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CA 02548065 2006-06-O1
WO 2005/061541 PCT/US2004/038230
ANTI-IGF-I RECEPTOR ANTIBODY
[0l] The present application is a continuation-in-part of parent application
number
10/170,390, filed June 14, 2002, incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[02] The present invention relates to antibodies that bind to human insulin-
like growth
factor-I receptor (IGF-I receptor). More particularly, the invention relates
to anti-IGF-I
receptor antibodies that inhibit the cellular functions of the IGF-I receptor.
Still more
particularly, the invention relates to anti-IGF-I receptor antibodies that
antagonize the effects
of IGF-I, IGF-II and serum on the growth and survival of tumor cells and which
are
substantially devoid of agonist activity. The invention also relates to
fragments of said
antibodies, humanized and resurfaced versions of said antibodies, conjugates
of said
antibodies, antibody derivatives, and the uses of same in diagnostic, research
and therapeutic
applications. The invention further relates to improved antibodies or
fragments thereof that
are made from the above-described antibodies and fragments thereof. In another
aspect, the
invention relates to a polynucleotide encoding the antibodies or fragments
thereof, and to
vectors comprising the polynucleotides.
BACKGROUND OF THE INVENTION
[03] Insulin-like growth factor-I receptor (IGF-I receptor) is a transmembrane
heterotetrameric protein, which has two extracellular alpha chains and two
membrane-
spanning beta chains in a disulfide-linked [3-oc-oc-[3 configuration. The
binding of the
ligands, which are insulin-like growth-factor-I (IGF-I) and insulin-like
growth factor-II (IGF-
II), by the extracellular domain of IGF-I receptor activates its intracellular
tyrosine kinase
domain resulting in autophosphorylation of the receptor and substrate
phosphorylation. The
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IGF-I receptor is homologous to insulin receptor, having a high sequence
similarity of 84% in
the beta chain tyrosine kinase domain and a low sequence similarity of 48% in
the alpha
chain extracellular cysteine rich domain (LTlrich, A. et al., 1986, EMBO, 5,
2503-2512; Fujita-
Yamaguchi, Y. et al., 1986, J. Biol. Chena., 261, 16727-16731; LeRoith, D. et
al., 1995,
Endocrine Reviews, 16, 143-163). The IGF-I receptor and its ligands (IGF-I and
IGF-II) play
important roles in numerous physiological processes including growth and
development
during embryogenesis, metabolism, cellular proliferation and cell
differentiation in adults
(LeRoith, D., 2000, Endocrinology, 141, 1287-1288; LeRoith, D., 1997, New
England J.
Med., 336, 633-640).
[04] IGF-I and IGF-II function both as endocrine hormones in the blood, where
they are
predominantly present in complexes with IGF-binding proteins, and as paracrine
and
autocrine growth factors that are produced locally (Humbel, R. E., 1990, Eur.
J. Biochem.,
190, 445-462; Cohick, W. S. and Clemmons, D. R., 1993, Annu. Rev. Physiol. 55,
131-153).
[OS] The IGF-I receptor has been implicated in promoting growth,
transformation and
survival of tumor cells (Baserga, R. et al., 1997, Biochem. Biophys. Acta,
1332, F105-F126;
Blakesley, V. A. et al., 1997, .Iournal of Endocrinology, 152, 339-344;
I~aleko, M., Rutter,
W. J., and Miller, A. D. 1990, Mol. Cell. Biol., 10, 464-473). Thus, several
types of tumors
are known to express higher than normal levels of IGF-I receptor, including
breast cancer,
colon cancer, ovarian carcinoma, synovial sarcoma and pancreatic cancer
(Khandwala, H. M.
et al., 2000, Endocrine Reviews, 21, 215-244; Werner, H. and LeRoith, D.,
1996, Adv.
Cancer~Res., 68,183-223; Happerfield, L. C. et al., 1997, J. Pathol., 183, 412-
417; Frier, S. et
al., 1999, C'rut, 44, 704-708; van Dam, P. A. et al., 1994, J. Clin. Pathol.,
47, 914-919; Xie, Y.
et al., 1999, Cancer Res., 59, 3588-3591; Bergmann, U. et al., 1995, Cancer
Res., 55, 2007-
2011). In vitro, IGF-I and IGF-II have been shown to be potent mitogens for
several human
2
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tumor cell lines such as lung cancer, breast cancer, colon cancer,
osteosarcoma and cervical
cancer (Ankrapp, D. P. and Bevan, D. R., 1993, Cancer Res., 53, 3399-3404;
Cullen, K. J.,
1990, Cancer Res., 50, 48-53; Hermanto, U. et al., 2000, Cell Growth &
Differentiation, 1 l,
655-664; Guo, Y. S. et al., 1995, J. Am. Coll. Surg., 181, 145-154; Kappel, C.
C. et al., 1994,
Cancer Res., 54, 2803-2807; Steller, M. A. et al., 1996, Caracer Res., 56,
1761-1765).
Several of these tumors and tumor cell lines also express high levels of IGF-I
or IGF-II,
which may stimulate their growth in an autocrine or paracrine manner (Quinn,
K. A. et al.,
1996, J. Biol. Chem., 271, 11477-11483).
[06] Epidemiological studies have shown a correlation of elevated plasma level
of IGF-I
(and lower level of IGF-binding protein-3) with increased risk for prostate
cancer, colon
cancer, lung cancer and breast cancer (Chan, J. M. et al., 1998, Science, 279,
563-566; Wolk,
A. et al., 1998, J. Natl. Cancer Inst., 90, 911-915; Ma, J. et al., 1999, J.
Natl. Cancer Inst.,
91, 620-625; Yu, H. et al., 1999, J. Natl. Cancer Inst., 91, 151-156;
Hankinson, S. E. et al.,
1998, Lancet, 351, 1393-1396). Strategies to lower the IGF-I level in plasma
or to inhibit the
function of IGF-I receptor have been suggested for cancer prevention (Wu, Y.
et al., 2002,
Cancer Res., 62, 1030-1035; Grimberg, A and Cohen P., 2000, J. Cell. Physiol.,
183, 1-9).
[07] The IGF-I receptor protects tumor cells from apoptosis caused by growth
factor
deprivation, anchorage independence or cytotoxic drug treatment (Navarro, M.
and Baserga,
R., 2001, EyZdocriraology, 142, 1073-1081; Baserga, R. et al., 1997, Biochem.
Biophys. Acta,
1332, F105-F126). The domains of IGF-I receptor that are critical for its
mitogenic,
transforming and anti-apoptotic activities have been identified by mutational
analysis.
[08] For example, the tyrosine 1251 residue of IGF-I receptor has been
identified as
critical for anti-apoptotic and transformation activities but not for its
mitogenic activity
(O'Connor, R. et al., 1997, Mol. Cell. Biol., 17, 427-435; Miura, M. et al.,
1995, J. Biol.
3
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Claem., 270, 22639-22644). The intracellular signaling pathway of ligand-
activated IGF-I
receptor involves phosphorylation of tyrosine residues of insulin receptor
substrates (IRS-1
and IRS-2), which recruit phosphatidylinositol-3-kinase (PI-3-kinase) to the
membrane. The
membrane-bound phospholipid products of PI-3-kinase activate a
serine/threonine kinase
Akt, whose substrates include the pro-apoptotic protein BAD which is
phosphorylated to an
inactive state (Datta, S. R., Brunet, A. and Greenberg, M. E., 1999, Genes &
Development,
13, 2905-2927; Kulik, G., Klippel, A. and Weber, M. J., 1997, Mol. Cell. Biol.
17, 1595-
1606). The mitogenic signaling of IGF-I receptor in MCF-7 human breast cancer
cells
requires PI-3-kinase and is independent of mitogen-activated protein kinase,
whereas the
survival signaling in differentiated rat pheochromocytoma PC12 cells requires
both PI-3-
kinase and rnitogen-activated protein kinase pathways (Dufourny, B. et al.,
1997, J. Biol.
Ghem., 272, 31163-31171; Parrizas, M., Saltiel, A. R. and LeRoith, D., 1997,
J. Biol. Cherra.,
272, 154-161).
[09] Down-regulation of IGF-I receptor level by anti-sense strategies has been
shown to
reduce the tumorigenicity of several tumor cell lines in vivo and in vitro,
such as melanoma,
lung carcinoma, ovarian cancer, glioblastoma, neuroblastoma and
rhabdomyosarcoma
(Resnicoff, M. et al., 1994, Cancer Res., 54, 4848-4850; Lee, C.-T. et al.,
1996, Cancer Res.,
56, 3038-3041; Muller, M. et al., 1998, Int. J. Caracer, 77, 567-571; Trojan,
J. et al., 1993,
Science, 259, 94-97; Liu, X. et al., 1998, Cancer Res., 58, 5432-5438;
Shapiro, D. N. et al.,
1994, J. Cliyz. Invest., 94, 1235-1242). Likewise, a dominant negative mutant
of IGF-I
receptor has been reported to reduce the tumorigenicity in vivo and growth in
vitro of
transformed Rat-1 cells overexpressing IGF-I receptor (Prager, D. et al.,
1994, Proc. Natl.
Acad. Sci. USA, 91, 2181-2185).
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[10] Tumor cells expressing an antisense to the IGF-I receptor mRNA undergo
massive
apoptosis when injected into animals in biodiffusion chambers. This
observation makes the
IGF-I receptor an attractive therapeutic target, based upon the hypothesis
that tumor cells are
more susceptible than normal cells to apoptosis by inhibition of IGF-I
receptor (Resnicoff, M.
et al., 1995, Cancer Res., 55, 2463-2469; Baserga, R., 1995, Cancer Res., 55,
249-252).
[1l] Another strategy to inhibit the function of IGF-I receptor in tumor cells
has been to
use anti-IGF-I receptor antibodies which bind to the extracellular domains of
IGF-I receptor
and inhibit its activation. Several attempts have been reported to develop
mouse monoclonal
antibodies against IGF-I receptor, of which two inhibitory antibodies - IR3
and 1H7 - are
available and their use has been reported in several IGF-I receptor studies.
[12] The IR3 antibody was developed using a partially purified placental
preparation of
insulin receptor to immunize mice, which yielded an antibody, IRl, that was
selective for
binding insulin receptor, and two antibodies,1R2 and 1R3, that showed
preferential
immunoprecipitation of IGF-I receptor (somatomedin-C receptor) but also weak
immunoprecipitation of insulin receptor (Kull, F. C. et al., 1983, J. Biol.
Chem., 258, 6561-
6566).
[13] The 1H7 antibody was developed by immunizing mice with purified placental
preparation of IGF-I receptor, which yielded the inhibitory antibody 1H7 in
addition to three
stimulatory antibodies (Li, S.-L. et al., 1993, Biochem. Biophys. Res.
Conzmuh., 196, 92-98;
Xiong, L. et al., 1992, P~oc. Natl. Acad. Sci. LISA, 89, 5356-5360).
[14] In another report, a panel of mouse monoclonal antibodies specific for
human IGF-I
receptor were obtained by immunization of mice with transfected 3T3 cells
expressing high
levels of IGF-I receptor, which were categorized into seven groups by binding
competition
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studies and by their inhibition or stimulation of IGF-I binding to transfected
3T3 cells (Soos,
M. A. et al., 1992, J. Biol. Chem., 267, 12955-12963).
[15] Thus, although IR3 antibody is the most commonly used inhibitory antibody
for IGF-I
receptor studies in vitf~o, it suffers from the drawback that it exhibits
agonistic activity in
transfected 3T3 and CHO cells expressing human IGF-I receptor (Kato, H. et
al., 1993, J.
Biol. Glaem., 268, 2655-2661; Steele-Perkins, G. and Roth, R. A., 1990,
Biochem. Biophys.
Res. CommurZ., 171, 1244-1251). Similarly, among the panel of antibodies
developed by
Soos et al., the most inhibitory antibodies 24-57 and 24-60 also showed
agonistic activities in
the transfected 3T3 cells (Soos, M. A. et al., 1992, J. Biol. Chern., 267,
12955-12963).
Although, IR3 antibody is reported to inhibit the binding of IGF-I (but not
IGF-II) to
expressed receptors in intact cells and after solubilization, it is shown to
inhibit the ability of
both IGF-I and IGF-II to stimulate DNA synthesis in cells irz vitro (Steele-
Perkins, G. and
Roth, R. A., 1990, Bioehem. Bioph~s. Res. Commuya., 171, 1244-1251). The
binding epitope
of IR3 antibody has been inferred from chimeric insulin-IGF-I receptor
constructs to be the
223-274 region of IGF-I receptor (Gustafson, T. A. and Rutter, W. J., 1990, J.
Biol. ChenZ.,
265, 18663-18667; Soos, M. A. et al., 1992, J. Biol. Chem., 267, 12955-12963).
[16] The MCF-7 human breast cancer cell line is typically used as a model cell
line to
demonstrate the growth response of IGF-I and IGF-II ira vitro (Dufourny, B. et
al., 1997, J.
Biol. Chem., 272, 31163-31171). In MCF-7 cells, the IR3 antibody incompletely
blocks the
stimulatory effect of exogenously added IGF-I and IGF-II in serum-free
conditions by
approximately 80%. Also, the 1R3 antibody does not significantly inhibit (less
than 25%) the
growth of MCF-7 cells in 10% serum (Cullen, K. J. et al., 1990, Cancer Res.,
50, 48-53).
This weak inhibition of sex-um-stimulated growth of MCF-7 cells by IR3
antibody in vitf°o
may be related to the results of an in vivo study in which IR3 antibody
treatment did not
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CA 02548065 2006-06-O1
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significantly inhibit the growth of a MCF-7 xenograft in nude mice (Arteaga,
C. L. et al.,
1989, J. Clifa. Ifzvest., 84, 1418-1423).
[17] Because of the weak agonistic activities of the IR3 and other reported
antibodies, and
their inability to significantly inhibit the growth of tumor cells such as MCF-
7 cells in the
more physiological condition of serum-stimulation (instead of stimulation by
exogenously
added IGF-I or IGF-II in serum-free condition), there is a need for new anti-
IGF-I receptor
antibodies which significantly inhibit the serum-stimulated growth of tumor
cells but which
do not show significant agonistic activity by themselves.
SUMMARY OF THE INVENTION
[18] Accordingly, it is an object of the invention to provide antibodies,
antibody fragments
and antibody derivatives that specifically bind to insulin-like growth factor-
I receptor and
inhibit the cellular activity of the receptor by antagonizing the receptor,
and are also
substantially devoid of agonist activity towards the receptor.
[19] Thus, in a first embodiment, there is provided murine antibody EM164,
which is fully
characterized herein with respect to the amino acid sequences of both its
light and heavy
chain variable regions, the cDNA sequences of the genes for the light and
heavy chain
variable regions, the identification of its CDRs (complementarity-determining
regions), the
identification of its surface amino acids, and means for its expression in
recombinant form.
[20] In a second embodiment, there are provided resurfaced or humanized
versions of
antibody EM164 wherein surface-exposed residues of the antibody or its
fragments are
replaced in both light and heavy chains to more closely resemble known human
antibody
surfaces. Such humanized antibodies may have increased utility, compared to
murine
EM164, as therapeutic or diagnostic agents. Humanized versions of antibody
EM164 are
also fully characterized herein with respect to their respective amino acid
sequences of both
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CA 02548065 2006-06-O1
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light and heavy chain variable regions, the DNA sequences of the genes for the
light and
heavy chain variable regions, the identification of the CDRs, the
identification of their
surface amino acids, and disclosure of a means for their expression in
recombinant form.
[21] In a third embodiment, there is provided an antibody that is capable of
inhibiting the
growth of a cancer cell by greater than about 80% in the presence of a growth
stimulant such
as, for example, serum, insulin-like growth factor-I and insulin-like growth
factor-II.
