Note: Descriptions are shown in the official language in which they were submitted.
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ErbB3 ANTIBODIES
BACKGROUND OF THE INVENTION
Field of the Invention
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.
Description of Related Art
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 erbBI gene. erbBI
has been causally implicated in human malignancy. In particular, increased
expression of this gene has been
l 5 observed in more aggressive carcinomas of the breast, bladder. lung and
stomach.
The second member of the class I subfamily, p185net, was originally identified
as the product of the
transforming gene from neuroblastornas 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.
A further related gene, called erbB3 or HER3, has also been described. See US
Pat. Nos. 5.183,884
and 5,480,968; Plowman et at, Proc. Nall. Acad. Sci. USA, 87:4905-4909 (1990);
Kraus et al., Proc. Natl.
Acad. Sci. USA, 86:9193-9197 (1989); EP Pat Appln No 444,961 A I ; and Kraus
et at, Proc. Natl.4cad. Sci.
USA, 90:2900-2904 (1993). Krause! al. (1989) discovered that markedly elevated
levels of erbB3 mRNA were
present in certain human mammary tumor cell lines indicating that erbB3, like
erbB 1 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 US Pat. Nos. 5.183.884 and 5,480,968.
The role of erbB3 in cancer has been explored by others It has been tound to
be overexpressed in
breast (Lemotne et at, Br..1. 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 at. J. Pathol. 168:269-273 (1992),
and Friess ei al., Clinical
Cancer Research. 1:1413-1420 (1995)).
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 el al- I Biol. Chem..
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269(20):14661-14665 (1994)) Additionally, the affinity of ErbB3 for heregulin
(HKG) 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. Nau. Acad. Sci. USA 92: 1431-1435 (1995): and
Lewis et at, Cancer Res..
56:1457-1465 (1996) with respect to the ErbB2-ErbB3 protein complex.
Kajkumar 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.
The class I subfamily of growth factor receptor protein tyrosine kinases has
been further extended to
include the HER4/p18OerbB4 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.
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 at., Genomics, 16:790-791
(1993); and Orr-Urtreger et al..
Proc. Natl. Acad. Sci. USA, Vol. 90 pp. 1867-1871 (1993).
Holmes et al. isolated and cloned a family of polypeptide activators for the
HER2 receptor which they
termed heregulin-a (HRG-a), heregulin- (31(HRG- R 1), heregulin- P2 (HRG-
(32), heregulin- P2-like (HRG- P2-
like), and heregulin-133 (HRG-(33). See Holmes et al., Science, 256:1205-1210
(1992); and WO 92/20798. The
45 kDa polypeptide, HRG-a, 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
45kDa soluble form. These pro-HRGs lack a N-terminal signal peptide.
While heregulins are substantially identical in the first 213 amino acid
residues, they are classified
into two major types, a and (3, based on two variant EGF-like domains which
differ in their C-terminal
portions. Nevertheless, these EGF-like domains are identical in the spacing of
six cysteine residues contained
therein, Based on an amino acid sequence comparison, Holmes er at. 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), 359/o identical to amphiregulin (AR), 32% identical to TGF-a, and 27%
identical to EGF.
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 at, 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 et 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 toe nomenclature of Holmes et al.. the NDFs are classified as either
a or (3 polypeptides based on
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the sequences of the EGF-like domains. Isoforms I 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.
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 (3-type heregulin
and lacks the entire "glyco" spacer (rich in glycosylation sites) present
between the Ig-like domain and EGF-
like domain, of HRGa, and HRG(31-p3.
Marchionni et at, 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.
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):S I 3-S24 (1994).
See also, Alimandi et al., Oncogene, 10:1813-1821 (1995); Beerli et al.,
Molecular and Cellular
Biology, 15:6496-6505 (1995); Karunagaran et at, EMBO J, 15:254-264 (1996);
Wallasch et al., EMBO J,
14:4267-4275 (1995); and Zhang et al., Journal of Biological Chemistry,
271:3884-3890 (1996), in relation
to the above receptor family
SUMMARY OF THE INVENTION
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
The invention also relates to an antibody which binds to ErbB3 protein and
reduces heregulin binding
thereto.
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 IOnM, more preferably at least about InM. 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.
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.
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 cel'i comprising
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the nucleic acid and, optionally, recovering the antibody from the cell
culture and. preferably. the cell culture
medium.
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.
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
!0 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
Fig. I depicts HRG binding to K562 ErbB3 coils in the presence of various anti-
ErbB3 monoclonal
antibodies. Purified anti-ErbB3 antibodies were incubated with a suspension of
K562 ErbB3 cells and 1251-
HRG (3 1(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 5 1 (177-244)( HRG). Antibodies against ErbB2
protein (2C4) and HSV
(5B6) were used as negative controls.
