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CA 02811747 2013-03-19
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GENERATION, CHARACTERIZATION AND USES THEREOF OF ANTI-HER3
ANTIBODIES
This Application claims the benefit of U.S. Provisional Patent Application No.
61/388,157; filed September 30, 2010, which is herein incorporated by
reference in its entirety.
FIELD OF THE INVENTION
The present invention relates generally to the fields of molecular biology.
More
specifically, the invention concerns anti-Her3 antibodies and antigen-bindnig
fragments thereof,
and uses of same. Methods for the prevention and treatment of diseases and
conditions
associated with aberrant Her3 activity are also included. Pharmaceutical
composition
comprising as an active agent an inhibitor of Her3 activity, particularly a
humanized anti-Her3
antibody are also included by the invention.
BACKGROUND OF THE INVENTION
Intrinsic, cell-autonomous factors as well as non-autonomous, short-range and
long-range signals guide cells through distinct developmental paths. An
organism frequently
uses the same signaling pathway within different cellular contexts to achieve
unique
developmental goals. Her3 signaling is an evolutionarily conserved mechanism
used to control
cell fates through local cell interactions. Signaling pathways between the
extracellular
environment and the nucleus of a cell involve the formation of many molecular
complexes in
which multiple proteins are assembled to directly or indirectly induce
molecular events, such as
enzyme activation or de-activation, Gomperts et al, Signal Transduction
(Academic Press, N.Y.,
2002). Such pathways and their components have been the subject of intense
investigation
because of the role aberrant pathway behavior plays in many disease
conditions, especially
cancer, e.g. McCormick, Trends in Cell Biology, 9: 53-56 (1999); Blume-Jensen
and Hunter,
Nature, 411: 355-365 (2001); Nicholson et al, Cellular Signalling, 14: 381-395
(2002). For
example, investigators have observed that many cancers are associated with an
accumulation of
mutations or other genetic alterations that affect components of signaling
pathways (e.g. by over
expression), particularly those pathways involved with cell proliferation,
cell motility,
differentiation, and cell death, e.g. Blume-Jensen and Hunter, supra. Indeed,
accumulating
evidence suggests that cancer in humans is linked to the activity of non-
viral, endogenous
oncogenes, and that a substantial portion of these oncogenes code for protein
tyrosine kinases.
Many of the growth factor receptor proteins function as tyrosine kinases and
it is by this process
that they effect signaling. The interaction of growth factors with these
receptors is a necessary
event in normal regulation of cell growth. However, under certain conditions,
as a result of
either mutation or over expression, these receptors can become deregulated,
the result of which
is uncontrolled cell proliferation which can lead to tumor growth and
ultimately to the disease
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known as cancer [Walks, A. F., Adv. Cancer Res., 60, 43 (1993) and Parsons, J.
T.; Parsons, S.
J., Important Advances in Oncology, DeVita, V. T. Ed., J. B. Lippincott Co.,
Phila, 3 (1993)].
The class of receptor tyrosine kinases is so named because when activated by
dimerization, the
intracellular domain of RTKs acquire tyrosine kinase activity that can, in
turn, activate a variety
signal transduction pathways. The predominant biological activity of some
receptor tyrosine
kinases is the stimulation of cell growth and proliferation, while other
receptor tyrosine kinases
are involved in arresting growth and promoting differentiation. In some
instances, a single
tyrosine kinase can inhibit, or stimulate cell proliferation depending on the
cellular environment
in which it is expressed.
Prominent among this class of enzymes implicated in the etiology of cancer are
the receptor tyrosine kinases, which are a subclass of cell-surface growth-
factor receptors with
an intrinsic, ligand-controlled tyrosine-kinase activity. Ligand-mediated
receptor tyrosine
kinases are believed to function as "master switches" for a coordinated
cellular communication
network that regulates the normal proliferation of eukaryotic cells. This is
generally
accomplished by catalyzing the transfer of a phosphate group from ATP to a
tyrosine residue
located on a protein substrate. Schlessinger, Cell, 103: 211-225 (2000). As
mentioned supra,
cellular growth is generally instigated via ligand-stimulated tyrosine
phosphorylation of
intracellular substrates. By binding to specific peptide ligands these
receptors are able to
integrate these external stimuli with internal signal transduction pathways,
and thereby instigate
an intracellular web of biochemical processes with a capacity to drive
dramatic cellular
transitions, such as proliferation and migration. (Schlessinger, J. and
Ullrich, A., Neuron,
9(3):383-391, 1992.).
A promising set of targets for therapeutic intervention in the treatment of
cancer
includes the members of the epidermal growth factor receptor since the
reversible
phosphorylation of tyrosine residues is required for activation of the EGFR
pathway. In other
words, EGFR-TKIs block a cell surface receptor responsible for triggering
and/or maintaining
the cell signaling pathway that induces tumor cell growth and division. The
epidermal growth
factor family can be subdivided into four groups based on their receptor-
binding specificities
(Hen, Her2, Her3, and Her4). These receptors are structurally related and
include three
functional domains: an extracellular ligand-binding domain, a transmembrane
domain, and a
cytoplasmic tyrosine kinase domain (Plowman, Culouscou et at. 1993). The
extracellular domain
can be further divided into four subdomains (I-IV), including two cysteine-
rich regions (II and
IV) and two flanking regions (I and III). Among the ErbB family, Her3 is
inimitable because of
its catalytically deficient kinase domain, its high propensity to self
associate in the absence of
ligand and the ability of the monomeric species of the extracellular domains
of Her3 to assume a
locked conformation, using an intramolecular tether.
Under normal physiological conditions, activation of the ERBB receptors is
controlled by the spatial and temporal expression of their ligands, which are
members of the
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EGF family of growth factors. Ligand binding to ERBB receptors induces the
formation of
receptor homo- and heterodimers. Dimerization consequently stimulates the
intrinsic tyrosine
kinase activity of the receptors and triggers autophosphorylation of specific
tyrosine residues
within the cytoplasmic tail. These phosphorylated residues serve as docking
sites for a range of
As one of the four members of the human epidermal growth factor receptor
(EGFR) family, Her2 distinguishes itself in several ways. First, Her2 is an
orphan receptor. The
765 (2003); Bianchi et al., J Cell Physiol 206: 702-708 (2006); Auora, supra].
The central role
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render tumor cells resistant to certain chemotherapeutics (Pegram, M., et al.,
1997, Oncogene
15;537). Both the erbB and erbB-2 genes have been shown to be activated as
oncogenes by
mechanisms involving overexpression or mutations that constitutively activate
the catalytic
activity of their encoded receptor proteins [Borgmann et al., Cell 45:649-657
(1986); Velu et al.,
Science 238:1408-1410 (1987)]. They are frequently upregulated in solid
epithelial tumors of,
by way of example, the prostate, lung and breast, and are also upregulated in
glioblastoma
tumors. Publications discussing EGFR and cancer are too numerous to disclose
herein, but
include Zeillinger et al., Clin. Biochem. 26:221-227, 1993; where it is
asserted: Increased
expression of this receptor [EGFR] has been found in various malignancies. In
carcinomas of the
cervix, ovaries, esophagus, and stomach, positive EGF-R status is definitely
associated with the
aggressiveness of the tumor. Likewise, over expression of the receptor kinase
product of the
erbB-2 oncogene has been associated with human breast and ovarian cancers
[Slamon, D. J., et.
al., Science, 244, 707 (1989) and Science, 235, 1146 (1987); David F. Stern,
J. mammary Gland
Biol. Neoplasm 13: 215-223 (2008).)]. Deregulation of EGF-R kinase has been
associated with
epidermoid tumors [Reiss, M., et al., Cancer Res., 51, 6254 (1991)], and
tumors involving other
major organs [Gullick, W. J., Brit. Med. Bull., 47, 87 (1991). Some reports
suggest hat Her2
protein overexpression occurs in approximately 30% of invasive human breast
cancers, with
Her2 gene amplification detected in 95% or more of the specimens found to over
express Her2
protein, [Gebhardt et al., Biochem. Biophys. Res. Commun., 247, 319-323
(1998)]. Other
reports suggest erbB-2 overexpression correlates with enhanced tumor
aggressiveness and a
high risk of relapse and death, and there is evidence that the overexpressed
Her2 receptor leads
to aggressive malignancies [ Slamon et al., Science 235, 177-182 (1987);
Dougall et al., DNA
Cell Biol. 15, 31-40 (1996); Yarden, Y., 2001, Oncology 1:1) and altered
sensitivity to hormonal
and chemotherapeutic agents in transfection studies in cellular and animal
models [Pegram et al.,
Oncogene, 15, 537-547 (1997); Witton et al., J Pathol 290-297 (2003); P
Arora, Oncogene
27: 4434-4445 (2008)]. As well, erb13-2 overexpression has been reported to be
an important
prognostic indicator of particularly aggressive tumors (Slamon et al, supra.].
Furthermore, in many tumours EGF-related growth factors are produced either by
the tumour cells themselves or are available from surrounding stromal cells,
leading to
constitutive EGFR activation. See Sunpaweravong et al., J. Cancer Res. Clin.
Oncol. 131, 111-
119 (2005); Salomon et al., Grit. Rev. Oncol. Hematol. 19, 183-232 (1995).
WO 00/31048 discloses a quinazoline derivative which acts as an inhibitor of
receptor tyrosine kinases such as EGFR, Her2 and Her4. An inhibition of Her3
is however not
disclosed.
WO 00/78347 discloses methods for arresting or inhibiting cell growth,
comprising preventing or reducing ErbB-2/ErbB-3 heterodimer formation. For
example, the
agent may be a combination of an anti-Her2 extracellular domain antibody and
an anti-Her3
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antibody, e.g. the Her3 antibody 113.105.5 purchased from Neomarkers. It is
however not clear
which type of anti-Her3 antibody is required to obtain desirable therapeutic
effects.
U.S. Pat. No. 5,804,396 describes a method for identifying an agent for
treatment
of a proliferative disorder, comprising the steps of assaying a potential
agent for activity in
inhibition of signal transduction by a Her2/Her3 or Her2/Her4 or Her3/Her4
heterodimer. The
patent is innocently silent relative to specific Her3 inhibitors.
Other references discussing cancer and EGFR include Karameris et al., Path.
Res.
Pract. 189:133-137, 1993; Hale et al., J. Clin, Pathol 46:149-153, 1993;
Caraglia et al., Cancer
Immunol Immunother 37:150-156, 1993; and Koenders et al., Breast Cancer
Research and
Treatment 25:21-27, 1993).
In the context of experimental cancer immunotherapy, the human epidermal
growth factor receptor 3 (also called Her3) signaling network is acquiring
increasing importance
for its possible roles in neoplastic cells and the immune system. Her3 is a
transmembrane
glycoprotein encoded by the c-erbB3 gene is a member of the epidermal growth
factor receptor
(EGFR) subfamily of type I receptor protein tyrosine kinase (RTK) family,
which also includes
EGER, Her2/neu, and Her4 (see, e.g. U.S. Pat. No. 5,183,884; Ullrich et al.,
(1984) Nature 309,
418-425; Schechter et al., (1985) Science 229, 976-978; Plowman et al.,.
(1993) Proc. Natl.
Acad. Sci. U.S.A. 90, 1746-1750.) The ErbB3 receptor, together with ErbB2, is
an important
receptor involved in cellular growth and differentiation. Particular attention
has focused on the
role of ErbB3 as a coreceptor of ErbB2 in the area of cancer research.
Her3 distinguishes itself from other epidermal growth factor receptors in that
it is
a kinase defective receptor, e.g., low tyrosine kinase activity (see, e.g. Guy
et al. (1994) Proc
Nat! Acad Sci USA 91(17), 8132-6; Carraway et al., (1994) J. Biol. Chem. 269,
14303-14306).
However, it makes up for this deficiency in that it functions most effectively
as a ligand binding
receptor for neuregulins NRG-1 and NRG-2. As well, since ErbB-2 is devoid of
an activating
ligand, it can act only in the context of a heterodimer with a ligand-bound
receptor.
Consequently, Her2 requires Her3 in order to transform normal cells into
cancer cells.1 Her3
signaling thus relies on the formation of signaling-competent heterodimers
with other ErbB
members. Amongst the numerous combinations that are possible, the most
rnitogenic "couple"
amongst the ErbB receptors is Her2/Her3. (Citri et al., 2003). Indeed, this
receptor pair
(Her2/Her3) forms the most potent signaling module of the ErbB-receptor family
in terms of cell
growth and transformation. [Kararnouzis et al., Intl J. of Biochemistry & Cell
Biol., 39: 851-856
(2007); Citri et al., Experimental, Cell Research, 284: 54-65 (2003)]. Hence,
both Her2 and
Her3 are active only in the context of ErbB heterodimers, and ErbB-2. This
dimerization enables
1-Ier2 to activate the PI3K signaling pathway. Indeed, increased expression of
Her3 increases the
signaling potency of Her2, whereas decreased Her3 expression results in the
loss of Her2
activity. Her3 is involved in Her2-mediated turnorigenesis through
dimerization with Her2.
Hsieh AC, Moasser MM. Br J Cancer. 2007;97;453-457. A characteristic feature
of attendant the
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potency of signaling by the ligand-activated ErbB-2 / ErbB-3 heterodimer lies
in the fact that this
dimer has the capacity to signal very potently, both through the Ras-Erk
pathway for
proliferation, and through the phosphatidylinosito1-3`-kinase (PI3K)-Akt
pathway for survival.
More, this receptor dimer evades downregulation mechanisms, leading to
prolonged signaling.
That the most potent signaling module is formed by partners that are incapable
of productively
signaling in isolation suggests that evolutionary forces formed these
mechanisms as a measure to
tightly control the output of the network.
Several lines of evidence have emerged in recent years that provide support
for
the pivotal role of Her3 in human tumorigenesis. An important observation
pertaining to ErbB
heterodimer collaboration during tumor development is that expression of ErbB3
is seen in many
of the same tumor types that overexpress ErbB2, including breast, bladder and
melanomas
[Rajkumar et al., Clin. Mol. Path., 49: M199-M202 (1996)1. Furthermore, many
ErbB2-
overexpressing breast tumors display elevated levels of phosphotyrosine on
ErbB3 probably as a
result of spontaneous dimerization with ErbB2.[Siegel PM, Ryan ED, Cardiff RD
and Muller
WS. (1999). EMBO J. 18,2140164.] Likewise, Mosesson et al., Seminars in Cancer
Biology,
14: 262-270 (2004) report that ErbB-3, is abundantly expressed in several
carcinomas (e.g.,
breast, colon and gastric tumors). Increased expression of EGFR and ErbB3 is
also correlated
with reduced breast cancer patient survival [Nicholson RI, Gee JM, Harper ME.
(2001). EGFR
and cancer prognosis. Eur J Cancer 37: S9¨S15; Witton et al. J Pathol 200: 290-
297 (2003).1
(Nicholson et al., 2001; Witton et al., 2003). Her2/Her3 heterodimers have
been shown to be
constitutively active in breast cancer cells with Her2 gene amplification.
Simultaneous
overexpression of Her2 and Her3 has been detected in 12-50% of invasive breast
cancers, and
the increased drug resistance in many Her2-overexpressing cancers depends on
augmented levels
of Her3 and/or EGFR [Abd El-Rehim et al. British Journal of Cancer pp. 1532-
1542 (2004)].
Her3 has been found to be overexpressed in various organs including breast,
lung, pancreas and
stomach. Furthermore, its overexpression has been documented in 20-30% of
invasive and in
approximately one third of in situ breast carcinomas, and is associated with
poor prognostic
factors [Badra et al., Cancer Letters, 244: 34-41 (2006)].
Presently, two approaches are being utilized to target the EGFR's. One
approach
proposes the use of monoclonal antibodies (mAbs) to target the extracellular
domain of the
EGFR to block natural ligand binding. See Wheeler et al., Onco gene (2008)
27,3944-3956;
doi:10.1038/onc.2008.19; published online 25 February 2008. Representative
examples include
Herceptin, and Cetuximab. The second approach involves the use of small
molecule tyrosine
kinase inhibitors (TKIs) that bind to the ATP-binding site in the tyrosine
kinase domain (TKD)
of the EGFR and Her2. Three anti-EGFR TKIs are representative of this group-
erlotinib (OSI-
774, Tarceva), gefitinib (ZD1839, Iressa) and lapatinib (GW572016, Tykerb).
Each has be
approved by the FDA for use in oncology.
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While both approaches to EGER inhibition show considerable clinical promise,
increasing evidence suggests that patients who initially respond to EGER
inhibitors may
subsequently become refractory [Wheeler et al. supra; Pao et al., PLoS Med 2:
1-11. (2005)].
With respect to the antibody approach, resistance to monoclonal antibodies
targeting Her2 are
well documented. [Lu Yet al., J Nati Cancer Inst 2001, 93: 1852-1857].
Additional limitations
relative to the use of antibody based therapy for targeting Her2 have been
noted. First, its
inhibitory effect is restricted to the Her2 displayed on the cell surface;
intracellular Her2
molecules are still available for mitogenic signaling. Second, Herceptin can
be bound and thus
"neutralized" by circulating ECDs that are released by proteolysis of membrane-
bound Her2
(Brodowicz, T., et al., 1997, Int. J. Cancer 73:875). Finally, as with many
other drugs, prolonged
treatment with Herceptin leads to acquired resistance (Kute, T., et al., 2004,
Cytometry Part A
57A:86). Another anti-Her2 antibody, pertuzumab, has been shown in a phase H
clinical trial to
have activity in ovarian cancer (Gordon, M. S., et al., 2006, J. Clin. Oncol.
24:4324). However,
only patients with highly amplified Her2 respond significantly to Herceptin
therapy, thus
limiting the number of patients suitable for therapy. In addition the
development of resistance to
drugs or a change in the expression or epitope sequence of Her2 on tumor cells
may render even
those approachable patients unreactive with the antibody and therefore
abrogating its therapeutic
benefits.
Along with the biological impact of Her3 signaling on ERBB2-amplified cellular
proliferative disorders such as breast cancer, increasing evidence links
active Her3 to resistance
to breast cancer therapeutics targeted at ERBB2 and ER. Indeed, a significant
limitation in using
these compounds is that recipients thereof may develop a resistance to their
therapeutic effects
after they initially respond to therapy, or they may not respond to EGFR-TKIs
to any measurable
degree ab initio. In fact, only 10-15 percent of advanced non-small cell lung
cancer patients
respond to EGER kinase inhibitors. Since Her3 has no catalytic activity, it
appears that Her3
promotes drug resistance by enabling autocrine or paracrine ligands (NRG1 and
NRG2) to
activate catalytically competent RTKs, and through its capacity to channel
signaling to PI3KJAkt
signaling pathways. Finally, it is formally possible that Her3 affects
response to ERBB
inhibitors indirectly, through protection of ERBB2 kinase domain or
extracellular domain in
heterodimers from phosphatases or inhibitors, or by reducing formation of
ERBB2 homodimers,
or dimers with other receptors such as ERBB4 that may have protective value
for patients.
[Stern, J. mammary Gland Biol. Neoplasm 13: 215-223 (2008)]. It appears that
when Her2 is
targeted using a tyrosine kinase inhibitor, tumor cells can compensate by
upregulating Her3
activation making it more difficult to reverse the process of Her2:Her3
phosphorylation. Thus,
Her3 does not remain "turned off" after tyrosine kinase inhibitor treatment
and is able to be
phosphorylated, which allows for activation of the PI3K/Akt pathway. [Sergina
et al., Nature.
2007;445:437-441.] For patients that develop resistance, this potentially life-
saving therapeutic
mechanism fails to achieve what they had hoped for and so desperately needed--
an active
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therapy for cancer. Thus, although the compounds may, at first, exhibit strong
anti-tumor
properties, they may soon become less potent or entirely ineffective in the
treatment of cancer.
Recent studies further support numerous prior epidemiologic reports indicating
that a considerable proportion of middle-aged women are at a high risk for
cardiovascular
-- disease (CVD) independent of prior history of breast cancer treatment.
Apparently, patients with
early-stage Her2/neu-positive breast cancer seem to be at an even greater risk
of CVD than these
women due to the direct and indirect toxic effects of adjuvant breast cancer
therapy. [Cancer
Epidemiology Biomarkers & Prevention 16: 1026 ¨ 1029 (2007); Lee W. Jones 1
Internal
Medicine News, Feb 15, 2006 by Bruce Jancin.
Murine or chimeric Her3 antibodies have been reported, such as in U.S. Pat.
No.
5,968,511, U.S. Pat. No. 5,480,968 and W003013602. A monoclonal antibody
against Her3
(Rajkumar et al., Br. J. Cancer 70 (1994), 459-456) had an agonistic effect on
the anchorage-
independent growth of cell lines expressing Her3. On the other hand, anti-Her3
antibodies
described in U.S. patent 5,968,511 are reported to reduce Heregulin-induced
formation of
-- Her2/Her3 heterodimers. Such an activity, however, is only demonstrated for
an antibody which
increases Heregulin binding to Her3. Likewise, while van der Horst EH, Murgia
M, Treder M, et
al; Int J Cancer 115:519-527, 2005 have demonstrated the inhibitory effect of
an antibody
directed against Her3 (N-Her3ECD) on Her3-mediated signaling, it is unclear if
the antibody had
any effect in-vivo. As well, the antibody is a murine antibody liable to
induce a HAMA response
-- in humans. Towards this end, the authors speculate that it would be very
interesting to see
whether the ck-Her3ECD in vitro data could be translated to an in vivo
setting. Thus, at present it
is not clear which type of anti-Her3-antibody - if any - has potential of
being used for therapeutic
applications.
As well, the prior art suggests that while interfering with signaling through
the
-- ERBB2¨Her3 dimer might offers an alternative therapeutic strategy to
targeting ERBB2 alone,
such inhibition appeared to be transitory in some cases and compensatory
mechanisms allowed
cells to restore PI3K--Akt pathway signaling and tumorigenesis. As well, the
prior art suggests
that there were concerns regarding the dose required to achieve a complete
blockade of the
signaling pathway. "A central role for HER3 in HER2-amplified breast cancer:
implications for
-- targeted therapy. Cancer Res. 68, 5878-5887 (2008); Nature Reviews Cancer
9, 463-475 (July
2009)
While the proposed partnership between Her2/Her3 have created opportunities
for improving efficacy of ERBB-targeted pharmaceuticals, by interfering with
coupling of
ERBB2 to ERBB3 through dimerization inhibitors, the art is completely silent
as to the
-- identification of a Her3 selective antagonist that is therapeutically
effective in treating Her3
mediated cellular proliferative disorders. Indeed, despite encouraging
results, the failure of
Herceptin therapies for many ERBB2-amplified breast cancers, the absence of a
Her3 selective
antibody that is therapeutically effective together with the eventual
development of therapeutic
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resistance in cases where robust responses occur at first, has created a need
for an alternative,
more potent, Her3 inhibitor. More, in spite of the discouraging landscape
attendant conventional
tyrosine kinase inhibitors, in particular, Her3 antibodies, the inventors
endeavored to develop
effective Her3 antagonists, the details of which are disclosed herein.
Disclosed herein are novel Her3 selective agents, e.g., antibodies that are
unencumbered by the deficiencies attendant current Her2/Her3 moieties. Also
disclosed is a
pharmaceutical composition comprising as an active agent a specific type of
inhibitor of Her3
activity, e.g., antibody and pharmaceutically acceptable carriers, diluents
and/or adjuvants.
Methods of using the Her3 antibodies are also disclosed.
SUMMARY OF THE INVENTION
Embodiments of this invention are made available by the development of
antibodies and antigen-bindnig fragments thereof, in particular humanized anti-
her3 antibodies
that retain favorable affinity to the Her3 receptor protein, particularly
human Her3 receptor
protein. Her3 presents as an important and advantageous therapeutic target and
the
identification of a variety of Her3 binding agents (such as immunoconjugates,
antibodies, and
fragments thereof) provide improved therapeutic and diagnostic agents for use
in targeting
pathological conditions associated with expression and/or activity of the Her3
mediated signaling
pathway. For example, the anti-Her3 antibodies described herein, ("Invention
Antibodies") offer
an important new approach to the treatment of various disorders of cell fate,
in particular
hyperproliferative disorders (e.g., cancer). Also provided are compositions
and methods based
on binding Her3. Kits and articles of manufacture related to the Her3 binding
are also included.
A broad aspect of the invention relates to at least one humanized monoclonal
antibody, or binding fragment thereof described herein that binds specifically
to an antigen
present in various cancers mediated by or related to Her3 activation or
dysregulation, wherein
the antigen is Her3. The monoclonal antibodies of the invention bind to the
human Her3
receptor (Her3) and can thus be useful in methods to treat or diagnose
pathological
hyperproliferative oncogenic disorders mediated by Her3 or Her2 expression or
dysplastic cells
associated with increased expression or activity of the Her3 receptor protein.
An embodiment of this invention relates to the antibodies and antigen-binding
fragments thereof described herein, including polypeptides comprising amino
acid sequences of
the immunoglobulin chains of the antibodies and fragments, e.g., of the VRs,
FRs and CDRs
polypeptides and the polynucleotides encoding them. Variant antibodies
exemplified by
diabody, bi-specific, trivalent & tetravalent antibodies or other antibodies
derived from the
herein described invention antibodies are also encompassed by the invention.
As mentioned, the present invention includes isolated polypeptides (e.g.,
antibodies and
antigen-binding fragments thereof) comprising one or more (e.g., 3) CDRs taken
from the light
and/or heavy chain immunoglobulin set forth herein (e.gõ 3 light chain CDRs
and 3 heavy chain
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CDRs) as defined by the convention set forth in Kabat, "Sequences of Proteins
of Immunological
Interest" (National Institutes of Health, Bethesda, Md., 1987 and 1991) or in
Chothia et al., J.
Mol. Biol. 196:901 (1987); Nature 342:878 (1989); and J. Mol. Biol. 186:651
(1989) (Kabat or
Chothia) or Al-Lazikani et al., J. Mol. Biol. 273: 927-948 (1997).
The present invention provides antibodies and antigen-bindnig fragments
thereof
that bind to Her3.
In certain embodiments, at least one invention described herein binds to the
ligand
binding domain of the Her3 receptor.
In yet another embodiment, the invention provides an antibody, in particular,
a
chimeric or humanized antibody that binds to the negative regulator region,
resident in the
extracellular domain of the Her3 receptor.
In one aspect, the invention provides an isolated antibody comprising at least
one
(at least 2, at least 3, at least 4, at least 5, and/or 6) hypervariable
sequence(s) (HVR(s))
comprising a sequence selected from the group consisting of HVR-L1, HVR-L2,
HVR-L3,
HVR-HL HVR-H2, and/or HVR-H3 of the antibody described herein, wherein said
isolated
antibody specifically binds human Her3.
In one aspect, the invention provides an isolated antibody that binds to the
same
epitope on human Her3 as any one or more of the humanized antibodies disclosed
herein.
In one aspect, the invention provides an isolated anti-Her3 antibody, wherein
a
full length IgG form of the antibody specifically binds human Her3 with a
binding affinity
sufficient to be therapeutically effective when administered to a patient. A
representative
affinity is 70 pM or better. As is well-established in the art, binding
affinity of a ligand to its
receptor can be determined using any of a variety of assays, and expressed in
terms of a variety
of quantitative values. Accordingly, in one embodiment, the binding affinity
is expressed as Kd
values and reflects intrinsic binding affinity (e.g., with minimized avidity
effects). Generally and
preferably, binding affinity is measured in vitro, whether in a cell-free or
cell-associated setting.
Any of a number of assays known in the art, including those described herein,
can be used to
obtain binding affinity measurements, including, for example, Biacore,
radioimmunoassay (RIA)
and ELISA.
In one aspect, the invention provides an anti-Her3 antibody comprising: at
least
one, two, three, four, five, and/or six hypervariable region (HVR) sequences
selected from the
group described in Appendix II, infra.
In one aspect, the invention provides an anti-Her3 antibody comprising: at
least
one, two, three, four, five, and/or six hypervariable region (HVR) sequences
selected from the
group described in Appendix II, infra. HVR-L1 comprising at least one HVR-
Llsequence from
those set forth in Appendix II.
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In one embodiment, an antibody of the invention comprises a light chain
comprising at least one, at least two or all three of HVR sequences selected
from the group as set
forth in Appendix II.
As is known in the art, the amino acid position/boundary delineating a
hypervariable region of an antibody can vary, depending on the context and the
various
definitions known in the art (as described below). Some positions within a
variable domain may
be viewed as hybrid hypervariable positions in that these positions can be
deemed to be within a
hypervariable region under one set of criteria while being deemed to be
outside a hypervariable
region under a different set of criteria. One or more of these positions can
also be found in
extended hypervariable regions.
It is understood that the term "antibody" includes "antibodies" such as one or
more of the Her3 specific antibodies described herein that bind Her3. As well,
it includes
monoclonal, polyclonal, multivalent, bispecific, and trivalent or optimized
antibodies including
fragments thereof. The invention also contemplates the use of single chains
such as the variable
heavy and light chains of the antibodies. Generation of any of these types of
antibodies or
antibody fragments is well known to those skilled in the art. In the present
case, monoclonal
antibodies to Her3 receptor proteins have been generated and have been
isolated and shown to
have high affinity to Her3.
The invention also includes modifications to the invention antibodies
including
variants thereof which do not significantly affect their binding properties.
Such variants may
have enhanced or decreased activity towards its binding partner.
Another embodiment of the invention encompasses monoclonal antibody or an
antigen-binding fragment thereof that may be Fab fragments, F(ab)2 fragments,
Fab' fragments,
F(ab')2 fragments, Fd fragments, Fd' fragments or Fv fragments, Fv, scFv, scFv-
Fc or diabodies
or any functional fragment whose half-life would have been increased by a
chemical
modification, especially by PEGylation, or by incorporation in a liposome. It
may also be an
anti-idiotypic antibody. Plasma protein binding can be an effective means of
improving the
pharmacokinetic properties of otherwise short lived molecules.
One general strategy of reducing a therapeutic proteins intrinsic rate of
clearance
is via amino acid substitutions. In case of a protein, this strategy may
entail amino acid
substitutions that reduce receptor binding affinity in intracellular endosomal
compartments,
thereby leading to increased recycling in the ligand-sorting process and
consequently resulting in
longer half-life in extracellular medium. See Sarkar C.A., Lowenhaupt K.,
Horan T., Boone
T.C., Tidor B., Lauffenburger D.A. Nat. Biotechnol. (2002) 20:908-913. A
second approach is
to express the therapeutic protein as a genetic fusion with a natural protein
that has a long serum
half-life; either 67 kDa serum albumin (SA) - Syed S., Schuyler P.D.,
Kulczycky M., Sheffield
W.P. Blood (1997) 89:3243-3252) or the Fc portion of an antibody, which adds
an additional
60-70 kDa in its natural dimeric form, depending on glycosylation (Mohler et
al., J. Immunol.,
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151:1548-1561 (1993). As a consequence, an embodiment of the invention
provides
modifications to at least one antibody disclosed herein resulting in a fusion
protein comprising
an antibody of the invention fused to albumin. See Dennis et al., "Albumin
binding as a general
strategy for improving the pharmacokinetics of proteins." 3" Biol Chem.,
277:35035-43 (2002).
Glycosylated variants (Glycoforms) of the invention antibodies are also
envisioned. In one embodiment of the invention, antibodies, or fragments
thereof, are modified
to reduce or eliminate potential glycosylation sites. The amino acids where
carbohydrate, such
as oligosaccharide, is attached are typically asparagine (N-linkage), serine
(0-linkage), and
threonine (0-linkage) residues. In order to identify potential glycosylation
sites within an
antibody or antigen-binding fragment, the sequence of the antibody is
examined, for example, by
using publicly available databases such as the webs ite provided by the Center
for Biological
Sequence Analysis (see http://www.ebs.dtu.dk/services/NetNGlye/ for predicting
N-linked
glycosylation sites) and http://www.ebs.dtu.didservices/Net0Glye/ for
predicting 0-linked
glycosylation sites). Additional methods for altering glycosylation sites of
antibodies are
described in U.S. Pat. Nos. 6,350,861 and 5,714,350, the entire content of
each of which is
incorporated herein in its entirety. In order to improve the binding affinity
of an antibody or
antigen-binding fragment thereof, glycosylation sites of the antibody can be
altered, for example,
by mutagenesis (e.g., site-directed mutagenesis). Such modified antibodies
having reduced
glycosylation sites or carbohydrates relative to the unmodified form are
referred to as
"aglycosylated" antibodies. An "afucosylated" anti-Her3 antibody derived from
one or more
antibodies described herein is representative of such an antibody that falls
within the scope of the
invention. See Li et al., Nat. Biotechnol., 24: 210-215 ( 2006); Shields, R.L.
et al. Lack of
fucose on human IgG1 N-linked oligosaccharide improves binding to human Fe
YRIII and
antibody-dependent cellular toxicity. J. Biol. Chem. 277, 26733-26740 (2002).
In alternative
embodiment, the invention antibodies or antigen binding fragments thereof are
modified to
enhance glycosylation.
Fe engineered variants of the invention antibodies are also encompassed by the
present invention. Such variants include antibodies or antigen binding
fragments thereof which
have been engineered so as to introduce mutations or substitutions in the Fe
region of the
antibody molecule so as to improve or modulate the effector functions of the
underlying
antibody molecule relative to the unmodified antibody. In general, improved
effector functions
is included o refer to such activities as CDC and ADCC. Further thereto, the
invention provides
Fe variants that have improved function and/or solution properties as compared
to the
aglycosylated form of the parent Fe polypeptide. Improved functionality herein
includes but is
not limited to binding affinity to an Fe ligand. Improved solution properties
herein include but
are not limited to stability and solubility. In one embodiment, the proposed
Fe variants bind to
an Fe* with an affinity that is within about 0.5-fold of the glycosylated form
of the parent Fe
polypeptide. In an alternate embodiment, the aglyeosylated Fe variants bind to
an FcyR with an
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affinity that is comparable to the glycosylated parent Fe polypeptide. In an
alternate
embodiment, the Fe variants bind to an FcyR with an affinity that is greater
than the glycosylated
form of the parent Fe polypeptide.
Another broad aspect of the invention comprises an antibody or a binding
fragment thereof that comprises a light chain comprising at least one
complementarity
determining region CDR having an amino acid sequence as set forth in one or
more appendices
detailed herein (Tables 1-4) or at least one CDR whose sequence has at least
80%, preferably
85%, 90%, 95% and 98% identity, after optimum alignment, with the sequences
set forth in one
or more appendices described herein or a heavy chain comprising at least one
CDR comprising
an amino acid sequence selected from the group set forth in one or more
appendices set forth
herein or at least one CDR whose sequence has at least 80%, preferably 85%,
90%, 95% and
98% identity, after optimum alignment, with said at least one CDR as set forth
in one or more
appendices set forth herein. Alternatively, the antibody of the invention
comprises at least one
heavy chain and/or a light chain comprising at least one amino acid sequence
as set forth in one
of Tables 1 or 2. Nucleic acid molecules comprising nucleotide sequences
encoding at least one
or more of the above referenced amino acid sequences are also contemplated.
See Appendices I
¨II.
The light chain may likewise comprise the amino acid sequence as set forth in
one
or more table detailed herein, while the heavy chain may comprise the amino
acid sequence as
set forth in one or more table as set forth herein. See Tables 1-6.
Antibodies that compete with any one or more of the antibodies described
herein
for binding with Her3 lare also within the scope of the invention.
In recent years, various strategies have been developed for preparing scFv as
a
multimeric derivative. This is intended to lead, in particular, to recombinant
antibodies with
improved pharmacokinetic and biodistribution properties as well as with
increased binding
avidity. In order to achieve multimerization of the scFv, scFv may be prepared
as fusion proteins
with multimerization domains. The multimerization domains may be, e.g. the CH3
region of an
IgG or coiled coil structure (helix structures) such as Leucine-zipper
domains. However, there
are also strategies in which the interaction between the \IFNI, regions of the
scFv are used for
the multimerization (e.g. di-, tri- and pentabodies).
Also considered are multivalent antibody constructs that are Her3 antagonists
or
agonists. In one embodiment, a multivalent antibody construct comprises at
least one antigen
recognition site specific for Her3 receptor protein. In certain embodiments,
at least one of the
antigen recognition sites is located on a scFv domain, while in other
embodiments; all antigen
recognition sites are located on scFv domains.
Also contemplated herein is a multivalent, multispecific antibody or fragment
thereof comprising more than one antigen binding site having an affinity
toward a Her3 target
antigen and one or more hapten binding sites having affinity towards hapten
molecules. Also
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preferred, the multivalent, multispecific antibody or fragment thereof further
comprises a
diagnostic/detection and/or therapeutic agent.
The phrase "multivalent antibody" or "multivalent antibody construct" refers
to an
antibody or antibody construct comprising more than one antigen recognition
site. For example,
a "bivalent" antibody construct has two antigen recognition sites, whereas a
"tetravalent"
antibody construct has four antigen recognition sites. The terms
"monospecific", "bispecific",
"trispecific", "tetraspecific", etc. refer to the number of different antigen
recognition site
specificities (as opposed to the number of antigen recognition sites) present
in a multivalent
antibody construct of the invention. For example, a "monospecific" antibody
construct's antigen
recognition sites all bind the same epitope. A "bispecific" antibody construct
has at least one
antigen recognition site that binds a first epitope and at least one antigen
recognition site that
binds a second epitope that is different from the first epitope. A
"multivalent monospecific"
antibody construct has multiple antigen recognition sites that all bind the
same epitope. A
"multivalent bispecific" antibody construct has multiple antigen recognition
sites, some number
of which bind a first epitope and some number of which bind a second epitope
that is different
from the first epitope.
In still another embodiment, the antibody construct is monospecific. In still
another embodiment, the multivalent antibody is tetravalent. In one embodiment
of the
invention, the antibody is a monospecific tetravalent antibody, wherein the
antibody comprises
four Her3 antigen recognition sites. In still another embodiment, the antibody
construct is
specific for an epitope on Her3.
In another embodiment of the invention, the antibody construct is bispecific.
In
one embodiment, the antibody construct has two Her3-specific antigen
recognition sites and two
Her3-specific recognition sites.
In another embodiment of the invention, the antibody construct is a trivalent
antibody construct specific for the Her3 receptor protein. In yet another
embodiment, the
invention contemplates an antibody construct having two Her3-specific antigen
recognition sites
and two Her3-specific recognition sites.
Other antibody constructs may be multispecific for different epitopes on human
Her3 receptor proteins. In any of the multispecific antibody constructs, at
least one antigen
recognition site may be located on a scFv domain, and in certain embodiments,
all antigen
recognition sites are located on scFv domains.
In one aspect, the invention provides an antibody fragment comprising: (i) a
first
polypeptide comprising a light chain variable domain (and in some embodiments
further
comprising a light chain constant domain), (ii) a second polypeptide
comprising a heavy chain
variable domain, a first Fe polypeptide sequence (and in some embodiments
further comprising a
non-Fc heavy chain constant domain sequence), and (iii) a third polypeptide
comprising a second
Fc polypeptide sequence. Generally, the second polypeptide is a single
polypeptide comprising a
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heavy chain variable domain, heavy chain constant domain (e.g., all or part of
all) and the first
Fc polypeptide. For example, the first Fe polypeptide sequence is generally
linked to the heavy
chain constant domain by a peptide bond [i.e., not a non-peptidyl bond]. In
one embodiment, the
third polypeptide comprises an N-terminally truncated heavy chain which
comprises at least a
portion of a hinge sequence at its N terminus. In one embodiment, the third
polypeptide
comprises an N-terminally truncated heavy chain which does not comprise a
functional or wild
type hinge sequence at its N terminus. In some embodiments, the two Fe
polypeptides of an
antibody of the invention or a fragment thereof are covalently linked. For
example, the two Fe
polypeptides may be linked through intermolecular disulfide bonds, for
instance through
intermolecular disulfide bonds between cysteine residues of the hinge region.
In one aspect, the invention provides a composition comprising a population of
immunoglobulins wherein at least (or at least about) 50%, 75%, 85%, 90%, 95%
of the
immunoglobulins are antibody fragments of the invention. A composition
comprising said
population of immunoglobulins can be in any of a variety of forms, including
but not limited to
host cell lysate, cell culture medium, host cell paste, or semi-purified or
purified forms thereof
Purification methods are well known in the art, some of which are described
herein.
Another embodiment of the invention provides a Her3-specific diabody antibody.
By diabody the skilled person means a bivalent homodimeric scFv derivative (Hu
et al., 1996,
PNAS 16: 5879-5883). The shortening of the Linker in a scFv molecule to 5-10
amino acids
leads to the formation of homodimers in which an inter-chain VH/NL-
superimposition takes
place. Diabodies may additionally be stabilized by the incorporation of
disulphide bridges.
Examples of diabody-antibody proteins from the prior art can be found in
Perisic et al. (1994,
Structure 2: 1217-1226).
By minibody the skilled person means a bivalent, homodimeric scFv derivative.
It
consists of a fusion protein which contains the CH3 region of an
immunoglobulin, preferably
IgG, most preferably IgG1 as the dimerization region which is connected to the
scFv via a Hinge
region (e.g. also from IgG1) and a Linker region. The disulphide bridges in
the Hinge region are
mostly formed in higher cells and not in prokaryotes. Preferably the minibody
is a Her3-specific
minibody antibody fragment. Examples of minibody-antibody proteins from the
prior art can be
found in Hu et al. (1996, Cancer Res. 56: 3055-61).
By triabody the skilled person means a: trivalent homotrimeric say derivative
(Kortt et al. 1997 Protein Engineering 10: 423-433). ScFv derivatives wherein
VI-VL are fused
directly without a linker sequence lead to the formation of trimers.
The skilled person will also be familiar with so-called miniantibodies which
have
a bi-, tri- or tetravalent structure and are derived from scFv. The
multimerization is carried out
by di-, tri- or tetrameric coiled coil structures (Pack et al., 1993
Biotechnology II:, 1271-1277;
Lovejoy et al. 1993 Science 259: 1288-1293; Pack et al., 1995 J. Mol. Biol.
246: 28-34).
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Therefore, an alternative embodiment proposes a Her3-specific multimerized
molecule based on the abovementioned antibody fragments and may be, for
example, a triabody,
a tetravalent miniantibody or a pentabody.
A related aspect of the invention provides monoclonal antibodies or functional
fragments thereof that specifically binds human Her3 with specified
affinities. In certain
embodiments, these antibodies bind human Her3 with an ED50 in the range of
about 10 pM to
about 500 nM. In certain embodiments, these antibodies bind human Her3 with an
ED50 in the
range of about 500 pM to about 300 nM.
In one embodiment, the antibody is a chimeric antibody, for example, an
antibody
comprising antigen binding sequences from a non-human donor grafted to a
heterologous non-
human, human or humanized sequence (e.g., framework and/or constant domain
sequences). In
one embodiment, the non-human donor is a mouse. In one embodiment, an antigen
binding
sequence is synthetic, e.g. obtained by mutagenesis (e.g., phage display
screening, etc.). In one
embodiment, a chimeric antibody of the invention has murine V regions and
human C region. In
one embodiment, the murine light chain V region is fused to a human kappa
light chain. In one
embodiment, the murine heavy chain V region is fused to a human IgG1 C region.
Humanized antibodies of the invention include those that have amino acid
substitutions in the FR and affinity maturation variants with changes in the
grafted CDRs. The
substituted amino acids in the CDR or FR are not limited to those present in
the donor or
recipient antibody. In other embodiments, the antibodies of the invention
further comprise
changes in amino acid residues in the Fc region that lead to improved effector
function including
enhanced CDC and/or ADCC function and B-cell killing. Other antibodies of the
invention
include those having specific changes that improve stability. Antibodies of
the invention also
include fucose deficient variants having improved ADCC function in vivo. In
other
embodiments, the antibodies of the invention comprise changes in amino acid
residues in the Fc
region that lead to decreased effector function, e.g. decreased CDC and/or
ADCC function
and/or decreased B-cell killing. In some embodiments, the antibodies of the
invention are
characterized by decreased binding (such as absence of binding) to human
complement factor
Clq and/or human Fc receptor on natural killer (NK) cells. In some
embodiments, the antibodies
of the invention are characterized by decreased binding (such as the absence
of binding) to
human Fc.gamma.RI, Fc.gamma.RIIA, and/or Fe.gamma.RIIIA.
In one aspect, the invention provides anti-Her3 polypeptides comprising any of
the antigen binding sequences provided herein, wherein the anti-Her3
polypeptides specifically
bind to the human Her3 receptor.
In one aspect, the invention provides methods for promoting the development,
proliferation, maintenance or regeneration of neurons, the methods comprising
administering an
effective amount of an anti-Her3 antibody or immunoconjugate (in some
embodiments, an anti-
Her3 antibody of the invention) to a subject in need of such treatment.
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In one aspect, the invention provides methods for inhibiting angiogenesis, the
methods comprising administering an effective amount of an anti-Her3 antibody
or
immunoconjugate (in some embodiments, an anti-Her3 antibody of the invention)
to a subject in
need of such treatment. In some embodiments, the site of angiogenesis is a
tumor or cancer.
Methods of the invention can be used to affect any suitable pathological
state.
Exemplary disorders are described herein, and include a cancer selected from
the group
consisting of small cell lung cancer, neuroblastomas, melanoma, breast
carcinoma, gastric
cancer, colorectal cancer (CRC), and hepatocellular carcinoma; and disorders
characterized by
colon adenomas.
In one embodiment, a cell that is targeted in a method of the invention is a
cancer
cell. For example, a cancer cell can be one selected from the group consisting
of a breast cancer
cell, a colorectal cancer cell, a lung cancer cell, a papillary carcinoma
cell, a colon cancer cell, a
pancreatic cancer cell, an ovarian cancer cell, a cervical cancer cell, a
central nervous system
cancer cell, an osteogenic sarcoma cell, a renal carcinoma cell, a
hepatocellular carcinoma cell, a
bladder cancer cell, a gastric carcinoma cell, a head and neck squamous
carcinoma cell, a
melanoma cell, a leukemia cell, and a colon adenoma cell. In one embodiment, a
cell that is
targeted in a method of the invention is a hyperproliferative and/or
hyperplastic cell. In one
embodiment, a cell that is targeted in a method of the invention is a
dysplastic cell. In yet another
embodiment, a cell that is targeted in a method of the invention is a
metastatic cell. In some
embodiments, the cell that is targeted is a colon adenoma cell. In some
embodiments, the cell
that is targeted expresses on the cell membrane at least about 20,000; 30,000;
40,000; 50,000;
60,000; 70,000; 80,000; 90,000; 100,000; 150,000; 200,000; 250,000; 300,000;
350,000;
400,000; 450,000; 500,000; 550,000; 600,000; 650,000; 700,000; 750,000;
800,000; 850,000, or
more Her3 molecules.
Methods of the invention can further comprise additional treatment steps. For
example, in one embodiment, a method further comprises a step wherein a
targeted cell and/or
tissue (for e.g., a cancer cell) is exposed to radiation treatment or a
chemotherapeutic agent.
In another aspect, the invention provides methods for detection of Her3, the
methods comprising detecting Her3-anti-Her3 antibody complex in the sample.
The term
"detection" as used herein includes qualitative and/or quantitative detection
(e.g., measuring
levels) with or without reference to a control. See infra for more details.
In another aspect, the invention provides methods for diagnosing a disorder
associated with Her3 expression and/or activity, the methods comprising
detecting Her3-anti-
Her3 antibody complex in a biological sample from a patient having or
suspected of having the
disorder. In some embodiments, the Her3 expression is increased expression or
abnormal
expression. In some embodiments, the disorder is a tumor, cancer, and/or a
cell proliferative
disorder.
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In another aspect, the invention provides any of the anti-Her3 antibodies
described herein, wherein the anti-Her3 antibody comprises a detectable label.
In another aspect, the invention provides a complex of any of the anti-Her3
antibodies described herein and Her3. In some embodiments, the complex is in
vivo or in vitro.
In some embodiments, the complex comprises a cancer cell. In some embodiments,
the anti-Her3
antibody is detectably labeled.
Also provided by the invention is a method of treating a Her3 mediated
neoplasm
or malignancy characterized by aberrant Her3 signaling or expression ,
comprising administering
to a patient suffering from the neoplasm or malignancy, a therapeutically
effective amount of a
Her3 binding antibody.
In one embodiment, the Her3 antibody is administered at dosages that are
therapeutically effective. In other embodiments, the Her3 antibody is
administered in
conjunction with chemotherapy.
In any of the embodiments of the methods, compositions and articles of
manufacture of the invention, the anti-Her3 antibody includes chimeric and
humanized antibody.
Purely for the purposes herein, "humanized Her3" refers to an intact antibody
or
antibody fragment comprising any one or more of the variable light or heavy
chain sequences as
set forth in Appendix II.
Another aspect of the invention relates to the screening of a patient
suspected of
having a Her3 related disease or condition to determine if such a patient
would benefit from
treatment with an anti-Her3 antibody. Such detection includes both cell
surface detection as well
as soluble Her3 found in the serum of said patient. See infra.
The invention also provides an isolated cell line that produces at least one
anti-
Her3 antibody as described herein. An embodiment of the invention thus
provides, in one
aspect, the use of a nucleic acid of the invention in the preparation of a
medicament for the
therapeutic and/or prophylactic treatment of a disorder, such as a cancer, a
tumor, and/or a cell
proliferative disorder. In another aspect, the invention provides use of an
expression vector of the
invention in the preparation of a medicament for the therapeutic and/or
prophylactic treatment of
a disorder, such as a cancer, a tumor, and/or a cell proliferative disorder.
In yet another aspect,
the invention provides use of a host cell of the invention in the preparation
of a medicament for
the therapeutic and/or prophylactic treatment of a disorder, such as a cancer,
a tumor, and/or a
cell proliferative disorder.
In one aspect, the invention provides compositions comprising one or more
nucleic acid of the invention and a carrier. In one embodiment, the carrier is
pharmaceutically
acceptable.
In one aspect, the invention provides host cells comprising a nucleic acid or
a
vector of the invention. A vector can be of any type, for example a
recombinant vector such as
an expression vector. Any of a variety of host cells can be used. In one
embodiment, a host cell
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is a prokaryotic cell, for example, B. coll. In one embodiment, a host cell is
a eukaryotic cell, for
example a mammalian cell such as Chinese Hamster Ovary (CHO) cell.
In one aspect, the invention provides methods of making an antibody or
immunoconjugate of the invention. For example, the invention provides methods
of making an
anti-Her3 antibody (which, as defined herein includes full length and
fragments thereof) or
immunoconjugate, said method comprising expressing in a suitable host cell a
recombinant
vector of the invention encoding said antibody (or fragment thereof), and
recovering said
antibody. The invention further provides methods of making an anti-Her3
immunoconjugate,
said method comprising expressing in a suitable host cell a recombinant vector
of the invention
encoding an anti-Her3 antibody (or fragment thereof) or the invention,
recovering said anti-Her3
antibody, and conjugating the anti-Her3 antibody to a drug.
In yet another embodiment, at least one antibody of the invention binds to the
negative regulator region, resident in the extracellular domain of the Her3
receptor.
The present invention further provides an antibody-recognized surface antigen
present on host cell, including but not limited to T-cell acute lymphoblastic
leukemia/lymphoma,
human colon cancer, melanoma, human lung cancer and human prostate cancer, the
antigen
being Her3 or a biologically equivalent variant or fragment thereof.
Also provided is a monoclonal antibody of the invention or a binding fragment
thereof that is bound to a solid matrix.
Antibodies to Her3 as described herein may also be used in production
facilities
or laboratories to isolate additional quantities of the proteins, such as by
affinity
chromatography. For example, the antibodies of the invention may also be
utilized to isolate
additional amounts of Her3.
In one aspect, the invention provides isolated, purified or recombinant
polypeptides having an amino acid sequence that is at least 90%, 95%, 98% or
99% identical to
an amino acid sequence as set forth in one or more appendices herein
described. In certain
embodiments the application provides an amino acid sequence that is at least
90%, 95%, 98%,
99%, 99.3%, 99.5% or 99.7% identical to the target amino acid sequence herein
described.
The present invention further relates to a polynucleotide encoding an antibody
of
the invention. In accordance therewith, the invention further provides:
isolated nucleic acid
encoding the inventive antibodies disclosed herein including the heavy and/or
light chain or
antigen-binding portions thereof. Thus, an aspect of the invention provides
isolated nucleic acid
molecules selected from the nucleotide sequences described in any one or more
of the
appendices detailed herein. A related aspect is drawn to (a) a nucleic aid
molecule described in
any one or more of the appendices detailed herein encoding one or more of the
sequence of
amino acids as set forth in one or more of the appendices described herein; or
(b) the nucleotide
sequence that hybridizes to the nucleotide sequence of (a) under moderately
stringent conditions,
or (c) a nucleic acid molecule comprising a nucleotide sequence that is a
degenerate sequence
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with respect to either (a) or (b) above, or (d) splice variant cDNA sequences
thereof or (e) a
nucleic acid of at least 18 nucleotides capable of hybridizing under
conditions of great stringency
with at least one of the CDRs of nucleic acid sequence described in one or
more of the
appendices described herein or with a sequence having at least 80%, preferably
85%, 90%, 95%
and 98%, identity after optimum alignment with the sequence as set forth in
one or more of the
appendices detailed herein.
A vector comprising the nucleic acid molecule described above, optionally,
operably linked to control sequences recognized by a host cell transformed
with the vector is also
provided as is a host cell transformed with the vector. The cells transformed
according to the
invention can be used in processes for preparation of recombinant antibody
disclosed herein. A
variety of host cells can be transformed with the nucleic acid molecules
encoding the antibody or
a fragment thereof. The host cell can be chosen from prokaryotic or eukaryotic
systems, for
example bacterial cells but likewise yeast cells or animal cells, in
particular mammalian cells. It
is likewise possible to use insect cells or plant cells. Methods of producing
a recombinant
protein are well known.
The invention likewise concerns animals, except man, which comprise at least
one cell transformed according to the invention. Thus, non-human transgenic
animals that
express the heavy and/or light chain or antigen-binding portions thereof of an
anti- Her3
antibody are also provided.
The present invention further provides a pharmaceutical composition comprising
the monoclonal antibody, or binding fragment thereof, according to the
invention, and a
pharmaceutically acceptable carrier, exeipient, or diluent. The pharmaceutical
composition may
further comprise another component, such as an anti-tumor agent or an imaging
reagent.
Certain embodiments of the invention relate to the use of the invention
antibodies
as targeted delivery systems for cytotoxic agents such as chemotherapeutic
drugs, peptides or
radionuclides, for immunological response promoters such as eytokines, for pro-
drugs or for
gene therapies. As well, the antibodies described herein may also
transport/deliver payloads
such as RNAi or shRNA. These payloads may be naked or chemically modified.
Immunoliposomes as potential delivery vehicles are also included.
As will be appreciated by one skilled in the art, the antibodies of the
invention or
binding fragments thereof will also find use in various medical or research
purposes, including
staging of various pathologies associated with expression of Her3. Indeed,
laboratory research
may also be facilitated through use of such antibodies. Identifying patients
at risk of a
pathological effect of an oncogenic disorder associated with expression of
Her3, particularly
hyperproliferative oncogenic disorders such as, but not limited to, cerebral
autosomal dominant
arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), T-
cell acute
lymphoblastic leukemia, lymphoma, Alagille syndrome, liver disease involving
aberrant
vascularization; diabetes, ovarian cancer, diseases involving vascular cell
fate, rheumatoid
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arthritis, pancreatic cancer, plasma cell neoplasms (such as multiple myeloma,
plasma cell
leukemia, and extramedullary plasmacytoma), and neuroblastoma is also
encompassed. As
would be recognized by one of ordinary skill in this art, the level of
antibody expression
associated with a particular disorder will vary depending on the nature and/or
the severity of the
As a consequence, additional embodiments of the invention pertain to the use
of
the invention antibodies for detecting dysplastic or neoplastic Her3 bearing
cells as well as
diagnosing, assessing and treating disorders associated with expression of
Her3 receptor protein
or aberrant activation of the Her3 cascade.
As used herein, the term "an oncogenic disorder associated with expression of
Her3" is intended to include diseases and other disorders in which the
presence of high levels or
abnormally low levels of Her3 receptor protein (aberrant) in a subject
suffering from the disorder
has been shown to be or is suspected of being either responsible for the
pathophysiology of the
disorder or a factor that contributes to a worsening of the disorder. Thus,
"neoplastic cells" or
In certain embodiments, "increased expression" as it relates to 1-Ier3 refers
to
protein or gene expression levels that demonstrate a statistically significant
increase in
expression (as measured by RNA expression or protein expression) relative to a
control. As well
"increased expression" is also used to encompass "increased activation of the
Her3 cascade".
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Administration of the antibodies of the present invention in any of the
conventional ways known to one skilled in the art (e.g., topical, parenteral,
intramuscular, IV,
subcutaneous etc.), will provide an extremely useful method of detecting
dysplastic cells in a
sample as well as allowing a clinician to monitor the therapeutic regiment of
a patient
undergoing treatment for a hyperproliferative disorder associated with or
mediated by expression
of Her3.
It is known in the art to use antibodies to detect the presence or expression
of a
specific protein. Because Her3 may be overexpressed in certain
hyperproliferative disorders
including, for example, cancer, Her3-specific antibodies of this invention may
be used to detect
the overexpression and, thus, to detect metastatic disease. As well, the
immunodetection
methods of the present invention may be of utility in the diagnosis of various
disease states. As
well, the invention antibodies may be exploited to detect differential
expression of Her3 in
metastatic cells. As will be apparent to the skilled artisan human Her3 or ICD
or any other
downstream target may be detected in a number of ways such as by various
assays.
Immunodetection techniques include but are not limited to immunohistological
staining, western
blotting, dot blotting, precipitation, agglutination, ELISA assays,
immunohistochemistry, in situ
hybridization, flow cytometry or radio-immunoassay (RIA) technique or
equivalent on a variety
of tissues and a variety of sandwich assays. These techniques are well known
in the art. See, for
example, U.S. Pat. No. 5,876,949, hereby incorporated by reference.
Another aspect of the invention relates to the use of these antibodies in
methods
or assays for detecting Iler3 activation or expression in patients suspected
of having a Her3
related disease or disorder. Such diseases or disorders may include, but not
limited to, cerebral
autosomal dominant arteriopathy with subcortical infarcts and
leukoencephalopathy
(CADASIL), T-cell acute lymphoblastic leukemia, lymphoma, Alagille syndrome,
liver disease
involving aberrant vascularization; diabetes, ovarian cancer, diseases
involving vascular cell fate,
rheumatoid arthritis, pancreatic cancer, plasma cell neoplasms (such as
multiple myeloma,
plasma cell leukemia, and extramedullary plasmacytoma), and neuroblastoma.
When used with suitable labels or other appropriate detectable biomolecule or
chemicals, the antibodies described herein are particularly useful for in
vitro and in vivo
diagnostic and prognostic applications. Suitable conditions for which the
antibody of the
invention will find particular use for include the detection and diagnosis of
neoplasias, such as,
but not limited to cancer of the ovary, prostate, colon and skin. Inflammatory
responses or
disorders triggered by Her3 receptor activation or cascade area also included.
Labels for use in immunoassays are generally known to those skilled in the art
and include enzymes, radioisotopes, and fluorescent, luminescent and
chrornogenic substances,
including colored particles such as colloidal gold or latex beads. Various
types of labels and
methods of conjugating the labels to the antibodies of the invention are well
known to those
skilled in the art, such as the ones set forth below.
