
herein may indicate that the antibody or composition is used to
treat cancer that does not respond, or respond poorly, to treatment
with HERCEPTIN.RTM.. In other embodiments, the package insert may
indicate that the antibody-maytansinoid conjugate or composition
can be used also to treat hormone independent cancer, prostate
cancer, colon cancer or colorectal cancer.
[0237] Moreover, the article of manufacture may comprise (a) a
first container with a composition contained therein, wherein the
composition comprises a maytansinoid conjugate of a first antibody
which binds ErbB2 and inhibits growth of cancer cells which
overexpress ErbB2; and (b) a second container with a composition
contained therein, wherein the composition comprises a second
antibody which binds ErbB2 and blocks ligand activation of an ErbB
receptor, or a conjugate of this second antibody with a
maytansinoid. The article of manufacture in this embodiment of the
invention may further comprises a package insert indicating that
the first and second compositions can be used to treat cancer.
Alternatively, or additionally, the article of manufacture may
further comprise a second (or third) container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution
and dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
[0238] Further details of the invention are illustrated in the
following non-limiting examples.
Example 1
[0239] Production, Characterization and Humanization of Anti-ErbB2
Monoclonal Antibody 4D5
[0240] The murine monoclonal antibody 4D5 which specifically binds
the extracellular domain of ErbB2 was produced as described in
Fendly et al., Cancer Research 50:1550-1558 (1990). Briefly, NIH
3T3/HER2-3.sub.400 cells (expressing approximately 1.times.10.sup.5
ErbB2 molecules/cell) produced as described in Hudziak et al Proc.
Natl. Acad. Sci. (USA) 84:7158-7163 (1987) were harvested with
phosphate buffered saline (PBS) containing 25 mM EDTA and used to
immunize BALB/c mice. The mice were given injections i.p. of
10.sup.7 cells in 0.5 ml PBS on weeks 0, 2, 5 and 7. The mice with
antisera that immunoprecipitated .sup.32P-labeled ErbB2 were given
i.p. injections of a wheat germ agglutinin-Sepharose (WGA) purified
ErbB2 membrane extract on weeks 9 and 13. This was followed by an
i.v. injection of 0.1 ml of the ErbB2 preparation and the
splenocytes were fused with mouse myeloma line X63-Ag8.653.
Hybridoma supernatants were screened for ErbB2-binding by ELISA and
radioimmunoprecipitation.
Epitope Mapping and Characterization
[0241] The ErbB2 epitope bound by monoclonal antibody 4D5 was
determined by competitive binding analysis (Fendly et al. Cancer
Research 50:1550-1558 (1990)). Cross-blocking studies were done by
direct fluorescence on intact cells using the PANDEX.TM. Screen
Machine to quantitate fluorescence. The monoclonal antibody was
conjugated with fluorescein isothiocyanate (FITC), using
established procedures (Wofsy et al. Selected Methods in Cellular
Immunology, p. 287, Mishel and Schiigi (eds.) San Francisco: W. J.
Freeman Co. (1980)). Confluent monolayers of NIH 3T3/HER2-3.sub.400
cells were trypsinized, washed once, and resuspended at
1.75.times.10.sup.6 cell/ml in cold PBS containing 0.5% bovine
serum albumin (BSA) and 0.1% NaN.sub.3. A final concentration of 1%
latex particles (IDC, Portland, Oreg.) was added to reduce clogging
of the PANDEX.TM. plate membranes. Cells in suspension, 20 .mu.l
and 20 .mu.l of purified monoclonal antibodies (100 .mu.g/ml to 0.1
.mu.g/ml) were added to the PANDEX.TM. plate wells and incubated on
ice for 30 minutes. A predetermined dilution of the FITC-labeled
monoclonal antibody in 20 .mu.l was added to each well, incubated
for 30 minutes, washed, and the fluorescence was quantitated by the
PANDEX.TM. Monoclonal antibodies were considered to share an
epitope if each blocked binding of the other by 50% or greater in
comparison to an irrelevant monoclonal antibody control. In this
experiment, monoclonal antibody 4D5 was assigned epitope I (amino
acid residues from about 529 to about 625, inclusive within the
ErbB2 extracellular domain (see SEQ NO: 3).
[0242] The growth inhibitory characteristics of monoclonal antibody
4D5 were evaluated using the breast tumor cell line, SK-BR-3 (see
Hudziak et al. Molec. Cell. Biol. 9(3):1165-1172 (1989)). Briefly,
SK-BR-3 cells were detached by using 0.25% (vol/vol) trypsin and
suspended in complete medium at a density of 4.times.10.sup.5 cells
per ml. Aliquots of 100 .mu.l (4.times.10.sup.4 cells) were plated
into 96-well microdilution plates, the cells were allowed to
adhere, and 100 .mu.l of media alone or media containing monoclonal
antibody (final concentration 5 .mu.g/ml) was then added. After 72
hours, plates were washed twice with PBS (pH 7.5), stained with
crystal violet (0.5% in methanol), and analyzed for relative cell
proliferation as described in Sugarman et al. Science 230:943-945
(1985). Monoclonal antibody 4D5 inhibited SK-BR-3 relative cell
proliferation by about 56%.
[0243] Monoclonal antibody 4D5 was also evaluated for its ability
to inhibit HRG-stimulated tyrosine phosphorylation of proteins in
the M.sub.r 180,000 range from whole-cell lysates of MCF7 cells
(Lewis et al. Cancer Research 56:1457-1465 (1996)). MCF7 cells are
reported to express all known ErbB receptors, but at relatively low
levels. Since ErbB2, ErbB3, and ErbB4 have nearly identical
molecular sizes, it is not possible to discern which protein is
becoming tyrosine phosphorylated when whole-cell lysates are
evaluated by Western blot analysis. However, these cells are ideal
for HRG tyrosine phosphorylation assays because under the assay
conditions used, in the absence of exogenously added HRG, they
exhibit low to undetectable levels of tyrosine phosphorylation
proteins in the M.sub.r 180,000 range.
[0244] MCF7 cells were plated in 24-well plates and monoclonal
antibodies to ErbB2 were added to each well and incubated for 30
minutes at room temperature; then rHRG.beta.1.sub.177-244 was added
to each well to a final concentration of 0.2 nM, and the incubation
was continued for 8 minutes. Media was carefully aspirated from
each well, and reactions were stopped by the addition of 100 .mu.l
of SDS sample buffer (5% SDS, 25 mM DTT, and 25 mM Tris-HCl, pH
6.8). Each sample (25 .mu.l) was electrophoresed on a 4-12%
gradient gel (Novex) and then electrophoretically transferred to
polyvinylidene difluoride membrane. Antiphosphotyrosine (4G10, from
UBI, used at 1 .mu.g/ml) immunoblots were developed, and the
intensity of the predominant reactive band at M.sub.r-180,000 was
quantified by reflectance densitometry, as described previously
(Holmes et al. Science 256:1205-1210 (1992); Sliwkowski et al. J.
Biol. Chem. 269:14661-14665 (1994))
[0245] Monoclonal antibody 4D5 significantly inhibited the
generation of a HRG-induced tyrosine phosphorylation signal at
M.sub.r 180,000. In the absence of HRG, but was unable to stimulate
tyrosine phosphorylation of proteins in the M.sub.r 180,000 range.
Also, this antibody does not cross-react with EGFR (Fendly et al.
Cancer Research 50:1550-1558 (1990)), ErbB3, or ErbB4. Monoclonal
antibody 4D5 was able to block HRG stimulation of tyrosine
phosphorylation by -50%.
[0246] The growth inhibitory effect of monoclonal antibody 4D5 on
MDA-MB-175 and SK-BR-3 cells in the presence or absence of
exogenous rHRG.beta.1 was assessed (Schaefer et al. Oncogene
15:1385-1394 (1997)). ErbB2 levels in MDA-MB-175 cells are 4-6
times higher than the level found in normal breast epithelial cells
and the ErbB2-ErbB4 receptor is constitutively tyrosine
phosphorylated in MDA-MB-175 cells. Monoclonal antibody 4D5 was
able to inhibit cell proliferation of MDA-MB-175 cells, both in the
presence and absence of exogenous HRG. Inhibition of cell
proliferation by 4D5 is dependent on the ErbB2 expression level
(Lewis et al. Cancer Immunol. Immunother. 37:255-263 (1993)). A
maximum inhibition of 66% in SK-BR-3 cells could be detected.
However this effect could be overcome by exogenous HRG.
Humanization
[0247] The murine monoclonal antibody 4D5 was humanized, using a
novel "gene conversion mutagenesis" strategy, as described in U.S.
Pat. No. 5,821,337, the entire disclosure of which is hereby
expressly incorporated by reference. The humanized monoclonal
antibody 4D5 used in the following experiments is designated
huMAb4D5-8. This antibody is of IgG1 isotype.
Example 2
HERCEPTIN.RTM.-DM1 Conjugates
[0248] 1. Purification of HERCEPTIN.RTM.
[0249] HERCEPTIN.RTM. (huMAb4D5-8, rhuMAb HER2, U.S. Pat. No.
5,821,337) (1 vial containing 440 mg antibody) was dissolved in 50
mL MES buffer (25 mM MES, 50 mM NaCl, pH 5.6). The sample was
loaded on a cation exchange column (Sepharose S, 15 cm.times.1.7
cm) that had been equilibrated in the same buffer. The column was
then washed with the same buffer (5 column volumes). HERCEPTIN.RTM.
was eluted by raising the NaCl concentration of the buffer to 200
mM. Fractions containing the antibody were pooled, diluted to 10
mg/mL, and dialyzed into a buffer containing 50 mm potassium
phosphate, 50 mM NaCl, 2 mM EDTA, pH 6.5.
[0250] 2. Modification of HERCEPTIN.RTM. with SPP
[0251] The purified HERCEPTIN.RTM. antibody was modified with
N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to introduce
dithiopyridyl groups. The antibody (376.0 mg, 8 mg/mL) in 44.7 mL
of 50 mM potassium phosphate buffer (pH 6.5) containing NaCl (50
mM) and EDTA (1 mM) was treated with SPP (5.3 molar equivalents in
2.3 mL ethanol). After incubation for 90 minutes under argon at
ambient temperature, the reaction mixture was gel filtered through
a Sephadex G25 column equilibrated with 35 mM sodium citrate, 154
mM NaCl, 2 mM EDTA. Antibody containing fractions were pooled and
assayed. The degree of modification of the antibody was determined
as described above. Recovery of the modified antibody
(HERCEPTIN.RTM.-SPP-Py) was 337 mg (89.7%) with 4.5 releasable
2-thiopyridine groups linked per antibody.
