Title of Invention

ANTIBODIES TO INSULIN-LIKE GROWTH FACTOR I RECEPTOR

Abstract The present invention relates to antibodies and antigen-binding protions thereof that specifically bind to insulin-like growth factor I receptor (IGF-IR), which is preferably human IGF-IR. The invention also relates to human anti-IGF-IR antibodies, including chimeric, bispecific, derivatized, single chain antibodies or portions of fusion proteins. The invention also relates to isolated heavy and light chain immunoglobulin molecules derived from anti-IGF-IR antibodies and nucleic acid molecules encoding such momlecules. The present invention also relates to methods of making anti-IGF-IR antibodies, pharmaceutical compositions comprising these antibodies and methods of using the antibodies and compositions thereof for diagnosis and treatment. The invention also provides gene therapy methods using nucleic acid molecules encoding the heavy and/or light immunoglobulin molecules that comprise the human anti-IGF-IR antibodies. The invention also relates to gene therapy methods and transgenic animals comprising nucleic acid molecules of the present invention.
Full Text 1
This application is divided out of the Indian Patent Application no.: 994/KOLNP/2003
This application claims the benefit of United States Provisional
Application 60/259,927, filed January 5,2001.
BACKGROUND OF THE INVENTION
Insulin-like growth factor (IGF-I) is a 7.5-kD polypeptide that
circulates in plasma in high concentrations and is detectable in most tissues. IGF-I
stimulates cell differentiation and cell proliferation, and is required by most
mammalian cell types for sustained proliferation. These cell types include, among
others, human diploid fibroblasts, epithelial cells, smooth muscle cells, T
lymphocytes, neural cells, myeloid cells, chondrocytes, osteoblasts and bone marrow
stem cells. For a review of the wide variety of cell types for which IGF-I/IGF-I
receptor interaction mediates cell proliferation, see Goldring et al., Eukar. Gene
Express., 1:31-326(1991).
The first step in the transduction pathway leading to IGF-I-stimulated
cellular proliferation or differentiation is binding of IGF-I or IGF-II (or insulin at
supraphysiological concentrations) to the IGF-I receptor. The IGF-I receptor is
composed of two types of subunits: an alpha subunit (a 130-135 kD protein that is
entirely extracellular and functions in ligand binding) and a beta subunit (a 95-kD
transmembrane protein, with transmembrane and cytoplasmic domains). The IGF-IR
belongs to the family of tyrosine kinase growth factor receptors (Ullrich et al., Cell
61: 203-212, 1990), and is structurally similar to the insulin receptor (Ullrich et al.,
EMBO J. 5: 2503-2512,1986). The IGF-IR is initially synthesized as a single chain

2
proreceptor polypeptide which is processed by glycosylation, proteolytic cleavage,
and covalent bonding to assemble into a mature 460-kD heterotetramer comprising
two alpha-subunits and two beta-subunits. The beta subunit(s) possesses ligand-
activated tyrosine kinase activity. This activity is implicated in the signaling pathways
mediating ligand action which involve autophosphorylation of the beta-subunit and
phosphorylation of IGF-IR substrates.
In vivo, serum levels of IGF-I are dependent upon the presence of
pituitary growth hormone (GH). Although the liver is a major site of GH-dependent
IGF-I synthesis, recent work indicates that the majority of normal tissues also produce
IGF-I. A variety of neoplastic tissues may also produce IGF-I. Thus IGF-I may act
as a regulator of normal and abnormal cellular proliferation via autocrine or paracrine,
as well as endocrine mechanisms. IGF-I and IGF-II bind to IGF binding proteins
(IGFBPs) in vivo. The availability of free IGF for interaction with the IGF-IR is
modulated by the IGFBPs. For a review of IGFBPs and IGF-I, see Grimberg et al., J.
Cell. Physiol. 183: 1-9,2000.
There is considerable evidence for a role for IGF-I and/or IGF-IR in
the maintenance of tumor cells in vitro and in vivo. IGF-IR levels are elevated in
tumors of lung (Kaiser et al., J. Cancer Res. Clin Oncol. 119: 665-668, 1993; Moody
et al., Life Sciences 52: 1161-1173, 1993; Macauley et al., Cancer Res., 50: 2511-
2517, 1990), breast (Pollak et al., Cancer Lett. 38: 223-230, 1987; Foekens et al.,
Cancer Res. 49: 7002-7009, 1989; Cullen et al., Cancer Res. 49: 7002-7009, 1990;
Arteaga et al., J. Clin. Invest. 84: 1418-1423, 1989), prostate and colon (Remaole-
Bennet et al., J. Clin. Endocrinol. Metab. 75: 609-616, 1992; Guo et al.,
Gastroenterol. 102: 1101-1108, 1992). Deregulated expression of IGF-I in prostate
epithelium leads to neoplasia in transgenic mice (DiGiovanni et al., Proc. Natl. Acad.
Sci. USA 97: 3455-60, 2000). In addition, IGF-I appears to be an autocrine stimulator
of human gliomas (Sandberg-Nordqvist et al., Cancer Res. 53: 2475-2478, 1993),
while IGF-I stimulated the growth of fibrosarcomas that overexpressed IGF-IR
(Butler et al., Cancer Res. 58: 3021-27, 1998). Further, individuals with "high
normal" levels of IGF-I have an increased risk of common cancers compared to
individuals with IGF-I levels in the "low normal" range (Rosen et al., Trends
Endocrinol. Metab. 10: 136-41, 1999). Many of these tumor cell types respond to
IGF-I with a proliferative signal in culture (Nakanishi et al., J. Clin. Invest. 82: 354-

