U.S. patent application number 13/431441 was filed with the patent office on 2013-08-01 for anti-erbb2 antibodies.
The applicant listed for this patent is Brian M. Fendly, Gail Dianne Phillips. Invention is credited to Brian M. Fendly, Gail Dianne Phillips.
Application Number | 20130195845 13/431441 |
Document ID | / |
Family ID | 39361578 |
Filed Date | 2013-08-01 |
United States Patent
Application |
20130195845 |
Kind Code |
A1 |
Fendly; Brian M. ; et
al. |
August 1, 2013 |
ANTI-ERBB2 ANTIBODIES
Abstract
Anti-ErbB2 antibodies are described which bind to an epitope in
Domain 1 of ErbB2 and induce cell death via apoptosis. Various uses
for these antibodies are also described.
Inventors: |
Fendly; Brian M.; (Half Moon
Bay, CA) ; Phillips; Gail Dianne; (San Carlos,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Fendly; Brian M.
Phillips; Gail Dianne |
Half Moon Bay
San Carlos |
CA
CA |
US
US |
|
|
Family ID: |
39361578 |
Appl. No.: |
13/431441 |
Filed: |
March 27, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12910590 |
Oct 22, 2010 |
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13431441 |
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11981602 |
Oct 31, 2007 |
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12910590 |
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08948149 |
Oct 9, 1997 |
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11981602 |
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60028811 |
Oct 18, 1996 |
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Current U.S.
Class: |
424/133.1 ;
424/139.1 |
Current CPC
Class: |
A61K 2039/505 20130101;
A61P 35/00 20180101; C07K 2317/73 20130101; C07K 16/32 20130101;
G01N 33/573 20130101; G01N 2333/912 20130101; C07K 2317/14
20130101; C07K 17/00 20130101; A61K 2039/507 20130101; C07K 2317/92
20130101; B65D 25/205 20130101; C07K 16/2863 20130101; C07K 16/3015
20130101; G01N 2333/71 20130101; G01N 33/5748 20130101; C07K
2317/732 20130101; A61K 39/3955 20130101; A61K 45/06 20130101; A61J
1/00 20130101; C07K 2317/567 20130101; C07K 16/3069 20130101; C07K
2317/565 20130101 |
Class at
Publication: |
424/133.1 ;
424/139.1 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Claims
1-41. (canceled)
42. A method of treating cancer comprising administering to a human
subject with cancer characterized by overexpression of the ErbB2
receptor a first antibody that binds Domain 1 (SEQ ID NO: 1) of
ErbB2 and thereby induces cell death in cancer cells overexpressing
ErbB2, or an antigen-binding fragment of said first antibody, and a
second antibody that binds ErbB2 at a different domain than Domain
1 and inhibits growth of cancer cells overexpressing ErbB2, or an
antigen-binding fragment of said second antibody.
43. The method of claim 42, wherein the first antibody binds
epitope 7C2/7F3 (SEQ ID NO: 2) of ErbB2.
44. The method of claim 42, wherein the first antibody comprises
CDRs of antibody 7C2 or 7F3.
45. The method of claim 42, wherein the first antibody induces
apoptosis in said cells.
46. The method of claim 42 wherein the second antibody comprises
CDRs of antibody 4D5.
47. The method of claim 42, wherein the first antibody comprises
CDRs of antibody 7C2 or 7F3 and the second antibody comprises CDRs
of antibody 4D5.
48. The method of claim 42, wherein the first and the second
antibodies are chimeric, humanized or human.
49. The method of claim 42, wherein the cancer is breast
cancer.
50. The method of claim 49, wherein administration is performed
intravenously as a bolus or by continuous infusion over a period of
time, by intramuscular, intraperitoneal, intracerebrospinal,
subcutaneous, intra-articular, intrasynovial, intrathecal, oral,
topical, or inhalation routes.
51. The method of claim 50 wherein administration is performed
intravenously.
Description
BACKGROUND OF THE INVENTION
[0001] This is a non-provisional application filed under 37 CFR
1.53(b)(1), claiming priority under USC Section 119(e) to
provisional Application Ser. No. 60/028,811, filed on 18 Oct.,
1996.
[0002] 1. Field of the Invention
[0003] This invention relates generally to antibodies which bind
the ErbB2 receptor. In particular, it pertains to anti-ErbB2
antibodies which bind to an epitope in Domain 1 of ErbB2 and induce
cell death via apoptosis.
[0004] 2. Description of Related Art
[0005] Transduction of signals that regulate cell growth and
differentiation is modulated in part by phosphorylation of various
cellular proteins. Protein tyrosine kinases are enzymes that are
involved in this process. Receptor protein tyrosine kinases are
believed to direct cellular growth via ligand-stimulated tyrosine
phosphorylation of intracellular substrates. The class I subfamily
of growth factor receptor protein tyrosine kinases includes the 170
kDa epidermal growth factor receptor (EGFR) encoded by the erbB1
gene. erbB1 has been causally implicated in human malignancy. In
particular, increased expression of this gene has been observed in
carcinomas of the breast, bladder, lung, head, neck and stomach.
Monoclonal antibodies directed against the EGFR have been evaluated
as therapeutic agents in the treatment of such malignancies. For a
review, see Baselga et al. Pharmac. Ther. 64:127-154 (1994). See
also Masui et al. Cancer Research 44:1002-1007 (1984).
[0006] Wu et al. J. Clin. Invest. 95:1897-1905 (1995) recently
report that the anti-EGFR monoclonal antibody (mAb) 225 (which
competitively inhibits EGF binding and blocks activation of this
receptor) could induce the human colorectal carcinoma cell line
DiFi (which expresses high levels of EGFR) to undergo G.sub.1 cell
cycle arrest and programmed cell death (apoptosis). Addition of
IGF-1 or high concentrations of insulin could delay apoptosis
induced by mAb 225, whereas G.sub.1 arrest could not be reversed by
addition of IGF-1 or insulin.
[0007] The second member of the class I subfamily, 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 protooncogene results from a point mutation (valine to
glutamic acid) in the transmembrane region of the encoded protein.
Amplification of the human homolog of neu (called erbB2 or HER2) is
observed in breast and ovarian cancers and correlates with a poor
prognosis (Slamon et al., Science, 235:177-182 (1987); and Slamon
et al., Science, 244:707-712 (1989)). Accordingly, Slamon et al. in
U.S. Pat. No. 4,968,603 describe various diagnostic assays for
determining erbB2 gene amplification or expression in tumor cells.
To date, no point mutation analogous to that in the neu
protooncogene has been reported for human tumors. Overexpression
(frequently but not uniformly due to amplification) of erbB2 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); Kem et al., Cancer Res., 50:5184 (1990); Park et
al., Cancer Res., 49:6605 (1989); Zhau et al., Mol. Carcinog.,
3:354-357 (1990); Aasland et al. Br. J. Cancer 57:358-363 (1988);
Williams et al. Pathiobiology 59:46-52 (1991); and McCann et al.,
Cancer, 65:88-92 (1990).
[0008] Antibodies directed against the rat neu and human erbB2
protein products have been described. Drebin et al., Cell
41:695-706 (1985) refer to an IgG2a monoclonal antibody which is
directed against the rat neu gene product. This antibody called
7.16.4 causes down-modulation of cell surface p185 expression on
B104-1-1 cells (NIH-3T3 cells transfected with the neu
protooncogene) and inhibits colony formation of these cells. Drebin
et al. say at page 699 that the antibody exerts a cytostatic effect
rather than an irreversible cytotoxic effect on neu-transformed
cells in soft agar. In Drebin et al. PNAS (USA) 83:9129-9133
(1986), the 7.16.4 antibody was shown to inhibit the tumorigenic
growth of neu-transformed NIH-3T3 cells as well as rat
neuroblastoma cells (from which the neu oncogene was initially
isolated) implanted into nude mice. Drebin et al. in Oncogene
2:387-394 (1988) discuss the production of a panel of antibodies
against the rat neu gene product. All of the antibodies were found
to exert a cytostatic effect on the growth of neu-transformed cells
suspended in soft agar. Antibodies of the IgM, IgG2a and IgG2b
isotypes were able to mediate significant in vitro lysis of
neu-transformed cells in the presence of complement, whereas none
of the antibodies were able to mediate high levels of
antibody-dependent cellular cytotoxicity (ADCC) of the
neu-transformed cells. Drebin et al. Oncogene 2:273-277 (1988)
report that mixtures of antibodies reactive with two distinct
regions on the p185 molecule result in synergistic anti-tumor
effects on neu-transformed NIH-3T3 cells implanted into nude mice.
Biological effects of anti-neu antibodies are reviewed in Myers et
al., Meth. Enzym. 198:277-290 (1991). See also WO94/22478 published
Oct. 13, 1994.
[0009] 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 SKBR3.
Relative cell proliferation of the SKBR3 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, including 7C2
and 7F3, reduced cellular proliferation to a lesser extent in this
assay. Hudziak et al. conclude that the effect of the 4D5 antibody
on SKBR3 cells was cytostatic rather than cytotoxic, since SKBR3
cells resumed growth at a nearly normal rate following removal of
the antibody from the medium. The antibody 4D5 was further found to
sensitize p185.sup.erB2-overexpressing breast tumor cell lines to
the cytotoxic effects of TNF-.alpha.. See also WO89/06692 published
Jul. 27, 1989. 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); and D'souza et al. Proc. Natl.
Acad. Sci. 91:7202-7206 (1994).
[0010] Tagliabue et al. Int. J. Cancer 47:933-937 (1991) describe
two antibodies which were selected for their reactivity on the lung
adenocarcinoma cell line (Calu-3) which overexpresses ErbB2. One of
the antibodies, called MGR3, was found to internalize, induce
phosphorylation of ErbB2, and inhibit tumor cell growth in
vitro.
[0011] McKenzie et al. Oncogene 4:543-548 (1989) generated a panel
of anti-ErbB2 antibodies with varying epitope specificities,
including the antibody designated TA1. This TA1 antibody was found
to induce accelerated endocytosis of ErbB2 (see Maier et al. Cancer
Res. 51:5361-5369 (1991)). Bacus et al. Molecular Carcinogenesis
3:350-362 (1990) reported that the TA1 antibody induced maturation
of the breast cancer cell lines AU-565 (which overexpresses the
erbB2 gene) and MCF-7 (which does not). Inhibition of growth and
acquisition of a mature phenotype in these cells was found to be
associated with reduced levels of ErbB2 receptor at the cell
surface and transient increased levels in the cytoplasm.
[0012] Stancovski et al. PNAS (USA) 88:8691-8695 (1991) generated a
panel of anti-ErbB2 antibodies, injected them i.p. into nude mice
and evaluated their effect on tumor growth of murine fibroblasts
transformed by overexpression of the erbB2 gene. Various levels of
tumor inhibition were detected for four of the antibodies, but one
of the antibodies (N28) consistently stimulated tumor growth.
Monoclonal antibody N28 induced significant phosphorylation of the
ErbB2 receptor, whereas the other four antibodies generally
displayed low or no phosphorylation-inducing activity. The effect
of the anti-ErbB2 antibodies on proliferation of SKBR3 cells was
also assessed. In this SKBR3 cell proliferation assay, two of the
antibodies (N12 and N29) caused a reduction in cell proliferation
relative to control. The ability of the various antibodies to
induce cell lysis in vitro via complement-dependent cytotoxicity
(CDC) and antibody-mediated cell-dependent cytotoxicity (ADCC) was
assessed, with the authors of this paper concluding that the
inhibitory function of the antibodies was not attributed
significantly to CDC or ADCC.
[0013] Bacus et al. Cancer Research 52:2580-2589 (1992) further
characterized the antibodies described in Bacus et al. (1990) and
Stancovski et al. of the preceding paragraphs. Extending the i.p.
studies of Stancovski et al., the effect of the antibodies after
i.v. injection into nude mice harboring mouse fibroblasts
overexpressing human ErbB2 was assessed. As observed in their
earlier work, N28 accelerated tumor growth whereas N12 and N29
significantly inhibited growth of the ErbB2-expressing cells.
Partial tumor inhibition was also observed with the N24 antibody.
Bacus et al. also tested the ability of the antibodies to promote a
mature phenotype in the human breast cancer cell lines AU-565 and
MDA-MB453 (which overexpress ErbB2) as well as MCF-7 (containing
low levels of the receptor). Bacus et al. saw a correlation between
tumor inhibition in vivo and cellular differentiation; the
tumor-stimulatory antibody N28 had no effect on differentiation,
and the tumor inhibitory action of the N12, N29 and N24 antibodies
correlated with the extent of differentiation they induced.
[0014] Xu et al. Int. J. Cancer 53:401-408 (1993) evaluated a panel
of anti-ErbB2 antibodies for their epitope binding specificities,
as well as their ability to inhibit anchorage-independent and
anchorage-dependent growth of SKBR3 cells (by individual antibodies
and in combinations), modulate cell-surface ErbB2, and inhibit
ligand stimulated anchorage-independent growth. See also WO94/00136
published Jan. 6, 1994 and Kasprzyk et al. Cancer Research
52:2771-2776 (1992) concerning anti-ErbB2 antibody combinations.
Other anti-ErbB2 antibodies are discussed in 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); and
Harwerth et al. J. Biol. Chem. 267:15160-15167 (1992).
[0015] A further gene related to erbB2, called erbB3 or HER3, has
also been described. See, e.g., U.S. Pat. Nos. 5,183,884 and
5,480,968. ErbB3 is unique among the ErbB receptor family in that
it possesses little or no intrinsic tyrosine kinase activity.
However, when ErbB3 is co-expressed with ErbB2, an active signaling
complex is formed and antibodies directed against ErbB2 are capable
of disrupting this complex (Sliwkowski et al., J. Biol. Chem.,
269(20):14661-14665 (1994)). Additionally, the affinity of ErbB3
for heregulin (HRG) is increased to a higher affinity state when
co-expressed with ErbB2. See also, Levi et al., Journal of
Neuroscience 15: 1329-1340 (1995); Morrissey et al., Proc. Natl.
Acad. Sci. USA 92: 1431-1435 (1995); and Lewis et al., Cancer Res.,
56:1457-1465 (1996) with respect to the ErbB2-ErbB3 protein
complex.
[0016] The class I subfamily of growth factor receptor protein
tyrosine kinases has been further extended to include the
HER4/p180.sup.erB4 receptor. See 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). Plowman et al. found
that increased HER4 expression correlated with certain carcinomas
of epithelial origin, including breast adenocarcinomas. This
receptor, like ErbB3, forms an active signalling complex with ErbB2
(Carraway and Cantley, Cell 78:5-8 (1994)).