[22] In a fourth embodiment, there is provided an antibody or antibody
fragment having a
heavy chain including CDRs having amino acid sequences represented by SEQ ID
NOS:1-3,
respectively:
SYWMH (SEQ ID NO:l),
EINPSNGRTNYNEKFKR (SEQ ID N0:2),
GRPDYYGSSKWYFDV (SEQ ID N0:3);
and having a light chain that comprises CDRs having amino acid sequences
represented by SEQ ID NOS:4-6:
RSSQSIVHSNVNTYLE (SEQ ID NO:4);
KVSNRFS (SEQ ID NO:S);
FQGSHVPPT (SEQ ID N0:6).
[23] In a fifth embodiment, there are provided antibodies having a heavy chain
that has an
amino acid sequence that shares at least 90% sequence identity with an amino
acid sequence
represented by SEQ ID N0:7:
QVQLQQSGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEI
NPSNGRTNYNEKFKRKATLTVDKSSSTAYMQLSSLTSEDSAVYYFARGRPDY
YGSSKWYFDVWGAGTTVTVSS (SEQ ID N0:7).
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[24] Similarly, there are provided antibodies having a light chain that has an
amino acid
sequence that shares at least 90% sequence identity with an amino acid
sequence represented
by SEQ ID N0:8:
DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNVNTYLEWYLQKPGQSPKLLIY
KVSNRFSGVPDRFSGSGSGTDFTLRISRVEAEDLGIYYCFQGSHVPPTFGGGTK
LEIKR (SEQ ID N0:8).
[25] In a sixth embodiment, antibodies are provided having a humanized or
resurfaced
light chain variable region having an amino acid sequence corresponding to one
of SEQ ID
NOS:- 9-12:
DVVMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYLQKPGQSPRLLIY
KVSNRFSGVPDRFSGSGAGTDFTLRISRVEAEDLGIYYCFQGSHVPPTFGGGT
KLEIKR (SEQ ID N0:9);
DVLMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYLQKPGQSPKLLIY
KVSNRFSGVPDRFSGSGAGTDFTLRISRVEAEDLGIYYCFQGSHVPPTFGGGT
KLEIKR (SEQ ID NO:10);
DVLMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYLQKPGQSPRLLIY
KVSNRFSGVPDRFSGSGAGTDFTLRISRVEAEDLGIYYCFQGSHVPPTFGGGT
KLEIKR (SEQ ID N0:11); or
DVVMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYLQKPGQSPKLLIY
KVSNRFSGVPDRFSGSGAGTDFTLRISRVEAEDLGIYYCFQGSHVPPTFGGGT
KLEIKR (SEQ ~ N0:12).
[26] Similarly, antibodies are provided having a humanized or resurfaced heavy
chain
variable region having an amino acid sequence corresponding to SEQ ID N0:13:
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QVQLVQSGAEVVKPGASVKLSCKASGYTFTSYWMIiWVKQRPGQGLEWIGEI
NP SNGRTNYNQKFQGKATLTVDKS S STAYMQLS SLTSED SAVYYFARGRPDY
YGSSKWYFDVWGQGTTVTVSS (SEQ ID N0:13).
[27] In a seventh embodiment, antibodies or antibody fragments of the present
invention
are provided that have improved properties. For example, antibodies or
antibody fragments
having improved affinity for IGF-I-receptor are prepared by affinity
maturation of an
antibody or fragment of the present invention.
[28] The present invention further provides conjugates of said antibodies,
wherein a
cytotoxic agent is covalently attached, directly or via a cleavable or non-
cleavable linker, to
an antibody or epitope-binding fragment of an antibody of the present
invention. 111 preferred
embodiments, the cytotoxic agent is a taxol, a maytansinoid, CC-1065 or a CC-
1065 analog.
[29] The present invention further provides for antibodies or fragments
thereof that are
further labeled for use in research or diagnostic applications. In preferred
embodiments, the
label is a radiolabel, a fluorophore, a chromophore, an imaging agent or a
metal ion.
[30] A method for diagnosis is also provided in which said labeled antibodies
or fragments
are administered to a subject suspected of having a cancer, and the
distribution of the label
within the body of the subject is measured or monitored.
[31] In an eighth embodiment, the invention provides methods for the treatment
of a
subject having a cancer by administering an antibody, antibody fragment or
antibody
conjugate of the present invention, either alone or in combination with other
cytotoxic or
therapeutic agents. The cancer can be one or more of, for example, breast
cancer, colon
cancer, ovarian carcinoma, osteosarcoma, cervical cancer, prostate cancer,
lung cancer,
synovial carcinoma, pancreatic cancer, or other cancer yet to be determined in
which IGF-I
receptor levels are elevated.
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[32] In a ninth embodiment, the invention provides methods for the treatment
of a subject
having a cancer by administering an antibody, antibody fragment or antibody
conjugate of the
present invention, either alone or in combination with other cytotoxic or
therapeutic agents.
In particular, preferred~cytotoxic and therapeutic agents include docetaxel,
paclitaxel,
doxorubicin, epirubicin, cyclophosphamide, trastuzumab (Herceptin),
capecitabine,
tamoxifen, toremifene, letrozole, anastrozole, fulvestrant, exemestane,
goserelin, oxaliplatin,
carboplatin, cisplatin, dexamethasone, amide, bevacizumab (Avastin), 5-
fluorouracil,
leucovorin, levamisole, irinotecan, etoposide, topotecan, gemcitabine,
vinorelbine,
estramustine, mitoxantrone, abarelix, zoledronate, streptozocin, rituximab
(Rituxan),
idarubicin, busulfan, chlorambucil, fludarabine, imatinib, cytarabine,
ibritumomab (Zevalin),
tositumomab (Bexxar), interferon alpha-2b, melphalam, bortezomib (Velcade),
altretamine,
asparaginase, gefitinib (Iressa), erlonitib (Tarceva), anti-EGF receptor
antibody (Cetuximab,
Abx-EGF), and an epothilone. More preferably, the therapeutic agent is a
platinum agent
(such as carboplatin, oxaliplatin, cisplatin), a taxane (such as paclitaxel,
docetaxel),
gemcitabine, or camptothecin.
[33] The cancer can be one or more of, for example, breast cancer, colon
cancer, ovarian
carcinoma, osteosarcoma, cervical cancer, prostate cancer, lung cancer,
synovial carcinoma,
pancreatic cancer, melanoma, multiple myeloma, neuroblastoma, and
rhabdomyosarcoma, or
other cancer yet to be determined in which IGF-I receptor levels are elevated.
[34] In a tenth embodiment, the invention provides kits comprising one or more
of the
elements described herein, and instructions for the use of those elements. In
a preferred
embodiment, a kit of the present invention includes antibody, antibody
fragment or conjugate
of the invention, and a therapeutic agent. The instructions for this preferred
embodiment
include instructions for inhibiting the growth of a cancer cell using the
antibody, antibody
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fragment or conjugate of the invention, and the therapeutic agent, and/or
instructions for a
method of treating a patient having a cancer using the antibody, antibody
fragment or
conjugate of the invention, and the therapeutic agent.
BRIEF DESCRIPTION OF THE FIGURES
[35] FIGURE 1 shows fluorescence activated cell sorting (FACS) analysis of the
specific
binding of purified EM164 antibody to cells overexpressing human Y1251F IGF-I
receptor or
human insulin receptor.
(36] FIGURE 2 shows a binding titration curve for the binding of EM164
antibody to
biotinylated human IGF-I receptor.
[37] FIGURE 3 shows the inhibition of the binding of biotinylated IGF-I to
human breast
cancer MCF-7 cells by EM164 antibody.
[38] FIGURE 4 shows the inhibition of IGF-I-stimulated autophosphorylation of
IGF-I
receptor in MCF-7 cells by EM164 antibody.
[39] FIGURE 5 shows the inhibition of IGF-I-stimulated IRS-1-phosphorylation
in MCF-7
cells by EM164 antibody.
[40] FIGURE 6 shows the inhibition of IGF-I-stimulated signal transduction in
SaOS-2
cells by EM164 antibody.
[41] FIGURE 7 shows the effect of EM164 antibody on the growth and survival of
MCF-7
cells under different growth conditions, as assessed by MTT assay.
[42] FIGURE 8 shows the effect of EM164 antibody on the growth and survival of
MCF-7
cells in the presence of various serum concentrations.
[43] FIGURE 9 shows the inhibition of IGF-I- and serum-stimulated growth and
survival
of NCI-H838 cells by EM164 antibody.
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[44] FIGURE 10 shows the effect of treatment with EM164 antibody, taxol, or a
combination of EM164 antibody and taxol, on the growth of a Calu-6 lung cancer
xenograft
m mice.
[45] FIGURE 11 shows competition between the binding of humanized EM164
antibody
(v.1.0) and marine EM164 antibody.
[46] FIGURE 12 shows the cDNA (SEQ ID N0:4.9) and amino acid sequences (SEQ ID
NO:50) of the light chain leader and variable region of the marine anti-IGF-I
receptor
antibody EM164. The arrow marks the start of framework 1. The 3 CDR sequences
according
to Kabat are underlined.
[47] FIGURE 13 shows the cDNA (SEQ ID NO:51) and amino acid sequences (SEQ ID
N0:52) of the heavy chain leader and variable region for the marine anti-IGF-I
receptor
antibody EM164. The arrow marks the start of framework 1. The 3 CDR sequences
according to Kabat are underlined.
[48] FIGURE 14 shows the light and heavy chain CDR amino acid sequences of
antibody
EM164 as determined from Chothia canonical class definitions. AbM modeling
software
definitions for the heavy chain CDRs are also shown. Light Chain: CDR1 is SEQ
ID N0:4,
CDR2 is SEQ ID NO:S, and CDR3 is SEQ ID N0:6. Heavy Chain: CDR1 is SEQ ID
NO:1,
CDR2 is SEQ ID N0:2, and CDR3 is SEQ ID N0:3 . AbM Heavy Chain: CDRl is SEQ ID
N0:53, CDR2 is SEQ ID N0:54, and CDR3 is SEQ m NO:55.
[49] FIGURE 15 shows the light chain and heavy chain amino acid sequences for
anti-
IGF-I-receptor antibody EM164 aligned with the germline sequences for the Crl
(SEQ ID
N0:56) and J558.c (SEQ ID N0:57) genes. Dashes (-) indicate sequence identity.
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[50] FIGURE 16 shows the plasmids used to build and express the recombinant
chimeric
and humanized EM164 antibodies. A) a light chain cloning plasmid, B) a heavy
chain
cloning plasmid, C) a mammalian antibody expression plasmid.
[51] FIGURE 17 shows the 10 most homologous amino acid sequences of the light
chains
screened from the 127 antibodies in the set of structure files used to predict
the surface
residues of EM164. em164 LC (SEQ ID NO:58), 2je1 (SEQ ID N0:59), 2pcp (SEQ ID
N0:60), lnqb (SEQ B? N0:61), lkel (SEQ ID N0:62), lhyx (SEQ ID N0:63), ligf
(SEQ ID
N0:64), ltet (SEQ ID N0:65), lclz (SEQ ~ N0:66), lbln (SEQ ID NO:67), lcly
(SEQ ID
N0:68), Consensus (SEQ ID N0:69).
[52] FIGURE 18 shows the 10 most homologous amino acid sequences of the heavy
chains screened from the 127 antibodies in the set of structure files used to
predict the surface
residues of EM164. em164 HC (SEQ ID N0:70), lnqb (SEQ ID N0:71), lngp (SEQ ID
N0:72), lf~i (SEQ ID N0:73), lafv (SEQ 11? N0:74), lyuh (SEQ ID N0:75), lplg
(SEQ ID
N0:76), 1 d5b (SEQ ID N0:77), 1 ae6 (SEQ 11? NO:78), 1 axs (SEQ ID N0:79),
3hf1 (SEQ ID
N0:80), Consensus (SEQ ID N0:81).
[53] FIGURE 19 shows the average accessibility for each of the (A) light, and
(B) heavy
chain variable region residues from the 10 most homologous structures. The
numbers
represent the Rabat antibody sequence position numbers.
[54] FIGURE 20 shows the light chain variable region amino acid sequences for
marine
EM164 (muEMl64) and humanized EM164 (huEMl64) antibodies. muEM164 (SEQ ID
N0:82), huEM164 V1.0 (SEQ ID N0:83), huEM164 Vl.l (SEQ 117 N0:84), huEM164
V1.2
(SEQ ID N0:85), huEMl64 V1.3 (SEQ ID N0:86).
[55] FIGURE 21 shows the heavy chain variable region amino acid sequences for
marine
(muEMl64, SEQ ID I~0:87) and humanized EM164 antibodies (huEMl64, SEQ ID
N0:88).
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[56] FIGURE 22 shows the huEM164 v1.0 variable region DNA and amino acid
sequences for both the light (DNA, SEQ ID N0:89, amino acid SEQ ID N0:90) and
heavy
chains (DNA, SEQ ID N0:91, amino acid SEQ ID N0:92).
[57] FIGURE 23 shows the light chain variable region DNA and amino acid
sequences for
humanized EM164 v1.1 (DNA, SEQ ID N0:93; amino acid SEQ LD N0:94), v1.2 (DNA,
SEQ ID N0:95; amino acid SEQ 117 N0:96) and v1 .3 (DNA, SEQ ID N0:97; amino
acid
SEQ ID N0:98).
[58] FIGURE 24 shows the inhibition of IGF-I-stimulated growth and survival of
MCF-7
cells by humanized EM164 v1.0 antibody and marine EM164 antibody.
[59] FIGURE 25 shows that EM164 suppresses IGF-1-stimulated cycling of MCF-7
cells.
[60] FIGURE 26 shows that EM164 suppresses the anti-apoptotic effect of IGF-l
and
serum. Treatment with EM164 results in apoptotic cell death as demonstrated by
the
increased levels of cleaved CI~18 protein.
[61] FIGURE 27 shows the effect of treatment with EM164 antibody, gemcitabine,
or a
combination of EM164 antibody and gemcitabine, on the growth of human BxPC-3
pancreatic cancer xenografts in immunodeficient mice.
DETAILED DESCRIPTION OF THE INVENTION
[62] The present inventors have discovered and improved novel antibodies that
specifically
bind to the human insulin-like growth factor-I receptor (IGF-IR) on the cell
surface. The
antibodies and fragments have the unique ability to inhibit the cellular
functions of the
receptor without the capacity to activate the receptor themselves. Thus, while
previously
known antibodies that specifically bind and inhibit IGF-IR also activate the
receptor even in
the absence of IGF-IR ligands, the antibodies or fragments of the present
invention
antagonize IGF-IR but are substantially devoid of agonist activity.
Furthermore, the
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antibodies and antibody fragments of the present invention inhibit the growth
of human
tumor cells such as MCF-7 cells in the presence of serum by greater than 80%,
which is a
higher degree of inhibition than is obtained using previously known anti-IGF-
1R antibodies.
[63] The present invention proceeds from a marine anti-IGF-IR antibody, herein
EM164,
that is fully characterized with respect to the amino acid sequences of both
light and heavy
chains, the identification of the CDRs, the identification of surface amino
acids, and means
for its expression in recombinant form.
[64] The germline sequences are shown in Figure 15 aligned with the sequence
of EM164.
The comparison identifies probable somatic mutations in EM164, including one
each in
CDRl in the light chain and in CDR2 in the heavy chain.
[65] The primary amino acid and DNA sequences of antibody EM 164 light and
heavy
chains, and of humanized versions, are disclosed herein. However, the scope of
the present
invention is not limited to antibodies and fragments comprising these
sequences. Instead, all
antibodies and fragments that specifically bind to an insulin-like growth
factor-I receptor and
antagonize the biological activity of the receptor, but which are
substantially devoid of
agonist activity, fall within the scope of the present invention. Thus,
antibodies and antibody
fragments may differ from antibody EM164 or the humanized derivatives in the
amino acid
sequences of their scaffold, CDRs, light chain and heavy chain, and still fall
within the scope
of the present invention.
[66] The CDRs of antibody EM164 are identified by modeling and their molecular
structures have been predicted. Again, while the CDRs are important for
epitope recognition,
they are not essential to the antibodies and fragments of the invention.
Accordingly,
antibodies and fragments are provided that have improved properties produced
by, for
example, affinity maturation of an antibody of the present invention.