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In accordance with the above, an illustrative embodiment provides a method for
detecting normal, benign, hyperplastic, and/or cancerous cells or a portion
thereof in a biological
sample comprising: providing a Her3 antibody or binding portion thereof which
recognizes an
antigen (Her3) on the surface of the cells, wherein the antibody or binding
portion thereof binds
to an epitope of Her3 which is also recognized by any one or more monoclonal
antibodies
detailed herein and wherein the antibody or binding portion thereof is bound
to a label effective
to permit detection of the cells or a portion thereof upon binding of the
antibody or binding
portion thereof to the antigen; contacting the biological sample with the
antibody or binding
portion thereof having a label under conditions effective to permit binding of
the antibody or
binding portion thereof to the antigen on any of the cells or a portion
thereof in the biological
sample; and detecting the presence of any of the cells or a portion thereof in
the biological
sample by detecting the label.
In certain embodiments, the step of contacting the antibody is carried out in
a
living mammal and comprises: administering the Nocthl antibody or binding
portion thereof to
the mammal under conditions effective to permit binding of the antibody or
binding portion
thereof to the antigen on any of the cells or a portion thereof in the mammal.
In certain embodiments, the invention antibodies may be labeled with a
detectable
moiety, such as a fluorophore, a ehromophore, a radionuclide, a
chemilumineseent agent, a
bioluminescent agent and an enzyme.
Yet another embodiment of the invention provides a method of diagnosis,
preferably in vitro, of illnesses connected with an overexpression or under
expression, preferably
overexpression of the Her3 receptor. Samples are taken from the patient and
subject to any
suitable immunoassay with Her3 specific antibodies to detect the presence of
Her3. Preferably,
the biological sample is preferably tissue sample or biopsies of human origin
which can be
conveniently assayed for the presence of a pathological hyperproliferative
oncogenic disorder
associated with expression of Her3.
Once a determination is made of the amount of Her3 present in the test sample,
the results can be compared with those of control samples, which are obtained
in a manner
similar to the test samples but from individuals that do not have or present
with a
hyperproliferative oncogenic disorder associated with expression of Her3,
e.g., ovarian cancer.
If the level of the Her3 receptor polypeptide is significantly elevated in the
test sample, it may be
concluded that there is an increased likelihood of the subject from which it
was derived has or
will develop said disorder, e.g., T-ALL, ovarian cancer etc. The diagnostic
uses of the
antibodies according to the present invention embrace primary tumors and
cancers, as well as
metastases. Preferably, the antibody, or one of its functional fragments, can
be present in the
form of an immunoconjugate or of a labeled antibody so as to obtain a
detectable and/or
quantifiable signal.
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An exemplary in vitro method of diagnosing pathological hyperproliferative
oncogenic disorder comprises: (a) determining the presence or absence of Her3
bearing cells in a
sample; and (b) diagnosing a pathological condition or a susceptibility to a
pathological
condition based on the presence or absence of said Her3 bearing cells. In the
clinical diagnosis
or monitoring of patients with an Her3 mediated neoplastic disease, the
detection of Her3
expressing cells or an increase in the levels of Her3, in comparison to the
levels in a
corresponding biological sample from a normal subject or non-cancerous tissue
is generally
indicative of a patient with or suspected of presenting with an Her3 mediated
disorder.
A representative in vitro method of diagnosing the presence of cancer in a
patient
or a susceptibility to a pathological condition associated therewith in a
subject, proposes: (a)
measuring the levels of Her3 receptor protein in cells or tissues of the
patient; and (b) comparing
the measured levels of the antigen of (a) with levels of the antigen (Her3
receptor protein) in
cells or tissues from a normal human control, wherein an increase in the
measured levels of the
antigen in the patient versus the normal control is associated with the
presence of the cancer. In
certain embodiments, decreased Her3 expression may be diagnostic of a
pathological condition
such as disorders of the skin._Alternatively, one may measure the level of the
ICD as a measure
of Her3 receptor activation.
Alternatively, the above method may be practiced over several time points. A
representative embodiment thus provides a method of diagnosing a pathological
oncogenic
disorder associated with aberrant expression of Her3 or increased Her3
receptor activation (Her3
cascade) comprising the steps of: a) detecting the presence and level of Her3
in a biological
sample obtained from the mammal at a plurality of time points, wherein Her3 is
detected by a
method selected from the group consisting of immunoblotting, western blotting,
immunoperoxidase staining, fluorescein labeling, diaminobenzadine and
biotinylation ; and b)
correlating change in Her3 expression with said diagnosis. It is understood
that other
conventional assays may be used instead of or in addition to those described
herein.
A method of monitoring metastatic potential of an oncogenic disorder
associated
with Her3 expression in a mammal is also encompassed. In accordance therewith,
provided
herein is a method for screening for metastatic potential of solid tumors
comprising: a) obtaining
a sample of tumor tissue from an individual in need of screening for
metastatic potential of a
solid tumor; b) reacting an antibody to Her3 with tumor tissue from the
patient; c) detecting the
extent of binding of the Her3 antibody to the tissue and d) correlating the
extent of binding of the
antibody with its metastatic potential. Preferably, the tumor is cancer
arising from large bowel
(colorectal cancer), prostate, breast or skin (ovarian cancer or T-ALL). In
certain embodiments,
step c) may be performed over a plurality of time points. As well, in certain
embodiments, Her3
expression is detected by a method selected from the group consisting of
immunohistochemical
staining, inamunoblotting, western blotting, immunoperoxidase staining,
fluorescein labeling,
diaminobenzadine and biotinylation.
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The invention further provides for a method for predicting susceptibility to
cancer
comprising detecting the expression level of Her3 in a tissue sample, its
presence indicating
susceptibility to cancer, wherein the degree of Her3 expression correlates to
the degree of
susceptibility. In certain embodiments, the expression of Her3 in, for
example, breast tissue,
-- prostate tissue, colon tissue, or any other tissue suspected of cells
expressing Her3 is examined,
with the presence of Her3 in the sample providing an indication of cancer
susceptibility or the
emergence or existence of a tissue specific tumor.
Stage determination has potential prognostic value and provides criteria for
designing optimal therapy. Simpson et al., J. Clin. Oncology 18:2059 (2000).
Generally,
-- pathological staging of breast cancer for example, is preferable to
clinical staging because the
former gives a more accurate prognosis. However, clinical staging would be
preferred if it were
as accurate as pathological staging because it does not depend on an invasive
procedure to obtain
tissue for pathological evaluation. Thus, methods for gauging tumor
aggressiveness are also
provided as are methods for observing the progression of a malignancy in an
individual over
time.
Accordingly, the invention provides an in vivo imaging reagent comprising an
antibody according to the invention, or one of its functional fragments,
preferably labeled,
especially radiolabeled, and its use in medical imaging, in particular for the
detection of Her3
mediated disorders e.g., cancer characterized by over expressing Her3 or other
pathologies in
-- which cells over express Her3.
The imaging reagents, e.g., diagnostic reagents can be administered by
intravenous injection into the body of the patient, or directly into a tissue
suspected of harboring
Her3 bearing cells, e.g., colon or ovary or the pancreas. The dosage of
reagent should be within
the same ranges as for treatment methods. Typically, the reagent is labeled,
although in some
-- methods, the primary reagent with affinity for Her3 is unlabelled and a
secondary labeling agent
is used to bind to the primary reagent. The choice of label depends on the
means of detection.
For example, a fluorescent label is suitable for optical detection. Use of
paramagnetic labels is
suitable for tomographic detection without surgical intervention. Radioactive
labels can also be
detected using PET or SPECT.
Diagnosis is performed by comparing the number, size, and/or intensity of
labeled
loci, to corresponding baseline values. The baseline values can, as an
example, represent the
mean levels in a population of undiseased individuals. Baseline values can
also represent
previous levels determined in the same patient. For example, baseline values
can be determined
in a patient before beginning treatment, and measured values thereafter
compared with the
-- baseline values. A decrease in values relative to baseline signals a
positive response to
treatment.
Thus, a general method embodied by the invention works by administering to a
patient an imaging-effective amount of an imaging reagent such as the above
described
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monoclonal antibodies or antigen-binding fragments which are labeled and a
pharmaceutically
effective carrier and then detecting the agent after it has bound to Her3
present in the sample.
In certain embodiments, the method works by administering an imaging-effective
amount of an imaging agent comprising a targeting moiety and an active moiety.
The targeting
moiety may be an antibody, Fab, Fab12, a single chain antibody or other
binding agent that
interacts with an epitope present in Her3. The active moiety may be a
radioactive agent, such as
radioactive technetium, radioactive indium, or radioactive iodine. The imaging
agent is
administered in an amount effective for diagnostic use in a mammal such as a
human and the
localization and accumulation of the imaging agent is then detected. The
localization and
accumulation of the imaging agent may be detected by radionuclide imaging,
radioscintigraphy,
nuclear magnetic resonance imaging, computed tomography, positron emission
tomography,
computerized axial tomography, X-ray or magnetic resonance imaging method,
fluorescence
detection, and chemiluminescent detection.
The in vivo imaging methods of the present invention are also useful for
providing
prognoses to cancer patients. For example, the presence of Her3 indicative of
an aggressive
cancer likely to metastasize or likely to respond to a certain treatment can
be detected. Thus, in
one aspect, the invention provides a method for observing the progression of a
malignancy in an
individual over time comprising determining the level of Her3 expressed by
cells in a sample of
the tumor, comparing the level so determined to the level of Her3 expressed in
an equivalent
tissue sample taken from the same individual at a different time, wherein the
degree of Her3
expression in the tumor sample over time provides information on the
progression of the cancer.
The in vivo imaging methods of the present invention can further be used to
detect
Her3 mediated cancers e.g., one that has metastasized in other parts of the
body.
A related embodiment relates to a pharmaceutical composition for in vivo
imaging
of an oncogenic disorder associated with expression of Her3 comprising the
invention antibodies
or binding fragment thereof which is labeled and which binds Her3 in vivo; and
a
pharmaceutically acceptable carrier.
The antibodies disclosed herein may also be used in methods of identifying
human tumors that can escape anti- Her3 treatment by observing or monitoring
the growth of the
tumor implanted into a rodent or rabbit after treatment with a conventional
anti- Her3 antibody.
The antibodies of the invention can also be used to study and evaluate
combination therapies with anti- Her3 antibodies of this invention and other
therapeutic agents.
The antibodies and polypeptides of this invention can be used to study the
role of Her3 in other
diseases by administering the antibodies or polypeptides to an animal
suffering from the disease
of a similar disease and determining whether one or more symptoms of the
disease are alleviated.
Those of skill in the art are very familiar with differentiating between
significant
expression of a target antigen, e.g., Her3, which represents a positive
identification, and low
level or background expression of the antigen. Indeed, background expression
levels are often
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used to form a "cut-offt above which increased staining will be scored as
significant or positive.
Significant expression may be represented by high levels of antigens in target
cells or tissues or
alternatively, by a high proportion of cells from within a tissue that each
give a positive signal.
The above diagnostic approaches can be combined with any one of a wide variety
of prognostic and diagnostic protocols known in the art. For example, another
embodiment of
the invention is directed to methods for observing a coincidence between the
expression of Her3
and a factor that is associated with malignancy, as a means for diagnosing and
prognosticating
the status of a tissue sample. A wide variety of factors associated with
malignancy can be
utilized, such as the expression of genes or gene products associated with
malignancy as well as
gross cytological observations. Methods for observing a coincidence between
the expression of
Her3 and another factor that is associated with malignancy are useful, for
example, because the
presence of a set of specific factors that coincide with disease provides
information crucial for
diagnosing and prognosticating the status of a tissue sample. The methods
proposed herein can
be useful to diagnose or confirm diagnosis of an oncogenic disorder associated
with expression
of Her3 or susceptibility thereof. For example, the methods can be used on a
patient presenting
with symptoms of an oncogenic disorder. If the patient has, for example
increased expression
levels of Her3 or aberrant Her3 receptor activation as evidenced by increased
expression levels
of any one or more downstream targets effected by or related to Her3 receptor
activation or
increased expression levels of ICD, then the patient is likely suffering from
a cancerous disorder.
The methods can also be used in asymptomatic patients. Presence of higher than
normal Her3
may indicate for example susceptibility to future symptomatic disease. As
well, the methods are
useful for monitoring progression and/or response to treatment in patients who
have been
previously diagnosed with an Her3 mediated cancer.
Generally speaking, malignancies are characterized by either increased Her3
receptor expression, increased or aberrant Her3 receptor activation or
mutations resident in the
Her3 receptor protein. Malignancies characterized by aberrant or increased
Her3 receptor
activation may be confirmed via determination of expression levels of ICD,
whose expression
level may be increased in the cytoplasm following activation of the Her3
cascade. Thus, in those
cases where a malignancy is characterized by increased Her3 receptor
activation, one is expected
to find increased expression of ICD in the cytoplasm. This increase in ICD
expression can be
traced to the translocation f the ICD into the cytoplasm upon Her3 receptor
activation. A similar
effect should be observed for downstream targets effected by aberrant Her3
receptor activation ¨
a decrease or increase relative to normal of a specific downstream Her3 target
occurring as a
result of Her3 receptor activation. Thus, measurement of Her3 in biopsied
tissue or other
biological sample can be corroborated by determining expression of downstream
target
expression as a means of identifying high risk patients. In certain
embodiments, single or
multiple determinations of increased Her3 expression and/or ICD expression
over time may
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serve as a marker for illness indicative of intervening medical intervention.
A positive test can
therefore supplement the clinician's judgment.
Increased Her3 levels also add prognostic accuracy to established severity of
illness scores. Such clinical judgments will benefit by a method of scoring
diseased cells. As
discussed herein, measurement of Her3 expression in a tissue sample can also
be used as an
indicator for additional monitoring or testing, or consideration for more
aggressive treatment,
especially when patients are found to have increased Her3 expression levels or
increased Her3
receptor activation as reflected by increased cytoplasmic ICD levels or any
other downstream
target.
Thus, for example, one would expect that a patient at risk of developing a
Her3
mediated cancer or presenting with such a cancer would likely have increased
Her3 expression
relative to a control sample. As such, in certain embodiments, for such
patients, a semi
quantitative estimation of Her3 immunoreactivity can be performed. Towards
this end, a score
can be given to each slide, considering the intensity of the stain. The slides
may be examined
and scored independently by two investigators, and discordances may be
reconciled by re-
examination of the slide, and the scores then averaged. The intensity of
immunostaining of
individual cells may be scored on a scale of 0 (no staining) to 4 (strongest
intensity) and the
percentage of cells with staining at each intensity estimated. If there is no
staining, a 0 score can
be given. A +1 score indicates weak staining, while a +4 score indicating
strong intensity of
staining. As will be appreciated, any scoring scheme used to compare staining
intensities may be
used as long as it takes into account the relative intensity of cytoplasmic
staining and allows
differentiation among degrees of intensity of staining, thus providing a way
to grade the
malignancies. Because of the novel staining aspects of the present invention
which results in
highly differentiated staining, the scoring or grading can be done visually,
thus allowing the
technique of the present invention to be widely used clinically without
sophisticated equipment.
It will be understood that the staining results can be analyzed by appropriate
sensitive optical
equipment and analyzed by computer.
In furtherance of the above objective, the invention provides a method for
diagnosing an oncogenic disorder associated with expression of Her3
comprising: a) measuring
by radioimmunoassay, competitive-binding assay, Western blot analysis, ELISA
assay, or
sandwich assay the amount of Her3 protein in a biological sample, e.g.,
biopsied tissue obtained
from a patient, using an antibody that specifically binds to Her3; and b)
comparing the amount of
antibody bound to said Her3 protein to a normal control tissue sample, wherein
increased
expression or over-expression of Her3 in the sample obtained from the patient
relative to the
normal control tissue sample is diagnostic of an oncogenic disorder associated
with expression of
Her3. Preferably, the Her3-specific antibody comprises sat least one antibody
detailed herein.
In certain embodiment, the same scoring criteria e.g., score of 0 to 4 may be
used
to score cytoplasmic 1CD staining as a means of corroborating the initial
diagnosis.
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Consequently, cells may be stained with an antibody specific for 1CD and the
intensity level
scored using the above criteria, where the intensity of immunostaining of
individual cells may be
scored on a scale of 0 (no staining) to 4 (strongest intensity) and the
percentage of cells with
staining at each intensity estimated. If there is no staining, a 0 score can
be given. A +1 score
indicates weak staining, while a +4 score indicating strong intensity of
staining.
In additional embodiments, a prognostic index is produced by preparing a
weighted scale of expression levels of the tumor markers, Her3 related to
progression observed
in a representative sample of a particular tumor type, wherein the different
values in the
weighted scale are related to increased invasiveness or metastatic spread in
the representative
sample.
The methods of the invention are also useful for identifying a human cancer
patient at risk for additional neoplastic disease, for staging malignant
disease in a human cancer
patient and assessing the relative risk of metastatic disease versus the risk
of toxicity (such as
leukocytopenia, for example) from chemotherapeutic treatment.
The invention thus provides methods wherein the results of the determination
of
the level of cell surface Her3 expression and the extent of cytoplasmic
localization of ICD are
used to prepare a prognostic or "risk" index for making a prognostic
determination. In this aspect
of the invention, a prognostic index is prepared using the above criteria,
wherein a value of 0
signifies a control, a value of +1 indicates weak staining etc, wherein a
prognosis of a likelihood
of progressing to metastatic disease is made when the staining intensity is
scored at +4.
An illustrative embodiment of the invention provides a diagnostic or
monitoring
method comprising: a) obtaining a sample of tissue from an individual in need
of diagnosis or
monitoring for cancer; b) detecting levels of Her3 protein in said sample, c)
scoring said sample
for Her3 protein levels; and d) comparing said scoring to that obtained from a
control tissue
sample to determine the prognosis associated with said cancer. Samples may be
scored using a
scale of 0 to 4, where 0 is negative (no detectable Her3 expression or level
comparable to a
control level), and 4 is high intensity staining in the majority of cells and
wherein a score of 1 to
4 indicates a poor prognosis while a score of 0 indicates a good prognosis.
A related aspect of the invention pertains to a method for screening for
metastatic
potential of a Her3 mediated hyperproliferative disorder comprising: a)
obtaining a sample of
diseased or target tissue from an individual in need of screening for
metastatic potential of a
Notch I mediated tumor, b) reacting an antibody to Her3 with tumor tissue from
the patient, c)
detecting the extent of binding of the Her3 antibody to said tissue and d)
correlating the extent of
binding of said antibody with its metastatic potential. In general, any of the
methods of the
invention involving analysis of the levels of Her3 or ICD may be used in
conjunction with
additional cancer markers readily known to those of skill in the art.
Also provided is a method of detecting the presence and extent of cancer in a
patient, comprising: determining the level of the antigen (Her3) in a sample
of cells or a tissue
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section from the patient and correlating the quantity of the antigen with the
presence and extent
of the cancer disease in the patient relative to a normal or control patient.
One of the major challenges facing the pharmaceutical industry in drug
development is to show efficacy associated with a potential therapeutic
candidate. This
drawback applies equally to the numerous efforts underway in the
pharmaceutical industry to
generate anti- Her3 inhibitory antibodies as anti-cancer therapeutics. One way
to do this is to
have a suitable marker that indicates when Her3 activity is inhibited.
Ideally, where a candidate
Her3 antagonist moiety is effective, one should observe a decrease in the
expression levels of
Her3 following treatment with the Her3 antagonist moiety. Alternatively, one
might expect an
increase in the levels of phosphorylated Her3, which signals activation of the
kinase domain
resident in Her3. It thus follows that favorable treatment with an Her3
antagonistic moiety would
predict a decrease in Her3 expression levels on tumor cells or any other cells
that express this
cell surface receptor, while an unfavorable outcome would predict either no
change in the
expression levels or an increase in expression levels of Her3. Thus, by
measuring Her3 protein
expression on a tumor cell, for example, with a suitable marker, decreased
expression levels may
be detected as an indicator of suppressed Her3 activity. The present invention
exploits the
ability of the Her3 antibodies of the invention to bind Her3 with high
affinity to be utilized in a
"biomarker strategy" for measuring Her3 activity and/or expression or
tumorigenic status by
specifically measuring the expression levels of Her3 on tumor/cancer cells. As
a consequence,
the present invention provides a rapid means, e.g., high affinity anti- Her3
antibodies, for
assessing the nature, severity and progression of a pathological
hyperproliferative oncogenic
disorder associated with expression of Her3.
In furtherance of the "biomarker strategy" noted above, the invention provides
a
method for determining onset, progression, or regression, of neoplasias
associated with
expression of Her3 in a subject, comprising: obtaining from a subject a first
biological sample at
a first time point, contacting the first sample with a effective amount of an
antibody described
herein under conditions allowing for binding of the antibody or a fragment
thereof to Her3
suspected to be contained in the sample and determining specific binding
between the antibody
in the first sample and Her3 bearing cells to thereby obtain a first value,
obtaining subsequently
from the subject a second biological sample at a second time point, and
contacting the second
biological sample with the Her3 antibody and determining specific binding
between the antibody
and Her3 in said sample to obtain a second value, and comparing the
determination of binding in
the first sample to the determination of specific binding in the second sample
as a determination
of the onset, progression, or regression of the colon cancer, wherein an
increase in expression
level of Her3 in said second or subsequent sample relative to the first sample
indicative of the
progression of said neoplasias, and wherein decrease in indicative of
regression of neoplasias in
said sample.
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In one embodiment, Her3 is detected by (1) adding an antibody of the invention
to
the sample or tissue section; (2) adding goat anti-mouse IgG antibody
conjugated with
peroxidase; (3) fixing with diaminobenzidene and peroxide, and (4) examining
the sample or
section, wherein reddish brown color indicates that the cells bear the
antigen. According to the
method, the effectiveness of a cancer treatment may be monitored by
periodically measuring
changes in the level of the antigen in a tissue sample taken from a patient
undergoing the
therapy, and correlating the change in level of the antigen with the
effectiveness of the therapy,
wherein a lower level of Her3 expression determined at a later time point
relative to the level of
Her3 determined at an earlier time point during the course of therapy
indicates effectiveness of
the therapy for the cancer disease.
In yet another embodiment, the application provides methods for determining
the
appropriate therapeutic protocol for a subject. Specifically, the antibodies
of the invention will
be very useful for monitoring the course of amelioration of malignancy in an
individual,
especially in those circumstances where the subject is being treated with a
Her3 antibody that
does not compete with the antibodies of the invention for binding Her3.
Essentially, presence or
absence or a change in the level of Her3 expression may be indicative as to
whether a subject is
likely to have a relapse or a progressive neoplasia or persistent neoplasias
such as cancer
associated with Her3. Thus, by measuring an increase in the number of cells
expressing Her3 or
changes in the concentration of Her3 present in various tissues or cells, it
is possible to determine
whether a particular therapeutic regimen aimed at ameliorating a malignancy
associated with
Her3 is effective. For those patients receiving conventional therapy, and
whose Her3 expression
has not changed over time, for example, these may, instead be treated with the
Her3 antibodies
of the invention and the change in Her3 expression is observed over time. If a
change if readily
evident over a period of time, then it may be possible to switch the patient
from conventional
therapy to therapy with one or more of the antibodies disclosed herein.
An antibody fragment of the invention is capable of specifically binding to a
target molecule of interest. For example, in some embodiments, an antibody
fragment
specifically binds a tumor antigen. In some embodiments, the antibody fragment
specifically
binds a cell surface receptor that is activated upon receptor multimerization
(e.g., dimerization).
In some embodiments, binding of an antibody of the invention to a target
molecule inhibits
binding of another molecule (such as a ligand, where the target molecule is a
receptor) to said
target molecule.
Thus, in one example, an antibody fragment of the invention when bound to a
target molecule inhibits binding of a cognate binding partner to the target
molecule. A cognate
binding partner can be a ligand, or a hetero or homodimerizing molecule. In
one embodiment,
an antibody fragment of the invention when bound to a target molecule inhibits
target molecule
receptor activation. For example, in some embodiments wherein an antibody or a
fragment
thereof is an antagonist, binding of the antibody fragment to a cell surface
receptor may inhibit
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dimerization of the receptor with another unit of the receptor, whereby
activation of the receptor
is inhibited (due at least in part to a lack of receptor dimerization). In one
embodiment, an
antibody fragment of the invention is capable of competing with a native Her3
receptor binding
partner, e.g., delta or Serrate to the Her3 receptor. In another embodiment,
an antibody of the
invention or a fragment thereof is capable of competing with an endogenous
Notch receptor
ligand for binding to a Her3 receptor.
In certain embodiments, the herein described antibodies antagonize, or
inhibit,
Her3 mediated signaling by either blocking or inhibiting Her3 binding to its
endogenous ligand
or preventing or delaying Her3 cascade activation (hereinafter "Antagonist
Therapeutics
Antibodies") are administered for therapeutic effect. Disorders which can thus
be treated can be
identified by in vitro assays such as those described herein or known to one
skilled in the art.
Such antagonist antibodies include anti-Her3 neutralizing antibodies and
competitive inhibitors
of EGER protein-protein interactions as detailed infra. In furtherance of the
above objective, an
antibody of the invention is administered to treat a cancerous condition, or
to prevent progression
from a pre-neoplastic or non-malignant state into a neoplastic or a malignant
state.
Another embodiment of the preset invention is the use of any of these
antibodies
for the preparation of a medicament or composition for the treatment of
diseases and disorders
associated with Her3 receptor activation.
Another embodiment of the preset invention is the use of any of these
antibodies
in the treatment of disorders associated with Her3 activation comprising the
inhibition of said
activation by, e.g., inhibiting Her3 signaling, or neutralization of the
receptor by blocking ligand
binding. Her3 related disorders may include, but are not limited to cancers,
lethal congenital
contractural syndrome type 2 (LCCS2).
Thus, in certain aspects, the invention provides a method for treating a
disease
comprising administering to a subject in need of such a treatment an effective
amount for
treating the disease of at least one antibody or antigenic or binding fragment
thereof ("fragment')
disclosed herein that binds to native human Her3 (hHer3) and abolishes or
attenuates the
function of the native hHer3 or Her3/Her2 heterodimer. A variety of diseases
may be treated
with the above described methods including cancer, in particular T-cell acute
lymphoblasticleuketnia/lymphoma, human breast cancer, human colorectal cancer,
melanoma,
human lung cancer, human head and neck cancers and human prostate cancer, an
immune or
inflammatory disorder an angiogenesis disorder or any other disorder mediated
by Her3
signaling (G. Sithanandam & LM Anderson (2008) Cancer Gene Therapy 15:413;
Jiang et al.
(2007) .113C 282:32689; Grivas etal. (2007) Eur J. Cancer 43:2602).
Yet another objective resides in the proposed use of constitutively active
Her3
receptor or an antagonist thereof, for the purpose of developing a medicament
that may find use
in the treatment of a condition which is responsive to constitutively active
Her3 receptor.
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In a further embodiment, the present invention provides a method of inhibiting
or
killing cancer cells, comprising: providing to a patient in need thereof the
monoclonal antibody,
or binding fragment thereof of the present invention, under conditions and in
an amount
sufficient for the binding to the cancer cells, thereby causing inhibition or
killing of the cancer
cells by the immune cells of the patient. Preferably, the method is for the
treatment of T-cell
acute lymphoblastic leukemia/lymphoma, human colon cancer, melanoma, human
lung cancer
and human prostate cancer. The monoclonal antibody is preferably conjugated
with a cytotoxic
moiety, such as a chemotherapeutic agent, a photoactivated toxin, an RNAi
molecule or a
radioactive agent. Preferably, the oytotoxic moiety may be a Ricin. An
alternative method
proposes treatment of a Her3 mediated disorder comprising the steps as
outlined above.
Representative disorders include an immune or inflammatory disorder like
colitis or asthma, an
infectious disease, an angiogenesis disorder, atherosclerosis, or a disorder
of the kidney or any
other disorder mediated by Her3 signaling.
In one embodiment, an oligonucleotide, such as an RNAi molecule inhibiting
Her3 expression may be conjugated to, or form the therapeutic agent portion of
an
immunoconjugate or antibody fusion protein of the present invention.
Alternatively, the
oligonucleotide may be administered concurrently or sequentially with a naked
or conjugated
anti-Her3 antibody or antibody fragment of the present invention. In one
embodiment, the
oligonucleotides are an antisense oligonucleotide (RNAi) that preferably is
directed against Her3
expression.
An alternative embodiment provides a method of treating a Her3 mediated
disorder by administering a pharmaceutical composition comprising at least one
Nocthl inhibitor
wherein the inhibitor is an anti-Her3 antibody conjugated to a Her3 specific
RNA inhibitor, and
a pharmaceutically acceptable carrier, RNA inhibition (RNAi) is based on
antisense modulation
of Her3 in cells and tissues comprising contacting the cells and tissues with
at least one Her3
antibody conjugated to a nucleic acid molecule that modulated transcription or
translation of
Her3 receptor protein, including but not limited to double stranded RNA,
(dsRNA), small
interfering RNA (siRNA), ribozymes and locked nucleic acids (LNAs), and a
pharmaceutically
acceptable carrier.
The present invention further provides a method for localizing cancer cells in
a
patient, comprising: (a) administering to the patient a detectably-labeled
monoclonal antibody of
the invention, or binding fragment thereof; (b) allowing the detectably-
labeled (e.g. radiolabeled;
fluorochrome labeled, or enzyme labeled, especially via ELISA) monoclonal
antibody, or
binding fragment thereof, to bind to the cancer cells within the patient; and
(c) determining the
location of the labeled monoclonal antibody or binding fragment thereof,
within the patient.
Another embodiment of the invention relates to the use of invention
antibodies,
and VRs, FRs and CDRs thereof, in directed molecular evolution technologies
such as phage
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display or bacterial or yeast cell surface display technologies in order to
generate polypeptides
with enhanced affinity, specificity, stability or other desired
characteristics.
Another embodiment of the present invention is a cancer cell targeting
diagnostic
immunoconjugate comprising an antibody component that comprises an antibody or
fragment
thereof of any one of the antibodies or fragments thereof of the present
invention, wherein the
antibody or fragment thereof is bound to at least one diagnostic/detection
agent.
Preferably, the diagnostic/detection agent is selected from the group
comprising a
radionuclide, a contrast agent, and a photoactive diagnostic/detection agent.
Still preferred, the
diagnostic/detection agent is a radionuclide with an energy between 20 and
4,000 keV or is a
,
radionuclide selected from the group consisting of' ICIIn,111In, 171u, isF
52Fe, 62cu, 64cu, 67cu,
67Ga, "Ga, "Y, 90Y, 89Zr, 94mTc, 94Tc, 99mTe, 1201, 1231, 1241, 1251, 131k,
154-158Gd, 32p, 11C, 13N, ISO,
186Re, 188Re,51-n,
M 521V1n, "Co, 72As, 75Br, 76Br, 82mRb, 83Sr, or other gamma-
, beta-, or positron-
emitters. Also preferred, the diagnostic/detection agent is a paramagnetic
ion, such as the a
metal comprising chromium (III), manganese (II), iron (III), iron (II), cobalt
(11), nickel (II),
copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium
(III), vanadium (II),
terbium (III), dysprosium (III), holmium (III) and erbium (III), or a
radioopaque material, such
as barium, diatrizoate, ethiodized oil, gallium citrate, meglumine,
metrizamide, metrizoate,
propyliodone, and thallous chloride.
Also preferred, the diagnostic/detection agent is a fluorescent labeling
compound
selected from the group comprising fluorescein isothiocyanate, rhodamine,
phycoerytherin,
phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine, a
chemiluminescent labeling
compound selected from the group comprising luminol, isoluminol, an aromatic
acridinium
ester, an imidazole, an acridinium salt and an oxalate ester, or a
bioluminescent compound
selected from the group comprising luciferin, luciferase and aequorin. In
another embodiment,
the diagnostic immunoconjugates of the present invention are used in
intraoperative, endoscopic,
or intravascular tumor diagnosis.
In one embodiment, the therapeutic agent is a cytotoxic agent, such as a drug
or a
toxin. Also preferred, the drug is selected from the group consisting of
nitrogen mustards,
ethylenimine derivatives, alkyl sulfonates, nitrosoureas, gemcitabine,
triazenes, folic acid
analogs, anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs, purine
analogs,
antibiotics, enzymes, enzyme inhibitors, epipodophyllotoxins, platinum
coordination complexes,
vinca alkaloids, substituted ureas, methyl hydrazine derivatives,
adrenocortical suppressants,
hormone antagonists, endostatin, taxols, camptothecins, SN-38, doxorubicins
and their analogs,
antimetabolites, alkylating agents, antimitotics, antiangiogenic, apoptotic
agents, methotrexate,
CPT-11, and a combination thereof.
In another embodiment, the therapeutic agent is an oligonucleotide. For
example,
the oligonucleotide may be an antisense oligonucleotide such as an antisense
oligonucleotide
against Her3 or an RNAi molecule against Her3 receptor expression.
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In another embodiment, the therapeutic agent is a toxin selected from the
group
consisting of ricin, abrin, alpha toxin, saporin, ribonuclease (RNase), DNase
I, Staphylococcal
enterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin,
Pseudomonas exotoxin, and
Pseudomonas endotoxin and combinations thereof, an immunomodulator is selected
from the
group consisting of a cytokine, a stem cell growth factor, a lymphotoxin, a
hematopoietic factor,
a colony stimulating factor (CSF), an interferon (IFN), a stem cell growth
factor, erythropoietin,
thrombopoietin and a combinations thereof, a radionuclide and combinations
thereof; or a
photoactive therapeutic agent selected from the group comprising chromogens
and dyes.
Still preferred, the therapeutic agent is an enzyme selected from the group
comprising malate dehydrogenase, staphylococcal nuclease, delta-V-steroid
isomerase, yeast
alcohol dehydrogenase, .alpha.-glycerophosphate dehydrogenase, triose
phosphate isomerase,
horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase,
.beta.-
galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate
dehydrogenase, glucoamylase
and acetylcholinesterase.
In one aspect, the invention provides an immunoconjugate (interchangeably
termed "antibody drug conjugate" or "ADC") comprising any of the anti-Her3
antibodies
disclosed herein conjugated to an agent, such as a drug. Another embodiment of
the present
invention is a cancer cell targeting therapeutic immunoconjugate comprising an
antibody
component that comprises an antibody or fragment thereof of any one of the
antibodies, fusion
proteins, or fragments thereof of the present invention, wherein the antibody,
fusion protein, or
fragment thereof is bound to at least one therapeutic agent.
Preferably, the therapeutic agent is selected from the group consisting of a
chemotherapeutic agent, a growth inhibitory agent, a toxin (e.g., an
enzymatically active toxin of
bacterial, fungal, plant, or animal origin, or fragments thereof),
radionuclide, an
immunomodulator, a hormone, a hormone antagonist, an enzyme, oligonucleotide,
an enzyme
inhibitor, a photoactive therapeutic agent, a cytotoxic agent, an angiogenesis
inhibitor, and a
combination thereof.
In some embodiments, the drug is a maytansinoid, an auristatin, a dolastatin,
or a
calicheamicin. In some embodiments, the drug is DM1, DM3, DM4, MMAE, or MMAF.
In the immunoconjugates of the invention, an antibody (Ab) is conjugated to
one
or more drug moieties (D), e.g. about 1 to about 20 drug moieties per
antibody, through a linker
(L). In some embodiments, the linker comprises linker components selected from
one or more of
6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-
cit"), alanine-
phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-Succinimidyl 4-
(2-
pyridylthio) pentanoate ("SPP"), N-Succinimidyl 4-(N-
maleimidomethyl)cyclohexane-1
carboxylate ("SMCC"), and/or N-Succinimidyl (4-iodo-acetyl) aminobenzoate
("STAB"). In
some embodiments, the linker comprises MC-val-cit-PAB.
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In some embodiments, the immunoconjugate comprises SPP-DM1, SMCC-DMI,
BMPEO-DM1, MC-vc-PAB-MMAF, MC-vc-PAB-MMAE, MC-MMAF, or MC-MMAE.
In one aspect, the invention provides an immunoconjugate comprising an anti-
Her3 antibody that kills tumor cells. In some embodiments, tumor cells are
killed in vitro while
in another aspect, tumor cells are killed in vivo. In some embodiments,
administration of the
immunoconjugate reduces tumor growth (in some embodiments, having about 90%,
80%, 70%,
60%, 50%, 40%, 30%, 20% or 10%, or less reduction in tumor growth compared to
a control
tumor) or reduces time to tumor doubling.
The antibodies and immunoconjugates of the invention bind (such as
specifically
bind) Her3, and in some embodiments, may modulate one or more aspects of Her3-
associated
effects, including but not limited to Her3 activation, Her3 downstream
molecular signaling, Her3
ligand activation, Her3 ligand downstream molecular signaling, disruption of
ligand binding to
Her3, Her2 phosphorylation and/or Her3 multimerization, and/or Her3 ligand
phosphorylation,
and/or disruption of any biologically relevant Her3 and/or Her3 ligand
biological pathway,
and/or treatment and/or prevention of a tumor, cell proliferative disorder or
a cancer; and/or
treatment or prevention of a disorder associated with Her3 expression and/or
activity (such as
increased Her3 expression and/or activity). In some embodiments, the antibody
or
immunoconjugate of the invention specifically binds to Her3. In some
embodiments, the
antibody or immunoconjugate specifically binds to the Her3 extracellular
domain (BCD). In
some embodiments, the antibody or immunoconjugate specifically binds Her3 with
a KD of 70
pM or stronger. In some embodiments, the antibody or immunoconjugate of the
invention
reduces, inhibits, and/or blocks Her3 activity in vivo and/or in vitro. In
some embodiments, the
antibody or immunoconjugate reduces, inhibits and/or blocks Her3
autophosphorylation. In some
embodiments, the antibody or immunoconjugate competes for binding with Her3-
ligand (reduces
and/or blocks Her3 ligand binding to Her3). In some embodiments, the antibody
or
immunoconjugate is internalized upon binding to Her3 expressed on a mammalian
cell. In some
embodiments, the antibody or immunoconjugate inhibits, reduces or prevents
Her3/her2
dimerization.
In one aspect, the invention provides use of an antibody or immunoconjugate of
the invention in the preparation of a medicament for the therapeutic and/or
prophylactic
treatment of a disorder, such as a cancer, a tumor, and/or a cell
proliferative disorder.
In one aspect, the invention provides compositions comprising one or more
antibodies or immunoconjugates of the invention and a carrier. In one
embodiment, the carrier is
pharmaceutically acceptable.
In one aspect, the invention provides use of an anti-Her3 antibody or
immunoconjugate (in some embodiments, an anti-Her3 antibody or immunoconjugate
of the
invention) in the preparation of a medicament for the therapeutic and/or
prophylactic treatment
of a disorder, such as a cancer, a tumor, and/or a cell proliferative
disorder.
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The invention provides methods and compositions useful for modulating disease
states associated with expression and/or activity of Her3, such as increased
expression and/or
activity or undesired expression and/or activity or decreased expression
and/or activity.
In one aspect, the invention provides methods for treating or preventing a
tumor, a
cancer, and/or a cell proliferative disorder associated with increased
expression and/or activity of
Her3, the methods comprising administering an effective amount of an anti-Her3
antibody or
immunoconjugate (in some embodiments, an anti-Her3 antibody of the invention)
to a subject in
need of such treatment.
In one aspect, the invention provides methods for killing a cell (such as a
cancer
or tumor cell), the methods comprising administering an effective amount of an
anti-Her3
antibody or immunoconjugate (in some embodiments, an anti-Her3 antibody of the
invention) to
a subject in need of such treatment.
In one aspect, the invention provides methods for reducing, inhibiting,
blocking,
or preventing growth of a tumor or cancer, the methods comprising
administering an effective
amount of an anti-Her3 antibody or immunoconjugate (in some embodiments, an
anti-Her3
antibody of the invention) to a subject in need of such treatment.
In one aspect, the invention provides methods for treating or preventing a
neuropathy or neurodegenerative disease, or repairing a damaged nerve cell,
the methods
comprising administering an effective amount of an anti-Her3 antibody or
immunoconjugate (in
some embodiments, an anti-Her3 antibody of the invention) to a subject in need
of such
treatment.
In a further embodiment, the invention concerns an article of manufacture,
comprising: a container; a label on the container; and a composition contained
within the
container, wherein the composition comprises one or more anti-Her3 antibodies
or
immunoconjugates of the invention; wherein the composition is effective for
the detection,
diagnosis or prognosis of neoplasia associated with expression of Her3 and the
label on the
container indicates that the composition can be used for the diagnosis or the
prognosis of
conditions characterized by overexpression of the Her3 protein receptor.
In one embodiment, a composition comprising an antibody or immunoconjugate
further comprises a carrier, which in some embodiments is pharmaceutically
acceptable. In one
embodiment, an article of manufacture of the invention further comprises
instructions for
administering the composition (for e.g., the antibody) to a subject (such as
instructions for any of
the methods described herein).
In one aspect, the invention provides use of a kit of the invention in the
preparation of a medicament for the therapeutic and/or prophylactic treatment
of a disorder, such
as a cancer, a tumor, and/or a cell proliferative disorder.
The article of manufacture or kit may also be used for determining whether an
embedded biological sample contains human Her3 protein comprising: (a) an Her3-
binding agent
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that specifically binds with an embedded human Her3 protein to form a binding
complex; and (b)
an indicator capable of signaling the formation of said binding complex,
wherein the Her3
binding agent is a monoclonal antibody or a binding fragment thereof as set
forth in the
application. Diagnostic procedures using anti- Her3 antibody of the invention
can be performed
by diagnostic laboratories, experimental laboratories, practitioners, or
private individuals. The
clinical sample is optionally pre-treated for enrichment of the target being
tested for. The user
then applies a reagent contained in the kit in order to detect the changed
level or alteration in the
diagnostic component.
It is, therefore, anticipated that each of the limitations of the invention
involving
any one element or combinations of elements can be included in each aspect of
the invention.
This invention is not limited in its application to the details of
construction and the arrangement
of components set forth in the following description or illustrated in the
drawings. The invention
is capable of other embodiments and of being practiced or of being carried out
in various ways.
Other characteristics and advantages of the invention appear in the
continuation of
the description with the examples and the Figures whose legends are
represented below.
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DETAILED DESCRIPTION OF THE INVENTION
Overview of the Invention
Provided herein are various human Her3 specific antibodies, preferably
monoclonal antibodies. Included are antagonist, inhibitory and neutralizing
anti-Her3 antibodies
that inhibit or decrease cancer cell growth or proliferation. Compositions
comprising one or
more of the herein described antibodies effective for use in the treating Her3
mediated hyper-
proliferative disorders are also included. An Her3 receptor antagonist
includes antigen-binding
fragments thereof that binds the Her3 receptor extracellularly and is
effective in blocking
cleavage of the receptor or activating the Her3 receptor mediated signaling
cascade or preventing
Her3 from dimerizing with Her2 and its attendant consequences. The
compositions can be
provided in an article of manufacture or a kit.
Another aspect of the invention is an isolated nucleic acid encoding any one
or
more of the anti-Her3 antibodies of the invention, as well as a vector
comprising the nucleic
acid. The human Her3 DNA sequence can be found using GenBank Accession Number
(GenBank accession number ¨ NM_001982). Methods of recombinant production of
the
invention antibodies are also within the scope of the invention. Another
aspect of the invention is
a method of inhibiting or decreasing the proliferation of cancer cells by
administering a Her3
antibody which results in blocking of the endogenous ligand to the Her3
receptor or
inactivation/deactivation of Her3 signaling. Another aspect of the invention
is a method of
destroying cancer and tumor cells which express a Her3 receptor by
administering to a patient in
need thereof, a therapeutically effective amount of a composition comprising a
Her3 receptor
binding partner, e.g., any one or more of the Her3 specific antibodies
disclosed herein effective
for that purpose. A further aspect of the invention is a method of alleviating
cancer by
administering an agonist or antagonist of Her3 receptor. For therapeutic
applications, the
modulators of Her3 signaling can be used alone, or in combination therapy
with, e.g., hormones,
antiangiogens, or radiolabeled compounds, or with surgery, cryotherapy, and/or
radiotherapy.
The Her3 receptor binding partner useful in destroying cancer cells includes
soluble ligands of the receptor, antibodies and fragments thereof that bind
the Her3 receptor. The
binding partners can be conjugated to a cytotoxic agent. The antibodies are
preferably growth
inhibitory antibodies, The cytotoxic agent can be a toxin, antibiotic,
radioactive isotope or
nucleolytic enzyme. A preferred cytotoxic agent is a toxin, preferably a small
molecule toxin
such as calicheamicin or a maytansinoid.
The antagonists and binding partners of Her3 receptor can be synthetically or
recombinantly produced or otherwise isolated.
The mention of particular references, patent application and patents
throughout
this application should be read as being incorporated by reference into the
text of the
specification herein in their entirety.
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Definitions
Before the present proteins, nucleotide sequences, and methods are described,
it is
to be understood that the present invention is not limited to the particular
methodologies,
protocols, cell lines, vectors, and reagents described, as these may vary. It
is also understood
that the terminology used herein is for the purpose of describing particular
embodiments only,
and is not to limit the scope of the present invention.
The singular forms "a," "an," and "the" include plural reference unless the
context
clearly dictates otherwise.
All technical and scientific terms used herein have the same meanings as
commonly understood by one of ordinary skill in the art to which this
invention pertains. The
practice of the present invention will employ, unless otherwise indicated,
conventional
techniques of protein chemistry and biochemistry, molecular biology,
microbiology and
recombinant DNA technology, which are within the skill of the art. Such
techniques are
explained fully in the literature.
Although any machines, materials, and methods similar or equivalent to those
described herein can be used to practice or test the present invention, the
preferred machines,
materials, and methods are now described. All patents, patent applications,
and publications
mentioned herein, whether supra or infra, are each incorporated by reference
in its entirety.
Terms used throughout this application are to be construed with ordinary and
typical meaning to those of ordinary skill in the art. However, Applicants
desire that the
following terms be given the particular definition as defined below:
Also, the phraseology and terminology used herein is for the purpose of
description and should not be regarded as limiting. The use of "including,"
"comprising," or
"having," "containing", "involving", and variations thereof herein, is meant
to encompass the
items listed thereafter and equivalents thereof as well as additional items.
"Nucleic acid" or a "nucleic acid molecule" "nucleic acid molecule encoding
Her3" have been used for convenience to encompass DNA encoding Her3, RNA
(including pre-
mRNA and rnRNA or portions thereof) transcribed from such DNA, and also cDNA
derived
from such RNA. As well it encompasses any DNA or RNA molecule, either single-
or double-
stranded and, if single-stranded, the molecule of its complementary sequence
in either linear or
circular form. As used herein, the terms "target nucleic acid" and. In
discussing nucleic acid
molecules, a sequence or structure of a particular nucleic acid molecule may
be described herein
according to the normal convention of providing the sequence in the 5' to 3'
direction. In some
embodiments of the invention, nucleic acids are "isolated." This term, when
applied to DNA,
refers to a DNA molecule that is separated from sequences with which it is
immediately
contiguous in the naturally occurring genome of the organism in which it
originated. For
example, an "isolated nucleic acid" may comprise a DNA molecule inserted into
a vector, such
as a plasrnid or virus vector, or integrated into the genomic DNA of a
prokaryotic or eukaryotic
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cell or host organism. When applied to RNA, the term "isolated nucleic acid"
refers primarily to
an RNA molecule encoded by an isolated DNA molecule as defined above.
Alternatively, the
term may refer to an RNA molecule that has been sufficiently separated from
other nucleic acids
with which it would be associated in its natural state (i.e., in cells or
tissues). An isolated nucleic
acid (either DNA or RNA) may further represent a molecule produced directly by
biological or
synthetic means and separated from other components present during its
production.
Similarly, "amino acid sequence" as used herein refers to an oligopeptide,
peptide,
polypeptide, or protein sequence and fragments or portions thereof, of a
naturally occurring or
synthetic molecule.
The terms "isolated", "purified", or "biologically pure" refer to material
that is
substantially or essentially free from components that normally accompany it
as found in its
native state. Purity and homogeneity are typically determined using analytical
chemistry
techniques such as polyacrylamide gel electrophoresis or high performance
liquid
chromatography. A protein or nucleic acid that is the predominant species
present in a
preparation is substantially purified. In particular, an isolated nucleic acid
is separated from some
open reading frames that naturally flank the gene and encode proteins other
than protein encoded
by the gene. The term "purified" in some embodiments denotes that a nucleic
acid or protein
gives rise to essentially one band in an electrophoretic gel. Preferably, it
means that the nucleic
acid or protein is at least 85% pure, more preferably at least 95% pure, and
most preferably at
least 99% pure. "Purify" or "purification" in other embodiments means removing
at least one
contaminant from the composition to be purified. In this sense, purification
does not require that
the purified compound be homogenous, e.g., 100% pure. An "isolated" nucleic
acid molecule is
a nucleic acid molecule that is identified and separated from at least one
contaminant nucleic
acid molecule with which it is ordinarily associated in the natural source of
the antibody nucleic
acid. An isolated nucleic acid molecule is other than in the form or setting
in which it is found in
nature. Isolated nucleic acid molecules therefore are distinguished from the
nucleic acid
molecule as it exists in natural cells. However, an isolated nucleic acid
molecule includes a
nucleic acid molecule contained in cells that ordinarily express the antibody
where, for example,
the nucleic acid molecule is in a chromosomal location different from that of
natural cells.
Likewise, 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 Comassie blue or, preferably, silver
stain. Isolated
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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.
Nucleic acid is "operably linked" when it is placed into a functional
relationship
with another nucleic acid sequence. This can be a gene and a regulatory
sequence(s) which are
connected in such a way as to permit gene expression when the appropriate
molecules (e.g.,
transcriptional activator proteins) are bound to the regulatory sequences(s).
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 affects
the transcription of
the sequence; or a ribosome binding site is operably linked to a coding
sequence if it is
positioned so as to facilitate translation. Generally, "operably linked" means
that the DNA
sequences being linked are contiguous, and, in the case of a secretory leader,
contiguous and in
reading phase. However, enhancers do not have to be contiguous. Linking is
accomplished by
ligation at convenient restriction sites. If such sites do not exist; the
synthetic oligonucleotide
adaptors or linkers are used in accordance with conventional practice.
The term "cell", "cell line" and "cell culture" are used interchangeably, and
all
such designations include progeny. 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 property, as screened for in the originally
transformed cell, are
included.
The "host cells" used in the present invention generally are prokaryotic or
eukaryotic hosts. Examples of suitable host cells are described in Section 13.
Vectors, Host Cells
and Recombinant Methods: (vii) Selection and transformation of host cells.
"Transformation" means introducing DNA into an organism so that the DNA is
replicable, either as an extra chromosomal element or by chromosomal
integration.
"Transfection" refers to the taking up of an expression vector by a host cell
whether or not any coding sequences are in fact expressed.
The terms "transfected host cell" and "transformed" refer to the introduction
of
DNA into a cell. The cell is termed "host cell" and it may be either
prokaryotic or eukaryotic.
Typical prokaryotic host cells include various strains of E. coll. Typical
eukaryotic host cells are
mammalian, such as Chinese hamster ovary or cells of human origin. The
introduced DNA
sequence may be from the same species as the host cell or a different species
from the host cell,
or it may be a hybrid DNA sequence, containing some foreign and some
homologous DNA.
In the context of the present invention, "modulation" and "modulation of
expression" mean either an increase (stimulation) or a decrease (inhibition)
in the amount or
levels of a nucleic acid molecule encoding the Her3 receptor or the level of
the protein Her3 or
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modulation of the activity attendant the native Her3 receptor ¨ signaling
cascade etc. Inhibition
is often the preferred form of modulation of expression and the protein
receptor is often a
preferred target nucleic acid.
The terms "replicable expression vector" and "expression vector" refer to a
piece
of DNA, usually double-stranded, which may have inserted into it a piece of
foreign DNA.
Foreign DNA is defined as heterologous DNA, which is DNA not naturally found
in the host
cell. The vector is used to transport the foreign or heterologous DNA into a
suitable host cell.
Once in the host cell, the vector can replicate independently of the host
chromosomal DNA and
several copies of the vector and its inserted (foreign) DNA may be generated.
The term "vector" means a DNA construct containing a DNA sequence which is
operably linked to a suitable control sequence capable of effecting the
expression of the DNA in
a suitable host. Such control sequences include a promoter to effect
transcription, an optional
operator sequence to control such transcription, a sequence encoding suitable
mRNA ribosome
binding sites, and sequences which control the termination of transcription
and translation. The
vector may be a plasmid, a phage particle, or simply a potential genomic
insert. Once
transformed into a suitable host, the vector may replicate and function
independently of the host
genome, or may in some instances, integrate into the genome itself. In the
present specification,
"plasmid" and "vector" are sometimes used interchangeably, as the plasmid is
the most
commonly used form of vector at present. However, the invention is intended to
include such
other form of vector which serve equivalent function as and which are, or
become, known in the
art. Typical expression vectors for mammalian cell culture expression, for
example, are based on
pRI(5 (EP 307,247), pSV16B (WO 91/08291), and pVL1392 (Pharmingen).
The expression "control sequences" refers to DNA sequences necessary for the
expression of an operably linked coding sequence in a particular host
organism. The control
sequences that are suitable for prokaryotes, for example, include a promoter,
optionally an
operator sequence, and a ribosome binding site. Eukaryotic cells are known to
utilize promoters,
polyadenylation signals, and enhancers.
The terms "protein" or "polypeptide" are intended to be used interchangeably.
They refer to a chain of two (2) or more amino acids which are linked together
with peptide or
amide bonds, regardless of post-translational modification (eg., glycosylation
or
phosphorylation). Antibodies are specifically intended to be within the scope
of this definition.
The polypeptides of this invention may comprise more than one subunit, where
each subunit is
encoded by a separate DNA sequence.
Amino acids may be referred to herein either by their commonly known three
letter symbols or by the one-letter symbols recommended by the IUPAC-IUB
Biochemical
Nomenclature Commission. Nucleotides, likewise, may be referred to by their
commonly
accepted single-letter codes.
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Amino acids may be grouped according to similarities in the properties of
their
side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth
Publishers, New
York (1975)):
(1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W),
Met (M)(2)
uncharged polar: Gly (G), Ser (5), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin
(0)(3) acidic: Asp
(D), Glu (E)(4) basic: Lys (K), Arg (R), His(H)
Alternatively, naturally occurring residues may be divided into groups based
on
common side-chain properties:
(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;
(3) acidic: Asp, Glu;
(4) basic; H is, Lys, Arg;
(5) residues that influence chain orientation: Gly, Pro;
(6) aromatic: Trp, Tyr, Phe.
"Percent (%) amino acid sequence identity" with respect to the ligand or
receptor
polypeptide sequences identified herein is defined as the percentage of amino
acid residues in a
candidate sequence that are identical with the amino acid residues in such a
ligand or receptor
sequence identified herein, after aligning the sequences and introducing gaps,
if necessary, to
achieve the maximum percent sequence identity, and not considering any
conservative
substitutions as part of the sequence identity. Alignment for purposes of
determining percent
amino acid sequence identity can be achieved in various ways that are within
the skill in the art,
for instance, using publicly available computer software such as BLAST, BLAST-
2, ALIGN,
ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine
appropriate
parameters for measuring alignment, including any algorithms needed to achieve
maximal
alignment over the full-length of the sequences being compared. For purposes
herein, however,
% amino acid sequence identity values are obtained as described below by using
the sequence
comparison computer program ALIGN-2, wherein the complete source code for the
ALIGN-2
program is provided in the table below. The ALIGN-2 sequence comparison
computer program
was authored by Genentech, Inc. and the source code shown in the table below
has been filed
with user documentation in the U.S, Copyright Office, Washington D.C., 20559,
where it is
registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2
program is
publicly available through Genentech, Inc., South San Francisco, Calif. or can
be compiled from
the source code provided in the table below. The ALIGN-2 program should be
compiled for use
on a UNIX operating system, preferably digital UNIX V4.0D. All sequence
comparison
parameters are set by the ALIGN-2 program and do not vary.
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A useful method for identification of certain residues or regions in a protein
that
are preferred locations for mutagenesis is called "alanine scanning
mutagenesis" as described by
Cunningham and Wells Science, 2441081-1085 (1989). A residue or group of
target residues are
identified (e.g., charged residues such as arg, asp, his, lys, and glu) and
replaced by a neutral or
negatively charged amino acid (most preferably alanine or polyalanine) to
affect the interaction
of the amino acids with a binding target. Those amino acid locations
demonstrating functional
sensitivity to the substitutions then are refined by introducing further or
other variants at, or for,
the sites of substitution. Thus, while the site for introducing an amino acid
sequence variation is
predetermined, the nature of the mutation per se need not be predetermined.
For example, to
analyze the performance of a mutation at a given site, ala scanning or random
mutagenesis is
conducted at the target codon or region and the expressed variants are
screened for the desired
activity.
The term, "dihedral angle" refers to a rotation about a bond. See e.g.,
Creighton,
T. E., (1993) Protein:Structures and Molecular Properties, 2 ed., W. H.
Freeman and Company,
New York, N.Y.
The term, "phi," is a dihedral angle that denotes a rotation about the N--
C.alpha. bond of an amino acid. See e.g., Creighton, T. E., (1993)
Protein:Structures and
Molecular Properties, 2 ed., W. H. Freeman and Company, New York, N.Y.
Type I beta turns are described in Hutchinson, E. G. & Thornton, J. M. (1994)
A
revised set of potentials for beta turn formation in proteins. Protein Science
3, 2207-2216.
The phrase "substantially identical" with respect to an antibody polypeptide
sequence shall be construed as an antibody exhibiting at least 70%, preferably
80%, more
preferably 90% and most preferably 95% sequence identity to the reference
polypeptide
sequence. The term with respect to a nucleic acid sequence shall be construed
as a sequence of
nucleotides exhibiting at least about 85%, preferably 90%, more preferably 95%
and most
preferably 97% sequence identity to the reference nucleic acid sequence. For
polypeptides, the
length of the comparison sequences will generally be at least 25 amino acids.
For nucleic acids,
the length will generally be at least 75 nucleotides. Methods and computer
programs for the
alignment are well known in the art. Sequence identity may be measured using
sequence analysis
software (e.g, Sequence Analysis Software Package, Genetics Computer Group,
University of
Wisconsin Biotechnology Center, 1710 University Ave., Madison, Wis. 53705).
This software
matches similar sequences by assigning degrees of homology to various
substitutions, deletions,
and other modifications.
The term "identity" or "homology" shall be construed to mean the percentage of
amino acid residues in the candidate sequence that are identical with the
residue of a
corresponding sequence to which it is compared, after aligning the sequences
and introducing
gaps, if necessary to achieve the maximum percent identity for the entire
sequence, and not
considering any conservative substitutions as part of the sequence identity. A
molecule is
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"substantially similar" to another molecule if both molecules have
substantially similar structures
or biological activity. Thus, provided that two molecules possess a similar
activity, they are
considered variants as that term is used herein even if the structure of one
of the molecules is not
found in the other, or if the sequence of amino acid residues is not
identical. Neither N- or C-
terminal extensions nor insertions shall be construed as reducing identity or
homology.