[0017] The quest for an ErbB2 activator has lead to the discovery
of a family of heregulin polypeptides. These proteins appear to
result from alternative splicing of a single gene and are called
neuregulins (NRGs), neu differentiation factors (NDFs), heregulins
(HRGs), glial growth factors (GGFs) and acetylcholine receptor
inducing activity (ARIA) in the literature. For a review, see
Groenen et al. Growth Factors 11:235-257 (1994); Lemke, G. Molec.
& Cell. Neurosci. 7:247-262 (1996) and Lee et al. Pharm. Rev.
47:51-85 (1995).
SUMMARY OF THE INVENTION
[0018] This invention relates, at least in part, to the surprising
discovery that certain anti-ErbB2 antibodies can induce death of an
ErbB2 overexpressing cell (e.g. a BT474, SKBR3, SKOV3 or Calu 3
cell) via apoptosis. In contrast to the apoptotic anti-EGFR
antibody described in Wu et al., J. Clin. Invest. 95:1897-1905
(1995), the anti-ErbB2 antibodies of interest herein are not
thought to induce apoptosis by disruption of an autocrine loop.
Antibodies herein with these cell death-inducing attributes will
normally bind to a region in the extracellular domain of ErbB2,
e.g. to an epitope in Domain 1 of ErbB2. Preferably, the antibodies
will bind to the ErbB2 epitope bound by the 7C2 and/or 7F3
antibodies described herein.
[0019] Preferred antibodies are monoclonal antibodies e.g.
humanized antibodies. Antibodies of particular interest are those
which, in addition to the above-described properties, bind the
ErbB2 receptor with an affinity of at least about 10 nM, more
preferably at least about 1 nM.
[0020] In certain embodiments, the antibody is immobilized on (e.g.
covalently attached to) a solid phase, e.g. for affinity
purification of the receptor or for diagnostic assays. For
diagnostic uses, it may also be beneficial to provide a labelled
antibody (i.e. the antibody bound to a detectable label).
[0021] The antibodies of the preceding paragraphs may be provided
in the form of a composition comprising the antibody and a
pharmaceutically acceptable carrier or diluent. Optionally, the
composition further comprises a second anti-ErbB2 antibody,
especially where the second anti-ErbB2 antibody is one which binds
to an epitope on the ErbB2 receptor which differs from that to
which the 7C2/7F3 antibodies disclosed herein bind. In a preferred
embodiment, the second antibody is one which inhibits growth of
SKBR3 cells in cell culture by 50%-100% (i.e. 4D5 antibody and
functional equivalents thereof). The composition for therapeutic
use will be sterile and may be lyophilized.
[0022] The invention also provides: an isolated nucleic acid
molecule encoding the antibody of the preceding paragraphs which
may further comprise a promoter operably linked thereto; an
expression vector comprising the nucleic acid molecule operably
linked to control sequences recognized by a host cell transformed
with the vector; a host cell comprising the nucleic acid (e.g. a
hybridoma cell line); and a process for making the antibody
comprising culturing a cell comprising the nucleic acid so as to
express the anti-ErbB2 antibody and, optionally, recovering the
antibody from the host cell culture and, preferably, the host cell
culture medium.
[0023] The invention also provides methods for using the anti-ErbB2
antibodies disclosed herein. For example, the invention provides a
method for inducing cell death comprising exposing a cell, such as
a cancer cell which overexpresses ErbB2, to anti-ErbB2 antibody
described herein in an amount effective to induce cell death. The
cell may be in cell culture or in a mammal, e.g. a mammal suffering
from cancer. The invention also provides a method for inducing
apoptosis of a cell which overexpresses ErbB2 comprising exposing
the cell to exogenous anti-ErbB2 antibody as described herein in an
amount effective to induce apoptosis of the cell. Thus, the
invention provides a method for treating a mammal suffering from a
condition characterized by overexpression of the ErbB2 receptor,
comprising administering a pharmaceutically effective amount of the
anti-ErbB2 antibodies disclosed herein to the mammal. According to
any of the above methods, a further anti-ErbB2 antibody may be
used, especially one which binds to a different ErbB2 epitope from
that to which the 7C2/7F3 antibodies disclosed herein bind (e.g.
one that does not bind Domain 1). In one embodiment, the second
antibody inhibits growth of SKBR3 cells in cell culture by 50%-100%
and optionally binds to the epitope on ErbB2 to which 4D5
binds.
[0024] In Example 2 below, it was found that the pro-apoptotic
antibody 7C2 almost completely eradicated the entire culture of
growth arrested cells. Therefore, it may be desirable to combine
the pro-apoptotic antibodies disclosed herein with a growth
inhibitory agent in the in vitro and in vivo methods discussed
above. In such embodiments, superior levels of apoptosis may be
achieved by administering the growth inhibitory agent prior to the
pro-apoptotic anti-ErbB2 antibody. However, simultaneous
administration or administration of the anti-ErbB2 antibody first
is also contemplated.
[0025] The invention also provides an article of manufacture for
use in the above in vivo methods which comprises a container
holding the anti-ErbB2 antibody and a label on or associated with
the container which indicates that the antibody can be used to
treat conditions characterized by ErbB2 overexpression, such as
cancer.
[0026] In a further aspect, the invention provides a method for
detecting ErbB2 in vitro or in vivo comprising contacting the
antibody with a cell suspected of containing ErbB2 and detecting if
binding has occurred. Accordingly, the invention provides an assay
for detecting a tumor characterized by amplified expression of
ErbB2 comprising the steps of exposing a cell to the antibody
disclosed herein and determining the extent of binding of the
antibody to the cell. Preferably the antibody for use in such an
assay will be labelled and will be supplied in the form of a kit
with instructions for using the antibody to detect ErbB2. The assay
herein may be an in vitro assay (such as an ELISA assay) or an in
vivo assay. For in vivo tumor diagnosis, the antibody is preferably
conjugated to a radioactive isotope and administered to a mammal,
and the extent of binding of the antibody to tissues in the mammal
is observed by external scanning for radioactivity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIGS. 1A & B show the effects of apoptosis on a cell and
a method for determining apoptosis, respectively. FIG. 1A shows the
physiological changes which occur to a cell which undergoes
programmed cell death (apoptosis). FIG. 1B shows translocation of
phosphatidyl serine (PS) from the inner leaflet of the plasma
membrane to the exterior of the cell. This PS translocation process
is specific for cells undergoing apoptosis. Annexin V binds
specifically to PS and thus provides a means for determining
apoptosis.
[0028] FIG. 2 shows the epitope specificity of anti-ErbB2
antibodies. MAbs were used to block the binding of 7C2 to BT474
cells. BT474 cells (0.5.times.10.sup.6) pretreated or not with 50
.mu.g anti-ErbB2 antibodies (15 minutes on ice) were washed twice,
resuspended in 0.1 ml 1% FBS/PBS and incubated with 5 .mu.g of
fluoresceinisothiocyanate (FITC) conjugated 7C2 antibody (15
minutes on ice). Following incubation, the cell suspensions were
washed twice with 1% FBS/PBS to remove unbound fluorochrome, fixed
with 1% paraformaldehyde/PBS and analyzed by flow cytometry.
[0029] FIGS. 3A-D depict the effect of anti-ErbB2 antibodies on
human breast cancer cells which overexpress ErbB2. Cells with
normal membrane functions at the time of harvest ("viable" cells)
exclude the DNA dye 7AAD and after membrane permeabilization are
stained preferentially with Hoechst. Analysis of these cells
reveals a classic DNA profile (far right panels) with cells in the
G.sub.0/G.sub.1 phase (major peak) and S-G.sub.2-M phase (indicated
with double headed arrow) of the cell cycle. In the center panels,
the population of cells indicated by the arrow represent dead
cells, some of which had abnormal permeability characteristics at
the time of harvest and had degraded their nuclear DNA.
4.times.10.sup.4 BT474 cells/ml were incubated for 72 hours in
medium containing an isotype-matched control Ig (FIG. 3A), 1 .mu.g
of monoclonal 4D5 (FIG. 3B), 50 .mu.g of monoclonal 7C2 (FIG. 3C),
or 1 .mu.g 4D5 antibody+50 .mu.g 7C2 antibody (FIG. 3D). The
percentage of dead cells is indicated in the middle panels (7AAD
fluorescence vs. Hoechst fluorescence) and the percentage of viable
cells in the combined S, G.sub.2, and M phases of the cell cycle is
indicated in the right panels (Hoechst fluorescence vs. cell
count). The lefthand panel shows the size of the cells as
determined by forward and right angle light scatter.
[0030] FIGS. 4A & B reveal the additive effect of 4D5 and 7C2
antibodies on DNA synthesis, and viability in BT474 cells. The
average of two experiments.+-.S.D. is shown. In FIG. 4A,
8.times.10.sup.3 cells/0.2 ml/well were treated with 4D5 (0.05
.mu.g/ml) and/or 7C2 (50 .mu.g/ml) for 72 hours and pulsed for the
last 12 hours with 1 .mu.Ci [.sup.3H]-thymidine (in triplicate). In
FIG. 4B, for cell viability, a total cell count was obtained and
viability was determined by FACS analysis. The standard deviation
for both 7C2 and 7C2 plus 4D5 treatment is too small
(1.times.10.sup.3 cells) to be seen in the figure.
[0031] FIGS. 5A-F show induction of apoptosis by anti-ErbB2 MAbs
7C2 and 4D5 in BT474 breast tumor cells. FIGS. 5A, 5C and 5E show
plots of forward scatter (FS), an indicator of cell size, vs. log
FITC (representing annexin V binding). FIGS. 5B, D and F are
quadrant plots of log FITC vs. log propidium iodine (PI), with
percent annexin V-positive cells shown in quadrant 4 and percent
annexin V/PI positive cells in quadrant 2. Untreated cells display
a uniform size and fluorescence signal, as seen within the drawn
circle, and are 85% viable (FIG. 5A). In addition to displaying low
annexin V-binding, these cells do not take up PI, indicating no
change in membrane integrity. Treatment for 3 days with 10 .mu.g/ml
MAb 7C2 results in a reduction in the percent of viable cells (to
25.3%, FIG. 5E) and a shift of almost the entire population to a
smaller, FITC-positive population of cells (FIG. 5E). As shown in
FIG. 5F, MAb 7C2 induces a 7-8 fold increase in the percent of
annexin V-positive/PI-positive cells, indicating apoptotic cell
death. The anti-proliferative MAb, 4D5, induces a small degree of
apoptosis (2.5 fold above control, FIGS. 5C and D).
[0032] FIG. 6 shows that the effects of MAb 7C2 are dose-dependent.
The induction of apoptosis in BT474 breast tumor cells by MAb 7C2,
as measured by an increase in the number of annexin V-positive and
PI-positive cells, is apparent at a concentration of 0.1 .mu.g/ml
and reaches a maximum at 1 .mu.g/ml.
[0033] FIGS. 7A and 7B are time-courses of MAb 7C2-induced
apoptosis in BT474 and SKBR3 breast tumor cells, respectively.
Treatment of BT474 cells (FIG. 7A) and SKBR3 cells (FIG. 78) with
10 .mu.g/ml MAb 7C2 results in a reduction in the percent of viable
cells (those cells which are annexin V- and PI-negative) as early
as 15 minutes after initiation of treatment and reaches a maximum
at 24 hours. The BT474 cell line is more sensitive to the
pro-apoptotic effect of MAb 7C2 compared to the SKBR3 cells.
[0034] FIGS. 8A-E show responses of different cell lines to
anti-ErbB2 MAbs. The BT474, SKBR3 and MCF7 breast tumor cell lines,
and normal human mammary epithelial cells (HMEC) (FIGS. 8A-D,
respectively) were incubated with the anti-ErbB2 MAbs 4D5, 3H4,
7F3, 7C2, 2H11, 3E8, and 7D3; the humanized version of muMAb 4D5
(hu4D5); or the isotype-matched irrelevant control MAb 1766.
Treatment was for 3 days at a MAb concentration of 10 .mu.g/ml. The
data are pooled from 2-9 separate experiments and are represented
as mean fold increase (+/-s.e.) in annexin V binding over control
cells. The response of the BT474 breast tumor cells to MAbs 7C2 and
4D5 is as described for FIG. 5 (9-fold and 2.5-fold above control,
respectively). Induction of apoptosis in the SKBR3 breast tumor
cell line, which expresses high levels of ErbB2 similar to the
BT474 cells, also occurs after treatment with MAb 7C2 (and to a
smaller degree, MAb 4D5). In addition, MAb 7F3 induces apoptosis in
both the BT474 and SKBR3 cell lines. The MCF7 breast tumor line,
which expresses normal ErbB2 levels, and the HMEC's showed no
change in annexin V binding after treatment with the anti-ErbB2
Mabs. These results suggest that overexpression of ErbB2 is
required for responsiveness to the anti-ErbB2 MAbs. In FIG. 8E, a
non-small cell lung carcinoma line overexpressing ErbB2, Calu 3,
was tested for induction of apoptosis by anti-ErbB2 MAbs. Treatment
with 7C2 or 7F3 resulted in enhanced binding of annexin V.
[0035] FIGS. 9A-I show the effects of MAbs 7C2 and 4D5 on cell
cycle progression and cell death. Untreated BT474 cells are largely
annexin V-negative and PI-negative (FIG. 9A, quadrant 3), and show
a normal cell cycle DNA histogram (FIGS. 9B &C). Cells treated
with 10 .mu.g/ml MAb 4D5 show some increase in uptake of PI and
annexin V-FITC binding (FIG. 9D, quadrant 2). The most pronounced
effect is on cell cycle progression, where MAb 4D5 almost
completely reduces the percent of cells in S phase (FIGS. 9E
&F). MAb 7C2 induces a significant amount of cell death in
BT474, as measured by PI uptake and annexin V-FITC binding (FIG.
9G, quadrant 2). Cell cycle analysis shows the presence of a
sub-G.sub.0/G.sub.1 or hypodiploid population (FIG. 9I),
characteristic of apoptotic cells, with the cells displaying high
levels of annexin V binding (FIG. 9H, quadrant 1).
[0036] FIGS. 10A-F are the results from curve-fitting analyses of
the DNA histograms of FIGS. 9A-I and show little change in the
percent of cells in S-phase (52%) after MAb 7C2 treatment compared
to control cells (61% S-phase cells), but a dramatic reduction in
the number of cells in S-phase (to 6%) in response to MAb 4D5
(FIGS. 10C, A and B, respectively). Analyses of the apoptotic
population of cells (annexin V/PI positive cells from quadrant 2,
9A, D and G) reveal no difference in the percent S-phase cells
compared to the total cell population (FIG. 10D control=55%; FIG.