[0252] 3. Conjugation of HERCEPTIN.RTM.-SPP-Py with DM1
[0253] The modified antibody (337.0 mg, 9.5 mmols of releasable
2-thiopyridine groups) was diluted with the above 35 mM sodium
citrate buffer, pH 6.5, to a final concentration of 2.5 mg/mL. DM1
(1.7 equivalents, 16.1 .mu.mols) in 3.0 mM dimethylacetamide (DMA,
3% v/v in the final reaction mixture) was then added to the
antibody solution. The structure of DM1 is shown in FIG. 3, where
the nature of the "R" group is not critical and can be occupied,
for example, by a variety of groups capable of forming a chemical
bond with a linker. DM1 used in the present reaction was stored as
an S--S form, which is more stable, and was reduced to the SH form
for conjugation with the HERCEPTIN.RTM. antibody. The reaction
proceeded at ambient temperature under argon for 20 hours. The
structure of HERCEPTIN.RTM.-DM1 conjugates is illustrated in FIG.
4.
[0254] The reaction was loaded on a Sephacryl S300 gel filtration
column (5.0 cm.times.90.0 cm, 1.77 L) equilibrated with 35 mM
sodium citrate, 154 mM NaCl, pH 6.5. The flow rate was 5.0 mL/min
and 65 fractions (20.0 mL each) were collected. A major peak
centered around fraction No. 47 (FIG. 5). The major peak comprises
monomeric HERCEPTIN.RTM.-DM1. Fractions 44-51 were pooled and
assayed. The number of DM1 drug molecules linked per antibody
molecule was determined by measuring the absorbance at 252 nm and
280 nm, and found to be 3.7 drug molecules per antibody
molecule.
[0255] 4. Anti-Proliferative Effect of HERCEPTIN.RTM.-DM1 Conjugate
In Vitro
[0256] SK-BR3 cells, which express 3+ level of HER2 on cell
surface, were treated with HERCEPTIN.RTM., HERCEPTIN.RTM.-DM1
conjugate, control mAb RITUXAN.RTM. or RITUXAN.RTM.-DM1 conjugates,
and the effect of these treatments on cell proliferation was
monitored. As shown in FIG. 6, the extent of cell growth inhibition
by treatment with HERCEPTIN.RTM.-DM1 was dramatically more
pronounced than that with HERCEPTIN.RTM., while the control
RITUXAN.RTM. antibody did not inhibit cell growth. Although the
RITUXAN.RTM.-DM1 did inhibit cell growth, it did so only at high
concentrations. For example, the RITUXAN.RTM.-DM1 conjugate did not
inhibit growth at concentration up to 1 .mu.g/ml. In contrast, the
HERCEPTIN.RTM.-DM1 conjugate was highly potent and significantly
inhibited cell growth starting from 0.01 .mu.g/ml and reaching a
plateau at 0.1 .mu.g/ml. The RITUXAN.RTM.-DM1 conjugate required
100 times higher concentration to achieve the same level of cell
growth inhibition as HERCEPTIN.RTM.-DM1 conjugate. This is also
reflected in a 100-fold difference in IC.sub.50 value,
concentration required to inhibit cell growth by 50%, of the
respective conjugates.
Example 3
Transgenic Animals
[0257] In order to improve the clinical activity of HERCEPTIN.RTM.,
a transgenic HER2 mouse model was developed in which novel
HER2-directed therapies could be tested preclinically. Tumors arise
readily in transgenic mice that express a mutationally activated
form of neu, the rat homolog of HER2, but the HER2 that is
overexpressed in breast cancers is not mutated and tumor formation
is much less robust in transgenic mice that overexpress nonmutated
HER2 (Webster et al., Semin. Cancer Biol. 5: 69-76 [1994]). To
improve tumor formation with nonmutated HER2, a strategy was used
to further enhance overexpression of nonmutated HER2 in a
transgenic mouse.
[0258] Any promoter that promotes expression of HER2 in epithelial
cells in the mouse mammary gland can be used in the disclosed
constructs. Many of the milk protein genes are transcribed by
promoter/enhancer elements that are specifically active in mammary
glands. Milk protein genes include those genes encoding caseins
(.alpha.-S.sub.1 and .beta.), .beta.-lactoglobulin,
.alpha.-lactalbumin, and whey acidic protein. The ovine
.beta.-lactoglobulin promoter is well characterized and widely used
in the art (Whitelaw et al., Biochem J. 286: 31-39, [1992]).
However, similar fragments of promoter DNA from other species are
also suitable. A preferred promoter is the promoter derived from
the Long Terminal Repeat (LTR) of the Mouse Mammary Tumor Virus
(MMTV). A HER2 transgene construct of the present invention was
generated using the MMTV LTR promoter.
[0259] To improve tumor formation with nonmutated HER2, we have
made transgenic mice using a HER2 cDNA plasmid in which an upstream
ATG was deleted in order to prevent initiation of translation at
such upstream ATG codons, which would otherwise reduce the
frequency of translation initiation from the downstream authentic
initiation codon of HER2 (for example, see Child et al., J. Biol.
Chem. 274: 24335-24341 [1999]). Additionally, a chimeric intron was
added to the 5' end, which should also enhance the level of
expression as reported earlier (Neuberger and Williams, Nucleic
Acids Res. 16: 6713 [1988]; Buchman and Berg, Mol. Cell. Biol. 8:
4395 [1988]; Brinster et al., Proc. Natl. Acad. Sci. USA 85: 836
[1988]). The chimeric intron was derived from a Promega vector,
pCI-neo mammalian expression vector (bp 890-1022). The cDNA 3'-end
is flanked by human growth hormone exons 4 and 5, and
polyadenylation sequences. Moreover, FVB mice were used because
this strain is more susceptible to tumor development. The promoter
from MMTV-LTR was used to ensure tissue-specific HER2 expression in
the mammary gland. Animals were fed the AIN 76A diet in order to
increase susceptibility to tumor formation (Rao et al., Breast
Cancer Res. and Treatment 45: 149-158 [1997]). The nucleotide
sequence of this transgene plasmid construct (SEQ ID NO: 1) is
shown in FIG. 7.
[0260] Animals suitable for transgenic experiments can be obtained
from standard commercial sources such as Taconic (Germantown,
N.Y.). Many strains are suitable, but FVB female mice are preferred
because of their higher susceptibility to tumor formation. FVB
males were used for mating and vasectomized CD.1 studs were used to
stimulate pseudopregnancy. Vasectomized mice can be obtained from
any commercial supplier. Founders were bred with either FVB mice or
with 129/BL6.times.FVB p53 heterozygous mice. The mice with
heterozygosity at p53 allele were used to potentially increase
tumor formation. However, this has proven unnecessary. Therefore,
some F1 tumors are of mixed strain. Founder tumors are FVB only.
Six founders were obtained with some developing tumors without
having litters.
Example 4
HER2 Transgenic Mouse as a Tumor Model to Evaluate HER2-Directed
Therapies
[0261] Mammary gland biopsies of one founder transgenic mouse made
as described in Example 3, showed 3+ expression of HER2, as
determined by immunohistochemical staining, at about 2 months of
age. The amount of HER2 extracellular domain (ECD) shed into serum
was measured and found to be about 1.2 ng/ml (Huang et al., supra).
This mouse subsequently developed a mammary tumor at 5 months of
age, after bearing 4 litters. The tumor was surgically resected
under aseptic conditions and minced into small pieces, 2 mm.sup.3,
which were then transplanted into the mammary fat pad of wild-type
FVB female mice. As can be seen in FIG. 8A, the amount of HER2 ECD
shed into serum increased over time following transplant and was
found to be directly proportional to the weight of the tumor that
developed (FIG. 8B). Tumors developed in 22 of 31 recipient mice,
with a latency of 5 weeks. With subsequent passage, tumors
developed with shorter latency and grew more rapidly, and tumor
incidence increased to >95% of recipients. HER2 expression, as
determined by immunohistochemical staining, was 3+ but
heterogeneous in the primary tumor, but became uniformly 3+ after
the first passage.
[0262] Treatment of tumor-bearing mice with HERCEPTIN.RTM. or 4D5,
the murine antibody from which humanized HERCEPTIN.RTM. was
derived, had only a modest effect on the growth of the transplanted
tumors (FIG. 9). HER2 expression was 3+ in tumors that grew during
HERCEPTIN.RTM. or 4D5 therapy, indicating that there was no
selection of HER2-negative tumors. Moreover, as can be seen in FIG.
10, cy3-HERCEPTIN.RTM. was detected decorating tumor cells after
injection into tumor-bearing mice, indicating that the lack of
efficacy was not due to failure of the antibody to access the
tumor. In addition, HER2 appears to be activated in the tumor
cells, as evidenced by the binding of an anti-tyrosine
phosphorylated HER2 antibody (FIG. 10).
[0263] Based on the persistent expression of HER2 and the failure
of this tumor model to respond to HERCEPTIN.RTM., a novel approach
was tested, using HERCEPTIN.RTM. conjugated to maytansinoid DM1 as
described in Example 3. FIG. 9 shows that the HERCEPTIN.RTM.-DM1
conjugate has dramatic anti-tumor activity in this model.
RITUXAN.RTM., an unrelated anti-CD20 monoclonal antibody, was used
as a negative control for these studies. There was little response
to HERCEPTIN.RTM. compared to the control antibody, RITUXAN.RTM.,
but there was striking anti-tumor activity of the maytansinoid
conjugate of HERCEPTIN.RTM.. As shown in FIG. 9, all of the mice
treated with HERCEPTIN.RTM.-maytansinoid showed striking shrinkage
of their tumors, though none of the tumors disappeared. After
approximately 4 weeks, tumors began to regrow. Five animals were
sacrificed at this time. Their tumors were found to express HER2 at
3+ levels. Thus, there was no selection for HER2-negative tumors.
Based on this observation, the remaining 3 mice were treated with
HERCEPTIN.RTM.-maytansinoid for 5 consecutive days. The tumors
again regressed in response to the treatment.