3
359,1988; Freed et al., J. Mol. Endocrinol. 3: 509-514,1989), and autocrine or
paracrine loops for proliferation in vivo have been postulated (LeRoith et al.,
Endocrine Revs. 16: 143-163,1995; Yee et al., Mol. Endocrinol. 3: 509-514,1989).
For a review of the role IGF-I/IGF-I receptor interaction plays in the growth of a
variety of human tumors, see Macaulay, Br. J. Cancer, 65: 311-320, 1992.
Increased IGF-I levels are also correlated with several noncancerous
pathological states, including acromegaly and gigantism (Barkan, Cleveland Clin. J.
Med. 65: 343, 347-349, 1998), while abnormal IGF-I/IGF-I receptor function has
been implicated in psoriasis (Wraight et al., Nat. Biotech. 18: 521-526, 2000),
atherosclerosis and smooth muscle restenosis of blood vessels following angioplasty
(Bayes-Genis et al., Circ. Res. 86: 125-130, 2000). Increased IGF-I levels also can be
a problem in diabetes or in complications thereof, such as microvascular proliferation
(Smith et al., Nat. Med. 5: 1390-1395, 1999). Decreased IGF-I levels, which occur,
inter alia, in cases when GH serum levels are decreased or when there is an
insensitivity or resistance to GH, is associated with such disorders as small stature
(Laron, Paediatr. Drugs 1: 155-159,1999), neuropathy, decrease in muscle mass and
osteoporosis (Rosen et al., Trends Endocrinol. Metab. 10: 136-141, 1999).
Using antisense expression vectors or antisense oligonucleotides to the
IGF-IR RNA, it has been shown that interference with IGF-IR leads to inhibition of
IGF-I-mediated or IGF-II-mediated cell growth (see, e.g., Wraight et al., Nat. Biotech.
18: 521-526, 2000). The antisense strategy was successful in inhibiting cellular
proliferation in several normal cell types and in human tumor cell lines. Growth can
also be inhibited using peptide analogues of IGF-I (Pietrzkowski et al., Cell Growth &
Diff. 3: 199-205, 1992; and Pietrzkowski et al., Mol. Cell. Biol., 12: 3883-3889,
1992), or a vector expressing an antisense RNA to the IGF-I RNA (Trojan et al.,
Science 259: 94-97, 1992). In addition, antibodies to IGF-IR (Arteaga et al., Breast
Cane. Res. Treatm., 22: 101-106, 1992; and Kalebic et al., Cancer Res. 54: 5531-
5534, 1994), and dominant negative mutants of IGF-IR (Pragcr ct al., Proc. Natl.
Acad. Sci. U.S.A. 91: 2181-2185, 1994; Li et al., J. Biol. Chem., 269: 32558-32564,
1994 and Jiang et al., Oncogene 18: 6071-77, 1999), can reverse the transformed
phenotype, inhibit tumorigenesis, and induce loss of the metastatic phenotype.
IGF-I is also important in the regulation of apoptosis. Apoptosis,
which is programmed eel] death, is involved in a wide variety of developmental

4
processes, including immune and nervous system maturation. In addition to its role in
development, apoptosis also has been implicated as an important cellular safeguard
against tumorigenesis (Williams, Cell 65:1097-1098,1991; Lane, Nature 362: 786-
787, 1993). Suppression of the apoptotic program, by a variety of genetic lesions,
may contribute to the development and progression of malignancies.
IGF-I protects from apoptosis induced by cytokine withdrawal in IL-3-
dependent hemopoietic cells (Rodriguez-Tarduchy, G. et al., J. Immunol. 149: 535-
540, 1992), and from serum withdrawal in Rat-1/mycER cells (Harrington, E., et al.,
EMBO J. 13: 3286-3295, 1994). The anti-apoptotic function of IGF-I is important in
the post-commitment stage of the cell cycle and also in cells blocked in cell cycle
progression by etoposide or thymidine. The demonstration that c-myc driven
fibroblasts are dependent on IGF-I for their survival suggests that there is an
important role for the IGF-IR in the maintenance of tumor cells by specifically
inhibiting apoptosis, a role distinct from the proliferative effects of IGF-I or IGF-IR.
This would be similar to a role thought to be played by other anti-apoptotic genes
such as bcl-2 in promoting tumor survival (McDonnell et al., Cell 57: 79-88, 1989;
Hockenberry et al., Nature 348: 334-336, 1990).
The protective effects of IGF-I on apoptosis are dependent upon
having IGF-IR present on cells to interact with IGF-I (Resnicoff et al., Cancer Res.
55: 3739-3741, 1995). Support for an anti-apoptotic function of IGF-IR in the
maintenance of tumor cells was also provided by a study using antisense
oligonucleotides to the IGF-ER that identified a quantitative relationship between IGF-
IR levels, the extent of apoptosis and the tumorigenie potential of a rat syngeneic
tumor (Rescinoff et al., Cancer Res. 55: 3739-3741, 1995). An overexpressed IGF-
IR has been found to protect tumor cells in vitro from etoposide-induced apoptosis
(Sell et al., Cancer Res. 55: 303-306,1995) and, even more dramatically, that a
decrease in IGF-IR levels below wild type levels caused massive apoptosis of tumor
cells in vivo (Resnicoff et al., Cancer Res. 55: 2463-2469, 1995).
Potential strategies for inducing apoptosis or for inhibiting cell
proliferation associated with increased IGF-I, increased IGF-II and/or increased IGF-
IR receptor levels include suppressing IGF-I levels or IGF-II levels or preventing the
binding of IGF-I to the IGF-IR. For example, the long acting somatostatin analogue
octreotide has been employed to reduce IGF synthesis and/or secretion. Soluble IGF-

5
IR has been used to induce apoptosis in tumor cells in vivo and inhibit tumorigenesis
in an experimental animal system (D'Ambrosio et al., Cancer Res. 56: 4013-20,
1996). In addition, IGF-IR antisense oligonucleotides, peptide analogues of IGF-I,
and antibodies to IGF-IR have been used to decrease IGF-I or IGF-IR expression (see
supra). However, none of these compounds has been suitable for long-term
administration to human patients. In addition, although IGF-I has been administered
to patients for treatment of short stature, osteoporosis, decreased muscle mass,
neuropathy or diabetes, the binding of IGF-I to IGFBPs has often made treatment with
IGF-I difficult or ineffective.
Accordingly, in view of the roles that IGF-I and IGF-IR have in such
disorders as cancer and other proliferative disorders when IGF-I and/or IGF-IR are
overexpressed, and the roles that too little IGF-I and IGF-IR have in disorders such as
short stature and frailty when either IGF-I and/or IGF-IR are underexpressed, it would
be desirable to generate antibodies to IGF-IR that could be used to either inhibit or
stimulate IGF-IR. Although anti-IGF-IR antibodies have been reported to have been
found in certain patients with autoimmune diseases, none of these antibodies has been
purified and none has been shown to be suitable for inhibiting IGF-I activity for
diagnostic or clinical procedures. See, e.g., Thompson et al., Pediat. Res. 32: 455-
459,1988; Tappy et al., Diabetes 37: 1708-1714, 1988; Weightman et al.,
Autoimmunity 16:251-257, 1993; Drexhage et al., Nether. J. of Med. 45:285-293,
1994. Thus, it would be desirable to obtain high-affinity human anti-IGF-IR
antibodies that could be used to treat diseases in humans.
BRIEF DESCRIPTION OF THE DRAWINGS
Figs. 1A-1C show alignments of the nucleotide sequences of the light
chain variable regions from six human anti-IGF-IR antibodies to each other and to
germline sequences. Fig. 1A shows the alignment of the nucleotide sequences of the
variable region of the light chain (VL) of antibodies 2.12.1 (SEQ ID NO: 1) 2.13.2
(SEQ ID NO: 5), 2.14.3 (SEQ ID NO: 9) and 4.9.2 (SEQ ID NO: 13) to each other
and to the germline VK A30 sequence (SEQ ID NO: 39). Fig. IB shows the
alignment of the nucleotide sequence of VL of antibody 4.17.3 (SEQ ID NO: 17) to
the germline VK 012 sequence (SEQ ID NO: 41). Fig. 1C shows the alignment of the
nucleotide sequence of VL of antibody 6.1.1 (SEQ ID NO: 21) to the germline VK