10E MAb 4D5=7%; FIG. 10F MAb 7C2=56%). Furthermore, the
G.sub.d/G.sub.1 and G.sub.2/M phases show no change (compare FIGS.
10A and D, B and E, C and F), indicating that cells are exiting the
cell cycle at all phases and that MAb-induced apoptotic cell death
is not cell cycle specific.
[0037] FIGS. 11A & B show MAb 7C2-induced apoptosis is enhanced
by growth arrest. In addition to the data from cell cycle studies,
it was observed, from video time-lapse recording, that a proportion
of MAb 7C2-treated cells continue to proliferate while others
undergo apoptosis. Therefore, experiments were performed to
determine if inhibition of cell growth would enhance the
pro-apoptotic activity of MAb 7C2. BT474 cells were serum-deprived
for 3 days to induce growth arrest, then treated with 10 .mu.g/ml
MAb 7C2 or 4D5 for 3 days and analyzed for viability (annexin
V-FITC binding and PI uptake) as well as cell cycle effects.
Serum-deprivation (by incubation in media supplemented with 0.1%
FBS) effectively reduces proliferation, as seen by a decrease in
the percent of S-phase cells from 33% (in 10% FBS) to 10% (FIG.
11A). The potent anti-proliferative activity of MAb 4D5 is not
further enhanced by prior growth arrest. The proportion of cells in
S-phase, with and without serum-deprivation, in MAb 7C2-treated
cells was similar to controls. FIG. 11B shows that serum-starvation
does not adversely affect cell viability of untreated cells.
However, viability is reduced to 55% in BT474 cells treated with 10
.mu.g/ml MAb 4D5 after a period of serum-deprivation. Moreover,
treatment of growth-arrested cells with 10 .mu.g/ml MAb 7C2 almost
completely eradicates the entire culture, in that the percent of
annexin V-negative and PI-negative cells is reduced to 10%.
[0038] FIG. 12 depicts with underlining the amino acid sequence of
Domain 1 of ErbB2 (SEQ ID NO:1). Bold amino acids indicate the
location of the epitope recognized by MAbs 7C2 and 7F3 as
determined by deletion mapping, i.e. the "7C2/7F3 epitope" (SEQ ID
NO:2).
[0039] FIG. 13 shows epitope-mapping of the extracellular domain of
ErbB2 as determined by truncation mutant analysis and site-directed
mutagenesis (Nakamura et al. J. of Virology 67(10):6179-6191
(October 1993); Renz et al. J. Cell Biol. 125(6):1395-1406 (June
1994)). Pro-apoptotic MAbs 7C2 and 7F3 bind an epitope at the
N-terminus of the receptor, whereas anti-proliferative MAbs 4D5 and
3H4 bind adjacent to the transmembrane domain. The various
ErbB2-ECD truncations or point mutations were prepared from cDNA
using polymerase chain reaction technology. The ErbB2 mutants were
expressed as gD fusion proteins in a mammalian expression plasmid.
This expression plasmid uses the cytomegalovirus promoter/enhancer
with SV40 termination and polyadenylation signals located
downstream of the inserted cDNA. Plasmid DNA was transfected into
293S cells. One day following transfection, the cells were
metabolically labeled overnight in methionine and cysteine-free,
low glucose DMEM containing 1% dialyzed fetal bovine serum and 25
.mu.Ci each of .sup.35S methionine and .sup.5S cysteine.
Supernatants were harvested either the ErbB2 MAbs or control
antibodies were added to the supernatant and incubated 2-4 hours at
4.degree. C. The complexes were precipitated, applied to a 10-20%
Tricine SDS gradient gel and electrophoresed at 100 V. The gel was
electroblotted onto a membrane and analyzed by autoradiography.
[0040] FIGS. 14A-E show that the effects of anti-ErbB2 MAbs are
epitope-specific. To determine if the anti-proliferative or
pro-apoptotic effects of anti-ErbB2 MAbs are related to epitope
specificity, BT474 cells were treated with 4 different MAbs for 3
days and stained with Hoechst 33342 for cell cycle analysis. The
MAbs were: 7C2 and 7F3, which bind amino acids 22-53 (SEQ ID NO:2)
on the ErbB2 extracellular domain (FIGS. 14B and C, respectively);
4D5, which binds residues 529-625 (SEQ ID NO:4) (FIG. 14D); and
3H4, which binds amino acids 541-599 (SEQ ID NO:3) (FIG. 14E). Both
7C2 and 7F3 induce apoptosis (to 60.5% and 53.4%, respectively, of
the cell population), but did not decrease the proportion of
S-phase cells (64.9% and 58.7%, respectively) compared to untreated
cells (52.5%; FIG. 14A). In contrast, MAbs 4D5 and 3H4, which bind
adjacent to the ErbB2 transmembrane region, show potent
anti-proliferative activity (% S=5.4 and 10.5, respectively,
control S=52.5%), but are not as effective as 7C2 or 7F3 in
promoting apoptotic cell death (% apoptosis for 4D5=41.9, for
3H4=26.3, controls=15.8%).
[0041] FIG. 15 shows induction of apoptosis by anti-HER2MAb 7C2 in
the SKOV3 ovarian carcinoma cell line as determined in Example
3.
[0042] FIG. 16 shows that combination treatment with anti-HER2MAbs
results in enhanced apoptotic effects on BT474 breast tumor cells
where anti-HER2MAb 7C2 is administered prior to anti-HER2MAb 4D5
(see Example 3).
[0043] FIG. 17 shows the effects of administration of anti-HER2MAbs
alone or in combination on mean tumor volume (mm.sup.3)+/-1 S.E. of
BT474M1 xenografts in nude mice as described in Example 4.
Antibodies were administered twice weekly beginning on day 6.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions
[0044] Unless indicated otherwise, the term "ErbB2" when used
herein refers to human ErbB2 protein and "erbB2" refers to human
erbB2 gene. The human erbB2 gene and ErbB2 protein are described in
Semba et al., PNAS (USA) 82:6497-6501 (1985) and Yamamoto et al.
Nature 319:230-234 (1986) (Genebank accession number X03363), for
example. ErbB2 comprises four domains (Domains 1-4). "Domain 1" at
the amino terminus of the extracellular domain of ErbB2 is shown in
FIG. 12 herein. See Plowman et al. Proc. Natl. Acad. Sci. USA
90:1746-1750 (1993).
[0045] 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 (see Example 2 below) to establish whether the
antibody binds to the 7C2/7F3 epitope on ErbB2 (i.e. any one or
more of residues in the region from about residue 22 to about
residue 53 of ErbB2 (SEQ ID NO:2)).
[0046] 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 region of ErbB2. To screen
for antibodies which bind to the 4D5 epitope, a routine
cross-blocking assay such as that described in Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and
David Lane (1988), can be performed. Alternatively, epitope mapping
can be performed (see Example 2 below) to assess whether the
antibody binds to the 4D5 epitope of ErbB2 (i.e. any one or more
residues in the region from about residue 529, e.g. about residue
561 to about residue 625, inclusive (SEQ ID NO:4)).
[0047] The term "induces cell death" refers to the ability of the
antibody to make a viable cell become nonviable. The "cell" here is
one which expresses the ErbB2 receptor, especially where the cell
overexpresses the ErbB2 receptor. A cell which "overexpresses"
ErbB2 has significantly higher than normal ErbB2 levels compared to
a noncancerous cell of the same tissue type.
[0048] Preferably, the cell is a cancer cell, e.g. a breast,
ovarian, stomach, endometrial, salivary gland, lung, kidney, colon,
thyroid, pancreatic or bladder cell. In vitro, the cell may be a
SKBR3, 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 cellular cytotoxicity (ADCC) or complement dependent
cytotoxicity (CDC). Thus, the assay for cell death may be performed
using heat inactivated serum (i.e. in the absence of complement)
and in the absence of immune effector cells. To determine whether
the antibody is able to induce cell death, loss of membrane
integrity as evaluated by uptake of propidium iodide (PI) (see
Example 2 below), trypan blue (see Moore et al. Cytotechnology
17:1-11 (1995)) or 7AAD (see Example 1 below) can be assessed
relative to untreated cells. Preferred cell death-inducing
antibodies are those which induce PI uptake in the "PI uptake assay
in BT474 cells" (see below)
[0049] The phrase "induces apoptosis" refers to the ability of the
antibody to induce programmed cell death as determined by binding
of annexin V, fragmentation of DNA, cell shrinkage, dilation of
endoplasmatic reticulum, cell fragmentation, and/or formation of
membrane vesicles (called apoptotic bodies). See FIGS. 1A & B
herein. The cell is one which overexpresses the ErbB2 receptor.
Preferably the "cell" is a tumor cell, e.g. a breast, ovarian,
stomach, endometrial, salivary gland, lung, kidney, colon, thyroid,
pancreatic or bladder cell. In vitro, the cell may be a SKBR3,
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 (see Example 2 below); DNA
fragmentation can be evaluated through DNA laddering as disclosed
in Example 2 herein; and nuclear/chromatin condensation along with
DNA fragmentation can be evaluated by any increase in hypodiploid
cells. Preferably, the antibody which induces apoptosis is one
which results in about 2 to 50 fold, preferably about 5 to 50 fold,
and most preferably about 10 to 50 fold, induction of annexin
binding relative to untreated cell in an "annexin binding assay
using BT474 cells" (see below).
[0050] Sometimes the pro-apoptotic antibody will be one which
blocks HRG binding/activation of the ErbB2/ErbB3 complex (e.g. 7F3
antibody). In other situations, the antibody is one which does not
significantly block activation of the ErbB2/ErbB3 receptor complex
by HRG (e.g. 7C2). Further, the antibody may be one like 7C2 which,
while inducing apoptosis, does not induce a large reduction in the
percent of cells in S phase (e.g. one which only induces about
0-10% reduction in the percent of these cells relative to control
as determined in FIG. 10).
[0051] The antibody of interest may be one like 7C2 which binds
specifically to human ErbB2 and does not significantly cross-react
with other proteins such as those encoded by the erbB1, erbB3
and/or erbB4 genes. Sometimes, the antibody may 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). In such embodiments, the extent of binding of
the antibody to these proteins (e.g., cell surface binding to
endogenous receptor) will be less than about 10% as determined by
fluorescence activated cell sorting (FACS) analysis or
radioimmunoprecipitation (RIA).
[0052] "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.1.sub.177-244).
[0053] The "ErbB2-ErbB3 protein complex" and "ErbB2-ErbB4 protein
complex" are noncovalently associated oligomers of the ErbB2
receptor and the ErbB3 receptor or ErbB4 receptor, respectively.
The complexes form when a cell expressing both of these receptors
is exposed to HRG 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).
[0054] "Antibodies" (Abs) and "immunoglobulins" (Igs) are
glycoproteins having the same structural characteristics. While
antibodies exhibit binding specificity to a specific antigen,
immunoglobulins include both antibodies and other antibody-like
molecules which lack antigen specificity. Polypeptides of the
latter kind are, for example, produced at low levels by the lymph
system and at increased levels by myelomas.
[0055] "Native antibodies" and "native immunoglobulins" 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- and heavy-chain variable
domains.
[0056] The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed throughout the variable
domains of antibodies. It is concentrated in three segments called
complementarity-determining regions (CDRs) or 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 (FR). The variable domains of native heavy and light
chains each comprise four FR regions, largely adopting .beta.-sheet
configuration, connected by three CDRs, which form loops
connecting, and in some cases forming part of, the .beta.-sheet
structure. The CDRs in each chain are held together in close
proximity by the FR regions and, with the CDRs from the other
chain, contribute to the formation of the antigen-binding site of
antibodies (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pages
647-669 (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 toxicity.
[0057] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
yields an F(ab').sub.2 fragment that has two antigen-combining
sites and is still capable of cross-linking antigen.
[0058] "Fv" is the minimum antibody fragment which contains a
complete antigen-recognition and -binding site. This region
consists of a dimer of one heavy- and one light-chain variable
domain in tight, non-covalent association. It is in this
configuration that the three CDRs of each variable domain interact
to define an antigen-binding site on the surface of the
V.sub.H-V.sub.L dimer. Collectively, the six CDRs confer
antigen-binding specificity to the antibody. However, even a single
variable domain (or half of an Fv comprising only three CDRs
specific for an antigen) has the ability to recognize and bind
antigen, although at a lower affinity than the entire binding
site.
[0059] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (CH1) of the heavy chain.
Fab fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear a free thiol group.
F(ab').sub.2 antibody fragments originally were produced as pairs
of Fab' fragments which have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.
[0060] The "light chains" of antibodies (immunoglobulins) from any
vertebrate species can be assigned to one of two clearly distinct
types, called kappa (K) and lambda (A), based on the amino acid
sequences of their constant domains.
[0061] Depending on the amino acid sequence of the constant domain
of their heavy chains, immunoglobulins can be assigned to different
classes. There are five major classes of immunoglobulins: 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 immunoglobulins are called .alpha., .delta., .epsilon.,
.gamma., and .mu., respectively. The subunit structures and
three-dimensional configurations of different classes of
immunoglobulins are well known.
[0062] The term "antibody" 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.
[0063] "Antibody fragments" comprise a portion of an intact
antibody, preferably the antigen binding or variable region of the
intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2, and Fv fragments; diabodies; linear antibodies
(Zapata et al. Protein Eng. 8(10):1057-1062 (1995)); single-chain
antibody molecules; and multispecific antibodies formed from
antibody fragments.
[0064] 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 conventional
(polyclonal) antibody preparations which typically 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 are synthesized
by the hybridoma culture, uncontaminated by other immunoglobulins.
The modifier "monoclonal" indicates the character of the antibody
as being obtained from a substantially homogeneous population of
antibodies, and is not to be construed as requiring production of
the antibody by any particular method. For example, the monoclonal
antibodies to be used in accordance with the present invention may
be made by 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.
[0065] The monoclonal antibodies herein specifically include
"chimeric" antibodies (immunoglobulins) 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; Morrison et
al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
[0066] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric immunoglobulins, immunoglobulin chains or fragments
thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies) which contain minimal
sequence derived from non-human immunoglobulin. For the most part,
humanized antibodies are human immunoglobulins (recipient antibody)
in which residues from a complementarity-determining region (CDR)
of the recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as mouse, rat or rabbit having the
desired specificity, affinity, and capacity. In some instances, Fv
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues which are found neither
in the recipient antibody nor in the imported CDR or framework
sequences. These modifications are made to further refine and
maximize 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 CDR
regions correspond to those of a non-human immunoglobulin and all
or substantially all of the FR regions are those of a human
immunoglobulin sequence. The humanized antibody optimally 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); Reichmann et
al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct.