Fig. 2 shows the effect of antibody concentration or 1"I RG 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 1251-HRG and increasing concentrations
of the 3-8D6 antibody. Data
from the experiment is shown plotted as cell bound counts versus antibody
concentration.
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 (BIT)
versus total HRG0 1(177-
244)
Detailed Description of the Preferred Embodiments
1. Definitions
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 US patent
5,480,968 and Plowman et
al., Proc. Natl. Acad. Sci. LISA, 87:4905-4909 (1990).
The antibody of interest may be one which does not significantly cross-react
with other proteins such
as those encoded by the erhB l . erbB2 and/or erbB4 genes. In such
embodiments, the extent of binding of the
antibody to these non-ErbB 3 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.
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"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(3 1 1 77-244 )
The "ErbB2-FrbB3 protein complex" is a noncovalent!y 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
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).
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 50 A, 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)
The antibody may he one which "increases the binding affinity of heregulin for
ErbB3 protein". This
means that, in the presence of the antibody (e.g. I00nM 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 HRG which binds to the K562 cell line
transfected with erbB3 as
described herein may be increased in the presence of IOOnM antibody by at
least 10% preferably at least 50%
and most preferably at least about 100% (see Fig. 1), relative to control.
The antibody which reduces HRG binding to ErbB3 protein teg ErbB3 present in a
cell) is one
which interferes with the HRG-binding site on ErbB3 protein such that it
statistically significantly decreases
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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.
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 FgM, IgG (e.g IgG1, igG7, IgG3 or IgG4), lgD, 1gA or IgE, for
example. Preferably however.
the antibody is not an IgM antibody.
"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')2. and Fv
fragments; diabodies; single-chain antibody molecules; and multispecific
antibodies formed from antibody
fragments.
The term "monoclonal antibody" as used herein refers to an antibody obtained
from a population of
substantially homogeneous antibodies, i.e., the individual antibodies
comprising the population are identical
except for possible naturally occurring mutations that may be present in minor
amounts. Monoclonal
antibodies are highly specific, being directed against a single antigenic
site, Furthermore, in contrast to
conventional (polyclonal) antibody preparations which typically include
different antibodies directed against
different determinants (epitopes), each monoclonal antibody is directed
against a single determinant on the
antigen. In addition to their specificity, the monoclonal antibodies are
advantageous in that they are
synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
The modifier "monoclonal"
indicates the character of the antibody as being obtained from a substantially
homogeneous population of
antibodies, and is not to be construed as requiring production of the antibody
by any particular method. For
example, the monoclonal antibodies to be used in accordance with the present
invention may be made by the
hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or
may be made by recombinant
DNA methods (see, e.g., U.S. Patent No. 4,816,567). The "monoclonal
antibodies" may also be isolated from
phage antibody libraries using the techniques described in Clackson et al.,
Nature, 352:624-628 (1991) and
Marks of al., J. Mol Biol, 222:581-597 (1991), for example.
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.
Patent No. 4,816,567: Morrison et al.,
Proc. Natl. Acad. Sci. USA, 81.6851-6855 (1984)).
"Humanized" forms of non-human (e.g., murine) antibodies are chimeric
immunoglobulins.
5 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
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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 ei ai., Nature, 321:522-525 (1986): Reichmann et a!
, Nature. 332:323-329 (1988):
and Presta, Curr. Op Struct. Biol , 2:593-596 (1992). The humanized antibody
includes a
PrimatizedT',Iantibody wherein the antigen-binding region of the antibody is
derived from an antibody produced
by immunizing macaque monkeys with the antigen of interest.
"Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains
of antibody,
wherein these domains are present in a single polypeptide chain, Generally.
the Fv polypeptide further
comprises a polypeptide linker between the VH and VL domains which enables the
sFv to form the desired
structure for antigen binding. For a review of sFv see Pluckthun in The
Pharmacology of Monoclonal
Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.
269-315 (1994).
The term "dabodies" refers to small antibody fragments with two antigen-
binding sites, which
fragments comprise a heavy-chain variable domain (VH) connected to a light-
chain variable domain (VL) in
the same polypeptide chain (VH - VL). 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).
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.
As used herein, the term "salvage receptor binding epitope" refers to an
epitope of the Fc region of
an I.gG molecule (e.g., IgG I, IgG-), IgG3, or IgG4) that is responsible for
increasing the in vivo serum half-life
of the igG molecule.
"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.
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"Mammal" for purposes of treatment refers to any animal classified as a
mammal. including humans,
domestic and farm animals, and zoo, spores, or pet animals. such as dogs.
horses. cats. cows. etc. Preferably.
the mammal is human,
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.