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[67] Diverse antibodies and antibody fragments, as well as antibody mimics may
be
readily produced by mutation, deletion and/or insertion within the variable
and constant
region sequences that flank a particular set of CDRs. Thus, for example,
different classes of
Ab are possible for a given set of CDRs by substitution of different heavy
chains, whereby,
for example, IgG1-4, IgM, IgAI-2, IgD, IgE antibody types and isotypes may be
produced.
Similarly, artificial antibodies within the scope of the invention may be
produced by
embedding a given set of CDRs within an entirely synthetic framework. The term
"variable"
is used herein to describe certain portions of the variable domains that
differ in sequence
among antibodies and are used in the binding and specificity of each
particular antibody for
its antigen. However, the variability is not usually evenly distributed
through the variable
domains of the antibodies. It is typically concentrated in three segments
called
complementarity determining regions (CDRs) or hypervariable regions both in
the light chain
and the heavy chain variable domains. The more highly conserved portions of
the variable
domains are called the framework (FR). The variable domains of heavy and light
chains each
comprise four framework 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 (E. A. Kabat et al. Sequences ofProteiyas
oflmmuraologicallntey~est, fifth
edition, 1991, NIH~. The constant domains axe not involved directly in binding
an antibody
to an antigen, but exhibit various effector functions, such as participation
of the antibody in
antibody-dependent cellular toxicity.
[68] Humanized antibodies, or antibodies adapted for non-rejection by other
mammals,
may be produced using several technologies such as resurfacing and CDR
grafting. In the
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resurfacing technology, molecular modeling, statistical analysis and
mutagenesis are
combined to adjust the non-CDR surfaces of variable regions to resemble the
surfaces of
known antibodies of the target host. Strategies and methods for the
resurfacing of
antibodies, and other methods for reducing immunogenicity of antibodies within
a different
host, are disclosed in US Patent 5,639,641, which is hereby incorporated in
its entirety by
reference. In the CDR grafting technology, the marine heavy and light chain
CDRs are
grafted into a fully human framework sequence.
[69] The invention also includes functional equivalents of the axitibodies
described in this
specification. Functional equivalents have binding characteristics that are
comparable to
those of the antibodies, and include, for example, chimerized, humanized and
single chain
antibodies as well as fragments thereof. Methods of producing such functional
equivalents
are disclosed in PCT Application WO 93/21319, European Patent Application No.
239,400;
PCT Application WO 89/09622; European Patent Application 338,745; and European
Patent
Application EP 332,424, which are incorporated in their respective entireties
by reference.
[70] Functional equivalents include polypeptides with amino acid sequences
substantially
the same as the amino acid sequence of the variable or hypervariable regions
of the
antibodies of the invention. "Substantially the same" as applied to an amino
acid sequence is
defined herein as a sequence with at least about 90%, and more preferably at
least about 95%
sequence identity to another amino acid sequence, as determined by the FASTA
search
method in accordance with Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85,
2444-2448
(1988).
[71] Chimerized antibodies preferably have constant regions derived
substantially or
exclusively from human antibody constant regions and variable regions derived
substantially
or exclusively from the sequence of the variable region from a mammal other
than a human.
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Humanized forms of the antibodies are made by substituting the complementarity
determining regions of, for example, a mouse antibody, into a human framework
domain,
e.g., see PCT Pub. No. W092/22653. Humanized antibodies preferably have
constant regions
and variable regions other than the complementarity determining regions (CDRs)
derived
substantially or exclusively from the corresponding human antibody regions and
CDRs
derived substantially or exclusively from a mammal other than a human.
[72] Functional equivalents also include single-chain antibody fragments, also
known as
single-chain antibodies (scFvs). These fragments contain at least one fragment
of an
antibody variable heavy-chain amino acid sequence (VH) tethered to at least
one fragment of
an antibody variable light-chain sequence (VL) with or without one or more
interconnecting
linkers. Such a linker may be a short, flexible peptide selected to assure
that the proper
three-dimensional folding of the (VL) and (VH) domains occurs once they are
linked so as to
maintain the target molecule binding-specificity of the whole antibody from
which the single-
chain antibody fragment is derived. Generally, the carboxyl terminus of the
(VL) or (VH)
sequence may be covalently linked by such a peptide linker to the amino acid
terminus of a
complementary (VL) and (VH) sequence. Single-chain antibody fragments may be
generated
by molecular cloning, antibody phage display library or similar techniques.
These proteins
may be produced either in eukaryotic cells or prokaryotic cells, including
bacteria.
[73] Single-chain antibody fragments contain amino acid sequences having at
least one of
the variable or complementarity determining regions (CDRs) of the whole
antibodies
described in this specification, but are lacking some or all of the constant
domains of those
antibodies. These constant domains are not necessary for antigen binding, but
constitute a
major portion of the structure of whole antibodies. Single-chain antibody
fragments may
therefore overcome some of the problems associated with the use of antibodies
containing a
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part or all of a constant domain. For example, single-chain antibody fragments
tend to be free
of undesired interactions between biological molecules and the heavy-chain
constant region,
or other unwanted biological activity. Additionally, single-chain antibody
fragments are
considerably smaller than whole antibodies and may therefore have greater
capillary
permeability than whole antibodies, allowing single-chain antibody fragments
to localize and
bind to target antigen-binding sites more efficiently. Also, antibody
fragments can be
produced on a relatively large scale in prokaryotic cells, thus facilitating
their production.
Furthermore, the relatively small size of single-chain antibody fragments
makes them less
likely to provoke an immune response in a recipient than whole antibodies.
[74] Functional equivalents further include fragments of antibodies that have
the same, or
comparable binding characteristics to those of the whole antibody. Such
fragments may
contain one or both Fab fragments or the F(ab')a fragment. Preferably the
antibody fragments
contain all six complementarity determining regions of the whole antibody,
although
fragments containing fewer than all of such regions, such as three, four or
five CDRs, are also
functional. Further, the functional equivalents may be or may combine members
of any one
of the following immunoglobulin classes: IgG, IgM, IgA, IgD, or IgE, and the
subclasses
thereof.
[75] The knowledge of the amino acid and nucleic acid sequences for the anti-
IGF-I
receptor antibody EM164 and its humanized variants, which are described
herein, can be
used to develop other antibodies which also bind to human IGF-I receptor and
inhibit the
cellular functions of the IGF-I receptor. Several studies have surveyed the
effects of
introducing one or more amino acid changes at various positions in the
sequence of an
antibody, based on the knowledge of the primary antibody sequence, on its
properties such as
binding and level of expression (Yang, W. P. et al., 1995, J. Mol. Biol., 254,
392-403; Rader,
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C. et al., 1998, Proc. Natl. Acad. Sci. USA, 95, 8910-8915; Vaughan, T. J. et
al., 1998, Nature
Biotechnology, 16, 535-539).
[76] In these studies, variants of the primary antibody have been generated by
changing the
sequences of the heavy and light chain genes in the CDRl, CDR2, CDR3, or
framework
regions, using methods such as oligonucleotide-mediated site-directed
mutagenesis, cassette
mutagenesis, error-prone PCR, DNA shuffling, or mutator-strains of E. coli
(Vaughan, T. J.
et al., 1998, Nature Biotechnology, 16, 535-539; Adey, N. B. et al., 1996,
Chapter 16, pp.
277-291, in "Phage Display of Peptides and Proteins ", Eds. Kay, B. K. et al.,
Academic
Press). These methods of changing the sequence of the primary antibody have
resulted in
improved affinities of the secondary antibodies (Gram, H. et al., 1992, Proc.
Natl. Acad. Sci.
USA, 89, 3576-3580; Boder, E. T. et al., 2000, Proc. Natl. Acad. Sci. USA, 97,
10701-10705;
Davies, J. and Riechmann, L., 1996, Imnaunotechnolgy, 2, 169-179; Thompson, J.
et al.,
1996, J. Mol. Biol., 256, 77-88; Short, M. K. et al., 2002, J. Biol. Chem.,
277, 16365-16370;
Furukawa, K. et al., 2001, J. Biol. Chem., 276, 27622-27628).
[77] By a similar directed strategy of changing one or more amino acid
residues of the
antibody, the antibody sequences described in this invention can be used to
develop anti-IGF-
I receptor antibodies with improved functions.
[78] The conjugates of the present invention comprise the antibody, fragments,
and their
analogs as disclosed herein, linked to a cytotoxic agent. Preferred cytotoxic
agents are
maytansinoids, taxanes and analogs of CC-1065. The conjugates can be prepared
by ifa vitro
methods. In order to link the cytotoxic agent to the antibody, a linking group
is used.
Suitable linking groups are well known in the art and include disulfide
groups, thioether
groups, acid labile groups, photolabile groups, peptidase labile groups and
esterase labile
groups. Preferred linking groups are disulfide groups and thioether groups.
For example,
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conjugates can be constructed using a disulfide exchange reaction or by
forming a thioether
bond between the antibody and the cytotoxic agent.
[79] Maytansinoids and maytansinoid analogs are among the preferred cytotoxic
agents.
Examples of suitable maytansinoids include maytansinol and maytansinol
analogs. Suitable
maytansinoids are disclosed in U.S. Patent Nos. 4,424,219; 4,256,746;
4,294,757; 4,307,016;
4,313,946; 4,315,929; 4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254;
4,322,348;
4,371,533; 6,333,410; 5,475,092; 5,585,499; and 5,846,545.
[80] Taxanes are also preferred cytotoxic agents. Taxanes suitable for use in
the present
invention are disclosed in U.S. Patent Nos. 6,372,738 and 6,340,701.
[81] CC-1065 and its analogs are also preferred cytotoxic drugs for use in the
present
invention. CC-1065 and its analogs are disclosed in U.S. Patent Nos.
6,372,738; 6,340,701;
5,846,545 and 5,585,499.
[82] An attractive candidate for the preparation of such cytotoxic conjugates
is CC-1065,
which is a potent anti-tumor antibiotic isolated from the culture broth of
Streptozzzyces
zelezzsis. CC-1065 is about 1000-fold more potent in vitro than are commonly
used anti-
cancer drugs, such as doxorubicin, methotrexate and vincristine (B.K. Bhuyan
et al., Cayzcer
Res., 42, 3532-3537 (1982)).
[83] Cytotoxic drugs such as methotrexate, daunorubicin, doxorubicin,
vincristine,
vinblastine, melphalan, mitomycin C, chlorambucil, and calicheamicin are also
suitable for
the preparation of conjugates of the present invention, and the drug molecules
can also be
linked to the antibody molecules through an intermediary carner molecule such
as serum
albumin.
[84] For diagnostic applications, the antibodies of the present invention
typically will be
labeled with a detectable moiety. The detectable moiety can be any one which
is capable of
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producing, either directly or indirectly, a detectable signal. For example,
the detectable
moiety may be a radioisotope, such as 3H, 14C' 32p, 3sS, or 1311; a
fluorescent or
chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or
luciferin; or
an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish
peroxidase.
[85] Any method known in the art for conjugating the antibody to the
detectable moiety
may be employed, including those methods described by Hunter, et al., Nature
144:945
(1962); David, et al., Biochemistry 13:1014 (1974); Pain, et al., J. Immunol.
Meth. 40:219
(1981); and Nygren, J. Histochem. and Cytochem. 30:407 (1982).
(86] The antibodies of the present invention can be employed in any known
assay method,
such as competitive binding assays, direct and indirect sandwich assays, and
immunoprecipitation assays (Zola, Monoclonal Antibodies: A Manual of
Techniques,
pp.147-158 (CRC Press, Inc., 1987)).
[87] The antibodies of the invention also are useful for ifa vivo imaging,
wherein an
antibody labeled with a detectable moiety such as a radio-opaque agent or
radioisotope is
administered to a subject, preferably into the bloodstream, and the presence
and location of
the labeled antibody in the host is assayed. This imaging technique is useful
in the staging
and treatment of malignancies. The antibody may be labeled with any moiety
that is
detectable in a host, whether by nuclear magnetic resonance, radiology, or
other detection
means known in the art.
[88] The antibodies of the invention also are useful as affinity purification
agents_ In this
process, the antibodies are immobilized on a suitable support, such a Sephadex
resin or filter
paper, using methods well known in the art.
[89] The antibodies of the invention also are useful as reagents in biological
reseaxch,
based on their inhibition of the function of IGF-I receptor in cells.
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[90] For therapeutic applications, the antibodies or conjugates of the
invention are
administered to a subject, in a pharmaceutically acceptable dosage form. They
can be
administered intravenously as a bolus or by continuous infusion over a period
of time, by
intramuscular, subcutaneous, infra-articular, intrasynovial, intrathecal,
oral, topical, or
inhalation routes. The antibody may also be administered by intratumoral,
peritumoral,
intralesional, or perilesional routes, to exert local as well as systemic
therapeutic effects.
Suitable pharmaceutically acceptable carriers, diluents, and excipients are
well known and
can be determined by those of skill in the art as the clinical situation
warrants. Examples of
suitable Garners, diluents and/or excipients include: (1) Dulbecco's phosphate
buffered saline,
pH about 7.4, containing about 1 mg/ml to 25 mg/ml human serum albumin, (2)
0.9% saline
(0.9% w/v NaCI), and (3) 5% (w/v) dextrose. The method of the present
invention can be
practiced irz vitro, in vivo, or ex vivo.
[91] In other therapeutic treatments, the antibodies, antibody fragments or
conjugates of
the invention are co-administered, or administered sequentially, with one or
more additional
therapeutic agents. Suitable therapeutic agents include, but are not limited
to, cytotoxic or
cytostatic agents. Taxol is a preferred therapeutic agent that is also a
cytotoxic agent.
[92] Cancer therapeutic agents are those agents that seek to kill or limit the
growth of
cancer cells while doing minimal damage to the host. Thus, such agents may
exploit any
difference in cancer cell properties (e.g. metabolism, vascularization or cell-
surface antigen
presentation) from healthy host cells. Differences in tumor morphology are
potential sites for
intervention: for example, the second therapeutic can be an antibody such as
an anti-VEGF
antibody that is useful in retarding the vascularization of the interior of a
solid tumor, thereby
slowing its growth rate. Other therapeutic agents include, but are not limited
to, adjuncts
such as granisetron HCl, androgen inhibitors such as leuprolide acetate,
antibiotics such as
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doxorubicin, antiestrogens such as tamoxifen, antimetabolites such as
interferon alpha-2a,
cytotoxic agents such as taxol, enzyme inhibitors such as ras farnesyl-
transferase inhibitor,
immunomodulators such as aldesleukin, and nitrogen mustard derivatives such as
melphalan
HCI, and the like.
[93] The therapeutic agents that can be combined with EM164 for improved anti-
cancer
efficacy include diverse agents used in oncology practice (Refererace: Cancer,
Principles &
Practice of Oncology, DeVita, V. T., Hellman, S., Rosenberg, S. A., 6th
edition, Lippincott-
Raven, Philadelphia, 2001), such as docetaxel, paclitaxel, doxorubicin,
epirubicin,
cyclophosphamide, trastuzumab (Herceptin), capecitabine, tamoxifen,
toremifene, letrozole,
anastrozole, fulvestrant, exemestane, goserelin, oxaliplatin, carboplatin,
cisplatin,
dexamethasone, amide, bevacizumab (Avastin), 5-fluorouracil, leucovorin,
levamisole,
irinotecan, etoposide, topotecan, gemcitabine, vinorelbine, estramustine,
mitoxantrone,
abarelix, zoledronate, streptozocin, rituximab (Rituxan), idarubicin,
busulfan, chlorambucil,
fludarabine, imatinib, cytarabine, ibritumomab (Zevalin), tositumomab
(Bexxar), interferon
alpha-2b, melphalam, bortezomib (Velcade), altretamine, asparaginase,
gefitinib (Iressa),
erlonitib (Tarceva), anti-EGF receptor antibody (Cetuximab, Abx-EGF),
epothilones, and
conjugates of cytotoxic drugs and antibodies against cell-surface receptors.