The terms "identical" or percent "identity," in the context of two or more
nucleic
acids or polypeptide sequences, refer to two or more sequences or subsequences
that are the
same or have a specified percentage of amino acid residues or nucleotides that
are the same (i.e.,
about 60% identity, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%,
97%, 98%, 99%, or higher identity over a specified region, when compared and
aligned for
maximum correspondence over a comparison window or designated region) as
measured using a
BLAST or BLAST 2.0 sequence comparison algorithms with default parameters
described
below, or by manual alignment and visual inspection (see, e.g., NCBI web site
located at
www.ncbi.nlm.nih.gov/BLAST/ or the like). Such sequences are then said to be
"substantially
identical." This definition also refers to, or may be applied to, the
compliment of a test sequence.
The definition also includes sequences that have deletions and/or additions,
as well as those that
have substitutions, as well as naturally occurring, e.g., polymorphic or
allelic variants, and man-
made variants. As described below, the preferred algorithms can account for
gaps and the like.
Preferably, identity exists over a region that is at least about 25 amino
acids or nucleotides in
length, or more preferably over a region that is 50-100 amino acids or
nucleotides in length.
For sequence comparison, typically one sequence acts as a reference sequence,
to
which test sequences are compared. When using a sequence comparison algorithm,
test and
reference sequences are entered into a computer, subsequence coordinates are
designated, if
necessary, and sequence algorithm program parameters are designated.
Preferably, default
program parameters can be used, or alternative parameters can be designated.
The sequence
comparison algorithm then calculates the percent sequence identities for the
test sequences
relative to the reference sequence, based on the program parameters.
A "comparison window", as used herein, includes reference to a segment of one
of the number of contiguous positions selected from the group consisting
typically of from 20 to
600, usually about 50 to about 200, more usually about 100 to about 150 in
which a sequence
may be compared to a reference sequence of the same number of contiguous
positions after the
two sequences are optimally aligned. Methods of alignment of sequences for
comparison are
well-known in the art. Preferred examples of algorithms that are suitable for
determining percent
sequence identity and sequence similarity include the BLAST and BLAST 2.0
algorithms,
BLAST and BLAST 2.0 are used, with the parameters described herein, to
determine percent
sequence identity for the nucleic acids and proteins of the invention.
An indication that two nucleic acid sequences or polypeptides are
substantially
identical is that the polypeptide encoded by the first nucleic acid is
immunologically cross
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reactive with the antibodies raised against the polypeptide encoded by the
second nucleic acid, as
described below. Thus, a polypeptide is typically substantially identical to a
second polypeptide,
e.g., where the two peptides differ only by conservative substitutions.
Another indication that
two nucleic acid sequences are substantially identical is that the two
molecules or their
another indication that two nucleic acid sequences are substantially identical
is that the same
primers can be used to amplify the sequences.
"Conservatively modified variants" applies to both amino acid and nucleic acid
sequences. With respect to particular nucleic acid sequences, conservatively
modified variants
sequences, or where the nucleic acid does not encode an amino acid sequence,
to essentially
identical or associated, e.g., naturally contiguous, sequences. Because of the
degeneracy of the
genetic code, a large number of functionally identical nucleic acids encode
most proteins. For
instance, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine.
Thus, at
25 The term "conservative" amino acid substitution as used within this
invention is
meant to refer to amino acid substitutions which substitute functionally
equivalent amino acids.
Conservative amino acid changes result in silent changes in the amino acid
sequence of the
resulting peptide. For example, one or more amino acids of a similar polarity
act as functional
equivalents and result in a silent alteration within the amino acid sequence
of the peptide. In
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or uncharged polar group may be conservative with respect to structure and
function. Residues
such as Pro, Gly, and Cys (disulfide form) can have direct effects on the main
chain
conformation, and often may not be substituted without structural distortions.
Conservative
substitution tables providing functionally similar amino acids are well known
in the art. Such
conservatively modified variants are in addition to and do not exclude
polymorphic variants,
interspecies homologs, and alleles of the invention. Typically conservative
substitutions for one
another; 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine
(N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine
(L), Methionine (M),
Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),
Threonine (T);
and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).
The term "amino acid sequence variant" refers to a polypeptide that has amino
acid sequences that differ to some extent from a native sequence polypeptide.
"Substitutional" "amino acid variant" refers to molecules with some
differences in
their amino acid sequences as compared to a native amino acid sequence. The
substitutions may
be single, where only one amino acid in the molecule as been substituted, or
they may be
multiple, where two or more amino acids have been substituted in the same
molecule.
Ordinarily, amino acid sequence variants of Her3 receptor will possess at
least about 70%
homology with the native sequence Her3 receptor, preferably, at least about
80%, more
preferably at least about 85%, even more preferably at least about 90%
homology, and most
preferably at least 95%. The amino acid sequence variants can possess
substitutions, deletions,
and/or insertions at certain positions within the amino acid sequence of the
native amino acid
sequence. "Insertional" variants are those with one or more amino acids
inserted immediately
adjacent to an amino acid at a particular position in a native sequence.
Immediately adjacent to
an amino acid means connected to either the .alpha.-carboxyl or .alpha.-amino
functional group
of the amino acid. "Deletional" variants are those with one or more amino
acids in the native
amino acid sequence removed. Ordinarily, deletional variants will have one or
two amino acids
deleted in a particular region of the molecule.
The term "immunoglobulin" or "antibody" (used interchangeably herein) is used
in the broadest sense and is meant to encompass an immunoglobulin molecule
obtained by in
vitro or in vivo generation of an immunogenic response. A broad scope refers
to an antigen-
binding protein having a basic four-polypeptide chain structure consisting of
two heavy and two
light chains, said chains being stabilized, for example, by interchain
disulfide bonds, which has
the ability to specifically bind antigen. Both heavy and light chains are
folded into domains. The
term "domain" refers to a globular region of a heavy or light chain
polypeptide comprising
peptide loops (e.g., comprising 3 to 4 peptide loops) stabilized, for example,
by .beta.-pleated
sheet and/or intraehain disulfide bond. Domains are further referred to herein
as "constant" or
"variable", based on the relative lack of sequence variation within the
domains of various class
members in the case of a "constant" domain, or the significant variation
within the domains of
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various class members in the case of a "variable" domain. "Constant" domains
on the light chain
are referred to interchangeably as "light chain constant regions", "light
chain constant domains",
"CL" regions or "CL" domains) "Constant" domains on the heavy chain are
referred to
interchangeably as "heavy chain constant regions", "heavy chain constant
domains", "CH"
regions or "CH" domains) "Variable" domains on the light chain are referred to
interchangeably
as "light chain variable regions", "light chain variable domains", "VL"
regions or "VL"
domains). "Variable" domains on the heavy chain are referred to
interchangeably as "heavy
chain variable regions", "heavy chain variable domains", "VH" regions or "VH"
domains).
Particular amino acid residues are believed to form an interface between the
light and heavy
chain variable domains (Clothia et al., J. Mol Biol. 186, 651-66, 1985);
Novotny and Haber,
Proc. Natl. Acad Sci. USA 82, 4592-4596 (1985). The term includes polyclonal,
monoclonal,
single chain and multivalent antibodies. Fragments e.g., Fab, Fab', F(ab)2, Fv
etc. are also
included. Representative members include for example, single anti-Her3
monoclonal antibodies
(including agonist, antagonist, and neutralizing antibodies), anti-Her3
antibody compositions
with polyepitopic specificity (e.g. bispecific antibodies so long as they
exhibit the desired
biological activity), polyclonal antibodies, single chain anti-Her3
antibodies, and fragments of
anti-Her3 antibodies as long as they exhibit the desired biological or
immunological activity.
The antibodies may be genetically engineered antibodies and/or produced by
recombinant DNA
techniques. Fully human antibodies can also be produced by phage display, gene
and
chromosome transfection methods, as well as by other means. The L chain from
any vertebrate
species can be assigned to one of two clearly distinct types, called kappa and
lambda, based on
the amino acid sequences of their constant domains. Depending on the amino
acid sequence of
the constant domain of their heavy chains (CH), immunoglobulins can be
assigned to different
classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE,
IgG, and IgM,
having heavy chains designated ct, 5, e, 7, and µ, respectively. The y and
cc classes are
further divided into subclasses on the basis of relatively minor differences
in CH sequence and
function, e.g., humans express the following subclasses: IgGl, IgG2, IgG3,
IgG4, IgA I, and
IgA2.
An "intact" antibody is one which comprises an antigen-binding site as well as
a
CL and at least heavy chain constant domains, Cm, CH2 and CF/3. The constant
domains may be
native sequence constant domains (e.g. human native sequence constant domains)
or amino acid
sequence variant thereof. Preferably, the intact antibody has one or more
effector functions.
The term "region" refers to a part or portion of an antibody chain and
includes
constant or variable domains as defined herein, as well as more discrete parts
or portions of said
domains. For example, light chain variable domains or regions include
"complementarity
determining regions" or "CDRs" interspersed among "framework regions" or
"FRs", as defined
herein.
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The term "antigen" as used herein, means a molecule which is reactive with a
specific antibody.
"Epitope" or "antigenic determinant" refers to a site on an antigen to which
an
antibody binds. Epitopes can be formed both from contiguous amino acids or
noncontiguous
amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from
contiguous amino
acids are typically retained on exposure to denaturing solvents whereas
epitopes formed by
tertiary folding are typically lost on treatment with denaturing solvents. An
epitope typically
includes at least 3, and more usually, at least 5 or 8-10 amino acids in a
unique spatial
conformation. Methods of determining spatial conformation of epitopes include,
for example, x-
ray crystallography and 2-dimensional nuclear magnetic resonance.
Antibodies of "IgG class" refers to antibodies of IgGl, IgG2, IgG3, and IgG4.
The numbering of the amino acid residues in the heavy and light chains is that
of the EU index
(Kabat, et al., "Sequences of Proteins of Immunological Interest", 5th ed.,
National Institutes of
Health, Bethesda, Md. (1991); the EU numbering scheme is used herein).
The term "antibody mutant" refers to an amino acid sequence variant of an
antibody wherein one or more of the amino acid residues have been modified.
Such mutant
necessarily have less than 100% sequence identity or similarity with the amino
acid sequence
having at least 75% amino acid sequence identity or similarity with the amino
acid sequence of
either the heavy or light chain variable domain of the antibody, more
preferably at least 80%,
more preferably at least 85%, more preferably at least 90%, and most
preferably at least 95%.
Since the method of the invention applies equally to both polypeptides,
antibodies and fragments
thereof, these terms are sometimes employed interchangeably.
Alternatively or additionally, the word "mutant", as used herein, is
interchangeable with "mutationally-altered" and "glycosylation site altered".
The terms refer to
an antibody that comprises at least one immunoglobulin variable region
containing at least one
mutation that modifies a V region glycosylation site. A mutant immunoglobulin
refers to an
immunoglobulin (e.g., F(a13')2, Fv, Fab, bifunctional antibodies, antibodies,
etc.) comprising at
least one immunoglobulin variable region containing at least one mutation that
modifies a V
region glycosylation site. A mutant immunoglobulin chain has at least one
mutation that
modifies a V region glycosylation site, typically in the V region framework.
Thus, the pattern
(i.e., frequency and or location(s) of occurrence) of V region glycosylation
sites is altered in a
mutant immunoglobulin.
The term "variable" in the context of variable domain of antibodies, refers to
the
fact that certain portions of the variable domains differ extensively in
sequence among antibodies
and are used in the binding and specificity of each particular antibody for
its particular antigen.
The V domain mediates antigen binding and define specificity of a particular
antibody for its
particular antigen. However, the variability is not evenly distributed across
the 110-amino acid
span of the variable domains. It is concentrated in three segments called
complementarity
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determining regions (CDRs) also known as hypervariable regions both in the
light chain and the
heavy chain variable domains. There are at least two techniques for
determining CDRs: (1) an
approach based on cross-species sequence variability (i.e., Kabat et al.,
Sequences of Proteins of
Immunological Interest (National Institute of Health, Bethesda, Md. 1987); and
(2) an approach
based on crystallographic studies of antigen-antibody complexes (Chothia, C.
el at. (1989),
Nature 342: 877). The more highly conserved portions of variable domains are
called the
framework (FR) of 15-30 amino acids separated by shorter "hypervariable
regions" (9-12 amino
acids long). The variable domains of native heavy and light chains each
comprise four FR
regions, largely adopting a .beta.-sheet configuration, connected by three
CDRs, which form
loops connecting, and in some cases forming part of, the .beta.-sheet
structure. The CDRs in
each chain are held together in close proximity by the FR regions and, with
the CDRs from the
other chain, contribute to the formation of the antigen binding site of
antibodies. See Kabat et
al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health
Service, National
Institutes of Health, Bethesda, Md. (1991). The constant domains are not
involved directly in
binding an antibody to an antigen, but exhibit various effector function, such
as participation of
the antibody in antibody-dependent cellular toxicity. The term "variable
domain residue
numbering as in Kabat" or "amino acid position numbering as in Kabat", and
variations thereof,
refers to the numbering system used for heavy chain variable domains or light
chain variable
domains of the compilation of antibodies in Kabat et al., Sequences of
Proteins of
Immunological interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda,
Md. (1991). Using this numbering system, the actual linear amino acid sequence
may contain
fewer or additional amino acids corresponding to a shortening of, or insertion
into, a FR or CDR
of the variable domain. For example, a heavy chain variable domain may include
a single amino
acid insert (residue 52a according to Kabat) after residue 52 of 112 and
inserted residues (e.g.
residues 82a, 82b, and 82c, etc according to Kabat) after heavy chain FR
residue 82. The Kabat
numbering of residues may be determined for a given antibody by alignment at
regions of
homology of the sequence of the antibody with a "standard" Kabat numbered
sequence.
The term "hypervariable region" when used herein refers to the amino acid
residues of an antibody which are responsible for antigen-binding. The
hypervariable region
generally comprises amino acid residues from a "compiementarity determining
region" or
"CDR" (e.g. around about residues 24-34 (Li), 50-56 (L2) and 89-97 (L3) in the
VL, and around
about 1-35 (H1), 50-65 (1-12) and 95-102 (113) in the VH; Kabate et al.,
Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda,
Md. (1991)) and/or those residues from a "hypervariable loop" (e.g. residues
26-32 (L1), 50-52
(L2) and 91-96 (L3) in the V1, and 26-32 (Hi), 53-55 (112) and 96-101 (1-13)
in the VH; Chothia
and LeskJ. Mol. Biol. 196:901-917 (1987)).
A "variable region" has the structure of an antibody variable region from a
heavy
or light chain. Antibody heavy and light chain variable regions contain three
complementarity
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determining regions ("CDRs") interspaced onto a framework ("FW"). The CDRs are
primarily
responsible for recognizing a particular epitope. It is well known that
epitopes usually consist of
chemically active surface groupings of molecules such as amino acids or sugar
side chains and
usually have specific three dimensional structural characteristics, as well as
specific charge
characteristics. Conformational and nonconformational epitopes are
distinguished in that the
binding to the former but not the latter is lost in the presence of denaturing
solvents.
The hypervariable regions are generally referred to as complementarity
determining regions ("CDR") and are interposed between more conserved flanking
regions
referred to as framework regions ("FW"). There are four (4) FW regions and
three (3) CDRs that
are arranged from the NH2 terminus to the COOH terminus as follows: FW1, CDRI,
FW2,
CDR2, FW3, CDR3, FW4. Amino acids associated with framework regions and CDRs
can be
numbered and aligned by approaches described by Kabat et al., Sequences of
Proteins of
Immunological Interest, U.S. Department of Health and Human Services, 1991; C.
Chothia and
A.M. Lesk, Canonical structures for the hypervariable regions of
immunoglobulins, Journal of
Molecular Biology 196(4):901 (1987); or B. Al-Lazikani, A.M. Lesk and C.
Chothia, Standard
conformations for the canonical structures of immunoglobulins, Journal of
Molecular Biology
273(4): 27, 1997. For example, the framework regions and CDRs can be
identified from
consideration of both the Kabat and Chothia definitions. The variable regions
of the heavy and
light chains contain a binding domain that interacts with an antigen.
CDRs are primarily responsible for binding to a particular epitope. Within a
particular CDR, there are a few specificity determining residues (SDRs) which
are of greater
importance for binding to an epitope (see Kashmiri et al., Methods 36:25-34,
2005; Presta,
Advanced Drug Delivery Reviews 58:640-656, 2006). SDRs can be identified, for
example,
through the help of target protein-antibody three-dimensional structures and
mutational analysis
of antibody combining sites. (Kashmiri et al., 2005, supra.) Thus, the PD-1
binding proteins of
the present invention do not always require both a variable heavy chain and
light chain domain
to render PD-1 specificity but may only need a single CDR loop or a fragment
of a functional
antibody (see, e.g., Xu and Davis, 2000, Immunity 13:37-45 and Levi et al.,
1993, Proc. Natl.
Acad. Sci. USA 90:4374-78 (for CDR3 specificity); Williams et al., 1989, Proc.
Natl. Acad. Sci.
USA 86:5537-41 (CDR2 specificity); and, Welling et al., 1991, J Chromatography
548:235-42
(10 amino acid miniantibody).
Immunoglobulins or antibodies can exist in monomeric or polymeric form. The
term "antigen-binding fragment" refers to a polypeptide fragment of an
immunoglobulin or
antibody binds antigen or competes with intact antibody (i.e., with the intact
antibody from
which they were derived) for antigen binding (i.e., specific binding). The
term "conformation"
refers to the tertiary structure of a protein or polypeptide (e.g., an
antibody, antibody chain,
domain or region thereof). For example, the phrase "light (or heavy) chain
conformation" refers
to the tertiary structure of a light (or heavy) chain variable region, and the
phrase "antibody
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conformation" or "antibody fragment conformation" refers to the tertiary
structure of an antibody
or fragment thereof. Preferably, the fragment exhibits qualitative biological
activity in common
with a full-length antibody. For example, a functional fragment or analog of
an anti-IgE
antibody is one which can bind to an IgE irnmunoglobulin in such a manner so
as to prevent or
substantially reduce the ability of such molecule from having the ability to
bind to the high
affinity receptor, Fc.epsilon.RI. Antibody fragments can be prepared by in
vitro manipulation of
antibodies (e.g., by limited proteolysis of an antibody), or via recombinant
DNA technology
(e.g., the preparation of single-chain antibodies from phage display
libraries). Examples of
antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies;
linear antibodies
(U.S. Patent. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):
1057-1062 (1995));
single-chain antibody molecules; and multispecific antibodies formed from
antibody fragments.
Binding fragments are produced by recombinant DNA techniques, or by
enzymatic or chemical cleavage of intact immunoglobulins. Binding fragments
include Fab,
Fab', F(abl)2, Fabc, Fv, single chains, and single-chain antibodies. Other
than "bispecific" or
"bifunctional" immunoglobulins or antibodies, an immunoglobulin or antibody is
understood to
have each of its binding sites identical.
"A functional variant" of the antibody molecule according to the invention is
an
antibody molecule which possesses a biological activity (either functional or
structural) that is
substantially similar to the antibody molecule according to the invention,
i.e. a substantially
similar substrate specificity or cleavage of the substrate. The term
"functional variant" also
includes "a fragment", "an allelic variant", "variant based on the
degenerative nucleic acid code"
or "chemical derivatives". Such a "functional variant" e.g. may carry one or
several point
mutations, one or several nucleic acid exchanges, deletions or insertions or
one or several amino
acid exchanges, deletions or insertions. Said functional variant is still
retaining its biological
activity such as antibody binding activity, at least in part or even going
along with an
improvement said biological activity.
An "allelic variant" is a variant due to the allelic variation, e.g.
differences in the
two alleles in humans. Said variant is still retaining its biological activity
such as antibody target
binding activity, at least in part or even going along with an improvement
said biological
activity.
A "variant based on the degenerative of the genetic code" is a variant due to
the
fact that a certain amino acid may be encoded by several different nucleotide
triplets. Said
variant is still retaining its biological activity such as antibody binding
activity, at least in part or
even going along with an improvement said biological activity.
A "fusion protein" and a "fusion polypeptide" refer to a polypeptide having
two
portions covalently linked together, where each of the portions is a
polypeptide having a
different property. The property may be a biological property, such as
activity in vitro or in vivo.
The property may also be a simple chemical or physical property, such as
binding to a target
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molecule, catalysis of a reaction, etc. The two portions may be linked
directly by a single peptide
bond or through a peptide linker containing one or more amino acid residues.
Generally, the two
portions and the linker will be in reading frame with each other.
As used herein, a "chemical derivative" according to the invention is an
antibody
molecule according to the invention chemically modified or containing
additional chemical
moieties not normally being part of the molecule. Such moieties may improve
the molecule's
activity such as target destruction (e.g. killing of tumor cells) or may
improve its solubility,
absorption, biological half life, etc.
Papain digestion of antibodies produces two identical antigen-binding
fragments,
called "Fab" fragments, and a residual "Fc" fragment, a designation reflecting
the ability to
crystallize readily. The Fab fragment consists of an entire L chain along with
the variable region
domain of the H chain (VH), and the first constant domain of one heavy chain
(CH 1). Each Fab
fragment is monovalent with respect to antigen binding, i.e., it has a single
antigen-binding site.
Pepsin treatment of an antibody yields a single large F(abi)2 fragment which
roughly
corresponds to two disulfide linked Fab fragments having divalent antigen-
binding activity and
is still capable of cross-linking antigen. Fab' fragments differ from Fab
fragments by having
additional few residues at the carboxy terminus of the CH1 domain including
one or more
cysteines from the antibody hinge region. Fab'-SH is the designation herein
for Fab' in which the
cysteine residue(s) of the constant domains bear a free thiol group. F(ab1)2
antibody fragments
originally were produced as pairs of Fab' fragments which have hinge cysteines
between them.
Other chemical couplings of antibody fragments are also known.
The Fe fragment comprises the carboxy-terminal portions of both H chains held
together by disulfides. The effector functions of antibodies are determined by
sequences in the
Fe region, which region is also the part recognized by Fe receptors (FcR)
found on certain types
of cells. By "Fe" or "Fe region" as used herein is meant the polypeptides
comprising the last two
constant region immunoglobulin domains of IgA, 1gD, and IgG, and the last
three constant
region immunoglobulin domains of IgE and IgM, and part of the flexible hinge N-
terminal to
these domains. Although the boundaries of the Fe region may vary, the human
IgG heavy chain
Fe region is usually defined to comprise residues C226 or P230 to its carboxyl-
terminus, wherein
the numbering is according to the EU numbering scheme. Fe may refer to this
region in isolation,
or this region in the context of a full length antibody or antibody fragment
Ergo, by "outside the
Fe region" as used herein is meant the region of an antibody that does not
comprise the Fe region
of the antibody. In accordance with the aforementioned definition of Fe
region, "outside the Fe
region" for an IgG1 antibody is herein defined to be from the N-terminus up to
and including
residue T225 or C229, wherein the numbering is according to the EU numbering
scheme. Thus
the Fab region and part of the hinge region of an antibody are outside the Fe
region.
"Fe receptor" or "FcR" describes a receptor that binds to the Fe region of an
antibody. The preferred FcR is a native sequence human FeR. Moreover, a
preferred RR. is one
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which binds an IgG antibody (a y receptor) and includes receptors of the
FcyRI, FcyRII and
FcyRIII subclasses, including allelic variants and alternatively spliced forms
of these receptors.
FcyRII receptors include FcyRIIA (an "activating receptor") and FcyRIIB (an
"inhibiting
receptor"), which have similar amino acid sequences that differ primarily in
the cytoplasmic
domains thereof. Activating receptor FcyRIIA contains an immunoreceptor
tyrosine-based
activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcyRIIB
contains an
immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic
domain. [M. in
Daeron, Annu. Rev. Immunol. 15:203-234 (1997)]. FeRs are reviewed in Ravetch
and Kind,
Annu. Rev. Immunol. 9:457-492 (1991); Capel et al., Immunomethods 4:25-34
(1994); and de
Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FoRs, including those
to be identified in
the future, are encompassed by the term "FcR" herein. The term also includes
the neonatal
receptor, FcRn, which is responsible for the transfer of maternal IgGs to the
fetus (Guyer et al., J.
Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).
An "Fv" fragment is the minimum antibody fragment which comprises the
variable domains of its heavy chain and light chain and thus contains a
complete antigen
recognition and binding site. This region consists of a dimer of one heavy and
one light chain
variable domain in a tight, non-covalent association (VH
dimer). It is in this configuration
that the three CDRs of each variable domain interact to define an antigen
binding site on the
surface of the VH -VL dimer. Collectively, the six CDRs confer antigen binding
specificity to the
antibody. However, even a single variable domain (or half of an Fv comprising
only three CDRs
specific for an antigen) has the ability to recognize and bind antigen,
although at a lower affinity
than the entire binding site. One or more scFv fragments may be linked to
other antibody
fragments (such as the constant domain of a heavy chain or a light chain) to
form antibody
constructs having one or more antigen recognition sites.
The term "single chain variable fragment or scFv" refers to an Fv fragment in
which the heavy chain domain and the light chain domain are linked. "Single-
chain Fv" also
abbreviated as "sFv" or "scFv" are antibody fragments that comprise the VH and
VL antibody
domains connected into a single polypeptide chain. Generally, the sFv
polypeptide further
comprises a polypeptide linker between the VFT and VI, 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); Borrebaeck 1995, infra.
The skilled person will also be familiar with so-called miniantibodies which
have
a bi-, tri- or tetravalent structure and are derived from scFv. The
multimerization is carried out
by di-, tri- or tetrameric coiled coil structures (Pack et al., 1993
Biotechnology II:, 1271-1277;
Lovejoy et al. 1993 Science 259: 1288-1293; Pack et al., 1995 J. Mol. Biol.
246: 28-34).
By minibody the skilled person means a bivalent, homodimeric scFv derivative.
It
consists of a fusion protein which contains the CH3 region of an
immunoglobulin, preferably
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IgG, most preferably IgG1 as the dimerization region which is connected to the
scFv via a hinge
region (e.g. also from IgG1) and a linker region. The disulphide bridges in
the hinge region are
mostly formed in higher cells and not in prokaryotes. In some embodiments an
antibody
according to the invention is a Her3-specific minibody antibody fragment.
Examples of
The term "diabodies" refers to a small antibody fragments with two antigen-
binding sites, which fragments comprise a heavy chain variable domain (VH)
connected to a light
chain variable domain (VI) in the same polypeptide chain (VH-VL). By using a
linker that is too
By triabody the skilled person means a: trivalent homotrimeric scFv derivative
(Kora et al. 1997 Protein Engineering 10: 423-433). ScFv derivatives wherein
VH-VL are fused
directly without a linker sequence lead to the formation of timers.
20 The Fab fragment [also designated as F(ab)] also contains the
constant domain of
the light chain and the first constant domain (CH1) of the heavy chain. Fab'
fragments differ
from Fab fragments by the addition of a few residues at the carboxyl terminus
of the heavy chain
CHI domain including one or more cysteines from the antibody hinge region.
Fab'-SH is the
designation herein for Fab' in which the cysteine residue(s) of the constant
domains have a free
A "bispecific" or "bifunctional antibody" is an artificial hybrid antibody
having
two different heavy/light chain pairs and two different binding sites.
Bispecific antibodies can
An antibody "which binds" an antigen of interest, e.g. a tumor-associated
polypeptide antigen target., e.g., Her3, is one that binds the antigen with
sufficient affinity such
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target protein as determined by fluorescence activated cell sorting (FACS)
analysis or
radioimmunoprecipitation (RIA). With regard to the binding of an antibody to a
target molecule,
the term "specific binding" or "specifically binds to" or is "specific for" a
particular polypeptide
or an epitope on a particular polypeptide target means binding that is
measurably different from a
non-specific interaction. Specific binding can be measured, for example, by
determining binding
of a molecule compared to binding of a control molecule, which generally is a
molecule of
similar structure that does not have binding activity. For example, specific
binding can be
determined by competition with a control molecule that is similar to the
target, for example, an
excess of non-labeled target. In this case, specific binding is indicated if
the binding of the
labeled target to a probe is competitively inhibited by excess unlabeled
target.
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 can
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 at, Nature,
256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S.
Pat. No.
4,816,567). The "monoclonal antibodies" may also be isolated from phage
antibody libraries
using the techniques described in Clackson et al., Nature, 352:624-628 (1991)
and Marks et al.,
J. Mol. Biol., 222:581-597 (1991), for example.
The monoclonal antibodies herein specifically include "chimeric" antibodies
(immunoglobulins) in which a portion of the heavy and/or light chain is
identical with or
homologous to corresponding sequences in antibodies derived from a particular
species or
belonging to a particular antibody class or subclass, while the remainder of
the chain(s) is
identical with or homologous to corresponding sequences in antibodies derived
from another
species or belonging to another antibody class or subclass, as well as
fragments of such
antibodies, so long as they exhibit the desired biological activity (U.S. Pat,
No. 4,816,567;
Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Methods of
making chimeric
antibodies are known in the art.
"Humanized" forms of non-human (e.g., murine) antibodies are chimeric
immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab,
Fab',
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F(ab')2 or other antigen-binding subsequences of antibodies) which
contain minimal
sequence derived from non-human immunoglobulin. For the most part, humanized
antibodies are
human immunoglobulins (recipient antibody) in which residues from a
complementarity-
determining region (CDR) of the recipient are replaced by residues from a CDR
of a non-human
species (donor antibody) such as mouse, rat or rabbit having the desired
specificity, affinity, and
capacity. In some instances, Fv framework region (FR) residues of the human
immunoglobulin
are replaced by corresponding non-human residues. Furthermore, humanized
antibodies may
comprise residues which are found neither in the recipient antibody nor in the
imported CDR or
framework sequences. These modifications are made to further refine and
maximize antibody
performance. In general, the humanized antibody will comprise substantially
all of at least one,
and typically two, variable domains, in which all or substantially all of the
hypervafiable loops
correspond to those of a non-human immunoglobulin and all or substantially all
of the FR
regions are those of a human immunoglobulin sequence although the FR regions
may include
one or more amino acid substitutions that improve binding affinity. The number
of these amino
acid substitutions in the FR are typically no more than 6 in the H chain, and
in the L chain, no
more than 3. The humanized antibody optimally also will comprise at least a
portion of an
immunoglobulin constant region (Fe), typically that of a human immunoglobulin.
For further
details, see Jones et al., Nature, 321:522-525 (1986); Reichmann et al.,
Nature, 332:323-329
(1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992). The humanized
antibody includes
a PRIMATIZED® antibody wherein the antigen-binding region of the antibody
is derived
from an antibody produced by, e.g., immunizing macaque monkeys with the
antigen of interest,
Methods of making humanized antibodies are known in the art.
Human antibodies can also be produced using various techniques known in the
art, including phage-display libraries. Hoogenboom and Winter, 3, Mol. Biol.,
227:381 (1991);
Marks et al., 3. Mal. Biol., 222:581 (1991). The techniques of Cole et al. and
Boerner et al. are
also available for the preparation of human monoclonal antibodies. Cole et
al., Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., 3.
Immunol.,
147(1):86-95 (1991).
"Functional fragments" or "antigen-binding fragments: of the antibodies of the
invention are those fragments that retain binding to Her3 , e.g,. that have
substantially the same
affinity as the intact full chain molecule from which they are derived.
An "effector" or "effector moiety" or "effector component" is a molecule that
is
bound (or linked, or conjugated), either covalently, through a linker or a
chemical bond, or
noncovalently, through ionic, van der Waals, electrostatic, or hydrogen bonds,
to an antibody.
The "effector" can be a variety of molecules including, e.g., detection
moieties including
radioactive compounds, fluorescent compounds, an enzyme or substrate, tags
such as epitope
tags, a toxin, activatable moieties, a chemotherapeutic or cytotoxic agent, a
chemoattractant, a
lipase; an antibiotic; or a radioisotope emitting "hard" e.g., beta radiation.
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The same criteria that make a target attractive for an ADC approach to cancer
therapy are also desirable for an antibody dependent cellular cytotoxicity
(ADCC) approach. In
an ADCC approach, a naked antibody to the target is used to recruit immune
effector cells
(cytotoxic T lymphocytes, natural killer cells, activated macrophages) to the
tumor. These
effector cells then specifically kill the targeted tumor cells.
Antibody "effector functions" refer to those biological activities
attributable to the
Fe region (a native sequence Fe region or amino acid sequence variant Fe
region) of an antibody,
and vary with the antibody isotype. Examples of antibody effector functions
include: C lq
binding and complement dependent cytotoxicity; Fe receptor binding; antibody-
dependent cell-
mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface
receptors (e.g. B
cell receptor); and B cell activation,
"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of
cytotoxicity in which secreted Jig bound onto Fe receptors (FcRs) present on
certain cytotoxic
cells (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) enable
these cytotoxic
effector cells to bind specifically to an antigen-bearing target cell and
subsequently kill the target
cell with cytotoxins. The antibodies "arm" the cytotoxic cells and are
absolutely required for
such killing. The primary cells for mediating ADCC, NK cells, express
Fe.gamma,RIII only,
whereas monocytes express Fc.gamma.RT, Fc.gamma.RII and Fc.gamma.RIII. FcR
expression
on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and
Kinet, Annu. Rev.
Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an
in vitro ADCC
assay, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may be
performed. Useful
effector cells for such assays include peripheral blood mononuclear cells
(PBMC) and Natural
Killer (NK) cells. Alternatively, or additionally, ADCC activity of the
molecule of interest may
be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes
at al. PNAS (USA)
95:652-656 (1998).
"Complement dependent cytotoxicity" or "CDC" refers to the lysis of a target
cell
in the presence of complement. Activation of the classical complement pathway
is initiated by
the binding of the first component of the complement system (Cl q) to
antibodies (of the
appropriate subclass) which are bound to their cognate antigen. To assess
complement
activation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J.
Irnmunol. Methods
202:163 (1996), may be performed.
"Humanized" forms of non-human (e.g., murine) antibodies are chimeric
immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab,
Fab', F(abt)2 or
other antigen-binding subsequences of antibodies) which contain minimal
sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies are human
immunoglobulins (recipient antibody) in which residues from a complementarity-
determining
region (CDR) of the recipient are replaced by residues from a CDR of a non-
human species
(donor antibody) such as mouse, rat or rabbit having the desired specificity,
affinity, and
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capacity. Thus, a humanized antibody is a recombinant protein in which the
CDRs from an
antibody from one species; e.g., a rodent antibody, is transferred from the
heavy and light
variable chains of the rodent antibody into human heavy and light variable
domains. The
constant domains of the antibody molecule is derived from those of a human
antibody. In some
instances, Fv framework region (FR) residues of the human immunoglobulin are
replaced by
corresponding non-human residues. Furthermore, humanized antibodies may
comprise residues
which are found neither in the recipient antibody nor in the imported CDR or
framework
sequences. These modifications are made to further refine and optimize
antibody performance.
In general, the humanized antibody will comprise substantially all of at least
one, and typically
two, variable domains, in which all or substantially all of the CDR regions
correspond to those of
a non-human immunoglobulin and all or substantially all of the FR regions are
those of a human
immunoglobulin sequence. The humanized antibody optimally also will comprise
at least a
portion of an immunoglobulin constant region (Fc), typically that of a human
immunoglobulin.
For further details, see Jones et al., Nature, 321:522-525 (1986); Reichmann
et al., Nature,
332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992). The
humanized
antibody includes a PrimatizedTM antibody wherein the antigen-binding region
of the antibody is
derived from an antibody produced by immunizing macaque monkeys with the
antigen of
interest.
Completely "human" antibodies may be desirable for therapeutic treatment of
human patients. Human antibodies can be made by a variety of methods known in
the art
including phage display methods described above using antibody libraries
derived from human
immunoglobulin sequences. See U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT
publications
WO 98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO 96/34096; WO 96/33735;
and
WO 91/10741, each of which is incorporated herein by reference in its
entirety. Human
antibodies can also be produced using transgenic mice which are incapable of
expressing
functional endogenous immunoglobulins, but which can express human
immunoglobulin genes.,
see, e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO
96/33735;
European Patent No. 0 598 877; US. Pat. Nos. 5,413,923; 5,625,126; 5,633,425;
5,569,825;
5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598, which
are incorporated
by reference herein in their entireties. In addition, companies such as
Abgenix, Inc, (Fremont,
Calif.) and Medarex (Princeton, N.J.) can be engaged to provide human
antibodies directed
against a selected antigen using technology similar to that described above.
Completely human antibodies that recognize a selected epitope can be generated
using a technique referred to as "guided selection." In this approach a
selected non-human
monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of
a completely
human antibody recognizing the same epitope (Jespers et al., Biotechnology
12:899-903 (1988).
A chimeric antibody is a recombinant protein that contains the variable
domains
including the complementarity determining regions (CDRs) of an antibody
derived from one
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species, preferably a rodent antibody, while the constant domains of the
antibody molecule is
derived from those of a human antibody. For veterinary applications, the
constant domains of the
chimeric antibody may be derived from that of other species, such as a cat or
dog.
A "species-dependent antibody," e.g., a mammalian anti-human Her3 antibody, is
an antibody which has a stronger binding affinity for an antigen from a first
mammalian species
than it has for a homologue of that antigen from a second mammalian species.
Normally, the
species-dependent antibody "bind specifically" to a human antigen (i.e., has a
binding affinity
(Kd) value of no more than about 1 X10-7 M, preferably no more than about 1 X
10-8 and most
preferably no more than about 1 X.10-9 M) but has a binding affinity for
homologue of the
antigen from a second non-human mammalian species which is at least about 500
fold, or at
least about 1000 fold, weaker than its binding affinity for the human antigen.
The species-
dependent antibody can be of any of the various types of antibodies as defined
above, but
preferably is a human antibody.
"Human effector cells" are leukocytes which express one or more FeRs and
perform effector functions. Preferably, the cells express at least FcyRIII and
perform ADCC
effector function. Examples of human leukocytes which mediate ADCC include
peripheral blood
mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T
cells and
neutrophils; with PBMCs and NK cells being preferred. The effector cells may
be isolated from
a native source, e.g., from blood.
The term "cytotoxic agent" as used herein refers to a substance that inhibits
or
prevents the function of cells and/or causes destruction of cells. The term is
intended to include
radioactive isotopes (e.g. At 211, 1131, I 125, Y 90, Re 186, Re 188, Sm
153,131212, P 32 and
radioactive isotopes of Lu), chemotherapeutic agents e.g. methotrexate,
adriamycin, vinca
alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan,
mitomycin C,
chlorambucil, daunorubicin or other intercalating agents, enzymes and
fragments thereof such as
nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or
enzymatically
active toxins of bacterial, fungal, plant or animal origin, including
fragments and/or variants
thereof, and the various anti-tumor or anticancer agents disclosed below.
Other cytotoxic agents
are described below.
A "chemotherapeutic agent" is a chemical compound useful in the treatment of
cancer. Examples of chemotherapeutic agents include alkylating agents such as
thiotepa and
cyclosphosphamide (CYTOXAN.TM.); alkyl sulfonates such as busulfan,
improsulfan and
piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa;
ethylenimines
and methylamelamines including altretamine, triethylenernelamine,
trietylenephosphoramide,
triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such
as
chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine,
prednimustine,
trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin,
fotemustine,
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lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins,
actinomycin,
authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin,
caminomycin,
carzinophilin, chromomycins, dactinomycin, daunorubic in, detorubicin, 6-diazo-
5-oxo-L-
norleucine, doxorubicin, epirubiein, esorubicin, idarubiein, marcellomycin,
mitomycins,
mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromyein,
puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,
zinostatin, zorubicin;
anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid
analogues such as
denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as
fludarabine, 6-
mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as
ancitabine, azacitidine, 6-
azauridine, earmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine,
floxuridine, 5-FU;
androgens such as calusterone, dromostanolone propionate, epitiostanol,
mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotarte, trilostane;
folic acid replenisher
such as frolinic acid; aceglatone; aldophosphamide glycoside; aminol evulinic
acid; amsacrine;
bestrabueil; bisantrene; edatraxate; defofamine; demeeolcine; diaziquone;
elfomithine;
elliptinium acetate; etoglueid; gallium nitrate; hydroxyurea; lentinan;
lonidamine; mitoguazone;
mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;
podophyllinie acid; 2-
ethylhydrazide; procarbazine; PSK®; razoxane; sizofuran; spirogermanium;
tenuazonic
acid; triaziquone; 2,2',2"-trichlorotriethylamine; urethan; vindesine;
dacarbazine; mannomustine;
mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");
eyelophosphamide;
thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology,
Princeton,
N.J.) and doxetaxel (TAXOTER®, Rhone-Poulenc Rorer, Antony, France);
chlorambucil;
gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs
such as eisplatin
and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;
mitomycin C;
mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide;
daunomycin;
aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;
difluoromethylornithine (DMFO); retinoic acid; esperamicins; capeeitabine; and
pharmaceutically acceptable salts, acids or derivatives of any of the above.
Also included in this
definition are anti-hormonal agents that act to regulate or inhibit hormone
action on tumors such
as anti-estrogens including for example tamoxifen, raloxifene, aromatase
inhibiting 4(5)-
imidazoles, 4-hydroxytarnoxifen, trioxifene, keoxifene, LY117018, onapristone,
and torernifene
(Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide,
leuprolide, and
goserelin; and pharmaceutically acceptable salts, acids or derivatives of any
of the above.
A "glycosylation variant" "Glycoform variant" antibody herein is an antibody
with one or more carbohydrate moieties attached thereto which differ from one
or more
carbohydrate moieties attached to a main species antibody. Examples of
glycosylation variants
herein include antibodies with a G1 or 02 oligosaccharide structure, instead
of a GO
oligosaccharide structure, attached to an Fe region thereof, antibody with one
or two
carbohydrate moieties attached to one or two light chains thereof, antibody
with no carbohydrate
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attached to one or two heavy chains of the antibody, etc, as well as
combinations of such
glycosylation alterations.
"Glycosylation sites" refer to amino acid residues which are recognized by a
eukaryotic cell as locations for the attachment of sugar residues. The amino
acids where
carbohydrate, such as oligosaccharide, is attached are typically asparagine (N-
linkage), serine
(0-linkage), and threonine (0-linkage) residues. The specific site of
attachment is typically
signaled by a sequence of amino acids, referred to herein as a "glycosylation
site sequence". The
glycosylation site sequence for N-linked glycosylation is: -Asn-X-Ser- or -Asn-
X-Thr-, where X
may be any of the conventional amino acids, other than proline. The
predominant glycosylation
site sequence for 0-linked glycosylation is: -(Thr or Ser)-X-X-Pro-, where X
is any conventional
amino acid. The recognition sequence for glycosaminoglycans (a specific type
of sulfated sugar)
is -Ser-Gly-X-Gly-, where X is any conventional amino acid. The terms "N-
linked" and
linked" refer to the chemical group that serves as the attachment site between
the sugar molecule
and the amino acid residue. N-linked sugars are attached through an amino
group; 0-linked
sugars are attached through a hydroxyl group. However, not all glycosylation
site sequences in a
protein are necessarily glycosylated; some proteins are secreted in both
glycosylated and
nonglycosylated forms, while others are fully glycosylated at one
glycosylation site sequence but
contain another glycosylation site sequence that is not glycosylated.
Therefore, not all
glycosylation site sequences that are present in a polypeptide are necessarily
glycosylation sites
where sugar residues are actually attached. The initial N-glycosylation during
biosynthesis
inserts the "core carbohydrate" or "core oligosaccharide" (Proteins,
Structures and Molecular
Principles, (1984) Creighton (ed.), W.H. Freeman and Company, New York, which
is
incorporated herein by reference).
A "V region glycosylation site" is a position in a variable region where a
carbohydrate, typically an oligosaccharide, is attached to an amino acid
residue in the
polypeptide chain via an N-linked or 0-linked covalent bond. Since not all
glycosylation site
sequences are necessarily glycosylated in a particular cell, a glycosylation
site is defined
operationally by reference to a designated cell type in which glycosylation
occurs at the site, and
is readily determined by one of ordinary skill in the art. Thus, a mutant
antibody has at least one
mutation that adds, subtracts, or relocates a V region glycosylation site,
such as, for example, an
N-linked glycosylation site sequence. Preferably, the mutation(s) are
substitution mutations that
introduce conservative amino acid substitutions, where possible, to modify a
glycosylation site.
Preferably, when the parent immunoglobulin sequence contains a glycosylation
site in a V region
framework, particularly in a location near the antigen binding site (for
example, near a CDR), the
glycosylation site sequence is mutated (e.g., by site-directed mutagenesis) to
abolish the
glycosylation site sequence, typically by producing a conservative amino acid
substitution of one
or more of the amino acid residues comprising the glycosylation site sequence.
When the parent
immunoglobulin sequence contains a glycosylation site in a CDR, and where the
parent
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immunoglobulin specifically binds an epitope that contains carbohydrate, that
glycosylation site
is preferably retained. If the parent immunoglobulin specifically binds an
epitope that comprises
only polypeptide, glycosylation sites occurring in a CDR are preferably
eliminated by mutation
(e.g., site-directed mutation).
"Glycosylation-reduced antibodies" and "glycosylation-reduced immunoglobulin
chains" are mutant antibodies and mutant immunoglobulin chains, respectively,
in which at least
one glycosylation site that is present in the parent sequence has been
destroyed by mutation and
is absent in the mutant sequence.
"Glycosylation-supplemented antibodies" and "glycosylation-supplemented
polynucleotide sequence" refer herein to a reference amino acid sequence or
polynucleotide
sequence, respectively. A parent polynucleotide sequence may encode a
naturally-occurring
well as molecules that contain both amino acid or protein portions and non-
protein portions.
Conjugates may be synthesized by a variety of techniques known in the art
including, for
example, recombinant DNA techniques, solid phase synthesis, solution phase
synthesis, organic
As used herein, the twenty conventional amino acids and their abbreviations
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93: 8618-23; Bernstein et al., Leukemia, 2000; 14: 474-5; Ross et al., Cancer
Res, 2002; 62:
2546-53; Bhaskar et al., Cancer Res, 2003; 63: 6387-94; Doronina et al., Nat
Biotechnol, 2003;
21: 778-84; Francisco et al., Blood, 2003; 102: 1458-65). The strategy of this
approach is to
deliver a toxic payload to the cancer cell via an antibody that targets a
cancer-specific antigen.
This strategy requires that the potent drug is internalized via the antibody-
antigen complex,
released within the cell and specifically kills the cancer cells (Bhaskar et
al., Cancer Res, 2003;
63: 6387-94; Doronina et al., Nat Biotechnol, 2003; 21: 778-84; Francisco et
al., Blood, 2003;
102: 1458-65). Ideally, the potent drug is internalized via the antibody-
antigen complex, released
within the cell and specifically kills the cancer cells. In order to minimize
toxic side effects it is
critical that the molecular target is not expressed in essential organs that
are accessible to
circulating antibodies. In addition, the target must be at the plasma membrane
of cancer cells to
allow antibody access.
The same criteria that make a target attractive for an ADC approach to cancer
therapy are also desirable for an antibody dependent cellular cytotoxicity
(ADCC) approach. In
an ADCC approach, a naked antibody to the target is used to recruit immune
effector cells
(cytotoxic T lymphocytes, natural killer cells, activated macrophages) to the
tumor. These
effector cells then specifically kill the targeted tumor cells.
As used herein, the term "antibody phage library" refers to the phage library
used
in the affinity maturation process described above and in Hawkins et at., J.
Mol Bio1.254: 889-
896 (1992), and in Lowman et al., Biochemistry 30(45): 10832-10838 (1991).
Each library
comprises a hypervariable region (eg. 6-7 sites) for which all possible amino
acid substitutions
are generated. The antibody mutants thus generated are displayed in a
monovalent fashion from
filamentous phage particles as fusions to the gene III product of M13 packaged
within each
particle and expressed on the exterior of the phage.
As used herein, "target molecule" means any molecule, not necessarily a
protein,
for which it is desirable to produce an antibody or ligand. Preferably,
however, the target will be
a protein and most preferably the target will be an antigen ¨EGFR or human
her3 or a Her2/Her3
dimer.
A "small molecule" is defined herein to have a molecular weight below about
500
Daltons.
The term "prodrug" as used in this application refers to a precursor or
derivative
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, "Prodnigs in Cancer Chemotherapy," Biochemical
Society
Transactions, 14, pp. 375-382, 615 Meeting, Belfast (1986) and Stella et al.,
(ed.), "Prodrugs: A
Chemical Approach to Targeted Drug Delivery," Directed Drug Delivery,
Borchardt el al., (ed.),
pp. 247-267, Human Press (1985. Examples of cytotoxic drugs that can be
derivatized into a
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prodrug form for use in this invention include, but are not limited to, those
chemotherapeutic
agents described above.
The terms "cancer" "neoplasia" and "cancerous" refer to or describe any
malignant neoplasm or spontaneous growth or proliferation of cells. The term
as used herein
encompasses both fully developed malignant neoplasms, as well as premalignant
lesions. A
subject having "cancer", for example, may have a tumor.
"Alleviation of cancer" refers to both therapeutic treatment and prophylactic
or
preventative measures, wherein the object is to prevent or slow down (lessen)
the targeted
pathologic condition or disorder. Those in need of alleviation include those
already with the
disorder as well as those prone to have the disorder or those in whom the
disorder is to be
prevented. A subject or mammal is "alleviated" for a Her3 receptor-expressing
cancer if, after
receiving a therapeutic amount of a Her3 receptor agonist according to the
methods of the
present invention, the patient shows observable and/or measurable reduction in
or absence of one
or more of the following: reduction in the number of cancer cells or absence
of the cancer cells,
reduction in the tumor size; inhibition (i.e., slow to some extent and
preferably stop) of cancer
cell infiltration into peripheral organs; inhibition (i.e., slow to some
extent and preferably stop)
of tumor metastasis; inhibition, to some extent, of tumor growth; and/or
relief to some extent,
one or more of the symptoms associated with the specific cancer, and reduced
morbidity and
mortality. To the extent the Her3 receptor antagonist or antibody may prevent
growth and/or kill
existing cancer cells, it may be cytostatic and/or cytotoxic. Reduction of
these signs or
symptoms may also be felt by the patient. Detection and measurement of these
above indicators
are known to those of skill in the art, including, but not limited for
example, reduction in tumor
burden, inhibition of tumor size, reduction in proliferation of secondary
tumors, expression of
genes in tumor tissue, presence of biomarkers, lymph node involvement,
histologic grade, and
nuclear grade.
The term "therapeutically effective amount" refers to an amount of an agonist
and/or antagonist antibody effective to "alleviate" a disease or disorder in a
subject or mammal.
A "therapeutically effective amount", in reference to the treatment of tumor,
refers to an amount
capable of invoking one or more of the following effects: (1) inhibition, to
some extent, of tumor
growth, including, slowing down and complete growth arrest; (2) reduction in
the number of
tumor cells; (3) reduction in tumor size; (4) inhibition (i.e., reduction,
slowing down or complete
stopping) of tumor cell infiltration into peripheral organs; (5) inhibition
(i.e., reduction, slowing
down or complete stopping) of metastasis; (6) enhancement of anti-tumor immune
response,
which may, but does not have to, result in the regression or rejection of the
tumor; and/or (7)
relief, to some extent, of one or more symptoms associated with the disorder.
A "therapeutically
effective amount" of a Her3 antibody for purposes of treatment of tumor may be
determined
empirically and in a routine manner.
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The term "inhibition of tumor volume" refers to any decrease or reduction in a
tumor volume. The term "tumor volume" refers to the total size of the tumor,
which includes the
tumor itself plus affected lymph nodes if applicable. Tumor volume may be
determined by a
variety of methods known in the art, such as, e.g. by measuring the dimensions
of the tumor
using calipers, computed tomography (CT) or magnetic resonance imaging (MR1)
scans, and
calculating the volume using equations based on, for example, the z-axis
diameter, or on
standard shapes such as the sphere, ellipsoid, or cube.
The term "biologically active" (synonymous with "bioactive") indicates that a
composition or compound itself has a biological effect, or that it modifies,
causes, promotes,
enhances, blocks, reduces, limits the production or activity of, or reacts
with or binds to an
endogenous molecule that has a biological effect. A "biological effect" may be
but is not limited
to one that stimulates or causes an immunreactive response; one that impacts a
biological process
in an animal; one that impacts a biological process in a pathogen or parasite;
one that generates
or causes to be generated a detectable signal; and the like. Biologically
active compositions,
complexes or compounds may be used in therapeutic, prophylactic and diagnostic
methods and
compositions. Biologically active compositions, complexes or compounds act to
cause or
stimulate a desired effect upon an animal. Non-limiting examples of desired
effects include, for
example, preventing, treating or curing a disease or condition in an animal
suffering therefrom;
limiting the growth of or killing a pathogen in an animal infected thereby;
augmenting or altering
the phenotype or genotype of an animal; and stimulating a prophylactic
immunoreactive
response in an animal.
In the context of therapeutic applications of the invention, the term
"biologically
active" indicates that the composition, complex or compound has an activity
that impacts an
animal suffering from a disease or disorder in a positive sense and/or impacts
a pathogen or
parasite in a negative sense. Thus, a biologically active composition, complex
or compound may
cause or promote a biological or biochemical activity within an animal that is
detrimental to the
growth and/or maintenance of a pathogen or parasite; or of cells, tissues or
organs of an animal
that have abnormal growth or biochemical characteristics, such as cancer
cells, or cells affected
by autoimmune or inflammatory disorders.
It will be understood by those skilled in the art that a given composition,
complex
or compound may be biologically active in therapeutic, diagnostic and
prophylactic applications.
A composition, complex or compound that is described as being "biologically
active in a cell" is
one that has biological activity in vitro (i.e., in a cell culture) or in vivo
(i.e., in the cells of an
animal). A "biologically active portion" of a compound or complex is a portion
thereof that is
biologically active once it is liberated from the compound or complex. It
should be noted,
however, that such a component may also be biologically active in the context
of the compound
or complex.
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In order to achieve a biological effect, invention constructs may comprise an
additional moiety to facilitate internalization and/or uptake by a target
cell.
A "patient" or "subject" or "host" refers to a mammal such as a human or non-
human animal.
"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. The above parameters for assessing
successful treatment
and improvement in the disease are readily measurable by routine procedures
familiar to a
physician. For cancer therapy, efficacy can be measured, for example, by
assessing the time to
disease progression (TTP) and/or determining the response rate (RR). For
prostate cancer, the
progress of therapy can be assessed by routine methods, usually by measuring
serum PSA
(prostate specific antigen) levels; the higher the level of PSA in the blood,
the more extensive the
cancer. Commercial assays for detecting PSA are available, e.g, Hybritech
Tandem-E and
Tandem-Re PSA assay kits, the Yang ProsChecke polyclonal assay (Yang Labs,
Bellevue,
Wash.), Abbott Imx (Abbott Labs, Abbott Park, Ill.) , etc. Metastasis can be
determined by
staging tests and by bone scan and tests for calcium level and other enzymes
to determine spread
to the bone. CT scans can also be done to look for spread to the pelvis and
lymph nodes in the
area. Chest X-rays and measurement of liver enzyme levels by known methods are
used to look
for metastasis to the lungs and liver, respectively. Other routine methods for
monitoring the
disease include transrectal ultrasonography (TRUS) and transrectal needle
biopsy (TRNB).
The phrase "pharmaceutically acceptable" is employed herein to refer to those
compounds, materials, compositions, and/or dosage forms which are, within the
scope of sound
medical judgment, suitable for use in contact with the tissues of human beings
and animals
without excessive toxicity, irritation, allergic response, or other problem or
complication,
commensurate with a reasonable benefit/risk ratio.
"Her3" encompasses all members of the Her3 receptor family and in particular,
the Her3 protein comprising the aminoa cid and cDNA sequence as set forth in
each of SEQ ID
NO: I and SEQ ID NO:2 in U.S Provisional Application Serial No. 61/250,060,
filed October 9
2009, which is incorporated by reference herein in its entirety. The Her3
ligands include
Jagged!, Jagged2, Deltal, Delta3, and Delta4. "Her3" cDNA and deduced amino
acid sequence
is as set forth in SEQ ID NO: 1. A "full length" Her3 receptor protein or
nucleic acid refers to a
polypeptide or polynucleotide sequence, or a variant thereof, that contains
all of the elements
normally contained in one or more naturally occurring, wild type Her3
polynucleotide or
polypeptide sequences. For example, a full length Her3 nucleic acid will
typically comprise all
of the exons that encode for the full length, naturally occurring protein. The
"full length" may be
prior to, or after, various stages of post-translation processing.
An "antibody that inhibits the growth of cancer cells expressing Her3 receptor
or
a "growth inhibitory" antibody is one which binds to and results in measurable
growth inhibition
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of cancer cells expressing or overexpressing Her3 receptor. Growth inhibition
of tumor cells in
vivo can be determined in various ways. The antibody is growth inhibitory in
vivo if
administration of the anti-Her3 antibody at a therapeutically effective dose
results in reduction in
tumor size or tumor cell proliferation within a measurable period of time from
the first
administration of the antibody. Growth inhibition can be measured at an
antibody concentration
of about 0.1 to 30 pg/ml or about 0.5 nM to 200 riM in cell culture where the
growth inhibition is
determined 1-10 days after exposure of the tumor cells to the antibody. An
antibody that binds
to "Her3" includes an antibody that preferably binds Her3 and prevents
dimerization with Her2.
A "Her3 receptor-expressing cancer" is a cancer comprising cells that have
Her3
receptor protein present on the cell surface. A "Her3 receptor-expressing
cancer" produces
sufficient levels of Her3 receptor on the surface of cells thereof, such that
a Her3 receptor
agonist/antagonist or antibody can bind thereto and have a therapeutic effect
with respect to the
cancer. A cancer which "overexpresses" Her3 receptor is one which has
significantly higher
levels of Her3 receptor at the cell surface thereof, compared to a
noncancerous cell of the same
tissue type. Her3 receptor overexpression may be determined in a diagnostic or
prognostic assay
by evaluating increased levels of the Her3 receptor protein present on the
surface of a cell (e.g.
via an immunohistochemistry assay; PACS analysis). Alternatively, or
additionally, one may
measure levels of Her3 receptor-encoding nucleic acid or mRNA in the cell,
e.g. via fluorescent
in situ hybridization; (FISH; see W098/45479 published October, 1998),
Southern blotting,
Northern blotting, or polymerase chain reaction (PCR) techniques, such as real
time quantitative
PCR (RT-PCR). One may also study Her3 receptor overexpression by measuring
shed antigen in
a biological fluid such as serum, e.g, using antibody-based assays (see also,
e.g., U.S. Pat. No.
4,933,294 issued Jun. 12, 1990; W091/05264 published Apr. 18, 1991: U.S. Pat.
No. 5,401,638
issued Mar. 28, 1995; and Sias et. al. J. Immunol. Methods 132: 73-80 (1990)).
Aside from the
above assays, various in vivo assays are available to the skilled
practitioner. For example, one
may expose cells within the body of the patient to an antibody which is
optionally labeled with a
detectable label, e.g. a radioactive isotope, and binding of the antibody to
cells in the patient can
be evaluated, e.g. by external scanning for radioactivity or by analyzing a
biopsy taken from a
patient previously exposed to the antibody.
An "antagonist" of Her3 receptor in addition to binding Her3 receptor, has a
direct
effect on a Her3 receptor bearing cell. The term "antagonist" is used in the
broadest sense, and
includes any molecule that partially or fully blocks, inhibits, or neutralizes
a biological activity
of a native Her3 receptor protein. The Her3 receptor agonist will bind Her3
receptor and or
her3/her2 complex, and as well, initiate or mediate the signaling event
associated with the Her3
receptor or the Her2/Her3 dimer. The ability to induce Her3 receptor
activation can be
quantified using techniques known in the art such as reporter constructs such
as Beta-
galactosidase, chloramphenicol acetyl transferase (CAT) or luciferase. The
Her3 receptor
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antagonist will inhibit signaling transmitted from the Her3 receptor or the
Her2/Her3 dimer or
prevent Her3 from associating with Her2 or another member of the EGFR family.
