U.S. patent application number 13/849218 was filed with the patent office on 2013-08-15 for erbb3 antibodies.
This patent application is currently assigned to GENENTECH, INC.. The applicant listed for this patent is Robert Akita, Mark Sliwkowski. Invention is credited to Robert Akita, Mark Sliwkowski.
Application Number | 20130209495 13/849218 |
Document ID | / |
Family ID | 26724364 |
Filed Date | 2013-08-15 |
United States Patent
Application |
20130209495 |
Kind Code |
A1 |
Akita; Robert ; et
al. |
August 15, 2013 |
ErbB3 ANTIBODIES
Abstract
Antibodies are disclosed which bind to ErbB3 protein and further
possess any one or more of the following properties: an ability to
reduce heregulin-induced formation of an ErbB2-ErbB3 protein
complex in a cell which expresses ErbB2 and ErbB3; the ability to
increase the binding affinity of heregulin for ErbB3 protein; and
the characteristic of reducing heregulin-induced ErbB2 activation
in a cell which expresses ErbB2 and ErbB3.
Inventors: |
Akita; Robert; (Hayward,
CA) ; Sliwkowski; Mark; (San Carlos, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Akita; Robert
Sliwkowski; Mark |
Hayward
San Carlos |
CA
CA |
US
US |
|
|
Assignee: |
GENENTECH, INC.
South San Francisco
CA
|
Family ID: |
26724364 |
Appl. No.: |
13/849218 |
Filed: |
March 22, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13181115 |
Jul 12, 2011 |
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13849218 |
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11943490 |
Nov 20, 2007 |
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13181115 |
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11051056 |
Feb 4, 2005 |
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11943490 |
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09825584 |
Apr 4, 2001 |
7285649 |
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11051056 |
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09316981 |
May 24, 1999 |
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09825584 |
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08827009 |
Mar 25, 1997 |
5968511 |
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09316981 |
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60046850 |
Mar 27, 1996 |
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Current U.S.
Class: |
424/178.1 ;
435/188; 435/334; 435/7.23; 530/389.7; 530/391.3; 530/391.7 |
Current CPC
Class: |
A61P 25/00 20180101;
C07K 2317/76 20130101; C07K 16/32 20130101; A61P 35/00 20180101;
C07K 2317/55 20130101; C07K 16/2863 20130101; G01N 33/74 20130101;
G01N 2333/71 20130101; A61K 2039/505 20130101; A61P 29/00
20180101 |
Class at
Publication: |
424/178.1 ;
530/391.7; 530/391.3; 530/389.7; 435/334; 435/7.23; 435/188 |
International
Class: |
C07K 16/28 20060101
C07K016/28; G01N 33/74 20060101 G01N033/74 |
Claims
1. An antibody which binds to ErbB3 protein and reduces
heregulin-induced formation of an ErbB2-ErbB3 protein complex in a
cell which expresses ErbB2 and ErbB3, wherein the antibody is
conjugated to a cytotoxic agent.
2. The antibody of claim 1 which further increases the binding
affinity of heregulin for ErbB3 protein.
3. The antibody of claim 1 which further reduces heregulin-induced
ErbB2 activation in the cell.
4. The antibody of claim 1 which is a monoclonal antibody.
5. The antibody of claim 1 which is humanized.
6. The antibody of claim 1 which is human.
7. The antibody of claim 1 which is an antibody fragment.
8. The antibody fragment of claim 7 which is a Fab.
9. The antibody of claim 1 which is labelled.
10. The antibody of claim 1 which is immobilized on a solid
phase.
11. (canceled)
12. An antibody which binds to ErbB3 protein and reduces
heregulin-induced ErbB2 activation in a cell which expresses ErbB2
and ErbB3.
13. An antibody which binds to ErbB3 protein and reduces heregulin
binding thereto.
14. The antibody of claim 13 which further reduces
heregulin-induced ErbB2 activation in a cell which expresses ErbB2
and ErbB3
15. The antibody of claim 1 which binds to the epitope bound by the
8B8 antibody.
16. The antibody of claim 1 which has the complementarity
determining regions of the 8B8 antibody.
17. A composition comprising the antibody of claim 1 and a
pharmaceutically acceptable carrier.
18. A cell line which produces the antibody of claim 1.
19. The cell line of claim 18 which is a hybridoma cell line
producing the 8B8 antibody.
20. A method for determining the presence of ErbB3 protein
comprising exposing a cell suspected of containing the ErbB3
protein to the antibody of claim 1 and determining binding of said
antibody to the cell.
21. A kit comprising the antibody of claim 1 and instructions for
using the antibody to detect the ErbB3 protein.
22. The antibody of claim 1, wherein the cytotoxic agent is
selected from the group consisting of a toxin, chemotherapeutic
agent, and radioactive isotope.
23. The antibody of claim 1, wherein the cytotoxic agent is a
toxin.
24. The antibody of claim 22 or 23, wherein the toxin is an
enzymatically active toxin, or fragment thereof.
25. The antibody of claim 24, wherein the enzymatically active
toxin is of bacterial origin.
26. The antibody of claim 24, wherein the enzymatically active
toxin is of fungal origin.
27. The antibody of claim 24, wherein the enzymatically active
toxin is of plant origin.
28. The antibody of claim 24, wherein the enzymatically active
toxin is of animal origin.
29. The antibody of claim 22, wherein the cytotoxic agent is a
chemotherapeutic agent.
30. The antibody of claim 22, wherein the cytotoxic agent is a
radioactive isotope.
Description
[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/______ (to be assigned) filed
on Mar. 27, 1996.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates generally to antibodies which bind
the ErbB3 receptor. In particular, it relates to anti-ErbB3
antibodies which, surprisingly, increase the binding affinity of
heregulin (HRG) for ErbB3 protein and/or reduce HRG-induced
formation of an ErbB2-ErbB3 protein complex in a cell which
expresses both these receptors and/or reduce heregulin-induced
ErbB2 activation in such a cell.
[0004] 2. Description of Related Art
[0005] Transduction of signals that regulate cell growth and
differentiation is regulated in part by phosphorylation of various
cellular proteins. Protein tyrosine kinases are enzymes that
catalyze this process. Receptor protein tyrosine kinases are
believed to direct cellular growth via ligand-stimulated tyrosine
phosphorylation of intracellular substrates. Growth factor receptor
protein tyrosine kinases of the class I subfamily include 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
more aggressive carcinomas of the breast, bladder, lung and
stomach.
[0006] 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 neu gene (also
called erbB2 and HER2) encodes a 185 kDa receptor protein tyrosine
kinase. Amplification and/or overexpression of the human HER2 gene
correlates with a poor prognosis in breast and ovarian cancers
(Slamon et al., Science, 235:177-182 (1987); and Slamon et al.,
Science, 244:707-712 (1989)). Overexpression of HER2 has also been
correlated with other carcinomas including carcinomas of the
stomach, endometrium, salivary gland, lung, kidney, colon and
bladder.