[0264] Despite its effectiveness at shrinking tumors and
suppressing tumor growth, HERCEPTIN.RTM.-DM1 does not kill normal
human cells, indicating a selective activity. The effect of various
concentrations of HERCEPTIN.RTM.-DM1 on human mammary epithelial
cells, human hepatocytes and human small airway epithelial cells
was investigated. At antibody concentrations of up to 10 .mu.g/ml,
the conjugate had no significant effect on cell number.
[0265] The pharmacokinetics of HERCEPTIN.RTM.-DM1 was evaluated in
mice and cynomolgus monkeys. It was determined that the
HERCEPTIN.RTM.-DM1 pharmacokinetics was linear with respect to dose
in both mouse and cynomolgus monkeys following i.v. administration.
Dose response analysis in mice indicated that tumor suppression
increased with increasing exposure to HERCEPTIN.RTM.-DM1 and
reached maximum suppression following a dose of at least 18 mg/kg
given once a week. The concentration-effect relationship will be
further characterized in future studies.
[0266] FIG. 11 shows the results of treating mice with HER-2
over-expressing tumors with HERCEPTIN.RTM.-DM1 once a week for 5
weeks. Each dose contained 300 .mu.g/kg of DM1 and 18 mg/kg of
HERCEPTIN.RTM.. Another group of animals received HERCEPTIN.RTM.
twice a week at a dose of 18 mg/kg body weight. Animals that did
not receive any treatment were used as control. As shown in FIG.
11, in animals that received HERCEPTIN.RTM.-DM1 tumor size was
dramatically controlled and, perhaps more importantly, the tumor
size was kept under control even after the therapy was stopped.
[0267] In yet another experiment, the amount of HERCEPTIN.RTM.-DM1
conjugate as well as the frequency of administration were varied
while keeping the total number of doses at five. The evaluation was
carried out in nude mice containing HER2 tumor transplants in
mammary pads. As shown in FIG. 12, some animals received
HERCEPTIN.RTM. DM1 at 300, 100, 30 or 10 .mu.g DM1/kg twice a week
for the total of 5 doses. Another group of animals received
HERCEPTIN.RTM.-DM1 at 300 or 100 .mu.g DM1/kg once a week for the
total of 5 doses. For comparison, HERCEPTIN.RTM. alone was
administered at 18 mg/kg twice a week or a control monoclonal
antibody (E25 directed against CD20, also known as RITUXAN.RTM.)
was administered twice a week. Consistent with earlier results in
this HERCEPTIN.RTM. insensitive model, HERCEPTIN.RTM. failed to
control the growth of mammary tumors. However, HERCEPTIN.RTM.-DM1
conjugate showed dramatic anti-tumor activity in a dose-dependent
manner. For example, higher doses showed more potent anti-tumor
activity than lower doses in both of the groups, i.e. those
receiving treatments twice a week and once a week. Furthermore,
twice a week treatment was more effective in keeping the tumor size
smaller than once a week treatment schedule. Interestingly,
however, there was no significant difference in the profile of
tumor growth inhibition at 300 .mu.g DM1/kg dose of
HERCEPTIN.RTM.-DM1 conjugate whether administered twice a week or
once a week. This suggests that at 300 .mu.g DM1/kg dose, once a
week schedule of treatment is effective in controlling tumor growth
and that a higher frequency of administration is not necessary.
[0268] The results of a similar experiment are depicted in FIG. 13.
The results of three different dosing regimens of
HERCEPTIN.RTM.-DM1 conjugate on tumor size are shown compared to
matching dosing regimens of RITUXAN.RTM.-DM1. Tumor size was
reduced and tumor growth was suppressed for at least about 50 days
by treatment with 5 doses of HERCEPTIN.RTM.-DM1 at a concentration
of 300 .mu.g DM1/kg. This was true both when the HERCEPTIN.RTM.-DM1
was administered once a week and when it was administered twice a
week. By contrast, administration of 5 doses of HERCEPTIN.RTM.-DM1
twice a week at a concentration of 100 .mu.g DM1/kg did not shrink
tumor size and suppressed tumor growth for somewhat less time.
Matched RITUXAN.RTM.-DM1 treatment showed little effect on tumor
size, indicating that the observed effect is specific to
HERCEPTIN.RTM.-DM1. Similarly, unconjugated RITUXAN.RTM. (control
MAb E25) showed no efficacy.
[0269] As can be seen clearly in FIG. 14, a dose of
HERCEPTIN.RTM.-DM1 (300 .mu.l DM1/kg) once a week for five weeks
caused tumors to shrink and prevented regrowth for more than 60
days. FIG. 14 also shows that a second round of treatment with
HERCEPTIN.RTM.-DM1 after tumor regrowth begins is capable of
shrinking tumors a second time. Neither unconjugated RITUXAN.RTM.
(control MAb E25) nor RITUXAN.RTM.-DM1 had any obvious effect on
tumor growth.
[0270] As shown here, the mammary tumor transplanted from HER2
transgenic mouse serves as a very useful model in evaluating
various anti-tumor compounds as well as in establishing efficacy of
different treatment regimens in preclinical studies. The model is
particularly unique as it shares an important attribute of a
fraction of human mammary tumors which are either completely or
partially refractory to the treatment of HERCEPTIN.RTM. inspite of
overexpression of HER2 at 2+ or 3+ levels. Thus, HER2 transgenic
model described herein provides a valuable tool not only to study
the mechanism of resistance to the anti-tumor activity of
HERCEPTIN.RTM., but also for screening compounds or modified
HERCEPTIN.RTM., including conjugates, for anti-tumor activity. The
insight gained from such studies is likely to help in developing
effective breast cancer therapies with broad coverage including
HERCEPTIN.RTM.-resistant metastatic HER2 overexpressing breast
carcinomas. The present HER2 transgenic model is particularly
suited for preclinical research as well as drug development, and is
a better alternative to in vitro studies carried out using breast
carcinoma cell lines. It is an in vivo system with normal stroma
and microenvironment of breast epithelium with cell-cell and
cell-matrix interactions that are typical of a tissue. It also
takes into account local factors and cytokines produced in the
normal course of mammary gland development and attendant regulatory
networks. It is also suitable to carry out pharmacokinetics studies
of drug candidates at a smaller scale, which can then be scaled up
for studies in non-human primate models. The results provide a
sound basis for actual clinical trials in human subjects. As per
the strategy described herein, the development of a HER2 transgenic
model does not need to involve any in vitro selection, and requires
minimal in vivo selection, the latter being limited to the extent
of serial passage of the mammary tissue in order to reduce the
duration of time needed to develop tumors and obtaining homogenous
overexpression of HER2 in mammary cells. Moreover, breeding of
these mice provides a continuous source of tissues for various
follow-up or supplemental studies. This is particularly significant
since the availability of clinical samples of tissues from breast
cancer patients is highly limited.
[0271] The HERCEPTIN.RTM.-DM1 conjugate as described herein was
found to have superior activity over HERCEPTIN.RTM. in this HER2
transgenic model that mimics HERCEPTIN.RTM.-resistant metastatic
HER2 overexpressing breast carcinomas. Approximately, 85% of breast
cancer patients either do not respond to HERCEPTIN.RTM. therapy or
respond poorly. The molecular basis of the resistance is not
clearly understood. However, it is not due to a lower level of HER2
expression since these tumors also overexpress HER2 at 2+ or 3+
levels. Nevertheless, this significant proportion of breast cancer
patients is not able to avail themselves of the powerful potential
of HERECEPTIN.RTM. therapy. Preclinical studies carried out using
the HERCEPTIN.RTM.-insensitive HER2 transgenic mouse model as
outlined in this application shows a dramatic response of these
tumors to HERCEPTIN.RTM.-DM1 conjugate as compared to
HERCEPTIN.RTM..
[0272] The HERCEPTIN.RTM.-DM1 conjugate was found to effectively
control the growth of HERCEPTIN.RTM.-resistant tumors in a
dose-dependent manner at a dosage of 100 .mu.g DM1/kg and above.
Administration of the tested conjugate at 300 .mu.g DM1/kg once a
week brought about a very impressive inhibition of tumor growth.
Five such doses completely prevented the emergence of tumor for
more than 60 days, and when the tumor did begin to reemerge, a
second round of HERCEPTIN.RTM.-DM1 was able to control the growth.
This is in contrast with a rapid growth of tumors in animals
treated with control monoclonal antibody (RITUXAN.RTM.),
maytansinoid conjugated control monoclonal antibody
(RITUXAN.RTM.-DM1) or unconjugated HERCEPTIN.RTM.. Thus, the
preclinical studies presented herein clearly show that the
HERCEPTIN.RTM.-DM1 conjugate is able to elicit a dramatic
anti-tumor response even in HERCEPTIN.RTM.-resistant breast tumors.
The better objective response rate obtained with the
HERCEPTIN.RTM.-DM1 conjugate will allow a higher fraction of breast
cancer patients to benefit from this powerful therapy. The fact
that the effect of HERCEPTIN.RTM.-DM1 is dose-dependent suggests
that in an actual clinical setting, the strategy is likely to
provide a considerable maneuver of doses to achieve the best
anti-tumor activity. Moreover, the duration of anti-tumor response
is significantly longer, permitting less frequent administration of
the conjugate without compromising the therapeutic efficacy. The
resultant cost-effectiveness and convenience is quite significant.
Furthermore, the conjugate is likely to improve survival rate among
the responders. For example, the median time to disease progression
in HERCEPTIN.RTM. treated patients was only 3.1 months. With the
superior therapeutic efficacy of HERCEPTIN.RTM.-DM1 as compared to
HERCEPTIN.RTM., survival rate is likely to be increased.
[0273] All references cited throughout the specification, and the
references cited therein, are hereby expressly incorporated by
reference.
Deposit of Biological Material
[0274] 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
[0275] This deposit was made under the provisions of the Budapest
Treaty on the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedure and the
Regulations thereunder (Budapest Treaty). This assures maintenance
of viable cultures for 30 years from the date of the deposit. The
organisms will be made available by ATCC under the terms of the
Budapest Treaty, and subject to an agreement between Genentech,
Inc. and ATCC, which assures permanent and unrestricted
availability of the progeny of the cultures to the public upon
issuance of the pertinent U.S. patent or upon laying open to the
public of any U.S. or foreign patent application, whichever comes
first, and assures availability of the progeny to one determined by
the U.S. Commissioner of Patents and Trademarks to be entitled
thereto according to 35 USC .sctn.122 and the Commissioner's rules
pursuant thereto (including 37 CFR .sctn.1.12 with particular
reference to 8860G 638).