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.
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. 1, Y, Pr),
chemotherapeutic agents, and toxins such as enzymatically active toxins of
bacterial, fungal, plant or animal
origin, or fragments thereof.
A "chemotherapeutic agent" is a chemical compound useful in the treatment of
cancer. Examples of
chemotherapeutic agents include Adriatnycin, Doxorubicin, 5-Fluorouracil,
Cytosine arabinoside ("Ara-C"),
Cyclophosphamide, Thiotepa, Busulfan, Cytoxin, Taxol, Methotrexate. Cisplatin,
Melphalan, Vinblastine,
Bleomvcin. Etoposide, lfosfamidc, Mitomycin C, Mitoxantrone, Vincreistine.
Vinorelbine. Carboplatin.
T'eniposide. Daunomycin, Carminomycin, Aminopterin, Dactinomycin, Mitomycins.
Esperamicins (see U.S.
Pat. No. 4,675,187), Melphalan and other related nitrogen mustards.
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; relax in; 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-a and -(3;
mullerian -inhibiting substance: mouse
3 gonadotropin-associated peptide: inhibin; activin: vascular endothelial
growth factor; integrin; thrombopoietin
(TPO); nerve growth factors such as NGF-(3; platelet-growth factor-:
transforming growth factors (TGFs) such
as TGF-a and TGF-(3: insulin-like growth factor-I and -Il; erythropoietin
(EPO); osteoinductive factors:
interferons such as interferon-a, -0, and -y; colony stimulating factors
(CSFs) such as macrophage-CSF (M-
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CSF), granulocyte-macrophage-CSF (GM-CSF); and granulocyte-C'SF (G-CSF):
mterleukins (lLs) such as 1L-
I, IL-I a. ll.-2. IL-3, IL-4, IL-5, IL-6, IL-7, IL-8. IL-9, IL-1 1. IL-I 2: a
tumor necrosis factor such as TNF-a
or TNF-p; and other polypeptide factors including LIF and kit ligand (KL). As
used herein, the term cvtokine
includes proteins from natural sources or from recombinant cell culture and
biologically active equivalents of
the native sequence cytokines.
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 Socierv Transactions, 14, pp.
375-382. 615th Meeting
Belfast (1986) and Stella et al., "Prodrugs: A Chemical Approach to Targeted
Drug Delivery," Directed Drug
Delivery, Borchardt et al., (ed.), pp 247-267, Humana Press (1985)- The
prodrugs of this invention include.
but are not limited to, phosphate-containing prodrugs, thiophosphate-
containing prodrugs, sulfate-containing
prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs,
glycosylated prodrugs, p-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.
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.
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), polyacry lam
ides, 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. Patent No.
4.275,149.
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.
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
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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.
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. polvadenylation signals. and
enhancers.
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
I S adaptors or linkers are used in accordance with conventional practice.
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.
11. Modes for Carrying out the Invention
A. Antibody Preparation
A description follows as to exemplary techniques for the production of the
claimed antibodies.
(1) Polyclonal antibodies
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, SOCI2, or
R1N=C=NR, where R and RI are
different alkyl groups.
Animals are immunized against the antigen, immunogenic conjugates, or
derivatives by combining,
e g- 100 pa or 5 pg 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
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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.
(ii) Monoclonal antibodies
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.
For example, the monoclonal antibodies may be made using the hybridoma method
first described by
Kohler et a!.. Nature, 256:495 (1975), or may he made by recombinant DNA
methods (U.S. Patent No.
4,816,567),
in the hybridoma method, a mouse or other appropriate host animal, such as a
hamster, is immunized
as hereinabove described to elicit lymphocytes that produce or are capable of
producing antibodies that will
specifically bind to the protein used for immunization. Alternatively,
lymphocytes may be immunized in vitro.
Lymphocytes then are fused with myeloma cells using a suitable fusing agent.
such as polyethylene glycol, to
form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice.
pp.59-103 (Academic Press,
1986)).
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.
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-1 I mouse tumors available from the Salk Institute Cell Distribution
Center, San Diego, California
USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture
Collection. Rockville,
Maryland 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)).
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 (RJA) or enzyme-linked immunoabsorbent assay (ELISA).
The binding affinity of the monoclonal antibody can, for example, be
determined by the Scatchard
analysis of Munson et a!., Anal. Biochem., 107:220 (1980).
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,
Monoclona/ Antibodies: Principles and Practice, pp. 59-1W (Academic Press,
1986)). Suitable culture media
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CA 02589421 2007-05-28
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.
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.