6
A27 sequence (SEQ ID NO: 37). The alignments also show the CDR regions of the
VL from each antibody. The consensus sequences for Figs. 1A-1C are shown in SEQ
ID NOS: 53-55, respectively.
Figs. 2A-2D show alignments of the nucleotide sequences of the heavy
chain variable regions from six human anti-IGF-IR antibodies to each other and to
germline sequences. Fig. 2A shows the alignment of the nucleotide sequence of the
VH of antibody 2.12.1 (SEQ ID NO: 3) to the germline VH DP-35 sequence (SEQ ID
NO: 29). Fig. 2B shows the alignment of the nucleotide sequence of the VH of
antibody 2.14.3 (SEQ ID NO: 11) to the germline VIV-4/4.35 sequence (SEQ ID NO:
43). Figs. 2C-1 and 2C-2 show the alignments of the nucleotide sequences of the VH
of antibodies 2.13.2 (SEQ ID NO: 7), 4.9.2 (SEQ ID NO: 15) and 6.1.1 (SEQ ID NO:
23) to each other and to the germline VH DP-47 sequence (SEQ ID NO: 31). Fig. 2D
shows the alignment of the nucleotide sequence of the VH of antibody 4.17.3 (SEQ
ID NO: 19) to the germline VH DP-71 sequence (SEQ ID NO: 35). The alignment
also shows the CDR regions of the antibodies. The consensus sequences for Figs. 2A-
2D are shown in SEQ ID NOS: 56-59, respectively.
Fig. 3 shows that anti-IGF-IR antibodies 2.13.2, 4.9.2 and 2.12.1
inhibit IGF-I binding to 3T3-IGF-IR cells.
Fig. 4 shows that anti-IGF-IR antibody 4.9.2 inhibits IGF-I-induced
receptor tyrosine phosphorylation (upper panel) and induces IGF-IR downregulation
at the cell surface (lower panel).
Fig. 5 shows that anti-IGF-IR antibodies 2.13.2 and 4.9.2 reduce IGF-
IR phosphotyrosine signal in 3T3-IGF-IR tumors.
Fig. 6 shows that anti-IGF-IR antibodies 2.13.2 and 4.9.2 reduce IGF-
IR in 3T3-IGF-IR tumors.
Fig. 7 shows that anti-IGF-IR antibody 2.13.2 inhibits 3T3-IGF-IR
tumor growth in vivo alone (left panel) or in combination with adriamycin (right
panel).
Fig. 8 shows the relationship between anti-IGF-IR antibody 2.13.2
serum levels and IGF-IR downregulation in 3T3-IGF-IR tumors.
Fig. 9 shows that multiple doses of anti-IGF-IR antibody 2.13.2 inhibit
3T3-IGF-IR rumor growth in vivo alone or in combination with adriamycin.

7
Fig. 10 shows that anti-IGF-IR antibody 2.13.2 inhibits large tumor
growth in vivo in combination with adriamycin.
Fig. 11 shows that anti-IGF-IR antibody 2.13.2 inhibits Colo 205
tumor growth in vivo alone or in combination with 5-deoxyuridine (5-FU).
Fig. 12 shows that multiple doses of anti-IGF-IR antibody 2.13.2
inhibit Colo 205 tumor growth in vivo alone or in combination with 5-FU.
Fig. 13 shows that multiple doses of anti-IGF-IR antibody 2.13.2
inhibit MCF-7 tumor growth in vivo alone or in combination with taxol.
Fig. 14 shows that anti-IGF-IR antibody 2.13.2 inhibits MCF-7 tumor
growth in vivo alone (left panel) or in combination with adriamycin (right panel).
Fig. 15 shows that multiple doses of anti-IGF-IR antibody 2.13.2
inhibit MCF-7 tumor growth in vivo alone or in combination with tamoxifen.
Fig. 16 shows that multiple doses of anti-IGF-IR antibody 2.13.2
inhibit A431 tumor growth in vivo alone or in combination with the epidermal growth
factor receptor (EGF-R) tyrosine kinase inhibitor CP-358,774.
Fig. 17 shows a pharmacokinetic evaluation of a single intravenous
injection of anti-IGF-IR antibody 2.13.2 in Cynomologus monkeys.
Fig. 18 shows that the combination of anti-IGF-IR antibody 2.13.2 and
adriamycin increases the downregulation of IGF-IR on 3T3-IGF-IR tumors in vivo.
Fig. 19A shows the number of mutations in different regions of the
heavy and light chains of 2.13.2 and 2.12.1 compared to the germline sequences.
Figs. 19A-D show alignments of the amino acid sequences from the
heavy and light chains of antibodies 2.13.2 and 2.12.1 with the germline sequences
from which they are derived. Fig. 19B shows an alignment of the amino acid
sequence of the heavy chain of antibody 2.13.2 (SEQ ID NO: 45) with that of
germline sequence DP-47(3-23)/D6-19/JH6 (SEQ ID NO: 46). Fig. 19C shows an
alignment of the amino acid sequence of the light chain of antibody 2.13.2 (SEQ ID
NO: 47) with that of germline sequence A30/Jk2 (SEQ ID NO: 48). Fig. 19D shows
an alignment of the amino acid sequence of the heavy chain of antibody 2.12.1 (SEQ
ID NO: 49) with that of germline sequence DP-35(3-ll)/D3-3/JH6 (SEQ ID NO: 50).
Fig. 19E shows an alignment of the amino acid sequence of the light chain of
antibody 2.12.1 (SEQ ID NO: 51) with that of germline sequence A30/Jkl (SEQ ID
NO: 52). For Figures 19B-E, the signal sequences are in italic, the CDRs are