Suitable antagonist molecules specifically include antagonist antibodies or
antibody fragments, fragments or amino acid sequence variants thereof etc.
Methods for
identifying agonists or antagonists of a Her3 receptor polypeptide are known
in the art. An
exemplary method proposes contacting a Her3 bearing cells or tissue with a
candidate antagonist
molecule and measuring a detectable change in one or more biological
activities normally
associated with the Her3 receptor.
"Mammal" for purposes of treatment refers to any animal classified as a
mammal,
including humans, domestic and farm animals, and zoo, sports, or pet animals,
such as dogs,
cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal
is human.
The term "administering" includes any method of delivery of a compound of the
present invention, including but not limited to, a pharmaceutical composition
or therapeutic
agent, into a subject's system or to a particular region in or on a subject.
The phrases "systemic
administration," "administered systemically," "peripheral administration" and
"administered
peripherally" as used herein mean the administration of a compound, drug or
other material other
than directly into the central nervous system, such that it enters the
patient's system and, thus, is
subject to metabolism and other like processes, for example, subcutaneous
administration.
"Farenteral administration" and "administered parenterally" means modes of
administration other
than enteral and topical administration, usually by injection, and includes,
without limitation,
intravenous, intramuscular, intraarterial, intrathecal, intracapsular,
intraorbital, intracardiac,
intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-
articular,
subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.
Compositions and methods of the invention include an anti-Her3 "in association
with" or
"in combination with" one or more further therapeutic agents or therapeutic
procedures (e.g.,
surgical tumorectomy or anti-cancer radiation therapy). The terms "in
association with" or "in
combination with" indicate that the components (e.g., anti-Her3 antibody along
with paclitaxel)
can be formulated into a single composition for simultaneous delivery or
formulated separately
into two or more compositions (e.g., a kit). Furthermore, each component can
be administered
to a subject at a different time than when the other component is
administered; for example, each
administration may be given non-simultaneously (e.g., separately or
sequentially) at several
intervals over a given period of time. Moreover, the separate components may
be administered
to a subject by the same or by a different route (e.g., wherein an anti-Her3
antibody formulation
is administered parenterally and gefitinib is administered orally).
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A "disorder" is any condition that would benefit from treatment with the
polypeptide. This includes chronic and acute disorders or diseases including
those pathological
conditions which predispose the mammal to the disorder in question.
"Chronic" administration refers to administration of the agent(s) in a
continuous
mode as opposed to an acute mode, so as to maintain the initial therapeutic
effect (activity) for
an extended period of time. "Intermittent" administration is treatment that is
not consecutively
done without interruption, but rather is cyclic in nature.
"Biological sample" as used herein is a sample of biological tissue or cells
that
contains nucleic acids or polypeptides, e.g., Her3 or Her3 protein,
polynucleotide or transcript.
Such samples include, but are not limited to, tissue isolated from primates
(e.g., humans) or from
rodents (e.g., mice, and rats). Biological samples may also include sections
of tissues such as
biopsy and autopsy samples, frozen sections taken for histologic purposes,
skin, etc. Biological
samples also include explants and primary and/or transformed cell cultures
derived from patient
tissues. A biological sample is typically obtained from a eukaryotic organism,
most preferably a
mammal such as a primate e.g., human.
"Providing a biological sample" means to obtain a biological sample for use in
methods described in this invention. Most often, this will be done by removing
a sample of cells
from a human, but can also be accomplished by using previously isolated cells
(e.g., isolated by
another person, at another time, and/or for another purpose), or by performing
the methods of the
invention in vivo.
"Tumor", as used herein, refers to all neoplastic cell growth and
proliferation,
whether malignant or benign, and all pre-cancerous and cancerous cells and
tissues.
A "label" or a "detectable moiety" is a composition detectable by
spectroscopic,
photochemical, biochemical, immunochemical, chemical, or other physical means.
The word
"label" when used herein refers to a detectable compound or composition which
is conjugated
directly or indirectly to the antibody so as to generate a "labeled" 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. For example, useful labels include fluorescent dyes, electron-
dense reagents,
enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, colloidal
gold, luminescent
nanocrystals (e.g. quantum dots), haptens and proteins or other entities which
can be made
detectable, e.g., by incorporating a radiolabel into the peptide or used to
detect antibodies
specifically reactive with the peptide. The radioisotope may be, for example,
311, 14C, 32-,
e 35s, or
125j. In some cases, particularly using antibodies against the proteins of the
invention, the
radioisotopes are used as toxic moieties, as described below. Any method known
in the art for
conjugating the antibody to the label may be employed. The lifetime of
radiolabeled peptides or
radiolabeled antibody compositions may be extended by the addition of
substances that stabilize
the radiolabeled peptide or antibody and protect it from degradation. Any
substance or
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combination of substances that stabilize the radiolabeled antibody may be used
including those
substances disclosed in U.S. Pat. No. 5,961,955.
During last few years, it has been show that the targeting of growth factor
receptors, like EGER or Herneu over-expressed on the tumoral cell surface,
with respectively
humanized (HerceptinTM) or chimeric (C225) antibodies results in an
significant inhibition of the
tumoral growth on patients and in a significant increase of the efficacy of
classical chemotherapy
treatments (Carter P., Nature Rev. Cancer, 2001, 1(2):118; Hortobagyi G. N.,
Semin. Oncol.,
2001, 28:43; Herbst R. S. et al., Semin. Oncol., 2002, 29:27). Other receptors
like IGF-IR or
VEGF-R (for vascular endothelial growth factor receptor) have been identified
as potential target
in several preclinical studies.
A "growth inhibitory agent" when used herein refers to a compound or
composition which inhibits growth of a cell, especially a Her3 expressing
cancer cell, either in
vitro or in vivo. Thus, the growth inhibitory agent may be one which
significantly reduces the
percentage of PSCA expressing cells in S phase. Examples of growth inhibitory
agents include
agents that block cell cycle progression (at a place other than S phase), such
as agents that induce
GI arrest and M-phase arrest. Classical M-phase blockers include the vincas
(vincristine and
vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin,
epirubicin,
datinorubicin, etoposide, and bleomycin. Those agents that arrest 01 also
spill over into S-phase
arrest, for example, DNA alkylating agents such as tamoxifen, prednisone,
dacarbazine,
mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further
information can be
found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter
1, entitled "Cell
cycle regulation, oncogenes, and antineoplastic drugs" by Murakami et al. (WB
Saunders:
Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel and docetaxel)
are anticancer drugs
both derived from the yew tree. Docetaxel (TAXOTERE®, Rhone-Poulenc
Rorer), derived
from the European yew, is a semisynthetic analogue of paclitaxel (TAXOL®,
Bristol-Myers
Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from
tubulin dimers and
stabilize microtubules by preventing depolymerization, which results in the
inhibition of mitosis
in cells.
"Carriers" as used herein include pharmaceutically acceptable carriers,
excipients,
or stabilizers which are nontoxic to the cell or mammal being exposed thereto
at the dosages and
concentrations employed. Often the physiologically acceptable carrier is an
aqueous pH buffered
solution. Examples of physiologically acceptable carriers include buffers such
as phosphate,
citrate, and other organic acids; antioxidants including ascorbic acid; low
molecular weight (less
than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin,
or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino
acids such as
glycine, glutamine, asparagine, arginine or lysine; monosaccharides,
disaccharides, and other
carbohydrates including glucose, mannose, or dextrins; chelating agents such
as EDTA; sugar
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alcohols such as mannitol or sorbitol; salt-forming counterions such as
sodium; and/or nonionic
surfactants such as TWEEN.TM., polyethylene glycol (PEG), and PLURONICS.TM..
Preparation of Chimeric, Humanized and Human Anti-Her3 Antibodies
The monoclonal antibodies herein include chimeric, hybrid and recombinant
antibodies produced by splicing a variable (including hypervariable) domain of
the antibody of
interest with a constant domain (e.g. "humanized" antibodies), or a light
chain with a heavy
chain, or a chain from one species with a chain from another species, or
fusions with
heterologous proteins, regardless of species of origin or immunoglobulin class
or subclass
designation, as well as antibody fragments (e.g., Fab, F(abl)2, and Fv), so
long as they exhibit the
desired biological activity or properties. See, e.g. U.S. Pat. No. 4,816,567
and Mage et al., in
Monoclonal Antibody Production Techniques and Applications, pp. 79-97 (Marcel
Dekker, Inc.:
New York, 1987). Thus, for the purposes of obtaining a chimeric Her3 antibody,
the CDR's
from the murine antibodies disclosed herein can be grafted onto a human "Y"
framework. The
resulting "chimeric" Her3 antibodies, in turn, can be humanized by techniques
known to one
skilled in the art. The affinity of a chimeric, humanized or human anti-Her3
antibody may be
evaluated using a direct binding assay or a competitive binding assay, as
exemplified below.
Antibody Structure
Naturally occurring (wildtype) antibody molecules are Y-shaped molecules
consisting of four polypeptide chains, two identical heavy chains and two
identical light chains,
which are covalently linked together by disulfide bonds. Both types of
polypeptide chains have
constant regions, which do not vary or vary minimally among antibodies of the
same class (i.e.,
IgA, IgM, etc.), and variable regions. The variable regions are unique to a
particular antibody
and comprise a recognition element for an epitope. The carboxy-terminal
regions of both heavy
and light chains are conserved in sequence and are called the constant regions
(also known as C-
domains). The amino-terminal regions (also known as V-domains) are variable in
sequence and
are responsible for antibody specificity. The antibody specifically recognizes
and binds to an
antigen mainly through six short complementarity-determining regions (CDRs)
located in their
V-domains.
Each light chain of an antibody is associated with one heavy chain, and the
two
chains are linked by a disulfide bridge formed between cysteine residues in
the carboxy-terminal
region of each chain, which is distal from the amino terminal region of each
chain that
constitutes its portion of the antigen binding domain. Antibody molecules are
further stabilized
by disulfide bridges between the two heavy chains in an area known as the
hinge region, at
locations nearer the carboxy terminus of the heavy chains than the locations
where the disulfide
bridges between the heavy and light chains are made. The hinge region also
provides flexibility
for the antigen-binding portions of an antibody.
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An antibody's specificity is determined by the variable regions located in the
amino terminal regions of the light and heavy chains. The variable regions of
a light chain and
associated heavy chain form an "antigen binding domain" that recognizes a
specific epitope; an
antibody thus has two antigen binding domains. The antigen binding domains in
a wild type
antibody are directed to the same epitope of an immunogenic protein, and a
single wild type
antibody is thus capable of binding two molecules of the immunogenic protein
at the same time.
Thus, a wild type antibody is monospecific (i.e., directed to a unique
antigen) and divalent (i.e.,
capable of binding two molecules of antigen).
Types of Antibodies
"Polyclonal antibodies" are generated in an immunogenic response to a protein
having many epitopes. A composition (e.g., serum) of polyclonal antibodies
thus includes a
variety of different antibodies directed to the same and to different epitopes
within the protein.
Methods for producing polyclonal antibodies are known in the art (see, e.g.,
Cooper et al.,
Section III of Chapter 11 in: Short Protocols in Molecular Biology, 2nd Ed.,
Ausubel et al., eds.,
John Wiley and Sons, New York, 1992, pages 11-37 to 11-41).
"Antipeptide antibodies" (also known as "monospecific antibodies") are
generated
in a humoral response to a short (typically, 5 to 20 amino acids) immunogenic
polypeptide that
corresponds to a few (preferably one) isolated epitopes of the protein from
which it is derived. A
plurality of antipeptide antibodies includes a variety of different antibodies
directed to a specific
portion of the protein, i.e, to an amino acid sequence that contains at least
one, preferably only
one, epitope. Methods for producing antipeptide antibodies are known in the
art (see, e.g.,
Cooper et al., Section III of Chapter 11 in: Short Protocols in Molecular
Biology, 2nd Ed.,
Ausubel et al., eds., John Wiley and Sons, New York, 1992, pages 11-42 to 11-
46).
A "Monoclonal antibody" is a specific antibody that recognizes a single
specific
epitope of an immunogenic protein. In a plurality of a monoclonal antibody,
each antibody
molecule is identical to the others in the plurality. In order to isolate a
monoclonal antibody, a
clonal cell line that expresses, displays and/or secretes a particular
monoclonal antibody is first
identified; this clonal cell line can be used in one method of producing the
antibodies of the
invention. Methods for the preparation of clonal cell lines and of monoclonal
antibodies
expressed thereby are known in the art (see, for example, Fuller et al.,
Section U of Chapter 11
in: Short Protocols in Molecular Biology, 2nd Ed., Ausubel et al., eds., John
Wiley and Sons,
New York, 1992, pages 11-22 to 11-11-36).
A "Naked antibody" is an antibody that lacks the Fe portion of a wildtype
antibody molecule.
However, it is possible that the Fe portion is not required for therapeutic
function
in every instance, as other mechanisms, such as apoptosis, can come into play.
Moreover, the Fe
region may be deleterious in some applications as antibodies comprising an Fe
region are taken
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up by Fe receptor-bearing cells, thereby reducing the amount of therapeutic
antibody taken up by
targeted cells. Vaswani and Hamilton, Humanized antibodies as potential
therapeutic drugs,
Ann. Allergy Asthma Immunol. 81:105-119, 1998. Components of the immune system
may
recognize and react to antibodies that are clumped together on the surface of
tumor cells. It is
thus envisioned that the resulting immune response will target and destroy, or
at least limit the
proliferation of, the tumor cells.
One way to get naked antibodies delivered to surfaces where they will chimp
together is to use a targetable construct or complex to bring different naked
antibodies together
on a targeted cellular surface. By way of non-limiting example, an anti-C20
antibody (e.g.,
Rituxan) and an anti-C22 antibody might be administered separately or
together, allowed to clear
so that unbound antibodies are removed from the system.
Naked antibodies are also of interest for therapy of diseases caused by
parasites,
such as malaria. Vukovic et al., Immunoglobulin G3 antibodies specific for the
19-kilodalton
carboxyl-terminal fragment of Plasmodium yoelii merozoite surface protein 1
transfer protection
to mice deficient in Fe-RI receptors, Infect. Immun. 68:3019-22, 2000.
"Single chain antibodies (scFv)" generally do not include portions of the Fe
region of antibodies that are involved in effector functions and are thus
naked antibodies,
although methods are known for adding such regions to known scFv molecules if
desired. See
Helfrich et al., A rapid and versatile method for harnessing scFv antibody
fragments with various
biological functions, J. Immunol. Meth. 237:131-145,2000; and de Haard et al.,
Creating and
engineering human antibodies for immunotherapy, Adv. Drug Delivery Rev. 31:5-
31, 1998.
Antibody Fragments
Proteolytic Antibody Fragments
Antibody fragments produced by limited proteolysis of wild type antibodies are
called proteolytic antibody fragments. These include, but are not limited to,
the following.
"F(abt)2 fragments" are released from an antibody by limited exposure of the
antibody to a proteolytic enzyme, e.g., pepsin or ficin. An F(ab)2 fragment
comprises two
"arms," each of which comprises a variable region that is directed to and
specifically binds a
common antigen. The two Fab' molecules are joined by interchain disulfide
bonds in the hinge
regions of the heavy chains; the Fab' molecules may be directed toward the
same (bivalent) or
different (bispecific) epitopes.
"Fab' fragments" contain a single anti-binding domain comprising a Fab and an
additional portion of the heavy chain through the hinge region.
"Fabl-SH fragments" are typically produced from F(a1302 fragments, which are
held together by disulfide bond(s) between the H chains in an F(a13')2
fragment. Treatment with a
mild reducing agent such as, by way of non-limiting example, beta-
mercaptoethylamine, breaks
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the disulfide bond(s), and two Fab' fragments are released from one F(ab)2
fragment. Fab'-SH
fragments are monovalent and monospecific.
"Fab fragments" (i.e., an antibody fragment that contains the antigen-binding
domain and comprises a light chain and part of a heavy chain bridged by a
disulfide bond) are
produced by papain digestion of intact antibodies. A convenient method is to
use papain
immobilized on a resin so that the enzyme can be easily removed and the
digestion terminated.
Fab fragments do not have the disulfide bond(s) between the H chains present
in an F(ab')2
fragment.
Recombinant Antibody Fragments
"Single-chain antibodies" are one type of antibody fragment. The term single
chain antibody is often abbreviated as "scFv" or "sFv." These antibody
fragments are produced
using molecular genetics and recombinant DNA technology. A single-chain
antibody consists of
a polypeptide chain that comprises both a VH and a VL portion. Unlike wildtype
antibodies,
wherein two separate heavy and light polypeptide chains are conjoined to form
a single antigen-
binding variable region, a single-chain antibody is a single polypeptide that
comprises an
antigen-binding variable region. That is, a single-chain antibody comprises
the variable, antigen-
binding determinative region of a single light and heavy chain of an antibody
linked together by
a chain of 10-25 amino acids.
The term "single-chain antibody" includes but is not limited to a disulfide-
linked
Fv (dsFv) in which two single-chain antibodies linked together by a disulfide
bond; a bispecific
sFv (a sFv or a dsPv molecule having two antigen-binding domains, each of
which may be
directed to a different epitope); a diabody (a dimerized sFv formed when the
VH domain of a first
sPv assembles with the VL domain of a second sFv and the VL domain of the
first sFv assembles
with the VH domain of the second sFv; the two antigen-binding regions of the
diabody may be
directed towards the same or different epitopes); and a triabody (a trimerized
sFv, formed in a
manner similar to a diabody, but in which three antigen-binding domains are
created in a single
complex; the three antigen binding domains may be directed towards the same or
different
epitopes).
"Fully human antibodies" are human antibodies that can be produced in
transgenic animals such as Xenomice. XenoMouse strains are genetically
engineered mice in
which the murine IgH and Igk loci have been functionally replaced by their
human 1g
counterparts on yeast artificial YAC transgenes. These human Ig transgenes can
carry the
majority of the human variable repertoire and can undergo class switching from
IgM to IgG
isotypes. The immune system of the xenomouse recognizes administered human
antigens as
foreign and produces a strong humoral response. The use of XenoMouse in
conjunction with
well-established hybridomas techniques, results in fully human IgG mAbs with
sub-nanomolar
affinities for human antigens (see U.S. Pat. No. 5,770,429, entitled
"Transgenic non-human
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animals capable of producing heterologous antibodies", U.S. Pat. No.
6,162,963, entitled
"Generation of xenogenetic antibodies"; U.S. Pat. No. 6,150,584, entitled
"Human antibodies
derived from immunized xenomice", U.S. Pat. No. 6,114,598, entitled
"Generation of
xenogeneic antibodies"; and U.S. Pat. No. 6,075,181, entitled "Human
antibodies derived from
immunized xenomice"; for reviews, see Green, Antibody engineering via genetic
engineering of
the mouse: XenoMouse strains are a vehicle for the facile generation of
therapeutic human
monoclonal antibodies, J. Immunol. Meth. 231:11-23, 1999; Wells, Eek, a
XenoMouse:
Abgenix, Inc., Chem. Biol. 7:R185-6, 2000; and Davis et al., Transgenic mice
as a source of filly
human antibodies for the treatment of cancer, Cancer Metastasis Rev. 18:421-5,
1999).
"Complementary determining region peptides" or "CDR peptides" are another
form of an antibody fragment. A CDR peptide (also known as "minimal
recognition unit") is a
peptide corresponding to a single complementarity-determining region (CDR),
and can be
prepared by constructing genes encoding the CDR of an antibody of interest.
Such genes are
prepared, for example, by using the polymerase chain reaction to synthesize
the variable region
from RNA of antibody-producing cells. See, for example, Larrick etal.,
Methods: A Companion
to Methods in Enzymology 2:106, 1991.
Compositions of the Invention and Methods of Making Same
This invention encompasses a plurality of substantially pure, isolated anti-
Her3
isolated antibodies and polynucleotide embodiments. Explicitly included are
compositions,
including pharmaceutical compositions, comprising an anti-Her3 antibody; and
polynucleotides
comprising sequences encoding an anti-Her3 antibody. As used herein,
compositions comprise
one or more antibodies that bind to Her3, and/or one or more polynucleotides
comprising
sequences encoding one or more antibodies that bind to Her3. These
compositions may further
comprise suitable carriers, such as pharmaceutically acceptable excipients
including buffers,
which are well known in the art.
The anti-Her3 antibodies of the invention are preferably monoclonal. Also
encompassed within the scope of the invention are Fab, Fab', Fab'-SH and
F(abt)2 fragments of
the anti-Her3 antibodies provided herein. Single chain anti-Her3 antibodies as
well as
multispecific and multivariant Her3 specific antibodies are also included.
These antibody
fragments can be created by traditional means, such as enzymatic digestion, or
may be generated
by recombinant techniques. These fragments are useful for the diagnostic and
therapeutic
purposes set forth below.
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.
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The anti-Her3 monoclonal antibodies of the invention are preferably made by
recombinant DNA methods (U.S. Pat. No. 4,816,567).
The binding specificity of monoclonal antibodies produced by recombinant means
is determined by immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay
(RIA) or enzyme-linked immunoadsorbent assay (ELISA).
The binding affinity of the monoclonal antibody can, for example, be
determined
by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).
The anti-Her3 antibodies of the invention can be made by using combinatorial
libraries to screen for synthetic antibody clones with the desired activity or
activities. In
principle, synthetic antibody clones are selected by screening phage libraries
containing phage
that display various fragments of antibody variable region (Fv) fused to phage
coat protein.
Such phage libraries are panned by affinity chromatography against the desired
antigen. Clones
expressing Fv fragments capable of binding to the desired antigen are adsorbed
to the antigen
and thus separated from the non-binding clones in the library. The binding
clones are then eluted
from the antigen, and can be further enriched by additional cycles of antigen
adsorption/elution.
Any of the anti-Her3 antibodies of the invention can be obtained by designing
a suitable antigen
screening procedure to select for the phage clone of interest followed by
construction of a full
length anti-Her3 antibody clone using the Fv sequences from the phage clone of
interest and
suitable constant region (Fc) sequences described in Kabat etal., Sequences of
Proteins of
Immunological Interest, Fifth Edition, NTH Publication 91-3242, Bethesda Md.
(1991), vols. 1-3.
The antigen-binding domain of an antibody is formed from two variable (V)
regions of about 110 amino acids, one each from the light (VI) and heavy (VH)
chains, that both
present three hypervariable loops or complementarity-determining regions
(CDRs). Variable
domains can be displayed functionally on phage, either as single-chain Fv
(scFv) fragments, in
which VH and V. are covalently linked through a short, flexible peptide, or as
Fab fragments, in
which they are each fused to a constant domain and interact non-covalently, as
described in
Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). As used herein, scFv
encoding phage
clones and Fab encoding phage clones are collectively referred to as "Fv phage
clones" or "Fv
clones".
Repertoires of VH and VL genes can be separately cloned by polym erase chain
reaction (PCR) and recombined randomly in phage libraries, which can then be
searched for
antigen-binding clones as described in Winter et al., Ann. Rev. Imrnunol., 12:
433-455 (1994).
Libraries from immunized sources provide high-affinity antibodies to the
immunogen without
the requirement of constructing hybridomas. Alternatively, the naive
repertoire can be cloned to
provide a single source of human antibodies to a wide range of non-self and
also self antigens
without any immunization as described by Griffiths et al., EMBO J. 12: 725-734
(1993). Finally,
naive libraries can also be made synthetically by cloning the unrearranged V-
gene segments
from stem cells, and using PCR primers containing random sequence to encode
the highly
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variable CDR3 regions and to accomplish rearrangement in vitro as described by
Hoogenboom
and Winter, J. Mal, Biol., 227: 381-388 (1992).
Filamentous phage is used to display antibody fragments by fusion to the minor
coat protein plII. The antibody fragments can be displayed as single chain Fv
fragments, in
which VH and VL domains are connected on the same polypeptide chain by a
flexible
polypeptide spacer, e.g. as described by Marks et al., J. Mol. Biol., 222: 581-
597 (1991), or as
Fab fragments, as described in Hoogenboom et al., Nucl. Acids Res., 19: 4133-
4137 (1991).
In general, nucleic acids encoding antibody gene fragments are obtained from
immune cells harvested from humans. If a library biased in favor of anti-Her3
clones is desired,
the subject is immunized with Her3 to generate an antibody response, and
spleen cells and/or
circulating B cells other peripheral blood lymphocytes (Pins) are recovered
for library
construction. In another embodiment, a human antibody gene fragment library
biased in favor of
anti-Her3 clones is obtained by generating an anti-Her3 antibody response in
transgenic mice
carrying a functional human immunoglobulin gene array (and lacking a
functional endogenous
antibody production system) such that Her3 immunization gives rise to B cells
producing human
antibodies against Her3. The generation of human antibody-producing transgenic
mice is
described below.
Additional enrichment for anti-Her3 reactive cell populations can be obtained
by
using a suitable screening procedure to isolate 13 cells expressing Her3-
specific membrane bound
antibody, e.g., by cell separation with Her3 affinity chromatography or
adsorption of cells to
fluorochrome-labeled Her3 followed by flow-activated cell sorting (FACS).
Alternatively, the use of spleen cells and/or 13 cells or other PBLs from an
unimmunized donor provides a better representation of the possible antibody
repertoire, and also
permits the construction of an antibody library using any animal (human or non-
human) species
in which Her3 is not antigenic. For libraries incorporating in vitro antibody
gene construction,
stem cells are harvested from the subject to provide nucleic acids encoding
unrearranged
antibody gene segments. The immune cells of interest can be obtained from a
variety of animal
species, such as human, mouse, rat, lagomorpha, lupine, canine, feline,
porcine, bovine, equine,
and avian species, etc.
Nucleic acid encoding antibody variable gene segments (including V and V1
segments) are recovered from the cells of interest and amplified. In the case
of rearranged VH
and VL gene libraries, the desired DNA can be obtained by isolating genomic
DNA or mRNA
from lymphocytes followed by polymerase chain reaction (PCR) with primers
matching the 5'
and 3' ends of rearranged VH and VL genes as described in Orlandi etal., Proc.
Natl. Acad. Sci.
(USA), 86: 3833-3837 (1989), thereby making diverse V gene repertoires for
expression. The V
genes can be amplified from cDNA and genomic DNA, with back primers at the 5'
end of the
exon encoding the mature V-domain and forward primers based within the J-
segment as
described in Orlandi et al. (1989) and in Ward et al., Nature, 341: 544-546
(1989). However, for
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amplifying from cDNA, back primers can also be based in the leader exon as
described in Jones
et al., Biotechnol., 9: 88-89 (1991), and forward primers within the constant
region as described
in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86: 5728-5732 (1989). To
maximize
complementarity, degeneracy can be incorporated in the primers as described in
Orlandi et at.
(1989) or Sastry et al. (1989). Preferably, the library diversity is maximized
by using PCR
primers targeted to each V-gene family in order to amplify all available VH
and VL arrangements
present in the immune cell nucleic acid sample, e.g. as described in the
method of Marks et al., J.
Mol. Biol., 222: 581-597 (1991) or as described in the method of Orum et al.,
Nucleic Acids
Res., 21: 4491-4498 (1993). For cloning of the amplified DNA into expression
vectors, rare
restriction sites can be introduced within the PCR primer as a tag at one end
as described in
Orlandi et al. (1989), or by further PCR amplification with a tagged primer as
described in
Clackson et al., Nature, 352: 624-628 (1991).
Repertoires of synthetically rearranged V genes can be derived in vitro from V
gene segments. Most of the human VH-gene segments have been cloned and
sequenced
(reported in Tomlinson et al., J. Mol, Biol., 227: 776-798 (1992)), and mapped
(reported in
Matsuda etal., Nature Genet., 3: 88-94 (1993); these cloned segments
(including all the major
conformations of the H1 and H2 loop) can be used to generate diverse VH gene
repertoires with
PCR primers encoding H3 loops of diverse sequence and length as described in
Hoogenboom
and Winter, J. Mol. Biol., 227: 381-388 (1992). VH repertoires can also be
made with all the
sequence diversity focused in a long H3 loop of a single length as described
in Barbas et al.,
Proc. Natl. Acad. Sei. USA, 89: 4457-4461 (1992). Human V.kappa. and V.lamda.
segments
have been cloned and sequenced (reported in Williams and Winter, Eur, I
Immunol., 23: 1456-
1461 (1993)) and can be used to make synthetic light chain repertoires.
Synthetic V gene
repertoires, based on a range of VH and VL folds, and L3 and H3 lengths, will
encode antibodies
of considerable structural diversity. Following amplification of V-gene
encoding DNAs,
germline V-gene segments can be rearranged in vitro according to the methods
of Hoogenboom
and Winter, J. Mo1.131o1., 227: 381-388 (1992).
Repertoires of antibody fragments can be constructed by combining VII and VL
gene repertoires together in several ways. Each repertoire can be created in
different vectors, and
the vectors recombined in vitro, e.g., as described in Hogrefe etal., Gene,
128: 119-126 (1993),
or in vivo by combinatorial infection, e.g., the loxP system described in
Waterhouse et al., Nucl.
Acids Res., 21: 2265-2266 (1993). The in vivo recombination approach exploits
the two-chain
nature of Fab fragments to overcome the limit on library size imposed by E.
coil transformation
efficiency. Naive VH and VL repertoires are cloned separately, one into a
phagemid and the other
into a phage vector. The two libraries are then combined by phage infection of
phagemid-
containing bacteria so that each cell contains a different combination and the
library size is
limited only by the number of cells present (about 1012 clones). Both vectors
contain in vivo
recombination signals so that the VH and VL genes are recombined onto a single
replicon and are
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co-packaged into phage virions. These huge libraries provide large numbers of
diverse
antibodies of good affinity (K,1-1 of about 10-8 M).
Alternatively, the repertoires may be cloned sequentially into the same
vector, e.g.
as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 88: 7978-7982
(1991), or assembled
together by PCR and then cloned, e.g. as described in Clackson et al., Nature,
352: 624-628
(1991). PCR assembly can also be used to join VH and VL DNAs with DNA encoding
a flexible
peptide spacer to form single chain Fv (say) repertoires. In yet another
technique, "in cell PCR
assembly" is used to combine VH and VL genes within lymphocytes by PCR and
then clone
repertoires of linked genes as described in Embleton et al., Nucl. Acids Res.,
20: 3831-3837
(1992).
The antibodies produced by naive libraries (either natural or synthetic) can
be of
moderate affinity (IQ-I of about 106 to 107 M-1), but affinity maturation can
also be mimicked in
vitro by constructing and reselecting from secondary libraries as described in
Winter et al.
(1994), supra, For example, mutation can be introduced at random in vitro by
using error-prone
polymerase (reported in Leung et al., Technique, 1: 11-15 (1989)) in the
method of Hawkins et
al., J. Mol, Biol., 226: 889-896 (1992) or in the method of Gram et al., Proc.
Natl. Acad. Sci.
USA, 89: 3576-3580 (1992). Additionally, affinity maturation can be performed
by randomly
mutating one or more CDRs, e.g. using PCR with primers carrying random
sequence spanning
the CDR of interest, in selected individual Fv clones and screening for higher
affinity clones.
WO 9607754 (published 14 Mar. 1996) described a method for inducing
mutagenesis in a
complementarily determining region of an immunoglobulin light chain to create
a library of light
chain genes. Another effective approach is to recombine the VH or VL domains
selected by phage
display with repertoires of naturally occurring V domain variants obtained
from unimmunized
donors and screen for higher affinity in several rounds of chain reshuffling
as described in Marks
et al., Biotechnol., 10: 779-783 (1992). This technique allows the production
of antibodies and
antibody fragments with affinities in the 10-9 M range.
Her3 nucleic acid and amino acid sequences are known in the art. A
representative nucleic acid and amino acid sequence of Her3 is detailed in SEQ
ID NOS. 1 and 2
respectively. Nucleic acid sequence encoding the Her3 can be designed using
the amino acid
sequence of the desired region of Her3. Alternatively, the cDNA sequence (or
fragments
thereof) of GenBank Accession Nos. NM-019074. Her3 is a transmembrane protein.
The
extracellular region contains 36 EGF-like repeats, as well as a DSL domain
that is conserved
among all Her3 ligands and is necessary for receptor binding. The predicted
protein also
contains a transmembrane region, and a cytoplasmic tail lacking any catalytic
motifs. Human
Her3 protein is a 685 amino acid protein. The accession number of human Her3
is NM-019074.
See Sarah J. Bray, "Her3 signaling: a simple pathway becomes complex" Nature
Reviews
Molecular Cell Biology, 7: 678-689 (2006), the entire content of which is
incorporated by
reference herein.
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DNAs encoding Her3 can be prepared by a variety of methods known in the art.
These methods include, but are not limited to, chemical synthesis by any of
the methods
described in Engels et al., Agnew. Chem, Int Ed. Engl., 28: 716-734 (1989),
such as the triester,
phosphite, phosphoramidite and H-phosphonate methods. In one embodiment,
codons preferred
by the expression host cell are used in the design of the Her3 encoding DNA.
Alternatively,
DNA encoding the Her3 can be isolated from a genomic or cDNA library.
Following construction of the DNA molecule encoding the Her3, the DNA
molecule is operably linked to an expression control sequence in an expression
vector, such as a
plasmid, wherein the control sequence is recognized by a host cell transformed
with the vector.
In general, plasmid vectors contain replication and control sequences which
are derived from
species compatible with the host cell. The vector ordinarily carries a
replication site, as well as
sequences which encode proteins that are capable of providing phenotypic
selection in
transformed cells. Suitable vectors for expression in prokaryotic and
eukaryotic host cells are
known in the art and some are further described herein. Eukaryotic organisms,
such as yeasts, or
cells derived from multicellular organisms, such as mammals, may be used.
Optionally, the DNA encoding the Her3 is operably linked to a secretory leader
sequence resulting in secretion of the expression product by the host cell
into the culture
medium. Examples of secretory leader sequences include still, ecotin, lamB,
herpes GD, lpp,
alkaline phosphatase, invertase, and alpha factor. Also suitable for use
herein is the 36 amino
acid leader sequence of protein A (Abrahmsen et al., EMBO J., 4: 3901 (1985)).
Host cells are transfected and preferably transformed with the above-described
expression or cloning vectors of this invention and cultured in conventional
nutrient media
modified as appropriate for inducing promoters, selecting transformants, or
amplifying the genes
encoding the desired sequences.
Transfection refers to the taking up of an expression vector by a host cell
whether
or not any coding sequences are in fact expressed. Numerous methods of
transfection are known
to the ordinarily skilled artisan, for example, CaPO4 precipitation and
electroporation.
Successful transfection is generally recognized when any indication of the
operation of this
vector occurs within the host cell. Methods for transfection are well known in
the art, and some
are further described herein.
Transformation means introducing DNA into an organism so that the DNA is
replicable, either as an extrachromosomal element or by chromosomal integrant.
Depending on
the host cell used, transformation is done using standard techniques
appropriate to such cells.
Methods for transformation are well known in the art, and some are further
described herein.
Prokaryotic host cells used to produce the Her3 can be cultured as described
generally in Sambrook et al., supra.
The mammalian host cells used to produce the Her3 can be cultured in a variety
of media, which is well known in the art and some of which is described
herein.
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The host cells referred to in this disclosure encompass cells in in vitro
culture as
well as cells that are within a host animal.
Purification of Her3 may be accomplished using art-recognized methods, some of
which are described herein.
The purified Her3 can be attached to a suitable matrix such as agarose beads,
acrylamide beads, glass beads, cellulose, various acrylic copolymers, hydroxyl
methacrylate
gels, polyacrylic and polymethacrylic copolymers, nylon, neutral and ionic
carriers, and the like,
for use in the affinity chromatographic separation of phage display clones.
Attachment of the
Her3 protein to the matrix can be accomplished by the methods described in
Methods in
Enzymology, vol. 44 (1976). A commonly employed technique for attaching
protein ligands to
polysaccharide matrices, e.g. agarose, dextran or cellulose, involves
activation of the carrier with
cyanogen halides and subsequent coupling of the peptide ligand's primary
aliphatic or aromatic
amines to the activated matrix.
Alternatively, Her3 can be used to coat the wells of adsorption plates,
expressed
on host cells affixed to adsorption plates or used in cell sorting, or
conjugated to biotin for
capture with streptavid in-coated beads, or used in any other art-known method
for panning
phage display libraries.
The phage library samples are contacted with immobilized Her3 under conditions
suitable for binding of at least a portion of the phage particles with the
adsorbent. Normally, the
conditions, including pH, ionic strength, temperature and the like are
selected to mimic
physiological conditions. The phages bound to the solid phase are washed and
then eluted by
acid, e.g. as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 88: 7978-
7982 (1991), or by
alkali, e.g. as described in Marks et al., J. Mol. Biol., 222: 581-597 (1991),
or by Her3 antigen
competition, e.g. in a procedure similar to the antigen competition method of
Clackson et al.,
Nature, 352: 624-628 (1991). Phages can be enriched 20-1,000-fold in a single
round of
selection. Moreover, the enriched phages can be grown in bacterial culture and
subjected to
further rounds of selection.
The efficiency of selection depends on many factors, including the kinetics of
dissociation during washing, and whether multiple antibody fragments on a
single phage can
simultaneously engage with antigen. Antibodies with fast dissociation kinetics
(and weak
binding affinities) can be retained by use of short washes, multivalent phage
display and high
coating density of antigen in solid phase. The high density not only
stabilizes the phage through
multivalent interactions, but favors rebinding of phage that has dissociated.
The selection of
antibodies with slow dissociation kinetics (and good binding affinities) can
be promoted by use
of long washes and monovalent phage display as described in Bass et al.,
Proteins, 8: 309-314
(1990) and in WO 92/09690, and a low coating density of antigen as described
in Marks et al.,
Biotechnol., 10: 779-783 (1992).
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It is possible to select between phage antibodies of different affinities,
even with
affinities that differ slightly, for Her3. However, random mutation of a
selected antibody (e.g. as
performed in some of the affinity maturation techniques described above) is
likely to give rise to
many mutants, most binding to antigen, and a few with higher affinity. With
limiting Her3, rare
high affinity phage could be competed out. To retain all the higher affinity
mutants, phages can
be incubated with excess biotinylated Her3, but with the biotinylated Her3 at
a concentration of
lower molarity than the target molar affinity constant for Her3. The high
affinity-binding phages
can then be captured by streptavidin-coated paramagnetic beads. Such
"equilibrium capture"
allows the antibodies to be selected according to their affinities of binding,
with sensitivity that
permits isolation of mutant clones with as little as two-fold higher affinity
from a great excess of
phages with lower affinity. Conditions used in washing phages bound to a solid
phase can also
be manipulated to discriminate on the basis of dissociation kinetics.
Anti-Her3 clones may be activity selected. In one embodiment, the invention
provides anti-Her3 antibodies that block the binding between a Her3 receptor,
preferably one of
a Her3 and/or Her3 receptor and its binding partner. Fv clones corresponding
to such anti-Her3
antibodies can be selected by (1) isolating anti-Fler3 clones from a phage
library as described
above, and optionally amplifying the isolated population of phage clones by
growing up the
population in a suitable bacterial host; (2) selecting Her3 and a second
protein against which
blocking and non-blocking activity, respectively, is desired; (3) adsorbing
the anti-Her3 phage
clones to immobilized Her3; (4) using an excess of the second protein to elute
any undesired
clones that recognize Her3-binding determinants which overlap or are shared
with the binding
determinants of the second protein; and (5) eluting the clones which remain
adsorbed following
step (4). Optionally, clones with the desired blocking/non-blocking properties
can be further
enriched by repeating the selection procedures described herein one or more
times.
DNA encoding, for example, phage display FA/ clones of the invention is
readily
isolated and sequenced using conventional procedures (e.g. by using
oligonucleotide primers
designed to specifically amplify the heavy and light chain coding regions of
interest from
hybridoma or phage DNA template). Once isolated, the DNA can 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 the desired monoclonal
antibodies in the
recombinant host cells. Review articles on recombinant expression in bacteria
of antibody-
encoding DNA include Skerra et al., Curr. Opinion in Immunol., 5: 256 (1993)
and Pluckthun,
Immunol. Revs, 130: 151 (1992).
DNA encoding the Fv clones of the invention can be combined with known DNA
sequences encoding heavy chain and/or light chain constant regions (e.g. the
appropriate DNA
sequences can be obtained from Kabat et at., supra) to form clones encoding
full or partial length
heavy and/or light chains. It will be appreciated that constant regions of any
isotype can be used
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for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and
that such constant
regions can be obtained from any human or animal species. A Fv clone derived
from the
variable domain DNA of one animal (such as human) species and then fused to
constant region
DNA of another animal species to form coding sequence(s) for "hybrid", full
length heavy chain
and/or light chain is included in the definition of "chimeric" and "hybrid"
antibody as used
herein. In a preferred embodiment, a Fv clone derived from human variable DNA
is fused to
human constant region DNA to form coding sequence(s) for all human, full or
partial length
heavy and/or light chains.
Antibody Fragments
In certain circumstances there are advantages of using antibody fragments,
rather
than whole antibodies. The smaller size of the fragments allows for rapid
clearance, and may
lead to improved access to solid tumors.
An antibody functional fragment refers to a portion of an antibody which
retains
some or all of its target-specific binding activity. Such functional fragments
can include, for
example, antibody functional fragments such as Fv, Fab, F(ab), F(ab)2, F(ab)2,
single chain Fv
(scFv), diabodies, triabodies, tetrabodies and minibody. Other functional
fragments can include,
for example, heavy (H) or light (L) chain polypeptides, variable heavy (VH)
and variable light
(VI) chain region polypeptides, complementarity determining region (CDR)
polypeptides, single
domain antibodies, and polypeptides that contain at least a portion of an
irnmunoglobulin that is
sufficient to retain target-specific binding activity. The present invention
encompasses antibody
fragments. In certain circumstances there are advantages of using antibody
fragments, rather than
whole antibodies. The smaller size of the fragments allows for rapid
clearance, and may lead to
improved access to solid tumors.
Various techniques have been developed for the production of antibody
fragments. Traditionally, these fragments were derived via proteolytic
digestion of intact
antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical
Methods 24:107-
117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these
fragments can now be
produced directly by recombinant host cells. Fab, Fv and ScFv antibody
fragments can all be
expressed in and secreted from E. coli, thus allowing the facile production of
large amounts of
these fragments. Antibody fragments can be isolated from the antibody phage
libraries discussed
above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli
and chemically
coupled to form F(abt)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. Fab and F(abt)2 fragment with increased in vivo half-life comprising
a salvage receptor
binding epitope residues are described in U.S. Pat. No. 5,869,046. Other
techniques for the
production of antibody fragments will be apparent to the skilled practitioner.
In other
embodiments, the antibody of choice is a single chain Fv fragment (scFv). See
WO 93/16185;
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U.S. Pat. Nos. 5,571,894; and 5,587,458. Fv and sFv are the only species with
intact combining
sites that are devoid of constant regions; thus, they are suitable for reduced
nonspecific binding
during in viva use. sFv fusion proteins may be constructed to yield fusion of
an effector protein
at either the amino or the carboxy terminus of an sFv. See Antibody
Engineering, ed.
Borrebaeck, supra. The antibody fragment may also be a "linear antibody",
e.g., as described in
U.S. Pat. No. 5,641,870 for example. Such linear antibody fragments may be
monospecific or
bispecific.
With respect to antibodies and functional fragments thereof that exhibit
beneficial
binding characteristics to a target molecule, various forms, alterations and
modifications are well
known in the art. Target-specific monoclonal antibodies for use in a
biopharmaceutical
formulation of the invention can include any of such various monoclonal
antibody forms,
alterations and modifications. Examples of such various forms and terms as
they are known in
the art are set forth below.
Polyclonal Antibodies
Polyclonal antibodies are preferably raised in animals by multiple
subcutaneous
(sc) or intraperitoneal (ip) injections of the relevant antigen and an
adjuvant. It may be useful to
conjugate the relevant antigen (especially when synthetic peptides are used)
to a protein that is
immunogenic in the species to be immunized. For example, the antigen can be
conjugated to
keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, or
soybean trypsin
inhibitor, using a bifunctional or derivatizing agent, e.g., maleimidobenzoyl
sulfosuccinimide
ester (conjugation through cysteine residues). N-hydroxysuccinimide (through
lysine residues),
glutaraldehyde, succinic anhydride, SOC1 2, or R IN=C=NR, where R and R I are
different alkyl
groups.
Animals are immunized against the antigen, immunogenic conjugates, or
derivatives by combining, e.g., 100 ug 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
Y5 to {fraction
(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. 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.
Monoclonal Antibodies
Monoclonal antibodies may be made using the hybridoma method first described
by Kohler et al., Nature, 256495 (1975), or may be made by recombinant DNA
methods (U.S.
Pat. No. 4,816,567).
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In the hybridoma method, a mouse or other appropriate host animal, such as a
hamster, is immunized as described above 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. After immunization,
lymphocytes are
isolated and then fused with a myeloma cell line 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 which medium preferably contains one or more substances that inhibit
the growth or
survival of the unfused, parental myeloma cells (also referred to as fusion
partner). For example,
if the parental myeloma cells lack the enzyme hypoxanthine guanine
phosphoribosyl transferase
(HGPRT or HPRT), the selective culture medium for the hybridomas typically
will include
hypoxanthine, aminopterin, and thymidine (HAT medium), which substances
prevent the growth
of HGPRT-deficient cells.
Preferred fusion partner 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 selective medium that selects against the unfused parental
cells. Preferred
myeloma cell lines are rnurine myeloma lines, such as those derived from MOPC-
21 and MPC-
11 mouse tumors available from the Salk Institute Cell Distribution Center,
San Diego, Calif.
USA, and SP-2 and derivatives e.g., X63-Ag8-653 cells available from the
American Type
Culture Collection, Rockville, Md. USA. Human myeloma and mouse-human
heteromyeloma
cell lines also have been described for the production of human monoclonal
antibodies (Kozbor,
Immunol., 133:3001 (1984); and 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 (RIA) or enzyme-linked
immunosorbent
assay (ELISA).
The binding affinity of the monoclonal antibody can, for example, be
determined
by the Scatehard analysis described in Munson et al., Anal, Biochem., 107:220
(1980).
Once hybridoma cells that produce antibodies of the desired specificity,
affinity,
and/or activity are identified, the clones may be subcloned by limiting
dilution procedures and
grown by standard methods (Goding, Monoclonal Antibodies: Principles and
Practice, pp.59-
103 (Academic Press, 1986)). Suitable culture media for this purpose include,
for example, D-
MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo
as ascites
tumors in an animal e.g, by i.p. injection of the cells into mice.
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The monoclonal antibodies secreted by the subclones are suitably separated
from
the culture medium, ascites fluid, or serum by conventional antibody
purification procedures
such as, for example, affinity chromatography (e. g., using protein A or
protein G-SepharoseZ)
or ion-exchange chromatography, hydroxylapatite chromatography, gel
electrophoresis, dialysis,
etc.
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. coil
cells, simian COS
cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not
otherwise produce
antibody protein, to obtain the synthesis of monoclonal antibodies in the
recombinant host cells.
Review articles on recombinant expression in bacteria of DNA encoding the
antibody include
Skerra et al., Curr. Opinion in Inununol., 5:256-262 (1993) and Pluckthun,
Inununol. Revs.,
130:151-188(1992).
In a further embodiment, monoclonal antibodies or antibody fragments can be
isolated from antibody phage libraries generated using the techniques
described in McCafferty et
al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991)
and Marks et al.,
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., Nue. 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 that encodes the antibody may be modified, for example, by
substituting human heavy chain and light chain constant domain (C H and CO
sequences for the
homologous murine sequences (U.S. Pat. No. 4,816,567; and Morrison, et al.,
Proc. Natl Acad.
Sci. USA, 81:6851(1984)), or by fusing the immunoglobulin coding sequence with
all or part of
the coding sequence for a non-immunoglobulin polypeptide. The non-
immunoglobulin
polypeptide sequences can substitute 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.
Humanized 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
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source which is non-human. These non-human amino acid residues are often
referred to as
"import" residues, which are typically taken from an "import" variable domain.
Humanization
can be essentially performed following the method of Winter and co-workers
(Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988);
Verhoeyen et al.,
Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences
for the
corresponding sequences of a human antibody. Accordingly, such "humanized"
antibodies are
chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially less than
an intact human
variable domain has been substituted by the corresponding sequence from a non-
human species.
In practice, humanized antibodies are typically human antibodies in which some
CDR residues
and possibly some FR residues are substituted by residues from analogous sites
in rodent
antibodies.
The choice of human variable domains, both light and heavy, to be used in
making the humanized antibodies is very important to reduce antigen icity.
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. Inmunol., 151:2296 (1993); Chothia et al.,
J. Mal. 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.
Mad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (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 art. Computer programs are
available which
illustrate and display probable three-dimensional conformational structures of
selected candidate
immunoglobulin sequences. Inspection of these displays permits analysis of the
likely role of the
residues in the functioning of the candidate immunoglobulin sequence, i.e.,
the analysis of
residues that influence the ability of the candidate immunoglobulin to bind
its antigen. In this
way, FR residues can be selected and combined from the recipient and import
sequences so that
the desired antibody characteristic, such as increased affinity for the target
antigen(s), is
achieved. In general, the CDR residues are directly and most substantially
involved in
influencing antigen binding.
Human Antibodies
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Human anti-Her3 antibodies of the invention can be constructed by combining Fv
clone variable domain sequence(s) selected from human-derived phage display
libraries with
known human constant domain sequences(s) as described above. Alternatively,
human
monoclonal anti-Her3 antibodies of the invention can be made by the hybridoma
method.
Human myeloma and mouse-human heteromyeloma cell lines for the production of
human
monoclonal antibodies have been described, for example, by Kozbor J. Immunol.,
133: 3001
(1984); Brodeur et al, Monoclonal Antibody Production Techniques and
Applications, pp. 51-63
(Marcel Dekker, Inc., New York, 1987); and Boerner et al, J. Immunol., 147: 86
(1991). Human
antibodies can also be derived from phage-display libraries (Hoogenboom et
al., 3. Mol. Biol.,
227:381 (1991); Marks et al., J. Mol. Biol., 222:581-597 (1991)).
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 immunoglobulin gene array in such germ-line mutant mice
will result in the
production of human antibodies upon antigen challenge. See, e.g., Jakobovits
et al., Proc. Natl.
Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993);
Bruggermann et
al., Year in Immuno., 7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825,
5,591,669 (all of
GenPharm); 5,545,807; and WO 97/17852.
Alternatively, phage display technology (McCafferty et al., Nature 348:552-553
(1990) can be used to produce human antibodies and antibody fragments in
vitro, from
immunoglobulin variable (V) domain gene repertoires from unimmunized donors.
According to
this technique, antibody V domain genes are cloned in-frame into either a
major or minor coat
protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed
as functional
antibody fragments on the surface of the phage particle. Because the
filamentous particle
contains a single-stranded DNA copy of the phage genome, selections based on
the functional
properties of the antibody also result in selection of the gene encoding the
antibody exhibiting
those properties. Thus, the phage mimics some of the properties of the B-cell.
Phage display
can be performed in a variety of formats, reviewed in, e.g., Johnson, Kevin S.
and Chiswell,
David .1., Current Opinion in Structural Biology 3:564-571 (1993). Several
sources of V-gene
segments can be used for phage display. Clackson et al., Nature, 352:624-628
(1991) isolated a
diverse array of anti-oxazolone antibodies from a small random combinatorial
library of V genes
derived from the spleens of immunized mice. A repertoire of V genes from
unimmunized
human donors can be constructed and antibodies to a diverse array of antigens
(including self-
antigens) can be isolated essentially following the techniques described by
Marks et al., ./. Mol.
Biol. 222:581-597 (1991), or Griffith etal., EMBO .1. 12:725-734 (1993). See,
also, U.S, Pat.
Nos. 5,565,332 and 5,573,905.
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Human antibodies may also be generated by in vitro activated B cells (see U.S.
Fat. Nos. 5,567,610 and 5,229,275).
Gene shuffling can also be used to derive human antibodies from non-human,
e.g.
rodent, antibodies, where the human antibody has similar affinities and
specificities to the
starting non-human antibody. According to this method, which is also called
"epitope
imprinting", either the heavy or light chain variable region of a non-human
antibody fragment
obtained by phage display techniques as described above is replaced with a
repertoire of human
V domain genes, creating a population of non-human chain/human chain scFv or
Fab chimeras.
Selection with antigen results in isolation of a non-human chain/human chain
chimeric scFv or
Fab wherein the human chain restores the antigen binding site destroyed upon
removal of the
corresponding non-human chain in the primary phage display clone, i.e. the
epitope governs
(imprints) the choice of the human chain partner. When the process is repeated
in order to
replace the remaining non-human chain, a human antibody is obtained (see PCT
WO 93/06213
published Apr. 1, 1993). Unlike traditional humanization of non-human
antibodies by CDR
grafting, this technique provides completely human antibodies, which have no
FR or CDR
residues of non-human origin.
Bispecific Antibodies
Bispecific antibodies are monoclonal antibodies that have binding
specificities for
at least two different antigens. In the present case, one of the binding
specificities is for Her3
and the other is for any other antigen. Exemplary bispecific antibodies may
bind to two different
epitopes of the Her3 protein. Bispecific antibodies may also be used to
localize cytotoxie agents
to cells which express Her3. These antibodies possess a Her3-binding arm and
an arm which
binds the cytotoxic agent (e.g. saporin, anti-interferon-Alpha., vinca
alkaloid, ricin A chain,
methotrexate or radioactive isotope hapten). Bispecific antibodies can be
prepared as full length
antibodies or antibody fragments (e.g. F(aW)2 bispecific antibodies).
Methods for making bispecific antibodies are known in the art. Traditionally,
the
recombinant production of bispecific antibodies is based on the co-expression
of two
immunoglobulin heavy chain-light chain pairs, where the two heavy chains have
different
specificities (Milstein and Cuello, Nature, 305: 537 (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. The 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 published May 13, 1993, and in Traunecker et al.,
EMBO J., 10:
3655 (1991).
According to a different and more preferred approach, antibody variable
domains
with the desired binding specificities (antibody-antigen combining sites) are
fused to
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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 one embodiment of this approach, the bispecific antibodies are composed of
a
hybrid immunoglobulin heavy chain with a first binding specificity in one arm,
and a hybrid
immunoglobulin heavy chain-light chain pair (providing a second binding
specificity) in the
other arm. It was found that this asymmetric structure facilitates the
separation of the desired
bispecific compound from unwanted immunoglobulin chain combinations, as the
presence of an
immunoglobulin light chain in only one half of the bispecific molecule
provides for a facile way
of separation. This approach is disclosed in WO 94/04690. For further details
of generating
bispecific antibodies see, for example, Suresh et al., Methods in Enzymology,
121:210 (1986).
According to another approach, the interface between a pair of antibody
molecules can be engineered to maximize the percentage of heterodimers which
are recovered
from recombinant cell culture. The preferred interface comprises at least a
part of the 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.
Techniques for generating bispecific antibodies from antibody fragments have
also been described in the literature. For example, bispecific antibodies can
be prepared using
chemical linkage. Brennan et al., Science, 229: 81(1985) describe a procedure
wherein intact
antibodies are proteolytically cleaved to generate F(ab)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 rnercaptoethylamine and is
mixed with an
equimolar amount of the other Fab'-TNB derivative to form the bispecific
antibody. The
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bispecific antibodies produced can be used as agents for the selective
immobilization of
enzymes.
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. See Kostelny et al., J.
Immunol.,
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun
proteins were
linked to the Fa& portions of two different antibodies by gene fusion. The
antibody homodimers
were reduced at the hinge region to form monomers and then re-oxidized to form
the antibody
heterodimers. This method can also be utilized for the production of antibody
homodimers.
Another strategy for making bispecific antibody fragments by the use of single-
chain Fv (scFv)
dimers has also been reported. See Gruber et at., J. Immunol., 152:5368
(1994).
Heteroconjugate
Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. As
such, heteroconjugate antibodies are also within the scope of the present
invention.
Heteroconjugate antibodies are composed of two covalently joined antibodies.
Such antibodies
have, for example, been proposed to target immune system cells to unwanted
cells, e.g., U.S. Pat.
No. 4,676,980, and for treatment of HIV infection, e.g., WO 91/00360; WO
92/200373; EP
03089. Heteroconjugate antibodies may be made using any convenient cross-
linking methods.
It is contemplated that the antibodies may be prepared in vitro using known
methods in synthetic
protein chemistry, including those involving cross-linking agents. For
example, immunotoxins
may be constructed using a disulfide exchange reaction or by forming a
thioether bond.
Examples of suitable reagents for this purpose include iminothiolate and
methy1-4-
mercaptobutyrimidate and those disclosed, for example, in U.S. Pat. No,
4,676,980.
Diabody
The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci.
USA, 90:6444-6448 (1993) has provided an alternative mechanism for making
bispecific
antibody fragments. A diabody is a bivalent dirner formed by the non-covalent
association of
two scFvs, yielding two Fv binding sites. Briefly, a diabody refers to an
engineered antibody
construct prepared by isolating the binding domains (both heavy and light
chain) of a binding
antibody, and supplying a linking moiety which joins or operably links the
heavy and light
chains on the same polypeptide chain thereby preserving the binding function
(see, Holliger et al.
(1993) Proc. Natl. Acad. Sci. USA 90:6444; Poljak (1994) Structure 2:1121-
1123). This forms,
in essence, a radically abbreviated antibody, having only the variable domain
necessary for
binding the antigen. 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. These dimeric antibody fragments,
or diabodies, are
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bivalent and bispecific. Thus, diabodies are dimers of two say molecules that
cannot fold
properly into one seFv molecule. Diabodies are built like scFv molecules, but
usually have a
short (less than 10, preferably 1-5 amino acids) peptide linker connecting
both V-domains,
whereby both domains can not interact intramolecular, and are forced to
interact intermolecular
(Holliger et al., 1993) (U.S. Pat. No. 5,837,242). A diabody thus may consist
of a VH-VL chain
that interacts with a similar VH-VL chain to form a dimer of the formula VH-
VL:VH-VL. The
diabody chain dimers bind the antigen specified by Vu and VL bivalent, Winter
described the
construction of bispecific diabodies by coupling the VH domain of a chosen
antibody A to the VL
domain of a chosen antibody B, using a peptide linker sufficiently short to
inhibit the interaction
of V(A) with VL(B). Also the reverse molecule VH(B)-Vi,(A) is made the same
way (Holliger,
Griffiths, Hoogenboom, Malmqvist, Marks, McGuinness, Pope, Prospero and
Winter:
"Multivalent and multispecific binding proteins, their manufacture and use",
U.S. Pat. No.
5,837,242, 1998). The skilled artisan will appreciate that any method to
generate diabodies can
be used. Suitable methods are described by Holliger, et at. (1993) supra,
Poljak (1994) supra,
Zhu, et al. (1996) Biotechnology 14:192-196, and U.S. Pat. No. 6,492,123,
incorporated herein
by reference.