[0007] A further related gene, called erbB3 or HER3, has also been
described. See U.S. Pat. Nos. 5,183,884 and 5,480,968; Plowman et
al., Proc. Nail. Acad. Sci. USA, 87:4905.4909 (1990); Kraus et al.,
Proc. Natl. Acad. Sci. USA, 86:9193-9197 (1989); EP Pat Appln No
444,961A1; and Kraus et al., Proc. Natl. Aced. Sci. USA,
90:2900-2904 (1993). Kraus et al. (1989) discovered that markedly
elevated levels of erbB3 mRNA were present in certain human mammary
tumor cell lines indicating that erbB3, like erbB1 and erbB2, may
play a role in some human malignancies. These researchers
demonstrated that some human mammary tumor cell lines display
significant elevation of steady-state ErbB3 tyrosine
phosphorylation, further indicating that this receptor may play a
role in human malignancies. Accordingly, diagnostic bioassays
utilizing antibodies which bind to ErbB3 are described by Kraus et
al. in U.S. Pat. Nos. 5,183,884 and 5,480,968.
[0008] The role of erbB3 in cancer has been explored by others. It
has been found to be overexpressed in breast (Lemoine et al., Br.
J. Cancer, 66:1116-1121 (1992)), gastrointestinal (Poller et al.,
J. Pathol., 168:275-280 (1992), Rajkumer et al., J. Pathol.,
170:271-278 (1993), and Sanidas et al., Int. J. Cancer, 54:935-940
(1993)), and pancreatic cancers (Lemoine et al., J. Pathol.,
168:269-273 (1992), and Friess et al., Clinical Cancer Research,
1:1413-1420 (1995)).
[0009] ErbB3 is unique among the ErbB receptor family in that it
possesses little or no intrinsic tyrosine kinase activity (Guy et
al., Proc. Natl. Acad. Sci. USA 91:8132-8136 (1994) and Kim et al.
J. Biol. Chem. 269:24747-55 (1994)). 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.
[0010] Rajkumar et al., British Journal Cancer, 70(3):459-465
(1994), developed a monoclonal antibody against ErbB3 which had an
agonistic effect on the anchorage-independent growth of cell lines
expressing this receptor.
[0011] The class I subfamily of growth factor receptor protein
tyrosine kinases has been, further extended to include the
HER4/p180.sup.erbB4 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 closely correlated with certain
carcinomas of epithelial origin, including breast adenocarcinomas.
Accordingly, diagnostic methods for detection of human neoplastic
conditions (especially breast cancers) which evaluate HER4
expression are described in EP Pat Appln No. 599,274.
[0012] The quest for an activator of the HER2 oncogene has lead to
the discovery of a family of heregulin polypeptides. These proteins
appear to result from alternative splicing of a single gene which
was Mapped to the short arm of human chromosome 8 by Lee et al.,
Genomics, 16:790-791. (1993); and Orr-Urtreger et al, Proc. Natl.
Acad. Sci. USA, 1952:1746-1750 (1993).
[0013] Holmes et al. isolated and cloned a family of polypeptide
activators for the HER2 receptor which they termed
heregulin-.alpha. (HRG-.alpha.), heregulin-.beta.1 (HRG-.beta.1),
heregulin-.beta.2 (HRG-.beta.2), heregulin-.beta.2-like
(HRG-.beta.2-like), and heregulin-.beta.3 (HRG-.beta.3). See Holmes
et al., Science, 256:1205-1210 (1992); and WO 92/20798. The 45 kDa
polypeptide, HRG-.alpha., was purified from the conditioned medium
of the MDA-MB-231 human breast cancer cell line. These researchers
demonstrated the ability of the purified heregulin polypeptides to
activate tyrosine phosphorylation of the HER2 receptor in MCF-7
breast tumor cells. Furthermore, the mitogenic activity of the
heregulin polypeptides on SK-BR-3 cells (which express high levels
of the HER2 receptor) was illustrated. Like other growth factors
which belong to the EGF family, soluble HRG polypeptides appear to
be derived from a membrane bound precursor (called pro-HRG) which
is proteolytically processed to release the 45 kDa soluble form.
These pro-HRGs lack a N-terminal signal peptide.
[0014] While heregulins are substantially identical in the first
213 amino acid residues, they are classified into two major types,
.alpha. and .beta., based on two variant EGF-like domains which
differ in their C-terminal portions. Nevertheless, these EGF-like
domains are identical in the spading of six cysteine residues
contained therein. Based on an amino acid sequence comparison,
Holmes et al. found that between the first and sixth cysteines in
the EGF-like domain, HRGs were 45% similar to heparin-binding
EGF-like growth factor (HB-EGF), 35% identical to amphiregulin
(AR), 32% identical to TGF-.alpha., and 27% identical to EGF.
[0015] The 44 kDa neu differentiation factor (NDF), which is the
rat equivalent of human HRG, was first described by Peles et al.,
Cell, 69:205-216 (1992); and Wen et al., Cell, 69:559-572 (1992).
Like the HRG polypeptides, NDF has an immunoglobulin (Ig) homology
domain followed by an EGF-like domain and lacks a N-terminal signal
peptide. Subsequently, Wen at al., Mol. Cell. Biol.,
14(3):1909-1919 (1994) carried out "exhaustive cloning" to extend
the family of NDFs. This work revealed six distinct fibroblastic
pro-NDFs. Adopting the nomenclature of Holmes at al., the NDFs are
classified as either .alpha. or .beta. polypeptides based on the
sequences of the EGF-like domains. Isoforms 1 to 4 are
characterized on the basis of the variable juxtamembrane stretch
(between the EGF-like domain and transmembrane domain). Also,
isoforms a, b and c are described which have variable length
cytoplasmic domains. These researchers conclude that different NDF
isoforms are generated by alternative splicing and perform distinct
tissue-specific functions.
[0016] Falls et al., Cell, 72:801-815 (1993) describe another
member of the heregulin family which they call acetylcholine
receptor inducing activity (ARIA) polypeptide. The chicken-derived
ARIA polypeptide stimulates synthesis of muscle acetylcholine
receptors. See also WO 94/08007. ARIA is a .beta.-type heregulin
and lacks the entire "glyco" spacer (rich in glycosylation sites)
present between the Ig-like domain and EGF-like domain of
HRG.alpha., and HRG.beta.1-.beta.3.
[0017] Marchionni at al., Nature, 362:312-318 (1993) identified
several bovine-derived proteins which they call glial growth
factors (GGFs). These GGFs share the Ig-like domain and EGF-like
domain with the other heregulin proteins described above, but also
have an amino-terminal kringle domain. GGFs generally do not have
the complete "glyco" spacer between the Ig-like domain and EGF-like
domain. Only one of the GGFs, GGFII, possessed a N-terminal signal
peptide.
[0018] Expression of the ErbB2 family of receptors and heregulin
polypeptides in breast cancer is reviewed in Bacus et al.,
Pathology Patterns, 102(4)(Supp. 1):S13-S24 (1994).