[0276] In respect of those designations in which a European patent
is sought, a sample of the deposited microorganism will be made
available until the publication of the mention of the grant of the
European patent or until the date on which the application has been
refused or withdrawn or is deemed to be withdrawn, only by the
issue of such a sample to an expert nominated by the person
requesting the sample. (Rule 28(4) EPC)
[0277] The assignee of the present application has agreed that if
the cultures on deposit should die or be lost or destroyed when
cultivated under suitable conditions, they will be promptly
replaced on notification with a viable specimen of the same
culture. Availability of the deposited strain is not to be
construed as a license to practice the invention in contravention
of the rights granted under the authority of any government in
accordance with its patent laws.
[0278] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The present invention is not to be limited in scope by
the constructs deposited, since the deposited embodiments are
intended to illustrate only certain aspects of the invention and
any constructs that are functionally equivalent are within the
scope of this invention. The deposit of material herein does not
constitute an admission that the written description herein
contained is inadequate to enable the practice of any aspect of the
invention, including the best mode thereof, nor is it to be
construed as limiting the scope of the claims to the specific
illustrations that they represent. Indeed, various modifications of
the invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
[0279] It is understood that the application of the teachings of
the present invention to a specific problem or situation will be
within the capabilities of one having ordinary skill in the art in
light of the teachings contained herein. Examples of the products
of the present invention and representative processes for their
isolation, use, and manufacture appear below, but should not be
construed to limit the invention.
Sequence CWU 1
1
111119PRTArtificial SequenceHumanized Antibody Sequence 1Glu Val
Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Thr1 5 10 15Ser
Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25
30Thr Met Asp Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45Gly Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg
Phe 50 55 60Lys Gly Lys Ala Ser Leu Thr Val Asp Arg Ser Ser Arg Ile
Val Tyr65 70 75 80Met Glu Leu Arg Ser Leu Thr Phe Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp
Tyr Trp Gly Gln Gly 100 105 110Thr Thr Leu Thr Val Ser Ser
1152119PRTArtificial SequenceHumanized Antibody Sequence 2Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25
30Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg
Phe 50 55 60Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp
Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
1153119PRTArtificial SequenceHumanized Antibody Sequence 3Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Val Ile Ser Gly Asp Gly Gly Ser Thr Tyr Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Arg Val Gly Tyr Ser Leu Tyr
Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
1154109PRTArtificial SequenceHumanized Antibody Sequence 4Asp Thr
Val Met Thr Gln Ser His Lys Ile Met Ser Thr Ser Val Gly 1 5 10
15Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly
20 25 30Val Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Lys Leu Leu
Ile 35 40 45Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe
Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser
Val Gln Ala65 70 75 80Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln Tyr
Tyr Ile Tyr Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys Arg Thr 100 1055109PRTArtificial SequenceHumanized Antibody
Sequence 5Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly 1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp
Val Ser Ile Gly 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Arg Thr 100 1056109PRTArtificial
SequenceHumanized Antibody Sequence 6Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn Tyr 20 25 30Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala
Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Leu Pro Trp
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 100
10579274DNAArtificial SequenceVector Sequence 7aagctcgatc
ggtgcacatt aattcatgat cgcgagctag cagcttgcat gcctgcagca 60gaaatggttg
aactcccgag agtgtcctac acctagggga gaagcagcca aggggttgtt
120tcccaccaag gacgacccgt ctgcgcacaa acggatgagc ccatcagaca
aagacatatt 180cattctctgc tgcaaacttg gcatagctct gctttgctgg
ggcattgggg gaagttgcgg 240ttcgtgctcg cagggctctc acccttgact
cttttaatag ctcttctgtg caagattaca 300atctaaacaa ttcggagaac
tcgaccttcc tctcctgagg caaggaccac agccaacttc 360ctcttacaag
ccgcatcgat tttgtccttc agaaatagaa ataagaatgc ttgctaaaaa
420ttatattttt accaataaga ccaatccaat aggtagatta ttagttacta
tgttaagaaa 480tgaatcatta tcttttagta ctatttttac tcaaattcag
aagttagaaa tgggaataga 540aaatagaaag agacgctcaa cctcaattga
agaacaggtg caaggactat tgaccacagg 600cctagaagta aaaaagggaa
aaaagagtgt ttttgtcaaa ataggagaca ggtggtggca 660accagggact
tataggggac cttacatcta cagaccaaca gatgccccct taccatatac
720aggaagatat gacttaaatt gggataggtg ggttacagtc aatggctata
aagtgttata 780tagatccctc ccttttcgtg aaagactcgc cagagctaga
cctccttggt gtatgttgtc 840tcaagaagaa aaagacgaca tgaaacaaca
ggtacatgat tatatttatc taggaacagg 900aatgcacttt tggggaaaga
ttttccatac caaggagggg acagtggctg gactaataga 960acattattct
gcaaaaactt atggcatgag ttattatgaa tagcctttat tggcccaacc
1020ttgcggttcc caaggcttaa gtaagttttt ggttacaaac tgttcttaaa
acgaggatgt 1080gagacaagtg gtttcctgac ttggtttggt atcaaaggtt
ctgatctgag ctctgagtgt 1140tctattttcc tatgttcttt tggaatttat
ccaaatctta tgtaaatgct tatgtaaacc 1200aagatataaa agagtgctga
ttttttgagt aaacttgcaa cagtcctaac attcacctct 1260tgtgtgtttg
tgtctgttcg ccatcccgtc tccgctcgtc acttatcctt cactttccag
1320agggtccccc cgcagacccc ggatcgctag ctcgcgaatc gataagcttg
cggccgctta 1380actgcagaag ttggtcgtga ggcactgggc aggtaagtat
caaggttaca agacaggttt 1440aaggagacca atagaaactg ggcttgtcga
gacagagaag actcttgcgt ttctgatagg 1500cacctattgg tcttactgac
atccactttg cctttctctc cacaggtgtc cactcccagg 1560ttcaattaca
gctcttaagc ggccgcaagc ttgatatcga attcctgcag cccgggggat
1620ccactagtgg atccaaagaa ttcaaaaagc ttctcgaggg cgcgcgcccg
gcccccaccc 1680ctcgcagcac cccgcgcccc gcgccctccc agccgggtcc
agccggagcc atggagctgg 1740cggccttgtg ccgctggggg ctcctcctcg
ccctcttgcc ccccggagcc gcgagcaccc 1800aagtgtgcac cggcacagac
atgaagctgc ggctccctgc cagtcccgag acccacctgg 1860acatgctccg
ccacctctac cagggctgcc aggtggtgca gggaaacctg gaactcacct
1920acctgcccac caatgccagc ctgtccttcc tgcaggatat ccaggaggtg
cagggctacg 1980tgctcatcgc tcacaaccaa gtgaggcagg tcccactgca
gaggctgcgg attgtgcgag 2040gcacccagct ctttgaggac aactatgccc
tggccgtgct agacaatgga gacccgctga 2100acaataccac ccctgtcaca
ggggcctccc caggaggcct gcgggagctg cagcttcgaa 2160gcctcacaga
gatcttgaaa ggaggggtct tgatccagcg gaacccccag ctctgctacc
2220aggacacgat tttgtggaag gacatcttcc acaagaacaa ccagctggct
ctcacactga 2280tagacaccaa ccgctctcgg gcctgccacc cctgttctcc
gatgtgtaag ggctcccgct 2340gctggggaga gagttctgag gattgtcaga
gcctgacgcg cactgtctgt gccggtggct 2400gtgcccgctg caaggggcca
ctgcccactg actgctgcca tgagcagtgt gctgccggct 2460gcacgggccc
caagcactct gactgcctgg cctgcctcca cttcaaccac agtggcatct
2520gtgagctgca ctgcccagcc ctggtcacct acaacacaga cacgtttgag