8
underlined, the constant domains are bold, the framework (FR) mutations are
highlighted with a plus sign ("+") above the amino acid residue and CDR mutations
are highlighted with an asterisk above the amino acid residue.
SUMMARY OF THE INVENTION
The present invention provides an isolated antibody or antigen-binding
portion thereof that binds IGF-ER, preferably one that binds to primate and human
IGF-IR, and more preferably one that is a human antibody. The invention provides an
anti-IGF-IR antibody that inhibits the binding of IGF-I or IGF-II to IGF-IR, and also
provides an anti-IGF-IR antibody that activates IGF-IR.
The invention provides a pharmaceutical composition comprising the
antibody and a pharmaceutically acceptable carrier. The pharmaceutical composition
may further comprise another component, such as an anti-tumor agent or an imaging
reagent.
Diagnostic and therapeutic methods are also provided by the invention.
Diagnostic methods include a method for diagnosing the presence or location of an
IGF-IR-expressing tissue using an anti-IGF-IR antibody. A therapeutic method
comprises administering the antibody to a subject in need thereof, preferably in
conjunction with administration of another therapeutic agent.
The invention provides an isolated cell line, such as a hybridoma, that
produces an anti-IGF-IR antibody.
The invention also provides nucleic acid molecules encoding the heavy
and/or light chain or antigen-binding portions thereof of an anti-IGF-IR antibody.
The invention provides vectors and host cells comprising the nucleic acid molecules,
as well as methods of recombinantly producing the polypeptides encoded by the
nucleic acid molecules.
Non-human transgenic animals that express the heavy and/or light
chain or antigen-binding portions thereof of an anti-IGF-IR antibody are also
provided. The invention also provides a method for treating a subject in need thereof
with an effective amount of a nucleic acid molecule encoding the heavy and/or light
chain or antigen-binding portions thereof of an anti-IGF-IR antibody.

9
DETAILED DESCRIPTION OF THE INVENTION
Definitions and General Techniques
Unless otherwise defined herein, scientific and technical terms used in
connection with the present invention shall have the meanings that are commonly
understood by those of ordinary skill in the art. Further, unless otherwise required by
context, singular terms shall include pluralities and plural terms shall include the
singular. Generally, nomenclatures used in connection with, and techniques of, cell
and tissue culture, molecular biology, immunology, microbiology, genetics and
protein and nucleic acid chemistry and hybridization described herein are those well
known and commonly used in the art. The methods and techniques of the present
invention are generally performed according to conventional methods well known in
the art and as described in various general and more specific references that are cited
and discussed throughout the present specification unless otherwise indicated. See,
e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et al.,
Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and
Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. (1990), which are incorporated herein by reference.
Enzymatic reactions and purification techniques are performed according to
manufacturer's specifications, as commonly accomplished in the art or as described
herein. The nomenclatures used in connection with, and the laboratory procedures
and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal
and pharmaceutical chemistry described herein are those well known and commonly
used in the art. Standard techniques are used for chemical syntheses, chemical
analyses, pharmaceutical preparation, formulation, and delivery, and treatment of
patients.
The following terms, unless otherwise indicated, shall be understood to
have the following meanings:
The term "polypeptide" encompasses native or artificial proteins,
protein fragments and polypeptide analogs of a protein sequence. A polypeptide may
be monomeric or polymeric.

10
The term "isolated protein" or "isolated polypeptide" is a protein or
polypeptide that by virtue of its origin or source of derivation (1) is not associated
with naturally associated components that accompany it in its native state, (2) is free
of other proteins from the same species (3) is expressed by a cell from a different
species, or (4) does not occur in nature. Thus, a polypeptide that is chemically
synthesized or synthesized in a cellular system different from the cell from which it
naturally originates will be "isolated" from its naturally associated components. A
protein may also be rendered substantially free of naturally associated components by
isolation, using protein purification techniques well known in the art.
A protein or polypeptide is "substantially pure," "substantially
homogeneous" or "substantially purified" when at least about 60 to 75% of a sample
exhibits a single species of polypeptide. The polypeptide or protein may be
monomeric or multimeric. A substantially pure polypeptide or protein will typically
comprise about 50%, 60, 70%, 80% or 90% WAV of a protein sample, more usually
about 95%, and preferably will be over 99% pure. Protein purity or homogeneity may
be indicated by a number of means well known in the art, such as polyacrylamide gel
electrophoresis of a protein sample, followed by visualizing a single polypeptide band
upon staining the gel with a stain well known in the art. For certain purposes, higher
resolution may be provided by using HPLC or other means well known in the art for
purification.
The term "polypeptide fragment" as used herein refers to a polypeptide
that has an amino-terminal and/or carboxy-terminal deletion, but where the remaining
amino acid sequence is identical to the corresponding positions in the naturally-
occurring sequence. Fragments typically are at least 5, 6, 8 or 10 amino acids long,
preferably at least 14 amino acids long, more preferably at least 20 amino acids long,
usually at least 50 amino acids long, even more preferably at least 70, 80, 90, 100,
150 or 200 amino acids long.
The term "polypeptide analog" as used herein refers to a polypeptide
that is comprised of a segment of at least 25 amino acids that has substantial identity
to a portion of an amino acid sequence and that has at least one of the following
properties: (1) specific binding to IGF-IR under suitable binding conditions, (2)
ability to block IGF-I or IGF-II binding to IGF-IR, or (3) ability to reduce IGF-IR cell
surface expression or tyrosine phosphorylation in vitro or in vivo. Typically,