Biol., 2:593-596 (1992). The humanized antibody includes a
PRIMATIZED.TM. antibody wherein the antigen-binding region of the
antibody is derived from an antibody produced by immunizing macaque
monkeys with the antigen of interest.
[0067] "Single-chain Fv" or "sFv" antibody fragments comprise the
V.sub.H and V.sub.L domains of antibody, wherein these domains are
present in a single polypeptide chain. Preferably, the Fv
polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the sFv to form the
desired structure for antigen binding. For a review of sFv see
Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315
(1994).
[0068] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy-chain
variable domain (V.sub.H) connected to a light-chain variable
domain (V) in the same polypeptide chain (V.sub.H-V.sub.L. By using
a linker that is too short to allow pairing between the two domains
on the same chain, the domains are forced to pair with the
complementary domains of another chain and create two
antigen-binding sites. Diabodies are described more fully in, for
example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.
Acad. Sci. USA, 90:6444-6448 (1993).
[0069] 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.
[0070] 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.
[0071] "Treatment" refers to both therapeutic treatment and
prophylactic or preventative measures. Those in need of treatment
include those already with the disorder as well as those in which
the disorder is to be prevented.
[0072] "Mammal" for purposes of treatment refers to any animal
classified as a mammal, including humans, domestic and farm
animals, and zoo, sports, or pet animals, such as dogs, horses,
cats, cows, etc. Preferably, the mammal is human.
[0073] A "disorder" is any condition that would benefit from
treatment with the anti-ErbB2 antibody. 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;
neuronal, glial, astrocytal, hypothalamic and other glandular,
macrophagal, epithelial, stromal and blastocoelic disorders; and
inflammatory, angiogenic and immunologic disorders.
[0074] 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.
More particular examples of such cancers include squamous cell
cancer, small-cell lung cancer, non-small cell lung cancer,
gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical
cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,
breast cancer, colon cancer, colorectal cancer, endometrial
carcinoma, salivary gland carcinoma, kidney cancer, liver cancer,
prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma
and various types of head and neck cancer.
[0075] 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. I.sup.131, I.sup.125, Y.sup.90 and
Re.sup.18), chemotherapeutic agents, and toxins such as
enzymatically active toxins of bacterial, fungal, plant or animal
origin, or fragments thereof.
[0076] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include Adriamycin, Doxorubicin, 5-Fluorouracil, Cytosine
arabinoside ("Ara-C"), Cyclophosphamide, Thiotepa; Busulfan,
Cytoxin, Taxol, Toxotere, Methotrexate, Cisplatin, Melphalan,
Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C,
Mitoxantrone, Vincreistine, Vinorelbine, Carboplatin, Teniposide,
Daunomycin, Caminomycin, Aminopterin, Dactinomycin, Mitomycins,
Esperamicins (see U.S. Pat. No. 4,675,187), Melphalan and other
related nitrogen mustards. Also included in this definition are
hormonal agents that act to regulate or inhibit hormone action on
tumors such as tamoxifen and onapristone.
[0077] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell, especially
an ErbB2-overexpressing cancer cell either in vitro or in vivo.
Thus, the growth inhibitory agent is one which significantly
reduces the percentage of ErbB2 overexpressing 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), taxol,
and topo II inhibitors such as doxorubicin, 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, oncogens, and
antineoplastic drugs by Murakami et al. (WB Saunders: Philadelphia,
1995), especially p. 13. The 4D5 antibody (and functional
equivalents thereof) can also be employed for this purpose.
[0078] The term "cytokine" is a generic term for proteins released
by one cell population which act on another cell as intercellular
mediators. Examples of such cytokines are lymphokines, monokines,
and traditional polypeptide hormones. Included among the cytokines
are growth hormone such as human growth hormone, N-methionyl human
growth hormone, and bovine growth hormone; parathyroid hormone;
thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein
hormones such as follicle stimulating hormone (FSH), thyroid
stimulating hormone (TSH), and luteinizing hormone (LH); hepatic
growth factor; fibroblast growth factor; prolactin; placental
lactogen; tumor necrosis factor-.alpha. and -.beta.;
mullerian-inhibiting substance; mouse gonadotropin-associated
peptide; inhibin; activin; vascular endothelial growth factor;
integrin; thrombopoietin (TPO); nerve growth factors such as
NGF-.beta.; platelet-growth factor; transforming growth factors
(TGFs) such as TGF-.alpha. and TGF-.beta.; insulin-like growth
factor-I and -II; erythropoietin (EPO); osteoinductive factors;
interferons such as interferon-.alpha., -.beta., and -.gamma.;
colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);
granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);
interleukins (ILs) such as IL-1, IL-1.alpha., IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor
such as TNF-.alpha. or TNF-.beta.; and other polypeptide factors
including LIF and kit ligand (KL). As used herein, the term
cytokine includes proteins from natural sources or from recombinant
cell culture and biologically active equivalents of the native
sequence cytokines.
[0079] 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.
[0080] The word "label" when used herein refers to a detectable
compound or composition which is conjugated directly or indirectly
to the antibody so as to generate a "labelled" antibody. The label
may be detectable by itself (e.g. radioisotope labels or
fluorescent labels) or, in the case of an enzymatic label, may
catalyze chemical alteration of a substrate compound or composition
which is detectable.
[0081] By "solid phase" is meant a non-aqueous matrix to which the
antibody of the present invention can adhere. Examples of solid
phases encompassed herein include those formed partially or
entirely of glass (e.g., controlled pore glass), polysaccharides
(e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol
and silicones. In certain embodiments, depending on the context,
the solid phase can comprise the well of an assay plate; in others
it is a purification column (e.g., an affinity chromatography
column). This term also includes a discontinuous solid phase of
discrete particles, such as those described in U.S. Pat. No.
4,275,149.
[0082] 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.
[0083] 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 antibody 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.
[0084] The expression "control sequences" refers to 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, polyadenylation
signals, and enhancers.
[0085] 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. Preferably, "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.
[0086] 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.
II. Modes for Carrying out the Invention
[0087] A. Antibody Preparation
[0088] A description follows as to exemplary techniques for the
production of the claimed antibodies. The ErbB2 antigen to be used
for production of antibodies may be, e.g., a soluble form of the
extracellular domain of ErbB2; a peptide such as a Domain 1 peptide
or a portion thereof (e.g. comprising the 7C2 or 7F3 epitope).
Alternatively, cells expressing ErbB2 at their cell surface (e.g.
NIH-3T3 cells transformed to overexpress ErbB2, see Examples 1
& 2 below; or a carcinoma cell line such as SKBR3 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.
[0089] (i) Polyclonal Antibodies
[0090] 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.
[0091] 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.
[0092] 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.
[0093] (ii) Monoclonal Antibodies
[0094] 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.
[0095] 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).
[0096] 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)).
[0097] 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.
[0098] 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); Brodeur et al., Monoclonal
Antibody Production Techniques and Applications, pp. 51-63 (Marcel
Dekker, Inc., New York, 1987)).
[0099] 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).
[0100] 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).
[0101] 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.
[0102] The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0103] 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 immunoglobulin 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).
[0104] In a further embodiment, 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.
[0105] The DNA also may be modified, for example, by substituting
the coding sequence for human heavy- and light-chain constant
domains in place of the homologous murine sequences (U.S. Pat. No.
4,816,567; 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.
[0106] 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.
[0107] (iii) Humanized and Human Antibodies
[0108] Methods for humanizing non-human antibodies are well known
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 rodent CDRs or CDR 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 CDR residues and possibly some FR residues
are substituted by residues from analogous sites in rodent
antibodies.
[0109] 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 (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 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)).
[0110] 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
CDR residues are directly and most substantially involved in
influencing antigen binding.
[0111] Alternatively, 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). Human antibodies can also be derived from
phage-display libraries (Hoogenboom et al., J. Mol. Biol., 227:381
(1991); Marks et al., J. Mol. Biol., 222:581-597 (1991)).
[0112] (iv) Antibody Fragments
[0113] 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.
[0114] (v) Bispecific Antibodies
[0115] 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. For example, one arm may bind an epitope in Domain 1
of ErbB2 such as the 7C2/7F3 epitope, the other may bind a
different ErbB2 epitope, e.g. the 4D5 epitope. Other such
antibodies may combine an ErbB2 binding site with binding site(s)
for EGFR, ErbB3 and/or ErbB4. Alternatively, an anti-ErbB2 arm may
be combined with an arm which binds to a triggering molecule on a
leukocyte such as a T-cell receptor molecule (e.g. CD2 or CD3), or
Fc receptors for IgG (Fc.gamma.R), such as Fc.gamma.RI (CD64),
Fc.gamma.RII (CD32) and Fc.gamma.RIII (CD16) so as to focus
cellular defense mechanisms to the ErbB2-expressing cell.
Bispecific antibodies may also be used to localize cytotoxic agents
to cells which express ErbB2. These antibodies possess an
ErbB2-binding arm and an arm which binds the cytotoxic agent (e.g.
saporin, anti-interferon-.alpha., vinca alkaloid, ricin A chain,
methotrexate or radioactive isotope hapten). Bispecific antibodies
can be prepared as full length antibodies or antibody fragments
(e.g. F(ab').sub.2 bispecific antibodies).
[0116] 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).
[0117] 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.
[0118] 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).
[0119] According to another approach described in WO96/27011, 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.
[0120] Bispecific antibodies include cross-linked or
"heteroconjugate" antibodies. For example, one of the antibodies in
the heteroconjugate can be coupled to avidin, the other to biotin.
Such antibodies have, for example, been proposed to target immune
system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for
treatment of HIV infection (WO 91/00360, WO 92/200373, and EP
03089). Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents are well known
in the art, and are disclosed in U.S. Pat. No. 4,676,980, along
with a number of cross-linking techniques.
[0121] 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.
[0122] 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.
[0123] 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).
[0124] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al. J.
Immunol. 147: 60 (1991).
[0125] (vi) Screening for Antibodies with the Desired
Properties
[0126] Techniques for generating antibodies have been described
above. Those antibodies having the characteristics described herein
are selected.
[0127] To select for antibodies which induce cell death, loss of
membrane integrity as indicated by, e.g., PI, trypan blue or 7AAD
uptake is 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 MAb. The cells are
incubated for a 3 day time period. Following each treatment,
monolayers are washed with PBS and detached by trypsinization.
Cells are then centrifuged at 1200 rpm for 5 minutes at 4.degree.
C., the pellet resuspended in 3 ml ice cold Ca.sup.2+ binding
buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl.sub.2) and
aliquoted into 35 mm strainer-capped 12.times.75 tubes (1 ml per
tube, 3 tubes per treatment group) for removal of cell clumps.
Tubes then receive PI (10 .mu.g/ml). Samples may be analyzed using
a FACSCAN.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 are
selected.
[0128] In order to select for antibodies which induce apoptosis, an
"annexin binding assay using BT474 cells" as described in Example 2
below 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 MAb. 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 labelled 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.
[0129] In addition to the annexin binding assay discussed in the
preceding paragraph, 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.
[0130] 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, epitope mapping described in Example 2
can be performed.
[0131] To identify anti-ErbB2 antibodies which inhibit growth of
SKBR3 cells in cell culture by 50-100%, the SKBR3 assay described
in WO89/06692 can be performed. According to this assay, SKBR3
cells are grown in a 1:1 mixture of F12 and DMEM medium
supplemented with 10% fetal bovine serum, glutamine and
penicillinstreptomycin. The SKBR3 cells are plated at 20,000 cells
in a 35 mm cell culture dish (2 mls/35 mm dish). 2.5 .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'' cell counter. Those antibodies which inhibit
growth of the SKBR3 cells by 50-100% are selected for combination
with the apoptotic antibodies as desired.
[0132] (vii) Effector Function Engineering
[0133] It may be desirable to modify the antibody of the invention
with respect to effector function, so as to enhance the
effectiveness of the antibody in treating cancer, for example. For
example 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);
[0134] (viii) Immunoconjugates
[0135] The invention also pertains to immunoconjugates comprising
the antibody described herein conjugated to a cytotoxic agent such
as a chemotherapeutic agent, toxin (e.g. an enzymatically active
toxin of bacterial, fungal, plant or animal origin, or fragments
thereof), or a radioactive isotope (i.e., a radioconjugate).
[0136] Chemotherapeutic agents useful in the generation of such
immunoconjugates have been described above. Enzymatically active
toxins and fragments thereof which can be used include diphtheria A
chain, nonbinding active fragments of diphtheria toxin, exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),
momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin and the tricothecenes. A variety of
radionuclides are available for the production of radioconjugated
anti-ErbB2 antibodies. Examples include .sup.212Bi, .sup.131I,
.sup.131In, .sup.90Y and .sup.186Re.
[0137] Conjugates of the antibody and cytotoxic agent are made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al. Science 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026.
[0138] In another embodiment, the antibody may be conjugated to a
"receptor" (such streptavidin) for utilization in tumor
pretargeting wherein the antibody-receptor conjugate is
administered to the patient, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g. avidin) which is conjugated to a
cytotoxic agent (e.g. a radionucleotide).
[0139] (ix) Immunoliposomes
[0140] The anti-ErbB2 antibodies disclosed herein may also be
formulated as immunoliposomes. Liposomes containing the antibody
are prepared by methods known in the art, such as described in
Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang
et al., Proc. Natl. Acad. Sci. USA, 77:4030 (1980); and U.S. Pat.
Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation
time are disclosed in U.S. Pat. No. 5,013,556.
[0141] Particularly useful liposomes can be generated by the
reverse phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes as described in Martin et al. J.
Biol. Chem. 257: 286-288 (1982) via a disulfide interchange
reaction. A chemotherapeutic agent (such as Doxorubicin) is
optionally contained within the liposome. See Gabizon et al. J.
National Cancer Inst. 81(19)1484 (1989)
[0142] (x) Antibody Dependent Enzyme Mediated Prodrug Therapy
(ADEPT)
[0143] The antibody of the present invention may also be used in
ADEPT by conjugating the antibody to a prodrug-activating enzyme
which converts a prodrug (e.g. a peptidyl chemotherapeutic agent,
see WO81/01145) to an active anti-cancer drug. See, for example, WO
88/07378 and U.S. Pat. No. 4,975,278.