tccatgccca 2580atcccgaggg ccggtataca ttcggcgcca gctgtgtgac
tgcctgtccc tacaactacc 2640tttctacgga cgtgggatcc tgcaccctcg
tctgccccct gcacaaccaa gaggtgacag 2700cagaggatgg aacacagcgg
tgtgagaagt gcagcaagcc ctgtgcccga gtgtgctatg 2760gtctgggcat
ggagcacttg cgagaggtga gggcagttac cagtgccaat atccaggagt
2820ttgctggctg caagaagatc tttgggagcc tggcatttct gccggagagc
tttgatgggg 2880acccagcctc caacactgcc ccgctccagc cagagcagct
ccaagtgttt gagactctgg 2940aagagatcac aggttaccta tacatctcag
catggccgga cagcctgcct gacctcagcg 3000tcttccagaa cctgcaagta
atccggggac gaattctgca caatggcgcc tactcgctga 3060ccctgcaagg
gctgggcatc agctggctgg ggctgcgctc actgagggaa ctgggcagtg
3120gactggccct catccaccat aacacccacc tctgcttcgt gcacacggtg
ccctgggacc 3180agctctttcg gaacccgcac caagctctgc tccacactgc
caaccggcca gaggacgagt 3240gtgtgggcga gggcctggcc tgccaccagc
tgtgcgcccg agggcactgc tggggtccag 3300ggcccaccca gtgtgtcaac
tgcagccagt tccttcgggg ccaggagtgc gtggaggaat 3360gccgagtact
gcaggggctc cccagggagt atgtgaatgc caggcactgt ttgccgtgcc
3420accctgagtg tcagccccag aatggctcag tgacctgttt tggaccggag
gctgaccagt 3480gtgtggcctg tgcccactat aaggaccctc ccttctgcgt
ggcccgctgc cccagcggtg 3540tgaaacctga cctctcctac atgcccatct
ggaagtttcc agatgaggag ggcgcatgcc 3600agccttgccc catcaactgc
acccactcct gtgtggacct ggatgacaag ggctgccccg 3660ccgagcagag
agccagccct ctgacgtcca tcgtctctgc ggtggttggc attctgctgg
3720tcgtggtctt gggggtggtc tttgggatcc tcatcaagcg acggcagcag
aagatccgga 3780agtacacgat gcggagactg ctgcaggaaa cggagctggt
ggagccgctg acacctagcg 3840gagcgatgcc caaccaggcg cagatgcgga
tcctgaaaga gacggagctg aggaaggtga 3900aggtgcttgg atctggcgct
tttggcacag tctacaaggg catctggatc cctgatgggg 3960agaatgtgaa
aattccagtg gccatcaaag tgttgaggga aaacacatcc cccaaagcca
4020acaaagaaat cttagacgaa gcatacgtga tggctggtgt gggctcccca
tatgtctccc 4080gccttctggg catctgcctg acatccacgg tgcagctggt
gacacagctt atgccctatg 4140gctgcctctt agaccatgtc cgggaaaacc
gcggacgcct gggctcccag gacctgctga 4200actggtgtat gcagattgcc
aaggggatga gctacctgga ggatgtgcgg ctcgtacaca 4260gggacttggc
cgctcggaac gtgctggtca agagtcccaa ccatgtcaaa attacagact
4320tcgggctggc tcggctgctg gacattgacg agacagagta ccatgcagat
gggggcaagg 4380tgcccatcaa gtggatggcg ctggagtcca ttctccgccg
gcggttcacc caccagagtg 4440atgtgtggag ttatggtgtg actgtgtggg
agctgatgac ttttggggcc aaaccttacg 4500atgggatccc agcccgggag
atccctgacc tgctggaaaa gggggagcgg ctgccccagc 4560cccccatctg
caccattgat gtctacatga tcatggtcaa atgttggatg attgactctg
4620aatgtcggcc aagattccgg gagttggtgt ctgaattctc ccgcatggcc
agggaccccc 4680agcgctttgt ggtcatccag aatgaggact tgggcccagc
cagtcccttg gacagcacct 4740tctaccgctc actgctggag gacgatgaca
tgggggacct ggtggatgct gaggagtatc 4800tggtacccca gcagggcttc
ttctgtccag accctgcccc gggcgctggg ggcatggtcc 4860accacaggca
ccgcagctca tctaccagga gtggcggtgg ggacctgaca ctagggctgg
4920agccctctga agaggaggcc cccaggtctc cactggcacc ctccgaaggg
gctggctccg 4980atgtatttga tggtgacctg ggaatggggg cagccaaggg
gctgcaaagc ctccccacac 5040atgaccccag ccctctacag cggtacagtg
aggaccccac agtacccctg ccctctgaga 5100ctgatggcta cgttgccccc
ctgacctgca gcccccagcc tgaatatgtg aaccagccag 5160atgttcggcc
ccagccccct tcgccccgag agggccctct gcctgctgcc cgacctgctg
5220gtgccactct ggaaagggcc aagactctct ccccagggaa gaatggggtc
gtcaaagacg 5280tttttgcctt tgggggtgcc gtggagaacc ccgagtactt
gacaccccag ggaggagctg 5340cccctcagcc ccaccctcct cctgccttca
gcccagcctt cgacaacctc tattactggg 5400accaggaccc accagagcgg
ggggctccac ccagcacctt caaagggaca cctacggcag 5460agaacccaga
gtacctgggt ctggacgtgc cagtgtgaac cagaaggcca agtccgcaga
5520agccctgatg tgtcctcagg gagcagggaa ggcggcctct gagctattcc
agaagtagtg 5580aggaggcttt tttggaggcc taggcttttg caaaaagctt
atcgataccg tcgactcgag 5640agtacttcta gagcggccgc gggcccatcg
cctctgacag caacgtctat gacctcctaa 5700aggacctaga ggaaggcatc
caaacgctga tggggaggct ggaagatggc agcccccgga 5760ctgggcagat
cttcaagcag acctacagca agttcgacac aaactcacac aacgatgacg
5820cactactcaa gaactacggg ctgctctact gcttcaggaa ggacatggac
aaggtcgaga 5880cattcctgcg catcgtgcag tgccgctctg tggagggcag
ctgtggcttc tagctgcccg 5940ggtggcatcc ctgtgacccc tccccagtgc
ctctcctggc cctggaagtt gccactccag 6000tgcccaccag ccttgtccta
ataaaattaa gttgcatcat tttgtctgac taggtgtcct 6060tctataatat
tatggggtgg aggggggtgg tatggagcaa ggggcccaag ttgggaagac
6120aacctgtagg gcctgcgggg tctattcggg aaccaagctg gagtgcagtg
gcacaatctt 6180ggctcactgc aatctccgcc tcctgggttc aagcgattct
cctgcctcag cctcccgagt 6240tgttgggatt ccaggcatgc atgaccaggc
tcagctaatt tttgtttttt tggtagagac 6300ggggtttcac catattggcc
aggctggtct ccaactccta atctcaggtg atctacccac 6360cttggcctcc
caaattgctg ggattacagg cgtgaaccac tgctcccttc cctgtccttc
6420tgattttaaa ataactatac cagcaggagg acgtccagac acagcatagg
ctacctgcca 6480tggcccaacc ggtgggacat ttgagttgct tgcttggcac
tgtcctctca tgcgttgggt 6540ccactcagta gatgcctgtt gaattacgat
cggtgcacat taattcatga aattcgtaat 6600catggtcata gctgtttcct
gtgtgaaatt gttatccgct cacaattcca cacaacatac 6660gagccggaag
cataaagtgt aaagcctggg gtgcctaatg agtgaggtaa ctcacattaa
6720ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
ctggattaat 6780gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
gcgctcttcc gcttcctcgc 6840tcactgactc gctgcgctcg gtcgttcggc
tgcggcgagc ggtatcagct cactcaaagg 6900cggtaatacg gttatccaca
gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 6960gccagcaaaa
ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
7020gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
aacccgacag 7080gactataaag ataccaggcg tttccccctg gaagctccct
cgtgcgctct cctgttccga 7140ccctgccgct taccggatac ctgtccgcct
ttctcccttc gggaagcgtg gcgctttctc 7200aatgctcacg ctgtaggtat
ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 7260tgcacgaacc
ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
7320ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
aggattagca 7380gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
gtggcctaac tacggctaca 7440ctagaaggac agtatttggt atctgcgctc
tgctgaagcc agttaccttc ggaaaaagag 7500ttggtagctc ttgatccggc
aaacaaacca ccgctggtag cggtggtttt tttgtttgca 7560agcagcagat
tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
7620ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
agattatcaa 7680aaaggatctt cacctagatc cttttaaatt aaaaatgaag
ttttaaatca atctaaagta 7740tatatgagta aacttggtct gacagttacc
aatgcttaat cagtgaggca cctatctcag 7800cgatctgtct atttcgttca
tccatagttg cctgactccc cgtcgtgtag ataactacga 7860tacgggaggg
cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
7920cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
agaagtggtc 7980ctgcaacttt atccgcctcc atccagtcta ttaattgttg
ccgggaagct agagtaagta 8040gttcgccagt taatagtttg cgcaacgttg
ttgccattgc tgctggcatc gtggtgtcac 8100gctcgtcgtt tggtatggct
tcattcagct ccggttccca acgatcaagg cgagttacat 8160gatcccccat
gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
8220gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
tctcttactg 8280tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
ctcaaccaag tcattctgag 8340aatagtgtat gcggcgaccg agttgctctt
gcccggcgtc atcacgggat aataccgcgc 8400cacatagcag aactttaaaa
gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 8460caaggatctt
accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
8520cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
aggcaaaatg 8580ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
actcatactc ttcctttttc 8640aatattattg aagcatttat cagggttatt
gtctcatgag cggatacata tttgaatgta 8700tttagaaaaa taaacaaata
ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg 8760tctaagaaac
cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