11
polypeptide analogs comprise a conservative amino acid substitution (or insertion or
deletion) with respect to the naturally-occurring sequence. Analogs typically are at
least 20 amino acids long, preferably at least 50, 60, 70, 80, 90, 100, 150 or 200
amino acids long or longer, and can often be as long as a full-length naturally-
occurring polypeptide.
Preferred amino acid substitutions are those which: (1) reduce
susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding
affinity for forming protein complexes, (4) alter binding affinities, and (4) confer or
modify other physicochemical or functional properties of such analogs. Analogs can
include various muteins of a sequence other than the naturally-occurring peptide
sequence. For example, single or multiple amino acid substitutions (preferably
conservative amino acid substitutions) may be made in the naturally-occurring
sequence (preferably in the portion of the polypeptide outside the domain(s) forming
intermolecular contacts. A conservative amino acid substitution should not
substantially change the structural characteristics of the parent sequence (e.g., a
replacement amino acid should not tend to break a helix that occurs in the parent
sequence, or disrupt other types of secondary structure that characterizes the parent
sequence). Examples of art-recognized polypeptide secondary and tertiary structures
are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H.
Freeman and Company, New York (1984)); Introduction to Protein Structure (C.
Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and
Thornton et at. Nature 354:105 (1991), which are each incorporated herein by
reference.
Non-peptide analogs are commonly used in the pharmaceutical
industry as drugs with properties analogous to those of the template peptide. These
types of non-peptide compound are termed "peptide mimetics" or "peptidomimetics".
Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger TINS p.392 (1985);
and Evans et al. J. Med. Chem. 30:1229 (1987), which are incorporated herein by
reference. Such compounds are often developed with the aid of computerized
molecular modeling. Peptide mimetics that are structurally similar to therapeutically
useful peptides may be used to produce an equivalent therapeutic or prophylactic
effect. Generally, peptidomimetics are structurally similar to a paradigm polypeptide
(i.e., a polypeptide that has a desired biochemical property or pharmacological

12
activity), such as a human antibody, but have one or more peptide linkages optionally
replaced by a linkage selected from the group consisting of: --CH2NH--, -CHiS--, -
CH2-CH2--, ~CH=CH«(cis and trans), -COCH2--, --CH(OH)CH2--, and-CH2SO--,
by methods well known in the art. Systematic substitution of one or more amino
acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in
place of L-lysine) may also be used to generate more stable peptides. In addition,
constrained peptides comprising a consensus sequence or a substantially identical
consensus sequence variation may be generated by methods known in the art (Rizo
and Gierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein by reference);
for example, by adding internal cysteine residues capable of forming intramolecular
disulfide bridges which cyclize the peptide.
An "immunoglobulin" is a tetrameric molecule. In a naturally-
occurring immunoglobulin, each tetramer is composed of two identical pairs of
polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy"
chain (about 50-70 kDa). The amino-terminal portion of each chain includes a
variable region of about 100 to 110 or more amino acids primarily responsible for
antigen recognition. The carboxy-terminal portion of each chain defines a constant
region primarily responsible for effector function. Human light chains are classified
as K and X light chains. Heavy chains are classified as n, A, y, a, or e, and define the
antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and
heavy chains, the variable and constant regions are joined by a "J" region of about 12
or more amino acids, with the heavy chain also including a "D" region of about 10
more amino acids. See generally. Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd
ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all
purposes). The variable regions of each light/eavy chain pair form the antibody
binding site such that an intact immunoglobulin has two binding sites.
Immunoglobulin chains exhibit the same general structure of
relatively conserved framework regions (FR) joined by three hypervariable regions,
also called complementarity determining regions or CDRs. The CDRs from the two
chains of each pair are aligned by the framework regions, enabling binding to a
specific epitope. From N-terminus to C-terminus, both light and heavy chains
comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The
assignment of amino acids to each domain is in accordance with the definitions of

13
Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health,
Bethesda, Md. {1987 and 1991)), or Chothia & LeskJ. Mol. Biol. 196:901-917
(1987); Chothia et al. Nature 342:878-883 (1989).
An "antibody" refers to an intact immunoglobulin or to an antigen-
binding portion thereof that competes with the intact antibody for specific binding.
Antigen-binding portions may be produced by recombinant DNA techniques or by
enzymatic or chemical cleavage of intact antibodies. Antigen-binding portions
include, inter alia, Fab, Fab', F(ab')2, Fv, dAb, and complementarity determining
region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies,
diabodies and polypeptides that contain at least a portion of an immunoglobulin that is
sufficient to confer specific antigen binding to the polypeptide.
As used herein, an antibody that is referred to as, e.g., 2.12.1, 2.13.2,
2.14.3, 4.9.2, 4.17.3 and 6.1.1, is an antibody that is derived from the hybridoma of
the same name. For example, antibody 2.12.1 is derived from hybridoma 2.12.1.
An Fab fragment is a monovalent fragment consisting of the VL, VH,
CL and CH I domains; a F(ab')2 fragment is a bivalent fragment comprising two Fab
fragments linked by a disulfide bridge at the hinge region; a Fd fragment consists of
the VH and CHI domains; an Fv fragment consists of the VL and VH domains of a
single arm of an antibody; and a dAb fragment (Ward et al., Nature 341:544-546,
1989) consists of a VH domain.
A single-chain antibody (scFv) is an antibody in which a VL and VH
regions are paired to form a monovalent molecules via a synthetic linker that enables
them to be made as a single protein chain (Bird et al., Science 242:423-426, 1988 and
Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883,1988). Diabodies are
bivalent, bispecific antibodies in which VH and VL domains are expressed on a single
polypeptide chain, but using a linker that is too short to allow for pairing between the
two domains on the same chain, thereby forcing the domains to pair with
complementary domains of another chain and creating two antigen binding sites (see
e.g., Holliger, P., et al., Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993, andPoIjak,
R. J., et al., Structure 2:1121-1123, 1994). One or more CDRs may be incorporated
into a molecule either covalently or noncovalently to make it an immunoadhesin. An
immunoadhesin may incorporate the CDR(s) as part of a larger polypeptide chain,
may covalently link the CDR(s) to another polypeptide chain, or may incorporate the