[0019] See also, Alimandi et al., Oncogene, 10:1813-1821 (1995);
Beerli et al., Molecular and Cellular Biology, 15:6496-6505 (1995);
Karunagaran et al., EMBO J, 15:254-264 (1996); Wallasch et al.,
EMBO J, 14:42674275 (1995); and Zhang et al., Journal of Biological
Chemistry, 271:3884-3890 (1996), in relation to the above receptor
family.
SUMMARY OF THE INVENTION
[0020] This invention provides antibodies which bind to ErbB3
protein and further possess any one or more of the following
properties: an ability to reduce heregulin-induced formation of an
ErbB2-ErbB3 protein complex in a cell which expresses ErbB2 and
ErbB3; the ability to increase the binding affinity of heregulin
for ErbB3 protein; and the characteristic of reducing
heregulin-induced ErbB2 activation in a cell which expresses ErbB2
and ErbB3.
[0021] The invention also relates to an antibody which binds to
ErbB3 protein and reduces heregulin binding thereto.
[0022] Preferred antibodies are monoclonal antibodies which bind to
an epitope in the extracellular domain of the ErbB3 receptor.
Generally, antibodies of interest will bind the ErbB3 receptor with
an affinity of at least about 10 nM, more preferably at least about
1 nM. 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.
[0023] 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.
[0024] 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 cell line comprising the nucleic acid (e.g. a
hybridoma cell line); and a process of using a nucleic acid
molecule encoding the antibody to effect production of the antibody
comprising culturing a cell comprising the nucleic acid and,
optionally, recovering the antibody from the cell culture and,
preferably, the cell culture medium.
[0025] The invention also provides a method for treating a mammal
comprising administering a therapeutically effective amount of the
antibody described herein to the mammal, wherein the mammal has a
disorder requiring treatment with the antibody.
[0026] In a further aspect, the invention provides a method for
detecting ErbB3 in vitro or in vivo comprising contacting the
antibody with a cell suspected of containing ErbB3 and detecting if
binding has occurred. Accordingly, the invention provides an assay
for detecting a tumor characterized by amplified expression of
ErbB3 comprising the steps of exposing a cell to the antibody
disclosed herein and determining the extent of binding of the
antibody to the cell. Generally the antibody for use in such an
assay will be labelled. 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 generally 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] FIG. 1 depicts HRG binding to K562 ErbB3 cells in the
presence of various anti-ErbB3 monoclonal antibodies. Purified
anti-ErbB3 antibodies were incubated with a suspension of K562
ErbB3 cells and .sup.125I-HRG.beta.1.sub.(177-244). After
approximately 18 hours on ice, cell bound counts were measured.
Counts are shown plotted as a percentage of binding in the absence
of antibody (control). Non-specific binding was determined using an
excess of unlabeled HRG.beta.1.sub.(177-244)(HRG). Antibodies
against ErbB2 protein (2C4) and HSV (5136) were used as negative
controls.
[0028] FIG. 2 shows the effect of antibody concentration on HRG
binding. A dose-response experiment was performed on the 3-8D6
antibody which was found to enhance HRG binding. K562 ErbB3 cells
were incubated with a fixed concentration of .sup.125I-HRG and
increasing concentrations of the 3-8D6 antibody. Data from the
experiment is shown plotted as cell bound counts versus antibody
concentration.
[0029] FIG. 3 illustrates HRG binding to K562 ErbB3 cells in the
presence and absence of the 3-8D6 antibody or a Fab fragment
thereof. Competitive ligand binding experiments were performed in
the absence (control) and presence of 100 nM 3-8D6 or Fab. The data
are plotted as bound/total (BPI) versus total
HRG.beta.1.sub.(177-244).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions
[0030] Unless indicated otherwise, the term "ErbB3" when used
herein refers to mammalian ErbB3 protein and "erbB3" refers to
mammalian erbB3 gene. The preferred ErbB3 protein is human ErbB3
protein present in the cell membrane of a cell. The human erbB3
gene is described in U.S. Pat. No. 6,480,968 and Plowman et al.,
Proc. Natl. Acad. Sci. USA, 87:4905-4909 (1990).
[0031] The antibody of interest may be one which does not
significantly cross-react with other proteins such as those encoded
by the erbB1, erbB2 and/or erbB4 genes. In such embodiments, the
extent of binding of the antibody to these non-ErbB3 proteins
(e.g., cell surface binding to endogenous receptor) will be less
than 10% as determined by fluorescence activated cell sorting
(FACS) analysis or radioimmunoprecipitation (RIA). However,
sometimes the antibody may be one which does cross-react with ErbB4
receptor, and, optionally, does not cross-react with the EGFR
and/or ErbB2 receptor, for example.
[0032] "Heregulin" (HRG) when used herein refers to a polypeptide
which activates the ErbB2-ErbB3 protein complex (i.e. induces
phosphorylation of tyrosine residues in the ErbB2-ErbB3 complex
upon binding thereto). Various heregulin polypeptides encompassed
by this term have been disclosed above. 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).
[0033] The "ErbB2-ErbB3 protein complex" is a noncovalently
associated oligomer of the ErbB2 receptor and the ErbB3 receptor.
This complex forms when a cell expressing both of these receptors
is exposed to HRG. The complex can be isolated by
immunoprecipitation and analyzed by SDS-PAGE as described in the
Example below.
[0034] The expression "reduces heregulin-induced formation of an
ErbB2-ErbB3 protein complex in a cell which expresses ErbB2 and
ErbB3" refers to the ability of the antibody to statistically
significantly reduce the number of ErbB2-ErbB3 protein complexes
which form in a cell which has been exposed to the antibody and HRG
relative to an untreated (control) cell The cell which expresses
ErbB2 and ErbB3 can be a naturally occurring cell or cell line
(e.g. Caov3 cell) or can be recombinantly produced by introducing
nucleic acid encoding each of these proteins into a host cell.
Preferably, the antibody will reduce formation of this complex by
at least 50%, and more preferably at least 70%, as determined by
reflectance scanning densitometry of Western blots of the complex
(see the Example below).
[0035] The antibody which "reduces heregulin-induced ErbB2
activation in a cell which expresses ErbB2 and ErbB3" is one which
statistically significantly reduces tyrosine phosphorylation
activity of ErbB2 which occurs when HRG binds to ErbB3 in the
ErbB2-ErbB3 protein complex (present at the surface of a cell which
expresses the two receptors) relative to an untreated (control)
cell. This can be determined based on phosphotyrosine levels in the
ErbB2-ErbB3 complex following exposure of the complex to HRG and
the antibody of interest. The cell which expresses ErbB2 and ErbB3
protein can be a naturally occurring cell or cell line (e.g. Caov3
cell) or can be recombinantly produced. ErbB2 activation can be
determined by Western blotting followed by probing with an
anti-phosphotyrosine antibody as described in the Example below.
Alternatively, the kinase receptor activation assay described in WO
95/14930 and Sadick et al., Analytical Biochemistry, 235:207-214
(1996) can be used to quantify ErbB2 activation. Preferably, the
antibody will reduce heregulin-induced ErbB2 activation by at least
60%, and more preferably at least 70%, as determined by reflectance
scanning densitometry of Western blots of the complex probed with
an anti-phosphotyrosine antibody (see the Example below).