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
Pltickthun, Immunol. Revs., 130:1'51-
188 (1992).
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.
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. Patent No. 4,816,567;
Morrison, et al., Proc. Nail 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.
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.
(iii) Humanized and human antibodies
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, 321:522-525 (1986); Riechmann et al.,
Nature, 332:323-327 (1988);
Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs
or CDR sequences for the
corresponding sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric
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antibodies (U.S. Patent No 4,816,567) wherein substantially less man 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.
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 Irnmunul , 151:2296
(1993): Chothia ei a1. 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. Immnol., 15 1:2623 (1993)).
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 an. 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.
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 (JH) gene in chimeric and germ-line mutant mice
results in complete inhibition of
endogenous antibody production. Transfer of the human germ-line tmmunoglobulin
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, 902551 (1993): Jakobovits et
al., Nature. 362:255-258 (1993);
Bruggermann et al.. Year in Immuno., 7:33 (1993). Human antibodies can also be
derived from phage- display
libraries (Hoogenboom et al.. J. Mal. Biol., 227:381 (1991); Marks et al., J.
Mol. Biol., 222:581-597 (1991)).
(iv) .4ntibodv fragments
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 er 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
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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')2
fragments (Carter et al.,
Bio/Technology 10:163-167 (1992)). According to another approach, F(ab')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.
(v) Bispecific antibodies
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 (FcyR), such as
FcyRI (CD64), FcyRII (CD32) and FcyRIII (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-a, 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')2 bispecific antibodies).
Methods for making bispecific antibodies are known in the all. 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 (Milstein 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).
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
(CHI) 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.
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
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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 ai Methods in Envvmologv,
121:210(1986).
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 [east a part of the CH3 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.
Bispecific antibodies include cross-finked 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 (US
Patent 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 crass-linking
agents are well known in the art,
and are disclosed in US Patent No. 4,676,980, along with a number of cross-
linking techniques.
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 a!.,
Science, 229: 81 (1985) describe a procedure wherein intact antibodies are
proteolytically cleaved to generate
F(ab')2 fragments. These fragments are reduced in the presence of the dithiol
complexing agent sodium
arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide
formation. The Fab' fragments
generated are then converted to thionitrobenzoate (TNB) derivatives One of the
Fab'-TNB derivatives is then
reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is
mixed with an equimolar amount
of the other Fab'-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced can be
used as agents for the selective immobilization of enzymes.
Recent progress has facilitated the direct recovery of Fab'-SH fragments from
E. coli, which can be
chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp.
Med., 175: 217-225 (1992) describe
the production of a fully humanized bispecific antibody F(ab')2 molecule. Each
Fab' fragment was separately
secreted from E. colt 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
'I' cells, as well as trigger the Ivtic activity of human cytotoxic
lymphocytes against human breast tumor targets.
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 a!., 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
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heterodimers. This method can also be utilized for the production of antibody
homodimers. The "diabody"
technology described by Hollinger et at, 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 (VH) connected to a light-chain variable domain (V1) by a
linker which is too short to allow
pairing between the two domains on the same chain Accordingly. the VH and VL
domains of one fragment
are forced to pair with the complementary V1 and VH 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 at, J Immune!., 152:5368
(1994).
Antibodies with more than two valencies are contemplated. For example,
trispecific antibodies can
be prepared. Tutt et al. J. lmmunol. 147: 60 (1991).
(vi) Screening for antibodies with the desired properties
Techniques for generating antibodies have been described above. 'Those
antibodies having the
characteristics described herein are selected.
To select for antibodies which reduce HRG-induced formation of the ErbB2-ErbB3
protein complex,
I5 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
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 a!., Analytical Biochemistry, 235:207-
214 (1996) can be used to
determine ErbB2 activation.
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-
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
CA 02589421 2007-05-28
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described in Antibodies, A Laboratory Manual. Cold Spring Harbor Laboratory.
Ed Harlow and David Lane
1988), can be performed.
(viii Effector function engineering
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 cvtotoxicity
(ADCC). See Caron et al., J.
Erp 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 a!. 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).
(viii) Immunoconjugates
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).
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-sarein, Aleuritesfordii
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-
ErbB3 antibodies. Examples include
21213i 1311, 1311n, 90Y and 186Re.
Conjugates of the antibody and cytotoxic agent are made using a variety of
bifunctional protein
coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
iminothiolane (IT),
bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL),
active esters (such as
disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido
compounds (such as bis (p-
azidobenzoyl)hexanediamine),bis-diazonium derivatives (such as bis-(p-
diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine
compounds (such as 1,5-difluoro-2,4-
dinitrobenzene). For example, a ricin inirnunotoxin can be prepared as
described in Vitetta eta!. Science 238:
1098 (1987). Carbon-l4-labeled I-isothiocyanatobenzyl-3-methyIdiethylent
triaminepentaacetic acid (MX-
DTPA) is an exemplary chelating agent for conjugation of radionucleotide to
the antibody. See W094/11026.