14
CDR(s) noncovalently. The CDRs permit the immunoadhesin to specifically bind to
a particular antigen of interest.
An antibody may have one or more binding sites. If there is more than
one binding site, the binding sites may be identical to one another or may be different.
For instance, a naturally-occurring immunoglobulin has two identical binding sites, a
single-chain antibody or Fab fragment has one binding site, while a "bispecific" or
"bifunctional" antibody has two different binding sites.
An "isolated antibody" is an antibody that (1) is not associated with
naturally-associated components, including other naturally-associated antibodies, that
accompany it in its native state, (2) is free of other proteins from the same species, (3)
is expressed by a cell from a different species, or (4) does not occur in nature.
Examples of isolated antibodies include an anti-IGF-IR antibody that has been affinity
purified using IGF-IR is an isolated antibody, an anti-IGF-IR antibody that has been
synthesized by a hybridoma or other cell line in vitro, and a human anti-IGF-IR
antibody derived from a transgenic mouse.
The term "human antibody" includes all antibodies that have one or
more variable and constant regions derived from human immunoglobulin sequences.
In a preferred embodiment, all of the variable and constant domains are derived from
human immunoglobulin sequences (a fully human antibody). These antibodies may
be prepared in a variety of ways, as described below.
A humanized antibody is an antibody that is derived from a non-human
species, in which certain amino acids in the framework and constant domains of the
heavy and light chains have been mutated so as to avoid or abrogate an immune
response in humans. Alternatively, a humanized antibody may be produced by fusing
the constant domains from a human antibody to the variable domains of a non-human
species. Examples of how to make humanized antibodies may be found in United
States Patent Nos. 6,054,297, 5,886,152 and 5,877,293.
The term "chimeric antibody" refers to an antibody that contains one or
more regions from one antibody and one or more regions from one or more other
antibodies. In a preferred embodiment, one or more of the CDRs are derived from a
human anti-IGF-IR antibody. In a more preferred embodiment, all of the CDRs are
derived from a human anti-IGF-IR antibody. In another preferred embodiment, the
CDRs from more than one human anti-IGF-IR antibodies are mixed and matched in a

15
chimeric antibody. For instance, a chimeric antibody may comprise a CDR1 from the
light chain of a first human anti-IGF-IR antibody may be combined with CDR2 and
CDR3 from the light chain of a second human anti-IGF-IR antibody, and the CDRs
from the heavy chain may be derived from a third anti-IGF-IR antibody. Further, the
framework regions may be derived from one of the same anti-IGF-IR antibodies, from
one or more different antibodies, such as a human antibody, or from a humanized
antibody.
A "neutralizing antibody" or "an inhibitory antibody" is an antibody
that inhibits the binding of IGF-IR to IGF-I when an excess of the anti-IGF-IR
antibody reduces the amount of IGF-I bound to IGF-IR by at least about 20%. In a
preferred embodiment, the antibody reduces the amount of IGF-I bound to IGF-IR by
at least 40%, more preferably 60%, even more preferably 80%, or even more
preferably 85%. The binding reduction may be measured by any means known to one
of ordinary skill in the art, for example, as measured in an in vitro competitive
binding assay. An example of measuring the reduction in binding of IGF-I to IGF-IR
is presented below in Example IV.
An "activating antibody" is an antibody that activates IGF-IR by at
least about 20% when added to a cell, tissue or organism expressing IGF-IR. In a
preferred embodiment, the antibody activates IGF-IR activity by at least 40%, more
preferably 60%, even more preferably 80%, or even more preferably 85%. In a more
preferred embodiment, the activating antibody is added in the presence of IGF-I or
IGF-II. In another preferred embodiment, the activity of the activating antibody is
measured by determining the amount of tyrosine autophosphorylation of IGF-IR.
Fragments or analogs of antibodies can be readily prepared by those of
ordinary skill in the art following the teachings of this specification. Preferred amino-
and carboxy-termini of fragments or analogs occur near boundaries of functional
domains. Structural and functional domains can be identified by comparison of the
nucleotide and/or amino acid sequence data to public or proprietary sequence
databases. Preferably, computerized comparison methods are used to identify
sequence motifs or predicted protein conformation domains that occur in other
proteins of known structure and/or function. Methods to identify protein sequences
that fold into a known three-dimensional structure are known. Bowie et al. Science
253:164(1991).

16
The term "surface plasmon resonance", as used herein, refers to an
optical phenomenon that allows for the analysis of real-time biospecific interactions
by detection of alterations in protein concentrations within a biosensor matrix, for
example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and
Piscataway, N.J.). For further descriptions, see Jonsson, U., et al. (1993) Ann. Biol.
Clin. 51:19-26; Jonsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson, B., et
al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al. (1991) Anal.
Biochem. 198:268-277.
The term "Koff" refers to the off rate constant for dissociation of an
antibody from the antibody/antigen complex.
The term "Koff" refers to the dissociation constant of a particular
antibody-antigen interaction.
The term "epitope" includes any protein determinant capable of
specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants
usually consist of chemically active surface groupings of molecules such as amino
acids or sugar side chains and usually have specific three dimensional structural
characteristics, as well as specific charge characteristics. An antibody is said to
specifically bind an antigen when the dissociation constant is nM and most preferably As used herein, the twenty conventional amino acids and their
abbreviations follow conventional usage. See Immunology - A Synthesis (2nd
Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass.
(1991)), which is incorporated herein by reference. Stereoisomers (e.g., D-amino
acids) of the twenty conventional amino acids, unnatural amino acids such as a-, a-
disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional
amino acids may also be suitable components for polypeptides of the present
invention. Examples of unconventional amino acids include: 4-hydroxyproline, y-
carboxyglutamate, e-N,N,N-trimethyllysine, e-N-acetyllysine, Oi-phosphoserine, N-
acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, s-N- .
methylarginine, and other similar amino acids and imino acids (e.g., 4-
hydroxyproline). In the polypeptide notation used herein, the lefthand direction is the
amino terminal direction and the righthand direction is the carboxy-terminal direction,
in accordance with standard usage and convention.

17
The term "polynucleotide" as referred to herein means a polymeric
form of nucleotides of at least 10 bases in length, either ribonucleotides or
deoxynucleotides or a modified form of either type of nucleotide. The term includes
single and double stranded forms of DNA.
The term "isolated polynucleotide" as used herein shall mean a
polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof,
which by virtue of its origin the "isolated polynucleotide" (1) is not associated with all
or a portion of a polynucleotide in which the "isolated polynucleotide" is found in
nature, (2) is operably linked to a polynucleotide which it is not linked to in nature, or
(3) does not occur in nature as part of a larger sequence.
The term "oligonucleotide" referred to herein includes naturally
occurring, and modified nucleotides linked together by naturally occurring, and non-
naturally occurring oiigonucleotide linkages. Oligonucleotides are a polynucleotide
subset generally comprising a length of 200 bases or fewer. Preferably
oligonucleotides are 10 to 60 bases in length and most preferably 12, 13, 14, 15, 16,
17, 18, 19, or 20 to 40 bases in length. Oligonucleotides are usually single stranded,
e.g. for probes; although oligonucleotides may be double stranded, e.g. for use in the
construction of a gene mutant. Oligonucleotides of the invention can be either sense
or antisense oligonucleotides.
The term "naturally occurring nucleotides" referred to herein includes
deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" referred
to herein includes nucleotides with modified or substituted sugar groups and the like.
The term "oligonucleotide linkages" referred to herein includes oligonucleotides
linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate,
phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, phosphoroamidate,
and the like. See e.g., LaPlanche et al. Nucl. Acids Res. 14:9081 (1986); Stec et al. J.
Am. Chem. Soc. 106:6077 (1984); Stein et al. Nucl. Acids Res. 16:3209 (1988); Zon et
al. Anti-Cancer Drug Design 6:539 (1991); Zon et al. Oligonucleotides and
Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University
Press, Oxford England (1991)); Stec et al. U.S. Patent No. 5,151,510; Uhlmann and
Peyman Chemical Reviews 90:543 (1990), the disclosures of which are hereby
incorporated by reference. An oligonucleotide can include a label for detection, if
desired.