[0036] The antibody may be one which "increases the binding
affinity of heregulin for ErbB3 protein". This means that, in the
presence of the antibody (e.g. 100 nM antibody), the amount of HRG
which binds to ErbB3 (e.g., endogenous ErbB3 present in a naturally
occurring cell or cell line or introduced into a cell by
recombinant techniques, see the Example below), relative to control
(no antibody), is statistically significantly increased. For
example, the amount of ELM which binds to the K562 cell line
transfected with erbB3 as described herein may be increased in the
presence of 100 nM antibody by at least 10% preferably at least 50%
and most preferably at least about 100% (see FIG. 1), relative to
control.
[0037] The antibody which reduces HRG binding to ErbB3 protein
(e.g. ErbB3 present in a cell) is one which interferes with the
HRG-binding site on ErbB3 protein such that it statistically
significantly decreases the amount of heregulin which is able to
bind to this site on the molecule. Exemplary such antibodies are
the 3-2F9, 3-3E9 and 3-6B9 antibodies described in the Example
herein.
[0038] 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. The
antibody may be an IgM, IgG (e.g. IgG.sub.1, IgG.sub.2, IgG.sub.3
or IgG.sub.4), IgD, IgA or IgE, for example. Preferably however,
the antibody is not an IgM antibody.
[0039] "Antibody fragments" comprise a portion of an intact
antibody, generally 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; single-chain antibody
molecules; and multispecific antibodies formed from antibody
fragments.
[0040] 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, 266:496 (1976), or may be made by recombinant DNA methods
(see, e.g., U.S. Pat. No. 4,816,667). The "monoclonal antibodies"
may also be isolated from phage antibody libraries using the
techniques described in Clackson et al., Nature, 362:624-628 (1991)
and Marks et al., J. Mol. Biol., 222:581-597 (1991), for
example.
[0041] 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. 4,816,667; Morrison et
al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
[0042] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric immunoglobulins, immunoglobulin chains or fragments
thereof (such as Fv, Fab, Fab', F(ab % 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
optimize 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, 321:622-526 (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.
[0043] "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. Generally, 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. 269-316
(1994).
[0044] 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.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L).
By using a linker that is too short to allow pairing between the
two domains on the same chain, the domains are forced to pair with
the complementary domains of another chain and create two
antigen-binding sites. Diabodies are described more fully in, for
example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.
Acad. Sci. USA, 90:6444-6448 (1993).
[0045] 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.
[0046] 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.
[0047] "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.
[0048] "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.
[0049] A "disorder" is any condition that would benefit from
treatment with the anti-ErbB3 antibody. This includes chronic and
acute disorders or diseases including those pathological conditions
which predispose the mammal to the disorder in question. Generally,
the disorder will be one in which excessive activation of the
ErbB2-ErbB3 protein complex by heregulin is occurring. 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.
[0050] 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, renal cancer,
prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma
and various types of head and neck cancer.
[0051] 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, Y, Pr), chemotherapeutic agents, and
toxins such as enzymatically active toxins of bacterial, fungal,
plant or animal origin, or fragments thereof.
[0052] 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, 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.
[0053] 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;
integrins 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-6, 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.
[0054] 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. 376-382,
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.
[0055] The word "label" when used herein refers to a detectable
compound or composition which is conjugated directly or indirectly
to the 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.
[0056] 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.
4,276,149.
[0057] 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-ErbB3 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.
[0058] 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.
[0059] 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.
[0060] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
[0061] 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
[0062] A. Antibody Preparation
[0063] A description follows as to exemplary techniques for the
production of the claimed antibodies.
[0064] (i) Polyclonal Antibodies
[0065] Polyclonal antibodies are generally 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.
[0066] Animals are immunized against the antigen, immunogenic
conjugates, or derivatives by combining, e.g., 100 .mu.g or 6 .mu.g
of the protein or conjugate (for rabbits or mice, respectively)
with 3 volumes of Freund's complete adjuvant and injecting the
solution intradermally at multiple sites. One month later the
animals are boosted with 1/5 to 1/10 the original amount of peptide
or conjugate in Freund's complete adjuvant by subcutaneous
injection at multiple sites. Seven to 14 days later the animals are
bled and the serum is assayed for antibody titer. Animals are
boosted until the titer plateaus. Preferably, the animal is boosted
with the conjugate of the same antigen, but conjugated to a
different protein and/or through a different cross-linking reagent.
Conjugates also can be made in recombinant cell culture as protein
fusions. Also, aggregating agents such as alum are suitably used to
enhance the immune response.
[0067] (iii) Monoclonal Antibodies
[0068] 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.
[0069] For example, the monoclonal antibodies may be made using the
hybridoma method first described by Kohler et al., Nature, 256:495
(1976), or may be made by recombinant DNA methods (U.S. Pat. No.
4,816,567).
[0070] 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
(coding, Monoclonal Antibodies: Principles and Practice, pp. 59-103
(Academic Press, 1986)).
[0071] 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.
[0072] 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 X63Ag8-653 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)).
[0073] 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).
[0074] 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).
[0075] 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.
[0076] 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.
[0077] 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).
[0078] 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.
[0079] 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.
[0080] 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.
[0081] (iii) Humanized and Human Antibodies
[0082] Methods for humanizing non-human antibodies are well known
in the art. Generally, 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, 321:522-625 (1986); Riechmann et al., Nature, 332:323-327
(1988); Verhoeyen et al., Science, 239:1634-1536 (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.
[0083] 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., 161:2296 (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., 161:2623 (1993)).
[0084] 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.
[0085] 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 (JO 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, 362:265-258 (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)).
[0086] (iv) Antibody Fragments
[0087] 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.
[0088] (v) Bispecific Antibodies
[0089] 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
ErbB3 protein. Other such antibodies may combine an ErbB3 binding
site with binding site(s) for EGFR, ErbB2 and/or ErbB4.
Alternatively, an anti-ErbB3 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 ErbB3-expressing cell. Bispecific antibodies may also be used
to localize cytotoxic agents to cells which express ErbB3. These
antibodies possess an ErbB3-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).
[0090] 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).
[0091] 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.
[0092] 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).
[0093] According to another approach, 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.
[0094] 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.
[0095] 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 victual 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.
[0096] 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., 176:
217-226 (1992) describe the production of a fully humanized
bispecific antibody F(ab').sub.2 molecule. Each Fab' fragment was
separately secreted from E. coli and subjected to directed chemical
coupling in vitro to form the bispecific antibody. The bispecific
antibody thus formed was able to bind to cells overexpressing the
HER2 receptor and normal human T cells, as well as trigger the
lytic activity of human cytotoxic lymphocytes against human breast
tumor targets.
[0097] 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., 162:6368 (1994).
[0098] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al. J.
Immunol. 147: 60 (1991).
[0099] (vi) Screening for Antibodies with the Desired
Properties
[0100] Techniques for generating antibodies have been described
above. Those antibodies having the characteristics described herein
are selected.