Preferred
therapeutic agents are platinum agents (such as carboplatin, oxaliplatin,
cisplatin), taxanes
(such as paclitaxel, docetaxel), gemcitabine, and camptothecin.
[94] The one or more additional therapeutic agents can be administered before,
concurrently, or after the antibody, antibody fragment or conjugate of the
invention. The
skilled artisan will understand that for each therapeutic agent there may be
advantages to a
particular order of administration. Similarly, the skilled artisan will
understand that for each
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therapeutic agent, the length of time between which the agent, and an
antibody, antibody
fragment or conjugate of the invention is administered, will vary.
[95] While the skilled artisan will understand that the dosage of each
therapeutic agent will
be dependent on the identity of the agent, the preferred dosages can range
from about 10
mg/square meter to about 2000 mg/square meter, more preferably from about 50
mg/square
meter to about 1000 mg/square meter. For preferred agents such as platinum
agents
(carboplatin, oxaliplatin, cisplatin), the preferred dosage is about 10
mg/square meter to about
400 mglsquare meter,1or taxanes (paclitaxel, docetaxel) the preferred dosage
is about 20
mglsquare meter to about 150 mg/square meter, for gemcitabine the preferred
dosage is about
100 mg/square meter to about 2000 mg/square meter, and for camptothecin the
preferred
dosage is about 50 mg/square meter to about 350 mg/square meter. The dosage of
this and
other therapeutic agents may depend on whether the antibody, antibody fragment
or
conjugate of the invention is administered concurrently or sequentially with a
therapeutic
agent.
[96] Administration of an antibody, antibody fragment or conjugate of the
invention, and
one or more additional therapeutic agents, whether co-administered or
administered
sequentially, may occur as described above for therapeutic applications.
Suitable
pharmaceutically acceptable carriers, diluents, and excipients for co-
administration will be
understood by the skilled artisan to depend on the identity of the particular
therapeutic agent
being co-administered.
[97] When present in an aqueous dosage form, rather than being lyophilized,
the antibody
typically will be formulated at a concentration of about 0.1 mg/ml to 100
mg/ml, although
wide variation outside of these ranges is permitted. For the treatment of
disease, the
appropriate dosage of antibody or conjugate will depend on the type of disease
to be treated,
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as defined above, the severity and course of the disease, whether the
antibodies are
administered for preventive or therapeutic purposes, the course of 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.
[98] Depending on the type and severity of the disease, preferably from about
1 mg/square
meter to about 2000 mg/square meter of antibody is an initial candidate dosage
for
administration to the patient, more preferably from about 10 mg/square meter
to about 1000
mg/square meter of antibody whether, for example, by one or more separate
administrations,
or by continuous infusion. For repeated administrations over several days or
longer,
depending on the condition, the treatment is repeated until a desired
suppression of disease
symptoms occurs. However, other dosage regimens may be useful and are not
excluded.
(99] The present invention also includes kits comprising one or more of the
elements
described herein, and instructions for the use of those elements. In a
preferred embodiment, a
kit of the present invention includes antibody, antibody fragment or conjugate
of the
invention, and a therapeutic agent. The instructions for this preferred
embodiment include
instructions for inhibiting the growth of a cancer cell using the antibody,
antibody fragment
or conjugate of the invention, and the therapeutic agent, and/or instructions
for a method of
treating a patient having a cancer using the antibody, antibody fragment or
conjugate of the
invention, and the therapeutic agent.
(100] Preferably, the antibody used in the kit has the same amino acid
sequence as the
murine antibody EM164 produced by mouse hybridoma EM164 (ATCC accession number
PTA-4457), or the antibody is an epitope-binding fragment thereof, wherein
both the
antibody and the fragment specifically bind to insulin-like growth factor-I
receptor. The
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antibody and antibody fragment used in the kit may also be a resurfaced
version of the
EM164 antibody, a humanized version of the EM164 antibody, or an altered
version of the
EM164 antibody having least one nucleotide mutation, deletion or insertion.
Antibodies and
antibody fragments of each of these three versions retain the same binding
specificity as the
EM164 antibody.
[101] Preferably, the therapeutic agent used in the kit is selected from the
group consisting
of docetaxel, paclitaxel, doxorubicin, epirubicin, cyclophosphamide,
trastuzumab
(Herceptin), capecitabine, tamoxifen, toremifene, letrozole, anastrozole,
fulvestrant,
exemestane, goserelin, oxaliplatin, carboplatin, cisplatin, dexamethasone,
amide,
bevacizumab (Avastin), 5-fluorouracil, leucovorin, levamisole, irinotecan,
etoposide,
topotecan, gemcitabine, vinorelbine, estramustine, mitoxantrone, abarelix,
zoledronate,
streptozocin, rituximab (Rituxan), idarubicin, busulfan, chlorambucil,
fludarabine, imatinib,
cytarabine, ibritumomab (Zevalin), tositumomab (Bexxar), interferon alpha-2b,
melphalam,
bortezomib (Velcade), altretamine, asparaginase, gefitinib (Iressa), erlonitib
(Tarceva), anti-
EGF receptor antibody (Cetuximab, Abx-EGF), and an epothilone. More
preferably, the
therapeutic agent is a platinum agent (such as carboplatin, oxaliplatin,
cisplatin), a taxane
(such as paclitaxel, docetaxel), gemcitabine, or camptothecin.
[102] The elements of the kits of the present invention are in a suitable form
for a kit, such
as a solution or lyophilized powder. The concentration or amount of the
elements of the kits
will be understood by the skilled artisan to varying depending on the identity
and intended
use of each element of the kit.
[103] The cancers and cells therefrom referred to in the instructions of the
kits include
breast cancer, colon cancer, ovarian carcinoma, osteosarcoma, cervical cancer,
prostate
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cancer, lung cancer, synovial carcinoma, pancreatic cancer, melanoma, multiple
myeloma,
neuroblastoma, and rhabdomyosarcoma.
EXAMPLES
[104J The invention is now described by reference to the following examples,
which are
illustrative only, and are not intended to limit the present invention.
EXAMPLE 1: Murine EM164 Antibody
[105] In this first example, the complete primary amino acid structure and
cDNA sequence
of a murine antibody of the present invention is disclosed, together with its
binding properties
and means for its expression in recombinant form. Accordingly, there is
provided a full and
complete disclosure of an antibody of the invention and its preparation, such
that one of
ordinary skill in the immunological arts would be able to prepare said
antibody without undue
experimentation.
A. Generation of Anti-IGF-I Receptor Monoclonal Antibody Hybridoma
[106] A cell line expressing human IGF-I receptor with a Y1251F mutation was
used for
immunization as it expressed a high number of IGF-I receptors (~10~ per cell).
The Y1251F-
mutation in the cytoplasmic domain of IGF-I receptor resulted in loss of
transformation and
anti-apoptotic signaling, but did not affect IGF-I binding and IGF-I-
stimulated mitogenic
signaling (O'Connor, R. et al., 1997, Mol. Cell. Biol., 17, 427-435; Miura, M.
et al., 1995, J.
Bi~l. Chem., 270, 22639-22644). The mutation did not otherwise affect antibody
generation
because the antibody of this example bound to the extracellular domain of IGF-
I receptor,
which was identical for both the Y1251F mutant and the wild type receptor.
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[107] A cell line expressing human IGF-I receptor with a Y1251F mutation was
generated
from 3T3-like cells of a IGF-I-receptor-deficient mouse by transfection with
Y1251F-mutant
human IGF-I-receptor gene together with a puromycin-resistance gene, and was
selected
using puromycin (2.5 microgram/mL) and by FACS sorting for high IGF-I receptor
expression (Miura, M. et al., 1995, J. Biol. Chem., 270, 22639-22644). A cell
line having a
high level of IGF-I receptor expression was further selected using a high
concentration of
puromycin such as 25 microgram/mL, which was toxic to most of the cells.
Surviving
colonies were picked and those displaying a high level of IGF-I receptor
expression were
selected.
[108 CAFl/J female mice, 6 months old, were immunized intraperitoneally on day
0 with
Y1251F-mutant-human-IGF-I-receptor-overexpressing cells (5x105 cells,
suspended in 0.2
mL PBS). The animals were boosted with 0.2 mL cell suspension as follows: day
2, 1x106
cells; day 5, 2x106 cells; days 7, 9, 12, and 23, 1x10' cells. On day 26, a
mouse was
sacrificed and its spleen removed.
[109] The spleen was ground between two frosted glass slides to obtain a
single cell
suspension, which was washed with serum-free RPMI medium containing penicillin
and
streptomycin (SFM). The spleen cell pellet was resuspended in 10 mL of 0.83%
(w/v)
ammonium chloride solution in water for 10 min on ice to lyse the red blood
cells, and was
then washed with serum-free medium (SFM). Spleen cells (1.2x108 ) were pooled
with
myeloma cells (4x100 from the non-secreting mouse myeloma cell line
P3X63Ag8.653
(ATCC, Rockville, MD; Cat. # CRL1580) in a tube, and washed with the serum-
free RPMI-
1640 medium (SFM). The supernatant was removed and the cell pellet resuspended
in the
residual medium. The tube was placed in a beaker of water at 37°C and
1.5 mL of
polyethylene glycol solution (50% PEG (w/v), average molecular weight 1500 in
75 mM
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HEPES, pH 8) was added slowly at a drop rate of 0.5 mL/minute while the tube
was gently
shaken. After a wait of one minute, 10 mL of SFM was added as follows: 1 mL
over the first
minute, 2 mL over the second minute, and 7 mL over the third minute. Another
10 mL was
then added slowly over one minute. Cells were pelleted by centrifugation,
washed in SFM
and resuspended in RPMI-1640 growth medium supplemented with 5% fetal bovine
serum
(FBS), hypoxanthine/aminopterin/ thymidine (HAT), penicillin, streptomycin,
and 10%
hybridoma cloning supplement (HCS). Cells were seeded into 96-well flat-bottom
tissue
culture plates at 2x105 spleen cells in 200 ~,L per well. After 5-7 days, 100
~,L per well were
removed and replaced with growth medium supplemented with
hypoxanthine/thymidine (HT)
and 5% FBS. The general conditions used for immunization and hybridoma
production were
as described by J. Langone and H. Vunakis (Eds., Methods in Enzymology, Vol.
121,
"Imlnunochemical Techniques, Part I"; 1986; Academic Press, Florida) and E.
Harlow and D.
Lane ("Antibodies: A Laboratory Manual"; 1988; Cold Spring Harbor Laboratory
Press,
New York). Other techniques of immunization and hybridoma production can also
be used,
as are well known to those of skill in the art.
[110] Culture supernatants from hybridoma clones were screened for binding to
purified
human IGF-I receptor by ELISA, for specific binding to cells overexpressing
human IGF-I
receptor, and for a lack of binding to cells overexpressing human insulin
receptor by ELISA
and FACS screening as described below. Clones exhibiting higher binding
affinity to cells
overexpressing human IGF-I receptor than to cells overexpressing human insulin
receptor
were expanded and subcloned. The culture supernatants of the subclones were
further
screened by the above binding assays. By this procedure, subclone 3F1-C8-D7
(EM164) was
selected, and the heavy and light chain genes were cloned and sequenced as
described below.
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[111] Human IGF-I receptor was isolated for use in the screening of
supernatants from
hybridoma clones for their binding to IGF-I receptor by the method below.
Biotinylated IGF-
I was prepared by modification of recombinant IGF-I using biotinylating
reagents such as
sulfo-NHS-LC-biotin, sulfo-NHS-SS-biotin, or NHS-PE04-biotin. Biotinylated IGF-
I was
absorbed on streptavidin-agarose beads and incubated with lysate from cells
that
overexpressed human wild type or Y1251F mutant IGFR. The beads were washed and
eluted
with a buffer containing 2 to 4 M urea and detergent such as triton X-100 or
octyl-(3-
glucoside. Eluted IGF-I receptor was dialyzed against PBS and was analyzed for
purity by
SDS-PAGE under reducing conditions, which showed alpha and beta chain bands of
IGF-I
receptor of molecular weights about 135 kDa and 95 kDa, respectively.
[112] To check for the binding of hybridoma supernatants to purified IGF-I
receptor, an
Immulon-4HB ELISA plate (Dynatech) was coated with a purified human IGF-I
receptor
sample (prepared by dialysis from urea/octyl-[3-glucoside elution of affinity
purified sample)
diluted in 50 mM CHES buffer at pH 9.5 (100 ~,I,; 4°C, overnight). The
wells were blocked
with 200 ~,L of blocking buffer (10 mg/mL BSA in TBS-T buffer containing 50 mM
Tris,
150 mM NaCI, pH 7.5, and 0.1% Tween-20) and incubated with supernatants from
hybridoma clones (100 ~,L; diluted in blocking buffer) for about 1 h to 12 h,
washed with
TBS-T buffer, and incubated with goat-anti-mouse-IgG-Fc-asltibody-horseradish
peroxidase
(HRP) conjugate (100 jvL; 0.8 ~,g/mL in blocking buffer; Jackson
ImmunoResearch
Laboratories), followed by washes and detection using ABTSlHaO2 substrate at
405 nm (0.5
mg/mL ABTS, 0.03% H202 in 0.1 M citrate buffer, pH 4.2). Typically, a
supernatant from a
3F1 hybridoma subclone yielded a signal of about 1.2 absorbance units within 3
min of
development, in contrast to values of 0.0 obtained for supernatants from some
other
hybridoma clones. General conditions for this ELISA were similar to the
standard ELISA
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conditions for antibody binding and detection as described by E. Harlow and D.
Lane ("Using
Antibodies: A Laboratory Manual"; 1999, Cold Spring Harbor Laboratory Press,
New York),
which conditions can also be used.
[113] Screening of hybridoma supernatants for specific binding to human IGF-I
receptor
and not to human insulin receptor was performed using ELISA on cell lines that
overexpressed human Y1251F-IGF-I receptor and on cell lines that overexpressed
human
insulin receptor. Both cell lines were generated from 3T3-like cells of IGF-I
receptor
deficient mice. The IGF-I receptor overexpressing cells and insulin receptor
overexpressing
cells were separately harvested from tissue culture flasks by quick
trypsin/EDTA treatment,
suspended in growth medium containing 10% FBS, pelleted by centrifugation, and
washed
with PBS. The washed cells (100 pL of about 1-3 x 106 cells/mL) were added to
wells of an
Immulon-2HB plate coated with phytohemagglutinin (100 pL of 20 pg/mL PHA),
centrifuged and allowed to adhere to PHA-coated wells for 10 min. The plate
with cells was
flicked to remove PBS and was then dried overnight at 37°C. The wells
were blocked with 5
mg/mL BSA solution in PBS for 1 h at 37°C and were then washed gently
with PBS.
Aliquots of the supernatants from hybridoma clones (100 ~L; diluted in
blocking buffer)
were then added to wells containing IGF-I-receptor-overexpressing cells and to
wells
containing insulin receptor-overexpressing cells and were incubated at ambient
temperature
for 1 h. The wells were washed with PBS, incubated with goat-anti-mouse-IgG-Fc-
antibody-
horseradish peroxidase conjugate (100 ~L; 0.8 ~.g/mL in blocking buffer) for 1
h, followed
by washes and then binding was detected using an ABTS/HZOa substrate. A
typical
supernatant from a 3F1 hybridoma subclone upon incubation with cells
overexpressing IGF-I
receptor yielded a signal of 0.88 absorbance units within 12 min of
development, in contrast
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to a value of 0.22 absorbance units obtained upon incubation with cells
overexpressing
human insulin receptor.
[114] The hybridoma was grown in Integra CL 350 flasks (Integra Biosciences,
Maryland),
according to manufacturer's specifications, to provide purified EM164
antibody. A yield of
about 0.5-1 mg/mL antibody was obtained in the harvested supernatants from the
Integra
flasks, based on quantitation by ELISA and by SDS-PAGE/Coomassie blue staining
using
antibody standards. The antibody was purified by affinity chromatography on
Protein A-
agarose bead column under standard purification conditions of loading and
washing in 100
mM Tris buffer, pH 8.9, containing 3 M NaCI, followed by elution in 100 mM
acetic acid
solution containing 150 mM NaCI. The eluted fractions containing antibody were
neutralized
with cold 2 M K.2HP04 solution and dialyzed in PBS at 4°C. The
concentration of the
antibody was determined by measuring absorbance at 280 nm (extinction
coefficient =1.4
mg 1 mL cm'1). The purified antibody sample was analyzed by SDS-PAGE under
reducing
conditions and Coornassie blue staining, which indicated only heavy and light
chain bands of
antibody at about 55 kDa and 25 kDa, respectively. The isotype of the purified
antibody was
IgGI with kappa light chain.