18
"Operably linked" sequences include both expression control
sequences that are contiguous with the gene of interest and expression control
sequences that act in trans or at a distance to control the gene of interest. The term
"expression control sequence" as used herein refers to polynucleotide sequences
which are necessary to effect the expression and processing of coding sequences to
which they are ligated. Expression control sequences include appropriate
transcription initiation, termination, promoter and enhancer sequences; efficient RNA
processing signals such as splicing and polyadenylation signals; sequences that
stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e.,
Kozak consensus sequence); sequences that enhance protein stability; and when
desired, sequences that enhance protein secretion. The nature of such control
sequences differs depending upon the host organism; in prokaryotes, such control
sequences generally include promoter, ribosomal binding site, and transcription
termination sequence; in eukaryotes, generally, such control sequences include
promoters and transcription termination sequence. The term "control sequences" is
intended to include, at a minimum, all components whose presence is essential for
expression and processing, and can also include additional components whose
presence is advantageous, for example, leader sequences and fusion partner
sequences.
The term "vector", as used herein, is intended to refer to a nucleic acid
molecule capable of transporting another nucleic acid to which it has been linked. One
type of vector is a "plasmid", which refers to a circular double stranded DNA loop
into which additional DNA segments may be ligated. Another type of vector is a viral
vector, wherein additional DNA segments may be ligated into the viral genome.
Certain vectors are capable of autonomous replication in a host cell into which they
are introduced (e.g., bacterial vectors having a bacterial origin of replication and
episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)
can be integrated into the genome of a host cell upon introduction into the host cell,
and thereby are replicated along with the host genome. Moreover, certain vectors are
capable of directing the expression of genes to which they are operatively linked.
Such vectors are referred to herein as "recombinant expression vectors" (or simply,
"expression vectors"). In general, expression vectors of utility in recombinant DNA
techniques are often in the form of plasmids. In the present specification, "plasmid"

19
and "vector" may be used interchangeably as the plasmid is the most commonly used
form of vector. However, the invention is intended to include such other forms of
expression vectors, such as viral vectors (e.g., replication defective retroviruses,
adenoviruses and adeno-associated viruses), which serve equivalent functions.
The term "recombinant host cell" (or simply "host cell"), as used
herein, is intended to refer to a cell into which a recombinant expression vector has
been introduced. It should be understood that such terms are intended to refer not only
to the particular subject cell but to the progeny of such a cell. Because certain
modifications may occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be identical to the parent
cell, but are still included within the scope of the term "host cell" as used herein.
The term "selectively hybridize" referred to herein means to detectably
and specifically bind. Polynucleotides, oligonucleotides and fragments thereof in
accordance with the invention selectively hybridize to nucleic acid strands under
hybridization and wash conditions that minimize appreciable amounts of detectable
binding to nonspecific nucleic acids. "High stringency" or "highly stringent"
conditions can be used to achieve selective hybridization conditions as known in the
art and discussed herein. An example of "high stringency" or "highly stringent"
conditions is a method of incubating a polynucleotide with another polynucleotide,
wherein one polynucleotide may be affixed to a solid surface such as a membrane, in
a hybridization buffer of 6X SSPE or SSC, 50% formamide, 5X Denhardt's reagent,
0.5% SDS, 100 ug/ml denatured, fragmented salmon sperm DNA at a hybridization
temperature of 42°C for 12-16 hours, followed by twice washing at 55°C using a
wash buffer of IX SSC, 0.5% SDS. See also Sambrook et al., supra, pp. 9.50-9.55.
The term "percent sequence identity" in the context of nucleic acid
sequences refers to the residues in two sequences which are the same when aligned
for maximum correspondence. The length of sequence identity comparison may be
over a stretch of at least about nine nucleotides, usually at least about 18 nucleotides,
more usually at least about 24 nucleotides, typically at least about 28 nucleotides,
more typically at least about 32 nucleotides, and preferably at least about 36,48 or
more nucleotides. There are a number of different algorithms known in the art which
can be used to measure nucleotide sequence identity. For instance, polynucleotide
sequences can be compared using FASTA, Gap or Bestfit, which are programs in

20
Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison,
Wisconsin. FAST A, which includes, e.g., the programs FASTA2 and FASTA3,
provides alignments and percent sequence identity of the regions of the best overlap
between the query and search sequences (Pearson, Methods Enzymol. 183: 63-98
(1990); Pearson, Methods Mol. Biol. 132: 185-219 (2000); Pearson, Metholds
Enzymol 266: 227-258 (1996); Pearson,/. Mol. Biol. 276: 71-84 (1998); herein
incorporated by reference). Unless otherwise specified, default parameters for a
particular program or algorithm are used. For instance, percent sequence identity
between nucleic acid sequences can be determined using FASTA with its default
parameters (a word size of 6 and the NOPAM factor for the scoring matrix) or using
Gap with its default parameters as provided in GCG Version 6.1, herein incorporated
by reference.
A reference to a nucleic acid sequence encompasses its complement
unless otherwise specified. Thus, a reference to a nucleic acid molecule having a
particular sequence should be understood to encompass its complementary strand,
with its complementary sequence.
In the molecular biology art, researchers use the terms "percent
sequence identity", "percent sequence similarity" and "percent sequence homology"
interchangeably. In this application, these terms shall have the same meaning with
respect to nucleic acid sequences only.
The term "substantial similarity" or "substantial sequence similarity,"
when referring to a nucleic acid or fragment thereof, indicates that, when optimally
aligned with appropriate nucleotide insertions or deletions with another nucleic acid
(or its complementary strand), there is nucleotide sequence identity in at least about
85%, preferably at least about 90%, and more preferably at least about 95%, 96%,
97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm
of sequence identity, such as FASTA, BLAST or Gap, as discussed above.
As applied to polypeptides, the term "substantial identity" means that
two peptide sequences, when optimally aligned, such as by the programs GAP or
BESTFIT using default gap weights, share at least 75% or 80% sequence identity,
preferably at least 90% or 95% sequence identity, even more preferably at least 98%
or 99% sequence identity. Preferably, residue positions which are not identical differ
by conservative amino acid substitutions. A "conservative amino acid substitution" is