Fab'-S1-1
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(abl)2
molecule. Each Fab' fragment was separately secreted from E. coli and
subjected to directed
chemical coupling in vitro to form the bispecific antibody. The bispecific
antibody thus formed
was able to bind to cells overexpressing the Her2 receptor and normal human T
cells, as well as
trigger the lytic activity of human cytotoxic lymphocytes against human breast
tumor targets.
Trispecific
Antibodies with more than two valences are contemplated. For example,
trispecific antibodies can be prepared. Tutt et at. J. Immunol. 147: 60
(1991).
Tetravalent
Tetravalent bispecific antibodies can be created by chemical cross-linking of
two
monoclonal antibodies (Bs(IgG)2) (Karpovsky et al., 1984) (U.S. Pat. No.
4,676,980). Problems
related to their rapid clearance in vivo via the kidney due to their small
size may be circumvented
by, for example, increasing their molecular weight size thereby increasing
their serum
permanence and product efficacy. (Wu, A. M., Chen, W., Raubitschek, A.,
Williams, L. E.,
Neumaier, M., Fischer, R., Hu, S. Z., Odom-Maryon, T., Wong, J. Y. and
Shively, J. E.: Tumor
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localization of anti-CEA single-chain Fvs: improved targeting by non-covalent
dimers.
Immunotechnology 2 (1996) 21-36).
Peptibodies
Peptibodies, which consist of an immunoglobulin constant region domain (Fe)
linked to two binding peptides through either the carboxyl- or amino termini
of the Fe domain,
also are included herein as an antibody functional fragment. Such antibody
binding fragments
can be found described in, for example, Harlow and Lane, supra; Mole . Biology
and
Biotechnology: A Comprehensive Desk Reference (Myers, R. A. (ed.), New York:
VCH
Publisher, Inc.); Huston et al., Cell Biophysics, 22:189-224 (1993); Pluekthun
and Skerra, Meth.
Enzymol., 178:497-515 (1989) and in Day, E. D., Advanced Immunochemistry,
Second Ed.,
Wiley-Liss, Inc., New York, N.Y. (1990).
Multivalent Antibodies
A multivalent antibody may be internalized (and/or catabolized) faster than a
bivalent antibody by a cell expressing an antigen to which the antibodies
bind. The antibodies of
the present invention can be multivalent antibodies (which are other than of
the IgM class) with
three or more antigen binding sites (e.g. tetravalent antibodies), which can
be readily produced
by recombinant expression of nucleic acid encoding the polypeptide chains of
the antibody. The
multivalent antibody can comprise a dimerization domain and three or more
antigen binding
sites. The preferred dimerization domain comprises (or consists of) an Fe
region or a hinge
region. In this scenario, the antibody will comprise an Fe region and three or
more antigen
binding sites amino-terminal to the Fe region. The preferred multivalent
antibody herein
comprises (or consists of) three to about eight, but preferably four, antigen
binding sites. The
multivalent antibody comprises at least one polypeptide chain (and preferably
two polypeptide
chains), wherein the polypeptide chain(s) comprise two or more variable
domains. For instance,
the polypeptide chain(s) may comprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a
first
variable domain, VD2 is a second variable domain, Fe is one polypeptide chain
of an Fe region,
X1 and X2 represent an amino acid or polypeptide, and n is 0 or 1. For
instance, the polypeptide
chain(s) may comprise: V11-CH1-flexible linker-VH-CHI-Fc region chain; or VH-
CH1-VH-CH1-
Fe region chain. The multivalent antibody herein preferably further comprises
at least two (and
preferably four) light chain variable domain polypeptides. The multivalent
antibody herein may,
for instance, comprise from about two to about eight light chain variable
domain polypeptides.
The light chain variable domain polypeptides contemplated here comprise a
light chain variable
domain and, optionally, further comprise a CL domain.
Antibody Variants
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In some embodiments, amino acid sequence modification(s) of the antibodies
described herein are contemplated. For example, it may be desirable to improve
the binding
affinity and/or other biological properties of the antibody. Amino acid
sequence variants of the
antibody are prepared by introducing appropriate nucleotide changes into the
antibody nucleic
acid, or by peptide synthesis. Such modifications include, for example,
deletions from, and/or
insertions into and/or substitutions of, residues within the amino acid
sequences of the antibody.
Any combination of deletion, insertion, and substitution is made to arrive at
the final construct,
provided that the final construct possesses the desired characteristics. The
amino acid alterations
may be introduced in the subject antibody amino acid sequence at the time that
sequence is
made.
A useful method for identification of certain residues or regions of the
antibody
that are preferred locations for mutagenesis is called "alanine scanning
mutagenesis" as
described by Cunningham and Wells (1989) Science, 244:1081-1085. Here, a
residue or group
of target residues are identified (e.g., charged residues such as arg, asp,
his, lys, and glu) and
replaced by a neutral or negatively charged amino acid (most preferably
alanine or polyalanine)
to affect the interaction of the amino acids with antigen. Those amino acid
locations
demonstrating functional sensitivity to the substitutions then are refined by
introducing further or
other variants at, or for, the sites of substitution. Thus, while the site for
introducing an amino
acid sequence variation is predetermined, the nature of the mutation per se
need not be
predetermined. For example, to analyze the performance of a mutation at a
given site, ala
scanning or random mutagenesis is conducted at the target codon or region and
the expressed
immunoglobulins are screened for the desired activity.
Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions
ranging in length from one residue to polypeptides containing a hundred or
more residues, as
well as intrasequence insertions of single or multiple amino acid residues.
Examples of terminal
insertions include an antibody with an N-terminal methionyl residue or the
antibody fused to a
cytotoxic polypeptide. Other insertional variants of the antibody molecule
include the fusion of
the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a
polypeptide which
increases the serum half-life of the antibody.
Glycosylation of polypeptides is typically either N-linked or 0-linked. N-
linked
refers to the attachment of the carbohydrate moiety to the side chain of an
asparagine residue.
The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where
X is any amino
acid except praline, are the recognition sequences for enzymatic attachment of
the carbohydrate
moiety to the asparagine side chain. Thus, the presence of either of these
tripeptide sequences in
a polypeptide creates a potential glycosylation site. 0-linked glycosylation
refers to the
attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to
a hydroxyamino
acid, most commonly serine or threonine, although 5-hydroxyproline or 5-
hydroxylysine may
also be used. Addition of glycosylation sites to the antibody is conveniently
accomplished by
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altering the amino acid sequence such that it contains one or more of the
above-described
tripeptide sequences (for N-linked glycosylation sites). The alteration may
also be made by the
addition of, or substitution by, one or more serine or threonine residues to
the sequence of the
original antibody (for 0-linked glycosylation sites). See section marked
"Effector Function
Engineering", infra.
Where the antibody comprises an Fe region, the carbohydrate attached thereto
may be altered. For example, antibodies with a mature carbohydrate structure
that lacks fucose
attached to an Pc region of the antibody are described in US Pat Appl No US
2003/0157108
(Presta, L.). See also US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd).
Antibodies with a
bisecting N-aeetylglucosamine (GIcNAc) in the carbohydrate attached to an Fe
region of the
antibody are referenced in WO 2003/011878, Jean-Mairet et al. and U.S. Pat.
No. 6,602,684,
Umana et al. Antibodies with at least one galactose residue in the
oligosaccharide attached to an
Fe region of the antibody are reported in WO 1997/30087, Patel et al. See,
also, WO 1998/58964
(Raju, S.) and WO 1999/22764 (Raju, S.) concerning antibodies with altered
carbohydrate
attached to the Fe region thereof. See also US 2005/0123546 (Umana et al.) on
antigen-binding
molecules with modified glycosylation.
At least one glycosylation variant herein comprises an Fe region, wherein a
carbohydrate structure attached to the Fe region lacks fucose. Such variants
have improved
ADCC function. Optionally, the Fe region further comprises one or more amino
acid
substitutions therein which further improve ADCC, for example, substitutions
at positions 298,
333, and/or 334 of the Fe region (Eu numbering of residues). Examples of
publications related
to "defucosylated" or "fucose-deficient" antibodies include: US 2003/0157108;
WO 2000/61739;
WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US
2004/0132140;
US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO
2003/084570;
WO 2005/035586; WO 2005/035778; W02005/053742; Okazaki et al. J. Mol. Biol.
336:1239-
1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of
cell lines
producing defucosylated antibodies include Lec13 CHO cells deficient in
protein fueosylation
(Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US
2003/0157108
Al, Presta, L; and WO 2004/056312 Al, Adams et al., especially at Example 11),
and knockout
cell lines, such as alpha-1,6-fixosyltransferase gene, FUT8, knockout CHO
cells (Yamane-
Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)). For further details, see
"Effector Function
Engineering" infra.
Another type of variant is an amino acid substitution variant. These variants
have
at least one amino acid residue in the antibody molecule replaced by a
different residue. The
sites of greatest interest for substitutional mutagenesis include the
hypervariable regions, but FR
alterations are also contemplated. Conservative substitutions are shown in
Table 1 under the
heading of "preferred substitutions". If such substitutions result in a change
in biological
activity, then more substantial changes, denominated "exemplary substitutions"
in Table 1, or as
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further described below in reference to amino acid classes, may be introduced
and the products
screened.
Exemplary Preferred Residue Substitutions Ala (A) Val; Leu; Ile Val Arg (R)
Lys; Gin; Asn Lys Asn (N) Gin; His; Asp, Lys; Arg Gin Asp (D) Glu; Asn Glu Cys
(C) Ser; Ala
Ser Gin (Q) Asn; Glu Asn Glu (E) Asp; Gin Asp Gly (G) Ala Ala His (H) Asn;
Gin; Lys; Arg
Arg Ile (I) Leu; Val; Met; Ala; Leu Phe; Norleucine Leu (L) Norleucine; Ile;
Val; Ile Met; Ala;
Phe Lys (K) Arg; Gin; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val;
Ile; Ala; Tyr
Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr
Tyr (Y) Trp;
Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Norleucine
Substantial modifications in the biological properties of the antibody are
accomplished by selecting substitutions that differ significantly in their
effect on maintaining (a)
the structure of the polypeptide backbone in the area of the substitution, for
example, as a sheet
or helical conformation, (b) the charge or hydrophobicity of the molecule at
the target site, or (c)
the bulk of the side chain. Naturally occurring residues are divided into
groups based on
common side-chain properties:
(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;
(3) acidic: Asp, Glu;
(4) basic: His, Lys, Arg;
(5) residues that influence chain orientation: Gly, Pro; and
(6) aromatic: Trp, Tyr, Phe.
Non-conservative substitutions will entail exchanging a member of one of these
classes for another class.
One type of substitutional variant involves substituting one or more
hypervariable
region residues of a parent antibody (e.g. human antibody). Generally, the
resulting variant(s)
selected for further development will have improved biological properties
relative to the parent
antibody from which they are generated. A convenient way for generating such
substitutional
variants involves affinity maturation using phage display. Briefly, several
hypervariable region
sites (e.g. 6-7 sites) are mutated to generate all possible amino acid
substitutions at each site.
The antibodies thus generated are displayed from filamentous phage particles
as fusions to the
gene III product of M13 packaged within each particle. The phage-displayed
variants are then
screened for their biological activity (e.g. binding affinity) as herein
disclosed. In order to
identify candidate hypervariable region sites for modification, alanine
scanning mutagenesis can
be performed to identify hypervariable region residues contributing
significantly to antigen
binding. Alternatively, or additionally, it may be beneficial to analyze a
crystal structure of the
antigen-antibody complex to identify contact points between the antibody and
antigen. Such
contact residues and neighboring residues are candidates for substitution
according to the
techniques elaborated herein. Once such variants are generated, the panel of
variants is
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subjected to screening as described herein and antibodies with superior
properties in one or more
relevant assays may be selected for further development.
Nucleic acid molecules encoding amino acid sequence variants of the antibody
are prepared by a variety of methods known in the art. These methods include,
but are not
limited to, isolation from a natural source (in the case of naturally
occurring amino acid sequence
variants) or preparation by oligonucleotide-mediated (or site-directed)
mutagenesis, PCR
mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-
variant version of
the antibody.
Effector Function Engineering
(A) Anti-Her3 Antibodies with Variant Fe Regions
Most immune functions of antibodies depend on their ability to act as flexible
adaptor molecules, linking pathogen with appropriate elimination mechanisms.
This 'bridging'
role entails two types of recognition, each involving contributions from
particular antibody
domains. The first involves highly specific recognition of the antigen target,
and is mediated
through the amino-terminal variable domains of the two Fab regions of the
antibody. The
second involves interaction of the constant domains of the Fe region of the
molecule with
various effector molecules, including complement and, perhaps most
importantly, Fe receptors
(FcRs) present on phagocytes and other immune cells. The dual recognition of
target and FcR
by immunoglobulin molecules has a key role in eliciting effector mechanisms to
rid the body of
bacteria, viruses, and parasites.
Briefly, therapeutic antibodies can exert potent biological functions through
two
major non-exclusive mechanisms: (i) they can block interactions between
receptors and their
ligands due to the exquisite epitope specificity of their variable domains
("neutralizing/antagonist antibodies") or trigger potent biological responses
such as apoptosis or
cell proliferation once they are bound to surface molecules ("agonist
antibodies"); (ii) induce
effector functions against pathogens and tumor cells following their
interactions with the
complement component Clq and/or with receptors for Fe region (FcyR). See Cragg
et al., Curr
Opin Immunol 11:541-547 (1999); Glennie et al., Immunol Today 21:403-410
(2000).
The effector functions of immunoglobulins e.g., IgG, which is the most common
immunoglobulin, are mediated by the antibody Fc region through two major
mechanisms: (1)
binding to the cell surface Fe receptors (FcyRs) can lead to ingestion of
pathogens by
phagocytosis or lysis by killer cells via the antibody-dependent cellular
cytotoxicity (ADCC)
pathway, or (2) binding to the Clq part of the first complement component Cl
initiates the
complement-dependent cytotoxicity (CDC) pathway, resulting in the lysis of
pathogens.
Reviewed in Daeron, Annu. Rev. Immunol. 15:203-234 (1997); Ward and Ghetie,
Therapeutic
Immunol. 2:77-94 (1995); Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492
(1991); Uananue
and Benacerraf, Textbook of Immunology, 2nd Edition, Williams & Wilkins, p.
218 (1984)).
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There are three known FcyRs, designated FcyRI (CD64), FeyRII (CD32), and
FeyRIII(CD16).
Anti-tumor efficacy can be due to a combination of these mechanisms, and their
relative
importance in clinical therapy appears to be cancer dependent. Notwithstanding
this arsenal of
anti-tumor weapons, the potency of antibodies as anti-cancer agents is
unsatisfactory,
particularly given their high cost. Currently for anti-cancer therapy, any
small improvement in
mortality rate defines success.
Thus, it may be desirable to introduce one or more amino acid modifications in
an
Fe region of the immunoglobulin polypeptides of the invention, thereby
generating an Fc region
variant. The Fe region variant may comprise a human Fc region sequence (e.g.,
a human IgGl,
IgG2, IgG3 or IgG4 Fe region) comprising an amino acid modification (e.g. a
substitution) at
one or more amino acid positions including that of a hinge cysteine.
ADCC involves the recognition of the antibody by immune cells that engage the
antibody-marked cells and either through their direct action, or through the
recruitment of other
cell types, leads to the tagged-cell's death. CDC is a process where a cascade
of different
complement proteins become activated, usually when several IgGs are in close
proximity to each
other, either with one direct outcome being cell lysis, or one indirect
outcome being attracting
other immune cells to this location for effector cell function.
A promising means for enhancing the anti-tumor potency of antibodies is via
enhancement of their ability to mediate cytotoxic effector functions such as
ADCC, ADCP, and
CDC, The importance of ADCC as a cytotoxic mechanism of anti-tumor mAbs has
been
demonstrated in animal studies. Ravetch et al., Annu. Rev. Immunol, 16:421-432
(1998)
showed that the tumoricidal effect of a humanized anti-Her2/neu mAb
(epithelial growth factor
receptor 2; Trastuzumab) was significantly reduced in FcyR knockout nude mice
as compared to
wild-type nude mice. Similarly, the tumor regression activity of a chimeric
anti-CD20 mAb
(Rituximab) was significantly reduced in FcyR deficient mice as compared to
wild-type mice.
Ravetch, supra; Clynes et al., Inhibitory Fc receptors modulate in vivo
cytotoxicity against tumor
targets. Nat. Med. 6:443-446 (2000). Further support for an important role for
ADCC was
provided by a study of Cartron et al., who found that in patients with a
polymorphism in
FcyRIIIa leading to increased binding of IgGI, therapy with an anti-CD20 mAb
produced a 90%
response rate (patients with complete remission or partial response) at 12
months, compared to a
51% response rate in individuals not expressing this polymorphism of FcyRIIIa.
Cartron et al.,
Therapeutic activity of humanized anti-CD20 monoclonal antibody and
polymorphism in IgG Fc
receptor FcyRIIIa gene, Blood 99:754-758 (2002). Others have shown that this
FcyRIIIa
polymorphism and also a polymorphism in FcyRIIa are associated with the
response rate to
therapeutic mAbs. W.K. Weng and R. Levy, Two imnriunoglobulin G fragment C
receptor
polymorphisms independently predict response to rituximab in patients with
follicular
lymphoma. J. Clin. Oncol. 21:3940-3947 (2003). The importance of FcyR-mediated
effector
functions for the anti-cancer activity of antibodies has also been
demonstrated in mice (Clynes et
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al., 1998, Proc Nati Acad Sci USA 95:652-656; Clynes et al., 2000, Nat Med
6:443-446), and the
affinity of interaction between Fe and certain FcyRs correlates with targeted
cytotoxicity in cell-
based assays (Shields et al., 2001, J Biol Chem 276:6591-6604; Presta et al.,
2002, Biochem Soc
Trans 30:487-490; Shields et al., 2002, I Biol Chem 277:26733-26740).
Additionally, a
correlation has been observed between clinical efficacy in humans and their
allotype of high
(V158) or low (F158) affinity polymorphic forms of FcyRIIIa (Cartron et al.,
2002, Blood
99:754-758). Together these data suggest that an antibody that is optimized
for binding to
certain FcyRs may better mediate effector functions and thereby destroy cancer
cells more
effectively in patients. The balance between activating and inhibiting
receptors is an important
consideration, and optimal effector function may result from an antibody that
has enhanced
affinity for activation receptors, for example FcyRI, FcyRIIa/c, and FcyRIlla,
yet reduced affinity
for the inhibitory receptor FcyRIIb. Furthermore, because FcyRs can mediate
antigen uptake and
processing by antigen presenting cells, enhanced FcyR affinity may also
improve the capacity of
antibody therapeutics to elicit an adaptive immune response. Fe variants have
been successfully
engineered with selectively enhanced binding to FcyRs, and furthermore these
Fe variants
provide enhanced potency and efficacy in cell-based effector function assays.
See for example
U.S. Ser. No. 10/672,280, U.S. Ser. No. 10/822,231, entitled "Optimized Fe
Variants and
Methods for their Generation", U.S. Ser, No. 60/627,774, entitled "Optimized
Fe Variants", and
U.S. Ser. No. 60/642,477, entitled "Improved Fe Variants", and references
cited therein.
7,276,585 Xencor, Inc. (Monrovia, CA) See also Patent No. 6,821,505.
Most mAbs that mediate antibody-dependent cellular cytotoxicity (ADCC) also
activate the complement system 1. A. Gorter and S. Merl, "Immune evasion of
tumor cells using
membrane-bound complement regulatory proteins." Immunol. Today, pp. 576-582
(1999).
Complement initiates three mechanisms that can be used against mAb-coated
tumor cells. The first is direct complement killing of tumor cells by the
membrane attack
complex (MAC), a process usually called 'complement-dependent cytotoxicity'
(CDC). The
second mechanism is complement receptor-dependent enhancement of ADCC. In this
case, CR3
binds to iC3b, thus enhancing FcyR-mediated effector cell binding. A third
mechanism used for
killing microorganisms, CR3-dependent cellular cytotoxicity (CR3-DCC), is
usually not
activated with tumors.
Based upon the results of chemical modifications and crystallographic studies,
Burton et al. (Nature, 288:338-344 (1980)) proposed that the binding site for
the complement
subcomponent Clq on IgG involves the last two (C-terminal) .beta.-strands of
the CH2 domain.
Burton later suggested (Molec. Immunol., 22(3):161-206 (1985)) that the region
comprising
amino acid residues 318 to 337 might be involved in complement fixation.
Duncan and Winter (Nature 332:738-40 (1988)), using site directed mutagenesis,
reported that Giu318, Lys320 and Lys322 form the binding site to Clq. The data
of Duncan and
Winter were generated by testing the binding of a mouse IgG2b isotype to
guinea pig C I q. The
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role of Glu318, Lys320 and Lys322 residues in the binding of Clq was confirmed
by the ability
of a short synthetic peptide containing these residues to inhibit complement
mediated lysis.
Similar results are disclosed in U.S. Pat. No. 5,648,260 issued on Jul. 15,
1997, and U.S. Pat. No.
5,624,821 issued on Apr. 29, 1997.
The residue Pro331 has been implicated in Clq binding by analysis of the
ability
of human IgG subclasses to carry out complement mediated cell lysis. Mutation
of Ser331 to
Pro331 in IgG4 conferred the ability to activate complement. (Tao et al., J.
Exp. Med., 178:661-
667 (1993); Brelcke et al., Eur. J. Immunol., 24:2542-47 (1994)).
From the comparison of the data of the Winter group, and the Tao et al. and
Brekke et al. papers, Ward and Ghetie concluded in their review article that
there are at least two
different regions involved in the binding of Clq: one on the .beta.-strand of
the CH2 domain
bearing the Glu318, Lys320 and Lys322 residues, and the other on a turn
located in close
proximity to the same .beta.-strand, and containing a key amino acid residue
at position 331.
Other reports suggest that human IgG1 residues Leti235, and Gly237, located in
the lower hinge region, play a critical role in complement fixation and
activation. Xu et al., J.
Immunol. 150:152A (Abstract) (1993). W094/29351 published Dec. 22, 1994
reports that amino
acid residues necessary for Clq and FcR binding of human IgG1 are located in
the N-terminal
region of the CH2 domain, i.e. residues 231 to 238.
It has further been proposed that the ability of IgG to bind Clq and activate
the
complement cascade also depends on the presence, absence, or modification of
the carbohydrate
moiety positioned between the two CH2 domains (which is normally anchored at
Asn297).
Ward and Ghetie, Therapeutic Immunology 2:77-94 (1995) at page 81.
The binding site on human and murine antibodies for FoyR have been previously
mapped to the so-called "lower hinge region" consisting of residues 233-239
(EU index
numbering as in Kabat et al., Sequences of Proteins of Immunological Interest,
5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md. (1991)). Woof et
al. Molec.
Immunol. 23:319-330 (1986); Duncan et al. Nature 332:563 (1988); Canfield and
Morrison, J.
Exp. Med. 173:1483-1491 (1991); Chappel et al., Proc. Natl. Acad. Sci USA
88:9036-9040
(1991). Of residues 233-239, P238 and S239 have been cited as possibly being
involved in
binding, but these two residues have never been evaluated by substitution or
deletion.
Other previously cited areas possibly involved in binding to FcyR: G316-K338
(human IgG) for human FeyRI (by sequence comparison only; no substitution
mutants were
evaluated) (Woof et al. Malec. Immunol 23:319-330 (1986)); K274-R301 (human
IgG1) for
human FcyRIII (based on peptides) (Sarmay et al. Molec. Immunol. 21:43-51
(1984)); Y407-
R416 (human IgG) for human FcyRIII (based on peptides) (Gergely et al.
Biochern. Soc. Trans.
12:739-743 (1984)).
U.S. Pat. No. 6,165,745 discloses a method of producing an antibody with a
decreased biological half-life by introducing a mutation into the DNA segment
encoding the
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antibody. The mutation includes an amino acid substitution at position 253,
310, 311, 433, or
434 of the Fe-hinge domain. The full disclosure of U.S. Pat. No. 6,165,745, as
well as the full
disclosure of all other U.S. patent references cited herein, are hereby
incorporated by reference.
U.S. Patent Application No. 20020098193 Al and PCT Publication No, WO
97/34621 disclose mutant IgG molecules having increased serum half-lives
relative to IgG
wherein the mutant IgG molecule has at least one amino acid substitution in
the Fc-hinge region.
However, no experimental support is provided for mutations at positions 250,
314, or 428.
U.S. Pat. No. 6,277,375 B1 discloses a composition comprising a mutant IgG
molecule having an increased serum half-life relative to the wild-type IgG,
wherein the mutant
IgG molecule comprises the amino acid substitutions: threonine to leucine at
position 252,
threonine to serine at position 254, or threonine to phenylalanine at position
256. A mutant IgG
with an amino acid substitution at position 433, 435, or 436 is also
disclosed.
U.S. Pat. No. 6,528,624 discloses a variant of an antibody comprising a human
IgG Fc region, which variant comprises an amino acid substitution at one or
more of amino acid
positions 270, 322, 326, 327, 329, 331, 333, and 334 of the human IgG Fe
region.
In accordance with the above description and the teachings of the art, it is
contemplated that in some embodiments, an antibody used in methods of the
invention may
comprise one or more alterations as compared to the wild type counterpart
antibody, e.g. in the
Fc region. These antibodies would nonetheless retain substantially the same
characteristics
required for therapeutic utility as compared to their wild type counterpart.
For example, it is
thought that certain alterations can be made in the Fc region that would
result in altered (i.e.,
either improved or diminished) Clq binding and/or Complement Dependent
Cytotoxicity (CDC),
e.g., as described in W099/51642. See also Duncan & Winter Nature 322:738-40
(1988); U.S.
Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and W094/29351 concerning other
examples of Fc
region variants. W000/42072 (Presta) and WO 2004/056312 (Lowman) describe
antibody
variants with improved or diminished binding to FcRs. The content of these
patent publications
are specifically incorporated herein by reference. See, also, Shields et al.
J. Biol. Chem. 9(2):
6591-6604 (2001). Antibodies with increased half lives and improved binding to
the neonatal Fc
receptor (FcRn), which is responsible for the transfer of maternal IgGs to the
fetus (Guyer et al.,
T. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are
described in
US2005/0014934 1 (Hinton et al.). These antibodies comprise an Fe region with
one or more
substitutions therein which improve binding of the Fe region to FeRn.
Polypeptide variants with
altered Fc region amino acid sequences and increased or decreased Clq binding
capability are
described in U.S. Pat. No. 6,194,551B1, W099/51642. The contents of those
patent publications
are specifically incorporated herein by reference. See, also, Idusogie et al.
J. Immunol.
164:4178-4184 (2000).
Functional Assays of Molecules with Variant Fe Regions
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Variant Fe Regions
The ability of any particular antibody e.g., any one or more of the anti-
antibodies
disclosed herein, to mediate lysis of the target cell by complement activation
and/or ADCC can
be assayed. Functional assays for identifying potent Fe variants of any one or
more of the anti-
Her3 antibodies of the invention are well known to one skilled in the art.
See, for example, U.S
Patent Application Publications 2005/0037000 and 2005/0064514, and
International Patent
Application Publication WO 04/063351 (each of which is hereby incorporated by
reference in its
entirety); that describe yeast display technology for characterizing an
antibody with a variant Fe
region. Likewise, R-Fe binding assays are disclosed in U.S Patent Application
Publications
2005/0037000 and 2005/0064514, and International Patent Application
Publication WO
04/063351 (each of which is hereby incorporated by reference in its entirety).
Examples of effector cell functions that can be assayed in accordance with the
invention, include but are not limited to, antibody-dependent cell mediated
cytotoxicity,
phagocytosis, opsonization, opsonophagocytosis, Clq binding, and complement
dependent cell
mediated cytotoxicity. Any cell-based or cell free assay known to those
skilled in the art for
determining effector cell function activity can be used (For effector cell
assays, see Perussia et
al., 2000, Methods Mol. Biol. 121: 179-92; Baggiolini et al., 1998
Experientia, 44(10): 841-8;
Lehmann et al., 2000 J. Immunol. Methods, 243(1-2): 229-42; Brown E J. 1994,
Methods Cell
Biol., 45: 147-64; Munn et al., 1990 J. Exp. Med., 172: 231-237, Abdul-Maj id
et al., 2002
Scand. J. Immunol, 55: 70-81; Ding et al., 1998, Immunity 8:403-411, each of
which is
incorporated by reference herein in its entirety).
Generally, the cells of interest are grown and labeled in vitro; the target
antibody
is added to the cell culture in combination with either serum complement or
immune cells which
may be activated by the antigen-antibody complexes. Cytolysis of the target
cells is detected by
the release of label from the lysed cells. In fact, antibodies can be screened
using the patient's
own serum as a source of complement and/or immune cells. The antibody that is
capable of
activating complement or mediating ADCC in the in vitro test can then be used
therapeutically in
that particular patient.
Preferably, the effector cells used in the ADCC assays of the invention are
peripheral blood mononuclear cells (PBMC) that are preferably purified from
normal human
blood, using standard methods known to one skilled in the art, e.g. using
Ficoll-Paque density
gradient centrifugation.
An exemplary assay for determining ADCC activity of such anti-Her3 antibodies
with variant Fe regions is based on a 51Cr release assay comprising of:
labeling target cells with
[51CriNa2Cr04 (this cell-membrane permeable molecule is commonly used for
labeling since it
binds cytoplasmic proteins and although spontaneously released from the cells
with slow
kinetics, it is released massively following target cell necrosis); opsonizing
the target cells with
the anti-antibodies with variant Fe region(s) of the invention; combining the
opsonized
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radiolabeled target cells with effector cells in a mierotitre plate at an
appropriate ratio of target
cells to effector cells; incubating the mixture of cells for 16-18 hours at 37
C.; collecting
supernatants; and analyzing radioactivity. The cytotoxieity of the anti-
antibodies with variant Fe
regions can then be determined using known formulae, for example using the
following formula:
% lysis¨(experimental cpm-target leak cpm)/(detergent lysis cpm-target leak
cpm)x100%.
Alternatively, % lysis=(ADCC-AICC)/(maximum release-spontaneous release).
Specific lysis
can be calculated using the formula: specific lysis=% lysis with the anti-
antibodies with variant
Fe region(s) of the invention-% lysis in the absence of the anti- antibodies
with variant Fe
region(s) of the invention. A graph can be generated by varying either the
target:effector cell
ratio or antibody concentration. Perussia et al., 2000, Methods Mol. Biol.
121: 179-92.
The affinities and binding properties of anti-antibodies with variant Fe
regions
for an FeyR may initially be determined using in vitro assays (biochemical or
immunological
based assays) known in the art for determining Fe-FeyR interactions, i.e.,
specific binding of an
Fe region to an FeyR including but not limited to ELISA assay, surface plasmon
resonance
assay, immunoprecipitation assays. Preferably, the binding properties of the
anti-antibodies with
variant Fe regions in accordance with the invention may also be characterized
by in vitro
functional assays for determining one or more FcyR mediator effector cell
functions. In some
embodiments, the anti-Her3 Fe variants of the invention have similar binding
properties in in
vivo models as those in in vitro based assays. However, the present invention
does not exclude
molecules of the invention that do not exhibit the desired phenotype in in
vitro based assays but
do exhibit the desired phenotype in vivo.
Methods for generating anti-antibodies with variant Fe regions are known. DNA
encoding an amino acid sequence variant of any one or more of the herein
disclosed starting anti-
Her3 antibodies may be prepared by a variety of methods known in the art.
These methods
include, but are not limited to, preparation by site-directed (or
oligonucleotide-mediated)
mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared
DNA encoding
the antibody. In an alternative embodiment of the invention, however, a
nucleic acid encoding
an Fe region of a parent antibody is available and this nucleic acid sequence
is altered to generate
a variant nucleic acid sequence encoding the Fe region variant.
Site-directed mutagenesis is a preferred method for preparing substitution
variants. This technique is well known in the art (see, e.g., Carter et al.
Nucleic Acids Res.
13:4431-4443 (1985) and Kunkel et al, Proc. Natl. Acad. Sci. USA 82:488
(1985)). Briefly, in
carrying out site-directed mutagenesis of DNA, the starting DNA is altered by
first hybridizing
an oligonucleotide encoding the desired mutation to a single strand of such
starting DNA. After
hybridization, a DNA polymerase is used to synthesize an entire second strand,
using the
hybridized oligonucleotide as a primer, and using the single strand of the
starting DNA as a
template. Thus, the oligonucleotide encoding the desired mutation is
incorporated in the
resulting double-stranded DNA.
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PCR mutagenesis is also suitable for making amino acid sequence variants of
the
starting polypeptide, See Higuchi, in PCR Protocols, pp. 177-183 (Academic
Press, 1990); and
Vallette et al., Nue. Acids Res. 17:723-733 (1989). Briefly, when small
amounts of template
DNA are used as starting material in a PCR, primers that differ slightly in
sequence from the
corresponding region in a template DNA can be used to generate relatively
large quantities of a
specific DNA fragment that differs from the template sequence only at the
positions where the
primers differ from the template.
Another method for preparing variants, cassette mutagenesis, is based on the
technique described by Wells et al., Gene 34:315-323 (1985). The starting
material is the
plasmid (or other vector) comprising the starting polypeptide DNA to be
mutated. The codon(s)
in the starting DNA to be mutated are identified. There must be a unique
restriction
endonuclease site on each side of the identified mutation site(s). If no such
restriction sites exist,
they may be generated using the above-described oligonucleotide-mediated
mutagenesis method
to introduce them at appropriate locations in the starting polypeptide DNA.
The plasmid DNA is
cut at these sites to linearize it. A double-stranded oligonucleotide encoding
the sequence of the
DNA between the restriction sites but containing the desired mutation(s) is
synthesized using
standard procedures, wherein the two strands of the oligonucleotide are
synthesized separately
and then hybridized together using standard techniques. This double-stranded
oligonucleotide is
referred to as the cassette. This cassette is designed to have 5' and 3' ends
that are compatible
with the ends of the linearized plasmid, such that it can be directly ligated
to the plasmid. This
plasmid now contains the mutated DNA sequence.
Alternatively, or additionally, the desired amino acid sequence encoding an
anti-
Fe variant can be determined, and a nucleic acid sequence encoding such amino
acid sequence
variant can be generated synthetically.
In certain embodiments, the modification entails one or more amino acid
substitutions. The substitution may, for example, be a "conservative
substitution",
In some embodiments, the molecules of the invention with altered affinities
for
activating and/or inhibitory receptors having variant Fe regions, have one or
more amino acid
modifications.
The Fe regions of any one or more of the herein disclosed anti-antibodies may
be
optimized for a variety of properties. Properties that may be optimized
include but are not
limited to enhanced or reduced affinity for an FcyR. In one embodiment, the Fe
variants are
optimized to possess enhanced affinity for a human activating FeyR, preferably
FcyRI, FcyRIIa,
FeyRne, FcyRIIIa, and FcyRillb, most preferably FcyRIIIa. In an alternative
embodiment, the
Fe region is optimized to possess reduced affinity for the human inhibitory
receptor FeyRIIb.
These embodiments are anticipated to provide anti-antibodies with enhanced
therapeutic
properties in humans, for example enhanced effector function and greater anti-
cancer potency.
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In an alternate embodiment, the Fe variants of the present invention are
optimized
to have reduced or ablated affinity for a human FeyR, including but not
limited to FcyRI,
FeyRIIa, FcyRIIb, FcyRIIc, FcyRIIIa, and FcyRIIIb. These embodiments are
anticipated to
provide anti-Her3 antibodies with enhanced therapeutic properties in humans,
for example
reduced effector function and reduced toxicity. As is known in the art, cancer
cells can be
grafted or injected into mice to mimic a human cancer, a process referred to
as xenografting.
Testing of any one or more of the anti-Her3 antibodies that comprise Fe
variants that are
optimized for one or more mouse FcyR, may provide valuable information with
regard to the
efficacy of the antibody, its mechanism of action, and the like.
The Fc variants of the present invention may also be optimized for enhanced
functionality and/or solution properties in aglycosylated form. In one
embodiment, the
aglycosylated Fe variants of the present invention bind an Fe ligand with
greater affinity than the
aglycosylated form of the parent Fe polypeptide. Exemplary Fe ligands include
but are not
limited to FeyRs, Clq, FeRn, and proteins A and G, and may be from any source,
preferably
human. In an alternative embodiment, the Fe variants of the invention are
optimized to be more
stable and/or more soluble than the aglycosylated form of the parent Fe
polypeptide.
Certain aspects of this invention thus involve antibodies which are (a)
directed
against a particular antigen and (b) belong to a subclass or isotype that is
capable of mediating
the lysis of cells to which the antibody molecule binds. More specifically,
these antibodies
should belong to a subclass or isotype that, upon complexing with cell surface
proteins, activates
serum complement and/or mediates antibody dependent cellular cytotoxicity
(ADCC) by
activating effector cells such as natural killer cells or macrophages. Towards
this end, 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, eysteine
residue(s) may be introduced in the Fe region, thereby allowing interchain
disulfide bond
formation in this region. The homodimeric antibody thus generated may have
improved
internalization capability and/or increased complement-mediated cell killing
and antibody-
dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176:1191-
1195 (1992)
and Shopes, 8. J. Immunol. 148:2918-2922 (1992). Homodimeric antibodies with
enhanced
anti-tumor activity may also be prepared using heterobifunctional cross-
linkers as described in
Wolff et al. Cancer Research 53:2560-2565 (1993). Alternatively, any one or
more of the anti-
antibodies of the invention can be engineered with dual Fe regions and may
thereby have
enhanced complement lysis and ADCC capabilities. See Stevenson et al. Anti-
Cancer Drug
Design 3:219-230 (1989).
In yet another embodiment, the antibodies with variant Fe region(s) of the
invention are characterized for antibody dependent cellular cytotoxicity
(ADCC) see, e.g., Ding
et al, Immunity, 1998, 8:403-11; which is incorporated herein by reference in
its entirety.
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In another example, one or more amino acids in the Fc region can be replaced
with a different amino acid residue such that the antibody has altered Clq
binding and/or reduced
or abolished complement dependent cytotoxicity (CDC). This approach is
described in further
detail in U.S. Pat. No. 6,194,551 by Idusogie et al.
In another example, one or more amino acid residues within amino acid
positions
231 and 239 are altered to thereby alter the ability of the antibody to fix
complement. This
approach is described further in PCT Publication WO 94/29351 by Bodmer et al.
A broad aspect of the invention thus relates to immunoglobulins (e.g., anti-
antibodies disclosed herein), comprising a variant Fc region, having one or
more amino acid
modifications (e.g., substitutions, but also including insertions or
deletions) in one or more
regions, which modifications alter, e.g., increase or decrease, the affinity
of the variant Fc region
for an FcyR. As binding to FeyRilb decreases ADCC, it is important to increase
binding to
FcyRIIIA and decrease binding to FcyRIIB. Thus, in certain embodiments, said
one or more
amino acid modification increases the affinity of the variant Fc region for
FcyRIIIA and/or
FeyRIIA.
In certain embodiments, the herein described anti-antibodies with a variant Fe
region further specifically bind FcyRIIB (via the Fc region) with a lower
affinity than a
comparable antibody molecule (i.e., having the same amino acid sequence as the
antibody with a
variant Fe region except for the one or more amino acid modifications in the
Fe region)
comprising the wild-type Fe region binds FcyRII13.
In some embodiments, the invention encompasses molecules comprising a variant
Fc region, wherein said variant Fe region comprises at least one amino acid
modification relative
to a wild type Fc region, which variant Fe region does not bind any FeyR or
binds with a reduced
affinity, relative to a comparable molecule comprising the wild-type Fc
region, as determined by
standard assays (e.g., in vitro assays) known to one skilled in the art. In a
specific embodiment,
the invention encompasses molecules comprising a variant Fe region, wherein
said variant Fe
region comprises at least one amino acid modification relative to a wild type
Fc region, which
variant Fe region only binds one FcyR, wherein the FcyR is FeyRIIIA. In
another specific
embodiment, the invention encompasses molecules comprising a variant Fe
region, wherein said
variant Fc region comprises at least one amino acid modification relative to a
wild type Fe
region, which variant Fc region only binds one FcyR wherein the FeyR is
FcyRIIA. In yet
another embodiment, the invention encompasses an anti-antibody molecule
comprising a variant
Fe region, wherein the variant Fe region comprises at least one amino acid
modification relative
to a wild type Fc region, which variant Fe region only binds one FeyR wherein
the FeyR is
FcyRIIB.
In yet another embodiment, provided herein is at least one or more anti-
antibodies which comprises an antigen binding region and a variant Fe region,
wherein the
variant Fe region: (A) differs from a wild-type Fe region by comprising at
least one amino acid
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modification according to the EU index as in Kabat, relative to the wild-type
Fe region
(unmodified), e.g., any one or more corresponding anti-antibodies disclosed
herein that include
the wild type Fe polypeptide; and (13) binds an FcyR with an increased
affinity relative to a said
wild-type Fe region.
The Fe variants of the present invention may be combined with other Fe
modifications, including but not limited to modifications that alter effector
function or
interaction with one or more Fe ligands. Such combination may provide
additive, synergistic, or
novel properties in antibodies or Fe fusions. In one embodiment, the Fe
variants of the present
invention may be combined with other known Fe variants (Duncan et al., 1988,
Nature 332:563-
564; Lund et al., 1991, J Immunol 147:2657-2662; Lund et al., 1992, Mol
Immunol 29:53-59;
Alegre et al., 1994, Transplantation 57:1537-1543; Hutchins et al., 1995, Proc
Nati Acad Sci
USA 92:11980-11984; Jefferis et al., 1995, Immunol Left 44:111-117; Lund et
al., 1995, Faseb
J9:115-119; Jefferis et al., 1996, Immunol Left 54:101-104; Lund et al., 1996,
J Immunol
157:4963-4969; Armour et al., 1999, Eur J Immunol 29:2613-2624; Idusogie et
al., 2000, J
Immunol 164:4178-4184; Reddy et al., 2000, J Immunol 164:1925-1933; Xu etal.,
2000, Cell
Immunol 200:16-26; Idusogie et al., 2001, J Immunol 166:2571-2575; Shields et
al., 2001, J Biol
Chem 276:6591-6604; Jefferis et al., 2002, Immunol Left 82:57-65; Presta et
al., 2002, Biochem
Soc Trans 30:487-490; Hinton et al., 2004, J Bid Chem 279:6213-6216) (U.S.
Pat. No.
5,624,821; U.S. Pat. No. 5,885,573; U.S. Pat. No. 6,194,551; PCT WO 00/42072;
PCT WO
99/58572; US 2004/0002587 Al). In an alternate embodiment, the Fe variants of
the present
invention are incorporated into an antibody or Fe fusion that comprises one or
more engineered
glyeoforms (infra). Thus combinations of the Fe variants of the present
invention with other Fe
modifications, as well as undiscovered Fe modifications, are contemplated with
the goal of
generating novel antibodies or Fe fusions with optimized properties.
Anti- Her3 Engineered Glyeoforms
The invention additionally, encompasses anti-antibodies including fragments
thereof which are differentially modified during or after translation, e.g.,
by glycosylation,
proteolytic cleavage etc. Any of numerous chemical modifications may be
carried out by known
techniques, including but not limited to, specific chemical cleavage by
trypsin, papain, metabolic
synthesis in the presence of tunicarnycin etc.
Antibodies are glycoproteins containing carbohydrate structures at conserved
positions in the heavy chain constant regions, with each isotype possessing a
distinct array of N-
linked carbohydrate structures, which variably affect protein assembly,
secretion or functional
activity. The structure of the attached N-linked carbohydrate varies
considerably and can
include high-mannose, multiply-branched as well as biantennary complex
oligosaceharides.
(Wright, A., and Morrison, S. L., Trends Biotech. 15:26-32 (1997)). The major
carbohydrate
units are attached to amino acid residues of the constant region of the
antibody. Carbohydrate is
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also known to attach to the antigen binding sites of some antibodies and may
affect the antibody-
binding characteristics by limiting access of the antigen to the antibody
binding site. Typically,
there is heterogeneous processing of the core oligosaccharide structures
attached at a particular
glycosylation site such that even monoclonal antibodies exist as multiple
glycoforms. Likewise,
it has been shown that major differences in antibody glycosylation occur
between cell lines, and
even minor differences are seen for a given cell line grown under different
culture conditions.
(Lifely, M. R. et al., Glycobiology 5(8):813-22 (1995)).
Monoclonal antibodies often achieve their therapeutic benefit through two
binding events. First, the variable domain of the antibody binds to a specific
tumor receptor on
the surface of the target cell. This is followed by recruitment of effector
cells such as natural
killer (NK) cells that bind to the constant region (Fc) of the antibody and
destroy cells to which
the antibody has bound. This process, known as antibody-dependent cell
cytotoxicity (ADCC),
partially depends on a specific N-glycosylation event at Asn 297 in the Fc
domain of the heavy
chain of IgGls. In general, antibodies that lack this N-glycosylation
structure still bind antigen
but cannot mediate ADCC, apparently as a result of reduced affinity of the Fc
domain of the
antibody for the Fc receptor Fc `YRIfla on the surface of NK cells.
Interestingly, there is a linear
increase of in vitro complement activation with increasing terminal
galactosylation of the
carbohydrate moiety in the Fc domain. There are a number of roles associated
with the
carbohydrate units. Glycosylation may affect overall solubility and the rate
of catabolism of the
antibody. It is also known that carbohydrate is necessary for cellular
secretion of some antibody
chains. It has been demonstrated that glycosylation of the constant region
plays a vital role in
the effector functioning of an antibody; without this glycosylation in its
correct configuration,
the antibody may be able to bind to the antigen but may not be able to bind
for example to
macrophages, helper and suppressor cells or complement, to carry out its role
of blocking or
lysing the cell to which it is bound. Hyperglycosylated proteins have been
shown to exhibit
increased serum half-life, are less sensitive to proteolysis and more heat-
stable compared with
the non-glycosylated forms. (Leatherbarrow et al., Mol. Immunol. 22:407
(1985)).
IgG1 type antibodies, which represent the most commonly used antibodies in
cancer immunotherapy, are glycoproteins that have a conserved N-linked
glycosylation site at
Asn297 in each CH2 domain. The two complex bi-antennary oligosaccharides
attached to
Asn297 are buried between the CH2 domains, forming extensive contacts with the
polypeptide
backbone, and their presence is essential for the antibody to mediate effector
functions such as
antibody dependent cellular cytotoxicity (ADCC) (Lifely, M. R., et al.,
Glycobiology 5:813-822
(1995); Jefferis, R., et al., Immunol Rev. 163:59-76 (1998); Wright, A. and
Morrison, S. L.,
Trends Biotechnol. 15:26-32 (1997)). Glycosylation of IgG at asparagine 297 in
the CH2 domain
is also required for full capacity of IgG to activate the classical pathway of
complement-
dependent cytolysis (Tao and Morrison, J. Immunol. 143:2595 (1989)).
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More, glycosylation of IgM at asparagine 402 in the CH 3 domain is necessary
for
proper assembly and eytolytic activity of the antibody (Muraoka and Shulman,
J. Immunol.
142:695 (1989)). Likewise, removal of glycosylation sites as positions 162 and
419 in the CHI
and CH3 domain of an IgA antibody has been shown to lead to intracellular
degradation and at
least 90% inhibition of secretion (Taylor and Wall, Mol. Cell. Biol.
8:4197(1988)).
Glycosylation of immunoglobulins in the variable (V) region has also been
observed. Sox and Hood, Proc. Natl. Acad. Sci. USA 66:975 (1970), reported
that about 20% of
human antibodies are glycosylated in the V region. Glycosylation of the V
domain is believed to
arise from fortuitous occurrences of the N-linked glycosylation signal Asn-Xaa-
Ser/Thr in the V
region sequence and has not been recognized in the art as playing an important
role in
immunoglobulin function.
It has also been reported that glycosylation at CDR2 of the heavy chain, in
the
antigen binding site, of a murine antibody specific for .alpha.-(1-6)dextran
increases its affinity
for dextran (Wallick et al., J. Exp. Med. 168:1099 (1988) and Wright et al.,
EMBO J. 10:2717
(1991)). See Patent No. 6,933,368. Some classes and subclasses also have 0-
linked sugars,
often in the hinge region, eg. IgD and IgA from some species.
For example, the absence of fucose or the presence of a bisecting N-
acetylglucosamine in the carbohydrate structure of the monoclonal antibody,
has been positively
correlated with the potency of ADCC. Specifically, defucosylated carbohydrate
residues on
monoclonal antibodies have been shown to enhance the ADCC capability of the
target antibody
more than threefold. "Glycosylation of therapeutic proteins in different
production systems"
Acta Paediatrica, 96: 17-22 (2007); Shields etal., "Lack of Fucose on Human
IgG I N-Linked
Oligosaccharide Improves Binding to Human FcyRIII and Antibody-dependent
Cellular
Toxicity", J. Biol. Chem., 277: 26733-26740 (2002). Likewise, specific
engineered glyeoforms
of monoclonal antibodies, which interact solely with the Fc7RIIIa receptor of
natural killer cells,
exhibit superior ADCC compared with heterogeneous glycoforms that interact
with different Fe
receptors. The collective data impel the conclusion that glycoengineering for
directed
glycosylation of therapeutic proteins can improve the therapeutic effect in
vivo. See Umaa, P. et
al., Nature Biotechnol. 17:176-180 (1999)). See also U.S Patent No. 5,624,821;
US Patent No.
6,602,684; WO 00/42072 and WO 07/048122, the content of each of which is
incorporated in its
entirety by reference herein. See also US serial No. 2006/0182744. Not only is
ADCC
dependent on glycosylation of the Fe domain, but the degree of cell-mediated
killing is also
sensitive to the composition of the glycans in the Fe region of the antibody.
As a consequence, the present invention, in related embodiments, provides
"Engineered Glycoforms" of any one or more of the anti-antibodies disclosed
herein including
fragments thereof, wherein the glycosylation profiles of the antibody are
altered to enhance their
use in the treatment of specific types of cancers or other disease states.
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By "engineered glycoform" as used herein is meant a carbohydrate composition
that is covalently attached to an Fe polypeptide, wherein the carbohydrate
composition differs
chemically from that of a parent Fc polypeptide. Engineered glycoforms may be
useful for a
variety of purposes, including but not limited to enhancing or reducing
effector function.
Engineered glycoforms may be generated by a variety of methods known in the
art (Umana et
al., 1999, Nat Biotechnol 17:176-180; Davies et al., 2001, Biotechnol Bioeng
74:288-294;
Shields et al., 2002, .1 Biol Chem 277:26733-26740; Shinkawa et al., 2003, J
Biol Chem
278:3466-3473); (U.S. Pat No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser.
No. 10/113,929;
PCT WO 00/61739A1; PCT WO 01/29246A1; PCT WO 02/31140A1; PCT WO 02/30954A1);
(Potelligentml technology [Biowa, Inc., Princeton, N.J.]; GlycoMAbTm
glycosylation
engineering technology[GLYCART biotechnology AG, Zurich, Switzerland)). Many
of these
techniques are based on controlling the level of fucosylated and/or bisecting
oligosaccharides
that are covalently attached to the Fc region, for example by expressing an Fc
polypeptide in
various organisms or cell lines, engineered or otherwise (for example Lec-13
CHO cells or rat
hybridoma YB2/0 cells), by regulating enzymes involved in the glycosylation
pathway (for
example FUT8 [.alpha.1,6-fucosyltranserase] and/or .beta.1-4-N-
acetylglucosaminyltransferase
III [GnTIII]), or by modifying carbohydrate(s) after the Fc polypeptide has
been expressed.
Engineered glycoform typically refers to the different carbohydrate or
oligosaccharide; thus an
Fc polypeptide, for example an antibody or Fc fusion, may comprise an
engineered glycoform.
Alternatively, engineered glycoform may refer to the Fc polypeptide that
comprises the different
carbohydrate or oligosaccharide.
Covalent modification of the target antibody included within the scope of this
invention comprises altering the native glycosylation pattern of the
polypeptide. "Altering the
native glycosylation pattern" is intended for purposes herein to mean deleting
one or more
carbohydrate moieties found in the native target antibody, and/or adding one
or more
glycosylation sites that are not present in the native target antibody.
In still another embodiment, the glycosylation of an antibody is modified. For
example, an aglycoslated antibody can be made (i.e., the antibody lacks
glycosylation).
Glycosylation can be altered to, for example, increase the affinity of the
antibody for antigen.
Such carbohydrate modifications can be accomplished by, for example, altering
one or more
sites of glycosylation within the antibody sequence. For example, one or more
amino acid
substitutions can be made that result in elimination of one or more variable
region framework
glycosylation sites to thereby eliminate glycosylation at that site. Such
aglycosylation may
increase the affinity of the antibody for antigen. Such an approach is
described in further detail in
U.S. Pat. Nos. 5,714,350 and 6,350,861 by Co et al.
Additionally or alternatively, an antibody can be made that has an altered
type of
glycosylation, such as a hypofucosylated antibody having reduced amounts of
fueosyl residues
or an antibody having increased bisecting GleNac structures. Such altered
glycosylation patterns
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have been demonstrated to increase the ADCC ability of antibodies. Such
carbohydrate
modifications can be accomplished by, for example, expressing the antibody in
a host cell with
altered glycosylation machinery. Cells with altered glycosylation machinery
have been
described in the art and can be used as host cells in which to express
recombinant antibodies of
the invention to thereby produce an antibody with altered glycosylation. For
example, EP
1,176,195 by Hanai et al. describes a cell line with a functionally disrupted
FUT8 gene, which
encodes a fucosyl transferase, such that antibodies expressed in such a cell
line exhibit
hypofucosylation. PCT Publication WO 03/035835 by Presta describes a variant
CHO cell line,
Lec13 cells, with reduced ability to attach fucose to Asn(297)-linked
carbohydrates, also
resulting in hypofucosylation of antibodies expressed in that host cell (see
also Shields, R. L. et
al. (2002) J. Biol. Chem. 277:26733-26740). PCT Publication WO 99/54342 by
Umana et al.
describes cell lines engineered to express glycoprotein-modifying glycosyl
transferases (e.g.,
beta(1,4)-N-acetylglucosaminyltransferase HI (GnTIII)) such that antibodies
expressed in the
engineered cell lines exhibit increased bisecting GIcNac structures which
results in increased
ADCC activity of the antibodies (see also Umana et al. (1999) Nat. Biotech.
17:176-180).
Antibodies disclosed herein can be glycosylated in both the C-regions and
in the V-regions. Obviously C-region glycosylation is dependent on the
particular sequence
which by definition defines the class and subclass of the antibody. As noted
elsewhere, many
classes of antibody have conserved N-linked glycosylation sites in the
constant domains. For
example all IgG antibodies have a conserved N-linked glycosylation site in the
CH2 domain at
residue Asn297.
Two basic types of glycosylation of therapeutic proteins are known: 0-linked
and
N-linked glycosylation. 0-linked glycosylation is initiated by the attachment
of N-acetyl-
galactosamine to a serine or threonine residue in the peptide backbone of the
therapeutic protein.
The proximal carbohydrate is the target for glycosyltransferases to form a
mature 0-glycan
structure. It is difficult to predict where 0-linked glycosylation will occur
in the protein as there
is no clear consensus amino acid sequence for 0-linked glycosylation (3, 4).
However, 0-linked
glycosylation is affected by secondary structural elements such as an extended
13-turn. In
contrast, consensus amino acid sequences are known for N-glycosylation. N-
glycosylation
occurs at a specific sequence motif, Asn¨X¨Thr/Ser (sequon or consensus
sequence; where X is
any amino acid except proline), and this consensus sequence must be accessible
to the precursor
transferring the enzyme. In the case where X = Pro, no glycosylation takes
place. Asn¨X¨
Thr/Ser sequences in 13-turns can influence the protein conformation by N-
linked glycosylation.
As glycosylation precedes final protein folding, the structure of the
resultant therapeutic protein
may be altered, resulting in differences in activity or stability compared
with the non-
glycosylated form. Thus, recombinant antibodies of the invention can be
modified to recreate or
create additional glycosylation sites if desired, which is simply achieved by
engineering the
appropriate amino acid sequences (such as Asn-X-Ser, Asn-X-Thr, Ser, or Thr)
into the primary
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sequence of the antibody. Glycosylation of polypeptides is typically either N-
linked or 0-linked.
N-linked refers to the attachment of the carbohydrate moiety to the side chain
of an asparagine
residue. The tri-peptide sequences asparagine-X-serine and asparagine-X-
threonine, where X is
any amino acid except proline, are the recognition sequences for enzymatic
attachment of the
carbohydrate moiety to the asparagine side chain. Thus, the presence of either
of these tri-
peptide sequences in a polypeptide creates a potential glycosylation site. 0-
linked glycosylation
refers to the attachment of one of the sugars N-acetylgalactosamine,
galactose, or xylose, to a
hydroxyamino acid, most commonly serine or threonine, although 5-
hydroxyproline or 5-
hydroxylysine may also be used.
Thus, in certain embodiments of the invention a mutant anti-antibody is
provided
that exhibits a higher affinity for its antigen e.g., receptor or endogenous
binding partner, than a
parent antibody that comprises a parent immunoglobulin chain, wherein the
mutant
immunoglobulin chain comprises an amino acid substitution that eliminates a
variable region
glycosylation site of the parent immunoglobulin chain, said elimination having
the effect of
increasing the affinity of the mutant antibody relative to the parent
antibody. Alternative
embodiments contemplate variants that are "aglycosylated."
"Glycosylation sites" refer to amino acid residues which are recognized by a
eukaryotic cell as locations for the attachment of sugar residues. The amino
acids where
carbohydrate, such as oligosaccharide, is attached are typically asparagine (N-
linkage), serine
(0-linkage), and threonine (0-linkage) residues. In order to identify
potential glycosylation sites
within an antibody or antigen-binding fragment, the sequence of the antibody
is examined, for
example, by using publicly available databases such as the website provided by
the Center for
Biological Sequence Analysis (see http://www.cbs.dtu.dk/services/NetNGlyc/ for
predicting N-
linked glycosylation sites) and http://www.cbs.dtu.dk/services/Net0Glyc/ for
predicting 0-
linked glycosylation sites). Additional methods for altering glycosylation
sites of antibodies are
described in U.S. Pat. Nos. 6,350,861 and 5,714,350.
Several approaches have been attempted to alter the glycosylation state of IgG
antibodies: inhibition of glycosylation by culturing cells in the presence of
the drug tunicamycin
(Leatherbarrow et al. 1985; Walker et al. 1989; Pound et al. 1993); treatment
of glycoproteins
with specific glycosidases that remove the entire oligosaccharide or specific
residues (Tsuchiya
et al. 1989; Boyd et al. 1995); or site-directed mutagenesis to remove either
the carbohydrate
addition site (Tao, Smith et al. 1993) or residues within the C112 region that
contact the core
oligosaccharide residues (Lund, Takahashi et al. 1996). These studies have
confirmed that the
presence of carbohydrate is essential to antibody function.
Glycosylation can be achieved by methods known in the art, e.g., by producing
the antibody in a mammalian host cell such as Chinese Hamster Ovary (CHO) cell
or in yeast.
Addition of glycosylation sites to the target antibody is conveniently
accomplished by altering
the amino acid sequence such that it contains one or more of the above-
described tri-peptide
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sequences (for N-linked glycosylation sites). The alteration may also be made
by the addition of,
or substitution by, one or more serine or threonine residues to the native
target antibody
sequence (for 0-linked glycosylation sites). For ease, the target antibody
amino acid sequence is
preferably altered through changes at the DNA level, particularly by mutating
the DNA encoding
Yeast provides substantial advantages over bacteria for the production of
immunoglobulin H and L chains. Yeasts carry out post-translational peptide
modifications
Another means of increasing the number of carbohydrate moieties on the target
antibody is by chemical or enzymatic coupling of glycosides to the
polypeptide. These
procedures are advantageous in that they do not require production of the
polypeptide in a host
cell that has glycosylation capabilities for N-and 0-linked glycosylation.