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).
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(ix) Immunoliposomes
The anti-ErbB3 antibodies disclosed herein may also be formulated as
imrnunoliposomes. Liposomes
containing the antibody are prepared by methods known in the an. such as
described in Epstein er al_ Proc.
Nail. Acad. Sci. USA, 82:3688 (1985); Hwang et al.. Proc..Nail ,Acad. Sci.
USA, 774030 (1980): and U.S.
Pat, Nos. 4,485,045 and 4.544,545. Liposomes with enhanced circulation time
are disclosed in U.S. Patent
No. 5,013,556.
Particularly useful fiposomes can be generated by the reverse phase
evaporation method with a lipid
composition comprising phosphatidylcholine, cholesterol and PF,G-derivatized
phosphatidylethanolamine
(PEG-PE). Liposomes are extruded through filters of defined pore size to yield
fiposomes with the desired
diameter. Fab' fragments of the antibody of the present invention can be
conjugated to the liposomes as
described in Martin et al. ,J. Biol. Chem. 257: 286-288 (1982) via a disulfide
interchange reaction. A
chemotherapeutic agent (such as Doxorubicin) is optionally contained within
the liposome. See Gabizon et a!.
J. National Cancer Inst. 81(19)1484 (1989)
(x) Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT)
1'he 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
W081/01145) to an active anti-cancer drug. See, for example, WO 88/07378 and
U.S. Patent No. 4,975,278.
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.
Enzymes that are useful in the method of this invention include, but are not
limited to, alkaline
phosphatase useful for converting phosphate-containing prodrugs into free
drugs; arylsulfatase useful for
converting sulfate-containing prodrugs into free drugs; cytosine deaminase
useful for converting non-toxic 5-
fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases, such as
serratia protease, thermolysin,
subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L),
that are useful for converting
peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases, useful
for converting prodrugs that
contain D-amino acid substituents: carbohydrate-cleaving enzymes such as P-
galactosidase and neuraminidase
useful for converting giycosylated prodrugs into free drugs; P-lactamase
useful for converting drugs derivatized
with ¾-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.
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 heterobifunctionai 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
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recombinant DNA techniques well known in the art (see, e.g., Neuberger et ul.,
Nature. 312: 604-608 (1984)).
(xi) 4ntibody-salvage receptor binding eprtope fusions
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).
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.
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.
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 CHI, CH3,
or VH 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 CL region or VL region, or
both, of the antibody fragment.
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: 5), particularly where the antibody fragment is a Fab or
F(ab')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 V!SSHLGQ (SEQ ID NO:
5) and the
sequence: PKNSSMISNTP (SEQ ID NO. I).
B. Vectors, Host Cells and Recombinant Methods
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.
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
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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.
(i) Signal sequence component
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 a-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.
The DNA for such precursor region is ligated in reading frame to DNA encoding
the anti-ErbB3
antibody.
(ii) Origin of replication component
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
2p plasmid origin is suitable for yeast, and various viral origins (SV40,
polvoma, 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).
(iii) Selection gene component
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.
One example of a selection scheme utilizes a drug to arrest growth of a host
cell. Those cells that are
successf ilv 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.
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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. metal loth ionein- I and -11, preferably primate metallothionein
genes. adenosine deaminase. ornithine
decarboxylase, etc.
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 activit).
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 G418. See U.S. Patent No. 4,965,199.
A suitable selection gene for use in yeast is the trp 1 gene present in the
yeast plasmid YRp7
(Stinchcomb et al., Nature, 282:39 (1979)). The trpl 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 trpl 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.
In addition, vectors derived from the 1.6 m circular plasmid pKDI can be used
for transformation
of Kluyveromyces yeasts. Alternatively, an expression system for large-scale
production of recombinant calf
chymosin was reported for K. lactic. 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).
(iv) Promoter component
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 . P-Iactamase and lactose promoter systems,
alkaline phosphatase. a
tryptophan (tip) promoter system, and hybrid promoters such as the tac
promoter. However, other known
bacterial promoters are suitable. Promoters for use in bacterial systems also
will contain a Shine-Dalgamo
(S.D.) sequence operably linked to the DNA encoding the anti-ErbB3 antibody.
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 Tend 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.
Examples of suitable promoting sequences for use with yeast hosts include the
promoters for 3-
phosphoglycerate kinase or other glycolytic enzymes, such as enolase.