[0101] To select for antibodies which reduce HRG-induced formation
of the ErbB2-ErbB3 protein complex, cells which express both these
receptors (e.g. Caov3 cells) can be pre-incubated with buffer
(control) or antibody, then treated with HRG or control buffer. The
cells are then lysed and the crude lysates can be centrifuged to
remove insoluble material. Supernatants may be incubated with an
antibody specific for ErbB2 covalently coupled to a solid phase.
Following washing, the immunoprecipitates may be separated by
SDS-PAGE. Western blots of the gels are then probed with anti-ErbB3
antibody. After visualization, the blots may be stripped and
re-probed with an anti-ErbB2 antibody. Reflectance scanning
densitometry of the gel can be performed in order to quantify the
effect of the antibody in question on HRG-induced formation of the
complex. Those antibodies which reduce formation of the ErbB2-ErbB3
complex relative to control (untreated cells) can be selected. See
the Example below.
[0102] To select for those antibodies which reduce HRG-induced
ErbB2 activation in a cell which expresses the ErbB2 and ErbB3
receptor, the cells can be pre-incubated with buffer (control) or
antibody, then treated with HRG or control buffer. The cells are
then lysed and the crude lysates can be centrifuged to remove
insoluble material. ErbB2 activation can be determined by Western
blotting followed by probing with an anti-phosphotyrosine antibody
as described in the Example below. ErbB2 activation can be
quantified via reflectance scanning densitometry of the gel, for
example. Alternatively, the kinase receptor activation assay
described in WO 95/14930 and Sadick et al., Analytical
Biochemistry, 235:207-214 (1996) can be used to determine ErbB2
activation.
[0103] The effect of the antibody on HRG binding to ErbB3 can be
determined by incubating cells which express this receptor (e.g.
4E9H3 cells transfected to express ErbB3) with radiolabelled HRG
(e.g. the EGF-like domain thereof), in the absence (control) or
presence of the anti-ErbB3 antibody, as described in the Example
below, for example. Those antibodies which increase the binding
affinity of HRG for the ErbB3 receptor can be selected for further
development. Where the antibody of choice is one which blocks
binding of HRG to ErbB3, those antibodies which do so in this assay
can be identified.
[0104] To screen for antibodies which bind to the epitope on ErbB3
bound by an antibody of interest (e.g., those which block binding
of the 3-8B8 antibody to ErbB3), 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.
[0105] (vii) Effector Function Engineering
[0106] 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).
[0107] (viii) Immunoconjugates
[0108] 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).
[0109] 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-ErbB8 antibodies. Examples include .sup.212Bi, .sup.131I,
.sup.90Y and .sup.186Re.
[0110] 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 his (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.
[0111] 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 radionuclide).
[0112] (ix) Immunoliposomes
[0113] The anti-ErbB3 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 4,644,646. Liposomes with enhanced circulation
time are disclosed in U.S. Pat. No. 6,013,566.
[0114] 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. 267: 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)
[0115] (x) Antibody Dependent Enzyme Mediated Prodrug Therapy
(ADEPT)
[0116] 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 WO81/01146) to an active anti-cancer drug. See, for example, WO
88/07378 and U.S. Pat. No. 4,976,278.
[0117] 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.
[0118] 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, thermolysis, 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 .beta.-galactosidase and neuraminidase useful for
converting glycosylated prodrugs into free drugs; .beta.-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.
[0119] The enzymes of this invention can be covalently bound to the
anti-ErbB3 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)).
[0120] (xi) Antibody-Salvage Receptor Binding Epitope Fusions.
[0121] 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).
[0122] 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.
[0123] 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.
[0124] The epitope generally 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.
[0125] In one most preferred embodiment, the salvage receptor
binding epitope comprises the sequence (5' to 3'): PKNSSMISNTP (SEQ
ID NO: 1), and optionally further comprises a sequence selected
from the group consisting of HQSLGTQ (SEQ ID NO: 2), HQNLSDGK (SEQ
ID NO: 3), HQNISDGK (SEQ ID NO: 4), or VISSHLGQ (SEQ ID NO: 6),
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: 3), HQNISDGK (SEQ ID NO: 4), or VISSHLGQ (SEQ
ID NO: 6) and the sequence: PKNSSMISNTP (SEQ ID NO: 1).
[0126] B. Vectors, Host Cells and Recombinant Methods
[0127] 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.
[0128] 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. 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 generally 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.
[0129] (i) Signal Sequence Component
[0130] The anti-ErbB3 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-ErbB3 antibody signal sequence, the signal
sequence is substituted by a prokaryotic signal sequence selected,
for example, from the group of the alkaline phosphatase,
penicillinase, lpp, 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.
[0131] The DNA for such precursor region is ligated in reading
frame to DNA encoding the anti-ErbB3 antibody.
[0132] (ii) Origin of Replication Component
[0133] Both expression and cloning vectors contain a nucleic acid
sequence that enables the vector to replicate in one or more
selected host cells. Generally, 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. Generally,
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).
[0134] (iii) Selection Gene Component
[0135] 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.
[0136] 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.
[0137] Another example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the anti-ErbB3 antibody nucleic acid, such as DHFR,
thymidine kinase, metallothionein-I and -II, preferably primate
metallothionein genes, adenosine deaminase, ornithine
decarboxylase, etc.
[0138] 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.
[0139] Alternatively, host cells (particularly wild-type hosts that
contain endogenous DHFR) transformed or co-transformed with DNA
sequences encoding anti-ErbB3 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
6418. See U.S. Pat. No. 4,965,199.
[0140] 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.
[0141] In addition, vectors derived from the 1.6 am 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).
[0142] (iv) Promoter Component
[0143] Expression and cloning vectors usually contain a promoter
that is recognized by the host organism and is operably linked to
the anti-ErbB3 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-Dalgarno (S.D.) sequence operably linked to the DNA encoding
the anti-ErbB3 antibody.
[0144] 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.
[0145] 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.
[0146] 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.
[0147] Anti-ErbB3 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.
[0148] 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.
[0149] (v) Enhancer Element Component
[0150] Transcription of a DNA encoding the anti-ErbB3 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-ErbB3
antibody-encoding sequence, but is preferably located at a site 5'
from the promoter.
[0151] (vi) Transcription Termination Component
[0152] 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-ErbB3 antibody. One useful transcription termination component
is the bovine growth hormone polyadenylation region. See WO94/11026
and the expression vector disclosed therein.
[0153] (vii) Selection and Transformation of Host Cells
[0154] 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 31,637), and E. coli W3110 (ATCC 27,326) are suitable.
These examples are illustrative rather than limiting.
[0155] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for anti-ErbB3 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.
lactic, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.
wickeramii (ATCC 24,178), K. waltii (ATCC 66,600), 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.
[0156] Suitable host cells for the expression of glycosylated
anti-ErbB3 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-6 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.
[0157] Plant cell cultures of cotton, corn, potato, soybean,
petunia, tomato, and tobacco can also be utilized as hosts.
[0158] 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); TRI 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).