Depending on the
Moreover, the main species antibody or variant thereof may further comprise
glycosylation variations, non-limiting examples of which include antibody
comprising a GI or
G2 oligosaccharide structure attached to the Fc region thereof, antibody
comprising a
carbohydrate moiety attached to a light chain thereof (e.g. one or two
carbohydrate moieties,
Immune effector functions are unnecessary or even deleterious in certain
clinical
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affinity of an antibody of the invention or antigen-binding fragment thereof
to the antigen while
minimizing or reducing its binding to the Fe receptor, the antibody can be
mutated at particular
regions necessary for Fc receptor (FcR) interactions (see e.g., Canfield, S.
M. and S. L. Morrison
(1991) J. Exp, Med. 173:1483-1491; and Lund, J. et al. (1991) J. of Immunol.
147:2657-2662).
Reduction in FeR binding ability of the antibody may also reduce other
effector functions which
rely on FcR interactions, such as opsonization and phagocytosis and antigen-
dependent cellular
cytotoxicity, glycosylation sites of the antibody can be altered, for example,
by mutagenesis
(e.g., site-directed mutagenesis). As a consequence, such antibodies do not
exhibit substantial
immune effector functions that are dependent on glycosylation of the Fe
region. Generally and
preferably, an aglycosylated antibody of the invention does not exhibit
substantial immune
effector functions except for binding to FeRn. In some embodiments, an
antibody of the
invention or a fragment thereof does not possess substantial or completely
lacks effector
functions other than FcRn binding. In one embodiment, said effector function
is complement
lysis. In one embodiment, said effector function is antibody dependent cell
cytotoxicity (ADCC).
In one embodiment, the antibody fragment binds FeRn.
Aglycosylated antibodies can be produced by a variety of methods known in the
art. A convenient method comprises expressing the antibody in a prokaryotic
host cell such as E.
coli. The amino acids where carbohydrate, such as oligosaccharide, is attached
are typically
asparagine (N-linkage), serine (0-linkage), and threonine (0-linkage)
residues. In order to
identify potential glycosylation sites within an antibody or antigen-binding
fragment, the
sequence of the antibody is examined, for example, by using publicly available
databases such as
the website provided by the Center for Biological Sequence Analysis (see
http://www.cbs.dtu.dk/services/NetNGlyc/ for predicting N-linked glycosylation
sites) and
http://www.cbs.dtu.dk/services/Net0Glyc/ for predicting 0-linked glycosylation
sites).
Additional methods for altering glycosylation sites of antibodies are
described in U.S. Pat. Nos.
6,350,861 and 5,714,350.
Removal of carbohydrate moieties present on the native target antibody may
also
be accomplished chemically or enzymatically. Chemical deglycosylation requires
exposure of
the polypeptide to the compound trifluoromethanesulfonic acid, or an
equivalent compound.
This treatment results in the cleavage of most or all sugars except the
linking sugar (N-
acetylglucosamine or N-acetylgalactosamine), while leaving the polypeptide
intact. Chemical
deglycosylation is described by Hakimuddin et al. (Arch. Biochem. Biophys.,
259:52 [1987])
and by Edge et al. (Anal. Biochem., 118:131 [1981]). Enzymatic cleavage of
carbohydrate
moieties on polypeptides can be achieved by the use of a variety of endo- and
exo-glycosidases
as described by Thotakura et al. (Meth. Enzymol. 138:350 [1987]). Glycosylati
011 at potential
glycosylation sites may be prevented by the use of the compound tunicamycin as
described by
Duskin et al. (J. Biol. Chem., 257:3105 [1982]). Tunicamycin blocks the
formation of protein-N-
glycoside linkages.
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Thus, in some embodiments, the antibodies of the invention or an antigen-
binding
fragment thereof is modified to reduce or eliminate potential glycosylation
sites. In still another
embodiment, the constant region of the antibody, or fragment thereof of the
invention is
modified to reduce at least one constant region-mediated biological effector
function relative to
an unmodified antibody.
Antibody-salvage receptor binding epitope 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 ease,
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 Fe 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 Fe 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
Fe domain are transferred. Still more preferred, the epitope is taken from the
CH2 domain of the
Fe region (e.g., of an IgG) and transferred to the CHI, CH3, or VII region, or
more than one such
region, of the antibody. Alternatively, the epitope is taken from the CH2
domain of the Fe region
and transferred to the CL region or VL region, or both, of the antibody
fragment.
Antibody Derivatives
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The antibodies of the present invention can be further modified to contain
additional nonproteinaceous moieties that are known in the art and readily
available. Preferably,
the moieties suitable for derivatization of the antibody are water soluble
polymers. Non-limiting
examples of water soluble polymers include, but are not limited to,
polyethylene glycol (PEG),
copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose,
dextran, polyvinyl
alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic
anhydride copolymer, polyaminoacids (either homopolymers or random
copolymers), and
dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol
homopolymers,
prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols
(e.g., glycerol),
polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde
may have
advantages in manufacturing due to its stability in water. The polymer may be
of any molecular
weight, and may be branched or unbranched. The number of polymers attached to
the antibody
may vary, and if more than one polymer is attached, they can be the same or
different molecules.
In general, the number and/or type of polymers used for derivatization can be
determined based
on considerations including, but not limited to, the particular properties or
functions of the
antibody to be improved, whether the antibody derivative will be used in a
therapy under defined
conditions, etc.
Other modifications of the antibody are contemplated herein. For example, the
antibody may be linked to one of a variety of nonproteinaceous polymers, e.g.,
polyethylene
glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene
glycol and
polypropylene glycol. The antibody also may be entrapped in microcapsules
prepared, for
example, by coacervation techniques or by interfacial polymerization (for
example,
hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules,
respectively), in colloidal drug delivery systems (for example, liposomes,
albumin microspheres,
microemulsions, nano-particles and nanocapsules), or in macroemulsions. Such
techniques are
disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed.,
(1980).
In vivo Stabilization Using Polymeric Stabilizing Moieties - PEGylation:
Another type of covalent modification of the target antibody, e.g., any one or
more of the anti-Her3 antibodies of the invention comprises linking the target
antibody to
various nonproteinaceous polymers, e.g. polyethylene glycol, polypropylene
glycol or
polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;
4,496,689; 4,301,144;
4,670,417; 4,791,192 or 4,179,337. Thus, in some embodiments, the antibodies
and antibody
fragments of the invention may be chemically modified to provide a desired
effect such as
increased solubility, stability and circulating time of the polypeptide, or
decreased
immunogenicity.
The antibody or fragments thereof polypeptides may be modified at random
positions within the molecule, or at predetermined positions within the
molecule and may
include one, two, three or more attached chemical moieties. For example,
PEGylation of
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antibodies and antibody fragments of the invention may be carried out by any
of the PEGylation
reactions known in the art, See for example, EP 0 154 316 by Nishimura et al.
and EP 0 401 384
by Ishikawa et at. Methods for determining the degree of substitution are
discussed, for example,
in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992). (each
of which is
incorporated by reference herein in its entirety). To PEGylate an antibody,
the antibody, or
fragment thereof, typically is reacted with polyethylene glycol (PEG), such as
a reactive ester or
aldehyde derivative of PEG, under conditions in which one or more PEG groups
become
attached to the antibody or antibody fragment. Preferably, the PEGylation is
carried out via an
acylation reaction or an alkylation reaction with a reactive PEG molecule (or
an analogous
reactive water-soluble polymer). As used herein, the term "polyethylene
glycol" is intended to
encompass any of the forms of PEG that have been used to defivatize other
proteins, such as
mono (CI-CIO) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-
maleimide. In
certain embodiments, the antibody to be PEGylated is an aglycosylated
antibody.
In another aspect, a single immunoglobulin variable domain derived from an
invention antibody containing composition is stabilized in vivo by linkage or
association with a
(non-polypeptide) polymeric stabilizing moiety. Examples of this type of
stabilization are
described, for example, in W099/64460 (Chapman et al.) and EP1,160,255 (King
et al.), each of
which is incorporated herein by reference. Specifically, these references
describe the use of
synthetic or naturally-occurring polymer molecules, such as polyalkylene,
polyalkenylenes,
polyoxyalkylenes or polysaccharides, to increase the in vivo half-life of
immunoglobulin
polypeptides. A typical example of a stabilizing moiety is polyethylene
glycol, or PEG, a
polyalkylene. The process of linking PEG to an immunoglobulin polypeptide is
described in
these references and is referred to herein as "PEGylation." As described
therein, an
immunoglobulin polypeptide can be PEGylated randomly, as by attachment of PEG
to lysine or
other amino acids on the surface of the protein, or site-specifically, e.g.,
through PEG attachment
to an artificially introduced surface cysteine residue. Depending upon the
immunoglobulin, it
may be preferred to use a non-random method of polymer attachment, because
random
attachment, by attaching in or near the antigen-binding site or sites on the
molecule often alters
the affinity or specificity of the molecule for its target antigen.
Polyethylene glycol can also be
attached to proteins using a number of different intervening linkers. For
example, U.S. Pat. No.
5,612,460, the entire disclosure of which is incorporated herein by reference,
discloses urethane
linkers for connecting polyethylene glycol to proteins. Protein-polyethylene
glycol conjugates
wherein the polyethylene glycol is attached to the protein by a linker can
also be produced by
reaction of proteins with compounds such as MPEG-succinimidylsuccinate, MPEG
activated
with I, r-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p-
nitrophenolcarbonate, and various MPEG-succinate derivatives. A number of
additional
polyethylene glycol derivatives and reaction chemistries for attaching
polyethylene glycol to
proteins are described in WO 98/32466, the entire disclosure of which is
incorporated herein by
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reference. PEGylated protein products produced using the reaction chemistries
set out herein are
included within the scope of the invention.
One system for attaching polyethylene glycol directly to amino acid residues
of
proteins without an intervening linker employs tresylated MPEG, which is
produced by the
modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride
(C1S02 CH2
CF3). Upon reaction of protein with tresylated MPEG, polyethylene glycol is
directly attached to
amine groups of the protein. Thus, the invention includes protein-polyethylene
glycol
conjugates produced by reacting proteins of the invention with a polyethylene
glycol molecule
having a 2,2,2-trifluoroethane sulphonyl group.
A general method for preparing PEGylated antibodies and antibody fragments of
the invention will generally comprise the steps of (a) reacting the antibody
or antibody fragment
with polyethylene glycol, such as a reactive ester or aldehyde derivative of
PEG, under
conditions whereby the antibody or antibody fragment becomes attached to one
or more PEG
groups, and (b) obtaining the reaction products. It will be apparent to one of
ordinary skill in the
art to select the optimal reaction conditions or the acylation reactions based
on known
parameters and the desired result.
There are a number of attachment methods available to those skilled in the
art,
e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF),
see also Malik et
al., Exp. Hematol. 20:1028-1035 (1992) (reporting PEGylation of GM-CSF using
tresyl
chloride). For example, polyethylene glycol may be covalently bound through
amino acid
residues via a reactive group, such as, a free amino or carboxyl group.
Reactive groups are those
to which an activated polyethylene glycol molecule may be bound. The amino
acid residues
having a free amino group may include lysine residues and the N-terminal amino
acid residues;
those having a free carboxyl group may include aspartic acid residues,
glutamic acid residues
and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a
reactive group
for attaching the polyethylene glycol molecules. Preferred for therapeutic
purposes is attachment
at an amino group, such as attachment at the N-terminus or lysine group.
As suggested above, polyethylene glycol may be attached to proteins via
linkage
to any of a number of amino acid residues. As indicated above, pegylation of
the proteins of the
invention may be accomplished by any number of means. For example,
polyethylene glycol
may be attached to the protein either directly or by an intervening linker.
Linkerless systems for
attaching polyethylene glycol to proteins are described in Delgado et al.,
Crit. Rev. Thera. Drug
Carrier Sys. 9:249-304 (1992); Francis et al., Intern. J. of Hematol. 68:1-18
(1998); U.S. Pat. No.
4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO 98/32466, the
disclosures of each of
which are incorporated herein by reference. For example, polyethylene glycol
can be linked to a
protein via covalent bonds to lysine, histidine, aspartic acid, glutamic acid,
or cysteine residues.
One or more reaction chemistries may be employed to attach polyethylene glycol
to specific
amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or
cysteine) of the
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protein or to more than one type of amino acid residue (e.g, lysine,
histidine, aspartic acid,
glutamic acid, cysteine and combinations thereof) of the protein.
One may specifically desire proteins chemically modified at the N-terminus.
Using polyethylene glycol as an illustration of the present composition, one
may select from a
variety of polyethylene glycol molecules (by molecular weight, branching,
etc.), the proportion
of polyethylene glycol molecules to protein (or peptide) molecules in the
reaction mix, the type
of PEGylation reaction to be performed, and the method of obtaining the
selected N-terminally
PEGylated protein. The method of obtaining the N-terminally PEGylated
preparation (i.e.,
separating this moiety from other mono-PEGylated moieties if necessary) may be
by purification
of the N-terminally PEGylated material from a population of PEGylated protein
molecules.
Selective proteins chemically modified at the N-terminus may be accomplished
by reductive
alkylation which exploits differential reactivity of different types of
primary amino groups
(lysine versus the N-terminal) available for defivatization in a particular
protein. Under the
appropriate reaction conditions, substantially selective derivatization of the
protein at the N-
terminus with a carbonyl group containing polymer is achieved.
The polymer may be of any molecular weight, and may be branched or
unbranched. Branched polyethylene glycols are described, for example, in U.S.
Pat. No.
5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72 (1996);
Vorobjev et al.,
Nucleosides Nucleotides 18:2745-2750(1999); and Caliceti et al., Bioconjug.
Chem, 10;638-646
(1999), the disclosures of each of which are incorporated herein by reference.
For polyethylene
glycol, the preferred molecular weight is between about 1 kDa and about 100
kDa (the term
"about" indicating that in preparations of polyethylene glycol, some molecules
will weigh more,
some less, than the stated molecular weight) for ease in handling and
manufacturing. Other sizes
may be used, depending on the desired therapeutic profile (e.g., the duration
of sustained release
desired, the effects, if any on biological activity, the ease in handling, the
degree or lack of
antigen icily and other known effects of the polyethylene glycol to a
therapeutic protein or
analog).
It is preferred that the addition of PEG or another polymer does not interfere
with
the antigen-binding affinity or specificity of the antibody variable domain
polypeptide. By "does
not interfere with the antigen-binding affinity or specificity" is meant that
the PEG-linked
antibody single variable domain has an 1050 or ND50 which is no more than 10%
greater than the
IC50 or ND50, respectively, of a non-PEG-linked antibody variable domain
having the same
antibody single variable domain. In the alternative, the phrase "does not
interfere with the
antigen-binding affinity or specificity" means that the PEG-linked form of an
antibody single
variable domain retains at least 90% of the antigen binding activity of the
non-PEGylated form
of the polypeptide.
PEGylated antibodies and antibody fragments may generally be used to treat
conditions that may be alleviated or modulated by administration of the
antibodies and antibody
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fragments described herein. Generally the PEGylated antibodies and antibody
fragments have
increased half-life, as compared to the non-PEGylated antibodies and antibody
fragments. The
PEGylated antibodies and antibody fragments may be employed alone, together,
or in
combination with other pharmaceutical compositions.
The target antibody may also be entrapped in microcapsules prepared, for
example, by coacervation techniques or by interfacial polymerization (for
example,
hydroxymethylcellulose or gelatin-microcapsules and poly-
[methylmethacylate]microcapsules,
respectively), in colloidal drug delivery systems (for example, liposomes,
albumin microspheres,
microemulsions, nano-particles and nanocapsules), or in macroemulsions. Such
techniques are
disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A., Ed.,
(1980).
Antibody-Coated Liposomes and Therapeutics (Immunoliposomes)
Liposomal formulations are often used in therapeutics and pharmaceuticals,
However, the biodistribution of liposomes in initial studies meant that such
formulations were
not widely applicable for use in humans. The technology of "stealth or
stealthed" liposomes and
formulations was thus developed, which allows liposomes to circulate for
longer durations. A
preferred agent for use in stealthing liposomes is polyethylene glycol (PEG),
and the resultant
liposomes are also termed PEGylated liposomes.
Any one of the antibodies or fragments thereof disclosed herein may also be
formulated as immunoliposomes. Liposomes containing the antibody are prepared
by methods
known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci.
USA 82: 3688 (1985);
Hwang et al., Proc. Natl. Acad Sci. USA 77: 4030 (1980); and U.S. Pat. Nos.
4,485,045 and
4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat.
No. 5,013,556.
Particularly useful liposomes can be generated by the reverse phase
evaporation
method with a lipid composition comprising phosphatidylcholine, cholesterol
and PEG-
derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of
defined pore size to yield liposomes with the desired diameter. Fab' fragments
of the antibody of
the present invention can be conjugated to the liposomes as described in
Martin et al., J. Biol.
Chem. 257: 286-288 (1982) via a disulfide interchange reaction. A
chemotherapeutic agent
(such as Doxorubicin) is optionally contained within the liposome. See Gabizon
et al., J.
National Cancer Inst. 81(19): 1484 (1989).
Stealth Liposomes have also been proposed for use in delivering cytotoxic
agents
to tumors in cancer patients. A range of drugs have been incorporated into
stealth liposomes,
including cisplatin (Rosenthal et al., 2002), TNE.alpha. (Kim et al., 2002),
doxorubicin (Symon
et al., 1999) and adriamycin (Singh et al., 1999), each reference being
specifically incorporated
herein by reference. However, recent reports have indicated unexpected low
efficacy of stealth
Liposomal doxorubicin and vinorelbine in the treatment of metastatic breast
cancer (Rimassa et
al., 2003). See also U.SPatent Application No. 20040170620, the content of
which is
incorporated in its entirety by reference herein.
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Thus, in certain embodiments, the invention provides improved stealthed
liposome formulations, in which the stealthed liposomes are functionally
associated or "coated"
with an antibody that binds to an aminophospholipid or anionic phospholipid,
preferably to PS or
PE. The 9D2, 3G4 (ATCC 4545) and like, competing antibodies of the invention
are preferred
for such uses, although any antibody, or antigen binding region thereof, which
binds to an
aminophospholipid or anionic phospholipid may be used.
Any stealthed liposome may form the basis of the new liposomal formulations,
and preferably a PEGylated liposome will be employed. The stealthed liposomes
are "coated",
i.e., operatively or functionally associated with the antibody that binds to
an aminophospholipid
or anionic phospholipid. The operative or functional association is made such
that the antibody
retains the ability to specifically bind to the target aminophospholipid or
anionic phospholipid,
preferably PS or PE, thereby delivering or targeting the stealthed liposome
and any contents
thereof to PS- and/or PE-positive cells, such as tumor cells and tumor
vascular endothelial cells.
The antibody-coated stealthed liposomes of the invention may be used alone.
Preferably, however, such liposomes will also contain one or more second
therapeutic agents,
such as anti-cancer or chemotherapeutic agents (the first therapeutic agent
being the antibody
itself). The second therapeutic agents are generally described as being within
the "core" of the
liposome. Any one or more of the second, anti-cancer or chemotherapeutic
agents known in the
art and/or described herein for conjugation to antibodies, or for combination
therapies, may be
used in the antibody-coated stealthed liposomes of the invention, for example,
any
chemotherapeutic or radiotherapeutic agent, cytokine, anti-angiogenic agent or
apoptosis-
inducing agent. In certain embodiments, preferred chemotherapeutic agents are
anti-tubulin
drugs, docetaxel and paclitaxel.
Moreover, the antibody-coated stealthed liposomes of the invention may also be
loaded with one or more anti-viral drugs for use in treating viral infections
and diseases. As with
the anti-cancer agents, any one or more of the second, anti-viral drugs known
in the art and/or
described herein for conjugation to antibodies, or for combination therapies,
may be used in the
antibody-coated stealthed liposomes of the invention.
In other embodiments of the invention the invention antibodies or antigen-
binding
fragments thereof are conjugated to albumen using art recognized techniques.
To increase the serum half life of the antibody, one may incorporate a salvage
receptor binding epitope into the antibody (especially an antibody fragment)
as described in U.S.
Pat. No. 5,739,277, for example. As used herein, the term "salvage receptor
binding epitope"
refers to an epitope of the Fe region of an IgG molecule (e.g., IgGI, IgG2,
IgG3, or Igal) that is
responsible for increasing the in vivo serum half-life of the IgG molecule.
Species and Molecule Selectivity
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The anti-Her3 antibody of the invention including binding fragments thereof
demonstrates both species and molecule selectivity. In one aspect, the anti-
Her3 antibody of the
invention binds to human Her3. Following the teachings of the specification,
one may determine
the species selectivity for the anti-Her3 antibody using methods well known in
the art. For
instance, one may determine species selectivity using Western blot, FACS,
ELISA or R1A. In a
preferred embodiment, one may determine the species selectivity using Western
blot.
Likewise, one may determine the selectivity of an anti-Her3 antibody for Her3
using methods well known in the art following the teachings of the
specification. For instance,
one may determine the molecule selectivity using Western blot, FACS, ELISA or
RIA. In a
preferred embodiment, one may determine the molecular selectivity using
Western blot.
Naked Antibody Therapy
A therapeutically effective amount of a naked fully human anti-Her3 antibody,
or
fragments thereof can be formulated in a pharmaceutically acceptable
excipient. The efficacy of
the naked fully human Her3 antibodies and their fragments can also be enhanced
by
supplementing these naked antibodies with one or more other naked antibodies,
with one or more
immunoconjugates of fully human invention Her3 antibodies, conjugated with one
or more
therapeutic agents, including drugs, toxins, immunomodulators, hormones,
oligonucleotides,
hormone antagonists, enzymes, enzyme inhibitors, therapeutic radionuclides, an
angiogenesis
inhibitor, etc., administered concurrently or sequentially or according to a
prescribed dosing
regimen, with the Her3 antibodies or fragments thereof.
Immunoliposomes
The anti-Her3 antibodies disclosed herein may also be formulated as
immunoliposomes. Liposomes containing the antibody are prepared by methods
known in the
art, such as described in Epstein et al., Proc. Natl. Acad. Sci, USA, 82:3688
(1985); Hwang et
al., Proc. Natl Acad. Sci. USA, 77:4030 (1980); and U.S. Pat. Nos. 4,485,045
and 4,544,545.
Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.
5,013,556.
Particularly useful liposomes can be generated by the reverse phase
evaporation
method with a lipid composition comprising phosphatidylcholine, cholesterol
and PEG-
derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of
defined pore size to yield liposomes with the desired diameter. Fab' fragments
of the antibody of
the present invention can be conjugated to the Liposomes as described in
Martin et al. J. Biol.
Chem, 257: 286-288 (1982) via a disulfide interchange reaction. A
chemotherapeutic agent (such
as Doxorubicin) is optionally contained within the liposome. See Gabizon et
al. J. National
Cancer Inst.81(19)1484 (1989)
Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT)
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The antibody of the present invention may also be used in ADEPT by conjugating
the antibody to a prodrug-activating enzyme which converts a prodrug (e.g., a
peptidyl
chemotherapeutic agent, see W081/01145) to an active anti-cancer drug. See,
for example, WO
88/07378 and U.S. Pat. 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 convert 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-fitiorocytosine into the anti-
cancer drug, 5-
fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin,
carboxypeptidases and
cathepsins (such as cathepsins 13 and L), that are useful for converting
peptide-containing
prodrugs into free drugs; D-alanylcarboxylpeptidascs, useful for converting
prodrugs that contain
D-amino acid substituents; carbohydrate-cleaving enzymes such as beta-
galactosidase and
neuraminidase useful for converting glycosylated prodrugs into free drugs;
beta-lactamase useful
for converting drugs derivatized with beta-lactams into free drugs; and
penicillin amidases, such
as penicillin V amidase or penicillin G amidase, useful for converting drugs
derivatized at their
amine nitrogens with phenoxylacetyl or phenylacetyl groups, respectively, into
free drugs.
Alternatively, antibodies with enzymatic activity, also known in the art as
"abzymes", can be
used to convert to prodrugs of the invention into free active drugs (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 antibody mutant
by
techniques well known in the art such as the use of the heterobifunctional
crosslinking reagents
discussed above. Alternatively, fusion proteins comprising at least the
antigen binding region of
an antibody of the invention linked to at least a functionally active portion
of an enzyme of the
invention can be constructed using recombinant DNA techniques well known in
the art
(Neubergeret et al., Nature 312: 604-608(1984)).
Antibodies with enzymatic activity, known as catalytic antibodies or
"abzymes",
can also be employed to convert prodrugs into active drugs. Abzymes based upon
the antibodies
of the invention, preferably the 9D2 and 3G4 and like antibodies, thus form
another aspect of the
present invention. The technical capacity to make abzymes also exists within
those of ordinary
skill in the art, as exemplified by Massey et al. (1987), specifically
incorporated herein by
reference for purposes of supplementing the abzyme teaching. Catalytic
antibodies capable of
catalyzing the breakdown of a prodrug at the carbamate position, such as a
nitrogen mustard aryl
carbamate, are further contemplated, as described in EP 745,673, specifically
incorporated herein
by reference.
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Screening for Antibodies with Desired Properties
Techniques for generating antibodies have been described above. The antibodies
of the present invention can be characterized for their physical/chemical
properties and
biological functions by various assays known in the art. In some embodiments,
antibodies are
characterized for any one or more of binding to Her3 receptor protein, and/or
reduction or
blocking of Her3 receptor activation; and/or reduction or blocking of Her3
receptor downstream
molecular signaling; and/or disruption or blocking of Her3 receptor binding to
its native ligand,
e.g, serrate or delta etc ; and/or promotion of endothelial cell
proliferation; and/or inhibition of
endothelial cell differentiation; and/or inhibition of arterial
differentiation; and/or inhibition of
tumor vascular perfusion; and/or treatment and/or prevention of a tumor, cell
proliferative
disorder or a cancer; and/or treatment or prevention of a disorder associated
with Her3
expression and/or activity; and/or treatment or prevention of a disorder
associated with Her3
receptor expression and/or activity.
In certain embodiments, antibodies may be selected based upon certain
biological
characteristics such as for example assessing the growth inhibitory effects of
an anti-Her3
antibody of the invention. This property may be assessed by methods known in
the art, e.g.,
using cells which express Her3 receptor either endogenously or following
transfeetion with the
Her3 receptor gene. For example, tumor cell lines and Her3 receptor-
transfected cells may be
treated with an anti-Her3 receptor monoclonal antibody of the invention at
various
concentrations for a few days (e.g., 2-7) days and stained with crystal violet
or MTT or analyzed
by some other colorimetric assay. Another method of measuring proliferation
would be by
comparing 3H-thymidine uptake by the cells treated in the presence or absence
of an anti-Her3
receptor antibody of the invention. After antibody treatment, the cells are
harvested and the
amount of radioactivity incorporated into the DNA quantitated in a
scintillation counter.
Appropriate positive controls include treatment of a selected cell line with a
growth inhibitory
antibody known to inhibit growth of that cell line. Preferably, the Her3
receptor agonist will
inhibit cell proliferation of a Her3 receptor-expressing tumor cell in vitro
or in vivo by about 25-
100% compared to the untreated tumor cell, more preferably, by about 30-100%,
and even more
preferably by about 50-100% or 70-100%, at an antibody concentration of about
0.5 to 30 Kg/ml.
Growth inhibition can be measured at an antibody concentration of about 0.5 to
30 1g/m1 or
about 0.5 nM to 200 nM in cell culture, where the growth inhibition is
determined 1-10 days
after exposure of the tumor cells to the antibody. The antibody is growth
inhibitory in vivo if
administration of the anti-Her3 receptor antibody at about 1 1.tWkg to about
100 mg/kg body
weight results in reduction in tumor size or tumor cell proliferation within
about 5 days to 3
months from the first administration of the antibody.
The purified antibodies can be further characterized by a series of assays
including, but not limited to, N-terminal sequencing, amino acid analysis, non-
denaturing size
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exclusion high pressure liquid chromatography (HPLC), mass spectrometry, ion
exchange
chromatography and papain digestion.
The assaying method for detecting Her3 using the antibodies of the invention
or
binding fragments thereof are not particularly limited. Any assaying method
can be used, so
long as the amount of antibody, antigen or antibody-antigen complex
corresponding to the
amount of antigen (e.g., the level of Her3) in a fluid to be tested can be
detected by chemical or
physical means and the amount of the antigen can be calculated from a standard
curve prepared
from standard solutions containing known amounts of the antigen.
Representative
immunoassays encompassed by the present invention include, but are not limited
to, those
described in U.S. Pat. Nos. 4,367,110 (double monoclonal antibody sandwich
assay); Wide etal.,
Kirkham and Hunter, eds. Radioimmunoassay Methods, E. and S. Livingstone,
Edinburgh
(1970); U.S. Pat. No. 4,452,901 (western blot); Brown et al., J. Biol. Chem,
255: 4980-4983
(1980) (irnmunoprecipitation of labeled ligand); and Brooks etal., Clin. Exp.
Immunol. 39;477
(1980) (immunocytochemistry); immunofluorescence techniques employing a
fluorescently
labeled antibody, coupled with light microscopic, flow cytometric, or
fluorometric detection etc.
See also Immunoassays for the 80's, A. Voller et al., eds., University Park,
1981, Zola,
Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc.
1987).
(1) Sandwich assays involve the use of two antibodies, each capable of binding
to
a different immunogenic portion, or epitope, of the protein to be detected. In
a sandwich assay,
the test sample analyte is bound by a first antibody which is immobilized on a
solid support, and
thereafter a second antibody binds to the analyte, thus forming an insoluble
three-part complex.
See, e.g., U.S. Pat. No. 4,376,110. The second antibody may itself be labeled
with a detectable
moiety (direct sandwich assays) or may be measured using an anti-
immunoglobulin antibody
that is labeled with a detectable moiety (indirect sandwich assay). For
example, one type of
sandwich assay is an ELISA assay, in which case the detectable moiety is an
enzyme.
In the sandwich assay, the immobilized antibody of the present invention is
reacted with a test fluid (primary reaction), then with a labeled form of
antibody of the present
invention (secondary reaction), and the activity of the labeling agent on the
immobilizing carrier
is measured, whereby the Her3 level in the test fluid can be quantified. The
primary and
secondary reactions may be performed simultaneously or with some time
intervals. The methods
of labeling and immobilization can be performed by modifications of those
methods described
above. In the immunoassay by the sandwich assay, the antibody used for
immobilized or labeled
antibody is not necessarily from one species, but a mixture of two or more
species of antibodies
may be used to increase the measurement sensitivity, etc. In the method of
assaying Her3 by the
sandwich assay, for example, when the antibodies used in the primary reaction
recognize the
partial peptides at the C-terminal region of Her3, the antibodies used in the
secondary reaction
are preferably those recognizing partial peptides other than the C-terminal
region (i.e., the N-
terminal region). When the antibodies used for the primary reaction recognize
partial peptides at
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the N-terminal region of Her3, the antibodies used in the secondary reaction,
antibodies
recognizing partial peptides other than the N-terminal region (i.e., the C-
terminal region) are
preferably employed.
Other types of "sandwich" assays, which can also be useful for detecting Her3,
are the so-called "simultaneous" and "reverse" assays. A simultaneous assay
involves a single
incubation step wherein the antibody bound to the solid support and labeled
antibody are both
added to the sample being tested at the same time. After the incubation is
completed, the solid
support is washed to remove the residue of fluid sample and uncomplexed
labeled antibody. The
presence of labeled antibody associated with the solid support is then
determined as it would be
in a conventional "forward" sandwich assay.
In the "reverse" assay, stepwise addition first of a solution of labeled
antibody to
the fluid sample followed by the addition of unlabeled antibody bound to a
solid support after a
suitable incubation period, is utilized. After a second incubation, the solid
phase is washed in
conventional fashion to free it of the residue of the sample being tested and
the solution of
unreacted labeled antibody. The determination of labeled antibody associated
with a solid
support is then determined as in the "simultaneous" and "forward" assays. In
one embodiment, a
combination of antibodies of the present invention specific for separate
epitopes can be used to
construct a sensitive three-site immunoradiometric assay.
This type of assays may also be used to quantify Her3 expression in whatever
"sample" it may present itself. Thus, in certain aspects, the sandwich assay
includes:
(1) a method for quantifying expression levels of Her3 in a test fluid,
comprising
reacting the antibody specifically reacting with a partial peptide at the N-
terminal region of the
Her3 immobilized on a carrier, a labeled form of the antibody specifically
reacting with a partial
peptide at the C-terminal region and the test fluid, and measuring the
activity of the label; or
(ii) a method for quantifying Her3 expression in a test fluid, comprising
reacting
the antibody specifically reacting with a partial peptide at the C-terminal
region of the Her3
immobilized onto a carrier, the antibody specifically reacting with a partial
peptide at the N-
terminal region of a labeled form of the Her3 and the test fluid, and
measuring the activity of the
label; etc.
(2) Competitive Assay
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 Her3
protein in the test sample is inversely proportional to the amount of standard
that becomes bound
to the antibodies. To facilitate determining the amount of standard that
becomes bound, the
antibodies generally are insolubilized before or after the competition, so
that the standard and
analyte that are bound to the antibodies may conveniently be separated from
the standard and
analyte which remain unbound.
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For quantifying the level of Her3 expression, one skilled in the art may
combine
and/or competitively react antibodies of the invention or fragments thereof, a
test fluid and a
labeled form of Her3, measure a ratio of the labeled Her3 bound to the
antibodies or fragments
thereof b to thereby quantify the Her3 in the test fluid.
(3) Immunometric Assay
In the immunometric assay, an antigen in a test fluid and a solid phase
antigen are
competitively reacted with a given amount of a labeled form of the antibody of
the present
invention followed by separating the solid phase from the liquid phase; or an
antigen in a test
fluid and an excess amount of labeled form of the antibody of the present
invention are reacted,
then a solid phase antigen is added to bind an unreacted labeled form of the
antibody of the
present invention to the solid phase and the solid phase is then separated
from the liquid phase.
Thereafter, the labeled amount of any of the phases is measured to determine
the antigen level in
the test fluid.
Typical, and preferred, immunometric assays include "forward" assays in which
the antibody bound to the solid phase is first contacted with the sample being
tested to extract the
Her3 from the sample by formation of a binary solid phase antibody-Her3
complex. After a
suitable incubation period, the solid support is washed to remove the residue
of the fluid sample,
including unreacted Her3, if any, and then contacted with the solution
containing a known
quantity of labeled antibody (which functions as a "reporter molecule"). After
a second
incubation period to permit the labeled antibody to complex with the Her3
bound to the solid
support through the unlabeled antibody, the solid support is washed a second
time to remove the
unreacted labeled antibody. This type of forward sandwich assay can be a
simple "yes/no" assay
to determine whether Her3 is present or can be made quantitative by comparing
the measure of
labeled antibody with that obtained for a standard sample containing known
quantities of Her3.
Such "two-site" or "sandwich" assays are described by Wide (Radioimmune Assay
Method,
Kirkham, ed., Livingstone, Edinburgh, 1970, pp. 199 206).
(4) Nephrometry
In the nephrometry, the amount of insoluble sediment, which is produced as a
result of the antigen-antibody reaction in a gel or in a solution, is
measured. Even when the
amount of an antigen in a test fluid is small and only a small amount of the
sediment is obtained,
a laser nephrotnetry utilizing laser scattering can be suitably used.
Examples of labeling agents, which may be used in the above referenced assay
methods (1) to (4) using labeling agents, include radioisotopes (e.g., 1251,
1311, 3H, 14C, 32p, 33p,
35S, etc., fluorescent substances, e.g., cyanine fluorescent dyes (e.g., Cy2,
Cy3, Cy5, Cy5.5,
Cy7), fluorescamine, fluorescein isothiocyanate, etc., enzymes (e.g., ,beta.-
galactosidase, .beta.-
glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc.),
luminescent
substances (e.g., luminol, a luminol derivative, luciferin, lucigenin, etc.),
biotin, lanthanides, etc.
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In addition, a biotin-avidin system may be used as well for binding an
antibody to a labeling
agent.
In the immobilization of antigens or antibodies, physical adsorption may be
used.
Alternatively, chemical binding that is conventionally used for immobilization
of proteins,
enzymes, etc. may be used as well. Examples of the carrier include insoluble
polysaccharides
such as agarose, dextran, cellulose, etc.; synthetic resins such as
polystyrene, polyacrylamide,
silicone, etc.; or glass; and the like.
In certain embodiments, the antibodies of the present invention are tested for
their
antigen binding activity. The antigen binding assays that are known in the art
and can be used
herein include without limitation any direct or competitive binding assays
using techniques such
as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent
assay), "sandwich"
immunoassays, immunoprecipitation assays, fluorescent immunoassays, and
protein A
immunoassays. Illustrative antigen binding assay are provided herein.
In some embodiments, the binding affinity of anti Her3 antibodies is
determined.
Antibodies of the invention preferably have a binding affinity(KD) to Her3 of
at least about lx
10-7 M, more preferably at least about lx1 0-8 M, more preferably at least
about lx10-9 M, and
most preferably at least about lx10-1 M. Preferred antibody-producing cells
of the invention
produce substantially only antibodies having a binding affinity to Her3 of at
least about lx1 0-7
M, more preferably at least about 1x10-8 M, more preferably at least about
1x10-9 M, and most
preferably at least about 1x10-1 M. Preferred compositions of the invention
comprise
substantially only antibodies having a binding affinity to Her3 of at least
about 1x10-7 M, more
preferably at least about 1x10' 8 M, more preferably at least about 1x10-9 M,
and most preferably
at least about lx1 0-1 M.
In another aspect of the invention, the antibodies of the invention bind to
Her3
with substantially the same KD as an antibody that comprises one of the amino
acid sequences
selected from the group as set forth in one of Appendix I or H. In another
embodiment, the
antibody binds to Her3 with substantially the same KD as an antibody that
comprises one or more
CDRs from an antibody that comprises one of the amino acid sequences set forth
herein.
Anti-Her3 antibodies according to the invention or identified using the
methods
disclosed herein have a low dissociation rate. In one embodiment, the anti-
Her3 antibody has a
Koff of 1 Xle or lower, preferably a Koff that is 5 X1 0-5 or lower.
In another embodiment, the antibodies of the invention or those identified or
produced using the methods of the invention bind to Her3 with substantially
the same K.off as an
antibody that comprises one or more CDRs disclosed herein. Illustrative assays
for affinity
analysis are described herein.
Affinity Analysis for Epitope(s)
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Affinity can be either absolute or relative. By absolute affinity, it is meant
that the
assay for affinity gives defined numerical determinations of the affinity of
one compound for
another. Comparison of the affinity of the complex being tested to that of a
reference compound
whose binding affinity is known allows for the determination of relative
binding affinity of the
test ligand.
Whether absolute or relative, affinity of one molecule for another can be
measured by any method known in the art. By way of non-limiting example, such
methods
include competition assays, surface plasmon resonance, half-maximal binding
assays,
competition assays, Scatchard analysis, direct force techniques (Wong et al.,
Direct force
measurements of the streptavidin-biotin interaction, Biomol. Eng. 16:45-55,
1999), and mass
spectrometry (Downard, Contributions of mass spectrometry to structural
immunology, J. Mass
Spectrom. 35:493-503, 2000).
The binding affinity and dissociation rate of an antibody to Her3 may be
determined by any method known in the art. For example, the binding affinity
can be measured
by competitive ELISAs, RIAs or surface plasmon resonance, such as BIAcore. The
dissociation
rate can also be measured by surface plasmon resonance. Alternatively, the
binding affinity and
dissociation rate is measured by surface plasmon resonance. More, the binding
affinity and
dissociation rate is measured using a BIAcore. See below for a brief
description, it being
understood the invention is not limited to the specific assays detailed
herein.
1. Absolute Affinity
As regards absolute affinity, "low affinity" refers to binding wherein the
dissociation constant (KD) between two molecules is about 10-8 M to le M.
"Moderate affinity"
refers to binding wherein the dissociation constant (KD) between two molecules
is at least about
10-7M to 10-8 M. "High affinity" refers to a binding wherein the association
constant between the
two molecules is at least about 10-8 M to about 10-14 M, and preferably about
10-9 M to about
10-14 M, more preferably about 10-10 M to about 10-14 M, and most preferably
greater than about
10-14 M.
The dissociation constant, KD, is an equilibrium constant for the dissociation
of
one species into two, e.g,, the dissociation of a complex of two or more
molecules into its
components, for example, dissociation of a substrate from an enzyme. Exemplary
KD values for
compositions of the present invention are from about 10-7 M (100 nM) to about
10-12 M (0.001
nM). The stability constant is an equilibrium constant that expresses the
propensity of a species
to form from its component parts. The larger the stability constant, the more
stable is the
species. The stability constant (formation constant) is the reciprocal of the
instability constant
(dissociation constant).
The affinity of an invention antibody for a target epitope, or the affinity of
a bi-
specific antibody for a carrier epitope, is driven by non-covalent
interactions. There are four
main non-covalent attractive forces between molecules: (i) electrostatic
forces, which occur
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between oppositely charged molecules such as amino groups and carboxylic
groups; (ii)
hydrogen bonds, which are formed when hydrogen atoms are shared between
electronegative
atoms such as nitrogen and oxygen; (iii) Van der Waals forces, which are
generated between
electron clouds around molecules oppositely polarized by neighboring atoms;
and (iv)
hydrophobic interactions, which are formed when water is excluded from the
interface allowing
hydrophobic molecules to interact in a waterless environment.
Non-covalent interactions can, but rarely do, have the strength of a covalent
linkage (i.e., a chemical bond). In some instances, the affinity of the
invention antibody for a
target epitope, although driven by non-covalent interactions, is so high as to
approach the
strength of a covalent bond. This provides for invention antibodies that are
very stable relative
to other Her3 receptor antibodies of the invention.
Preferably, the affinity of an invention antibody for its cognate target
epitope, is a
KD of about 100 nM to about 0.01 nM; more preferably, greater than about 100
nM, or greater
than about 10 nM; most preferably, greater than about 1 nM, or greater than
about 0.1 nM.
Typical KD for target epitopes are from about 0.1 nM to 100 nM, preferably
from about 0.1 nM
to 10 nM, more preferably from about 0.5 nM to 5 nM, or about 1 nM.
In the invention, when multiple copies of a carrier epitope are present on the
antibody, the affinity of an antibody for its cognate carrier epitope may be
greater than the
affinity of an antibody for a free carrier epitope or for a monovalent
antibody comprising the
carrier epitope. Additionally or alternatively, a multivalent targetable
construct having x carrier
epitopes has a greater affinity for its target epitope than would x number of
constructs. Put
another way, the compositions of the invention also provides for synergistic,
rather than merely
additive, binding effects.
2. Surface Plasmon Resonance
Binding parameters such as KD may be measured using surface plasmon
resonance on a chip, for example, with a BIAcoreTM chip coated with
immobilized binding
components. Surface plasmon resonance is used to characterize the microscopic
association and
dissociation constants of reaction between an antibody or antibody fragment
and its ligand. Such
methods are generally described in the following references which are
incorporated herein by
reference, (Vely et al., BLAcore analysis to test phosphopeptide-SH2 domain
interactions, Meth.
Mol. Biol. 121:313-21, 2000; Liparoto et al., Biosensor analysis of the
interleukin-2 receptor
complex, J. Mol. Reeog. 12:316-21, 1999; Lipsehultz et al., Experimental
design for analysis of
complex kinetics using surface plasmon resonance, Methods 20:310-8, 2000;
Malmqvist.,
BIACORE: an affinity biosensor system for characterization of biornolecular
interactions,
Biochem, Soc. Transactions 27:33540, 1999; Alfthan, Surface plasmon resonance
biosensors as
a tool in antibody engineering, Biosensors & Bioelectronics 13:653-63, 1998;
Fivash et al.,
BIAeore for macromolecular interaction, Curr. Opin. Biotech. 9:97-101, 1998;
Price et al.,
Summary report on the ISOBM TD-4 Workshop: analysis of 56 monoclonal
antibodies against
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PCT/US2011/053436
the MUC I mucin, Tumour Biol. 19 Supp11:1-20, 1998; Malmqvist et al.,
Bionaolecular
interaction analysis: affinity biosensor technologies for functional analysis
of proteins, Curr.
Opin. Chem. Biol. 1:378-83, 1997; O'Shannessy et al., Interpretation of
deviations from pseudo-
first-order kinetic behavior in the characterization of ligand binding by
biosensor technology,
Anal. Biochem. 236:275-83, 1996; Malmborg et al., BIAcore as a tool in
antibody engineering,
J. Immunol. Meth. 183:7-13, 1995; Van Regenmortel, Use of biosensors to
characterize
recombinant proteins, Dev. Biol. Standardization 83:143-51, 1994; O'Shannessy,
Determination
of kinetic rate and equilibrium binding constants for macromolecular
interactions: a critique of
the surface plasmon resonance literature, Curr. Opin. Biotechnol. 5:65-71,
1994). Models using
BIAcore to examine the binding of fixed ligands to multivalent compounds have
been described
(Muller et al., Model and simulation of multivalent binding to fixed ligands,
Anal. Biochem.
261:149-158, 1998).
BIAcorel'm uses the optical properties of surface plasmon resonance (SPR) to
detect alterations in protein concentration bound within to a dextran matrix
lying on the surface
of a gold/glass sensor chip interface, a dextran biosensor matrix. In brief,
proteins are covalently
bound to the dextran matrix at a known concentration and a ligand for the
protein (e.g., antibody)
is injected through the dextran matrix. Near infrared light, directed onto the
opposite side of the
sensor chip surface is reflected and also induces an evanescent wave in the
gold film, which in
turn, causes an intensity dip in the reflected light at a particular angle
known as the resonance
angle. If the refractive index of the sensor chip surface is altered (e.g., by
ligand binding to the
bound protein) a shift occurs in the resonance angle. This angle shift can be
measured and is
expressed as resonance units (RUs) such that 1000 RUs is equivalent to a
change in surface
protein concentration of 1 ng/mm 2. These changes are displayed with respect
to time along the
y-axis of a sensorgram, which depicts the association and dissociation of any
biological reaction.
Additional details may be found in Jonsson et al., Introducing a biosensor
based
technology for real-time biospecific interaction analysis, Ann. Biol. Clin.
51:19-26, 1993;
Jonsson et al., Real-time biospecific interaction analysis using surface
plasmon resonance and a
sensor chip technology, Biotechniques 11:620-627, 1991; Johnsson et al.,
Comparison of
methods for immobilization to carboxymethyl dextran sensor surfaces by
analysis of the specific
activity of monoclonal antibodies, J. Mol. Recog. 8:125-131, 1995; and
Johnsson,
Immobilization of proteins to a carboxymethyldextran-modified gold surface for
biospecific
interaction analysis in surface plasmon resonance sensors, Anal. Biochem.
198:268-277, 1991;
Karlsson et al., Kinetic analysis of monoclonal antibody-antigen interactions
with a new
biosensor based analytical system, J. Immunol. Meth. 145:229, 1991; Weinberger
et al., Recent
trends in protein biochip technology, Pharmacogenomics 1:395-416, 2000;
Lipschultz et al.,
Experimental design for analysis of complex kinetics using surface plasmon
resonance, Methods
20:310-8, 2000.
3. Relative Affinity
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Affinity may also be defined in relative terms, e.g., by 1050. In the context
of
affinity, the IC50 of a compound is the concentration of that compound at
which 50% of a
reference ligand is displaced from a target epitope in vitro or targeted
tissue in vivo. Typically,
IC50 is determined by competitive ELISA. In still another embodiment, the
invention provides
anti-Her3 monoclonal antibodies that compete with a conventional anti-Her3
antibody for
binding to Her3 receptor protein. Such competitor antibodies include
antibodies that recognize a
Her3 epitope that is the same as or overlaps with the Her3 epitope recognized
by any one of a
conventional antibody. Such competitor antibodies can be obtained by assay
well known to one
skilled in the art. For example, they can b obtained by screening anti-Her3
hybridoma
supernatants for binding to immobilized Her3 in competition with labeled 26.6,
26.14, 26.20,
26.34, and/or 26.82 antibodies. Alternatively, they can be used in a binding
assay A hybridoma
supernatant containing competitor antibody will reduce the amount of bound,
labeled antibody
detected in the subject competition binding mixture as compared to the amount
of bound, labeled
antibody detected in a control binding mixture containing irrelevant (or no)
antibody. Any of the
competition binding assays described herein are suitable for use in the
foregoing procedure.
Anti-Her3 antibodies of the invention possessing the unique properties
described
herein can be obtained by screening anti-Her3 hybridoma clones for the desired
properties by
any convenient method. For example, if an anti-Her3 monoclonal antibody that
blocks or does
not block the binding of Her3 receptors to its binding partner e.g., a Her3
ligand is desired, the
candidate antibody can be tested in a binding competition assay, such as a
competitive binding
ELISA, wherein plate wells are coated with the binding partner, and a solution
of antibody in an
excess of the Her3 receptor of interest is layered onto the coated plates, and
bound antibody is
detected enzymatically, e.g. contacting the bound antibody with HRP-conjugated
anti-Ig
antibody or biotinylated anti-Ig antibody and developing the HRF color
reaction., e.g. by
developing plates with streptavidin-HRP and/or hydrogen peroxide and detecting
the HRP color
reaction by spectrophotometry at 490 nm with an ELISA plate reader.
In one embodiment, the present invention contemplates an altered antibody that
possesses some but not all effector functions, which make it a desired
candidate for many
applications in which the half life of the antibody in vivo is important yet
certain effector
functions (such as complement and ADCC) are unnecessary or deleterious. In
certain
embodiments, the Fc activities of the produced immunoglobulin are measured to
ensure that only
the desired properties are maintained. In vitro and/or in vivo cytotoxicity
assays can be
conducted to confirm the reduction/depletion of CDC and/or ADCC activities.
For example, Fe
receptor (FcR) binding assays can be conducted to ensure that the antibody
lacks FeyR binding
(hence likely lacking ADCC activity), but retains FeRn binding ability. The
primary cells for
mediating ADCC, NK cells, express FcyRIII only, whereas monacytes express Fe
?RI, Fc7RII
and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on
page 464 of
Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). An example of an in
vitro assay to
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assess ADCC activity of a molecule of interest is described in U.S. Pat, No.
5,500,362 or
5,821,337. Useful effector cells for such assays include peripheral blood
mononuclear cells
(PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC
activity of the
molecule of interest may be assessed in vivo, e.g., in an animal model such as
that disclosed in
Clynes et al. Proc. Natl. Acad. Sci. A 95:652-656 (1998). Clq binding assays
may also be
carried out to confirm that the antibody is unable to bind Clq and hence lacks
CDC activity. To
assess complement activation, a CDC assay, e.g. as described in Gazzano-
Santoro et al., J.
Immunol. Methods 202:163 (1996), may be performed. FeRn binding and in vivo
clearance/half
life determinations can also be performed using methods known in the art, e.g.
those described in
the Examples section.
Identification of Her3 Epitopes Recognized by Anti-Her3 Antibody
One may determine whether an anti-Her3 antibody derived from the antibodies of
the invention or produced in accordance with the methods described above binds
to the same
antigen as another antibody, e.g., conventional antibody using a variety of
methods known in the
art. For instance, one may determine whether a test anti-Her3 antibody binds
to the same antigen
by using an anti-Her3 antibody to capture an antigen that is known to bind to
the anti-Her3
antibody, eluting the antigen from the antibody, and then determining whether
the test antibody
will bind to the eluted antigen.
One may determine whether a test antibody binds to the same epitope as an anti-
Her3 antibody by binding the anti-Her3 antibody to Her3 receptor protein under
saturating
conditions, and then measuring the ability of the test antibody to bind to
Her3. If the test
antibody, e.g., anti-Her3 antibodies derived from the invention antibodies or
in accordance with
the methods of the invention is able to bind to the Her3 receptor protein at
the same time as the
reference anti-Her3 antibody, then the test antibody binds to a different
epitope as the anti-Her3
antibody. However, if the test antibody is not able to bind to Her3 receptor
protein at the same
time, then the test antibody binds to the same epitope as the human anti-Her3
antibody. This
experiment may be performed using ELISA, RR or surface plasmon resonance. In
certain
embodiments, the experiment is performed using surface plasmon resonance,
supra. In another
embodiment, BIAcore is used, see supra. One may also determine whether an anti-
Her3
antibody cross-competes with a reference anti-Her3 antibody. For example, one
may determine
whether a test anti-Her3 antibody cross-competes with another by using the
same method that is
used to measure whether the anti-Her3 antibody is able to bind to the same
epitope as another
anti-Her3 antibody.
The diagnostic method may also be used to determine whether a tumor is
potentially cancerous, if it expresses high levels of Her3, or benign, if it
expresses low levels of
Her3. Thus, for example, biological samples obtained from patients suspected
of exhibiting an
oncogenic disorder mediated by Her3 may be assayed for the presence of Her3
expressing cells.
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As noted, the anti-Her3 antibodies of the invention may be used to determine
the
levels of Her3 receptor protein in a tissue or in cells derived from the
tissue. In one embodiment,
the tissue is a diseased tissue. In a more preferred embodiment, the tissue is
a tumor or a biopsy
thereof. In a preferred embodiment of the method, a tissue or a biopsy thereof
is excised from a
patient. The tissue or biopsy is then used in an immunoassay to determine,
e.g., Her3 levels, cell
surface levels of Her3, levels of tyrosine phosphorylation of Her3, or
localization of Her3 by the
methods discussed herein. The method can be used to determine tumors that
express Her3.
In a related embodiment, the present invention provides methods for diagnosing
cancers by assaying for changes in the level of Her3 in cells, tissues or body
fluids compared
with the levels in cells, tissues, or body fluids, preferably of the same type
in a control sample.
A change, especially an increase, in levels of Her3 in the patient versus the
control is associated
with the presence of cancer. Typically, for a quantitative diagnostic assay, a
positive result
indicating that the patient being tested has cancer is one in which levels of
Her3 in or on cells,
tissues or body fluid are at least two times higher, and preferably three to
five times higher, or
greater, than the levels of the antigens in or on the same cells, tissues, or
body fluid of the
control. Normal controls include a human without cancer and/or non-cancerous
samples from
the patient.
The in vitro diagnostic methods may include any method known to one skilled in
the art including immunohistological or immunohistochemical detection of tumor
cells (e.g., on
human tissue, or on cells dissociated from excised tumor specimens), or
serological detection of
tumor associated antigens (e.g., in blood samples or other biological fluids).
Immunohistochemical techniques involve staining a biological specimen, such as
a tissue
specimen, with one or more of the antibodies of the invention and then
detecting the presence on
the specimen of antibody-antigen complexes comprising antibodies bound to the
cognate
antigen. The formation of such antibody-antigen complexes with the specimen
indicates the
presence of cancer in the tissue.
Detection of the antibody on the specimen can be accomplished using techniques
known in the art such as immunoenzymatic techniques, e.g., immunoperoxidase
staining
technique, or the avidin-biotin technique, or immunofluorescence techniques
(see, e.g., Ciocca et
al., 1986, "Immunohistochemical Techniques Using Monoclonal Antibodies", Meth.
Enzymol.,
121:562 79 and Introduction to Immunology, Ed. Kimball, (2nd Ed), Macmillan
Publishing
Company, 1986, pp. 113 117). Those skilled in the art can determine operative
and optimal
assay conditions by routine experimentation.
In another embodiment, the present invention assists in the diagnosis of
cancers
and tumors by the identification and measurement of the Her3 receptor protein
levels in
biological samples. If Her3 receptor protein is normally present, and the
development of the
oncogenic disorder is caused by an abnormal quantity of the cell surface
receptor (1-ler3), e.g.,
expression relative to normal, the assay should compare Her3 levels in the
biological sample to
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the range expected in normal, non-oncogenic tissue of the same cell type.
Thus, a statistically
significant increase in the amount of Her3 bearing cells or Her3 expression
level in the subject
relative to the control subject or subject's baseline, can be a factor that
may lead to a diagnosis of
an oncogenic disorder that is progressing or at risk for such a disorder.
Likewise, the presence of
high levels of Her3 indicative of cancers likely to metastasize can also be
detected. For those
cancers that express the antigen recognized by the antibodies of the
invention, e.g., Her3, the
ability to detect the antigen provides early diagnosis, thereby affording the
opportunity for early
treatment. Early detection is especially important for cancers difficult to
diagnose in their early
stages.
Moreover, the level of antigen detected and measured in a body fluid sample
such
as for example diseased tissue provides a means for monitoring the course of
therapy for the
cancer or tumor, including, but not limited to, surgery, chemotherapy,
radiation therapy, the
therapeutic methods of the present invention, and combinations thereof. By
correlating the level
of the antigen in the tissue sample with the severity of disease, the level of
such antigen can be
used to indicate successful removal of the primary tumor, cancer, and/or
metastases, for
example, as well as to indicate and/or monitor the effectiveness of other
therapies overtime. For
example, a decrease in the level of the cancer or tumor-specific antigen over
time indicates a
reduced tumor burden in the patient. By contrast, no change, or an increase,
in the level of
antigen over time indicates ineffectiveness of therapy, or the continued
growth of the tumor or
cancer.
A typical in vitro immunoassay for detecting Her3 comprises incubating a
biological sample in the presence of a detectably labeled anti-Her3 antibody
or antigen binding
fragment of the present invention capable of selectively binding to Her3, and
detecting the
labeled fragment or antibody which is bound in a sample. The antibody is bound
to a label
effective to permit detection of the cells or portions (e.g., Her3 or
fragments thereof liberated
from hyperplastic, dysplastic and/or cancerous cells) thereof upon binding of
the antibody to the
cells or portions thereof. The presence of any cells or portions thereof in
the biological sample is
detected by detection of the label.
The biological sample may be brought into contact with, and immobilized onto,
a
solid phase support or carrier, such as nitrocellulose, or other solid support
or matrix, which is
capable of immobilizing cells, cell particles, membranes, or soluble proteins.
The support may
then be washed with suitable buffers, followed by treatment with the
detectably-labeled anti-
Her3 antibody. The solid phase support may then be washed with buffer a second
time to
remove unbound antibody. The amount of bound label on the solid support may
then be detected
by conventional means. Accordingly, in another embodiment of the present
invention,
compositions are provided comprising the monoclonal antibodies, or binding
fragments thereof,
bound to a solid phase support, such as described herein.
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In vitro assays in accordance with the present invention also include the use
of
isolated membranes from cells expressing a recombinant Her3, soluble fragments
comprising the
ligand binding segments of Her3, or fragments attached to solid phase
substrates. These assays
allow for the diagnostic determination of the effects of either binding
segment mutations and
modifications, or ligand mutations and modifications, e.g., ligand analogues.
In certain embodiments the monoclonal antibodies and binding fragments thereof
of the present invention may be used in in vitro assays designed to screen
compounds for binding
affinity to Her3. See Fodor et al. Science 251: 767-773 (1991), incorporated
herein by reference.
In accordance with this objective, the invention contemplates a competitive
drug screening
assay, where the monoclonal antibodies or fragments thereof of the invention
compete with a test
compound for binding to Her3. In this manner the monoclonal antibodies and
fragments thereof
are used to detect the presence of any polypeptide which shares one or more
binding sites of the
Her3 and can be used to occupy binding sites on the receptor which might
otherwise be occupied
by the antibody.