[0144] The enzyme component of the immunoconjugate useful for ADEPT
includes any enzyme capable of acting on a prodrug in such a way so
as to covert it into its more active, cytotoxic form.
[0145] Enzymes that are useful in the method of this invention
include, but are not limited to, alkaline phosphatase useful for
converting phosphate-containing prodrugs into free drugs;
arylsulfatase useful for converting sulfate-containing prodrugs
into free drugs; cytosine deaminase useful for converting non-toxic
5-fluorocytosine into the anti-cancer drug, 5-fluorouracil;
proteases, such as serratia protease, thermolysin, subtilisin,
carboxypeptidases and cathepsins (such as cathepsins B and L), that
are useful for converting peptide-containing prodrugs into free
drugs; D-alanylcarboxypeptidases, useful for converting prodrugs
that contain D-amino acid substituents; carbohydrate-cleaving
enzymes such as (3-galactosidase and neuraminidase useful for
converting glycosylated prodrugs into free drugs; (3-lactamase
useful for converting drugs derivatized with .beta.-lactams into
free drugs; and penicillin amidases, such as penicillin V amidase
or penicillin G amidase, useful for converting drugs derivatized at
their amine nitrogens with phenoxyacetyl or phenylacetyl groups,
respectively, into free drugs. Alternatively, antibodies with
enzymatic activity, also known in the art as "abzymes", can be used
to convert the prodrugs of the invention into free active drugs
(see, e.g., Massey, Nature 328: 457-458 (1987)). Antibody-abzyme
conjugates can be prepared as described herein for delivery of the
abzyme to a tumor cell population.
[0146] The enzymes of this invention can be covalently bound to the
anti-ErbB2 antibodies by techniques well known in the art such as
the use of the heterobifunctional crosslinking reagents discussed
above. Alternatively, fusion proteins comprising at least the
antigen binding region of an antibody of the invention linked to at
least a functionally active portion of an enzyme of the invention
can be constructed using recombinant DNA techniques well known in
the art (see, e.g., Neuberger et al., Nature, 312: 604-608
(1984)).
[0147] (xi) Antibody-Salvage Receptor Binding Epitope Fusions.
[0148] In certain embodiments of the invention, it may be desirable
to use an antibody fragment, rather than an intact antibody, to
increase tumor penetration, for example. In this case, it may be
desirable to modify the antibody fragment in order to increase its
serum half life. This may be achieved, for example, by
incorporation of a salvage receptor binding epitope into the
antibody fragment (e.g. by mutation of the appropriate region in
the antibody fragment or by incorporating the epitope into a
peptide tag that is then fused to the antibody fragment at either
end or in the middle, e.g., by DNA or peptide synthesis).
[0149] A systematic method for preparing such an antibody variant
having an increased in vivo half-life comprises several steps. The
first involves identifying the sequence and conformation of a
salvage receptor binding epitope of an Fc region of an IgG
molecule. Once this epitope is identified, the sequence of the
antibody of interest is modified to include the sequence and
conformation of the identified binding epitope. After the sequence
is mutated, the antibody variant is tested to see if it has a
longer in vivo half-life than that of the original antibody. If the
antibody variant does not have a longer in vivo half-life upon
testing, its sequence is further altered to include the sequence
and conformation of the identified binding epitope. The altered
antibody is tested for longer in vivo half-life, and this process
is continued until a molecule is obtained that exhibits a longer in
vivo half-life.
[0150] The salvage receptor binding epitope being thus incorporated
into the antibody of interest is any suitable such epitope as
defined above, and its nature will depend, e.g., on the type of
antibody being modified. The transfer is made such that the
antibody of interest still possesses the biological activities
described herein.
[0151] The epitope preferably constitutes a region wherein any one
or more amino acid residues from one or two loops of a Fc domain
are transferred to an analogous position of the antibody fragment.
Even more preferably, three or more residues from one or two loops
of the Fc domain are transferred. Still more preferred, the epitope
is taken from the CH2 domain of the Fc region (e.g., of an IgG) and
transferred to the CH1, CH3, or V.sub.H region, or more than one
such region, of the antibody. Alternatively, the epitope is taken
from the CH2 domain of the Fc region and transferred to the C.sub.L
region or V.sub.L region, or both, of the antibody fragment.
[0152] In one most preferred embodiment, the salvage receptor
binding epitope comprises the sequence (5' to 3'): PKNSSMISNTP (SEQ
ID NO:5), and optionally further comprises a sequence selected from
the group consisting of HQSLGTQ (SEQ ID NO:6), HQNLSDGK (SEQ ID
NO:7), HQNISDGK (SEQ ID NO:8), or VISSHLGQ (SEQ ID NO:9),
particularly where the antibody fragment is a Fab or F(ab').sub.2.
In another most preferred embodiment, the salvage receptor binding
epitope is a polypeptide containing the sequence(s)(5' to 3'):
HQNLSDGK (SEQ ID NO:7), HQNISDGK (SEQ ID NO:8), or VISSHLGQ (SEQ ID
NO:9) and the sequence: PKNSSMISNTP (SEQ ID NO:5).
[0153] B. Vectors, Host Cells and Recombinant Methods
[0154] The invention also provides isolated nucleic acid encoding
an antibody as disclosed herein, vectors and host cells comprising
the nucleic acid, and recombinant techniques for the production of
the antibody. In addition to recombinant production of the
antibody, the nucleic acid encoding the antibodies disclosed herein
may be used to inhibit cell surface expression of the ErbB2 protein
according to the teachings of WO96/07321, published Mar. 14, 1996,
for example. For example, the antibody may be a single chain Fv
fragment provided in an expression vector (such as a viral or
plasmid vector), which vector is introduced into a cell so as to
bind to the ErbB2 protein intracellularly and thereby induce death
of the cell.
[0155] For recombinant production of the antibody, the nucleic acid
encoding it is isolated and inserted into a replicable vector for
further cloning (amplification of the DNA) or for expression.
[0156] DNA encoding the monoclonal antibody 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 the antibody). Many
vectors are available. The vector components preferably include,
but are not limited to, one or more of the following: a signal
sequence, an origin of replication, one or more marker genes, an
enhancer element, a promoter, and a transcription termination
sequence.
[0157] (i) Signal Sequence Component
[0158] The anti-ErbB2 antibody of this invention may be produced
recombinantly not only directly, but also as a fusion polypeptide
with a heterologous polypeptide, which is preferably a signal
sequence or other polypeptide having a specific cleavage site at
the N-terminus of the mature protein or polypeptide. The
heterologous signal sequence selected preferably is one that is
recognized and processed (i.e., cleaved by a signal peptidase) by
the host cell. For prokaryotic host cells that do not recognize and
process the native anti-ErbB2 antibody signal sequence, the signal
sequence is substituted by a prokaryotic signal sequence selected,
for example, from the group of the alkaline phosphatase,
penicillinase, Ipp, or heat-stable enterotoxin II leaders. For
yeast secretion the native signal sequence may be substituted by,
e.g., the yeast invertase leader, a factor leader (including
Saccharomyces and Kluyveromyces .alpha.-factor leaders), or acid
phosphatase leader, the C. albicans glucoamylase leader, or the
signal described in WO 90/13646. In mammalian cell expression,
mammalian signal sequences as well as viral secretory leaders, for
example, the herpes simplex gD signal, are available.
[0159] The DNA for such precursor region is ligated in reading
frame to DNA encoding the anti-ErbB2 antibody.
[0160] (ii) Origin of Replication Component
[0161] Both expression and cloning vectors contain a nucleic acid
sequence that enables the vector to replicate in one or more
selected host cells. Preferably, in cloning vectors this sequence
is one that enables the vector to replicate independently of the
host chromosomal DNA, and includes origins of replication or
autonomously replicating sequences. Such sequences are well known
for a variety of bacteria, yeast, and viruses. The origin of
replication from the plasmid pBR322 is suitable for most
Gram-negative bacteria, the 2.mu. plasmid origin is suitable for
yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or
BPV) are useful for cloning vectors in mammalian cells. Preferably,
the origin of replication component is not needed for mammalian
expression vectors (the SV40 origin may typically be used only
because it contains the early promoter).
[0162] (iii) Selection Gene Component
[0163] Expression and cloning vectors may contain a selection gene,
also termed a selectable marker. Typical selection genes encode
proteins that (a) confer resistance to antibiotics or other toxins,
e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)
complement auxotrophic deficiencies, or (c) supply critical
nutrients not available from complex media, e.g., the gene encoding
D-alanine racemase for Bacilli.
[0164] One example of a selection scheme utilizes a drug to arrest
growth of a host cell. Those cells that are successfully
transformed with a heterologous gene produce a protein conferring
drug resistance and thus survive the selection regimen. Examples of
such dominant selection use the drugs neomycin, mycophenolic acid
and hygromycin.
[0165] Another example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the anti-ErbB2 antibody nucleic acid, such as DHFR,
thymidine kinase, metallothionein-I and -II, preferably primate
metallothionein genes, adenosine deaminase, ornithine
decarboxylase, etc.
[0166] For example, cells transformed with the DHFR selection gene
are first identified by culturing all of the transformants in a
culture medium that contains methotrexate (Mtx), a competitive
antagonist of DHFR. An appropriate host cell when wild-type DHFR is
employed is the Chinese hamster ovary (CHO) cell line deficient in
DHFR activity.
[0167] Alternatively, host cells (particularly wild-type hosts that
contain endogenous DHFR) transformed or co-transformed with DNA
sequences encoding anti-ErbB2 antibody, wild-type DHFR protein, and
another selectable marker such as aminoglycoside
3'-phosphotransferase (APH) can be selected by cell growth in
medium containing a selection agent for the selectable marker such
as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or
G418. See U.S. Pat. No. 4,965,199.
[0168] A suitable selection gene for use in yeast is the trp1 gene
present in the yeast plasmid YRp7 (Stinchcomb et al., Nature,
282:39 (1979)). The trp1 gene provides a selection marker for a
mutant strain of yeast lacking the ability to grow in tryptophan,
for example, ATCC No. 44076 or PEP4-1. Jones, Genetics, 85:12
(1977). The presence of the trp1 lesion in the yeast host cell
genome then provides an effective environment for detecting
transformation by growth in the absence of tryptophan. Similarly,
Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are
complemented by known plasmids bearing the Leu2 gene.
[0169] In addition, vectors derived from the 1.6 .mu.m circular
plasmid pKD1 can be used for transformation of Kluyveromyces
yeasts. Alternatively, an expression system for large-scale
production of recombinant calf chymosin was reported for K. lactis.
Van den Berg, Bio/Technology, 8:135 (1990). Stable multi-copy
expression vectors for secretion of mature recombinant human serum
albumin by industrial strains of Kluyveromyces have also been
disclosed. Fleer et al., Bio/Technology, 9:968-975 (1991).
[0170] (iv) Promoter Component
[0171] Expression and cloning vectors usually contain a promoter
that is recognized by the host organism and is operably linked to
the anti-ErbB2 antibody nucleic acid. Promoters suitable for use
with prokaryotic hosts include the phoA promoter, .beta.-lactamase
and lactose promoter systems, alkaline phosphatase, a tryptophan
(trp) promoter system, and hybrid promoters such as the tac
promoter. However, other known bacterial promoters are suitable.
Promoters for use in bacterial systems also will contain a
Shine-Dalgamo (S.D.) sequence operably linked to the DNA encoding
the anti-ErbB2 antibody.
[0172] Promoter sequences are known for eukaryotes. Virtually all
eukaryotic genes have an AT-rich region located approximately 25 to
30 bases upstream from the site where transcription is initiated.
Another sequence found 70 to 80 bases upstream from the start of
transcription of many genes is a CNCAAT region where N may be any
nucleotide. At the 3' end of most eukaryotic genes is an AATAAA
sequence that may be the signal for addition of the poly A tail to
the 3' end of the coding sequence. All of these sequences are
suitably inserted into eukaryotic expression vectors.
[0173] Examples of suitable promoting sequences for use with yeast
hosts include the promoters for 3-phosphoglycerate kinase or other
glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate
dehydrogenase, hexokinase, pyruvate decarboxylase,
phospho-fructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase.
[0174] Other yeast promoters, which are inducible promoters having
the additional advantage of transcription controlled by growth
conditions, are the promoter regions for alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase, degradative enzymes associated
with nitrogen metabolism, metallothionein,
glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible
for maltose and galactose utilization. Suitable vectors and
promoters for use in yeast expression are further described in EP
73,657. Yeast enhancers also are advantageously used with yeast
promoters.
[0175] Anti-ErbB2 antibody transcription from vectors in mammalian
host cells is controlled, for example, by promoters obtained from
the genomes of viruses such as polyoma virus, fowlpox virus,
adenovirus (such as Adenovirus 2), bovine papilloma virus, avian
sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and
most preferably Simian Virus 40 (SV40), from heterologous mammalian
promoters, e.g., the actin promoter or an immunoglobulin promoter,
from heat-shock promoters, provided such promoters are compatible
with the host cell systems.
[0176] The early and late promoters of the SV40 virus are
conveniently obtained as an SV40 restriction fragment that also
contains the SV40 viral origin of replication. The immediate early
promoter of the human cytomegalovirus is conveniently obtained as a
HindIII E restriction fragment. A system for expressing DNA in
mammalian hosts using the bovine papilloma virus as a vector is
disclosed in U.S. Pat. No. 4,419,446. A modification of this system
is described in U.S. Pat. No. 4,601,978. See also Reyes et al.,
Nature, 297:598-601 (1982) on expression of human .beta.-interferon
cDNA in mouse cells under the control of a thymidine kinase
promoter from herpes simplex virus. Alternatively, the rous sarcoma
virus long terminal repeat can be used as the promoter.
[0177] (v) Enhancer Element Component
[0178] Transcription of a DNA encoding the anti-ErbB2 antibody of
this invention by higher eukaryotes is often increased by inserting
an enhancer sequence into the vector. Many enhancer sequences are
now known from mammalian genes (globin, elastase, albumin,
.alpha.-fetoprotein, and insulin). Typically, however, one will use
an enhancer from a eukaryotic cell virus. Examples include the SV40
enhancer on the late side of the replication origin (bp 100-270),
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin, and adenovirus
enhancers. See also Yaniv, Nature, 297:17-18 (1982) on enhancing
elements for activation of eukaryotic promoters. The enhancer may
be spliced into the vector at a position 5' or 3' to the anti-ErbB2
antibody-encoding sequence, but is preferably located at a site 5'
from the promoter.