B. Binding characterization of EM164 Antibody
[115J The specific binding of the purified EM 164 antibody was demonstrated by
fluorescence activated cell sorting (FAGS) using cells overexpressing human
IGF-I receptor
and by using cells that overexpressed human insulin receptor (Figure 1).
Incubation of EM
164 antibody (50-100 nM) in 100 ~,L cold FACS buffer (1 mg/mL BSA in
Dulbecco's MEM
medium) was performed using cells overexpressing IGF-I receptor and using
cells
overexpressing insulin receptor (2x105 cells/mL) in a round-bottom 96-well
plate fox 1 h.
The cells were pelleted by centrifugation and washed with cold FACS buffer by
gentle
34
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flicking, followed by incubation with goat-anti-mouse-IgG-antibody-FITC
conjugate (100
~L; 10 ~,g/mL in FACS buffer) on ice for 1 h. The cells were pelleted, washed,
and
resuspended in 120 ~,L of 1% formaldehyde solution in PBS. The plate was
analyzed using a
FACSCalibur reader (BD Biosciences).
[116] A strong fluorescence shift was obtained upon incubation of IGF-I
receptor
overexpressing cells with EM 164 antibody, in contrast to an insignificant
shift upon
incubation of insulin receptor overexpressing cells with EM 164 antibody
(Figure 1), which
demonstrated that the EM 164 antibody was selective in its binding to IGF-I
receptor and did
not bind to insulin receptor., The control antibodies, anti-IGF-I receptor
antibody 1H7 (Santa
Cruz Biotechnology) and anti-insulin receptor alpha antibody (BD Pharmingen
Laboratories),
yielded fluorescence shifts upon incubations with cells that overexpressed IGF-
I receptor and
insulin receptor, respectively (Figure 1). A strong fluorescence shift was
also observed by
FAGS assay using EM 164 antibody and human breast cancer MCF-7 cells, which
expressed
IGF-I receptor (Dufourny, B. et al., 1997, J. Biol. Chefra., 272, 31163-
31171), which showed
that EM164 antibody bound to human IGF-I receptor on the surface of human
tumor cells.
[117) The dissociation constant (Ka) for the binding of EM164 antibody with
human IGF-I
receptor was determined by ELISA titration of the binding of antibody at
several
concentrations with either directly coated IGF-I receptor (affinity purified
using biotinylated
IGF-I, as above) or indirectly captured biotinylated IGF-I receptor.
Biotinylated IGF-I
receptor was prepared by biotinylation of detergent-solubilized lysate from
IGF-I receptor
overexpressing cells using PEO-maleimide-biotin reagent (Pierce, Molecular
Biosciences),
which was affinity purified using an anti-IGF-I receptor beta chain antibody
immobilized on
NHS-agarose beads and was eluted with 2-4 M urea in buffer containing NP-40
detergent and
dialyzed in PBS.
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[118] The Kd determination for the binding of EM164 antibody with biotinylated
IGF-I
receptor was carried out by coating Immulon-2HB plates with 100 p,L of 1
p,g/mL
streptavidin in carbonate buffer (150 mM sodium carbonate, 350 mM sodium
bicarbonate) at
4°C overnight. The streptavidin-coated wells were blocked with 200 ~L
of blocking buffer
(10 mg/mL BSA in TBS-T buffer), washed with TBS-T buffer and incubated with
biotinylated IGF-I receptor (10 to 100 ng) for 4 h at ambient temperature. The
wells
containing indirectly captured biotinylated IGF-I receptor were then washed
and incubated
with EM164 antibody in blocking buffer at several concentrations (5.1x10'13 M
to 200 nM)
for 2 h at ambient temperature and were then incubated overnight at
4°C. The wells were
next washed with TBS-T buffer and incubated with goat-anti-mouse-IgGH+L-
antibody-
horseradish peroxidase conjugate (100 p,L; 0.5 p.g/mL in blocking buffer),
followed by
washes and detection using ABTS/HZOa substrate at 405 nm. The value of Kd was
estimated
by non-linear regression for one-site binding.
[119] A similar binding titration was carned out using the Fab fragment of
EM164
antibody, prepared by papain digestion of the antibody as described by E.
Harlow and D.
Lane ("Using Antibodies: A Laboratory Manual"; 1999, Cold Spring Harbor
Laboratory
Press, New York).
[120] The binding titration curve for the binding of EM164 antibody to
biotinylated human
IGF-I receptor yielded a Kd value of 0.1 nM (Figure 2). The Fab fragment of
EM164
antibody also bound the human IGF-I receptor very tightly with a Ka value of
0.3 nM, which
indicated that the monomeric binding of the EM164 antibody to IGF-I receptor
was also very
strong.
[121] This extremely low value of dissociation constant for the binding of IGF-
I receptor by
EM164 antibody was in part due to a very slow k°ff rate as verified by
the strong binding
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CA 02548065 2006-06-O1
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signals observed after prolonged 1-2 day washes of the antibody bound to
immobilized IGF-I
receptor.
[122] EM164 antibody can be used for'immunoprecipitation of IGF-I receptor, as
demonstrated by incubation of detergent-solubilized lysate of the human breast
cancer MCF-
7 cells with EM164 antibody immobilized on protein G-agarose beads (Pierce
Chemical
Company). A Western blot of the EM164 antibody immunoprecipitate was detected
using a
rabbit polyclonal anti-IGF-I receptor beta chain (C-terminus) antibody (Santa
Cruz
Biotechnology) and a goat-anti-rabbit-IgG-antibody-horseradish peroxidase
conjugate,
followed by washes and enhanced chemiluminescence (ECL) detection. The Western
blot of
EM164 immunoprecipitate from MCF-7 cells exhibited bands corresponding to the
beta
chain of IGF-I receptor at about 95 kDa and the pro-IGF-I receptor at about
220 kDa. Similar
immunoprecipitations were carned out for other cell types to check species
specificity of the
binding of EM164 antibody, which also bound to IGF-I receptor from cos-7 cells
(African
green monkey), but did not bind to IGF-I receptor of 3T3 cells (mouse), CHO
cells (Chinese
hamster) or goat fibroblast cells (goat). The EM164 antibody did not detect
SDS-denatured
human IGF-I receptor in Western blots of lysates from MCF-7 cells, which
indicated that it
bound to a conformational epitope of native, non-denatured human IGF-I
receptor.
[123] The binding domain of EM164 antibody was further characterized using a
truncated
alpha chain construct, which comprised the cysteine rich domain flanked by L1
and L2
domains (residues 1-46~) fused with the 16-mer-C-terminus piece (residues 704-
719) and
which was terminated by a C-terminus epitope tag. This smaller IGF-I receptor,
which
lacked residues 469-703, has been reported to bind IGF-I, although less
tightly compared to
the native full-length IGF-I receptor (Molina, L. et al., 2000, FEBS Letters,
467, 226-230;
I~ristensen, C. et al., 1999, J. Biol. Chem., 274, 37251-37356). Thus, a
truncated IGF-I
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receptor alpha chain construct was prepared comprising residues 1-46~ fused to
the C-
terminus piece that is residues 704-719 and flanked by a C-terminus myc
epitope tag. A
stable cell line which expressed this construct, and which also expresses the
construct
transiently in human embryonic kidney 293T cells, was constructed. A strong
binding of
EM164 antibody to this truncated IGF-I receptor alpha chain construct was
observed. Of the
two antibodies tested, IR3 (Calbiochem) also bound to this truncated alpha
chain, but 1H7
antibody (Santa Cruz Biotechnology) did not bind, which indicated that the
epitope of
EM164 antibody was clearly distinct from that of 1H7 antibody.
C. Inhibition of binding of IGF-I to MCF-7 cells by EM164 antibody
[124] The binding of IGF-I to human breast cancer MCF-7 cells was inhibited by
EM164
antibody (Figure 3). MCF-7 cells were incubated with or without 5 p,g/mL EM164
antibody
for 2 h in serum-free medium, followed by incubation with 50 ng/mL
biotinylated IGF-I for
20 min at 37°C. The cells were then washed twice with serum-free medium
to remove
unbound biotin-IGF-I, and were then lysed in 50 mM HEPES, pH 7.4, containing
1% NP-40
and protease inhibitors. An Immulon-2HB ELISA plate was coated with a mouse
monoclonal anti-IGF-I receptor beta chain antibody and was used to capture the
IGF-I
receptor and bound biotin-IGF-I from the lysate. The binding of the coated
antibody to the
cytoplasmic C-terminal domain of the beta chain of IGF-I receptor did not
interfere with the
binding of biotin-IGF-I to the extracellular domain of IGF-I receptor. The
wells were
washed, incubated with streptavidin-horseradish peroxidase conjugate, washed
again, and
then detected using ABTS/H2O2 substrate. The inhibition of IGF-~ binding to
MCF-7 cells
by 5 ~g/mL EM164 antibody was essentially quantitative, and was almost
equivalent to that
of the ELISA background obtained using a control lacking biotin-IGF-I.
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[125] In addition to the assay described above for the inhibition of binding
of IGF-I to
MCF-7 cells by EM164 antibody, the following assay demonstrated that EM164
antibody
was highly effective at displacing bound IGF-I from MCF-7 cells, as desired
under
physiological conditions for an antagonistic anti-IGF-I receptor antibody to
displace the
bound endogenous physiological ligand (such as IGF-I or IGF-II). In this IGF-I
displacement
assay, MCF-7 cells grown in a 12-well plate were serum-starved and then
incubated with
biotinylated IGF-I (20-50 ng/mL) in serum-free medium at 37°C (or at
4°C) for 1 to 2 h. The
cells with bound biotinylated IGF-I were then treated with EM164 antibody or a
control
antibody (10-100 ~g/mL) at 37°C (or at 4°C) for 30 min to 4 h.
Cells were then washed with
PBS and lysed in lysis buffer containing 1 % NP-40 at 4°C. ELISA was
carried out as
described above to capture the IGF-I receptor from the lysate and then detect
the biotinylated
IGF-I bound to the receptor using streptavidin-horseradish peroxidase
conjugate. This
ELISA demonstrated that EM164 antibody was able to displace pre-bound
biotinylated IGF-I
from cells nearly completely (90% within 30 min and 100% within 4 h) at
37°C and by
about 50% in 2 h at 4°C. In another experiment, NCI-H838 lung cancer
cells were incubated
with biotin-IGF-I, then washed and incubated with EM164 antibody at 4°C
for 2 h, which
resulted in a 80% decrease in the bound biotin-IGF-I. Therefore, EM164
antibody was
highly effective at displacing pre-bound IGF-I from cancer cells, which would
be important
therapeutically for the antagonism of the IGF-I receptor by displacement of
the bound
endogenous physiological ligand.
[126] The incubation of MCF-7 cells with EM164 antibody at 4°C for 2 h
(or at 37°C for 30
min) did not result in a significant downregulation of the IGF-I receptor
based on Western
blot analysis using anti-IGF-I receptor beta chain antibody (Santa Cruz
Biotechnology; sc-
713), although a longer incubation with EM164 antibody at 37°C for 2 h
resulted in a 25%
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downregulation of the IGF-I receptor. Therefore, the inhibition of binding of
IGF-I and the
displacement of bound IGF-I by EM164 antibody at both 4°C and
37°C in these short-term
experiments may not be explained by the down-regulation of the receptor due to
the binding
of the EM164 antibody. The mechanism for the potent inhibition of the binding
of IGF-I to
IGF-I receptor and for the displacement of the pre-bound IGF-I by EM164
antibody is likely
to be competition for binding, either through sharing of the binding site or
through steric
occlusion or through allosteric effects.
D. Inhibition of IGF-I receptor mediated cell signaling by EM164 antibody
[127] Treatment of breast cancer MCF-7 cells and osteosarcoma SaOS-2 cells
with EM164
antibody almost completely inhibited intracellular IGF-I receptor signaling,
as shown by the
inhibition of IGF-I receptor autophosphorylation and by the inhibition of
phosphorylation of
its downstream effectors such as insulin receptor substrate-1 (IRS-1), Akt and
Erkl/2
(Figures 4-6).
[128] In Figure 4, the MCF-7 cells were grown in a 12-well plate in regular
medium for 3
days, and were then treated with 20 ~,g/mL EM164 antibody (or anti-B4 control
antibody) in
serum-free medium for 3 h, followed by stimulation with 50 ng/mL IGF-I for 20
min at 37°C.
The cells were then lysed in ice-cold lysis buffer containing protease and
phosphatase
inhibitors (50 mM HEPES buffer, pH 7.4, 1% NP-40, 1 mM sodium orthovanadate,
100 mM
sodium fluoride, 10 mM sodium pyrophosphate, 2.5 mM EDTA, 10 p,M leupeptin, 5
~M
pepstatin, 1 mM PMSF, 5 mM benzamidine, and 5 p.g/mL aprotinin). An ELISA
plate was
pre-coated with anti-IGF-I receptor beta chain C-terminus monoclonal antibody
TC 123 and
was incubated with the lysate samples for 5 h at ambient temperature to
capture IGF-I
receptor. The wells containing the captured IGF-I receptor were then washed
and incubated
with biotinylated anti-phosphotyrosine antibody (PY20; 0.25 ~,g/mL; BD
Transduction
CA 02548065 2006-06-O1
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Laboratories) for 30 min, followed by washes and incubation with streptavidin-
horseradish
peroxidase conjugate (0.8 ~,g/mL) for 30 min. The wells were washed and
detected with
ABTS/HZOZ substrate. Use of a control anti-B4 antibody showed no inhibition of
the IGF-I
stimulated autophosphorylation of IGF-I receptor. In contrast, a complete
inhibition of the
IGF-I stimulated autophosphorylation of IGF-I receptor was obtained upon
treatment with
EM164 antibody (Figure 4).
[129] To demonstrate inhibition of phosphorylation of insulin receptor
substrate-1 (IRS-1),
an ELISA using immobilized anti-IRS-1 antibody to capture IRS-1 from lysates
was used,
followed by measurement of the associated p85 subunit of phosphatidylinositol-
3-kinase (PI-
3-kinase) that binds to the phosphorylated IRS-1 (Jackson, J. G. et al., 1998,
J. Biol. Chem.,
273, 9994-10003). In Figure 5, MCF-7 cells were treated with 5 ~.g/mL antibody
(EM164 or
IR3) in serum-free medium for 2 h, followed by stimulation with 50 ng/mL IGF-I
for 10 min
at 37°C. Anti-IRS-1 antibody (rabbit polyclonal; Upstate Biotechnology)
was indirectly
captured by incubation with coated goat-anti-rabbit-IgG antibody on an ELISA
plate, which
was then used to capture IRS-1 from the cell lysate samples by overnight
incubation at 4°C.
The wells were then incubated with mouse monoclonal anti-p85-PI-3-kinase
antibody
(Upstate Biotechnology) for 4 h, followed by treatment with goat-anti-mouse-
IgG-antibody-
HRP conjugate for 30 min. The wells were then washed and detected using ABTS/
Ha02
substrate (Figure 5). As shown in Figure 5, EM164 antibody was more effective
at inhibiting
the IGF-I-stimulated IRS-1 phosphorylation than was IR3 antibody, and EM164
antibody did
not show any agonistic activity on IRS-1 phosphorylation when incubated with
cells in the
absence of IGF-I.