[0159] Host cells are transformed with the above-described
expression or cloning vectors for anti-ErbB3 antibody production
and cultured in conventional nutrient media modified as appropriate
for inducing promoters, selecting transformants, or amplifying the
genes encoding the desired sequences.
[0160] (viii) Culturing the Host Cells
[0161] The host cells used to produce the anti-ErbB3 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. No. 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.
[0162] (ix) Purification of Anti-ErbB3 Antibody
[0163] 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 3.6),
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 generally 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.
[0164] 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 (Cuss et
al., EMBO J. 6:16671675 (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. re-sin (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.
[0165] 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 2.5-4.6, preferably performed
at low salt concentrations (e.g. from about 0-0.25M salt).
[0166] C. Pharmaceutical Formulations
[0167] Therapeutic formulations of the antibody are prepared for
storage by mixing the antibody having the desired degree of purity
with optional physiologically 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).
[0168] 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, ErbB4, or
vascular endothelial factor (VEGF) in the one formulation.
Alternatively, or in addition, the composition may comprise a
chemotherapeutic agent or a cytokine. Such molecules are suitably
present in combination in amounts that are effective for the
purpose intended.
[0169] 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).
[0170] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0171] 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.
[0172] D. Non-Therapeutic Uses for the Antibody
[0173] 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 ErbB3 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 ErbB3 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 ErbB3 protein from the antibody.
[0174] Anti-ErbB3 antibodies may also be useful in diagnostic
assays for ErbB3 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).
[0175] For diagnostic applications, the antibody typically will be
labeled with a detectable moiety. Numerous labels are available
which can be generally grouped into the following categories:
[0176] (a) Radioisotopes, such as .sup.35S, .sup.14C, .sup.125H,
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.
[0177] (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.
[0178] (c) Various enzyme-substrate labels are available and U.S.
Pat. No. 4,275,149 provides a review of some of these. The enzyme
generally 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. 4,737,466), luciferin,
2,3-dihydrophthalazinediones, malate dehydrogenase, urease,
peroxidase such as horseradish peroxidase (HRPO), alkaline
phosphatase, 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).
[0179] Examples of enzyme-substrate combinations include, for
example:
[0180] (i) Horseradish peroxidase (URPO) 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));
[0181] (ii) alkaline phosphatase (AP) with para-Nitrophenyl
phosphate as chromogenic substrate; and
[0182] (iii) .beta.-D-galactosidase (.beta.-D-Gal) with a
chromogenic substrate (e.g.
.beta.-nitrophenyl-.beta.-D-galactosidase) or fluorogenic substrate
4-methylumbelliferyl-.beta.-D-galactosidase.
[0183] 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.
[0184] 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.
[0185] In another embodiment of the invention, the anti-ErbB3
antibody need not be labeled, and the presence thereof can be
detected using a labeled antibody which binds to the ErbB3
antibody.
[0186] 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).
[0187] 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 ErbB3 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 generally 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.
[0188] 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 thereafter a 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.
[0189] 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.
[0190] The antibodies may also be used for in vivo diagnostic
assays. Generally, the antibody is labelled with a radionuclide
(such as .sup.111In, .sup.14C, .sup.131I, .sup.125I, .sup.3H,
.sup.32P or .sup.35S) so that the tumor can be localized using
immunoscintiography.
[0191] E. Diagnostic Hits
[0192] 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.
[0193] F. Therapeutic Uses for the Antibody
[0194] It is contemplated that the anti-ErbB3 antibody of the
present invention may be used to treat conditions in which
excessive activation of the ErbB2-ErbB3 complex is occurring,
particularly where such activation is mediated by a heregulin
polypeptide. Exemplary conditions or disorders to be treated with
the ErbB3 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.
[0195] 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.
[0196] Other therapeutic regimens may be combined with the
administration of the anti-ErbB3 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.
[0197] It may be desirable to also administer antibodies against
other tumor associated antigens, such as antibodies which bind to
the EGFR, ErbB2, ErbB4, or vascular endothelial factor (VEGF). Two
or more anti-ErbB3 antibodies may be co-administered to the
patient. Alternatively, or in addition one or more cytokines may be
administered to the patient.
[0198] 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.
[0199] 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.
[0200] G. Articles of Manufacture
[0201] 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-ErbB3 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, Ringer's solution and dextrose solution. It may further
include other materials desirable from a commercial and user
standpoint, including other buffers, diluents, filters, needles,
syringes, and package inserts with instructions for use.
[0202] H. Deposit of Materials
[0203] The following hybridoma cell line has been deposited with
the American Type Culture Collection, 12301 Parklawn Drive,
Rockville, Md., USA (ATCC):
TABLE-US-00001 Hybridoma/Antibody Designation ATCC No. Deposit Date
8B8 HB12070 Mar. 22, 1996
[0204] This deposit was made under the provisions of the Budapest
Treaty on the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedure and the
Regulations thereunder (Budapest Treaty). This assures maintenance
of a viable culture for 30 years from the date of deposit. The cell
line 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 culture 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 culture so deposited will be
irrevocably removed upon the granting of the patent.
[0205] The assignee of the present application has agreed that if
the culture on deposit should die or be lost or destroyed when
cultivated under suitable conditions, it will be promptly replaced
on notification with a viable specimen of the same culture.
Availability of the deposited cell line 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.
[0206] 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 culture deposited, since the deposited embodiment is intended
as a single illustration of one aspect of the invention and any
culture 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.
[0207] 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
Production of Anti-ErbB3 Antibodies
[0208] This example describes the production of the anti-ErbB3
antibodies having the characteristics described herein.
Materials and Methods
[0209] Cell Lines.
[0210] The human myeloid leukemia cell line K562 (which lacks class
I subfamily receptor protein tyrosine kinases as determined by
Northern blotting) and human ovarian carcinoma cell line Caov3 were
obtained from the American Type Culture Collection (Rockville,
Md.). Both were cultured in RPMI 1640 medium supplemented with 10%
fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin, 100
.mu.g/mL streptomycin, and 10 mM HEPES ("growth medium").
[0211] Stable Transfection of K562 Cells.
[0212] The K662 cell line was transfected and ErbB3 expressing
clones were selected for. Briefly, erbB3 cDNA was subcloned into
the pcDNA-3 mammalian cell expression vector (Invitrogen) and
introduced into K562 cells by electroporation (1180 mF, 350 V).
Transfected cells were cultured in growth medium containing 0.8
mg/mL G418. Resistant clones were obtained by limiting dilution and
tested for ErbB3 expression by Western blot and heregulin (HRG)
binding assays. The ErbB3 expressing clone 4E9H3 was used in the
experiments described in this report. Phorbol ester stimulation was
found to significantly enhance ErbB3 expression in the K662
transfectants. Therefore, the 4E9H3 cells were placed in growth
medium containing 10 ng/mL phorbol-12-myristate acetate (PMA)
overnight prior to use in the various assays described below.
[0213] Antibodies.