[0179] (vi) Transcription Termination Component
[0180] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human, or nucleated cells from other
multicellular organisms) will also contain sequences necessary for
the termination of transcription and for stabilizing the mRNA. Such
sequences are commonly available from the 5' and, occasionally 3',
untranslated regions of eukaryotic or viral DNAs or cDNAs. These
regions contain nucleotide segments transcribed as polyadenylated
fragments in the untranslated portion of the mRNA encoding
anti-ErbB2 antibody. One useful transcription termination component
is the bovine growth hormone polyadenylation region. See WO94/11026
and the expression vector disclosed therein.
[0181] (vii) Selection and Transformation of Host Cells
[0182] Suitable host cells for cloning or expressing the DNA in the
vectors herein are the prokaryote, yeast, or higher eukaryote cells
described above. Suitable prokaryotes for this purpose include
eubacteria, such as Gram-negative or Gram-positive organisms, for
example, Enterobacteriaceae such as Escherichia, e.g., E. coli,
Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g.,
Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and
Shigella, as well as Bacilli such as B. subtilis and B.
licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710
published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and
Streptomyces. One preferred E. coli cloning host is E. coli 294
(ATCC 31,446), although other strains such as E coli B, E coli
X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.
These examples are illustrative rather than limiting.
[0183] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for anti-ErbB2 antibody-encoding vectors. Saccharomyces cerevisiae,
or common baker's yeast, is the most commonly used among lower
eukaryotic host microorganisms. However, a number of other genera,
species, and strains are commonly available and useful herein, such
as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K.
lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.
wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum
(ATCC 36,906), K. thermotolerans, and K. marxianus; yarrowia (EP
402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia
(EP 244,234); Neurospora crassa; Schwanniomyces such as
Schwanniomyces occidentalis; and filamentous fungi such as, e.g.,
Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such
as A. nidulans and A. niger.
[0184] Suitable host cells for the expression of glycosylated
anti-ErbB2 antibody are derived from multicellular organisms.
Examples of invertebrate cells include plant and insect cells.
Numerous baculoviral strains and variants and corresponding
permissive insect host cells from hosts such as Spodoptera
frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes
albopictus (mosquito), Drosophila melanogaster(fruitfly), and
Bombyx mori have been identified. A variety of viral strains for
transfection are publicly available, e.g., the L-1 variant of
Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,
and such viruses may be used as the virus herein according to the
present invention, particularly for transfection of Spodoptera
frugiperda cells.
[0185] Plant cell cultures of cotton, corn, potato, soybean,
petunia, tomato, and tobacco can also be utilized as hosts.
[0186] However, interest has been greatest in vertebrate cells, and
propagation of vertebrate cells in culture (tissue culture) has
become a routine procedure. Examples of useful mammalian host cell
lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC
CRL 1651); human embryonic kidney line (293 or 293 cells subcloned
for growth in suspension culture, Graham et al., J. Gen Virol.,
36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);
Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl.
Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather,
Biol. Reprod., 23:243-251 (1980)); monkey kidney cells (CV1 ATCC
CCL 70); African green monkey kidney cells (VERO-76, ATCC
CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2);
canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells
(BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75);
human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT
060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad.
Sci., 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human
hepatoma line (Hep G2).
[0187] Host cells are transformed with the above-described
expression or cloning vectors for anti-ErbB2 antibody production
and cultured in conventional nutrient media modified as appropriate
for inducing promoters, selecting transformants, or amplifying the
genes encoding the desired sequences.
[0188] (viii) Culturing the Host Cells
[0189] The host cells used to produce the anti-ErbB2 antibody of
this invention may be cultured in a variety of media. Commercially
available media such as Ham's F10 (Sigma), Minimal Essential Medium
((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's
Medium ((DMEM), Sigma) are suitable for culturing the host cells.
In addition, any of the media described in Ham et al. Meth. Enz.,
58:44 (1979), Barnes et al., Anal. Biochem., 102:255 (1980), U.S.
Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469;
WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be used as
culture media for the host cells. Any of these media may be
supplemented as necessary with hormones and/or other growth factors
(such as insulin, transferrin, or epidermal growth factor), salts
(such as sodium chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleotides (such as adenosine and
thymidine), antibiotics (such as GENTAMYCIN.TM. drug), trace
elements (defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations that would be known to
those skilled in the art. The culture conditions, such as
temperature, pH, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
[0190] (ix) Purification of Anti-ErbB2 Antibody
[0191] When using recombinant techniques, the antibody can be
produced intracellularly, in the periplasmic space, or directly
secreted into the medium. If the antibody is produced
intracellularly, as a first step, the particulate debris, either
host cells or lysed fragments, is removed, for example, by
centrifugation or ultrafiltration. Carter et al., Bio/Technology
10:163-167 (1992) describe a procedure for isolating antibodies
which are secreted to the periplasmic space of E. coli. Briefly,
cell paste is thawed in the presence of sodium acetate (pH 3.5),
EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
Cell debris can be removed by centrifugation. Where the antibody is
secreted into the medium, supernatants from such expression systems
are preferably first concentrated using a commercially available
protein concentration filter, for example, an Amicon or Millipore
Pellicon ultrafiltration unit. A protease inhibitor such as PMSF
may be included in any of the foregoing steps to inhibit
proteolysis and antibiotics may be included to prevent the growth
of adventitious contaminants.
[0192] The antibody composition prepared from the cells can be
purified using, for example, hydroxylapatite chromatography, gel
electrophoresis, dialysis, and affinity chromatography, with
affinity chromatography being the preferred purification technique.
The suitability of protein A as an affinity ligand depends on the
species and isotype of any immunoglobulin Fc domain that is present
in the antibody. Protein A can be used to purify antibodies that
are based on human .gamma.1, .gamma.2, or .gamma.4 heavy chains
(Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is
recommended for all mouse isotypes and for human .gamma.3 (Guss et
al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity
ligand is attached is most often agarose, but other matrices are
available. Mechanically stable matrices such as controlled pore
glass or poly(styrenedivinyl)benzene allow for faster flow rates
and shorter processing times than can be achieved with agarose.
Where the antibody comprises a C.sub.H3 domain, the Bakerbond
ABX.TM. resin (J. T. Baker, Phillipsburg, N.J.) is useful for
purification. Other techniques for protein purification such as
fractionation on an ion-exchange column, ethanol precipitation,
Reverse Phase HPLC, chromatography on silica, chromatography on
heparin SEPHAROSE.TM. chromatography on an anion or cation exchange
resin (such as a polyaspartic acid column), chromatofocusing,
SDS-PAGE, and ammonium sulfate precipitation are also available
depending on the antibody to be recovered.
[0193] Following any preliminary purification step(s), the mixture
comprising the antibody of interest and contaminants may be
subjected to low pH hydrophobic interaction chromatography using an
elution buffer at a pH between about 2.5-4.5, preferably performed
at low salt concentrations (e.g. from about 0-0.25M salt).
[0194] C. Pharmaceutical Formulations
[0195] Therapeutic formulations of the antibody are prepared for
storage by mixing the 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).
[0196] The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
preferably those with complementary activities that do not
adversely affect each other. For example, it may be desirable to
further provide antibodies which bind to EGFR, ErbB2 (e.g. an
antibody which binds a different epitope on ErbB2), ErbB3, ErbB4,
or vascular endothelial factor (VEGF) in the one formulation.
Alternatively, or in addition, the composition may comprise a
cytotoxic agent, cytokine or growth inhibitory agent. Such
molecules are suitably present in combination in amounts that are
effective for the purpose intended.
[0197] 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).
[0198] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0199] 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. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods. When encapsulated antibodies remain in
the body for a long time, they may denature or aggregate as a
result of exposure to moisture at 37.degree. C., resulting in a
loss of biological activity and possible changes in immunogenicity.
Rational strategies can be devised for stabilization depending on
the mechanism involved. For example, if the aggregation mechanism
is discovered to be intermolecular S--S bond formation through
thio-disulfide interchange, stabilization may be achieved by
modifying sulfhydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.
[0200] D. Non-therapeutic Uses for the Antibody
[0201] The antibodies of the invention may be used as affinity
purification agents. In this process, the antibodies are
immobilized on a solid phase such a Sephadex resin or filter paper,
using methods well known in the art. The immobilized antibody is
contacted with a sample containing the ErbB2 protein (or fragment
thereof) to be purified, and thereafter the support is washed with
a suitable solvent that will remove substantially all the material
in the sample except the ErbB2 protein, which is bound to the
immobilized antibody. Finally, the support is washed with another
suitable solvent, such as glycine buffer, pH 5.0, that will release
the ErbB2 protein from the antibody.
[0202] Anti-ErbB2 antibodies may also be useful in diagnostic
assays for ErbB2 protein, e.g., detecting its expression in
specific cells, tissues, or serum. Thus, the antibodies may be used
in the diagnosis of human malignancies (see, for example, U.S. Pat.
No. 5,183,884).
[0203] For diagnostic applications, the antibody typically will be
labeled with a detectable moiety. Numerous labels are available
which can be preferably grouped into the following categories:
[0204] (a) Radioisotopes, such as .sup.35S, .sup.14C, .sup.125I,
.sup.3H, and .sup.131I. The antibody can be labeled with the
radioisotope using the techniques described in Current Protocols in
Immunology, Volumes 1 and 2, Coligen et al., Ed.,
Wiley-Interscience, New York, N.Y., Pubs., (1991) for example and
radioactivity can be measured using scintillation counting.
[0205] (b) Fluorescent labels such as rare earth chelates (europium
chelates) or fluorescein and its derivatives, rhodamine and its
derivatives, dansyl, Lissamine, phycoerythrin and Texas Red are
available. The fluorescent labels can be conjugated to the antibody
using the techniques disclosed in Current Protocols in Immunology,
supra, for example. Fluorescence can be quantified using a
fluorimeter.
[0206] (c) Various enzyme-substrate labels are available and U.S.
Pat. No. 4,275,149 provides a review of some of these. The enzyme
preferably catalyses a chemical alteration of the chromogenic
substrate which can be measured using various techniques. For
example, the enzyme may catalyze a color change in a substrate,
which can be measured spectrophotometrically. Alternatively, the
enzyme may alter the fluorescence or chemiluminescence of the
substrate. Techniques for quantifying a change in fluorescence are
described above. The chemiluminescent substrate becomes
electronically excited by a chemical reaction and may then emit
light which can be measured (using a chemiluminometer, for example)
or donates energy to a fluorescent acceptor. Examples of enzymatic
labels include luciferases (e.g., firefly luciferase and bacterial
luciferase; U.S. Pat. No. 4,737,456), luciferin,
2,3-dihydrophthalazinediones, malate dehydrogenase, urease,
peroxidase such as horseradish peroxidase (HRPO), alkaline
phosphatase, .beta.-galactosidase, glucoamylase, lysozyme,
saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and
glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as
uricase and xanthine oxidase), lactoperoxidase, microperoxidase,
and the like. Techniques for conjugating enzymes to antibodies are
described in O'Sullivan et al., Methods for the Preparation of
Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in
Methods in Enzym. (ed J. Langone & H. Van Vunakis), Academic
press, New York, 73: 147-166 (1981).
[0207] Examples of enzyme-substrate combinations include, for
example:
[0208] (i) Horseradish peroxidase (HRPO) with hydrogen peroxidase
as a substrate, wherein the hydrogen peroxidase oxidizes a dye
precursor (e.g. orthophenylene diamine (OPD) or
3,3',5,5'-tetramethyl benzidine hydrochloride (TMB));
[0209] (ii) alkaline phosphatase (AP) with para-Nitrophenyl
phosphate as chromogenic substrate; and
[0210] (iii) .beta.-D-galactosidase (.beta.-D-Gal) with a
chromogenic substrate (e.g. p-nitrophenyl-.beta.-D-galactosidase)
or fluorogenic substrate
4-methylumbelliferyl-.beta.-D-galactosidase.
[0211] Numerous other enzyme-substrate combinations are available
to those skilled in the art. For a general review of these, see
U.S. Pat. Nos. 4,275,149 and 4,318,980.
[0212] Sometimes, the label is indirectly conjugated with the
antibody. The skilled artisan will be aware of various techniques
for achieving this. For example, the antibody can be conjugated
with biotin and any of the three broad categories of labels
mentioned above can be conjugated with avidin, or vice versa.
Biotin binds selectively to avidin and thus, the label can be
conjugated with the antibody in this indirect manner.
Alternatively, to achieve indirect conjugation of the label with
the antibody, the antibody is conjugated with a small hapten (e.g.
digoxin) and one of the different types of labels mentioned above
is conjugated with an anti-hapten antibody (e.g. anti-digoxin
antibody). Thus, indirect conjugation of the label with the
antibody can be achieved.
[0213] In another embodiment of the invention, the anti-ErbB2
antibody need not be labeled, and the presence thereof can be
detected using a labeled antibody which binds to the ErbB2
antibody.
[0214] The antibodies of the present invention may be employed in
any known assay method, such as competitive binding assays, direct
and indirect sandwich assays, and immunoprecipitation assays. Zola,
Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC
Press, Inc., 1987).
[0215] Competitive binding assays rely on the ability of a labeled
standard to compete with the test sample analyte for binding with a
limited amount of antibody. The amount of ErbB2 protein in the test
sample is inversely proportional to the amount of standard that
becomes bound to the antibodies. To facilitate determining the
amount of standard that becomes bound, the antibodies preferably
are insolubilized before or after the competition, so that the
standard and analyte that are bound to the antibodies may
conveniently be separated from the standard and analyte which
remain unbound.
[0216] Sandwich assays involve the use of two antibodies, each
capable of binding to a different immunogenic portion, or epitope,
of the protein to be detected. In a sandwich assay, the test sample
analyte is bound by a first antibody which is immobilized on a
solid support, and thereaftera second antibody binds to the
analyte, thus forming an insoluble three-part complex. See, e.g.,
U.S. Pat. No. 4,376,110. The second antibody may itself be labeled
with a detectable moiety (direct sandwich assays) or may be
measured using an anti-immunoglobulin antibody that is labeled with
a detectable moiety (indirect sandwich assay). For example, one
type of sandwich assay is an ELISA assay, in which case the
detectable moiety is an enzyme.
[0217] For immunohistochemistry, the tumor sample may be fresh or
frozen or may be embedded in paraffin and fixed with a preservative
such as formalin, for example.
[0218] The antibodies may also be used for in vivo diagnostic
assays. Preferably, the antibody is labelled with a radionuclide
(such as .sup.111In, .sup.99Tc, .sup.14C, .sup.131I, .sup.125I,
.sup.3H, .sup.32P or .sup.35S) so that the tumor can be localized
using immunoscintiography.