In certain embodiments, the anti-Her3 antibodies of the invention may be used
to
determine or quantify the amount of Her3 on the cell surface after treatment
of the cells with
various compounds. This method can be used to test compounds that may be used
to activate or
inhibit Her3. In this method, one sample of cells is treated with a test
compound for a period of
time while another sample is left untreated. If the total level of Her3 is to
be measured, the cells
are lysed and the total Her3 level is measured using one of the immunoassays
described herein.
A preferred immunoassay for measuring total Her3 receptor protein levels is an
ELISA or Western blot. If only the cell surface level of Her3 is to be
measured, the cells are not
lysed, and the cell surface levels of Her3 are measured using any one or more
of the assays
known to the skilled artisan, e.g., one of the immunoassays described herein.
A preferred
immunoassay for determining cell surface levels of Her3 includes the steps of
labeling the cell
surface proteins with a detectable label, such as biotin or 1251,
immunoprecipitating the Her3 with
an anti-Her3 antibody and then detecting the labeled Her3. Another preferred
immunoassay for
determining the localization of Her3, e.g., cell surface levels, is by using
immunohistochernistry.
As well, provided herein is a method to determine whether a conventional anti-
Her3 antibody decreases Her3 expression on a target tumor tissue or cell. The
term
"conventional Her3 antagonist" "conventional treatment with a Her3 moiety" is
used
interchangeably to mean Her3 specific monoclonal antibodies currently
available that
specifically target Her3 expression and do not bind to the same epitope as the
antibodies of the
invention.
A further aspect of the invention is an assessment of the susceptibility that
an
individual has for developing cancer mediated by Her3. The method comprises
the steps of
measuring the level of expression of Her3 in a cell or tissue of interest,
incubating the cell or
tissue with an anti-Her3 antibody or antigen-binding portion thereof, then re-
measuring the level
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of Her3 expression with an anti-Her3 antibody or antigen binding fragment of
the invention in
the cell or tissue. Alternatively, 1CD expression levels may be measured in
the above example.
A diagnosis that levels of Her3 are low could be used for predicting that the
patient is responding
to treatment with the conventional anti-Her3 antibody regiment. On the
contrary, no change in
the level of Her3 or an increase in expression of Her3 after treatment with a
conventional anti-
Her3 antibody indicate that the patient is either unresponsive to the current
treatment protocol or
unlikely to respond to further treatment with the conventional anti-Her3
antibody, thereby
allowing for earlier intervention. The anti-Her3 antibodies of the invention
may be used in the
above diagnostic assays either simultaneously with administration of the
conventional Her3
antibody or after treatment with the conventional anti-Her3 antibody.
Preferably, the
conventional Her3 antibody does not compete with the anti-Her3 antibody of the
invention for
binding Her3 protein, The above assays can be performed iteratively over a
period of time to
assess the therapeutic efficacy of a conventional anti-Her3 antibody based
therapeutic protocol.
In this way, the anti-Her3 antibody of the invention can be used as a
"negative biomarker"
allowing it to be used to assess the treatment and therapeutic protocol of a
conventional anti-
Her3 antibody based therapy.
Vectors, Host Cells and Recombinant Methods
The invention also includes nucleic acids encoding the heavy chain and/or
light
chain of the anti-Her3 antibodies of the invention. Nucleic acids of the
invention also include
fragments of the nucleic acids of the invention. A "fragment" refers to a
nucleic acid sequence
that is preferably of sufficient length to encode a functionally active
fragment of the invention
antibodies, e.g., light or heavy chain. A "fragment" can also mean the whole
coding sequence of
a gene and may include 5' and 3' untranslated regions.
Constructs of any one or more polynucleotides having sequences as set forth
herein can be generated synthetically. Alternatively, single-step assembly of
a gene and entire
plasmid from large numbers of oligodeoxyribonucleotides is described by, e.g.,
Stemmer et al.,
Gene (Amsterdam) (1995) 164(1):49-53. In this method, assembly PCR (the
synthesis of long
DNA sequences from large numbers of oligodeoxyribonucleotides) is derived from
DNA
shuffling (Stemmer, Nature (1994) 370:389-391).
Appropriate polynucleotide constructs are purified using standard recombinant
DNA techniques as described in, for example, Sambrook et al., Molecular
Cloning: A
Laboratory Manual, 2nd Ed., (1989) Cold Spring Harbor Press, Cold Spring
Harbor, N.Y. The
gene product encoded by a polynucleotide of the invention is expressed in any
expression
system, including, for example, bacterial, yeast, insect, amphibian and
mammalian systems.
Vectors, host cells and methods for obtaining expression in same are well
known in the art.
Suitable vectors and host cells are described in U.S. Pat. No. 5,654,173.
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Polynucleotide molecules comprising a polynucleotide sequence provided herein
are generally propagated by placing the molecule in a vector. Viral and non-
viral vectors are
used, including plasmids. The choice of plasmid will depend on the type of
cell in which
propagation is desired and the purpose of propagation. Certain vectors are
useful for amplifying
and making large amounts of the desired DNA sequence. Other vectors are
suitable for
expression in cells in culture. Still other vectors are suitable for transfer
and expression in cells in
a whole animal or person. The choice of appropriate vector is well within the
skill of the art.
Many such vectors are available commercially. Methods for preparation of
vectors comprising a
desired sequence are well known in the art.
The polynucleotides set forth in any one or more of SEQ ID NOs set forth in
one
or more appendices disclosed herein or their corresponding full-length
polynucleotides are linked
to regulatory sequences as appropriate to obtain the desired expression
properties. These can
include promoters (attached either at the 5' end of the sense strand or at the
3' end of the
antisense strand), enhancers, terminators, operators, repressors, and
inducers. The promoters can
be regulated or constitutive. In some situations it may be desirable to use
conditionally active
promoters, such as tissue-specific or developmental stage-specific promoters.
These are linked
to the desired nucleotide sequence using the techniques described above for
linkage to vectors.
Any techniques known in the art can be used.
When any appropriate host cells or organisms are used to replicate and/or
express
the polynucleotides or nucleic acids of the invention, the resulting
replicated nucleic acid, RNA,
expressed protein or polypeptide, is within the scope of the invention as a
product of the host cell
or organism. The product is recovered by any appropriate means known in the
art.
Expression of a target gene, e.g., corresponding to any one or more of the
nucleic
acid molecules set forth herein can be regulated in the cell to which the gene
is native. For
example, an endogenous gene of a cell can be regulated by an exogenous
regulatory sequence as
disclosed in U.S. Pat. No, 5,641,670.
The encoded antibody heavy chain preferably comprises an amino acid sequence
selected from the group consisting of SEQ ID Nos as set forth in one of
Appendix I-III. The
encoded antibody light chain preferably comprises an amino acid sequence a set
forth in one of
the appendices set forth herein.
In some embodiments, the invention provides nucleic acids encoding both a
heavy chain and a light chain of an antibody of the invention. For example, a
nucleic acid of the
invention may comprise a nucleic acid sequence (Appendix I) encoding an amino
acid sequence
as set forth in one of Appendix 1i or III and a nucleic acid sequence
(Appendix I) encoding an
amino acid sequence as set forth in one of Appendix II or III.
Nucleic acids of the invention include nucleic acids having at least 80%, more
preferably at least about 90%, more preferably at least about 95%, and most
preferably at least
about 98% homology to nucleic acids of the invention. The terms "percent
similarity", "percent
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identity" and "percent homology" when referring to a particular sequence, are
used as set forth in
the University of Wisconsin GCG software program. Nucleic acids of the
invention also include
complementary nucleic acids. In some instances, the sequences will be fully
complementary (no
mismatches) when aligned. In other instances, there may be up to about a 20%
mismatch in the
sequences.
The invention also provides a nucleic acid molecule encoding the variable
region
of the light chain (VL) as described herein as well as an amino acid sequence
that is at least 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to one of the amino
acid
sequences encoding a VL as described herein, particularly to a VL that
comprises an amino acid
sequence of one of the sequences as set forth in Appendix II or III. The
invention also provides
a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%, 98% or
99% identical to a nucleic acid sequence of one of the sequences as set forth
in Appendix I. In
another embodiment, the nucleic acid molecule encoding a VL is one that
hybridizes under
highly stringent conditions to a nucleic acid sequence encoding a VL as
described above.
The invention also provides a nucleic acid molecule encoding the variable
region
of the heavy chain (Vii) as described herein as well as an amino acid sequence
that is at least
70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to one of the
amino acid
sequences encoding a Vii as described herein, particularly to a Vii that
comprises an amino acid
sequence of one of the sequences set forth in Appendix II or III. The
invention also provides a
nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98% or 99%
identical to a nucleic acid sequence of any one or more of the sequences set
forth in Appendix I.
In another embodiment, the nucleic acid molecule encoding a VH is one that
hybridizes under
highly stringent conditions to a nucleic acid sequence encoding a VH as
described above.
In the context of this invention, "hybridization" means the pairing of
complementary strands of oligomeric compounds. In the present invention, the
preferred
mechanism of pairing involves hydrogen bonding, which may be Watson-Crick,
Hoogsteen or
reversed Hoogsteen hydrogen bonding, between complementary nucleoside or
nucleotide bases
(nucleobases) of the strands of oligomeric compounds. For example, adenine and
thymine are
complementary nucleobases which pair through the formation of hydrogen bonds.
Hybridization
can occur under varying circumstances.
The term "selectively hybridize" referred to herein means to detectably and
specifically bind. Polynucleotides, oligonucleotides and fragments thereof in
accordance with
the invention selectively hybridize to nucleic acid strands under
hybridization and wash
conditions that minimize appreciable amounts of detectable binding to
nonspecific nucleic acids.
"High stringency" or "highly stringent" conditions can be used to achieve
selective hybridization
conditions as known in the art and discussed herein. An example of "high
stringency" or "highly
stringent" conditions is a method of incubating a polynucleotide with another
polynucleotide,
wherein one polynucleotide may be affixed to a solid surface such as a
membrane, in a
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hybridization buffer of 6x. SSPE or SSC, 50% formamide, 5x. Denhardt's
reagent, 0.5% SDS,
100 p.g/m1 denatured, fragmented salmon sperm DNA at a hybridization
temperature of 42 C.
for 12-16 hours, followed by twice washing at 55 C. using a wash buffer of
1xSSC, 0.5% SDS.
See also Sambrook et al., supra, pp. 9.50-9.55.
The nucleic acid molecule encoding either or both of the entire heavy and
light
chains of an anti-Her3 antibodies or the variable regions thereof may be
obtained from any
source that produces an anti-Her3 antibody. Methods of isolating mRNA encoding
an antibody
are well-known in the art (See, e.g., Sambrook et al.) The mRNA may be used to
produce cDNA
for use in the polymerase chain reaction (PCR) or cDNA cloning of antibody
genes.
A nucleic acid molecule encoding the entire heavy chain of an anti-Her3
antibody
disclosed herein, may be constructed by fusing a nucleic acid molecule
encoding the variable
domain of a heavy chain or an antigen-binding domain thereof with a constant
domain of a
heavy chain. Similarly, a nucleic acid molecule encoding the light chain of
the anti-Her3
antibody of the invention, may be constructed by fusing a nucleic acid
molecule encoding the
variable domain of a light chain or an antigen-binding domain thereof with a
constant domain of
a light chain. The nucleic acid molecules encoding the VH and VL chain may be
converted to
full-length antibody genes by inserting them into expression vectors already
encoding heavy
chain constant and light chain constant regions, respectively, such that the
VH segment is
operatively linked to the heavy chain constant region (CH) segment(s) within
the vector and the
VL segment is operatively linked to the light chain constant region (CL)
segment within the
vector. Alternatively, the nucleic acid molecules encoding the VH or V1 chains
are converted
into full-length antibody genes by linking, e.g., ligating, the nucleic acid
molecule encoding a VH
chain to a nucleic acid molecule encoding a CH chain using standard molecular
biological
techniques. The same may be achieved using nucleic acid molecules encoding VL
and CL
chains. The sequences of human heavy and light chain constant region genes are
known in the
art. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest,
5th Ed., NIH Publ.
No. 91 3242, 1991. Nucleic acid molecules encoding the full-length heavy
and/or light chains
may then be expressed from a cell into which they have been introduced and the
anti-Her3
antibody isolated.
The nucleic acid molecules may be used to recombinantly express large
quantities
of anti-Her3 antibodies using techniques known to one skilled in the art of
recombinant biologist.
Likewise, the herein described nucleic acid molecules can also be used to
recombinantly produce
any one of the anti-Her3 antibody variants, mutants, fragments thereof or
derivatives, including
single chain antibodies, bispecific, scFv etc immunoadhesins, diabodies,
mutated antibodies and
antibody derivatives, as described further below.
In another embodiment, the nucleic acid molecules of the invention may be used
as probes or PCR primers for specific antibody sequences. For instance, a
nucleic acid molecule
probe may be used in diagnostic methods or a nucleic acid molecule PCR primer
may be used to
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amplify regions of DNA that could be used, inter alia, to isolate nucleic acid
sequences for use in
producing variable domains of anti-Her3 antibodies. In a preferred embodiment,
the nucleic acid
molecules are oligonucleotides. In a more preferred embodiment, the
oligonucleotides are from
highly variable regions of the heavy and light chains of the antibody of
interest. In an even more
preferred embodiment, the oligonucleotides encode all or a part of one or more
of the CDRs,
A "vector" is a replicon, such as a plasmid, cosmid, bacmid, phage, artificial
chromosome (BAC, YAC) or virus, into which another genetic sequence or element
(either DNA
or RNA) may be inserted so as to bring about the replication of the attached
sequence or
element. A "replicon" is any genetic element, for example, a plasmid, cosmid,
bacmid, phage,
artificial chromosome (BAC, YAC) or virus, which is capable of replication
largely under its
own control. A replicon may be either RNA or DNA and may be single or double
stranded. In
some embodiments, the expression vector contains a constitutively active
promoter segment
(such as but not limited to CMV, SV40, Elongation Factor or LTR sequences) or
an inducible
promoter sequence such as the steroid inducible pIND vector (Invitrogen),
where the expression
of the nucleic acid can be regulated. The expression vector can be introduced
into a cell by
transfection,
In addition to the antibody chain genes, the recombinant expression vectors of
the
invention carry regulatory sequences that control the expression of the
antibody chain genes in a
host cell. It will be appreciated by those skilled in the art that the design
of the expression
vector, including the selection of regulatory sequences may depend on such
factors as the choice
of the host cell to be transformed, the level of expression of protein
desired, etc. Preferred
regulatory sequences for mammalian host cell expression include viral elements
that direct high
levels of protein expression in mammalian cells, such as promoters and/or
enhancers derived
from retroviral LTRs, cytomegalovirus (CMV) (such as the CMV
promoter/enhancer), Simian
Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., the
adenovirus major
late promoter (AdMIP)), polyoma and strong mammalian promoters such as native
immunoglobulin and actin promoters. For further description of viral
regulatory elements, and
sequences thereof, see e.g., U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No.
4,510,245 by Bell
et al. and U.S. Pat. No. 4,968,615 by Schaffner et al.
In addition to the antibody chain genes and regulatory sequences, the
recombinant
expression vectors of the invention may carry additional sequences, such as
sequences that
regulate replication of the vector in host cells (e.g., origins of
replication) and selectable marker
genes. The selectable marker gene facilitates selection of host cells into
which the vector has
been introduced (see e.g., U.S. Pat. Nos. 4,399,2)6, 4,634,665 and 5,179,017,
all by Axel et al.).
For example, typically the selectable marker gene confers resistance to drugs,
such as G418,
hygromycin or methotrexate, on a host cell into which the vector has been
introduced. Preferred
selectable marker genes include the dihydrofolate reductase (DHFR) gene (for
use in dhfr-host
cells with methotrexate selection/amplification) and the neo gene (for G418
selection).
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For recombinant production of an antibody of the invention, 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 antibody is readily isolated and
sequenced using
conventional procedures (e.g., by using oligonucleotide probes that are
capable of binding
specifically to genes encoding the heavy and light chains of the antibody).
Many vectors are
available. The choice of vector depends in part on the host cell to be used.
Generally, preferred
host cells are of either prokaryotic or eukaryotic (generally mammalian)
origin. It will be
appreciated that constant regions of any isotype can be used for this purpose,
including IgG,
IgM, IgA, IgD, and IgE constant regions, and that such constant regions can be
obtained from
any human or animal species.
a. Generating Antibodies Using Prokaryotic Host Cells
i. Vector Construction
Polynucleotide sequences encoding polypeptide components of the antibody of
the invention can be obtained using standard recombinant techniques. Desired
polynucleotide
sequences may be isolated and sequenced from antibody producing cells such as
hybridoma
cells. Alternatively, polynucleotides can be synthesized using nucleotide
synthesizer or PCR
techniques. Once obtained, sequences encoding the polypeptides are inserted
into a recombinant
vector capable of replicating and expressing heterologous polynucleotides in
prokaryotic hosts.
Many vectors that are available and known in the art can be used for the
purpose of the present
invention. Selection of an appropriate vector will depend mainly on the size
of the nucleic acids
to be inserted into the vector and the particular host cell to be transformed
with the vector. Each
vector contains various components, depending on its function (amplification
or expression of
heterologous polynucleotide, or both) and its compatibility with the
particular host cell in which
it resides. The vector components generally include, but are not limited to:
an origin of
replication, a selection marker gene, a promoter, a ribosome binding site
(RBS), a signal
sequence, the heterologous nucleic acid insert and a transcription termination
sequence.
In general, plasmid vectors containing replicon and control sequences which
are
derived from species compatible with the host cell are used in connection with
these hosts. The
vector ordinarily carries a replication site, as well as marking sequences
which are capable of
providing phenotypic selection in transformed cells. For example, E. coil is
typically transformed
using pBR322, a plasmid derived from an E. colt species, pBR322 contains genes
encoding
ampicillin (Amp), kanamycin (Kn) and tetracycline (Tet) resistance and thus
provides easy
means for identifying transformed cells. pBR322, its derivatives, or other
microbial plasmids or
bacteriophage may also contain, or be modified to contain, promoters which can
be used by the
microbial organism for expression of endogenous proteins. Examples of pBR322
derivatives
used for expression of particular antibodies are described in detail in Carter
et al., U.S. Pat. No.
5,648,237.
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A convenient vector is one that encodes a functionally complete human CH or CL
immunoglobulin sequence, with appropriate restriction sites engineered so that
any VII or VL
sequence can be easily inserted and expressed, as described above. In such
vectors, splicing
usually occurs between the splice donor site in the inserted J region and the
splice acceptor site
preceding the human C region, and also at the splice regions that occur within
the human CH
exons. Polyadenylation and transcription termination occur at native
chromosomal sites
downstream of the coding regions. The recombinant expression vector can also
encode a signal
peptide that facilitates secretion of the antibody chain from a host cell. The
antibody chain gene
may be cloned into the vector such that the signal peptide is linked in-frame
to the amino
terminus of the antibody chain gene. The signal peptide can be an
immunoglobulin signal
peptide or a heterologous signal peptide (i.e., a signal peptide from a non-
immunoglobulin
protein).
In addition, phage vectors containing replicon and control sequences that are
compatible with the host microorganism can be used as transforming vectors in
connection with
these hosts. For example, bacteriophage such as 2,GEMTm-11 may be utilized in
making a
recombinant vector which can be used to transform susceptible host cells such
as E. coil LE392.
The expression vector of the invention may comprise two or more promoter-
cistron pairs, encoding each of the polypeptide components. A promoter is an
untranslated
regulatory sequence located upstream (5') to a cistron that modulates its
expression. Prokaryotic
promoters typically fall into two classes, inducible and constitutive.
Inducible promoter is a
promoter that initiates increased levels of transcription of the cistron under
its control in response
to changes in the culture condition, e.g. the presence or absence of a
nutrient or a change in
temperature.
A large number of promoters recognized by a variety of potential host cells
are
well known. The selected promoter can be operably linked to cistron DNA
encoding the light or
heavy chain by removing the promoter from the source DNA via restriction
enzyme digestion
and inserting the isolated promoter sequence into the vector of the invention.
Both the native
promoter sequence and many heterologous promoters may be used to direct
amplification and/or
expression of the target genes. In some embodiments, heterologous promoters
are utilized, as
they generally permit greater transcription and higher yields of expressed
target gene as
compared to the native target polypeptide promoter.
Promoters suitable for use with prokaryotic hosts include the PhoA promoter,
the
.beta.-galactamase and lactose promoter systems, a tryptophan (trp) promoter
system and hybrid
promoters such as the tae or the tre promoter. However, other promoters that
are functional in
bacteria (such as other known bacterial or phage promoters) are suitable as
well. Their
nucleotide sequences have been published, thereby enabling a skilled worker
operably to ligate
them to cistrons encoding the target light and heavy chains (Siebenlist et al.
(1980) Cell 20: 269)
using linkers or adaptors to supply any required restriction sites.
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In one aspect of the invention, each cistron within the recombinant vector
comprises a secretion signal sequence component that directs translocation of
the expressed
polypeptides across a membrane. In general, the signal sequence may be a
component of the
vector, or it may be a part of the target polypeptide DNA that is inserted
into the vector. The
signal sequence selected for the purpose of this invention should be 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 signal sequences native to the heterologous
polypeptides, the
signal sequence is substituted by a prokaryotic signal sequence selected, for
example, from the
group consisting of the alkaline phosphatase, penicillinase, Ipp, or heat-
stable enterotoxin II
(ST11) leaders, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of the
invention, the
signal sequences used in both cistrons of the expression system are STII
signal sequences or
variants thereof.
In another aspect, the production of the immunoglobulins according to the
invention can occur in the cytoplasm of the host cell, and therefore does not
require the presence
of secretion signal sequences within each cistron. In that regard,
immunoglobulin light and
heavy chains are expressed, folded and assembled to form functional
immunoglobulins within
the cytoplasm. Certain host strains (e.g., the E. coli trx13-strains) provide
cytoplasm conditions
that are favorable for disulfide bond formation, thereby permitting proper
folding and assembly
of expressed protein subunits. Proba and Pluckthun. Gene, 159:203 (1995).
Prokaryotic host cells suitable for expressing antibodies of the invention
include
Archaebacteria and Eubactefia, such as Gram-negative or Gram-positive
organisms. Examples
of useful bacteria include Escherichia (e.g., E. colt), Bacilli (e.g., B.
subtilis), Enterobacteria,
Pseudomonas species (e.g., P. aeruginosa), Salmonella typhimurium, Serratia
mareeseans,
Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus. In one
embodiment, gram-
negative cells are used. In one embodiment, E. con cells are used as hosts for
the invention.
Examples of E. coli strains include strain W3110 (Bachmann, Cellular and
Molecular Biology,
vol. 2 (Washington, D.C.: American Society for Microbiology, 1987), pp. 1190-
1219; ATCCTm
Deposit No. 27,325) and derivatives thereof, including strain 33D3 having
genotype W3110
.DELTA.fhu.DELTA. (.DELTA.tonA) ptr3 lac Iq lacL8 .DELTA.ompT.DELTA. (nmpc-
fepE)
degP41 kanR (U.S. Pat. No. 5,639,635). Other strains and derivatives thereof,
such as E. coli
294 (ATCC 31,446), E. coli B, E. coli .lambda. 1776 (ATCC 31,537) and E. coli
RV308 (ATCC
31,608) are also suitable. These examples are illustrative rather than
limiting. Methods for
constructing derivatives of any of the above-mentioned bacteria having defined
genotypes are
known in the art and described in, for example, Bass et al., Proteins, 8:309-
314 (1990). It is
generally necessary to select the appropriate bacteria taking into
consideration replicability of the
replicon in the cells of a bacterium. For example, E. coli, Serrano, or
Salmonella species can be
suitably used as the host when well known plasmids such as pBR322, pBR325,
pACYC177, or
pKN410 are used to supply the replicon. Typically the host cell should secrete
minimal amounts
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of proteolytic enzymes, and additional protease inhibitors may desirably be
incorporated in the
cell culture.
Antibody Production
Host cells are transformed with the above-described expression vectors and
cultured in conventional nutrient media modified as appropriate for inducing
promoters,
selecting transformants, or amplifying the genes encoding the desired
sequences.
Transformation means introducing DNA into the prokaryotic host so that the
DNA is replicable, either as an extrachromosomal element or by chromosomal
integrant.
Depending on the host cell used, transformation is done using standard
techniques appropriate to
such cells. The calcium treatment employing calcium chloride is generally used
for bacterial
cells that contain substantial cell-wall barriers. Another method for
transformation employs
polyethylene glycol/DMSO. Yet another technique used is electroporation.
Prokaryotic cells used to produce any one or more of the anti-Her3 antibodies
of
the invention are grown in media known in the art and suitable for culture of
the selected host
cells. Examples of suitable media include Luria broth (L13) plus necessary
nutrient supplements.
In some embodiments, the media also contains a selection agent, chosen based
on the
construction of the expression vector, to selectively permit growth of
prokaryotic cells
containing the expression vector. For example, ampicillin is added to media
for growth of cells
expressing ampicillin resistant gene.
Any necessary supplements besides carbon, nitrogen, and inorganic phosphate
sources may also be included at appropriate concentrations introduced alone or
as a mixture with
another supplement or medium such as a complex nitrogen source. Optionally the
culture
medium may contain one or more reducing agents selected from the group
consisting of
glutathione, cysteine, cystamine, thioglycollate, dithioerythritol and
dithiothreitol.
The prokaryotic host cells are cultured at suitable temperatures. For E. coli
growth, for example, the preferred temperature ranges from about 20 C to about
39 C, more
preferably from about 25 C to about 37 C, even more preferably at about 30 C.
The pH of the
medium may be any pH ranging from about 5 to about 9, depending mainly on the
host
organism. For E. coil, the pH is preferably from about 6.8 to about 7.4, and
more preferably
about 7Ø
If an inducible promoter is used in the expression vector of the invention,
protein
expression is induced under conditions suitable for the activation of the
promoter. In one aspect
of the invention, PhoA promoters are used for controlling transcription of the
polypeptides.
Accordingly, the transformed host cells are cultured in a phosphate-limiting
medium for
induction. Preferably, the phosphate-limiting medium is the C.R.A.P medium
(see, e.g,,
Simmons et al., J. Immunol. Methods (2002), 263:133-147). A variety of other
inducers may be
used, according to the vector construct employed, as is known in the art.
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In one embodiment, the expressed polypeptides of the present invention are
secreted into and recovered from the periplasm of the host cells. Protein
recovery typically
involves disrupting the microorganism, generally by such means as osmotic
shock, sonication or
lysis. Once cells are disrupted, cell debris or whole cells may be removed by
centrifugation or
filtration. The proteins may be further purified, for example, by affinity
resin chromatography.
Alternatively, proteins can be transported into the culture media and isolated
therein. Cells may
be removed from the culture and the culture supernatant being filtered and
concentrated for
further purification of the proteins produced. The expressed polypeptides can
be further isolated
and identified using commonly known methods such as polyacrylamide gel
electrophoresis
(PAGE) and Western blot assay.
Another aspect of the invention contemplates antibody production in large
quantity by a fermentation process. Various large-scale fed-batch fermentation
procedures are
available for production of recombinant proteins. Large-scale fermentations
have at least 1000
liters of capacity, preferably about 1,000 to 100,000 liters of capacity.
These fermentors use
agitator impellers to distribute oxygen and nutrients, especially glucose (the
preferred
carbon/energy source). Small scale fermentation refers generally to
fermentation in a ferrnentor
that is no more than approximately 100 liters in volumetric capacity, and can
range from about 1
liter to about 100 liters.
In a fermentation process, induction of protein expression is typically
initiated
after the cells have been grown under suitable conditions to a desired
density, e.g., an 0D550 of
about 180-220, at which stage the cells are in the early stationary phase. A
variety of inducers
may be used, according to the vector construct employed, as is known in the
art and described
above. Cells may be grown for shorter periods prior to induction. Cells are
usually induced for
about 12-50 hours, although longer or shorter induction time may be used.
To improve the production yield and quality of the polypeptides of the
invention,
various fermentation conditions can be modified. For example, to improve the
proper assembly
and folding of the secreted antibody polypeptides, additional vectors
overexpressing chaperone
proteins, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (a
peptidylprolyl cis,trans-isomerase with chaperone activity) can be used to co-
transform the host
prokaryotic cells. The chaperone proteins have been demonstrated to facilitate
the proper folding
and solubility of heterologous proteins produced in bacterial host cells. Chen
et al. (1999) J Bio
Chem 274:19601-19605; Georgiou et al., U.S. Pat. No. 6,083,715; Georgiou et
al., U.S. Pat. No.
6,027,888; Bothmann and Pluckthun (2000) J. Biol. Chem, 275;17100-17105; Ramm
and
Pluckthun (2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol.
Microbiol. 39;199-
210.
To minimize proteolysis of expressed heterologous proteins (especially those
that
are proteolytically sensitive) certain host strains deficient for proteolytic
enzymes can be used for
the present invention. For example, host cell strains may be modified to
effect genetic
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mutation(s) in the genes encoding known bacterial proteases such as Protease
III, OmpT, DegP,
Tsp, Protease I, Protease Mi, Protease V, Protease VI and combinations
thereof. Some E. coli
protease-deficient strains are available and described in, for example, Joly
et al. (1998), supra;
Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et at., U.S. Pat. No.
5,508,192; Hara et al.,
Microbial Drug Resistance, 2:63-72 (1996).
In one embodiment, E. coil strains deficient for proteolytic enzymes and
transformed with plasmicis overexpressing one or more chaperone proteins are
used as host cells
in the expression system of the invention.
iii. Antibody Purification
Standard protein purification methods known in the art can be employed. 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, are
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 Amicofin 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 following procedures are exemplary of suitable purification procedures:
fractionation on immunoaffinity or ion-exchange columns, ethanol
precipitation, reverse phase
HPLC, chromatography on silica or on a cation-exchange resin such as DEAE,
chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration
using, for
example, Sephadex G-75.
The suitability of protein A as an affinity ligand depends on the species and
isotype of any immunoglobulin Pc domain that is present in the antibody.
Protein A can be used
to purify antibodies that are based on human yl, 72, or -y4 heavy chains
(Lindmark et al., J.
Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse
isotypes and for
human 73 (Gusset al., EMBO J. 5:15671575 (1986)). The matrix to which the
affinity ligand is
attached is most often agarose, but other matrices are available. Mechanically
stable matrices
such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster
flow rates and
shorter processing times than can be achieved with agarose. Where the antibody
comprises a C
H3 domain, the Bakerbond ABX resin (J. T. Baker, Phillipsburg, N. J.) is
useful for
purification. Other techniques for protein purification such as fractionation
on an ion-exchange
column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica,
chromatography
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on heparin SEPHAROSES 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.
In one aspect, Protein A immobilized on a solid phase is used for
immunoaffinity
purification of the full length antibody products of the invention. Protein A
is a 41 kD cell wall
protein from Staphylococcus aureus which binds with a high affinity to the Fe
region of
antibodies. Lindmark et al (1983) J. Immunol, Meth. 62:1-13. The solid phase
to which Protein
A is immobilized is preferably a column comprising a glass or silica surface,
more preferably a
controlled pore glass column or a silicic acid column. In some applications,
the column has been
coated with a reagent, such as glycerol, in an attempt to prevent nonspecific
adherence of
contaminants.
As the first step of purification, the preparation derived from the cell
culture as
described above is applied onto the Protein A immobilized solid phase to allow
specific binding
of the antibody of interest to Protein A. The solid phase is then washed to
remove contaminants
non-specifically bound to the solid phase. Finally the antibody of interest is
recovered from the
solid phase by elution,
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).
Generating Antibodies Using Eukaryotie Host Cells
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
A vector for use in a eukaryotic host cell may also contain a signal sequence
or
other polypeptide having a specific cleavage site at the N-terminus of the
mature protein or
polypeptide of interest. The heterologous signal sequence selected preferably
is one that is
recognized and processed (i.e., cleaved by a signal peptidase) by the host
cell. 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 antibody.
(ii) Origin of Replication
Generally, an origin of replication component is not needed for mammalian
expression vectors. For example, the SV40 origin may typically be used only
because it contains
the early promoter.
(iii) Selection Gene Component
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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, where relevant, or (c) supply critical
nutrients not
available from complex media.
One example of a selection scheme utilizes a drug to arrest growth of a host
cell.
Those cells that are successfully transformed with a heterologous gene produce
a protein
conferring drug resistance and thus survive the selection regimen. Examples of
such dominant
selection use the drugs neomycin, mycophenolic acid and hygromycin.
Another example of suitable selectable markers for mammalian cells are those
that enable the identification of cells competent to take up the antibody
nucleic acid, such as
DHFR, thymidine kinase, metallothionein-I and -II, preferably primate
metallothionein genes,
adenosine deaminase, omithine 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 activity (e.g.,
ATCC CRL-9096).
Alternatively, host cells (particularly wild-type hosts that contain
endogenous
DHFR) transformed or co-transformed with DNA sequences encoding an antibody,
wild-type
DHFR protein, and another selectable marker such as aminoglycoside T-
phosphotransferase
(APH) can be selected by cell growth in medium containing a selection agent
for the selectable
marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or
G418. See U.S. Pat.
No. 4,965,199.
(iv) Promoter Component
Expression and cloning vectors usually contain a promoter that is recognized
by
the host organism and is operably linked to the antibody polypeptide nucleic
acid. Promoter
sequences are known for eukaryotes. Virtually all eukaryotic genes have an AT-
rich region
located approximately 25 to 30 bases upstream from the site where
transcription is initiated.
Another sequence found 70 to 80 bases upstream from the start of transcription
of many genes is
a CNCAAT region where N may be any nucleotide. At the 3' end of most
eukaryotic genes is an
AATAAA sequence that may be the signal for addition of the poly A tail to the
3' end of the
coding sequence. All of these sequences are suitably inserted into eukaryotic
expression vectors.
Antibody polypeptide transcription from vectors in mammalian host cells is
controlled, for example, by promoters obtained from the genomes of viruses
such as polyorna
virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma
virus, avian sarcoma
virus, cytomegalovirus, a retrovirus, hepatitis-B virus and 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.
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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
Hindill E restriction
fragment. A system for expressing DNA in mammalian hosts using the bovine
papilloma virus
as a vector is disclosed in U.S. Pat. No. 4,419,446. A modification of this
system is described in
U.S. Pat. No. 4,601,978. Alternatively, the Rous Sarcoma Virus long terminal
repeat can be used
as the promoter.
(v) Enhancer Element Component
Transcription of DNA encoding the antibody polypeptide of this invention by
higher eukaryotes is often increased by inserting an enhancer sequence into
the vector. Many
enhancer sequences are now known from mammalian genes (globin, elastase,
albumin, .alpha.-
fetoprotein, and insulin). Typically, however, one will use an enhancer from a
eukaryotic cell
virus. Examples include the SV40 enhancer on the late side of the replication
origin (bp 100-
270), the cytornegalovirus 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 antibody polypeptide-encoding
sequence, but is preferably
located at a site 5' from the promoter.
(vi) Transcription Termination Component
Expression vectors used in eukaryotic host cells will typically also contain
sequences necessary for the termination of transcription and for stabilizing
the mRNA. Such
sequences are commonly available from the 5' and, occasionally 3',
untranslated regions of
eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments
transcribed as
polyadenylated fragments in the untranslated portion of the inRNA encoding an
antibody. One
useful transcription termination component is the bovine growth hormone
polyadenylation
region, See W094/11026 and the expression vector disclosed therein.
(vii) Selection and Transformation of Host Cells
Suitable host cells for cloning or expressing the DNA in the vectors herein
include higher eukaryote cells described herein, including vertebrate host
cells. Propagation of
vertebrate cells in culture (tissue culture) has become a routine procedure.
Examples of useful
mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-
7, ATCC
CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth
in suspension
culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells
(BHK, ATCC
CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl.
Acad. Sci. USA
77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251
(1980); monkey
kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76,
ATCC CRL-
1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells
(MDCK,
ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung
cells (W138,
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ATCC CCL 75); human liver cells (Hep 02, HB 8065); mouse mammary tumor (MMT
060562,
ATCC CCL51); TM cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68
(1982)); MRC 5
cells; FS4 cells; and a human hepatoma line (Hep 02).
Host cells are transformed with the above-described expression or cloning
vectors
for 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 Suitable host cells for producing an antibody of this invention may be
cultured in a variety of media. Commercially available media such as Ham's HO
(Sigma),
Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's
Modified
Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In
addition, any of
the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al.,
Anal. Biochem.
102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or
5,122,469; WO
90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be used as culture media
for the host cells.
Any of these media may be supplemented as necessary with hormones and/or other
growth
factors (such as insulin, transferrin, or epidermal growth factor), salts
(such as sodium chloride,
calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such
as adenosine
and thymidine), antibiotics (such as GENTAMYONTm drug), trace elements
(defined as
inorganic compounds usually present at final concentrations in the micrornolar
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 Antibody
When using recombinant techniques, the antibody can be produced
intracellularly,
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, are removed, for
example, by
centrifugation or ultrafiltration. 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 PelliconTM
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, hydroxylapatite chromatography, gel electrophoresis, dialysis, and
affinity
chromatography, with affinity chromatography being the preferred purification
technique. The
suitability of protein A as an affinity ligand depends on the species and
isotype of any
immunoglobulin Fe domain that is present in the antibody. Protein A can be
used to purify
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antibodies that are based on human yl, y2, or y4 heavy chains (Lindmark et
al., J. Immunol.
Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for
human y3
(Gusset 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 ABXTM resin (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
polyaspaitic 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 0-0.25 M salt).
Immunoconjugates
The invention also pertains to immunoconjugates (interchangeably termed
"antibody-drug conjugates" or "ADC")comprising at least one invention antibody
conjugated to
a cytotoxic agent such as a chemotherapeutic agent, a growth inhibitory agent,
a 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).
The use of antibody-drug conjugates for the local delivery of cytotoxic or
cytostatic agents, i.e. drugs to kill or inhibit tumor cells in the treatment
of cancer (Syrigos and
Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer
(1997) Adv.
Drg Del. Rev. 26:151-172; U.S. Pat. No. 4,975,278) allows targeted delivery of
the drug moiety
to tumors, and intracellular accumulation therein, where systemic
administration of these
unconjugated drug agents may result in unacceptable levels of toxicity to
normal cells as well as
the tumor cells sought to be eliminated (Baldwin et al., (1986) Lancet pp.
(Mar. 15, 1986):603-
05; Thorpe, (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review," in
Monoclonal Antibodies '84: Biological And Clinical Applications, A. Pinchera
et al. (ed.$), pp.
475-506). Maximal efficacy with minimal toxicity is sought thereby. Both
polyclonal antibodies
and monoclonal antibodies have been reported as useful in these strategies
(Rowland et al.,
(1986) Cancer Immunol. Immunother., 21:183-87). Drugs used in these methods
include
daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., (1986)
supra). Toxins
used in antibody-toxin conjugates include bacterial toxins such as diphtheria
toxin, plant toxins
such as ricin, small molecule toxins such as geldanamycin (Mandler et al
(2000) Jour. of the Nat.
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Cancer Inst. 92(19):1573-1581; Mandler et al (2000) Bioorganie & Med. Chem,
Letters 10:
1025-1028; Mandler et al (2002) Bioconjugate Chem. 13:786-791), maytansinoids
(EP 1391213;
Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93;8618-8623), and calicheamicin
(Lode et al
(1998) Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342). The
toxins may
effect their cytotoxic and eytostatic effects by mechanisms including tubulin
binding, DNA
binding, or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive
or less active
when conjugated to large antibodies or protein receptor ligands.
ZEVALINTIvi (ibritumomab tiuxetan, Biogen/Idec) is an antibody-radioisotope
conjugate composed of a murine IgG1 kappa monoclonal antibody directed against
the CD20
antigen found on the surface of normal and malignant B lymphocytes and or 9
Y
radioisotope bound by a thiourea linker-chelator (Wiseman et al (2000) Eur.
Jour. Nucl. Med.
27(7):766-77; Wiseman et al (2002) Blood 99(12):4336-42; Witzig et al (2002)
J. Clin. Oncol.
20(10):2453-63; Witzig eta! (2002) J. Clin. Oncol, 20(15):3262-69). Although
ZEVALINTm
has activity against B-cell non-Hodgkin's Lymphoma (NHL), administration
results in severe
and prolonged cytopenias in most patients. MYLOTARGTm (gemtuzumab ozogamicin,
Wyeth
Pharmaceuticals), an antibody drug conjugate composed of a hu CD33 antibody
linked to
calicheamicin, was approved in 2000 for the treatment of acute myeloid
leukemia by injection
(Drugs of the Future (2000) 25(7):686; U.S. Pat. Nos. 4,970,198; 5,079,233;
5,585,089;
5,606,040; 5,693,762; 5,739,116; 5,767,285; 5,773,001). Cantuzumab mertansine
(Immunogen,
Inc.), an antibody drug conjugate composed of the huC242 antibody linked via
the disulfide
linker SPP to the maytansinoid drug moiety, DM1, is advancing into Phase II
trials for the
treatment of cancers that express CanAg, such as colon, pancreatic, gastric,
and others. MLN-
2704 (Millennium Pharm., BZL Biologies, Immunogen Inc.), an antibody drug
conjugate
composed of the anti-prostate specific membrane antigen (PSMA) monoclonal
antibody linked
to the maytansinoid drug moiety, DM1, is under development for the potential
treatment of
prostate tumors. The auristatin peptides, auristatin E (AE) and
monomethylauristatin (MMAE),
synthetic analogs of dolastatin, were conjugated to chimeric monoclonal
antibodies cBR96
(specific to Lewis Y on carcinomas) and cAC10 (specific to CD30 on
hematological
malignancies) (Doronina et al (2003) Nature Biotechnology 21(7):778-784) and
are under
therapeutic development.
Chemotherapeutic agents useful in the generation of immunoconjugates are
described herein (eg., above). Enzymatically active toxins and fragments
thereof that can be used
include diphtheria A chain, nonbinding active fragments of diphtheria toxin,
exotoxin A chain
(from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,
alpha-sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins
(PAPI, PAPII, and
PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis
inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. See,
e.g., WO 93/21232
published Oct. 28, 1993. A variety of radionuclides are available for the
production of
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radioconjugated antibodies. Examples include 212Bi, .131I, 13I1n, .90Y, and
I86Re. Conjugates of
the antibody and cytotoxic agent are made using a variety of bifunctional
protein-coupling agents
such as N-succinimidy1-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane
(IT), bifunctional
derivatives of imidoesters (such as dimethyl adipimidate HCI), active esters
(such as
disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido
compounds (such as his
(p-azidobenzoyl) hexanediarnine), bis-diazonium derivatives (such as bis-(p-
diazoniumbenzoyI)-
ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-
active fluorine
compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin
immunotoxin can be
prepared as described in Vitetta et al., Science, 238: 1098 (1987). Carbon-14-
labeled 1-
isothiocyanatobenzy1-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is
an exemplary
chelating agent for conjugation of radionucleotide to the antibody. See
W094/11026.
Conjugates of an antibody and one or more small molecule toxins, such as a
calicheamicin, maytansinoids, dolastatins, auristatins, a trichothecene, and
CC1065, and the
derivatives of these toxins that have toxin activity, are also contemplated
herein.
i. Maytansine and Maytansinoids
In some embodiments, the immunoconjugate comprises an antibody (full length
or fragments) of the invention conjugated to one or more maytansinoid
molecules.
Maytansinoids are mitotoxic inhibitors which act by inhibiting tubulin
polymerization. Maytansine was first isolated from the east African shrub
Maytenus serrata (U.S.
Pat. No. 3,896,111). Subsequently, it was discovered that certain microbes
also produce
maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No.
4,151,042).
Synthetic maytansinol and derivatives and analogues thereof are disclosed, for
example, in U.S.
Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757;
4,307,016;
4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;
4,331,598;
4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533.
Maytansinoid drug moieties are attractive drug moieties in antibody drug
conjugates because they are: (i) relatively accessible to prepare by
fermentation or chemical
modification, derivatization of fermentation products, (ii) amenable to
derivatization with
functional groups suitable for conjugation through the non-disulfide linkers
to antibodies, (iii)
stable in plasma, and (iv) effective against a variety of tumor cell lines.
Immunoconjugates containing maytansinoids, methods of making same, and their
therapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020,
5,416,064 and European
Patent EP 0 425 235 Bl, the disclosures of which are hereby expressly
incorporated by
reference. Liu etal., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) described
irnmunoconjugates comprising a maytansinoid designated DM I linked to the
monoclonal
antibody C242 directed against human colorectal cancer. The conjugate was
found to be highly
cytotoxic towards cultured colon cancer cells, and showed antitumor activity
in an in vivo tumor
growth assay. Chari et al., Cancer Research 52:127-131(1992) describe
inununoconjugates in
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which a maytansinoid was conjugated via a disulfide linker to the murine
antibody A7 binding to
an antigen on human colon cancer cell lines, or to another rnurine monoclonal
antibody TA.1
that binds the Her2/neu oncogene. The cytotoxicity of the TA.1-maytansinoid
conjugate was
tested in vitro on the human breast cancer cell line SK-BR-3, which expresses
3x105 Her2
surface antigens per cell. The drug conjugate achieved a degree of
cytotoxicity similar to the
free maytansinoid drug, which could be increased by increasing the number of
maytansinoid
molecules per antibody molecule. The A7-maytansinoid conjugate showed low
systemic
cytotoxicity in mice.
Antibody-maytansinoid conjugates are prepared by chemically linking an
antibody to a maytansinoid molecule without significantly diminishing the
biological activity of
either the antibody or the maytansinoid molecule. See, e.g., U.S. Pat. No.
5,208,020 (the
disclosure of which is hereby expressly incorporated by reference). An average
of 3-4
maytansinoid molecules conjugated per antibody molecule has shown efficacy in
enhancing
cytotoxicity of target cells without negatively affecting the function or
solubility of the antibody,
although even one molecule of toxin/antibody would be expected to enhance
cytotoxicity over
the use of naked antibody. Maytansinoids are well known in the art and can be
synthesized by
known techniques or isolated from natural sources. Suitable maytansinoids are
disclosed, for
example, in U.S. Pat. No. 5,208,020 and in the other patents and nonpatent
publications referred
to hereinabove. Preferred maytansinoids are maytansinol and maytansinol
analogues modified in
the aromatic ring or at other positions of the maytansinol molecule, such as
various maytansinol
esters.
There are many linking groups known in the art for making antibody-
maytansinoid conjugates, including, for example, those disclosed in U.S. Pat.
No. 5,208,020 or
EP Patent 0 425 235 B1, Chari et al., Cancer Research 52:127-131 (1992), and
U.S. patent
application Set. No. 10/960,602, filed Oct. 8, 2004, the disclosures of which
are hereby expressly
incorporated by reference. Antibody-maytansinoid conjugates comprising the
linker component
SMCC may be prepared as disclosed in U.S. patent application Ser. No.
10/960,602, filed Oct. 8,
2004. The linking groups include disulfide groups, thioether groups, acid
labile groups,
photolabile groups, peptidase labile groups, or esterase labile groups, as
disclosed in the above-
identified patents, disulfide and thioether groups being preferred. Additional
linking groups are
described and exemplified herein.
Conjugates of the antibody and maytansinoid may be made using a variety of
bifunctional protein coupling agents such as N-succinimidy1-3-(2-
pyridyldithio) propionate
(SPDP), succinimidy1-4-(N-maleimidomethypcyclohexane-1-carboxylate (SMCC),
iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl
adipimidate HC1),
active esters (such as disuccinimidyl suberate), aldehydes (such as
glutaraldehyde), bis-azido
compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as
bis-(p-diazoniumbenzoy1)-ethylenediamine), diisocyanates (such as toluene 2,6-
diisocyanate),
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and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
Particularly
preferred coupling agents include N-suceinimidy1-3-(2-pyridyldithio)
propionate (SPDP)
(Carlsson et al., Biochem. J. 173:723-737 (1978)) and N-succinimidy1-4-(2-
pyridyithio)pentanoate (SPP) to provide for a disulfide linkage.
The linker may be attached to the maytansinoid molecule at various positions,
depending on the type of the link. For example, an ester linkage may be formed
by reaction with
a hydroxyl group using conventional coupling techniques. The reaction may
occur at the C-3
position having a hydroxyl group, the C-14 position modified with
hydroxymethyl, the C-15
position modified with a hydroxyl group, and the C-20 position having a
hydroxyl group. In a
preferred embodiment, the linkage is formed at the C-3 position of maytansinol
or a maytansinol
analogue.
Auristatins and Dolastatins
In some embodiments, the inununoconjugate comprises an antibody of the
invention conjugated to dolastatins or dolostatin peptidic analogs and
derivatives, the auristatins
(U.S. Pat. Nos. 5,635,483; 5,780,588). Dolastatins and auristatins have been
shown to interfere
with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division
(Woyke et al
(2001) Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anticancer
(U.S. Pat.
No. 5,663,149) and antifungal activity (Pettit et al (1998) Antimicrob. Agents
Chemother.
42:2961-2965). The dolastatin or auristatin drug moiety may be attached to the
antibody through
the N (amino) terminus or the C (carboxyl) terminus of the peptidic drug
moiety (WO
02/088172).
Exemplary auristatin embodiments include the N-terminus linked
monomethylauristatin drug moieties DE and DF, disclosed in "Monomethylvaline
Compounds
Capable of Conjugation to Ligands", U.S. Ser. No. 10/983,340, filed Nov. 5,
2004, the disclosure
of which is expressly incorporated by reference in its entirety.
Typically, peptide-based drug moieties can be prepared by forming a peptide
bond between two or more amino acids and/or peptide fragments. Such peptide
bonds can be
prepared, for example, according to the liquid phase synthesis method (see E.
Schroder and K.
Lubke, "The Peptides", volume 1, pp 76-136, 1965, Academic Press) that is well
known in the
field of peptide chemistry. The auristatin/dolastatin drug moieties may be
prepared according to
the methods of: U.S. Pat. No. 5,635,483; U.S. Pat. No. 5,780,588; Pettit et al
(1989) J. Am.
Chem. Soc. 111:5463-5465; Pettit et al (1998) Anti-Cancer Drug Design 13:243-
277; Pettit, G.
R., et al. Synthesis, 1996, 719-725; and Pettit et al (1996) J. Chem. Soc.
Perkin Trans. I 5:859-
863. See also Doronina (2003) Nat Biotechnol 21(7):778-784; "Monomethylvaline
Compounds
Capable of Conjugation to Ligands", U.S. Ser. No. 10/983,340, filed Nov. 5,
2004, hereby
incorporated by reference in its entirety (disclosing, e.g., linkers and
methods of preparing
monomethylvaline compounds such as MMAE and MMAF conjugated to linkers).
Calicheamicin
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In other embodiments, the immunoconjugate comprises an antibody of the
invention conjugated to one or more calicheamicin molecules. The calicheamicin
family of
antibiotics are capable of producing double-stranded DNA breaks at sub-
picomolar
concentrations. For the preparation of conjugates of the calicheamicin family,
see U.S. Pat. Nos.
5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001,
5,877,296 (all to
American Cyanamid Company). Structural analogues of calicheamicin which may be
used
include, but are not limited to, .gamma sub.lI, .alpha.2I,
.alpha3I, N-
acetykgamma.1I, PSAG and .theta.I1 (Hinman et al., Cancer
Research
53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998) and the
aforementioned U.S. patents to American Cyanamid). Another anti-tumor drug
that the antibody
can be conjugated is QFA which is an antifolate. Both calicheamicin and QFA
have intracellular
sites of action and do not readily cross the plasma membrane. Therefore,
cellular uptake of these
agents through antibody mediated internalization greatly enhances their
cytotoxic effects.
iv. Other Cytotoxic Agents
Other antitumor agents that can be conjugated to the antibodies of the
invention
include BCNU, streptozoicin, vincristine and 5-fluorouracil, the family of
agents known
collectively LL-E33288 complex described in U.S. Pat. Nos, 5,053,394,
5,770,710, as well as
esperamicins (U.S. Pat. No. 5,877,296).
Enzymatically active toxins and fragments thereof which can be used include
diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin
A chain (from
Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-
sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins
(PAPI, PAPII, and
PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis
inhibitor, gelonin,
mitogellin, restfictocin, phenomycin, enomycin and the tricothecenes. See, for
example, WO
93/21232 published Oct. 28, 1993.
Procedures for conjugating the biological agents with the cytotoxic agents
have
been previously described. Procedures for conjugating chlorambucil with
antibodies are
described by Flechner, I, European Journal of Cancer, 9:741-745 (1973); Ghose,
T. et al., British
Medical Journal, 3;495-499 (1972); and Szekerke, M., et al., Neoplasma, 19:211-
215(1972),
which are hereby incorporated by reference. Procedures for conjugating
daunomycin and
adriamycin to antibodies are described by Hurwitz, E. et al., Cancer Research,
35:1175-1181
(1975) and Arnon, R. et al. Cancer Surveys, 1:429-449 (1982), which are hereby
incorporated by
reference. Procedures for preparing antibody-ricin conjugates are described in
U.S. Pat. No.
4,414,148 and by Osawa, T., et al. Cancer Surveys, 1:373-388 (1982) and the
references cited
therein, which are hereby incorporated by reference. Coupling procedures are
also described in
EP 86309516.2, which is hereby incorporated by reference.
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The present invention further contemplates an immunoconjugate formed between
an antibody and a compound with nucleolytic activity (e.g., a ribonuclease or
a DNA
endonuclease such as a deoxyribonuclease; DNase).
For selective destruction of the tumor, the antibodies of the invention can be
emitter, which, when localized at the tumor site, results in a killing of
several cell diameters.
See, e.g., S. E. Order, "Analysis, Results, and Future Prospective of the
Therapeutic Use of
Radiolabeled Antibody in Cancer Therapy", Monoclonal Antibodies for Cancer
Detection and
Therapy, R. W. Baldwin et al. (eds.), pp 303-316 (Academic Press 1985), which
is hereby
The radio- or other labels may be incorporated in the conjugate in known ways.
For example, the peptide may be biosynthesized or may be synthesized by
chemical amino acid
synthesis using suitable amino acid precursors involving, for example,
fluorine-19 in place of
and In" can be attached via a cysteine
residue in the peptide. Yttrium-90 can be attached via a lysine residue. The
IODOGEN method
(Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57) can be used to
incorporate
iodine-123. "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press
1989)
describes other methods in detail.
25
Conjugates of the antibody and cytotoxic agent may be made using a variety of
bifunctional protein coupling agents such as N-succinimidy1-3-(2-
pyridyldithio) propionate
(SPDP), succinimidy1-4-(N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC),
iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl
adipimidate HC1),
active esters (such as disuccinimidyl suberate), aldehydes (such as
glutaraldehyde), bis-azido
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dimethyl linker or disulfide-containing linker (Chari et al., Cancer Research
52:127-131 (1992);
U.S. Pat. No. 5,208,020) may be used.
The compounds of the invention expressly contemplate, but are not limited to,
ADC prepared with cross-linker reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS,
1VIPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS,
sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidy1-(4-
vinylsulfone)benzoate) which are commercially available (e.g., from Pierce
Biotechnology, Inc.,
Rockford, Ill., U.S.A). See pages 467-498, 2003-2004 Applications Handbook and
Catalog.
v. Preparation of Antibody Drug Conjugates
In the antibody drug conjugates (ADC) of the invention, an antibody (Ab) is
conjugated to one or more drug moieties (D), e.g. about 1 to about 20 drug
moieties per
antibody, through a linker (L). The ADC of Formula I may be prepared by
several routes,
employing organic chemistry reactions, conditions, and reagents known to those
skilled in the
art, including: (1) reaction of a nucleophilic group of an antibody with a
bivalent linker reagent,
to form Ab-L, via a covalent bond, followed by reaction with a drug moiety D;
and (2) reaction
of a nucleophilic group of a drug moiety with a bivalent linker reagent, to
form D-L, via a
covalent bond, followed by reaction with the nucleophilic group of an
antibody. Additional
methods for preparing ADC are described herein.
Ab-(L-D)p I
The linker may be composed of one or more linker components. Exemplary linker
components include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"),
valine-citrulline
("val-cit"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl
("PAB"), N-
Succinimidyl 4-(2-pyridylthio) pentanoate ("SPP"), N-Suceinimidyl 4-(N-
maleimidomethyl)cyclohexane-1 carboxylate ("SMCC"), and N-Succinimidyl (4-iodo-
acetyl)aminobenzoate ("SIAB"). Additional linker components are known in the
art and some
are described herein. See also "Monomethylvaline Compounds Capable of
Conjugation to
Ligands", U.S. Ser. No. 10/983,340, filed Nov. 5, 2004, the contents of which
are hereby
incorporated by reference in its entirety.
In some embodiments, the linker may comprise amino acid residues. Exemplary
amino acid linker components include a dipeptide, a tripeptide, a tetrapeptide
or a pentapeptide.
Exemplary dipeptides include: valine-citrulline (ye or val-cit), alanine-
phenylalanine (af or ala-
phe). Exemplary tripeptides include: glycine-valine-citrulline (gly-val-cit)
and glycine-glycine-
glycine (gly-gly-gly). Amino acid residues which comprise an amino acid linker
component
include those occurring naturally, as well as minor amino acids and non-
naturally occurring
amino acid analogs, such as citrulline. Amino acid linker components can be
designed and
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optimized in their selectivity for enzymatic cleavage by a particular enzyme,
for example, a
tumor-associated protease, cathepsin B, C and D, or a plasmin protease.
Nucleophilic groups on antibodies include, but are not limited to: (i) N-
terminal
amine groups, (ii) side chain amine groups, e.g. lysine, (iii) side chain
thiol groups, e.g. cysteine,
and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated.
Amine, thiol, and
hydroxyl groups are nucleophilic and capable of reacting to form covalent
bonds with
electrophilic groups on linker moieties and linker reagents including: (1)
active esters such as
NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl
halides such as
haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups.
Certain antibodies
have reducible interchain disulfides, i.e. cysteine bridges. Antibodies may be
made reactive for
conjugation with linker reagents by treatment with a reducing agent such as
DTT (dithiothreitol).
Each cysteine bridge will thus form, theoretically, two reactive thiol
nucleophiles. Additional
nucleophilic groups can be introduced into antibodies through the reaction of
lysines with 2-
iminothiolane (Trout's reagent) resulting in conversion of an amine into a
thiol. Reactive thiol
groups may be introduced into the antibody (or fragment thereof) by
introducing one, two, three,
four, or more cysteine residues (e.g., preparing mutant antibodies comprising
one or more non-
native cysteine amino acid residues).
Antibody drug conjugates of the invention may also be produced by modification
of the antibody to introduce electrophilic moieties, which can react with
nucleophilic substituents
on the linker reagent or drug. The sugars of glycosylated antibodies may be
oxidized, e.g. with
periodate oxidizing reagents, to form aldehyde or ketone groups which may
react with the amine
group of linker reagents or drug moieties. The resulting imine Schiff base
groups may form a
stable linkage, or may be reduced, e.g. by borohydride reagents to form stable
amine linkages. In
one embodiment, reaction of the carbohydrate portion of a glycosylated
antibody with either
galactose oxidase or sodium meta-periodate may yield carbonyl (aldehyde and
ketone) groups in
the protein that can react with appropriate groups on the drug (Hermanson,
Bioconjugate
Techniques). In another embodiment, proteins containing N-terminal serine or
threonine
residues can react with sodium meta-periodate, resulting in production of an
aldehyde in place of
the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138-146;
U.S. Pat. No.