8820ttcgtcttca agaatactgc ctcgcgcgtt tcggtgatga cggtgaaaac
ctctgacaca 8880tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
tgccgggagc agacaagccc 8940gtcagggcgc gtcagcgggt gttggcgggt
gtcggggcgc agccatgacc cagtcacgta 9000gcgatagcgg agttggctta
actatgcggc atcagagcag attgtactga gagtgcacca 9060tatgcggtgt
gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccattc
9120gccattcagg ctacgcaact gttgggaagg gcgatcggtg cgggcctctt
cgctattacg 9180ccagctggcg aaggggggat gtgctgcaag gcgattaagt
tgggtaacgc cagggttttc 9240ccagtcacga cgttgtaaaa cgacggccag tgcc
927483768DNAHomo sapiens 8atggagctgg cggccttgtg ccgctggggg
ctcctcctcg ccctcttgcc ccccggagcc 60gcgagcaccc aagtgtgcac cggcacagac
atgaagctgc ggctccctgc cagtcccgag 120acccacctgg acatgctccg
ccacctctac cagggctgcc aggtggtgca gggaaacctg 180gaactcacct
acctgcccac caatgccagc ctgtccttcc tgcaggatat ccaggaggtg
240cagggctacg tgctcatcgc tcacaaccaa gtgaggcagg tcccactgca
gaggctgcgg 300attgtgcgag gcacccagct ctttgaggac aactatgccc
tggccgtgct agacaatgga 360gacccgctga acaataccac ccctgtcaca
ggggcctccc caggaggcct gcgggagctg 420cagcttcgaa gcctcacaga
gatcttgaaa ggaggggtct tgatccagcg gaacccccag 480ctctgctacc
aggacacgat tttgtggaag gacatcttcc acaagaacaa ccagctggct
540ctcacactga tagacaccaa ccgctctcgg gcctgccacc cctgttctcc
gatgtgtaag 600ggctcccgct gctggggaga gagttctgag gattgtcaga
gcctgacgcg cactgtctgt 660gccggtggct gtgcccgctg caaggggcca
ctgcccactg actgctgcca tgagcagtgt 720gctgccggct gcacgggccc
caagcactct gactgcctgg cctgcctcca cttcaaccac 780agtggcatct
gtgagctgca ctgcccagcc ctggtcacct acaacacaga cacgtttgag
840tccatgccca atcccgaggg ccggtataca ttcggcgcca gctgtgtgac
tgcctgtccc 900tacaactacc tttctacgga cgtgggatcc tgcaccctcg
tctgccccct gcacaaccaa 960gaggtgacag cagaggatgg aacacagcgg
tgtgagaagt gcagcaagcc ctgtgcccga 1020gtgtgctatg gtctgggcat
ggagcacttg cgagaggtga gggcagttac cagtgccaat 1080atccaggagt
ttgctggctg caagaagatc tttgggagcc tggcatttct gccggagagc
1140tttgatgggg acccagcctc caacactgcc ccgctccagc cagagcagct
ccaagtgttt 1200gagactctgg aagagatcac aggttaccta tacatctcag
catggccgga cagcctgcct 1260gacctcagcg tcttccagaa cctgcaagta
atccggggac gaattctgca caatggcgcc
1320tactcgctga ccctgcaagg gctgggcatc agctggctgg ggctgcgctc
actgagggaa 1380ctgggcagtg gactggccct catccaccat aacacccacc
tctgcttcgt gcacacggtg 1440ccctgggacc agctctttcg gaacccgcac
caagctctgc tccacactgc caaccggcca 1500gaggacgagt gtgtgggcga
gggcctggcc tgccaccagc tgtgcgcccg agggcactgc 1560tggggtccag
ggcccaccca gtgtgtcaac tgcagccagt tccttcgggg ccaggagtgc
1620gtggaggaat gccgagtact gcaggggctc cccagggagt atgtgaatgc
caggcactgt 1680ttgccgtgcc accctgagtg tcagccccag aatggctcag
tgacctgttt tggaccggag 1740gctgaccagt gtgtggcctg tgcccactat
aaggaccctc ccttctgcgt ggcccgctgc 1800cccagcggtg tgaaacctga
cctctcctac atgcccatct ggaagtttcc agatgaggag 1860ggcgcatgcc
agccttgccc catcaactgc acccactcct gtgtggacct ggatgacaag
1920ggctgccccg ccgagcagag agccagccct ctgacgtcca tcgtctctgc
ggtggttggc 1980attctgctgg tcgtggtctt gggggtggtc tttgggatcc
tcatcaagcg acggcagcag 2040aagatccgga agtacacgat gcggagactg
ctgcaggaaa cggagctggt ggagccgctg 2100acacctagcg gagcgatgcc
caaccaggcg cagatgcgga tcctgaaaga gacggagctg 2160aggaaggtga
aggtgcttgg atctggcgct tttggcacag tctacaaggg catctggatc
2220cctgatgggg agaatgtgaa aattccagtg gccatcaaag tgttgaggga
aaacacatcc 2280cccaaagcca acaaagaaat cttagacgaa gcatacgtga
tggctggtgt gggctcccca 2340tatgtctccc gccttctggg catctgcctg
acatccacgg tgcagctggt gacacagctt 2400atgccctatg gctgcctctt
agaccatgtc cgggaaaacc gcggacgcct gggctcccag 2460gacctgctga
actggtgtat gcagattgcc aaggggatga gctacctgga ggatgtgcgg
2520ctcgtacaca gggacttggc cgctcggaac gtgctggtca agagtcccaa
ccatgtcaaa 2580attacagact tcgggctggc tcggctgctg gacattgacg
agacagagta ccatgcagat 2640gggggcaagg tgcccatcaa gtggatggcg
ctggagtcca ttctccgccg gcggttcacc 2700caccagagtg atgtgtggag
ttatggtgtg actgtgtggg agctgatgac ttttggggcc 2760aaaccttacg
atgggatccc agcccgggag atccctgacc tgctggaaaa gggggagcgg
2820ctgccccagc cccccatctg caccattgat gtctacatga tcatggtcaa
atgttggatg 2880attgactctg aatgtcggcc aagattccgg gagttggtgt
ctgaattctc ccgcatggcc 2940agggaccccc agcgctttgt ggtcatccag
aatgaggact tgggcccagc cagtcccttg 3000gacagcacct tctaccgctc
actgctggag gacgatgaca tgggggacct ggtggatgct 3060gaggagtatc
tggtacccca gcagggcttc ttctgtccag accctgcccc gggcgctggg
3120ggcatggtcc accacaggca ccgcagctca tctaccagga gtggcggtgg
ggacctgaca 3180ctagggctgg agccctctga agaggaggcc cccaggtctc
cactggcacc ctccgaaggg 3240gctggctccg atgtatttga tggtgacctg
ggaatggggg cagccaaggg gctgcaaagc 3300ctccccacac atgaccccag
ccctctacag cggtacagtg aggaccccac agtacccctg 3360ccctctgaga
ctgatggcta cgttgccccc ctgacctgca gcccccagcc tgaatatgtg
3420aaccagccag atgttcggcc ccagccccct tcgccccgag agggccctct
gcctgctgcc 3480cgacctgctg gtgccactct ggaaagggcc aagactctct
ccccagggaa gaatggggtc 3540gtcaaagacg tttttgcctt tgggggtgcc
gtggagaacc ccgagtactt gacaccccag 3600ggaggagctg cccctcagcc
ccaccctcct cctgccttca gcccagcctt cgacaacctc 3660tattactggg
accaggaccc accagagcgg ggggctccac ccagcacctt caaagggaca
3720cctacggcag agaacccaga gtacctgggt ctggacgtgc cagtgtga
376891255PRTHomo sapiens 9Met Glu Leu Ala Ala Leu Cys Arg Trp Gly
Leu Leu Leu Ala Leu Leu1 5 10 15Pro Pro Gly Ala Ala Ser Thr Gln Val
Cys Thr Gly Thr Asp Met Lys 20 25 30Leu Arg Leu Pro Ala Ser Pro Glu
Thr His Leu Asp Met Leu Arg His 35 40 45Leu Tyr Gln Gly Cys Gln Val
Val Gln Gly Asn Leu Glu Leu Thr Tyr 50 55 60Leu Pro Thr Asn Ala Ser
Leu Ser Phe Leu Gln Asp Ile Gln Glu Val65 70 75 80Gln Gly Tyr Val
Leu Ile Ala His Asn Gln Val Arg Gln Val Pro Leu 85 90 95Gln Arg Leu
Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr 100 105 110Ala
Leu Ala Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro 115 120
125Val Thr Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser
130 135 140Leu Thr Glu Ile Leu Lys Gly Gly Val Leu Ile Gln Arg Asn
Pro Gln145 150 155 160Leu Cys Tyr Gln Asp Thr Ile Leu Trp Lys Asp
Ile Phe His Lys Asn 165 170 175Asn Gln Leu Ala Leu Thr Leu Ile Asp
Thr Asn Arg Ser Arg Ala Cys 180 185 190His Pro Cys Ser Pro Met Cys
Lys Gly Ser Arg Cys Trp Gly Glu Ser 195 200 205Ser Glu Asp Cys Gln
Ser Leu Thr Arg Thr Val Cys Ala Gly Gly Cys 210 215 220Ala Arg Cys
Lys Gly Pro Leu Pro Thr Asp Cys Cys His Glu Gln Cys225 230 235
240Ala Ala Gly Cys Thr Gly Pro Lys His Ser Asp Cys Leu Ala Cys Leu
245 250 255His Phe Asn His Ser Gly Ile Cys Glu Leu His Cys Pro Ala
Leu Val 260 265 270Thr Tyr Asn Thr Asp Thr Phe Glu Ser Met Pro Asn
Pro Glu Gly Arg 275 280 285Tyr Thr Phe Gly Ala Ser Cys Val Thr Ala
Cys Pro Tyr Asn Tyr Leu 290 295 300Ser Thr Asp Val Gly Ser Cys Thr
Leu Val Cys Pro Leu His Asn Gln305 310 315 320Glu Val Thr Ala Glu
Asp Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys 325 330 335Pro Cys Ala
Arg Val Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu 340 345 350Val
Arg Ala Val Thr Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys 355 360
365Lys Ile Phe Gly Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp
370 375 380Pro Ala Ser Asn Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln
Val Phe385 390 395 400Glu Thr Leu Glu Glu Ile Thr Gly Tyr Leu Tyr
Ile Ser Ala Trp Pro 405 410 415Asp Ser Leu Pro Asp Leu Ser Val Phe
Gln Asn Leu Gln Val Ile Arg 420 425 430Gly Arg Ile Leu His Asn Gly
Ala Tyr Ser Leu Thr Leu Gln Gly Leu 435 440 445Gly Ile Ser Trp Leu
Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly 450 455 460Leu Ala Leu
Ile His His Asn Thr His Leu Cys Phe Val His Thr Val465 470 475
480Pro Trp Asp Gln Leu Phe Arg Asn Pro His Gln Ala Leu Leu His Thr