2lyceraldehyde-3-phosphate
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dehydrogenase. hexokinase. pyruvate decarhoxylase, phosphofructokmase, glucose-
6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase. triosephosphate isomerase,
phosphoglucose isomerase, and
glucokinase.
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.
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.
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 Hindlll E restriction fragment.
A system for expressing DNA
in mammalian hosts using the bovine papilloma virus as a vector is disclosed
in U.S. Patent No. 4,419,446.
A modification of this system is described in U.S Patent No. 4,601,978. See
also Reyes et al., Nature,
297:598-601 (1982) on expression of human G3-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.
(v) Enhancer element component
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, a-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.
NO Transcription termination component
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 polvadenviated fragments in the untranslated portion
of the mRNA encoding anti-
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ErbB3 antibody. One useful transcription termination component is the bovine
growth hormone
polyadenylation region. See W094/11026 and the expression vector disclosed
therein.
(vu) Selection and transformation of host cells
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 Eschertchia, e.g.. E. colt,
Enterobacter. Erwinia, Klebstella, Proteus. Salmonella, e.g., Salmonella
typhimurium, Serratia, ag.. Serratia
marcescans. and Shigella. as well as Bacilli such as B. subtilis and B.
licheniformis (e.g.. B. licheniformis 41 P
disclosed in DD 266,710 published 12 April 1989), Pseudomonas such as P
aeruginosa. and Strepromvices.
One preferred E. colt cloning host is E colt 294 (ATCC 31.446), although other
strains such as E. colt B, E.
colt X1776 (ATCC 31,537), and E. coil W3110 (ATCC 27.325) are suitable. These
examples are illustrative
rather than limiting.
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
Schizosaccharomvices pombe;
Kluvveromyces hosts such as, e.g., K. lochs, K. fragilis (ATCC 12,424), K.
bulgarieus (ATCC 16,045), K.
wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC
36,906), K. thermotolerans,
and K. marxianus: yarrowia (EP 402,226); Pichia pastoris (EP 183,070);
Candida; Trichoderma reesia (EP
244.234); Neurospora crassa; Schwanniomyces such as Schwanniomyces
occidentalis; and filamentous fungi
such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts
such as A. nidulans and A. niger.
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 marl have been identified. A variety of viral strains for transfection
are publicly available. e.g.. the
L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori
NPV, and such viruses may
be used as the virus herein according to the present invention, particularly
for transfection of Spodoptera
frugiperda cells.
Plant cell cultures of cotton. corn, potato. soybean, petunia. tomato, and
tobacco can also he utilized
as hosts.
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 CV 1 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
Viral., 36:59 (1977)); baby hamster
kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cellsi-DHFR (CHC).
Urlaub et a! Proc. Nad.
Acad. Sct. USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol
Reprod.. 23:243-251 (1980)):
monkey kidney cells (CV I ATCC CCI. 70): African green monkey kidney cells
(VERO-76. ATCC CRL-
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1587): human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells
(MDCK. ATCC CCL 34):
buffalo rat liver cells (BRL 3A, ATCC CRI.. 1442); human lung cells (W 138.
ATCC CCL 75): human liver
cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562. ATCC CCL` 1): TRI
cells (Mather et at.
Annals A Y Acad Sci., 383 44-68 (1982)); MRC 5 cells: FS4 cells: and a human
hepatoma line (Hep G2).
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.
(viii) Culturing the host cells
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. Nos. 4,767,704; 4,657,866;
4,927,762; 4,560,655; or
5,122,469; WO 90/03430; WO 87/00195; or U.S. Patent 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
GENTAMYCINTmdrug), 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.
(ix) Purification of anti-ErbB3 antibody
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. coll. Briefly, cell paste is
thawed in the presence of sodium
acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30
min. Cell debris can be
removed by centrifugation. Where the antibody is secreted into the medium,
supernatants from such expression
systems are 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.
The antibody composition prepared from the cells can be purified using, for
example. hvdroxylapatite
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 Fe domain that is present in the antibody.
Protein A can be used to purify
antibodies that are based on human y I. y2, or y4 heavy chains (I.indmark el
a,, J. Immunol Meth. 62:1-13
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WO 9735885 PCTIUS97/03546
(1983)). Protein G is recommended for all mouse isotypes and for human -(3
(cuss el al., EMBO J.
5:15671575 (1986)). The matrix to which the affinity ligand is attached is
most often agarose, but other
matrices are available Mechanically stable matrices such as controlled pore
glass or
poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing
times than can be achieved with
agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABXTMresin
(J. T. Baker, Phillipsburg,
NJ) 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 SepharoseTM 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
Following any preliminary purification step(s), the mixture comprising the
antibody of interest and
contaminants may be subjected to low pH hydrophobic interaction chromatography
using an elution buffer at
a pH between about 2.5-4.5, preferably performed at low salt concentrations
(e.g. from about 0-0.25M salt).