[130] The activation of other downstream effectors, such as Akt and Erk1/2,
was also
inhibited in a dose-dependent manner by EM164 antibody in SaOS-2 cells (Figure
6) and in
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MCF-7 cells, as was shown using Western blots of lysates and phosphorylation-
specific
antibodies (rabbit polyclonal anti-phospho-Ser4~3 Akt polyclonal and anti-
phospho-ERKl/2
antibodies; Cell Signaling Technology). A pan-ERK antibody demonstrated equal
protein
loads in all lanes (Figure 6). Treatment of SaOS-2 cells with EM164 antibody
did not inhibit
the EGF-stimulated phosphorylation of Erkl/2, thus demonstrating the
specificity of
inhibition of IGF-I receptor signaling pathway by EM164 antibody.
E. Inhibition of IGF-I-, IGF-II- and serum-stimulated growth and survival of
human tumor cells by EM164 antibody
[131] Several human tumor cell lines were tested in serum-free conditions for
their growth
and survival response to IGF-I. These cell lines were treated with EM164
antibody in the
presence of IGF-I, IGF-II, or serum, and their growth and survival responses
were measured
using an MTT assay after 2-4 days. Approximately 1500 cells were plated in a
96-well plate
in regular medium with serum, which was replaced with serum-free medium the
following
day (either serum-free RPMI medium supplemented with transferrin and BSA, or
phenol-red
free medium as specified by Dufourny, B. et al., 1997, J. Biol. 'hem., 272,
31163-31171).
After one day of growth in serum-free medium, the cells were incubated with
about 75 ~L of
~g/mL antibody for 30 min.-3 h, followed by the addition of 25 ~.L of IGF-I
(or IGF-II or
serum) solution to obtain a final concentration of 10 ng/mL IGF-I, or 20 ng/mL
IGF-lI, or
0.04-10% serum. In some experiments, the cells were stimulated first with IGF-
I for 15 min
before the addition of EM164 antibody, or both IGF-I and EM164 antibody were
added
together. The cells were then allowed to grow for another 2-3 days. A solution
of MTT (3-
(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; 25 p,L of a 5
mg/mL solution in
PBS) was then added and the cells were returned to the incubator for 2-3 h.
The medium was
then removed and replaced by 100 ~.L DMSO, mixed, and the absorbance of the
plate was
measured at 545 nm. Several human tumor cell lines showed a growth and
survival response
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upon addition of IGF-I or IGF-II or serum that was significantly inhibited by
EM164
antibody, irrespective of whether the antibody was added before IGF-I, or if
IGF-I was added
before the antibody, or if both IGF-I and antibody were added together (Table
1).
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TABLE 1. Inhibition of IGF-I-stimulated growth and survival of tumor cells by
EM164 antibody
Tumor Cell Type Fold growth response% Inhibition Inhibition by
to IGF-I (MTT by EM164 antibody
assay: EM164 antibodyof
ratio for IGF-I of IGF-I- Growth/survival
treated stimulated of
vs untreated growth cells in 1.25-10
cells in in serum-free %
serum-free medium)medium serumb
MCF-7 reast 1.7-2.8 100 % 85 %
HT-3 cervical 2 70-90 % ND
Colo 205 colon 2.3 50 % Yes
HT-29 1.5 60 % Yes
NCI-H838 un 3 100 % 85-90 %
Calu-6 1.6-1.8 85 % Yes
SK-LU-1 1.4 100% No
NCI-H596 1.4 100 % Weald
A 549 1.2 80 % ND
A 375 melanoma 1.6 90 % No
SK-Mel-37 1.4 85 % ND
RD rhabdom ocarcoma1.7 85-100 % Yes
SaOS-2 osteosarcoma2.5 100 % Yes
A 431 a idermoid 2.2 85 % Yes
SK-N-SH neuroblastoma2 85 % 30-50 %
M'1-1' assay of 3- to 4-day growth/survival of cells in response to 10 ng/mL
IGF-I in serum-free
medium containing 5-10 ~,g/mL EM164 antibody.
b Inhibition of growth of cells in 1.25-10 % serum in the presence of 5-10
~,g/mL EM164 antibody by
MTT assay or colony formation assay based on comparison with the control (with
serum but without
antibody); the extent of inhibition was quantitatively measured for MCF-7, NCI-
H838 and SK-N-SH
cells based on controls (without serum but with antibody, and with serum but
without antibody) to
account for autocrine/paracrine IGF-stimulation by cells. ND indicates no data
or poor data due to
staining difficulties.
[132] The EM164 antibody strongly inhibited IGF-I-or serum-stimulated growth
and
survival of breast cancer MCF-7 cells (Figures 7 and 8). In a separate
experiment, the
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EM164 antibody strongly inhibited IGF-II-stimulated growth and survival of MCF-
7 cells.
Previous reports using commercially available antibodies such as IR3 antibody
showed only
weak inhibition of serum-stimulated growth and survival of MCF-7 cells, as
confirmed in
Figure 7 for the IR3 and 1H7 antibodies (Cullen, K. J. et al., 1990, Cahcer
Res., 50, 48-53).
In contrast, EM164 antibody was a potent inhibitor of the serum- or IGF-
stimulated growth
of MCF-7 cells. As shown in Figure 8, EM164 antibody was equally effective in
inhibiting
the growth and survival of MCF-7 cells over a wide range of serum
concentrations (0.04-10%
serum).
[133] The growth inhibition of MCF-7 cells by EM164 antibody was measured by
counting
cells. Thus, in a 12-well plate, about 7500 cells were plated in RPMI medium
with 10%
FBS, in the presence or absence of 10 ~,g/mL EM164 antibody. After 5 days of
growth, the
cell count for the untreated control sample was 20.5 a~ 104 cells, in contrast
to a cell count of
only 1.7 x 104 cells for the EM164 antibody-treated sample. Treatment with the
EM164
antibody inhibited the growth of MCF-7 cells by about 12-fold in 5 days. This
inhibition by
EM164 antibody was significantly greater than was a reported 2.5-fold
inhibition using IR3
antibody in a 6-day assay for MCF-7 cells (Rohlik, Q. T, et a1.,1987, Biochem.
Biophys. Res.
Commute., 149, 276-281).
[134] The IGF-I- and serum-stimulated growth and survival of a non-small cell
lung cancer
line NCI-H838 were also strongly inhibited by EM164 antibody, compared to a
control anti-
B4 antibody (Figure 9). Treatment with EM164 antibody in serum-free medium
produced a
smaller signal than the untreated sample for both NCI-H838 and MCF-7 cells,
presumably
because EM 164 antibody also inhibited the autocrine and paracrine IGF-I and
IGF-II
stimulation of these cells (Figures 7 and 9). The colony size of HT29 colon
cancer cells was
also greatly reduced upon treatment with EM164 antibody.
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[135 EM164 antibody is therefore unique among all known anti-IGF-I receptor
antibodies
in its effectiveness to inhibit the serum-stimulated growth of tumor cells
such as MCF-7 cells
and NCI-H838 cells by greater than 80%.
[136] The EM164 antibody caused growth arrest of cells in GO/G1 phase of cell
cycle and
abrogated the mitogenic effect of IGF-I. For cell cycle analysis, MCF-7 cells
were treated
with IGF-I (20 ng/mL) in the presence or absence of EM164 (20 ~ug/mL) for 1
day and then
analyzed by propidium iodide staining and flow cytometry. As shown in Figure
25, the
cycling of cells in response to IGF-I stimulation in the absence of EM164
(with 41% of the
cells in the S phase and 50% in the GO/G1 phase) was suppressed in EM164-
treated cells
(with only 9% in the S phase and 77% of the cells in the GO/G1 phase).
[137] In addition to its inhibition of cell proliferation, EM164 antibody
treatment resulted in
apoptosis of cells. For measurement of apoptosis, cleavage of the cytokeratin
CK18 protein
by caspase was measured in NCI-H838 lung cancer cells incubated with IGF-I or
serum in
the presence or absence of EM164 for 1 day (Figure 26). In the absence of
EM164, the
addition of IGF-I or serum resulted in a lower caspase-cleaved CK18 signal
compared to the
no IGF-I control, indicating that IGF-I and serum prevent the activation of
caspase.
Treatment with EM164 suppressed the anti-apoptotic effects of IGF-I and serum,
as indicated
by the greater cleaved CK18 levels obtained in the presence of EM164 than in
the absence of
EM164 (Figure 26).
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F. Synergistic inhibition by EM164 antibody of growth and survival of human
tumor cells in combinations with other cytotoxic and cytostatic agents
[138] The combined administration of EM164 antibody with taxol was
significantly more
inhibitory to the growth and survival of non-small cell lung cancer Calu6
cells than was taxol
alone. Similarly, the combination of EM164 antibody with camptothecin was
significantly
more inhibitory than camptothecin alone toward the growth and survival of
colon cancer
HT29 cells. Because EM164 antibody alone was not expected to be as toxic to
cells as
organic chemotoxic drugs, the synergism between the predominantly cytostatic
effect of
EM164 antibody and the cytotoxic effect of the chemotoxic drug may be highly
efficacious in
combination cancer therapies in clinical settings.
[139] The combined effect of EM164 antibody with an anti-EGF receptor antibody
(KS77)
was significantly more inhibitory than either EM164 antibody or KS77 antibody
alone on the
growth and survival of several tumor cell lines such as HT-3 cells, RD cells,
MCF-7 cells,
and A431 cells. Therefore, the synergistic effect of combining neutralizing
antibodies for
two growth factor receptors such as IGF-I receptor and EGF receptor may also
be useful in
clinical cancer treatment.
[140] Based on the efficacy of EM164 antibody as a single agent in inhibiting
the
proliferation and survival of diverse human cancer cell lines as shown in
Table 1, additional
efficacy studies were carried out using combinations of EM164 antibody with
other anti-
cancer therapeutic agents. In these studies on diverse cancer cell lines, the
combined
treatment of EM164 antibody and other anti-cancer therapeutic agents resulted
in an even
greater anti-cancer efficacy than with either EM164 or the other therapeutic
agent alone.
These combinations of EM164 with other therapeutic agents are therefore highly
effective in
inhibiting the proliferation and survival of cancer cells. The therapeutic
agents that can be
combined with EM164 for improved anti-cancer efficacy include diverse agents
used in
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oncology practice (RefeYe~ce: Cancer, Principles & Practice of Oncology,
DeVita, V. T.,
Hellman, S., Rosenberg, S. A., 6th edition, Lippincott-Raven, Philadelphia,
2001), such as
docetaxel, paclitaxel, doxorubicin, epirubicin, cyclophosphamide, trastuzumab
(Herceptin),
capecitabine, tamoxifen, toremifene, letrozole, anastrozole, fulvestrant,
exemestane,
goserelin, oxaliplatin, carboplatin, cisplatin, dexamethasone, amide,
bevacizumab (Avastin),
5-ffuorouracil, leucovorin, levamisole, irinotecan, etoposide, topotecan,
gemcitabine,
vinorelbine, estramustine, mitoxantrone, abarelix, zoledronate, streptozocin,
rituximab
(Rituxan), idarubicin, busulfan, chlorambucil, fludarabine, imatinib,
cytarabine, ibritumomab
(Zevalin), tositumomab (Bexxar), interferon alpha-2b, melphalam, bortezomib
(Velcade),
altretamine, asparaginase, gefitinib (Iressa), erlonitib (Tarceva), anti-EGF
receptor antibody
(Cetuximab, Abx-EGF), epothilones, and conjugates of cytotoxic drugs and
antibodies
against cell-surface receptors.
[141] For these combination therapies, EM164 is combined with one or more anti-
cancer
agents of diverse mechanisms of action such as alkylating agents, platinum
agents, hormonal
therapies, antimetabolites, topoisomerase inhibitors, antimicrotubule agents,
differentiation
agents, antiangiogenic or antivascularization therapies, radiation therapy,
agonists and
antagonists of leuteinizing hormone releasing hormone (LHRH) or gonadotropin-
releasing
hormone (GnRH), inhibitory antibodies or small molecule inhibitors against
cell-surface
receptors, and other chemotherapeutic agents (Reference: Cancer, Principles &
Practice of
Oncology, DeVita, V. T., Hellman, S., Rosenberg, S. A., 6th edition,
Lippincott-Raven,
Philadelphia, 2001). In one example, the combination of an LHRH antagonist
amide (0.1 to
micromolar) and EM164 antibody (0.1 to 10 nanomolar) inhibited the
proliferation of
MCF-7 breast cancer cells significantly more than that with either EM164 or
amide alone. In
an example of a combination therapy with a platinum agent, the combined
treatment with
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EM164 antibody (10 microgram/ml) and cisplatin (0.1-60 microgram/ml) resulted
in a greater
inhibition of the proliferation and survival of MCF-7 breast cancer cells in
comparison to the
inhibition by either EM164 antibody or cisplatin alone.
[142] These combinations of EM164 antibody with other therapeutic agents are
effective
against several types of cancers including breast, lung, colon, prostate,
pancreatic, cervical,
ovarian, melanoma, multiple myeloma, neuroblastoma, rhabdomyosarcoma and
osteosarcoma. The EM164 antibody and the therapeutic agent can be administered
for cancer
therapy either simultaneously or in sequence.
[143] Conjugates of EM164 antibody with cytotoxic drugs are also valuable in
targeted
delivery of the cytotoxic drugs to the tumors overexpressing IGF-I receptor.
Conjugates of
EM164 antibody with radiolabels or other labels can be used in the treatment
and imaging of
tumors that overexpress IGF-I receptor.
G. Effect of EM164 treatment, as a single agent or in combination with anti-
cancer
agents, in human cancer xenografts in immunodeficient mice
[144] Human non-small cell lung cancer Calu-6 xenografts were established in
immunodeficient mice by subcutaneous injections of 1x10 Calu-6 cells. As shown
in Figure
10, these mice containing established 100 rnm3 Calu-6 xenografts were treated
with EM 164
antibody alone (6 injections of O.S mg/mouse, i. v., two per week) or with
taxol alone (five
injections of taxol, i.p. every two days; 15 mg/kg), or with a combination of
taxol and EM164
antibody treatments, or PBS alone (200 ~,L/mouse, 6 injections, two per week,
i.v.) using five
mice per treatment group. The growth of tumors was significantly slowed by
EM164
antibody treatment compared to a PBS control. No toa~icity of EM164 antibody
was
observed, based on measurements of the weights of the mice. Although taxol
treatment alone
was effective until day 14, the tumor then started to grow back. However, the
growth of the
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tumor was delayed significantly in the group that was treated by a combination
of taxol and
EM164 antibody, compared to the group that was treated with taxol alone.
[145] Human pancreatic cancer xenografts were established in 5 week-old,
female
SCm/ICR mice (Taconic) by subcutaneous injections of 10' BxPC-3 cells in PBS
(day 0).
The mice bearing established tumors of 80 mm3 were then treated with EM164
alone (13
injections of 0.8 mg/mouse, i.v., lateral tail vein, on days 12,16, 19, 23,
26, 29, 36, 43, 50,
54, 58, 61 and 64), with gemcitabine alone (two injections of 150 mg/kg/mouse,
i.p., on days
12 and 19), with a combination of gemcitabine and EM164 following the above
schedules,
PBS alone, and a control antibody alone (following the same schedule as EM164)
using five
mice in each of the five treatment groups. As shown in Figure 27, treatment
with EM164
alone, or in combination with gemcitabine, resulted initially in total
regression of tumor
xenografts in 4 of 5 animals in the EM164 treatment group and in all 5 animals
in the
combination treatment group. Measurable tumor regrowth was only seen in more
than one
animal on day 43 in the EM164 group and on day 68 in the combination treatment
group,
resulting in significantly smaller mean tumor volumes on day 74 in comparison
with the
control treatments (P = 0.029 and 0.002, respectively; two-tailed T test;
Figure 27). In
another study, EM164 antibody treatment (alone or in combination with an anti-
EGF receptor
antibody; intraperitoneal injections) inhibited the growth of established BxPC-
3 xenografts in
mice.