485 490 495Ala Asn Arg Pro Glu Asp Glu Cys Val Gly Glu Gly Leu Ala
Cys His 500 505 510Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly
Pro Thr Gln Cys 515 520 525Val Asn Cys Ser Gln Phe Leu Arg Gly Gln
Glu Cys Val Glu Glu Cys 530 535 540Arg Val Leu Gln Gly Leu Pro Arg
Glu Tyr Val Asn Ala Arg His Cys545 550 555 560Leu Pro Cys His Pro
Glu Cys Gln Pro Gln Asn Gly Ser Val Thr Cys 565 570 575Phe Gly Pro
Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr Lys Asp 580 585 590Pro
Pro Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro Asp Leu 595 600
605Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln
610 615 620Pro Cys Pro Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp
Asp Lys625 630 635 640Gly Cys Pro Ala Glu Gln Arg Ala Ser Pro Leu
Thr Ser Ile Val Ser 645 650 655Ala Val Val Gly Ile Leu Leu Val Val
Val Leu Gly Val Val Phe Gly 660 665 670Ile Leu Ile Lys Arg Arg Gln
Gln Lys Ile Arg Lys Tyr Thr Met Arg 675 680 685Arg Leu Leu Gln Glu
Thr Glu Leu Val Glu Pro Leu Thr Pro Ser Gly 690 695 700Ala Met Pro
Asn Gln Ala Gln Met Arg Ile Leu Lys Glu Thr Glu Leu705 710 715
720Arg Lys Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys
725 730 735Gly Ile Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro Val
Ala Ile 740 745 750Lys Val Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn
Lys Glu Ile Leu 755 760 765Asp Glu Ala Tyr Val Met Ala Gly Val Gly
Ser Pro Tyr Val Ser Arg 770 775 780Leu Leu Gly Ile Cys Leu Thr Ser
Thr Val Gln Leu Val Thr Gln Leu785 790 795 800Met Pro Tyr Gly Cys
Leu Leu Asp His Val Arg Glu Asn Arg Gly Arg 805 810 815Leu Gly Ser
Gln Asp Leu Leu Asn Trp Cys Met Gln Ile Ala Lys Gly 820 825 830Met
Ser Tyr Leu Glu Asp Val Arg Leu Val His Arg Asp Leu Ala Ala 835 840
845Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr Asp Phe
850 855 860Gly Leu Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr His
Ala Asp865 870 875 880Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu
Glu Ser Ile Leu Arg 885 890 895Arg Arg Phe Thr His Gln Ser Asp Val
Trp Ser Tyr Gly Val Thr Val 900 905 910Trp Glu Leu Met Thr Phe Gly
Ala Lys Pro Tyr Asp Gly Ile Pro Ala 915 920 925Arg Glu Ile Pro Asp
Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro 930 935 940Pro Ile Cys
Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met945 950 955
960Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val Ser Glu Phe
965 970 975Ser Arg Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile Gln
Asn Glu 980 985 990Asp Leu Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe
Tyr Arg Ser Leu 995 1000 1005Leu Glu Asp Asp Asp Met Gly Asp Leu
Val Asp Ala Glu Glu Tyr Leu 1010 1015 1020Val Pro Gln Gln Gly Phe
Phe Cys Pro Asp Pro Ala Pro Gly Ala Gly1025 1030 1035 1040Gly Met
Val His His Arg His Arg Ser Ser Ser Thr Arg Ser Gly Gly 1045 1050
1055Gly Asp Leu Thr Leu Gly Leu Glu Pro Ser Glu Glu Glu Ala Pro Arg
1060 1065 1070Ser Pro Leu Ala Pro Ser Glu Gly Ala Gly Ser Asp Val
Phe Asp Gly 1075 1080 1085Asp Leu Gly Met Gly Ala Ala Lys Gly Leu
Gln Ser Leu Pro Thr His 1090 1095 1100Asp Pro Ser Pro Leu Gln Arg
Tyr Ser Glu Asp Pro Thr Val Pro Leu1105 1110 1115 1120Pro Ser Glu
Thr Asp Gly Tyr Val Ala Pro Leu Thr Cys Ser Pro Gln 1125 1130
1135Pro Glu Tyr Val Asn Gln Pro Asp Val Arg Pro Gln Pro Pro Ser Pro
1140 1145 1150Arg Glu Gly Pro Leu Pro Ala Ala Arg Pro Ala Gly Ala
Thr Leu Glu 1155 1160 1165Arg Ala Lys Thr Leu Ser Pro Gly Lys Asn
Gly Val Val Lys Asp Val 1170 1175 1180Phe Ala Phe Gly Gly Ala Val
Glu Asn Pro Glu Tyr Leu Thr Pro Gln1185 1190 1195 1200Gly Gly Ala
Ala Pro Gln Pro His Pro Pro Pro Ala Phe Ser Pro Ala 1205 1210
1215Phe Asp Asn Leu Tyr Tyr Trp Asp Gln Asp Pro Pro Glu Arg Gly Ala
1220 1225 1230Pro Pro Ser Thr Phe Lys Gly Thr Pro Thr Ala Glu Asn
Pro Glu Tyr 1235 1240 1245Leu Gly Leu Asp Val Pro Val 1250
1255109274DNAArtificial SequenceVector Sequence 10ttcgagctag
ccacgtgtaa ttaagtacta gcgctcgatc gtcgaacgta cggacgtcgt 60ctttaccaac
ttgagggctc tcacaggatg tggatcccct cttcgtcggt tccccaacaa
120agggtggttc ctgctgggca gacgcgtgtt tgcctactcg ggtagtctgt
ttctgtataa 180gtaagagacg acgtttgaac cgtatcgaga cgaaacgacc
ccgtaacccc cttcaacgcc 240aagcacgagc gtcccgagag tgggaactga
gaaaattatc gagaagacac gttctaatgt 300tagatttgtt aagcctcttg
agctggaagg agaggactcc gttcctggtg tcggttgaag 360gagaatgttc
ggcgtagcta aaacaggaag tctttatctt tattcttacg aacgattttt
420aatataaaaa tggttattct ggttaggtta tccatctaat aatcaatgat
acaattcttt 480acttagtaat agaaaatcat gataaaaatg agtttaagtc
ttcaatcttt acccttatct 540tttatctttc tctgcgagtt ggagttaact
tcttgtccac gttcctgata actggtgtcc 600ggatcttcat tttttccctt
ttttctcaca aaaacagttt tatcctctgt ccaccaccgt 660tggtccctga
atatcccctg gaatgtagat gtctggttgt ctacggggga atggtatatg
720tccttctata ctgaatttaa ccctatccac ccaatgtcag ttaccgatat
ttcacaatat 780atctagggag ggaaaagcac tttctgagcg gtctcgatct
ggaggaacca catacaacag 840agttcttctt tttctgctgt actttgttgt
ccatgtacta atataaatag atccttgtcc 900ttacgtgaaa acccctttct
aaaaggtatg gttcctcccc tgtcaccgac ctgattatct 960tgtaataaga
cgtttttgaa taccgtactc aataatactt atcggaaata accgggttgg
1020aacgccaagg gttccgaatt cattcaaaaa ccaatgtttg acaagaattt
tgctcctaca 1080ctctgttcac caaaggactg aaccaaacca tagtttccaa
gactagactc gagactcaca 1140agataaaagg atacaagaaa accttaaata
ggtttagaat acatttacga atacatttgg 1200ttctatattt tctcacgact
aaaaaactca tttgaacgtt gtcaggattg taagtggaga 1260acacacaaac
acagacaagc ggtagggcag aggcgagcag tgaataggaa gtgaaaggtc
1320tcccaggggg gcgtctgggg cctagcgatc gagcgcttag ctattcgaac
gccggcgaat 1380tgacgtcttc aaccagcact ccgtgacccg tccattcata
gttccaatgt tctgtccaaa 1440ttcctctggt tatctttgac ccgaacagct
ctgtctcttc tgagaacgca aagactatcc 1500gtggataacc agaatgactg
taggtgaaac ggaaagagag gtgtccacag gtgagggtcc 1560aagttaatgt
cgagaattcg ccggcgttcg aactatagct taaggacgtc gggcccccta
1620ggtgatcacc taggtttctt aagtttttcg aagagctccc gcgcgcgggc
cgggggtggg 1680gagcgtcgtg gggcgcgggg cgcgggaggg tcggcccagg
tcggcctcgg tacctcgacc 1740gccggaacac ggcgaccccc gaggaggagc
gggagaacgg ggggcctcgg cgctcgtggg 1800ttcacacgtg gccgtgtctg
tacttcgacg ccgagggacg gtcagggctc tgggtggacc 1860tgtacgaggc
ggtggagatg gtcccgacgg tccaccacgt ccctttggac cttgagtgga
1920tggacgggtg gttacggtcg gacaggaagg acgtcctata ggtcctccac
gtcccgatgc 1980acgagtagcg agtgttggtt cactccgtcc agggtgacgt
ctccgacgcc taacacgctc 2040cgtgggtcga gaaactcctg ttgatacggg
accggcacga tctgttacct ctgggcgact 2100tgttatggtg gggacagtgt
ccccggaggg gtcctccgga cgccctcgac gtcgaagctt 2160cggagtgtct
ctagaacttt cctccccaga actaggtcgc cttgggggtc gagacgatgg
2220tcctgtgcta aaacaccttc ctgtagaagg tgttcttgtt ggtcgaccga
gagtgtgact 2280atctgtggtt ggcgagagcc cggacggtgg ggacaagagg
ctacacattc ccgagggcga 2340cgacccctct ctcaagactc ctaacagtct
cggactgcgc gtgacagaca cggccaccga 2400cacgggcgac gttccccggt
gacgggtgac tgacgacggt actcgtcaca cgacggccga 2460cgtgcccggg
gttcgtgaga ctgacggacc ggacggaggt gaagttggtg tcaccgtaga
2520cactcgacgt gacgggtcgg gaccagtgga tgttgtgtct gtgcaaactc
aggtacgggt 2580tagggctccc ggccatatgt aagccgcggt cgacacactg
acggacaggg atgttgatgg 2640aaagatgcct gcaccctagg acgtgggagc
agacggggga cgtgttggtt ctccactgtc 2700gtctcctacc ttgtgtcgcc
acactcttca cgtcgttcgg gacacgggct cacacgatac 2760cagacccgta
cctcgtgaac gctctccact cccgtcaatg gtcacggtta taggtcctca
2820aacgaccgac gttcttctag aaaccctcgg accgtaaaga cggcctctcg
aaactacccc 2880tgggtcggag gttgtgacgg ggcgaggtcg gtctcgtcga
ggttcacaaa ctctgagacc 2940ttctctagtg tccaatggat atgtagagtc
gtaccggcct gtcggacgga ctggagtcgc 3000agaaggtctt ggacgttcat
taggcccctg cttaagacgt gttaccgcgg atgagcgact 3060gggacgttcc