C. Pharmaceutical Formulations
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
octadecyidimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol,
butyl or benzyl alcohol; alkyl
parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptides; proteins, such as
serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino
acids such as glycine, glutamine,
asparagine, histiidine, aginine, 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 TweenTM, PluronicsTM or polyethylene glycol (PEG).
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 aesirable 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.
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-(methvlmethacylate) 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).
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The formulations to be used for in vivo administration must be sterile. This
is readily accomplished
by tiltration through sterile filtration membranes.
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-methacrvlate), or
poly(vinylalcohol)), polylactides
(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and y ethyl-L-
glutamate, non-degradable ethylene-
vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the
Lupron DepotTM(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 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.
D. Non-therapeutic Uses for the Antibody
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 5Ø that will
release the ErbB3 protein from the antibody.
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, US Patent 5,183,884).
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:
(a) Radioisotopes, such as 35S, 14C, 1251, 3H, and 1311. The antibody can be
labeled with the
radioisotope using the techniques described in Current Protocols in
Immunology, Volumes I and 2. Coligen
et al., Ed.. Wiley-Interscience. New York, New York, Pubs., (1991) for example
and radioactivity can be
measured using scintillation counting.
(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
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(c) Various enzyme-substrate labels are available and U.S. Patent 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. Patent
No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase,
urease, peroxidase such as
horseradish peroxidase (HRPO), alkaline phosphatase, p-galactosidase,
giucoamylase, 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).
Examples of enzyme-substrate combinations include, for example:
(i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate,
wherein the hydrogen
peroxidase oxidizes a dye precursor (e.g. orthophenylene diamine (OPD) or
3,3',5,5'-tetramethyl benzidine
hydrochloride (TMB));
(ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate as chromogenic
substrate; and
(iii) p-D-galactosidase (13-D-Gal) with a chromogenic substrate (e.g. p-
nitrophenyl-(3-D-
galactosidase) or fluorogenic substrate 4-methylumbelliferyi-(3-D-
galactosidase
Numerous other enzyme-substrate combinations are available to those skilled in
the art. For a general
review of these, see U.S. Patent Nos. 4,275,149 and 4,318,980.
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.
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.
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
immunoorecipitation assays. Zola,
Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc ,
1987).
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
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WO 97/35885 PCT/US97/03546
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.
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.. US
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.
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.
The antibodies may also be used for in vivo diagnostic assays. Generally, the
antibody is labelled with
a radionuclide (such as 111In999Tc, 14C11311, 1251, 3H, 32P or 35S) so that
the tumor can be localized using
immunoscintiography.
E. Diagnostic Kits
As a matter of convenience, the antibody of the present invention can oe
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.
F. Therapeutic Uses for the Antibody
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; gliobiastomas: 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.
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. mtra-
articular, intrasvnovial. intrathecal,
oral. topical, or inhalation routes Intravenous administration of the antibody
is preferred.
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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
EE.. 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.
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.
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.
Depending on the type and severity of the disease, about I .tg/kg to 15 mg/kg
(e.g. 0.1-20mg/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 I gg/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.
G. Articles of Manufacture
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 pierceabte 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.
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CA 02589421 2007-05-28
H. Deposit of Materials
The following hybridoma cell line has been deposited with the American Type
Culture Collection,
12301 Parklawn Drive, Rockville, MD, USA (ATCC):
Hybridoma/Antibody Designation ATCC No. Deposit Date
8138 HB-12070 March 22, 1996
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
1.14 and 35 USC :: 2, 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.
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.
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.
In respect of those designations in which a European patent is sought, a
sample of the deposited
microorganism will be made available until the publication of the mention of
the grant of the European patent
or until the date on which the application has been refused or withdrawn or is
deemed to be withdrawn, only
by the issue of such a sample to an expert nominated by the person requesting
the sample. (Rule 28(4) EPC)
The following examples are offered by way of illustration and not by way of
limitation.
EXAMPLE
PRODUCTION OF ANTI-ErbB3 ANTIBODIES
This example describes the production of the anti-Erb133 antibodies having the
characteristics
described herein.
Materials and Methods
Cell Lines. 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
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WO 97/35885 PCTIUS97/03546
were obtained from the American Type Culture Collection (Rockville, MD). Both
were cultured in RPM1 1640
medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U'mL
penicillin. 100 g/mL
streptomycin, and 10 mM HEPES ("growth medium").