[146] The murine EM164 and the humanized EM164 antibodies showed equivalent
inhibition of the growth of established BxPC-3 xenografts in mice, thus
demonstrating that
the potency of the humanized EM164 is equivalent to that of the murine EM164
i~ vivo. In a
comparison of different modes of administration of EM164 antibody, both
intraperitoneal and
intravenous administrations of EM164 antibody showed equivalent inhibition of
the growth
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of established BxPC-3 xenografts in mice. In another xenograft study,
treatment with EM164
antibody showed significant growth delay of established A-673 human
rhabdornyosarcoma/Ewing's sarcoma xenografts in mice.
H. Cloning and sequencing of the light and heavy chains of EM164 antibody
[147] Total RNA was purified from EM164 hybridoma cells. Reverse transcriptase
reactions were performed using 4-5 ~g total RNA and either oligo dT or random
hexamer
primers.
[148] PCR reactions were performed using a RACE method described in Co et al.
(J.
Immunol., 148, 1149-1154 (1992)) and using degenerate primers as described in
Wang et al.,
(J. Immunol. Methods, 233, 167-177 (2000)). The RACE PCR method required an
intermediate step to add a poly G tail on the 3' ends of the first strand
cDNAs. RT reactions
were purified with Qianeasy (Qiagen) columns and eluted in 50 ~l 1 X NEB
buffer 4. A dG
tailing reaction was performed on the eluate with 0.25, mM CoCl2,1 mM dGTP,
and 5 units
terminal transferase (NEB), in 1 X NEB buffer 4. The mixture was incubated at
37°C for 30
minutes and then 1/5 of the reaction (10 ~,1) was added directly to a PCR
reaction to serve as
the template DNA.
[149] The RACE and degenerate PCR reactions were identical except for
differences in
primers and template. The terminal transferase reaction was used directly for
the RACE PCR
template, while the RT reaction mix was used directly for degenerate PCR
reactions.
[150] In both RACE and degenerate PCR reactions the same 3' light chain
primer:
HindKI. - tatagagctcaagcttggatggtgggaagatggatacagttggtgc (SEQ ID NO: 14)
and 3' heavy chain primer:
Bgl2IgG1- ggaagatctatagacagatgggggtgtcgttttggc (SEQ ID NO: 15)
were used.
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[151] In the RACE PCR, one poly C 5' primer was used for both the heavy and
light chain:
EcoPolyC - TATATCTAGAATTCCCCCCCCCCCCCCCCC (SEQ ID NO: 16),
while the degenerate 5' end PCR primers were:
SaclMK - GGGAGCTCGAYATTGTGMTSACMCARWCTMCA (SEQ ID NO: 17) for the
light chain, and an equal mix of:
EcoRIMHI - CTTCCGGAATTCSARGTNMAGCTGSAGSAGTC (SEQ ID NO: 18) and
EcoRIMH2 - CTTCCGGAATTCSARGTNMAGCTGSAGSAGTCWGG (SEQ ID NO: 19)
for the heavy chain.
[152] In the above primer sequences, mixed bases are defined as follows:
H=A+T+C,
S=g+C, Y=C+T, K= G+T, M=A+C, R=A+g, W=A+T, V = A+C+G.
[153] The PCR reactions were performed using the following program: 1) 94
°C 3 min, 2)
94 °C 15 sec, 3) 45 °C 1 min, 4) 72 °C 2 min, 5) cycle
back to step #2 29 times, 6) finish with
a final extension step at 72 °C for 10 min.
[154] The PCR products were cloned into pBluescript II SK+ (Stratagene) using
restriction
enzymes created by the PCR primers.
[155] Several individual light and heavy chain clones were sequenced by
conventional
means to identify and avoid possible polymerise generated sequence errors
(Figures 12 and
13). Using Chothia canonical classification definitions, the three light chain
and heavy chain
GDRs were identified (Figures 12-14).
[156] A search of the NCBI IgBlast database indicated that the anti-IGF-I
receptor antibody
light chain variable region probably derived from the mouse IgVk Cr1 germline
gene while
the heavy chain variable region probably derived from the IgVh J558.c germline
gene (Figure
15).
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[157] Protein sequencing of murine EM164 antibody was performed to confirm the
sequences shown in Figures 12 and 13. The heavy and light chain protein bands
of purified
EM164 antibody were transferred to a PVDF membrane from a gel (SDS-PAGE,
reducing
conditions), excised from the PVDF membrane and analyzed by protein
sequencing. The N-
terminal sequence of the light chain was determined by Edman sequencing to be:
DVLMTQTPLS (SEQ ID N0:20), which matches the N-terminal sequence of the cloned
light chain gene obtained from the EM164 hybridoma.
[158] The N-terminus of the heavy chain was found to be blocked for Edman
protein
sequencing. A tryptic digest peptide fragment of the heavy chain of mass
1129.5 (M+H+,
monoisotopic) was fragmented via post-source decay (PSD) and its sequence was
determined
to be GRPDYYGSSK (SEQ ID N0:21). Another tryptic digest peptide fragment of
the
heavy chain of mass 2664.2 (M+H+, monoisotopic) was also fragmented via post-
source
decay (PSD) and its sequence was identified as: SSSTAYMQLSSLTSEDSAVYYFAR (SEQ
ID N0:22). Both of these sequences match perfectly those of CDR3 and framework
3 (FR3)
of the cloned heavy chain gene obtained from the EM164 hybridoma.
I. Recombinant expression of EM164 antibody
[159] The light and heavy chain paired sequences were cloned into a single
mammalian
expression vector (Figure 16). The PCR primers for the human variable
sequences created
restriction sites that allowed the human signal sequence to be attached while
in the
pBluescriptII cloning vector, and the variable sequences were cloned into the
mammalian
expression plasmid using EcoRI and BsiWI or HindIII and ApaI sites for the
light chain or
heavy chain, respectively (Figure 16). The light chain variable sequences were
cloned in-
frame onto the human IgK constant region and the heavy chain variable
sequences were
cloned into the human Iggammal constant region sequence. In the final
expression plasmids,
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human CMV promoters drove the expression of both the light and heavy chain
cDNA
sequences. Expression and purification of the recombinant mouse EM164 antibody
proceeded according to methods that are well-known in the art.
EXAMPLE 2: Humanized versions of EM164 antibody
[160] Resurfacing of the EM164 antibody to provide humanized versions suitable
as
therapeutic or diagnostic agents generally proceeds according to the
principles and methods
disclosed in LJ.S. Patent 5,639,641, and as follows.
A. Surface prediction
[161] The solvent accessibility of the variable region residues for a set of
antibodies with
solved structures was used to predict the surface residues for the murine anti-
IGF-I receptor
antibody (EM164) variable region. The amino acid solvent accessibility for a
set of 127
unique antibody structure files (Table 2) were calculated with the MC software
package
(Pedersen et al., 1994, J. Mol. Biol., 235, 959-973). The ten most similar
light chain and
heavy chain amino acid sequences from this set of 127 structures were
determined by
sequence alignment. The average solvent accessibility for each variable region
residue was
calculated, and positions with greater than a 30% average accessibility were
considered to be
surface residues. Positions with average accessibilities of between 25% and
35% were
further examined by calculating the individual residue accessibility for only
those structures
with two identical flanking residues.
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TABLE 2 -127 antibody structures from the Brookhaven database used to predict
the
surface of anti-IGF-I-receptor antibody (EM164)
127 Brookhaven structure files used for surface predictions
2rcs 3hfl 3hfm 1 1 1 43c9 4fab 6fab 7fab
aif air bbj
2gfb 2h1 2hfl 1 1 1 2hrp 2jel 2mcp 2pcp
p a6t axt bog
1yuh 2bfv 2cgr 8fab 1ae6 lbvl 2dbl 2f19 2fb4 2fbj
1sm3 1tet 1vfa glb2 1a4j 1cly 1vge 1yec 1yed 1yee
1 1 1 1 1 1 1 1 psk 1 1
nsn opg osp aj7 ay1 clz plg rmf sbs
1 1 1 1 1 1 1 1 nma 1 1
ncd nfd ngp acy afv cbv nld nmb nqb
1 1 1 15c8 1 1 1 1 mpa 1 1
mcp mfb mim a5f axs mlb nbv ncb
1 1 1 1 1 1 1 1 kir 1 1
jrh kb5 kel apt b2w adq kip Ive mam
1 1 1 1 1 1 1 1 ikf 1 1
igi igm igt ado baf cfv igy jel jhl
1 1 1 1 1 1 1 1 ibg 1 1
gpo hil hyx a0q bjm clo iai igc igf
1fpt 1frg 1fvc laqk 1bln 1d5b 1gaf 1ggi 1ghf 1gig
1fai 1fbi 1fdl 1ad9 1bbd 1f58 lfgv lfig 1flr 1for
1 1 1 1 1 1 1 dvf
dbl dfb a31 bfo eap dsf
B. Molecular modeling:
[162] A molecular model of murine EM164 was generated using the Oxford
Molecular
software package AbM. The antibody framework was built from structure files
for the
antibodies with the most similar amino acid sequences, which were 2je1 for the
light chain
and lnqb for the heavy chain. The non-canonical CDRs were built by searching a
C-a
structure database containing non-redundant solved structures. Residues that
lie within 5 6
of a CDR were determined.
C. Human Ab selection
[163] The surface positions of murine EM164 were compared to the corresponding
positions in human antibody sequences in the Kabat database (Johnson, G. and
Wu, T. T.
(2001) Nucleic Acids Research, 29: 205-206). The antibody database management
software
SR (Searle 1998) was used to extract and align the antibody surface residues
from natural
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heavy and light chain human antibody pairs. The human antibody surface with
the most
identical surface residues, with special consideration given to positions that
come within 5 6
of a CDR, was chosen to replace the murine anti-IGF-I receptor antibody
surface residues.
D. PCR mutagenesis
[164] PCR mutagenesis was performed on the murine EM164 cDNA clone (above) to
build
the resurfaced, human EM164 (herein huEM164). Primer sets were designed to
make the 8
amino acid changes required for all tested versions of huEM164, and additional
primers were
designed to alternatively make the two 5 6 residue changes (Table 3). PCR
reactions were
performed with the following program: 1) 94 °C 1 min, 2) 94 °C
15 sec, 3) 55 °C 1 min, 4)
72 °C 1 min, 5) cycle back to step #2 29 times, 6) finish with a final
ea~tension step at 72 °C
for 4 min. The PCR products were digested with their corresponding restriction
enzymes and
were cloned into the pBluescript cloning vectors as described above. Clones
were sequenced
to confirm the desired amino acid changes. ,
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TABLE 3 - PCR primers used to build 4 humanized EM164 antibodies
Primer Sequence SEQ ID
NO:
Em164hcw CAGGTGTACACTCCCAGGTCCAACTGGTGCAGTCTGGGG 23
' CTGAAGTGGTGAAGCCTG
Em164hc CAATCAGAAGTTCCAGGGGAAGGCCACAC 24
o11
Em164hc CCTTCCCCTGGAACITCTGATTGTAGTTAGTACG 25
o12
Em1641cv3 CAGGTGTACACTCCGATGTTGTGATGACCCAAACTCC 26
Em1641c13 CAGGTGTACACTCCGATGTTTTGATGACCCAAACTCC 27
Em1641c GACTAGATCTGCAAGAGATGGAGGGTGGATCTCCAAGAC 28
18
Em1641cb TTGCAGATCTAGTCAGAGCATAGTACATAGTAATG 29
12
Em164x45 GAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAGGCTC 30
CTGATCrAC
Em164a67o11GTGGCAGTGGAGCAGGGACAGATTTCAC 31
Em164a67o12GAAATCTGTCCCTGCTCCACTGCCACTG 32
E. Variable region surface residues
[165] The antibody resurfacing techniques described by Pedersen et al. (3.
Mol. Biol., 235,
959-973,1994) and Roguska et al. (Protein Eng., 9, 895-904,1996) begin by
predicting the
surface residues of the murine antibody variable sequences. A surface residue
is defined as
an amino acid that has at least 30% of its total surface area accessible to a
water molecule.
[166] The 10 most homologous antibodies in the set of 127 antibody structure
files were
identified (Figures 17 and 18). The solvent accessibility for each Kabat
position was
averaged for these aligned sequences and the distribution of the relative
accessibilities for
each residue were as shown in Figure 19. Both the light and heavy chain have
26 residues
with average relative accessibilities of at least 30% (Figure 19): these
residues were therefore
the predicted surface residues for EM164. Several residues had average
accessibilities of
between 25% and 35%, and these were further examined by averaging only the
antibodies
with two identical residues flanking either side of the residue (Tables 4 and
5). After this
additional analysis, the original set of surface residues that was identified
above remained
unchanged.
57
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TABLE 4 - Surface residues and average accessibility (ave. acc.) for the 1
fight and heavy
chain variable sequences of EM164 antibody
EM164
Surface
Residues
Li ht Hea
Chain Chain
EM164 Kabat Ave. Acc.EM164 Kabat Ave.
# # Acc.
D 1 45.89 Q 1 58.19
L 3 41.53 Q 3 34.08
T 7 31.40 Q 5 34.36
L 9 50.08 A 9 38.01
L 15 35.45 L 11 47.72
Q 18 39.79 K 13 46.51
R 24 34.36 P 14 31.49
S 26 32.63 G 15 31.42
Q 27 34.35 K 19 34.41
N 28 6.38 K 23 31.23
3
P 40 _ T 28 36.24
- 43.05
-. _
G 41 46.56 P 41 _
44.01
Q 42 34.92 G 42 42.62
~5 30.58 Q 43 46.85
-. -
52 30.40 E __ 31 46.68
~
S 56 41.46 K 62 44.87
57 42.41 K _ _ _ 64 38.92
-- _
D 60 45.96 R ' 65 40.06
S 67 38.20 K 73 35.92
R 77 42.61 S 74 48.91
E 81 38.46 S 82B 32.72
V 95E 34.83 S 84 35.21
K 103 31.10 E 85 39.62
K 107 36.94 D 98 36.00
R 108 60.13 A 106 37.65
A 109 53.65 S 113 43.42
58
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TABLE 5
Borderline
Surface
Residues
Li ht Hea
Chain Chain
EM164 Kabat Ave. Acc. EM164 Kabat Ave. Acc.
# #
T 5 28.68 Q 3 31.62
T 7 30.24 Q 5 36.07
P 12 26.59 P 14 29.88
G 16 25.20 G 15 30.87
D 17 25.73 S 17 25.64
S 20 25.37 K 19 35.06
R 24 36.73 K 23 31.48
S 26 31.00 G 26 30.53
Q 27 32.29 S 31 27.12
S 27A 29.78 R 56 NA
V 27C 29.05 T 68 27.71
V 29 NA T 70 24.65
Q 42 34.92 S 75 18.80
K 45 32.24 S 82B 32.87
S 52 30.02 P 97 NA
R 54 29.50 Y 99 NA
D 70 26.03 V 103 NA
R 74 NA T 111 25.95
E 79 26.64
A 80 29.61
V 95E 42.12
G 100 29.82
K 103 31.10
I_E __1_05 - - 25.78 I I ( -
_I I
Residues which had average accessibilities between 25% and 35% were further
analyzed by averaging
a subset of antibodies that had two identical residues flanking either side of
the residue in question.
These borderline surface positions and their new average accessibilities are
given. The NA's refer to
residues with no identical flanking residues in the 10 most similar
antibodies.
59
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F. Molecular modeling to determine which residues fall within 5 ~ of a CDR
[167] The molecular model above, generated with the AbM software package, was
analyzed
to determine which EM164 surface residues were within 5 6 of a CDR. In order
to resurface
the murine EM164 antibody, all surface residues outside of a CDR should be
changed to the
human counterpart, but residues within 5 6 of a CDR are treated with special
care because
they may also contribute to antigen specificity. Therefore, these latter
residues must be
identified and carefully considered throughout the humanization process. The
CDR
definitions used for resurfacing combine the AbM definition for heavy chain
CDR2 and
Kabat definitions for the remaining 5 CDRs (Figure 14). Table 6 shows the
residues that
were within 5 6 of any CDR residue in either the light or heavy chain sequence
of the EM164
model.