cgacccgtag tcgaccgacc ccgacgcgag tgactccctt gacccgtcac
3120ctgaccggga gtaggtggta ttgtgggtgg agacgaagca cgtgtgccac
gggaccctgg 3180tcgagaaagc cttgggcgtg gttcgagacg aggtgtgacg
gttggccggt ctcctgctca 3240cacacccgct cccggaccgg acggtggtcg
acacgcgggc tcccgtgacg accccaggtc 3300ccgggtgggt cacacagttg
acgtcggtca aggaagcccc ggtcctcacg cacctcctta 3360cggctcatga
cgtccccgag gggtccctca tacacttacg gtccgtgaca aacggcacgg
3420tgggactcac agtcggggtc ttaccgagtc actggacaaa acctggcctc
cgactggtca 3480cacaccggac acgggtgata ttcctgggag ggaagacgca
ccgggcgacg gggtcgccac 3540actttggact ggagaggatg tacgggtaga
ccttcaaagg tctactcctc ccgcgtacgg 3600tcggaacggg gtagttgacg
tgggtgagga cacacctgga cctactgttc ccgacggggc 3660ggctcgtctc
tcggtcggga gactgcaggt agcagagacg ccaccaaccg taagacgacc
3720agcaccagaa cccccaccag aaaccctagg agtagttcgc tgccgtcgtc
ttctaggcct 3780tcatgtgcta cgcctctgac gacgtccttt gcctcgacca
cctcggcgac tgtggatcgc 3840ctcgctacgg gttggtccgc gtctacgcct
aggactttct ctgcctcgac tccttccact 3900tccacgaacc tagaccgcga
aaaccgtgtc agatgttccc gtagacctag ggactacccc 3960tcttacactt
ttaaggtcac cggtagtttc acaactccct tttgtgtagg gggtttcggt
4020tgtttcttta gaatctgctt cgtatgcact accgaccaca cccgaggggt
atacagaggg 4080cggaagaccc gtagacggac tgtaggtgcc acgtcgacca
ctgtgtcgaa tacgggatac 4140cgacggagaa tctggtacag gcccttttgg
cgcctgcgga cccgagggtc ctggacgact 4200tgaccacata cgtctaacgg
ttcccctact cgatggacct cctacacgcc gagcatgtgt 4260ccctgaaccg
gcgagccttg cacgaccagt tctcagggtt ggtacagttt taatgtctga
4320agcccgaccg agccgacgac ctgtaactgc tctgtctcat ggtacgtcta
cccccgttcc 4380acgggtagtt cacctaccgc gacctcaggt aagaggcggc
cgccaagtgg gtggtctcac 4440tacacacctc aataccacac tgacacaccc
tcgactactg aaaaccccgg tttggaatgc 4500taccctaggg tcgggccctc
tagggactgg acgacctttt ccccctcgcc gacggggtcg 4560gggggtagac
gtggtaacta cagatgtact agtaccagtt tacaacctac taactgagac
4620ttacagccgg ttctaaggcc ctcaaccaca gacttaagag ggcgtaccgg
tccctggggg 4680tcgcgaaaca ccagtaggtc ttactcctga acccgggtcg
gtcagggaac ctgtcgtgga 4740agatggcgag tgacgacctc ctgctactgt
accccctgga ccacctacga ctcctcatag 4800accatggggt cgtcccgaag
aagacaggtc tgggacgggg cccgcgaccc ccgtaccagg 4860tggtgtccgt
ggcgtcgagt
agatggtcct caccgccacc cctggactgt gatcccgacc 4920tcgggagact
tctcctccgg gggtccagag gtgaccgtgg gaggcttccc cgaccgaggc
4980tacataaact accactggac ccttaccccc gtcggttccc cgacgtttcg
gaggggtgtg 5040tactggggtc gggagatgtc gccatgtcac tcctggggtg
tcatggggac gggagactct 5100gactaccgat gcaacggggg gactggacgt
cgggggtcgg acttatacac ttggtcggtc 5160tacaagccgg ggtcggggga
agcggggctc tcccgggaga cggacgacgg gctggacgac 5220cacggtgaga
cctttcccgg ttctgagaga ggggtccctt cttaccccag cagtttctgc
5280aaaaacggaa acccccacgg cacctcttgg ggctcatgaa ctgtggggtc
cctcctcgac 5340ggggagtcgg ggtgggagga ggacggaagt cgggtcggaa
gctgttggag ataatgaccc 5400tggtcctggg tggtctcgcc ccccgaggtg
ggtcgtggaa gtttccctgt ggatgccgtc 5460tcttgggtct catggaccca
gacctgcacg gtcacacttg gtcttccggt tcaggcgtct 5520tcgggactac
acaggagtcc ctcgtccctt ccgccggaga ctcgataagg tcttcatcac
5580tcctccgaaa aaacctccgg atccgaaaac gtttttcgaa tagctatggc
agctgagctc 5640tcatgaagat ctcgccggcg cccgggtagc ggagactgtc
gttgcagata ctggaggatt 5700tcctggatct ccttccgtag gtttgcgact
acccctccga ccttctaccg tcgggggcct 5760gacccgtcta gaagttcgtc
tggatgtcgt tcaagctgtg tttgagtgtg ttgctactgc 5820gtgatgagtt
cttgatgccc gacgagatga cgaagtcctt cctgtacctg ttccagctct
5880gtaaggacgc gtagcacgtc acggcgagac acctcccgtc gacaccgaag
atcgacgggc 5940ccaccgtagg gacactgggg aggggtcacg gagaggaccg
ggaccttcaa cggtgaggtc 6000acgggtggtc ggaacaggat tattttaatt
caacgtagta aaacagactg atccacagga 6060agatattata ataccccacc
tccccccacc atacctcgtt ccccgggttc aacccttctg 6120ttggacatcc
cggacgcccc agataagccc ttggttcgac ctcacgtcac cgtgttagaa
6180ccgagtgacg ttagaggcgg aggacccaag ttcgctaaga ggacggagtc
ggagggctca 6240acaaccctaa ggtccgtacg tactggtccg agtcgattaa
aaacaaaaaa accatctctg 6300ccccaaagtg gtataaccgg tccgaccaga
ggttgaggat tagagtccac tagatgggtg 6360gaaccggagg gtttaacgac
cctaatgtcc gcacttggtg acgagggaag ggacaggaag 6420actaaaattt
tattgatatg gtcgtcctcc tgcaggtctg tgtcgtatcc gatggacggt
6480accgggttgg ccaccctgta aactcaacga acgaaccgtg acaggagagt
acgcaaccca 6540ggtgagtcat ctacggacaa cttaatgcta gccacgtgta
attaagtact ttaagcatta 6600gtaccagtat cgacaaagga cacactttaa
caataggcga gtgttaaggt gtgttgtatg 6660ctcggccttc gtatttcaca
tttcggaccc cacggattac tcactccatt gagtgtaatt 6720aacgcaacgc
gagtgacggg cgaaaggtca gccctttgga cagcacggtc gacctaatta
6780cttagccggt tgcgcgcccc tctccgccaa acgcataacc cgcgagaagg
cgaaggagcg 6840agtgactgag cgacgcgagc cagcaagccg acgccgctcg
ccatagtcga gtgagtttcc 6900gccattatgc caataggtgt cttagtcccc
tattgcgtcc tttcttgtac actcgttttc 6960cggtcgtttt ccggtccttg
gcatttttcc ggcgcaacga ccgcaaaaag gtatccgagg 7020cggggggact
gctcgtagtg tttttagctg cgagttcagt ctccaccgct ttgggctgtc
7080ctgatatttc tatggtccgc aaagggggac cttcgaggga gcacgcgaga
ggacaaggct 7140gggacggcga atggcctatg gacaggcgga aagagggaag
cccttcgcac cgcgaaagag 7200ttacgagtgc gacatccata gagtcaagcc
acatccagca agcgaggttc gacccgacac 7260acgtgcttgg ggggcaagtc
gggctggcga cgcggaatag gccattgata gcagaactca 7320ggttgggcca
ttctgtgctg aatagcggtg accgtcgtcg gtgaccattg tcctaatcgt
7380ctcgctccat acatccgcca cgatgtctca agaacttcac caccggattg
atgccgatgt 7440gatcttcctg tcataaacca tagacgcgag acgacttcgg
tcaatggaag cctttttctc 7500aaccatcgag aactaggccg tttgtttggt
ggcgaccatc gccaccaaaa aaacaaacgt 7560tcgtcgtcta atgcgcgtct
ttttttccta gagttcttct aggaaactag aaaagatgcc 7620ccagactgcg
agtcaccttg cttttgagtg caattcccta aaaccagtac tctaatagtt
7680tttcctagaa gtggatctag gaaaatttaa tttttacttc aaaatttagt
tagatttcat 7740atatactcat ttgaaccaga ctgtcaatgg ttacgaatta
gtcactccgt ggatagagtc 7800gctagacaga taaagcaagt aggtatcaac
ggactgaggg gcagcacatc tattgatgct 7860atgccctccc gaatggtaga
ccggggtcac gacgttacta tggcgctctg ggtgcgagtg 7920gccgaggtct
aaatagtcgt tatttggtcg gtcggccttc ccggctcgcg tcttcaccag
7980gacgttgaaa taggcggagg taggtcagat aattaacaac ggcccttcga
tctcattcat 8040caagcggtca attatcaaac gcgttgcaac aacggtaacg
acgaccgtag caccacagtg 8100cgagcagcaa accataccga agtaagtcga
ggccaagggt tgctagttcc gctcaatgta 8160ctagggggta caacacgttt
tttcgccaat cgaggaagcc aggaggctag caacagtctt 8220cattcaaccg
gcgtcacaat agtgagtacc aataccgtcg tgacgtatta agagaatgac
8280agtacggtag gcattctacg aaaagacact gaccactcat gagttggttc
agtaagactc 8340ttatcacata cgccgctggc tcaacgagaa cgggccgcag
tagtgcccta ttatggcgcg 8400gtgtatcgtc ttgaaatttt cacgagtagt
aaccttttgc aagaagcccc gcttttgaga 8460gttcctagaa tggcgacaac
tctaggtcaa gctacattgg gtgagcacgt gggttgacta 8520gaagtcgtag
aaaatgaaag tggtcgcaaa gacccactcg tttttgtcct tccgttttac
8580ggcgtttttt cccttattcc cgctgtgcct ttacaactta tgagtatgag
aaggaaaaag 8640ttataataac ttcgtaaata gtcccaataa cagagtactc
gcctatgtat aaacttacat 8700aaatcttttt atttgtttat ccccaaggcg
cgtgtaaagg ggcttttcac ggtggactgc 8760agattctttg gtaataatag
tactgtaatt ggatattttt atccgcatag tgctccggga 8820aagcagaagt
tcttatgacg gagcgcgcaa agccactact gccacttttg gagactgtgt
8880acgtcgaggg cctctgccag tgtcgaacag acattcgcct acggccctcg
tctgttcggg 8940cagtcccgcg cagtcgccca caaccgccca cagccccgcg
tcggtactgg gtcagtgcat 9000cgctatcgcc tcaaccgaat tgatacgccg
tagtctcgtc taacatgact ctcacgtggt 9060atacgccaca ctttatggcg
tgtctacgca ttcctctttt atggcgtagt ccgcggtaag 9120cggtaagtcc
gatgcgttga caacccttcc cgctagccac gcccggagaa gcgataatgc
9180ggtcgaccgc ttccccccta cacgacgttc cgctaattca acccattgcg
gtcccaaaag 9240ggtcagtgct gcaacatttt gctgccggtc acgg
92741116PRThuman 11Cys Val Asp Leu Asp Asp Lys Gly Cys Pro Ala Glu
Gln Arg Ala Ser1 5 10 15
* * * * *