Stable Transfection of K562 Cells. The K562 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 K562 transfectants. 'Therefore,
the 4E9H3 cells were placed in
growth medium containing 10 ng/mL phorbol-l2-myristate acetate (PMA) overnight
prior to use in the various
assays described below.
Antibodies. Monocional 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 (IISV 1) glycoprotein D (gD) epitope for
the monoclonal antibody 5136, 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 Balblc
mice (Charles River) were initially injected via footpad with 5 g of
gD.ErbB3.ECD in 100 I RIBI'sTM adjuvant
(Ribi ImmunochemResearch, Inc., Hamilton, MT). The animals were boosted 2
times with 5 g of
gD.ErbB3.ECD in their footpad every two weeks followed by a fmal footpad
injection of 5 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.
Monoclonal antibodies were purified and tested by immobilized and solution
phase ELISA for
cross-reactivity with ErbB2 and ErbB4. For the immobilized ELISA, I 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 I pg/ml was added
and incubated for I hour at room temperature (RT), washed and followed by goat
anti-mouse (gam) IgG
conjugated to HRPO. The ELISA was developed and read at 490nm. For the
solution phase ELISA, I g/ml
of gam IgG (Fc specific) was used to coat a 96 well microtiter plate
overnight. Anti-ErbB3 Mab at l ltgiml was
added and incubated for I 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 490nm. In this assay, none of the anti-
ErbB3 antibodies cross-reacted
with ErbB2 or ErbB4.
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.
HRG Binding Assays. All HRG binding experiments were carried out using the EGF-
like domain
of the p I isoform. i. e. HRG 13 1 177 244 (Sliwkowski et al., J. Biol Chem.
269: 14661-5 (1994)) The ErbB3
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WO 97/35885 PCT/US97/03546
antibody panel was screened for an effect on HRG binding by incubating 5.0 x
10'14E9H3 cells with 100 pM
1251-HRG overnight at 0 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
(RPM1 medium containing 10 mM HEPES, pH = 7.2) in a 96 well filtration device
(Millipore). The filters
were then removed and counted.
For the antibody dose-response experiments, 4E9H3 cells were incubated with
100 pM 1251-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 1251-HRG. For the control experiment (no antibody) I x 105 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 x 104 cells per
sample.
Antibody reduction of HRG stimulated phosphorylation. 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 HRG 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 er al.,
Science, 256:1205-1210 (1992).
Antibody reduction of ErbB2-ErbB3 protein complex formation. 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 25 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA,
1.0% Triton X-100TM
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)), covalentiy coupled to an insoluble support (Affi Prep-10TM, Bio-Rad).
The incubation was carried
out overnight at 4 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 poiyclonal 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
chemi luminescent 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.
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Results
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 1251-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 1751-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 1251-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 EC50 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.
Scatchard analysis of HRG binding was determined in the presence of these
antibodies and the results
are shown in Table I
Table 1
Data Set Kd Sites/Cell
Control 1.2 x Io-9 3.6 x I05
MAb 3-8D6 2.1 x 10-10 2.4 x 105
FAb 3-8D6 2.8 x 10-10 2.9 x 105
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.
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 in 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
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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.
The effect of the 3-8D6 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 3-8D6 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 -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 3-8D6 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.
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.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(ii APPLICANT: Genentech, Inc.
iii) TITLE OF INVENTION. E:rbB3 Antibodies
(iii) NUMBER OF SEQUENCES: 5
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Genentech, Inc.
(B) STREET: 460 Point San Bruno Blvd
(C) CITY: South San Francisco
(D) STATE: California
(E) COUNTRY: USA
(F) ZIP: 94080
(v) COMPUTER READABLE FORM;
(A) MEDIUM TYPE: 3.5 inch, 1.44 Mb floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: WinPatin (Genentech)
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Lee, Wendy M.
(B) REGISTRATION NUMBER: 40,378
(C) REFERENCE/DOCKET NUMBER: P1003PCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 415/225-1994
(B) TELEFAX: 415/952-9881
(C) TELEX: 910/371-71.68
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: L1 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Pro Lys Asn Ser Se_ Met. lie Ser Asn Thr Pro
1 10 11
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
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WO 97/35885 PCT/US97103546
(xi) SEQUENCE DESCRIPTION: SEQ ID NC:2:
His Gln Ser Leu G-.y Thr Gln
1 5
;2 INFORMATION FOF SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: B amino acids
(B) TYPE; Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
His Gln Asn Leu Seer Asp Gly Lys
1 5 8
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
His Gln Asn Ile Ser Asp Gly Lys
1 5 8
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Val Ile Ser Ser His Leu Gly Gln
1 5 8
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