TABLE 6 EM164 antibody framework surface residues within 5 6 of a CDR
EM164 Surface Residues
within 5~ of a CDR
Li ht chainHeav chain
D1 T28
L3 K73
T7 S74
P40
Q42
K45
G57
D60
X81
G. Identification of the most homologous human surfaces
[168] Candidate human antibody surfaces for resurfacing EM164 were identified
within the
Kabat antibody sequence database using SR software, which provided for the
searching of
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only specified residue positions against the antibody database. To preserve
the natural
pairings, surface residues of both the light and heavy chains were compared
together. The
most homologous human surfaces from the Kabat database were aligned in rank
order of
sequence identity. The top 5 surfaces are given in Table 7. These surfaces
were then
compared to identify which of them would require the least changes within 5 6
of a CDR.
The Leukemic B-cell antibody, CLL 1.69, required the least number of surface
residue
changes (10 in total) and only two of these residues were within 5 6 of a CDR.
[169] The full length variable region sequence for EM164 was also aligned
against the
Kabat human antibody database and CLL 1.69 was again identified as the most
similar
human variable region sequence. Together, these sequence comparisons
identified the human
Leukemic B-cell antibody CLL 1.69 as the preferred choice as a human surface
for EM164.
TABLE 7 - The top 5 human sequences extracted from the Kabat database
5 gous
Most Human
Homolo Antibody
Surfaces
Antibody- Light SEQ >D
Chain NO:
MuEM164 D T L LQ P G Q K S _EKK R 33
L G D R A
CLL1.69 D T L LP P G Q R A E KK R 34
V G D R -
MSL5 D S L IP P G Q K S D KK R 3S
Q G D R A
CDP571 D S S VR P G Q K S D KK R 36
M G S S -
LC3aPB E S G PR P G Q R S E KK R 37
V G D R -
SSbPB E S G PR P G Q R S E KK R 3$
V G D R -
Antibody Heavy SEQ m NO:
Chain
MuEMl64 Q QQ K P GK K T P G K R KS S E S 39
A Q E K S A
L
CLL1.69 Q QV K P GK K T P G K G KS S E S 4Q
A Q Q Q S Q
V
MSL5 Q QQ K P GK K T P G D G TS N E S 41
P K D K N Q
L
CDP571 Q QV K P GK K T P G K G KS S E S 42
A Q Q K S Q
V
LC3aPB - QV K P GK K T P G K G KS S E S 43
A Q Q Q S Q
V
SSbPB - QV K P GK K T P G K G ES S E S 44
A Q Q Q S Q
V
Abignments were generated by SR (Pedersen 1993). The EM164 surface residues
that come within
of a CDR are underlined.
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H. Construction of humanized EM164 genes
[170] The ten surface residue changes for EM164 (Table 7) were made using PCR
mutagenesis techniques as described above. Because eight of the surface
residues for CLL
1.69 were not within 5 6 of a CDR, these residues were changed from marine to
human in all
versions of humanized EM164 (Tables 8 and 9). The two light chain surface
residues that
were within 5 6 of a CDR (Kabat positions 3 and 45) were either changed to
human or were
retained as marine. Together, these options generate the four humanized
versions of EM164
that were constructed (Figures 22 and 23).
[171] Of the four humanized versions, version 1.0 has all 10 human surface
residues. The
most conservative version with respect to changes in the vicinity of the CDR
is version 1.1,
which retained both of the marine surface residues that were within 5 6 of a
CDR. All four
humanized EM164 antibody genes were cloned into an antibody expression plasmid
(Figure
16) for use in transient and stable transfections.
TABLE 8 - Residue changes for versions 1.0-1.3 of humanized EM164 antibody
Changes in all versions
ht Chain: muQl8 to huPl8; muS67 to huA67
Chain: muQ5 to huVS; muLi 1 to huV11; muE61 to huQ6l; muK64 to huQ64;
muR65 to huG65; muA106 to huW106
huEM164
Li ht Chain Lic ht Chain aa45 Total
aa3 5A
~..>",.,.. ,:::,:, :..:<::.;;:,:::<~-::,.
,:,.:::, ._
,:~:::%.i: ,"~,>:
". :::;;:.,:;, . . :::: ouse
<:;: : Res
. .~
. a hu
~ mG:.
~ G
;:; hu
~1
.
; :.
,: ,.~:
;,.:,; ~::
. ..,. ~., ,
.:,.,;,... ~.:
,. .... . . ...;,
~":.,;;;:,r.." .:...::.,;:. ,:
;. ..:,:.,
,
%;~~
~
~
~
.0 . ,..:::: ~ R 0
..
.:>
,
;, ~.,,, V
:, ;
.1 f h~ ,~: .I,~ %~ ; 2
' ,
:;;;;;;:;; ...
._.:_.:r' ,
;::;;;;:-.::._:;:::;::::;.: -:.-.:,;.:
: .:..
~::::,:: ;>:;.. r.:;,.;:/.,~'..
:>
;: :::~r::;a<:
i%
.2 , , 1
; :
:: :
: ;..:: R
.:. ;~;,: !.:: i..~:<
>;::;~:::;;;: >::,;,::i.:f..,:...~
.,...,,.:; ~~.,;. .:
.:..
::I~.,.,:,..:..:.:..:
::,,;::...:;":.;,.:,:..;::: :.;,::;., :<::~-::,.,,:"
3 c,., .:-;,:,:.,.i.:.;~
. "..a/ ,,:
. ~
a
<
~
V
,.,,,
. ..: ..::.~./ ,I~ 1
, ..
.
::<::::
,
;:i::, ~, ,..:~;:
62
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I. Comparison of the affinities of humanized EM164 antibody versions with
murine
EM164 antibody for binding to full-length IGF-I receptor and to truncated IGF-
I receptor alpha chain
[172] The affinities of the humanized EM164 antibody versions 1.0-1.3 were
compared to
those of murine EM164 antibody through binding competition assays using
biotinylated full-
length human IGF-I receptor or myc-epitope tagged truncated IGF-I receptor
alpha chain, as
described above. Humanized EM164 antibody samples were obtained by transient
transfection of the appropriate expression vectors in human embryonic kidney
293T cells,
and antibody concentrations were determined by ELISA using purified humanized
antibody
standards. For ELISA binding competition measurements, mixtures of humanized
antibody
samples and various concentrations of murine EM164 antibody were incubated
with
indirectly captured biotinylated full-length IGF-I receptor or myc-epitope
tagged truncated
IGF-I receptor alpha chain. After equilibration, the bound humanized antibody
was detected
using a goat-anti-human-Fab'~-antibody-horseradish peroxidase conjugate. Plots
of ([bound
murine Ab]/[bound humanized Ab]) vs ([murine Ab]/[humanized Abp), which
theoretically -
yield a straight line with slope = (Kd h"m~d A» / ~d m,~r~e ab), were used to
determine the
relative affinities of the humanized and murine antibodies.
[173] An exemplary competition assay is shown in Figure 11. An Immulon-2HB
ELISA
plate was coated with 100 ~uL of 5 ~,g/mL streptavidin per well in carbonate
buffer at ambient
temperature for 7 h. The streptavidin-coated wells were blocked with 200 ~,L
of blocking
buffer (10 mg/mL BSA in TBS-T buffer) for 1 h, washed with TBS-T buffer and
incubated
with biotinylated IGF-I receptor (5 ng per well) overnight at 4°C. The
wells containing
indirectly captured biotinylated IGF-I receptor were then washed and incubated
with
mixtures of humanized EM164 antibody (15.5 ng) and murine antibody (0 ng, or
16.35 ng, or
32.7 ng, or 65.4 ng, or 163.5 ng) in 100 ~,L blocking buffer for 2 h at
ambient temperature
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and were then incubated overnight at 4°C. The wells were then washed
with TBS-T buffer
and incubated with goat-anti-human-Fab'2-antibody-horseradish pero~idase
conjugate for 1 h
(100 ~,L; 1 ~,g/mL in blocking buffer), followed by washes and detection using
ABTS/H~O~
substrate at 405 nm.
[174] The plot of ([bound murine Ab]/[bound humanized Ab]) vs ([murine
Ab]/[humanized
Ab]) yielded a straight line (r2 = 0.996) with slope (= Kd h"g,~d.v~ / Ka mere
ab) of 0.52. The
humanized antibody version 1.0 therefore bound to IGF-I receptor more tightly
than did
murine EM164 antibody. Similar values for the gradient, ranging from about 0.5
to 0.8, were
obtained for competitions of versions 1.0,1.1,1.2 and 1.3 of humanized EM164
antibodies
with murine EM164 antibody for binding to full-length IGF-I receptor or to
truncated IGF-I
receptor alpha chain, which indicated that all of the humanized versions of
EM164 antibody
had similar affinities, which were all better than that of the parent murine
EM164 antibody.
A chimeric version of EM164 antibody with 92F-~ C mutation in heavy chain
showed a slope
of about 3 in a similar binding competition with murine EM164 antibody, which
indicated
that the 92F-; C mutant of EM 164 had a 3-fold lower affinity than did murine
EM164
antibody for binding to IGF-I receptor. The humanized EM164 v1.0 antibody
showed a
similar inhibition of IGF-I -stimulated growth and survival of MCF-7 cells as
did the murine
EM164 antibody (Figure 24). The inhibition of serum-stimulated growth and
survival of
MCF-7 cells by humanized EM164 v1.0 antibody was similar to the inhibition by
murine
EM164 antibody.
64
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TABLE 9
Se went Li ht Chain Hea Chain
FR1 1-23 (with an occasional1-30 (with an occasional residue
residue at 0)
at 0, and a deletion
at 10 in
V~, chains
CDR1 24-34 (with possible 31-35 (with possible insertions
insertions numbered
numbered as 27A, B, C, as 35A, B
D, E, F
FR2 35-49 36-49
CDR2 50-56 50-65 (with possible insertions
numbered
as 52A, B, C
FR3 57-88 66-94 (with possible insertions
numbered
as 82A, B, C
CDR3 89-97 (with possible 95-102 (with possible insertions
insertions numbered
numbered as 95A, B, C, as 100A, B, C, D, E, F, G,
D, E, H, I, J, K
FR4 98-107 (with a possible 103-113
insertion
numbered as 106A
The Kabat numbering system is used for the light chain and heavy chain
variable region polypeptides
of the different versions of the EM164 Ab. The amino acid residues are grouped
into Framework (FR)
and Complementarity Determining Regions (CDR) according to position in the
polypeptide chain.
Taken from Kabat et al. Sequences of Proteihs of Immunological Interest, Fifth
Edition, 1991, NIH
Publication No. 91-3242
J. Process of providing improved anti-IGF-I-receptor antibodies starting from
the
murine and humanized antibody sequences described herein:
[175] The amino acid and nucleic acid sequences of the anti-IGF-I receptor
antibody
EM164 and its humanized variants were used to develop other antibodies that
have improved
properties and that are also within the scope of the present invention. Such
improved
properties include increased affinity for the IGF-I receptor. Several studies
have surveyed the
effects of introducing one or more amino acid changes at various positions in
the sequence of
an antibody, based on the knowledge of the primary antibody sequence, on its
properties such
as binding and level of expression (Yang, W. P. et al., 1995, J. Mol. Baol.,
254, 392-403;
Rader, C. et al., 1998, Proc. Natl. Acad. Sci. USA, 95, 8910-8915; Vaughan, T.
J. et al., 1998,
Nature Baotechrcology,16, 535-539).
[176] In these studies, variants of the primary antibody have been generated
by changing the
sequences of the heavy and light chain genes in the CDR1, CDR2, CDR3, or
framework
CA 02548065 2006-06-O1
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regions, using methods such as oligonucleotide-mediated site-directed
mutagenesis, cassette
mutagenesis, error-prone PCR, DNA shuffling, or mutator-strains of E. coli
(Vaughan, T. J.
et a1.,1998, NatuYe Biotech~ology,16, 535-539; Adey, N. B. et al., 1996,
Chapter 16, pp.
277-291, in "Phage l7isplay of Peptades aid Proteahs ", Eds. Kay, B. K. et
al., Academic
Press). These methods of changing the sequence of the primary antibody have
resulted,
through the use of standard screening techniques, in improved affinities of
such secondary
antibodies (Gram, H. et a1.,1992, Proc. Natl. Acad. Sci. ZISA, 89, 3576-3580;
Boder, E. T. et
al., 2000, Proc. Natl. Acad. Sci. ZISA, 97,10701-10705; Davies, J. and
Riechmann, L., 1996,
Immunotechholgy, 2,169-179; Thompson, J. et al., 1996, J. llTol. Biol., 256,
77-88; Short, M.
K. et al., 2002, J. Biol. Chem., 277, 16365-16370; Furukawa, K. et al., 2001,
J. Biol. Chem.,
276, 27622-27628).
[177] By a similar directed strategy of changing one or more amino acid
residues of the
antibody, the antibody sequences described in this invention can be used to
develop anti-IGF-
I receptor antibodies with improved functions, such as antibodies having
suitable groups such
as free amino groups or thiols at convenient attachment points for covalent
modification for
use, for example, in the attachment of therapeutic agents.
K. Alternative expression system for murine, chimeric and other anti-IGF-I
receptor antibodies
[178] The murine anti IGF-I receptor antibody was also expressed from
mammalian
expression plasmids similar to those used to express the humanized antibody
(above).
Expression plasmids are known that have murine constant regions including the
light chain
kappa and heavy chain gamma-1 sequences (McLean et al., 2000, Mol Immu~ol.,
37, 837-
845). These plasmids were designed to accept any antibody variable region,
such as for
example the murine anti-IGF-I receptor antibody, by a simple restriction
digest and cloning.
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Additional PCR of the anti-IGF-1 receptor antibody was usually required to
create the
restriction compatible with those in the expression plasmid.
[179] An alternative approach for expressing the fully murine anti-IGF-I
receptor antibody
was to replace the human constant regions in the chimeric anti-IGF-I receptor
antibody
expression plasmid. The chimeric expression plasmid (Figure 16) was
constructed using
cassettes for the variable regions and for both the light and heavy chain
constant regions. Just
as the antibody variable sequences were cloned into this expression plasmid by
restriction
digests, separate restriction digests were used to clone in any constant
region sequences. The
kappa light chain and gamma-1 heavy chain cDNAs were cloned, for example, from
murine
hybridoma RNA, such as the RNA described herein for cloning of the anti-IGF-1
antibody
variable regions. Similarly, suitable primers were designed from sequences
available in the
Kabat database (see Table 10). For example, RT-PCR was used to clone the
constant region
sequences and to create the restriction sites needed to clone these fragments
into the chimeric
anti-IGF-I receptor antibody expression plasmid. This plasmid was then used to
express the
fully murine anti-IGF-I receptor antibody in standard mammalian expression
systems such as
the CHO cell line.
TABLE 10 - Primers designed to clone the murine gamma-1 constant region and
murine
kappa constant region respectively.
Murine Constant
Re ion Primers
Primer name Primer Sequence SEQ ID NO:
MuIgG1 C3endX TTTTGAGCTCTTATTTACCAGGAGAGTGGGAGA 45
GGCTCTT
MuI G1 CSendH TTTTAAGCTTGCCAAAACGACACCCCCATCTGTCTAT46
MuI Ka C3endB TTTTGGATCCTAACACTCATTCCTGTTGAAGC 47
MuIgKap CSendETTTTGAATTCGGGCTGATGCTGCACCAACTG I 4g
~
The primers were designed from sequences available in the Kabat database
(Johnson, G and Wu, T.T.
(2001) Nucleic Acids Research, 29: 205-206).
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Statement of Deposit
[180] The hybridoma that makes murine EM164 antibody was deposited with the
American
Type Culture Collection, PO Box 1549, Manassas, VA 20108, on June 14, 2002,
under the
Terms of the Budapest Treaty and assigned ATCC accession number PTA-4457.
[181] Certain patents and printed publications have been referred to in the
present
disclosure, the teachings of which are hereby each incorporated in their
respective entireties
by reference.
[182] While the invention has been described in detail and with reference to
specific
embodiments thereof, it will be apparent to one of skill in the art that
various changes and
modifications can be made thereto without departing from the spirit and scope
thereof.
68
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