[0219] E. Diagnostic Kits
[0220] As a matter of convenience, the antibody of the present
invention can be provided in a kit, i.e., a packaged combination of
reagents in predetermined amounts with instructions for performing
the diagnostic assay. Where the antibody is labelled with an
enzyme, the kit will include substrates and cofactors required by
the enzyme (e.g. a substrate precursor which provides the
detectable chromophore or fluorophore). In addition, other
additives may be included such as stabilizers, buffers (e.g. a
block buffer or lysis buffer) and the like. The relative amounts of
the various reagents may be varied widely to provide for
concentrations in solution of the reagents which substantially
optimize the sensitivity of the assay. Particularly, the reagents
may be provided as dry powders, usually lyophilized, including
excipients which on dissolution will provide a reagent solution
having the appropriate concentration.
[0221] F. Therapeutic Uses for the Antibody
[0222] It is contemplated that the anti-ErbB2 antibody of the
present invention may be used to treat various conditions,
including those characterized by overexpression and/or activation
of the ErbB2 receptor. Exemplary conditions or disorders to be
treated with the ErbB2 antibody include benign or malignant tumors
(e.g. renal, liver, kidney, bladder, breast, gastric, ovarian,
colorectal, prostate, pancreatic, ling, vulval, thyroid, hepatic
carcinomas; sarcomas; glioblastomas; and various head and neck
tumors); leukemias and lymphoid malignancies; other disorders such
as neuronal, glial, astrocytal, hypothalamic and other glandular,
macrophagal, epithelial, stromal and blastocoelic disorders; and
inflammatory, angiogenic and immunologic disorders.
[0223] The antibodies of the invention are administered to a
mammal, preferably a human, in accord with known methods, such as
intravenous administration as a bolus or by continuous infusion
over a period of time, by intramuscular, intraperitoneal,
intracerobrospinal, subcutaneous, intra-articular, intrasynovial,
intrathecal, oral, topical, or inhalation routes. Intravenous
administration of the antibody is preferred.
[0224] Other therapeutic regimens may be combined with the
administration of the anti-ErbB2 antibodies of the instant
invention. For example, the patient to be treated with the
antibodies disclosed herein may also receive radiation therapy.
Alternatively, or in addition, a chemotherapeutic agent may be
administered to the patient. 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). The
chemotherapeutic agent may precede, or follow administration of the
antibody or may be given simultaneously therewith. The antibody may
be combined with an anti-oestrogen compound such as tamoxifen or an
anti-progesterone such as onapristone (see, EP 616812) in dosages
known for such molecules.
[0225] It may be desirable to also administer antibodies against
other tumor associated antigens, such as antibodies which bind to
the EGFR, ErbB3, ErbB4, or vascular endothelial factor (VEGF).
Alternatively, or in addition, two or more anti-ErbB2 antibodies
may be co-administered to the patient. Sometimes, it may be
beneficial to also administer one or more cytokines to the patient.
In a preferred embodiment, the ErbB2 antibody is co-administered
with a growth inhibitory agent. For example, the growth inhibitory
agent may be administered first, followed by the ErbB2 antibody.
However, simultaneous administration or administration of the ErbB2
antibody first is also contemplated. Suitable dosages for the
growth inhibitory agent are those presently used and may be lowered
due to the combined action (synergy) of the growth inhibitory agent
and anti-ErbB2 antibody.
[0226] For the prevention or treatment of disease, the appropriate
dosage of antibody 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 is suitably administered to the patient at
one time or over a series of treatments.
[0227] 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 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. However, other dosage
regimens may be useful. The progress of this therapy is easily
monitored by conventional techniques and assays.
[0228] G. Articles of Manufacture
[0229] 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. Suitable containers include, for
example, bottles, vials, syringes, and test tubes. 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). The active
agent in the composition is the anti-ErbB2 antibody. The label on,
or associated with, the container indicates that the composition is
used for treating the condition of choice. The article of
manufacture may further comprise a second container comprising a
pharmaceutically-acceptable buffer, such as phosphate-buffered
saline, Ringers solution and dextrose solution. It may further
include other materials desirable from a commercial and user
standpoint, including other buffers, diluents, filters, needles,
syringes, and package inserts with instructions for use.
[0230] H. Deposit of Materials
[0231] The following hybridoma cell lines have been deposited with
the American Type Culture Collection, 12301 Parklawn Drive,
Rockville, Md., USA (ATCC):
TABLE-US-00001 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
[0232] These deposits were made under the provisions of the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedure and the
Regulations thereunder (Budapest Treaty). This assures maintenance
of viable cultures for 30 years from the date of deposit. The cell
lines 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 (a) that access to the cultures will
be available during pendency of the patent application to one
determined by the Commissioner to be entitled thereto under 37 CFR
.sctn.1.14 and 35 USC .sctn.122, and (b) that all restrictions on
the availability to the public of the cultures so deposited will be
irrevocably removed upon the granting of the patent.
[0233] 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 cell lines 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.
[0234] 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 antibodies deposited, since any antibody that is functionally
equivalent is 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 illustration that it represents. 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.
[0235] The following examples are offered by way of illustration
and not by way of limitation. The disclosures of all citations in
the specification are expressly incorporated herein by
reference.
Example 1
Induction of Cell Death
[0236] Cell Lines.
[0237] The established human breast tumor cells BT474 and
MDA-MB-231 (which are available from ATCC) were grown in minimum
essential medium (Gibco, Grand Island, N.Y.) supplemented with 10%
heat-inactivated fetal bovine serum (FBS) (HyClone, Logan, Utah),
sodium pyruvate, L-glutamine (2 mM), non-essential amino acids and
2.times. vitamin solution and maintained at 37.degree. C. in 5%
CO.sub.2 (Zhang et al. Invas. & Metas. 11(4):204-215 (1991) and
Price et al. Cancer Res. 50(3):717-721 (1990)).
[0238] Antibodies.
[0239] The anti-ErbB2 IgG.sub.1K murine monoclonal antibodies 4D5
and 7C2, specific for the extracellular domain of ErbB2, were
produced as described in Fendly et al. Cancer Research 50:1550-1558
(1990) and WO89/06692. 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) 84:7159 (1987) were harvested with phospate 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. MOPC-21 (IgG1), (Cappell, Durham, N.C.),
was used as an isotype-matched control.
[0240] Analysis of Cell Cycle Status and Viability.
[0241] Cells were simultaneously examined for viability and cell
cycle status by flow cytometry on a FACSTAR PLUS.TM. (Becton
Dickinson Immunocytometry Systems USA, San Jose, Calif.). Breast
tumor cells were harvested by washing the monolayer with phosphate
buffered saline (PBS), incubating cells in 0.05% trypsin and 0.53
mM EDTA (Gibco) and resuspending them in culture medium. The cells
were washed twice with PBS containing 1% FBS and the pellet was
incubated for 30 minutes on ice with 50 .mu.l of 400 .mu.M 7 amino
actinomycin D (7AAD) (Molecular Probes, Eugene, Oreg.), a vital dye
which stains all permeable cells. Cells were then fixed with 1.0 ml
of 0.5% paraformaldehyde in PBS and simultaneously permeabilized
and stained for 16 hours at 4.degree. C. with 220 .mu.l of 10
.mu.g/ml HOECHST 33342.TM. dye (also a DNA binding dye) containing
5% TWEEN 20.TM..
[0242] The data from 1.times.10.sup.4 cells were collected and
stored using LYSYS II.TM. software and analyzed using
PAINT-A-GATE.TM. software (Becton Dickinson) (Darzynkiewica et al.
Cytometry 13:795-808 (1992) and Picker et al. J. Immunol.
150(3):1105-1121 (1993)). The viability and percentage of cells in
each stage of the cell cycle were determined on gated single cells
using 7AAD and Hoechst staining, respectively. (Cell doublets were
excluded by pulse analysis of width vs. area of the Hoechst
signal.) Cell numbers were determined using a hemocytometer.
[0243] DNA Synthesis.
[0244] Triplicate cultures of 8.times.10.sup.3 cells/well were
plated in 96-well flat bottom plates, allowed to adhere overnight,
then continuously incubated in the presence or absence of
anti-ErbB2 or control Ig for different periods of time. During the
last 12 hours of culture, wells were pulsed with 1 .mu.Ci
.sup.3H-thymidine (Amersham, Arlington, Va.).
[0245] Affinity of Binding to the Extracellular Domain of the
ErbB2.
[0246] Radioiodinated anti-ErbB2 antibodies were prepared by the
Iodogen method (Fracker et al. Biochem. Biophys. Res. Comm.
80:849-857 (1978)). Binding assays were performed using monolayers
of BT474 cells cultured in 96-well tissue culture plates (Falcon,
Becton Dickenson Labware, Lincoln Park, N.J.). The cells were
trypsinized and seeded in wells of 96-well plates at a density of
10.sup.4 cells/well and allowed to adhere overnight. The monolayers
were washed with cold culture medium supplemented with 0.1% sodium
azide and then incubated in triplicate with 100 .mu.l of serial
dilutions of .sup.125I-anti-ErbB2 antibodies in cold culture medium
with 0.1% azide for 4 hours on ice. Non-specific binding was
estimated by the preincubation of each sample with a 100-fold molar
excess of nonradioactive antibodies in a total volume of 100 .mu.l.
Unbound radioactivity was removed by two washes with cold medium
with 0.1% sodium azide. The cell-associated radioactivity was
detected in a gamma counter after solubilization of the cells with
150 .mu.l 0.1 M NaOH/well. The anti-ErbB2 binding constants
(K.sub.d) were determined by Scatchard analysis.
[0247] Results.
[0248] The binding affinities of anti-ErbB2 antibodies (7C2 and
4D5) were determined by Scatchard analysis. The binding constants
(K.sub.d) were 6.5.times.10.sup.-9 M (4D5) and 2.9.times.10.sup.9 M
(7C2). Blocking experiments were carried out using unlabelled
antibodies followed by FITC-7C2. As shown in FIG. 2, 4D5 reacts
with a different epitope than 7C2.
[0249] The effect of these antibodies on the growth of the BT474
human breast cancer cells which overexpress ErbB2 was then
investigated. FIG. 3A shows the results of flow cytometric analysis
of cells incubated with an isotype-matched control. 10-12% of the
cells were dead and 28% of the viable cells were in the S-G.sub.2-M
phases of the cell cycle. Similar results were obtained when the
cells were incubated in medium alone. Treatment with 4D5 (FIG. 3B)
induced a decrease in cell size as measured by forward light
scatter, a moderate increase in the proportion of dead cells
(27.0%) and a marked decrease of viable cells in S-G.sub.2-M (6.3%)
with a concurrent increase of cells in G.sub.0/G.sub.1 (94%) as
compared to the control cells. Cell counts were reduced by 46.7%.
Without being bound to any one theory, it appears that 4D5 induces
primarily cell cycle arrest (CCA) in G.sub.0/G.sub.1 but that a
significant proportion of cells also die. FIG. 3C shows the results
of incubating the BT474 cell with 50 .mu.g/ml of 7C2. There was no
change in forward light scatter of the residual viable cells, a
marked increase in the proportion of dead cells (72%) and a 70%
decrease in cell count compared to control cells (data not shown).
Hence, viable cells were decreased by 85%. There was a slight
reduction in cycling cells (21% vs. an average of 29% for controls)
but because of extensive cell death, it was difficult to
distinguish CCA from a preferential loss of cycling cells. Hence,
7C2 and 4D5 affect cells differently; 7C2 induces predominantly
cell death while 4D5 induces predominantly CCA. FIG. 3D shows the
results of adding 50 .mu.g/ml 7C2 and 1 .mu.g/ml 4D5 to BT474 cells
simultaneously. There was no increase in the proportion of dead
cells compared to cells treated with 7C2 alone (FIG. 3C). However,
the number of residual viable cells was reduced by an additional
50%. In addition, there was a marked increase in cells having both
permeable membranes and significantly degraded DNA (<1 X). An
analysis of the small number of residual viable cells showed that
there was a similar reduction in cycling cells compared to cells
treated with 4D5 alone (FIG. 3D compared to FIG. 3B).
[0250] As an additional control, MDA-MB-231 breast cancer cells,
which express normal levels of ErbB2 (Lewis et al. Cancer Immunol.
Immunther. 37:255-263 (1993)), were treated with 405 or 7C2. As
compared to the control, neither antibody affected the growth of
these cells (27-28% of viable cells in S-G.sub.2-M and 12-13% dead
cells).
[0251] The additive effects of both antibodies was dearly
demonstrated when a suboptimal dose of 4D5 (0.05 .mu.g/ml) was
used. FIG. 4A shows that thymidine incorporation was reduced by
22.3% and 23% in cells treated with 7C2 and 4D5 respectively, and
by 58% in cells treated with 4D5 and 7C2. An additional experiment
utilizing 20 .mu.g 7C2 and 0.1 .mu.g 4D5 gave similar results,
i.e., thymidine incorporation was reduced by 41%, 25% and 72% in
cells treated with 7C2 alone, 4D5 alone, or 4D5 plus 7C2,
respectively. In FIG. 4B, the viable cell counts are shown. A total
cell count was determined and FACS analysis was used to establish
the number of viable cells. Viable cell counts were reduced by 64%,
29% and 84% in cells treated with 7C2, 4D5 or the combination,
respectively, when compared to untreated controls.
Example 2
Induction of Apoptosis
[0252] Materials and Cell Culture.
[0253] All tumor cell lines were obtained from the American Type
Culture Collection (Rockville, Md.). Cells were cultured in
Dulbecco's Modified Eagle Medium (D-MEM):Ham's F-12 (50:50)
supplemented with 10% heat-inactivated fetal bovine serum (FBS)
(Hyclone) and 2 mM L-glutamine. Human mammary epithelial cells
(HMEC) were obtained from Clonetics and grown in MEGM (mammary
epithelial growth medium, Clonetics) containing bovine pituitary
extract. Biochemicals used were: annexin V-FTIC (BioWhittaker,
Inc.), propidium iodide (PI, Molecular Probes, Inc.), and HOECHST
33342.TM. (Calbiochem). Anti-ErbB2 monoclonal antibodies (MAbs)
were produced as described in Fendly et al. Cancer Research
50:1550-1558 (1990) and WO89/06692 (see Example 1 above). The
anti-ErbB2 MAbs tested are designated: 4D5, 7C2, 7F3, 3H4, 2C4,
2H11, 3E8, and 7D3. The isotype-matched control MAb 1766 is
directed against the herpes simplex virus (HSV-1) glycoprotein
D.