5,362,852). Such aldehyde can be reacted with a drug moiety or linker
nucleophile.
Likewise, nucleophilic groups on a drug moiety include, but are not limited
to:
amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone,
hydrazine carboxylate,
and arylhydrazide groups capable of reacting to form covalent bonds with
electrophilic groups
on linker moieties and linker reagents including: (i) active esters such as
NHS esters, HOBt
esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as
haloacetamides; (iii)
aldehydes, ketones, carboxyl, and maleimide groups.
Alternatively, a fusion protein comprising the antibody and cytotoxic agent
may
be made, e.g., by recombinant techniques or peptide synthesis. The length of
DNA may
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comprise respective regions encoding the two portions of the conjugate either
adjacent one
another or separated by a region encoding a linker peptide which does not
destroy the desired
properties of the conjugate.
In yet another embodiment, the antibody may be conjugated to a "receptor"
(such
streptavidin) for utilization in tumor pre-targeting wherein the antibody-
receptor conjugate is
administered to the patient, followed by removal of unbound conjugate from the
circulation
using a clearing agent and then administration of a "ligand" (e.g., avidin)
which is conjugated to
a cytotoxic agent (e.g., a radionucleotide).
RNAi
In certain embodiments, the cytotoxic agent is a gene modifier, e.g, RNAi
molecule. Methods for making specific RNAi (RNA interference) nucleic acids
are described in
the art (see, e.g., U.S. Pat. No. 6,506,559; WO 01/75164; WO 99/32619;
Elbashir et al., Nature
411:494-98 (2001); Zhang et al., Curr. Pharm. Biotech. 5:1-7 (2004); Paddison
et al., Curr. Opin.
Md. Ther. 5:217-24 (2003).
RNA interference refers to the process of sequence-specific post
transcriptional
gene silencing in animals mediated by short interfering RNAs (siRNA) (Fire et
al., 1998, Nature,
391, 806). The corresponding process in plants is commonly referred to as post
transcriptional
gene silencing or RNA silencing and is also referred to as quelling in fungi.
The process of post
transcriptional gene silencing is thought to be an evolutionarily conserved
cellular defense
mechanism used to prevent the expression of foreign genes which is commonly
shared by
diverse flora and phyla (Fire et al., 1999, Trends Genet., 15, 358). Such
protection from foreign
gene expression may have evolved in response to the production of double
stranded RNAs
(dsRNA) derived from viral infection or the random integration of transposon
elements into a
host genome via a cellular response that specifically destroys homologous
single stranded RNA
or viral genomic RNA. The presence of dsRNA in cells triggers the RNAi
response though a
mechanism that has yet to be fully characterized. This mechanism appears to be
different from
the interferon response that results from dsRNA mediated activation of protein
kinase PKR and
2',5'-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by
ribonuclease L.
The presence of long dsRNAs in cells stimulates the activity of a ribonuclease
III
enzyme referred to as dicer. Dicer is involved in the processing of the dsRNA
into short pieces
of dsRNA known as short interfering RNAs (siRNA) (Berstein et al., 2001,
Nature, 409, 363).
Short interfering RNAs derived from dicer activity are typically about 21-23
nucleotides in
length and comprise about 19 base pair duplexes. Dicer has also been
implicated in the excision
of 21 and 22 nucleotide small temporal RNAs (stRNA) from precursor RNA of
conserved
structure that are implicated in translational control (Hutvagner et al.,
2001, Science, 293, 834).
The RNAi response also features an endonuclease complex containing a siRNA,
commonly
referred to as an RNA-induced silencing complex (RISC), which mediates
cleavage of single
stranded RNA having sequence complementary to the antisense strand of the
siRNA duplex.
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Cleavage of the target RNA takes place in the middle of the region
complementary to the
antisense strand of the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15,
188).
Pharmaceutical Formulations
Therapeutic formulations comprising an antibody of the invention are prepared
for storage by mixing the antibody having the desired degree of purity with
optional
physiologically acceptable carriers, excipients or stabilizers (Remington: The
Science and
Practice of Pharmacy 20th edition (2000)), in the form of aqueous solutions,
lyophilized or other
dried formulations. Acceptable carriers, excipients, or stabilizers are
nontoxic to recipients at the
dosages and concentrations employed, and include buffers such as phosphate,
citrate, histidine
and other organic acids; antioxidants including ascorbic acid and methionine;
preservatives (such
as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium
chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl
parabens such as methyl
or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-
cresol); low molecular
weight (less than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrmlidone; amino acids
such as
glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides,
and other carbohydrates including glucose, mannose, or dextrins; chelating
agents such as
EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming
counter-ions such as
sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic
surfactants such as
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. Such molecules are
suitably present in
combination in amounts that are effective for the purpose intended.
The active ingredients may also be entrapped in microcapsule prepared, for
example, by coacervation techniques or by interfacial polymerization, for
example,
hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate)
microcapsule,
respectively, in colloidal drug delivery systems (for example, liposomes,
albumin microspheres,
microernulsions, nano-particles and nanocapsules) or in macroemulsions. Such
techniques are
disclosed in Remington: The Science and Practice of Pharmacy 20th edition
(2000).
The formulations to be used for in vivo administration must be sterile. This
is
readily accomplished by filtration through sterile filtration membranes.
Therapeutic and Non-Therapeutic Uses
Any one or more of the anti-Her3 antibodies described herein can be used in a
method for binding an antigen, preferably Her3 receptor protein in a subject
suffering from a
disorder associated with increased antigen expression and/or activity,
comprising administering
to the subject an antibody of the invention such that the antigen in the
subject is bound.
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Preferably, the antigen is a human protein molecule and the subject is a human
subject.
Consequently, an antibody of the invention can be administered to a human
subject for
therapeutic purposes. As well, an antibody of the invention can be
administered to a non-human
mammal expressing an antigen with which the immunoglobulin cross-reacts (e.g.,
a primate, pig
or mouse) for veterinary purposes or as an animal model of human disease.
Regarding the latter,
such animal models may be useful for evaluating the therapeutic efficacy of
antibodies of the
invention (e.g., testing of dosages and time courses of administration).
The present Her3 receptor antagonist and neutralizing antibodies are useful as
therapeutic reagents for treating a Her3 receptor expressing cancer or
alleviating one or more
symptoms of the cancer in a mammal. The antibodies of the invention can also
be used to treat
other Her3-medaisted disorders such as inflammatory disorders etc. The
antibody is able to bind
to at least a portion of the cancer cells that express a Her3 receptor in the
mammal and preferably
destroy or kill Her3 receptor-expressing tumor cells or inhibit the growth of
such tumor cells, in
vitro, ex vivo or in vivo, Such an antibody include a naked anti-Her3 receptor
antibody (not
conjugated to any agent). Naked antibodies that have cytotoxic or cell growth
inhibition
properties can be further harnessed with a cytotoxic agent to render them even
more potent in
tumor cell destruction. Cytotoxic properties can be conferred to an anti-Her3
receptor antibody
by, e.g., conjugating the antibody with a cytotoxic agent, to form an
immunoconjugate as
described herein. Alternative embodiments include Her3 specific agonist
antibodies.
In one aspect, the invention provides methods for treating or preventing a
tumor, a
cancer, and/or a cell proliferative disorder associated with increased
expression and/or activity of
Her3, the methods comprising administering an effective amount of an anti-Her3
antibody to a
subject in need of such treatment.
In one aspect, the invention provides methods for reducing, inhibiting,
blocking,
or preventing growth of a tumor or cancer, the methods comprising
administering an effective
amount of an anti-Her3 antibody to a subject in need of such treatment.
In one aspect, the invention provides methods for treating a tumor, a cancer,
and/or a cell proliferative disorder comprising administering an effective
amount of an anti-Her3
antibody to a subject in need of such treatment.
In one aspect, the invention provides methods for inhibiting cellular
proliferative
disorders including angiogenesis comprising administering an effective amount
of an anti-Her3
antibody to a subject in need of such treatment.
In one aspect, the invention provides methods for treating a pathological
condition associated with a cellular proliferative disorder comprising
administering an effective
amount of an anti-Her3 antibody to a subject in need of such treatment. In
some embodiments,
the pathological condition associated with said cellular proliferative
disorder is a tumor and/or
cancer.
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The antibodies in accordance with the present invention may be used to deliver
a
variety of cytotoxic drugs including therapeutic drugs, a compound emitting
radiation, molecules
of plants, fungal, or bacterial origin, biological proteins, and mixtures
thereof. The cytotoxic
drugs can be intracellularly acting cytotoxic drugs, such as short-range
radiation emitters,
including, for example, short-range, high-energy .alpha.-emitters.
The antibodies of the invention can be used to treat, inhibit, delay
progression of,
prevent/delay recurrence of, ameliorate, or prevent diseases, disorders or
conditions associated
with expression and/or activity of one or more antigen molecules.
Exemplary disorders include those described supra. Also included are Her3-
receptor mediated leukemias, disorders involving neovascularization,
cardiovascular disease
associated with Her3 polymorphism etc.
In certain embodiments, an immunoconjugate comprising an antibody conjugated
with one or more cytotoxic agent(s) is administered to the patient. In some
embodiments, the
immunoconjugate and/or antigen to which it is bound is/are internalized by the
cell, resulting in
increased therapeutic efficacy of the immunoconjugate in killing the target
cell to which it binds.
In one embodiment, the cytotoxic agent targets or interferes with nucleic acid
in the target cell.
In one embodiment, the cytotoxic agent targets or interferes with microtubule
polymerization.
Examples of such cytotoxic agents include any of the chemotherapeutic agents
noted herein
(such as a maytansinoid, auristatin, dolastatin, or a calicheamicin), a
radioactive isotope, or a
ribonuclease or a RNAi or a DNA endonuclease.
The anti-Her3 antibodies or immunoconjugates are administered to a human
patient, in accord with known methods, such as intravenous administration,
e.g., as a bolus or by
continuous infusion over a period of time, by intramuscular, intraperitoneal,
intracerebrospinal,
subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or
inhalation routes.
Intravenous or subcutaneous administration of the antibody is preferred.
Other therapeutic regimens may be combined with the administration of the anti-
Her3 receptor antibody. The combined administration includes co-
administration, using separate
formulations or a single pharmaceutical formulation, and consecutive
administration in either
order, wherein preferably there is a time period while both (or all) active
agents simultaneously
exert their biological activities. Preferably such combined therapy results in
a synergistic
therapeutic effect.
It may also be desirable to combine administration of the anti-Her3 receptor
antibody or antibodies, with administration of an antibody directed against
another tumor antigen
associated with the particular cancer. In another embodiment, the invention
antibody is a
bispecific construct targeting two distinct epitopes. The epitopes may be on
the same antigen or
on separate antigens, one of which is Her3 receptor protein.
Antibodies of the invention can be used either alone or in combination with
other
compositions in a therapy. For instance, an antibody of the invention may be
co-administered
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with another antibody, chemotherapeutic agent(s) (including cocktails of
chemotherapeutic
agents), other eytotoxic agent(s), anti-angiogenic agent(s), cytokines, and/or
growth inhibitory
agent(s). Where an antibody of the invention inhibits tumor growth, it may be
particularly
desirable to combine it with one or more other therapeutic agent(s) which also
inhibits tumor
growth, e.g. anti-EGFR agents including antibodies to any one or more of the
EGFR family of
receptors. Alternatively, or additionally, the patient may receive combined
radiation therapy
(e.g. external beam irradiation or therapy with a radioactive labeled agent,
such as an antibody).
Such combined therapies noted above include combined administration (where the
two or more
agents are included in the same or separate formulations), and separate
administration, in which
case, administration of the antibody of the invention can occur prior to,
and/or following,
administration of the adjunct therapy or therapies.
It is to be further understood that a cocktail of different monoclonal
antibodies,
such as a mixture of the specific monoclonal antibodies described herein, or
their binding
fragments, may be administered, if necessary or desired, for cancer treatment.
Indeed, using a
mixture of monoclonal antibodies, or binding fragments thereof, in a cocktail
to target several
antigens, or different epitopes, on cancer cells, is an advantageous approach,
particularly to
prevent evasion of tumor cells and/or cancer cells due to down regulation of
one of the antigens.
Combination Therapies
As indicated above, the invention provides combined therapies in which an anti-
Her3 antibody or antigen-binding fragment thereof is administered in
association with another
therapeutic agents; as well as compositions or combinations comprising said
antibody or
fragment in association with a therapeutica agent. For example, anti-Her3
antibodies are used in
combinations with anti-cancer therapeutics or anti-neovascularization
therapeutics to treat
various neoplastic or non-neoplastic conditions. In one embodiment, the
neoplastic or non-
neoplastic condition is characterized by pathological disorder associated with
aberrant or
undesired angiogenesis. The anti-Her3 antibody can be administered serially or
in combination
with another agent that is effective for those purposes, either in the same
composition or as
separate compositions. Alternatively, or additionally, multiple inhibitors of
Her3 can be
administered.
The administration of the anti-Her3 antibody can be done simultaneously, e.g.,
as
a single composition or as two or more distinct compositions using the same or
different
administration routes. Alternatively, or additionally, the administration can
be done sequentially,
in any order. In certain embodiments, intervals ranging from minutes to days,
to weeks to
months, can be present between the administrations of the two or more
compositions. For
example, the anti-cancer agent may be administered first, followed by the Her3
inhibitor.
However, simultaneous administration or administration of the anti-Her3
antibody first is also
contemplated.
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The effective amounts of therapeutic agents administered in combination with
an
anti-Her3 antibody will be at the physician's or veterinarian's discretion.
Dosage administration
and adjustment is done to achieve maximal management of the conditions to be
treated. The
dose will additionally depend on such factors as the type of therapeutic agent
to be used and the
specific patient being treated. Suitable dosages for the anti-cancer agent are
those presently used
and can be lowered due to the combined action (synergy) of the anti-cancer
agent and the anti-
Her3 antibody. In certain embodiments, the combination of the inhibitors
potentiates the
efficacy of a single inhibitor. The term "potentiate" refers to an improvement
in the efficacy of a
therapeutic agent at its common or approved dose. See also the section
entitled "Pharmaceutical
Compositions" herein.
Typically, the anti-Her3 antibodies and anti-cancer agents are suitable for
the
same or similar diseases to block or reduce a pathological disorder such as
tumor growth or
growth of a cancer cell. In one embodiment the anti-cancer agent is an anti-
angiogenesis agent.
"Hyper cellular proliferative" therapy in relationship to cancer is a cancer
treatment strategy aimed at inhibiting the neoplastie growth of tumor cells
expressing the Her3
receptor protein as well as preventing metastasis of tumors at the secondary
sites, therefore
allowing attack of the tumors by other therapeutics.
The anti-Her3 antibodies may also be used in addition to or in conjunction
with
anti-angiogenic agents that inhibit excessive tumor vascular development.
Consequently, hyper-
cellular proliferative therapy contemplated herein may be combined with an
"antiangiogenic"
therapy comprising anti-angiogenic compounds, of which many have been
identified and are
known in the arts, including those listed herein, e.g., listed under
Definitions, and by, e.g.,
Carmeliet and Jain, Nature 407:249-257 (2000); Ferrara et al., Nature Reviews.
Drug Discovery,
3:391-400 (2004); and Sato Int. J. Clin. Oncol., 8:200-206 (2003). In one
embodiment, an anti-
Her3 antibody is used in combination with an anti-Her2 antibody (or fragment)
and/or another
Her2 antagonist. Alternatively, or additionally, the anti-Her3 antibody may be
used in
combination with an anti-IGF-lr antibody or antagonist. See, US patent No.
7,244, 444. In
addition or alternatively, two or more angiogenesis inhibitors may optionally
be co-administered
to the patient, an anti-her3 antibody and another antiangiogenic moiety. In
certain embodiment,
one or more additional therapeutic agents, e.g., anti-cancer agents, can be
administered in
combination with anti-Her3 antibody, the Her2 or IGF-1R antagonist, and an
anti-angiogenesis
agent.
In certain aspects of the invention, other therapeutic agents useful for
combination
tumor therapy with an anti-Her3 antibody include other cancer therapies,
(e.g., surgery,
radiological treatments (e.g., involving irradiation or administration of
radioactive substances),
chemotherapy, treatment with anti-cancer agents listed herein and known in the
art, or
combinations thereof). Alternatively, or additionally, two or more antibodies
binding the same or
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two or more different antigens disclosed herein can be co-administered to the
patient.
Sometimes, it may be beneficial to also administer one or more cytokines to
the patient.
Yet another embodiment provides a method for treating an Her3 mediated cancer
comprising: a) obtaining a sample of diseased tissue from a patient in need of
treatment of said
cancer; b) determining the level of expression of Her3 levels in the tissue
sample; c) scoring the
samples for expression of Her3 levels; d) correlating the score to identify
patients likely to
benefit from treatment with an Her3 antagonist, wherein the step of
correlating comprises
comparing said scoring to that obtained from a control sample, e) treating the
patient with a
therapeutic regime known to improve the prognosis for the particular cancer.
In certain
embodiments, the method further proposes f) repeating steps "a" and "b", and
g) adjusting the
therapeutic regime known to improve the prognosis for the cancer; h) repeating
steps a-f as
frequently as deemed appropriate. Exemplary and non-limiting methods of
"detecting" Her3
expression for staging or scoring purposes is provided here below. Refer to,
for example, section
marked "Detection of Her3 antigen."
Additional combination therapies include combining any one or more of the
invention antibodies with glucocorticoids (US 20060003927) and gamma-secretase
inhibitors
(20080206753 including references cited therein e.g., Lanz, T. A., Hosley, J.
D., Adams, W. J.,
and Merchant, K. M. 2004. Studies of Abeta pharmacodynamies in the brain,
cerebrospinal fluid,
and plasma in young (plaque-free) Tg2576 mice using the gamma-secretase
inhibitor N2-[(2S)-
2-(3,5-difluoropheny1)-2-hydroxyethanoyl]-N1-[(7S)-5-methyl-6-ox- o-6,7-
dihydro-5H-
dibenzo[b,d]azepin-7-yll-L-alaninamide (LY-411575). I Pharmacol Exp Ther
309:49-55.)
Chemotherapeutic Agents
In certain aspects, the invention provides a method of blocking or reducing
tumor
growth or growth of a cancer cell, by administering effective amounts of an
antagonist of Her3
and/or an angiogenesis inhibitor(s) and one or more chemotherapeutic agents to
a patient
susceptible to, or diagnosed with, cancer. A variety of chemotherapeutic
agents may be used in
the combined treatment methods of the invention. An exemplary and non-limiting
list of
chemotherapeutic agents contemplated is provided herein under "Definitions."
Likewise, in certain aspects, the invention provides for the use of "agonist"
anti-
Her3 antibodies.
As will be understood by those of ordinary skill in the art, the appropriate
doses
of chemotherapeutic agents will be generally around those already employed in
clinical therapies
wherein the chemotherapeutics are administered alone or in combination with
other
chemotherapeuties. Variation in dosage will likely occur depending on the
condition being
treated. The physician administering treatment will be able to determine the
appropriate dose for
the individual subject.
The invention also provides methods and compositions for inhibiting or
preventing relapse tumor growth or relapse cancer cell growth. Relapse tumor
growth or relapse
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cancer cell growth is used to describe a condition in which patients
undergoing or treated with
one or more currently available therapies (e.g., cancer therapies, such as
chemotherapy, radiation
therapy, surgery, hormonal therapy and/or biological therapy/immunotherapy,
anti-VEGF
antibody therapy, particularly a standard therapeutic regimen for the
particular cancer) is not
effect from the therapy such that these patients need additional effective
therapy. As used
herein, the phrase can also refer to a condition of the "non-
responsive/refractory" patient, e.g.,
which describe patients who respond to therapy yet suffer from side effects,
develop resistance,
do not respond to the therapy, do not respond satisfactorily to the therapy,
etc. In various
The invention provides methods of blocking or reducing relapse tumor growth or
relapse cancer cell growth in a subject by administering one or more anti-Her3
antibodies to
35 The antibody of the invention (and adjunct therapeutic agent)
is/are administered
by any suitable means, including parenteral, subcutaneous, intraperitoneal,
intrapulmonary, and
intranasal, and, if desired for local treatment, intralesional or intravitreal
administration.
Parenteral infusions include intramuscular, intravenous, intraarterial,
intraperitoneal, or
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subcutaneous administration. In addition, the antibody is suitably
administered by pulse
infusion, particularly with declining doses of the antibody. Dosing can be by
any suitable route,
e.g. by injections, such as intravenous or subcutaneous injections, depending
in part on whether
the administration is brief or chronic.
The antibody composition of the invention will be formulated, dosed, and
administered in a fashion consistent with good medical practice. Factors for
consideration in this
context include the particular disorder being treated, the particular mammal
being treated, the
clinical condition of the individual patient, the cause of the disorder, the
site of delivery of the
agent, the method of administration, the scheduling of administration, and
other factors known to
medical practitioners. The antibody need not be, but is optionally formulated
with one or more
agents currently used to prevent or treat the disorder in question. The
effective amount of such
other agents depends on the amount of antibodies of the invention present in
the formulation, the
type of disorder or treatment, and other factors discussed above. These are
generally used in the
same dosages and with administration routes as used hereinbefore or about from
I to 99% of the
heretofore employed dosages.
For the prevention or treatment of disease, a therapeutically effective dosage
of an
anti-Her3 antibody or antigen-binding fragment thereof of the invention (when
used alone or in
combination with other agents such as chemotherapeutic agents) will depend on
the type of
disease to be treated, the type of antibody, 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 1 gg/kg to 15 mg/kg
(e.g., 0.1 mg/kg-10
mg/kg) of antibody is an initial candidate dosage for administration to the
patient, whether, for
example, by one or more separate administrations, or by continuous infusion.
One typical daily
dosage might range from about 1 rig/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.
One exemplary dosage of the antibody would be in the range from about 0.05
mg/kg to about 10
mg/kg, Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10
mg/kg (or any
combination thereof) may be administered to the patient. Such doses may be
administered
intermittently, e.g. every week or every three weeks (e.g. such that the
patient receives from
about two to about twenty, e.g. about six doses of the antibody). An initial
higher loading dose,
followed by one or more lower doses may be administered. An exemplary dosing
regimen
comprises administering an initial loading dose of about 4 mg/kg, followed by
a weekly
maintenance dose of about 2 mg/kg of the antibody. However, other dosage
regimens may be
useful. The progress of this therapy is easily monitored by conventional
techniques and assays.
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Detection of Her3 antigen
It is well accepted that cell surface growth receptor proteins, especially
those
whose expression correlates with an oncogenic disorder, e.g., Her3 are
excellent targets for drug
candidates or tumor (e.g., cancer) treatment. The state of the art now
concludes that such
proteins may also find use in non-therapeutic applications. The exquisite
specificity of the anti-
Her3 antibodies for the Her3 receptor protein can be exploited for various
uses including
diagnostic and prognostic reagents. The proposed uses exploit the observation
that (i) the anti-
Her3 antibodies of the invention including antigen binding fragments thereof
specifically bind
Her3 and (ii) the target receptor bound by the antibodies of the invention is
highly expressed on
cancerous cells. Expressly contemplated also are the use of the invention
antibodies in detecting,
monitoring, and quantifying Her3 expression (e.g. in an EL1SA or a Western
blot); purification
or immunoprecipitation of Her3 from cells, to kill and eliminate Her3-
expressing cells from a
population of mixed cells as a step in the purification of other cells.
Proposed methods of
diagnosis can be performed in vitro using a cellular sample (e.g., lymph node
biopsy or tissue)
from a patient or be performed by in vivo imaging. Diagnostic and prognostic
applications
include scoring tumors as well as staging Her3-expressing cancers (e.g., in
radioimaging). They
may be used alone or in combination with other Her3 related cancer markers.
The diagnostic
uses of the antibodies according to the present invention embrace primary
tumors and cancers, as
well as metastases. Other cancers and tumors bearing the antigen are also
amenable to these
diagnostic and imaging procedures.
In one embodiment, the invention antibody or binding fragments thereof will be
very useful in cancer diagnosis and prognosis by effectively allowing one
skilled in the art to
quantitate or quantify the expression levels of Her3 in whatever kind of
"sample" it may occur.
This can be achieved, for example, by immunofluorescence techniques employing
a
fiuorescently labeled antibody, coupled with light microscopic, flow
cytometric, or fluorometric
detection. In addition, the antibodies, or binding fragments thereof,
according to the present
invention may additionally be employed histologically, as in
immunofluorescence,
immunoelectron microscopy, or non-immuno assays, for in situ detection of the
cancer-specific
antigen on cells, such as for use in monitoring, diagnosing, or detection
assays. See, for example,
Zola, Monoclonal Antibodies: A Manual of Techniques, pp.147 158 (CRC Press,
Inc. 1987).
In another aspect, the invention provides methods for detection of Her3, the
methods comprising detecting Her3-anti-Her3 antibody complex in the sample.
The term
"detection" as used herein includes qualitative and/or quantitative detection
(measuring levels)
with or without reference to a control.
In another aspect, the invention provides methods for diagnosing a disorder
associated with Her3 expression and/or activity, the methods comprising
detecting Her3-anti-
Her3 antibody complex in a biological sample from a patient having or
suspected of having the
disorder. In some embodiments, the Her3 expression is increased expression or
abnormal
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(undesired) expression. In some embodiments, the disorder is a tumor, cancer,
and/or a cell
proliferative disorder.
In another aspect, the invention provides any of the anti-Her3 antibodies
described herein, wherein the anti-Her3 antibody comprises a detectable label.
In another aspect, the invention provides a complex of any of the anti-Her3
antibodies described herein and Her3. In some embodiments, the complex is in
vivo or in vitro.
In some embodiments, the complex comprises a cancer cell. In some embodiments,
the anti-Her3
antibody is detectably labeled.
Anti-Her3 antibodies can be used for the detection of Her3 in any one of a
number of well known detection assay methods. For example, a biological sample
may be
assayed for Her3 by obtaining the sample from a desired source, admixing the
sample with anti-
Her3 antibody to allow the antibody to form antibody/Her3 complex with any
Her3 present in
the mixture, and detecting any antibody/Her3 complex present in the mixture.
The biological
sample may be prepared for assay by methods known in the art which are
suitable for the
particular sample. The methods of admixing the sample with antibodies and the
methods of
detecting antibody/Her3 complex are chosen according to the type of assay
used. Such assays
include immunohistochemistry, competitive and sandwich assays, and steno
inhibition assays.
Analytical methods for Her3 all use one or more of the following reagents:
labeled Her3 analogue, immobilized Her3 analogue, labeled anti-Her3 antibody,
immobilized
anti-Her3 antibody and steric conjugates. The labeled reagents also are known
as "tracers."
For diagnostic and imaging applications, the antibodies of the invention may
be
labeled. The label used is any detectable functionality that does not
interfere with the binding of
Her3 and anti-Her3 antibody and it can bind to the antibodies by means of
physical binding,
chemical binding or the like, thus allowing them to be detected. Numerous
labels are known for
use in immunoassay, examples including moieties that may be detected directly,
such as
fluorochrome, chemiluminescent, and radioactive labels, as well as moieties,
such as enzymes,
that must be reacted or derivatized to be detected. Specific examples of
labeling substances
include enzymes, fluorescent substances, chemiluminescent substances, biotin,
avidin,
radioactive isotopes and the like. When the fluorescently labeled antibody is
exposed to light of
the proper wavelength, its presence can then be detected due to fluorescence.
The radioactive
isotopes and fluorescent substances detailed herein independently produce
detectable signals, but
the enzymes, chemiluminescent substances, biotin and avidin do not
independently produce
detectable signals, but instead produce detectable signals when they react
with at least one other
substance. For example, in the case of an enzyme at least a substrate is
required, and a variety of
substrates are used depending on the method of measuring enzyme activity
(colorimetry,
fluorescence method, bioluminescence method or chemoluminescence method). In
the case of
biotin generally at least avidin or enzyme-modified avidin is reacted. A
variety of colorants
dependent on the substrate can also be used as necessary.
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Examples of such labels include: Examples of such labels include the
radioisotopes32P, 14C, 125j, 3H, and 1311, fluorophores such as rare earth
chelates or fluorescein
and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone,
luciferases, e.g., firefly
luciferase and bacterial luciferase (U.S. Pat, No. 4,737,456), lueiferin, 2,3-
dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase,
.beta.-
galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose
oxidase, galactose
oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as
unease and
xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to
oxidize a dye
precursor such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin,
spin labels,
bacteriophage labels, stable free radicals, and the like. The label may be
directly conjugated to
the antibodies or fragments thereof or indirectly conjugated. Indeed, numerous
ways to
detectably label protein molecules are known and practiced in the art. Means
of indirect
conjugation of a protein to a label are also well known. Indirect conjugation
of the label to the
antibody may, for example, be achieved by conjugating antibody to a small
hapten (e.g.,
digoxin) and one of the different types of labels mentioned herein is
conjugated with an anti-
hapten antibody mutant (e.g., anti-digoxin antibody). See, e.g., Wagner et
al., J. Nucl. Med. 20:
428 (1979) and Saha et al., J. Nucl. Med. 6:542 (1976), hereby incorporated by
reference.
Conventional methods are available to bind these labels covalently to proteins
or
polypeptides. For instance, coupling agents such as dialdehydes,
carbodiimides, dimaleimides,
bis-imidates, bis-diazotized benzidine, and the like may be used to tag the
antibodies with the
above-described fluorescent, chemiluminescent, and enzyme labels. See, for
example, U.S. Pat.
Nos. 3,940,475 (fluorimetry) and 3,645,090 (enzymes); Hunter et al., Nature,
144: 945 (1962);
David et al., Biochemistry, 13: 1014-1021 (1974); Pain et al., J. Immunol.
Methods, 40: 219-230
(1981); and Nygren, J. Histochem. and Cytochem., 30: 407-412 (1982). Preferred
labels herein
are enzymes such as horseradish peroxidase and alkaline phosphatase. The
conjugation of such
label, including the enzymes, to the antibody is a standard manipulative
procedure for one of
ordinary skill in immunoassay techniques. See, for example, O'Sullivan et al.,
"Methods for the
Preparation of Enzyme-antibody Conjugates for Use in Enzyme Immunoassay," in
Methods in
Enzymology, ed. J. J. Langone and H. Van Vunakis, Vol. 73 (Academic Press, New
York, N.Y.,
1981), pp. 147-166.
Another way to label the antibodies of the invention is by linking the
antibody to
an enzyme, e.g., for use in an enzyme immunoassay (EIA), (A. Voller et al.,
1978, "The Enzyme
Linked Immunosorbent Assay (ELISA)", Diagnostic Horizons, 2:1 7;,
Microbiological
Associates Quarterly Publication, Walkersville, Md.; A. Voller et al., 1978,
J. Clin. Pathol.,
31:507 520; J. E. Butler et al., 1981, Meths. Enzymol., 73:482 523; Enzyme
Immunoassay,
1980, (Ed.) E. Maggio, CRC Press, Boca Raton, Fla.; Enzyme Immunoassay, 1981,
(Eds.) E.
Ishikawa et al., Kgaku Shoin, Tokyo, Japan). The enzyme that is bound to the
antibody reacts
with an appropriate substrate, preferably a chromogenic substrate, so as to
produce a chemical
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moiety which can be detected, for example, by spectrophotometric,
fluorometric, or by visual
detection means. Nonlimiting examples of enzymes which can be used to
detectably label the
antibodies include malate dehydrogenase, staphylococcal nuclease, delta-5-
steroid isomerase,
yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose
phosphate
isomerase, horseradish peroxidase, alkaline phosphatase, ribonuclease, urease,
catalase, glucose-
6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The
detection can be
accomplished by calorimetric methods, which employ a chromogenic substrate for
the enzyme,
or by visual comparison of the extent of enzymatic reaction of a substrate
compared with
similarly prepared standards or controls. Numerous other enzyme-substrate
combinations are
available to those skilled in the art. For a general review of these, see U.S.
Pat. Nos. 4,275,149
and 4,318,980.
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) .beta.-D-galactosidase (.beta.-D-Gal) with a chromogenic substrate
(e.g., p-
nitrophenyl-.beta.-D-galactosidase) or fluorogenic substrate 4-
methylumbelliferyl,beta.-D-
galactosidase.
Immobilization of reagents is required for certain assay methods.
Immobilization
entails separating the anti-Her3 antibody from any Her3 that remains free in
solution. This
conventionally is accomplished by either insolubilizing the anti-Her3 antibody
or Her3 analogue
before the assay procedure, as by adsorption to a water-insoluble matrix or
surface (Bennich et
al., U.S. Pat. No. 3,720,760), by covalent coupling (for example, using
glutaraldehyde cross-
linking), or by insolubilizing the anti-Her3 antibody or Her3 analogue
afterward, e.g., by
immunoprecipitation.
Suitable subjects include those who are suspected of being at risk of a
pathological effect of any hyperproliferative oncogenic disorders,
particularly carcinoma and
sarcomas mediated by Her3, are suitable for the detection, diagnosis and
prognosis paradigms of
the invention. Those with a history of cancer are especially suitable.
Suitable human subjects
for the diagnostic and prognostic therapies may comprise two groups, which can
be
distinguished by clinical criteria. Patients with "advanced disease" or "high
tumor burden" are
those who bear a clinically measurable tumor. A clinically measurable tumor is
one that can be
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detected on the basis of tumor mass (e.g., by palpation, CAT scan, or X-Ray;
positive
biochemical or histopathological markers on their own may be insufficient to
identify this
population).
A second group of suitable subjects is known in the art as the "adjuvant
group".
These are individuals who have had a history of cancer, but have been
responsive to another
mode of therapy. The prior therapy may have included, but is not restricted
to, surgical
resection, radiotherapy, and traditional chemotherapy. As a result, these
individuals have no
clinically measurable tumor. However, they are suspected of being at risk for
progression of the
disease, either near the original tumor site, or by metastases.
This group can be further subdivided into high-risk and low-risk individuals.
The
subdivision is made on the basis of features observed before or after the
initial treatment. These
features are known in the clinical arts, and are suitably defined for each
different cancer.
Features typical of high risk subgroups are those in which the tumor has
invaded neighboring
tissues, or who show involvement of lymph nodes.
Another suitable group of subjects is those with a genetic predisposition to
cancer
but who have not yet evidenced clinical signs of cancer. For instance, women
with a family
history of breast cancer, but still of childbearing age, may avail themselves
of having their breast
tissue examined for expression levels of Her3 and those testing positive,
e.g., having higher than
normal expression level of Her3 may wish to be monitored for presenting with
breast cancer or
alternatively avail themselves of preventive treatment with a conventional
Her3 specific
monoclonal therapy.
A variety of other immunoassays are available for detecting Her3. For example,
by labeling the antibodies, or binding fragments thereof, with a radioisotope,
a
radioimmunoassay (R1A) can be used to detect cancer-specific antigens (e.g.,
Current Protocols
in Immunology, Volumes I and 2, Coligen et al., Ed. Wiley-Interscience, New
York, N.Y., Pubs.
(19910, Colcher et al., 1981, Cancer Research, 41, 1451 1459; Weintraub,
"Principles of
Radioimmunoassays", Seventh Training Course on Radioligand Techniques, The
Endocrine
Society, March, 1986). The radioactive isotope label can be detected by using
a gamma counter
or a scintillation counter or by radiography. Representative radioisotopes
include 35S, 14C, 1251,
3, and 1311. Procedures for labeling biological agents with the radioactive
isotopes are generally
known in the art. Tritium labeling procedures are described in U.S. Pat. No.
4,302,438, which is
hereby incorporated by reference. Iodinating, tritium labeling, and 35S
labeling procedures
especially adapted for murine monoclonal antibodies are well known. Other
procedures for
iodinating biological agents, such as antibodies, binding portions thereof,
probes, or ligands, are
described by Hunter and Greenwood, Nature 144:945 (1962), David et al.,
Biochemistry
13:1014-1021 (1974), and U.S. Pat. Nos. 3,867,517 and 4,376,110, which are
hereby
incorporated by reference. Procedures for iodinating biological agents are
described by
Greenwood, F. et al., Biochern. J. 89:114-123 (1963); Marchalonis, J.,
Biochem. J. 113:299-305
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(1969); and Morrison, M. et al., Immunochernistry, 289-297 (1971), which are
hereby
incorporated by reference. Procedures for 99mTc-labeling are described by
Rhodes, B. et al. in
Burchiel, S. et al. (eds.), Tumor Imaging: The Radioimmunochemical Detection
of Cancer, New
York: Masson 111-123 (1982) and the references cited therein, which are hereby
incorporated by
reference. Procedures suitable for '11n-labeling biological agents are
described by Hnatowieh,
D. J. et al., J. Immul. Methods, 65:147-157 (1983), Hnatowich, D. et al., J.
Applied Radiation,
35:554-557 (1984), and Buckley, R. G. et al., F.E.B.S. 166:202-204 (1984),
which are hereby
incorporated by reference.
The presently universally-accepted method for the diagnosis of solid cancer is
the
histologic determination of abnormal cellular morphology in surgically
biopsied or resected
tissue. Once removed, the tissue is preserved in a fixative, embedded in
paraffin wax, cut into 5
1.tm-thick sections, and stained with two dyes: hematoxylin for the nucleus
and eosin for the
cytoplasm ("H&E staining"). This approach is simple, fast, reliable, and
inexpensive.
Histopathology allows the diagnosis of a variety of tissue and cell types. By
providing an
estimation of tumor "Grade" (cellular differentiation/tissue architecture) and
"Stage" (depth of
organ penetration) it also makes prognosis possible.
Immunohistochemistiy ('IIIC") techniques utilize an antibody to probe and
visualize cellular antigens in situ, generally by chromogenic or fluorescent
methods. For sample
preparation, a tissue or cell sample from a mammal (typically a human patient)
may be used.
Examples of samples include, but are not limited to, cancer cells such as
colon, breast, prostate,
ovary, lung, stomach, pancreas, lymphoma, and leukemia cancer cells. The
sample can be
obtained by a variety of procedures known in the art including, but not
limited to surgical
excision, aspiration or biopsy. The tissue may be fresh or frozen. In one
embodiment, the
sample is fixed and embedded in paraffin or the like. The tissue sample may be
fixed (i.e.
preserved) by conventional methodology. One of ordinary skill in the art will
appreciate that the
choice of a fixative is determined by the purpose for which the sample is to
be histologically
stained or otherwise analyzed. One of ordinary skill in the art will also
appreciate that the length
of fixation depends upon the size of the tissue sample and the fixative used.
IHC may be performed in combination with additional techniques such as
morphological staining and/or fluorescence in-situ hybridization. Two general
methods of IHC
are available; direct and indirect assays. According to the first assay,
binding of antibody to the
target antigen (e.g., Her3) is determined directly. This direct assay uses a
labeled reagent, such
as a fluorescent tag or an enzyme-labeled primary antibody, which can be
visualized without
further antibody interaction. In a typical indirect assay, unconjugated
primary antibody binds to
the antigen and then a labeled secondary antibody binds to the primary
antibody. Where the
secondary antibody is conjugated to an enzymatic label, a chromogenic or
fluorogenic substrate
is added to provide visualization of the antigen. Signal amplification occurs
because several
secondary antibodies may react with different epitopes on the primary
antibody.
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The primary and/or secondary antibody used for immunohistochemistry typically
will be labeled with a detectable moiety. Numerous labels are available, some
of which are
detailed herein.
Aside from the sample preparation procedures discussed above, further
treatment
of the tissue section prior to, during or following IHC may be desired, for
example, epitope
retrieval methods, such as heating the tissue sample in citrate buffer may be
carried out (see, e.g.,
Leong et al. Appl. Immunohistochem. 4(3):201 (1996)).
Following an optional blocking step, the tissue section is exposed to primary
antibody for a sufficient period of time and under suitable conditions such
that the primary
antibody binds to the target protein antigen in the tissue sample. Appropriate
conditions for
achieving this can be determined by routine experimentation. The extent of
binding of antibody
to the sample is determined by using any one of the detectable labels
discussed above.
Preferably, the label is an enzymatic label (e.g. FIRPO) which catalyzes a
chemical alteration of
the chromogenic substrate such as 3,3t-diaminobenzidine ehromogen. Preferably
the enzymatic
label is conjugated to antibody which binds specifically to the primary
antibody (e.g. the primary
antibody is rabbit polyclonal antibody and secondary antibody is goat anti-
rabbit antibody).
Specimens thus prepared may be mounted and coverslipped. Slide evaluation is
then determined, e.g. using a microscope.
Alternatively, one may also utilize microscope-based cell imaging, which uses
conventional light microscopy combined with monochromatic light filters and
computer
software programs. The wavelengths of the light filters are matched to the
colors of the antibody
stain and the cell counterstain. The filters allow the microscopist to
identify, classify and then
measure differences in the optical density of specific colors of light
transmitted through
immunostained portions of tissue sections. See U.S. Pat. Nos. 5,235,522 and
5,252,487, both of
which are incorporated herein by reference, for applications of these methods
to tumor protein
measurement. Yet other cell imaging systems (image cytometers) permit
automated recognition
of features, and combine this with automated calculation of feature areas,
automated calibration,
and automatic calculation of average and integrated (SOD) optical density.
(See, e.g., U.S. Pat.
Nos. 5,548,661, 5,787,189, both of which are incorporated herein by reference,
and references
therein.)
Since immunohistochemical staining of tissue sections has been shown to be a
reliable method of assessing or detecting presence of proteins in a sample.
Consequently, use of
the antibodies described herein to score staining and/or detection levels are
also contemplated.
Protein expression may be determined using a validated scoring method
(Dhanasekaran et al., 2001, Nature 412, 822-826; Rubin et al., 2002, supra;
Varambally et al.,
2002, Nature 419, 624-629) where staining was evaluated for intensity and the
percentage of
cells staining positive. In cases where benign tissue and cancer are present,
only one or the other
tissue type is evaluated for purposes of analysis. Any of the methods of the
invention may score
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the analysis by using a scale of 0 to 4, where 0 is negative (no detectable
Her3 or level of
expression same as that of a control sample) and 4 is high intensity staining
in the majority of
cells. In certain embodiments, the scoring may be used for diagnostic or
prognostic purposes.
For example, a score of 1, while a positive score, may indicate better
prognosis than, say, a score
of 3 or 4.
The information gathered in accordance with the invention will aid the
physician
in determining a course of treatment for a patient presenting with a Her3-
mediated oncogenic
disorder. For example, in the case of tumor cells comprising Her3 receptor
expressing, a low
score might dictate that additional intervention, e.g., surgery is not
warranted. Typically, a
staining pattern score of about 3+ or higher in an IHC assay is diagnostic
and/or prognostic. In
some embodiments, a staining pattern score of about 1+ or higher is diagnostic
and/or
prognostic. In other embodiments, a staining pattern score of about 2+ or
higher is diagnostic
and/or prognostic. It is understood that when cells and/or tissue from a tumor
are examined using
IHC, staining is generally determined or assessed in tumor cell and/or tissue
(as opposed to
stromal or surrounding tissue that may be present in the sample).
Other assay methods, known as competitive or sandwich assays, are well
established and widely used in the commercial diagnostics industry.
Competitive assays rely on the ability of a tracer Her3 analogue to compete
with
the test sample Her3 for a limited number of anti-Her3 antibody antigen-
binding sites. The anti-
Her3 antibody generally is insolubilized before or after the competition and
then the tracer and
Her3 bound to the anti-Her3 antibody are separated from the unbound tracer and
Her3. This
separation is accomplished by decanting (where the binding partner was
preinsolubilized) or by
centrifuging (where the binding partner was precipitated after the competitive
reaction). The
amount of test sample Her3 is inversely proportional to the amount of bound
tracer as measured
by the amount of marker substance. Dose-response curves with known amounts of
Her3 are
prepared and compared with the test results to quantitatively determine the
amount of Her3
present in the test sample. These assays are called ELISA systems when enzymes
are used as the
detectable markers.
Another species of competitive assay, called a "homogeneous" assay, does not
require a phase separation. Here, a conjugate of an enzyme with the Her3 is
prepared and used
such that when anti-Her3 antibody binds to the Her3 the presence of the anti-
Her3 antibody
modifies the enzyme activity. In this case, the Her3 or its immunologically
active fragments are
conjugated with a bifunctional organic bridge to an enzyme such as peroxidase.
Conjugates are
selected for use with anti-Her3 antibody so that binding of the anti-Her3
antibody inhibits or
potentiates the enzyme activity of the label. This method per se is widely
practiced under the
name of EMIT.
Steric conjugates are used in steric hindrance methods for homogeneous assay.
These conjugates are synthesized by covalently linking a low-molecular-weight
hapten to a small
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Her3 fragment so that antibody to hapten is substantially unable to bind the
conjugate at the
same time as anti-Her3 antibody. Under this assay procedure the Her3 present
in the test sample
will bind anti-Her3 antibody, thereby allowing anti-hapten to bind the
conjugate, resulting in a
change in the character of the conjugate hapten, e.g., a change in
fluorescence when the hapten is
a fluorophore.
Sandwich assays particularly are useful for the determination of Her3 or anti-
Her3
antibodies. In sequential sandwich assays an immobilized anti-Her3 antibody is
used to adsorb
test sample Her3, the test sample is removed as by washing, the bound Her3 is
used to adsorb a
second, labeled anti-Her3 antibody and bound material is then separated from
residual tracer.
The amount of bound tracer is directly proportional to test sample Her3. In
"simultaneous"
sandwich assays the test sample is not separated before adding the labeled
anti-Her3. A
sequential sandwich assay using an anti-Her3 monoclonal antibody as one
antibody and a
polyclonal anti-Her3 antibody as the other is useful in testing samples for
Her3.
In another embodiment, the invention provides a method for determining the
effect of a therapeutic regimen for alleviating a Her3 mediated disorder,
wherein the regimen
comprises the use of an Her3 antagonist or an agonist antibody, the method
comprising the steps
of: a) obtaining a cell or tissue sample from an individual undergoing the
therapeutic regimen b)
measuring the levels of Her3 in the cell or tissue sample; c) scoring the
sample for Her3 protein
levels, and d) comparing the levels to that of a control sample to predict the
responsiveness of
the Her3 mediated disorder to the therapeutic regimen. Thus, a low score,
e.g., 0 or a lowering
score over time suggests that the treatment regimen comprising a Her3
antagonist, e.g., Her3
specific antibody, is effective in reducing tumor burden or Her3 expressing
cells or level of Her3
expression.
In certain embodiments, the methods of the invention propose contacting the
sample of interest with an antibody to Her3. In certain embodiments, the
detecting is done on
histological or tissue sections or cytological preparations by
immunohistoehemistry or
immunocytochemistry. As well, detecting Her3 may be done by immunoblotting or
by
Fluorescence-Activated Cell Sorting (PACS).
The invention is also directed to a method for predicting disease-free
survival
and/or overall survival in a patient diagnosed with an oncogenic disorder
associated with Her3
expression comprising: a) obtaining a sample of diseased or cancerous tissue
from an individual
presenting with an oncogenic disorder, b) detecting levels of Her3 expressing
cells in the cancer
cells or cancer tissue of the sample, c) scoring the samples for expression of
Her3 levels; and d)
comparing the scoring to that obtained from a control sample to determine
likelihood of disease-
free survival and overall survival associated with Her3. Preferably, the
scoring comprises using
a scale of 0 to 4, where 0 is negative (no detectable Her3 or level of Her3
comparable to a
control level), and 4 is high intensity staining in the majority of cells and
wherein a score of Ito
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4 (i.e. a positive score) indicates a poor prognosis for disease free and
overall survival in patients
with said disorder.
A method for screening for metastatic potential of solid tumors is also
provided.
The method comprises a) obtaining a sample of tumor tissue from an individual
in need of
screening for metastatic potential of a solid tumor; b) reacting an antibody
to Her3 with tumor
tissue from the patient; c) detecting the extent of binding of the antibody to
the tissue and d)
correlating the extent of binding of the antibody with its metastatic
potential.
The present invention further encompasses in vivo imaging methods useful for
visualizing the presence of Her3 expressing cells indicative of an oncogenic
disorder. Such
techniques allow for a diagnosis without the use of an unpleasant biopsy or
other invasive
diagnostic technique. The concentration of detectably labeled anti-Her3
antibody of the
invention which is administered should be sufficient such that the binding to
those cells having
or expressing the Her3 antigen is detectable compared to the background.
Further, it is desirable
that the detectably labeled anti-Her3 antibody of the invention be rapidly
cleared from the
circulatory system in order to give the best target-to-background signal
ratio.
Imaging analysis is well known in the medical art, and includes, without
limitation, x-ray analysis, magnetic resonance imaging (MR1) or computed
tomography (CE).
As indicated supra, preferably, the Her3 antibodies used in the in vivo (and
also in vitro)
diagnostic methods are directly or indirectly labeled with a detectable
substance/label that can be
imaged in a patient. Suitable detectable substances include various enzymes,
prosthetic groups,
fluorescent materials, luminescent materials and radioactive materials. As a
rule, the dosage of
detectably labeled anti-Her3 antibody of the invention for in vivo diagnosis
is somewhat patient-
specific and depends on such factors as age, sex, and extent of disease.
Dosages may also vary,
for example, depending on number of injections given, tumor burden, and other
factors known to
those of skill in the art. For instance, tumors have been labeled in vivo
using cyanine-conjugated
Mabs. Ballou et al. (1995) Cancer Imrnunol. Immunother. 41:257 263.
In the case of a radiolabeled biological agent, the biological agent is
administered
to the patient and is localized to the tumor bearing the antigen (Her3
receptor protein) with
which the biological agent reacts, and is detected or "imaged" in vivo using
known techniques
such as radionuclear scanning using e.g., a gamma camera or emission
tomography. See e.g., A.
R. Bradwell et al., "Developments in Antibody Imaging", Monoclonal Antibodies
for Cancer
Detection and Therapy, R. W. Baldwin et al., (eds.), pp. 65-85 (Academic Press
1985), which is
hereby incorporated by reference. Alternatively, a positron emission
transaxial tomography
scanner, such as designated Pet VI located at Brookhaven National Laboratory,
can be used
where the radiolabel emits positrons (e.g., 11C, 18F, 150, and 13N).
In one embodiment the invention provides for the use of the Her3 antibodies in
the diagnosis of cancer, by specifically allowing one to detect and visualize
tissues that express
Her3 or contain Her3 expressing cells (e.g., cancer). The method comprises:
(i) administering to
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a subject (and optionally a control subject) a diagnostically effective amount
of detectably
labeled anti-Her3 antibody of the invention or an antigen-binding fragment
thereof or a
pharmaceutical composition thereof comprising as an active component the
antibodies of the
invention or binding fragments thereof that specifically bind Her3, under
conditions that allow
interaction of the antibodies to Her3 to occur; and (ii) detecting the binding
agent, for example,
to locate Her3 expressing tissues or otherwise identify Her3 expressing cells.
The term
"diagnostically effective" means that the amount of detectably labeled anti-
Her3 antibody of the
invention is administered in sufficient quantity to enable detection of
neoplasia.
In certain embodiments, the antibodies of the invention may be labeled with a
contrast agent, such as barium, which can be used for x-ray analysis, or a
magnetic contrast
agent, such as a gadolinium chelate, which can be used for MRI or CE.
In another embodiment of the method, a biopsy is obtained from the patient to
determine whether the tissue of interest expresses Her3 rather than subjecting
the patient to
imaging analysis.
A radiolabeled antibody or immunoconjugate may comprise a gamma.-emitting
radioisotope or a positron-emitter useful for diagnostic imaging. The label
used will depend on
the imaging modality chosen. The use of antibodies for in vivo diagnosis is
well known in the
art. Sumerdon et al., (Nucl. Med. Biol 17:247-254 (1990)) have described an
optimized
antibody-chelator for the radioimmunoscintographic imaging of tumors using
Indium-Ill as the
label. Griffin et al., (I Clin One 9:631-640 [1991]) have described the use of
this agent in
detecting tumors in patients suspected of having recurrent colorectal cancer.
The methods of the present invention may also use paramagnetic isotopes for
purposes of in vivo detection. The use of similar agents with paramagnetic
ions as labels for
magnetic resonance imaging is also known in the art -Lauffer, Magnetic
Resonance in Medicine
22:339-342 (1991).
Radioactive labels such as Indium-111, Technetium-99m, or Iodine-131 can be
used for planar scans or single photon emission computed tomography (SPECT).
Positron
emitting labels such as Fluorine-19 can also be used for positron emission
tomography (PET).
For MRI, paramagnetic ions such as Gadolinium (III) or Manganese (II) can be
used.
For in vivo diagnostic imaging, the type of detection instrument available is
a
major factor in selecting a given radioisotope. The radioisotope chosen must
have a type of
decay which is detectable for a given type of instrument. Still another
important factor in
selecting a radioisotope for in vivo diagnosis is that the half-life of the
radioisotope be long
enough so that it is still detectable at the time of maximum uptake by the
target, but short enough
so that deleterious radiation with respect to the individual is minimized.
Ideally, a radioisotope
used for in vivo imaging lacks a particle emission, but produces a large
number of photons in the
140 250 keV range, to be readily detected by conventional gamma cameras.
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Radioactive metals with half-lives ranging from 1 hour to 3.5 days are
available
for conjugation to antibodies, such as scandium-47 (3.5 days) gallium-67 (2.8
days), gallium-68
(68 minutes), technetiium-99m (6 hours), and indium-ill (3.2 days), of which
gallium-67,
technetium-99m, and indium-111 are preferable for gamma camera imaging,
gallium-68 is
preferable for positron emission tomography. Labels such as Indium-111,
Technetium-99m, or
Iodine-131 can be used for planar scans or single photon emission computed
tomography
(SPECT).
In the case of the radiometals conjugated to the specific antibody, it is
likewise
desirable to introduce as high a proportion of the radiolabel as possible into
the antibody
molecule without destroying its immunospecificity. A further improvement may
be achieved by
effecting radiolabeling in the presence of the specific cancer marker of the
present invention, to
insure that the antigen binding site on the antibody will be protected. The
antigen is separated
after labeling.
Suitable radioisotopes, particularly in the energy range of 60 to 4,000keV,
include, 51Cr, 57Co, 58Co, 59Fe, 1311, 1211, 1241, 86y, 62cu, 64-u,
U
67Ga, 68Ga, 99mTeõ. 94mTc, 111p,
"C, "N, 150, "Br, "Se, 97Ru, 99mTc, 'In,
123/, 125/, 1311, 169yb, 197,-rrig, and 201T1, and the
like. See for example, U.S. Patent Application entitled "Labeling Targeting
Agents with
Gallium-68"--Inventors G. L. Griffiths and W..1. McBride, (U.S. Provisional
Application No.
60/342,104), which discloses positron emitters, such as 18F, 68Ga, 94mTc, and
the like, for
imaging purposes and which is incorporated in its entirety by reference.
Particularly useful
diagnostic/detection radionuclides include, but are not limited to, 18F, 52Fe,
62c.u, 64
Cu,
67Ga, "Go, 86Y, "Zr, 94mTc, 94mTc, 99mTc, 1I, 123I, i241, l25 154-158Gd,
321),
Y '"Re,
and 175Lu.
Decay energies of useful gamma-ray emitting radionuclides are preferably 20
2000 keV, more preferably 60 600 keV, and most preferably 100 300 keV.
Radionuclides useful for positron emission tomography include, but are not
limited to: 18F, 2mmn, 52Fe, 55co, 62cti, 64cu, 68Ga, 72 s,
A "Br, 76Br, 82mRb, "Sr, 86Y, "Zr,
94mTc, Hohi, 120,,
and 1241. Total decay energies of useful positron-emitting radionuclides are
preferably <2,000 keV, more preferably under 1,000 keV, and most preferably
<700 keV.
Also contemplated by the present invention is the use of non-radioactive
agents as
diagnostic reagents. A suitable non-radioactive diagnostic agent is a contrast
agent suitable for
magnetic resonance imaging, computed tomography or ultrasound. Magnetic
imaging agents
include, for example, non-radioactive metals, such as manganese, iron and
gadolinium,
complexed with metal-chelate combinations that include 2-benzyl-DTPA and its
monomethyl
and cyclohexyl analogs, when used along with the antibodies of the invention.
See U.S. Ser. No.
09/921,290 filed on Oct. 10, 2001, which is incorporated in its entirety by
reference.
Bispecific antibodies are also useful in targeting methods and provide a
preferred
way to deliver two diagnostic agents to a subject. U.S. Ser. Nos. 09/362,186
and 09/337,756
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discloses a method of pretargeting using a bispeeific antibody, in which the
bispecific antibody is
labeled with 2511 and delivered to a subject, followed by a divalent peptide
labeled with 99mTc
and are incorporated herein by reference in their entirety. Pretargeting
methods are also
described in U.S. Pat. No. 6,962,702 (Hansen et al.), U.S. Ser. Nos.
10/150,654 (Goldenberg et
al.), and Ser. No. 10/768,707 (McBride et al.), which are all also
incorporated herein by
reference in their entirety. The delivery results in excellent tumor/normal
tissue ratios for 1251
and 99mTc, thus showing the utility of two diagnostic radioisotopes. Any
combination of known
diagnostic agents can be used to label the antibodies. The binding specificity
of the antibody
component of the MAb conjugate, the efficacy of the therapeutic agent or
diagnostic agent and
the effector activity of the Fe portion of the antibody can be determined by
standard testing of
the conjugates.
A diagnostic agent can be attached at the hinge region of a reduced antibody
component via disulfide bond formation. As an alternative, such peptides can
be attached to the
antibody component using a heterobifunctional cross-linker, such as N-suecinyl
3-(2-
pyridyldithio)propionate (SPDP). Yu et al., Int. J. Cancer 56: 244 (1994).
General techniques for
such conjugation are well-known in the art. See, for example, Wong, CHEMISTRY
OF
PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslaeis et al.,
"Modification of Antibodies by Chemical Methods," in MONOCLONAL ANTIBODIES:
PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages 187 230 (Wiley-Liss,
Inc.
1995); Price, "Production and Characterization of Synthetic Peptide-Derived
Antibodies," in
MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL
APPLICATION, Ritter et al. (eds.), pages 60 84 (Cambridge University Press
1995).
Methods for conjugating peptides to antibody components via an antibody
carbohydrate moiety are also well-known to those of skill in the art. See, for
example, Shih et al.,
Int. J. Cancer 41: 832 (1988); Shih et al., hit, J. Cancer 46: 1101 (1990);
and Shih et al., U.S. Pat.
No. 5,057,313, all of which are incorporated in their entirety by reference.
The general method
involves reacting an antibody component having an oxidized carbohydrate
portion with a carrier
polymer that has at least one free amine function and that is loaded with a
plurality of peptide.
This reaction results in an initial Schiff base (imine) linkage, which can be
stabilized by
reduction to a secondary amine to form the final conjugate.
The Fe region is absent if the antibody used as the antibody component of the
immunoconjugate is an antibody fragment. However, it is possible to introduce
a carbohydrate
moiety into the light chain variable region of a full length antibody or
antibody fragment. See,
for example, Leung etal., J. Immunol. 154: 5919 (1995); Hansen et al., U.S.
Pat. No. 5,443,953
(1995), Leung et al, U.S. Pat. No. 6,254,868, all of which are incorporated in
their entirety by
reference. The engineered carbohydrate moiety is used to attach the
therapeutic or diagnostic
agent.
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