[0214] Monoclonal antibodies specific for ErbB3 protein were
generated against a recombinant fragment of the receptor
corresponding to the extracellular domain (ECD) thereof fused at
its amino terminus to the herpes simplex virus type I (HSV
glycoprotein D (gD) epitope for the monoclonal antibody 5B6. The
coding sequence for the signal sequence of ErbB3 was replaced with
a sequence encoding amino acids 1-53 of the gD polypeptide. Amino
acids 1-25 encode the signal sequence of gD while amino acids 26-53
contain an epitope for the monoclonal antibody 5B6. See WO
95/14776. The resulting construct, gD.Erb3.ECD, was purified using
an anti-gD antibody affinity column. Immunizations were performed
as follows. Female Balb/c mice (Charles River) were initially
injected via footpad with 5 .mu.g of gD.ErbB3.ECD in 100 .mu.l
RIBI's.TM. adjuvant (Ribi ImmunochemResearch, Inc., Hamilton,
Mont.). The animals were boosted 2 times with 5 .mu.g of
gD.ErbB3.ECD in their footpad every two weeks followed by a final
footpad injection of 5 .mu.g of gD.ErbB3.ECD. Three days after the
last immunization, popliteal lymph nodes were removed and a single
cell suspension was prepared for PEG fusion.
[0215] Monoclonal antibodies were purified and tested by
immobilized and solution phase ELISA for cross-reactivity with
ErbB2 and ErbB4. For the immobilized ELISA, 1 .mu.g/ml of
ErbB2.ECD, gD.ErbB3.ECD or gD.ErbB4.ECD was used to coat a 96 well
microtiter plate overnight. Anti-ErbB3 Mab at 1 .mu.g/ml was added
and incubated for 1 hour at room temperature (RT), washed and
followed by goat anti-mouse (gam) IgG conjugated to HRPO. The ELISA
was developed and read at 490 nm. For the solution phase ELISA, 1
.mu.g/ml of gam IgG (Fc specific) was used to coat a 96 well
microtiter plate overnight. Anti-ErbB3 Mab at 1 .mu.g/ml was added
and incubated for 1 hour at RT, washed and followed by biotinylated
ErbB2.ECD, gD.ErbB3.ECD or gD.ErbB4.ECD. This reaction was
incubated for 1 hour at RT, washed and followed by HRPO
strepavidin. The ELISA was developed and read at 490 nm. In this
assay, none of the anti-ErbB3 antibodies cross-reacted with ErbB2
or ErbB4.
[0216] Fab fragments of the 3-8D6 antibody were generated by papain
digestion. Undigested IgG and Fc fragments were removed by protein
A affinity chromatography followed by gel filtration
chromatography. No IgG was detectable in the Fab pool by SDS-PAGE
and by a Western blot probed with an Fc specific antibody.
[0217] HRG Binding Assays.
[0218] All HRG binding experiments were carried out using the
EGF-like domain of the .beta.1 isoform, i.e.
HRG.beta.1.sub.177-244, (Sliwkowski et al., J. Biol. Chem. 269:
14661-5 (1994)). The ErbB3 antibody panel was screened for an
effect on HRG binding by incubating 5.0.times.10.sup.4 4E9H3 cells
with 100 pM .sup.125I HRG overnight at 0.degree. C., in the absence
(control) or presence of 100 nM anti-ErbB3 antibody. Irrelevant
IgGs were used as negative controls. The cells were harvested and
rapidly washed with ice cold assay buffer (RPMI medium containing
10 mM HEPES, pH=7.2) in a 96 well filtration device (Millipore).
The filters were then removed and counted.
[0219] For the antibody dose-response experiments, 4E9H3 cells were
incubated with 100 pM .sup.125I-HRG in the presence of increasing
concentrations of antibody. HRG affinity measurements were
determined in the absence (control) or presence of either 100 nM
antibody or Fab fragment. These experiments were carried out in a
competitive inhibition format with increasing amounts of unlabeled
HRG and a fixed concentration (35 pM) of .sup.125I-HRG. For the
control experiment (no antibody) 1.times.10.sup.5 4E9H3 cells were
used for each sample. Due to limitations in the dynamic range of
the assay, the number of 4E9H3 cells used for binding in the
presence of either the antibody or the Fab was reduced to
2.5.times.10.sup.4 cells per sample.
[0220] Antibody Reduction of HRG Stimulated Phosphorylation.
[0221] Caov3 cells, which naturally express ErbB2 and ErbB3, were
pre-incubated with 250 nM anti-ErbB3 antibody 3-8D6, Fab fragments
of this antibody, or buffer (control), for 60 minutes at room
temperature. The anti-ErbB2 antibody, 2C4 (Fendly et al., Cancer
Res., 50:1550-1558 (1990)), which was previously shown to block HRG
stimulated phosphorylation of ErbB2 was included as a positive
control. The cells were then stimulated with UM at a final
concentration of 10 nM for 8 minutes at room temperature, or left
unstimulated. The reaction was stopped by removing the supernatants
and dissolving the cells in SDS sample buffer. The lysates were
then run on SDS-PAGE. Western blots of the gels were probed with
anti-phosphotyrosine conjugated to horseradish peroxidase
(Transduction Labs), and the blots were visualized using a
chemiluminescent substrate (Amersham). The blots were scanned with
a reflectance scanning densitometer as described in Holmes et al.,
Science, 256:1205-1210 (1992).
[0222] Antibody Reduction of ErbB2-ErbB3 Protein Complex
Formation.
[0223] Caov3 cells were pre-incubated with buffer (control), 250 nM
anti-ErbB3 antibody 3-8D6, or Fab fragments of this antibody, or
the anti-ErbB2 antibody (2C4) for 60 minutes at room temperature,
then treated with 10 nM HRG or control buffer for 10 minutes. The
cells were lysed in 26 mM Tris, pH=7.6, 150 mM NaCl, 1 mM EDTA,
1.0% Triton X-100.TM., 1.0% CHAPS, 10% v/v glycerol, containing 0.2
mM PMSF, 50 mTU/mL aprotinin, and 10 mM leupeptin ("lysis buffer"),
and the crude lysates were centrifuged briefly to remove insoluble
material. Supernatants were incubated with 3E8, a monoclonal
antibody specific for ErbB2 (Fendly et al., Cancer Res.,
50:1550-1558 (1990)), covalently coupled to an insoluble support
(Affi Prep-10.TM., Bio-Rad). The incubation was carried out
overnight at 4.degree. C. The immunoprecipitates were washed twice
with ice cold lysis buffer, re-suspended in a minimal volume of SDS
sample buffer, and run on SDS-PAGE. Western blots of the gels were
then probed with a polyclonal anti-ErbB3 (Santa Cruz Biotech). The
blots were scanned with a reflectance scanning densitometer as
described in Holmes et al, Science, 256:1205-1210 (1992). After
visualization with the ECL chemiluminescent substrate, the blots
were stripped and re-probed with a polyclonal anti-ErbB2 (Santa
Cruz Biotech). A duplicate plot probed with anti-ErbB2 showed that
equal amounts of ErbB2 were immunoprecipitated from each
sample.