[0254] Flow Cytometry Experiments for Measuring Induction of
Apoptosis.
[0255] Cells were 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 was then removed and replaced with fresh medium alone or
medium containing 10 .mu.g/ml of the appropriate MAb. For most
experiments, cells were incubated for a 3 day time period. For time
course studies, cells were treated for 0.25, 0.5, 1, 2, 24, 72, 96
hr, 7 or 10 days. MAb concentrations used in the dose-response
experiments were 0.01, 0.1, 1 and 10 .mu.g/ml. Following each
treatment, supernatants were individually collected and kept on
ice, monolayers were detached by trypsinization and pooled with the
corresponding supernatant. Cells were 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 aggregates. Each group of 3 tubes then received
annexin V-FTIC (1 .mu.g/ml) or PI (10 .mu.g/ml) or annexin V-FTIC
plus PI. Samples were analyzed using a FACSCAN.TM. flow cytometer
and FACSCONVERT.TM. CellQuest software (Becton Dickinson). For cell
cycle analysis, cells were 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).
[0256] Serum-deprivation experiments were performed in the
following way. BT474 breast tumor cells were seeded in culture
medium at a density of 5.times.10.sup.6 per dish in 100.times.20 mm
dishes. The following day, the medium was replaced with medium
containing 0.1% FBS and the cells were incubated for 3 days. Cells
then received 10 .mu.g/ml of MAb 7C2 or 4D5 in fresh medium
supplemented with 0.1% FBS. After a 3 day incubation, analyses of
annexin V binding, PI uptake and cell cycle progression were
performed as described above. In order to compare growth of
serum-starved cells to non-deprived cells, separate dishes of BT474
cells, incubated in medium supplemented with 10% FBS for all time
points, were studied in parallel.
[0257] Detection of DNA Ladder Formation.
[0258] For measuring internucleosomal fragmentation of DNA, BT474
breast tumor cells were plated and treated for 3 days with 10
.mu.g/ml MAb 4D5 or 7C2 as described above. DNA was extracted,
.sup.32P-end-labeled, and run on a 2% agarose gel containing 5
.mu.g/ml ethidium bromide. The gel was then dried and exposed to
Kodak film. The formation of DNA ladders, a hallmark of apoptosis,
was observed in BT474 breast tumor cells treated with 10 .mu.g/ml
MAb 7C2 or MAb 4D5 for 3 days.
[0259] Electron Micrography Studies.
[0260] BT474 cells were treated with 10 .mu.g/ml MAb 7C2 for 3
days, then fixed in 1.25% formaldehyde/1% glutaraldehyde in 0.1M
cacodylate buffer. Post-fixation was performed in 2% osmium
tetroxide in cacodylate buffer. The fixed cells were then end-block
stained in uranyl acetate, dehydrated in graded. concentrations of
ethanol, and embedded in Eponet. Sections were cut utilizing a
microtome and observed under a Philips CM12.TM. electron
microscope. Highly shrunken cells displaying nuclear and
cytoplasmic condensation, typical of apoptotic cells, were observed
after treatment with MAb 7C2. The apoptotic cells eventually become
phagocytosed by underlying cells.
[0261] The results of the experiments performed are shown in FIGS.
5-14. Certain anti-ErbB2 MAbs induce apoptosis in human tumor cell
lines which overexpress ErbB2 as evidenced by electron microscopy,
annexin-V binding, cell cycle analysis of DNA content, DNA
laddering and time-lapse videomicrography. Anti-ErbB2 MAbs 7C2 and
7F3, which recognize the same epitope on the ErbB2 extracellular
domain, display the most potent pro-apoptotic effects. Anti-ErbB2
MAb 4D5, which recognizes a different ErbB2 epitope, induces a
small amount of apoptosis in addition to its potent reduction in
proliferation. Induction of apoptotic cell death by 7C2 or 4D5
appears to be independent of cell cycle. Inhibition of growth,
either by serum deprivation or by treatment with 4D5, followed by
treatment with 7C2 can result in complete cell death of the
culture.
Example 3
Apoptosis of Ovarian Cells and Combination Treatment
[0262] Methods. SKOV3 ovarian cancer cells were seeded at a density
10.sup.6 per dish in 20.times.100 mm dishes and allowed to attach
for 2-3 days. The medium was then removed and replaced with fresh
medium alone or medium containing the appropriate anti-HER2MAb. For
studies on treatment with single MAb, cells were incubated with 10
.mu.g/ml MAb 7C2 or 4D5 for 3 days. For antibody combination
treatments, BT474 cells were treated first for 24 hr with 0.25 or
0.5 .mu.g/ml MAb 7C2. Following this treatment, 10 .mu.g/ml MAb 4D5
was added and the cells were incubated for 3 more days. Following
each treatment, supernatants were individually collected and kept
on ice, monolayers were detached by trypsinization and pooled with
the corresponding supernatant. Cells were then centrifuged at 1200
rpm for 5 min at 4.degree. C., the pellet resuspended in 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. Cells were stained with 1 .mu.g/ml annexin V-FITC and 10
.mu.g/ml propidium iodide (PI) and analyzed on a FACScan flow
cytometer using FACSCONVERT CELLQUEST.TM. software (Becton
Dickinson).
[0263] Induction of Apoptosis in Ovarian Cells.
[0264] The percent of remaining viable (annexin V negative/PI
negative) cells after 3 days of treatment with MAb 7C2 was reduced
by 48% (3.8 fold increase in annexin V binding) in the SKOV3
ovarian carcinoma line (FIG. 15).
[0265] Combination Treatment.
[0266] Induction of apoptosis by the sequential addition of MAbs
7C2 and 4D5 leads to additive effects compared to either antibody
alone (FIG. 16). Treatment of BT474 breast tumor cells with 0.5
.mu.g/ml MAb 7C2 followed by 10 .mu.g/ml MAb 4D5 results in a
reduction in viable (annexin V negative/PI negative) cells to 12%,
compared to 68% or 29.1% with MAb 4D5 or 7C2 alone, respectively.
This additive effect is also seen with a suboptimal dose of MAb
7C2, where treatment with 0.25 .mu.g/ml 7C2 plus 10 .mu.g/ml 4D5
leads to a decrease in viable cells to 37.5%, compared to 68% for
MAb 4D5 alone and 77% for 7C2 alone. Simultaneous addition of MAbs
7C2 and 4D5 or addition of MAb 4D5 prior to 7C2 does not appear to
lead to additive effects on cell death. Thus, without being bound
by any one theory, the sequential application of MAb 7C2, then MAb
4D5, may be important for achieving an enhanced apoptotic
effect.
Example 4
In Vivo Effects of Anti-HER2 Antibodies
[0267] A xenograft model of HER2 overexpressing human breast cancer
was established to assess the effect of anti-HER2MAbs in relation
to HER2-overexpressing tumors. The model uses the BT474 cell line
selected in vivo for growth in nude mice, BT474M1. Tumor bearing
mice were treated twice weekly with monoclonal antibodies, 4D5, 7C2
or 7F3 or combinations of 4D5 with 7C2 or 7F3. Tumor growth was
assessed by measuring tumor size twice weekly. The 4D5 monoclonal
antibody had significant growth inhibitory effects on HER2
overexpressing xenograft tumors from the BT474M1 cell line. These
results are similar to previously published results. The monoclonal
antibodies 7C2 and 7F3 had modest growth inhibitory effects on
their own, at the doses tested, and significantly enhanced the
growth inhibitory effect of 4D5. None of the antibodies exhibited
any toxic effect on the animals.
[0268] Animal Model.
[0269] NCR.nu/nu mice (homozygous females, 4 weeks of age) were
implanted subcutaneously with 0.72 mg sustained release
17.beta.estradiol pellets to support the growth of tumor. Animals
were inoculated by subcutaneous injection with 5 million BT474M1
tumor cells in MATRIGEL.TM. 24 hours after estrogen implantation.
Animals were monitored daily for well being and tumors were
measured twice weekly. Reports on tumor measurements were supplied
as they were collected. Animals were weighed weekly to assess
toxicity during the study. One hundred five (105) animals were
inoculated and all animals were evaluated in the study.
[0270] Treatment Protocol.
[0271] Animals were randomized to one of 7 treatment groups (15
animals per group). Treatment was initiated 6 days after
inoculation of tumors. Tumor sizes for all animals and mean tumor
sizes for each of the treatment groups were examined prior to
beginning treatment to ensure consistency between groups. Treatment
groups were:
1) Vehicle control injection--100 .mu.l by intraperitoneal (IP)
injection twice weekly 2) Irrelevant antibody (agp120) (isotype
matched)--10 mg/kg in 100 .mu.l IP twice weekly 3) MAb 7C.sub.2-10
mg/kg in 100 .mu.l by IP injection twice weekly 4) MAb 7F3--10
mg/kg in 100 .mu.l by IP injection twice weekly 5) MAb 4D5--10
mg/kg in 100 .mu.l by IP injection twice weekly 6) MAb 7C2 (10
mg/kg)+MAb 4D5 (10 mg/kg)--in 100 .mu.l by IP injection weekly 7)
MAb 7F3 (10 mg/kg)+MAb 4D5 (10 mg/kg)--in 100 .mu.l by IP injection
weekly
[0272] All treatment groups were treated twice weekly for a total
of 10 treatments by intraperitoneal injection. Treatment groups 1-4
were euthanized at this point due to large tumor size. Treatment
groups 5, 6 and 7 were continued and received a total of 16
antibody treatments. Throughout the study, animals were monitored
daily for well being, twice weekly for tumor measurements and
weekly for body weights. Individual animals data and mean data by
treatment group was supplied as it became available.
[0273] Termination of Experiment.
[0274] After conclusion of treatment, animals in treatment groups
1-4 were euthanized due to some large tumor sizes within these
treatment groups. Tumor volume was not allowed to exceed 4 gms
(4,000 mm.sup.3). No animals were observed with significant weight
loss and weight loss greater than 15% loss of body weight from the
weight at onset of treatment was never observed. At the conclusion
of the experiment, all animals were euthanized.
[0275] Results.
[0276] The monoclonal antibodies, 4D5, 7C2 and 7F3, directed
against the HER2 growth factor receptor, were used in a mouse
xenograft model which overexpresses the HER2 receptor. Antibodies
were used alone and in combinations of growth inhibiting antibody
(4D5) with apoptotic antibodies (7C2 and 7F3). The apoptotic
antibodies (7C2 and 7F3) had a growth inhibitory effect early on in
the study that was lost at later time points (FIG. 17). The growth
inhibitory antibody, 4D5, had a marked growth inhibitory effect
throughout the study, as has been reported in previous studies. The
combination of 4D5 with either 7C2 or 7F3 potentiated the growth
inhibitory effect significantly, with 4D5/7C2 being the best
combination. There was one complete remission in the 4D5 alone
treatment group and one complete remission in the 4D5/7C2 treatment
group. The antibody control group (anti-gp120 MAb) was equivalent
to the saline treated control group. Body weights initially
increased after tumor inoculation, then were maintained through the
remainder of the study. None of the antibodies exhibited any toxic
effect on the animals.
Sequence CWU 1
1
91166PRTHomo sapiens 1Cys Thr Gly Thr Asp Met Lys Leu Arg Leu Pro
Ala Ser Pro Glu Thr 1 5 10 15 His Leu Asp Met Leu Arg His Leu Tyr
Gln Gly Cys Gln Val Val Gln 20 25 30 Gly Asn Leu Glu Leu Thr Tyr
Leu Pro Thr Asn Ala Ser Leu Ser Phe 35 40 45 Leu Gln Asp Ile Gln
Glu Val Gln Gly Tyr Val Leu Ile Ala His Asn 50 55 60 Gln Val Arg
Gln Val Pro Leu Gln Arg Leu Arg Ile Val Arg Gly Thr 65 70 75 80 Gln
Leu Phe Glu Asp Asn Tyr Ala Leu Ala Val Leu Asp Asn Gly Asp 85 90
95 Pro Leu Asn Asn Thr Thr Pro Val Thr Gly Ala Ser Pro Gly Gly Leu
100 105 110 Arg Glu Leu Gln Leu Arg Ser Leu Thr Glu Ile Leu Lys Gly
Gly Val 115 120 125 Leu Ile Gln Arg Asn Pro Gln Leu Cys Tyr Gln Asp
Thr Ile Leu Trp 130 135 140 Lys Asp Ile Phe His Lys Asn Asn Gln Leu
Ala Leu Thr Leu Ile Asp 145 150 155 160 Thr Asn Arg Ser Arg Ala 165
232PRTHomo sapiens 2Ser Thr Gln Val Cys Thr Gly Thr Asp Met Lys Leu
Arg Leu Pro Ala 1 5 10 15 Ser Pro Glu Thr His Leu Asp Met Leu Arg
His Leu Tyr Gln Gly Cys 20 25 30 359PRTHomo sapiens 3Val Glu Glu
Cys Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn 1 5 10 15 Ala
Arg His Cys Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly 20 25
30 Ser Val Thr Cys Phe Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala
35 40 45 His Tyr Lys Asp Pro Pro Phe Cys Val Ala Arg 50 55
497PRTHomo sapiens 4Val Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys
Val Glu Glu Cys 1 5 10 15 Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr
Val Asn Ala Arg His Cys 20 25 30 Leu Pro Cys His Pro Glu Cys Gln
Pro Gln Asn Gly Ser Val Thr Cys 35 40 45 Phe Gly Pro Glu Ala Asp
Gln Cys Val Ala Cys Ala His Tyr Lys Asp 50 55 60 Pro Pro Phe Cys
Val Ala Arg Cys Pro Ser Gly Val Lys Pro Asp Leu 65 70 75 80 Ser Tyr
Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln 85 90 95
Pro 511PRTArtificial SequenceSynthetic Salvage Receptor Binding
Epitope 5Pro Lys Asn Ser Ser Met Ile Ser Asn Thr Pro 1 5 10
67PRTArtificial SequenceSynthetic Salvage Receptor Binding Epitope
6His Gln Ser Leu Gly Thr Gln 1 5 78PRTArtificial SequenceSynthetic
Salvage Receptor Binding Epitope 7His Gln Asn Leu Ser Asp Gly Lys 1
5 88PRTArtificial SequenceSynthetic Salvage Receptor Binding
Epitope 8His Gln Asn Ile Ser Asp Gly Lys 1 5 98PRTArtificial
SequenceSynthetic Salvage Receptor Binding Epitope 9Val Ile Ser Ser
His Leu Gly Gln 1 5
* * * * *