Results
[0224] A panel of monoclonal antibodies directed against the
extracellular domain of ErbB3 were evaluated for their ability to
affect HRG binding to ErbB3. The initial screen was carried out by
incubating each of the purified antibodies at a final concentration
of 100 nM with 4E9H3 cells in the presence of .sup.125I-HRG. 4E9H3
cells are ErbB3 transfectants of the human myeloid leukemia cell
line K562. The K562 cell line does not express endogenous ErbB
receptors or HRG. Therefore, heregulin binding to 4E9H3 cells
occurs exclusively through ErbB3. After incubating the samples
overnight on ice, cell associated counts were measured. As shown in
FIG. 1, two of the anti-ErbB3 monoclonal antibodies (2F9 and 3E9)
reduced the amount of .sup.125I-HRG bound to 4E9H3 cells relative
to control (no antibody). However, several others significantly
enhanced ligand binding. These results suggested that these
anti-ErbB3 antibodies were able to increase the affinity for HRG
binding and/or increase the availability of HRG binding sites. To
further characterize the influence of these antibodies on HRG
binding to ErbB3, dose-response experiments were performed using
the 3-8D6 antibody that increased HRG binding. 4E9H3 cells were
incubated with 100 pM of .sup.125I-HRG in the presence of
increasing concentrations of the 3-8D6 antibody. Cell associated
counts were then measured after an overnight incubation on ice. The
results are shown in FIG. 2 as plots of cell associated counts
versus antibody concentrations. There is a correlation between
increased HRG binding and increasing antibody concentration.
Heregulin binding reached saturation between 10 and 100 nM IgG. The
EC.sub.50 value for the 3-8D6 antibody was 722 pM. No decrease in
the dose-response curves at high antibody concentrations were
observed for either antibody.
[0225] Scatchard analysis of HRG binding was determined in the
presence of these antibodies and the results are shown in Table
1.
TABLE-US-00002 TABLE 1 Data Set K.sub.d Sites/Cell Control 1.2
.times. 10.sup.-9 3.6 .times. 10.sup.5 MAb 3-8D6 2.1 .times.
10.sup.-10 2.4 .times. 10.sup.5 FAb 3-8D6 2.8 .times. 10.sup.-10
2.9 .times. 10.sup.5
[0226] In the absence of the antibody, a Kd of 1200 pM was measured
for HRG binding to ErbB3, which is in agreement with a previously
measured affinity measurement of HRG binding to ErbB3. The number
of binding sites per cell was determined to be 36,000. In the
presence of the antibody, 3-8D6, the measured binding constant for
HRG binding is significantly increased to 210 pM. However, the
number of HRG binding sites is not increased in the presence of
3-8D6.
[0227] To determine whether the increase in ErbB3 ligand binding
affinity was dependent on the antibody being divalent, HRG binding
experiments were performed in the presence of 100 nM of a Fab
fragment prepared by papain digestion of the 3-8D6 antibody. Fab
fragments used for these experiments were purified by Protein A
affinity chromatography and by gel filtration chromatography. No
intact IgG was detected is this purified preparation by SDS-PAGE.
As shown in FIG. 3, binding of HRG in the presence of the intact
antibody or the resulting Fab is nearly identical. Scatchard
analysis of these data yield a dissociation constant for HRG
binding in the presence of Fab of 280 pM and the number of
receptors per cell determined from this experiment was also
essentially the same as that of the control. These data are
consistent with those presented in FIG. 2, where the dose response
curves with the intact antibodies showed a plateau rather than a
bell-shaped curve at higher antibody concentration, where univalent
antibody binding might be occurring. Without being bound by any
theory, these data suggest that the alteration in HRG binding
observed in the presence of these antibodies does not require a
divalent antibody.
[0228] The effect of the 3-806 antibody in a receptor tyrosine
phosphorylation assay, using the ovarian tumor cell line Caov3
which co-expresses ErbB2 and ErbB3 was next examined. Cells were
stimulated with 10 nM HRG following a 60 minute pre-incubation with
either the 3-806 antibody (at 250 nM) or buffer (control). Whole
cell lysates were analyzed on a Western blot probed with
anti-phosphotyrosine. HRG treatment did not stimulate
phosphorylation in 4E9H3 cells. Treatment of 4E9H3 cells with the
3-8D6 antibody did not induce phosphorylation of ErbB3 by itself
nor did it have any effect on tyrosine phosphorylation in Caov3
cells. A marked tyrosine phosphorylation signal was detected on a
protein with a molecular size .about.180 kDa following HRG
stimulation. Treatment of Caov3 cells with 2C4, an antibody
specific for ErbB2, was able to block the HRG-mediated tyrosine
phosphorylation signal. When cells were treated with the anti-ErbB3
antibody, 3-8D6, prior to HRG stimulation, tyrosine phosphorylation
was also decreased. By scanning densitometry of the
anti-phosphotyrosine blots of whole cell lysates, it was observed
that 3-8D6 inhibits the phosphotyrosine signal at 180-185 kDa by
about 80% (range 76-84%). This signal is contributed by tyrosine
phosphate residues on both ErbB3 and ErbB2. Treatment of Caov3
cells with the Fab fragments prepared from the 8-806 antibody, also
reduced the HRG stimulated phosphorylation of the 180 kDa band
relative to control. However, the inhibitory activity of the Fab
was slightly less potent than the intact antibody.
[0229] The 3-8D6 antibody-mediated increase in receptor affinity on
cells which express ErbB3 alone is analogous to the increase in
affinity associated with co-expression of ErbB2 with ErbB3.
Moreover, this antibody blocks the HRG stimulated ErbB2 kinase
activity in cells which express both receptors. To determine
whether the anti-ErbB3 antibody competes directly with ErbB2 for
binding to ErbB3, a series of co-immunoprecipitation experiments
were performed using Caov3 cells. Cells were pre-incubated with
either antibody, or buffer (control) and then treated with 10 nM
HRG for 10 minutes. Lysates of the cells were then
immunoprecipitated with a monoclonal antibody against ErbB2.
Immunoprecipitates were then analyzed by Western blot for the
presence of ErbB3. The results of these experiments indicated that
ErbB3 was present in the ErbB2 immunoprecipitate of the HRG
stimulated cell lysate, but not in the immunoprecipitate of
unstimulated lysate. These data suggests that HRG drives the
formation of an ErbB2-ErbB3 complex in Caov3 cells. ErbB3 was not
detectable in the immunoprecipitate of the sample treated with the
anti-ErbB2 monoclonal antibody, 2C4. A significant diminution in
the ErbB3 signal was observed when the cells were pre-incubated
with the 3-8D6 antibody or its resulting Fab prior to HRG
stimulation. These data indicate that the 3-8D6 antibody inhibits
the formation of a ErbB2-ErbB3 complex following HRG treatment.
Scanning densitometry of the anti-ErbB3 Western blots of anti-ErbB2
immunoprecipitates revealed that the anti-ErbB3 signal (which
indicates the number of ErbB2-ErbB3 complexes present) is also
diminished by 3-8D6 by about 80% (range 71-90%). When duplicate
blots were probed with anti-ErbB2, equivalent amounts of ErbB2 were
present in all lanes.
Sequence CWU 1
1
* * * * *