21
one in which an amino acid residue is substituted by another amino acid residue
having a side chain (R group) with similar chemical properties (e.g., charge or
hydrophobicity). In general, a conservative amino acid substitution will not
substantially change the functional properties of a protein. In cases where two or
more amino acid sequences differ from each other by conservative substitutions, the
percent sequence identity or degree of similarity may be adjusted upwards to correct
for the conservative nature of the substitution. Means for making this adjustment are
well-known to those of skill in the art. See, e.g., Pearson, Methods Mol. Biol. 24:
307-31 (1994), herein incorporated by reference. Examples of groups of amino acids
that have side chains with similar chemical properties include 1) aliphatic side chains:
glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains:
serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4)
aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains:
lysine, arginine, and histidine; and 6) sulfur-containing side chains are cysteine and
methionine. Preferred conservative amino acids substitution groups are: valine-
leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-
aspartate, and asparagine-glutamine.
Alternatively, a conservative replacement is any change having a
positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al.,
Science 256: 1443-45 (1992), herein incorporated by reference. A "moderately
conservative" replacement is any change having a nonnegative value in the PAM250
log-likelihood matrix.
Sequence similarity for polypeptides, which is also referred to as
sequence identity, is typically measured using sequence analysis software. Protein
analysis software matches similar sequences using measures of similarity assigned to
various substitutions, deletions and other modifications, including conservative amino
acid substitutions. For instance, GCG contains programs such as "Gap" and "Bestfit"
which can be used with default parameters to determine sequence homology or
sequence identity between closely related polypeptides, such as homologous .
polypeptides from different species of organisms or between a wild type protein and a
mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be
compared using FASTA using default or recommended parameters, a program in
GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and

22
percent sequence identity of the regions of the best overlap between the query and
search sequences (Pearson (1990); Pearson (2000). Another preferred algorithm
when comparing a sequence of the invention to a database containing a large number
of sequences from different organisms is the computer program BLAST, especially
blastp or tblastn, using default parameters. See, e.g., Altschul et ah, J. Mol. Biol. 215:
403-410 (1990); Altschul et al., Nucleic Acids Res. 25:3389-402 (1997); herein
incorporated by reference.
The length of polypeptide sequences compared for homology will
generally be at least about 16 amino acid residues, usually at least about 20 residues,
more usually at least about 24 residues, typically at least about 28 residues, and
preferably more than about 35 residues. When searching a database containing
sequences from a large number of different organisms, it is preferable to compare
amino acid sequences.
As used herein, the terms "label" or "labeled" refers to incorporation of
another molecule in the antibody. In one embodiment, the label is a detectable
marker, e.g., incorporation of a radiolabeled amino acid or attachment to a
polypeptide of biotinyl moieties that can be detected by marked avidin (e.g.,
streptavidin containing a fluorescent marker or enzymatic activity that can be detected
by optical or colorimetric methods). In another embodiment, the label or marker can
be therapeutic, e.g., a drug conjugate or toxin. Various methods of labeling
polypeptides and glycoproteins are known in the art and may be used. Examples of
labels for polypeptides include, but are not limited to, the following: radioisotopes or
radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, wTc, '"in, I25I, mI), fluorescent labels
(e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish
peroxidase, P-galactosidase, luciferase, alkaline phosphatase), chemiluminescent
markers, biotinyl groups, predetermined polypeptide epitopes recognized by a
secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary
antibodies, metal binding domains, epitope tags), magnetic agents, such as gadolinium
chelates, toxins such as pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium
bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine,
colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine,
tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.

23
In some embodiments, labels are attached by spacer arms of various lengths to reduce
potential steric hindrance.
The term "agent" is used herein to denote a chemical compound, a
mixture of chemical compounds, a biological macromolecule, or an extract made
from biological materials. The term "pharmaceutical agent or drug" as used herein
refers to a chemical compound or composition capable of inducing a desired
therapeutic effect when properly administered to a patient. Other chemistry terms
herein are used according to conventional usage in the art, as exemplified by The
McGraw-Hill Dictionary of Chemical Terms (Parker, S.', Ed., McGraw-Hill, San
Francisco (1985)), incorporated herein by reference).
The term "antineoplastic agent" is used herein to refer to agents that
have the functional property of inhibiting a development or progression of a neoplasm
in a human, particularly a malignant (cancerous) lesion, such as a carcinoma,
sarcoma, lymphoma, or leukemia. Inhibition of metastasis is frequently a property of
antineoplastic agents.
The term patient includes human and veterinary subjects.
Human Anti-IGF-IR Antibodies
and Characterization Thereof
Human antibodies avoid certain of the problems associated with
antibodies that possess mouse or rat variable and/or constant regions. The presence of
such mouse or rat derived sequences can lead to the rapid clearance of the antibodies
or can lead to the generation of an immune response against the antibody by a patient.
Therefore, in one embodiment, the invention provides humanized anti-IGF-IR
antibodies. In a preferred embodiment, the invention provides fully human anti-IGF-
IR antibodies by introducing human immunoglobulin genes into a rodent so that the
rodent produces fully human antibodies. More preferred are fully human anti-human
IGF-IR antibodies. Fully human anti-IGF-IR antibodies are expected to minimize the
immunogenic and allergic responses :ntrinsic to mouse or mouse-derivatized
monoclonal antibodies (Mabs) and thus to increase the efficacy and safety of "the
administered antibodies. The use of fully human antibodies can be expected to
provide a substantial advantage in the treatment of chronic and recurring human
diseases, such as inflammation and cancer, which may require repeated antibody

24
administrations. In another embodiment, the invention provides an anti-IGF-IR
antibody that does not bind complement.
In a preferred embodiment, the anti-IGF-IR antibody is 2.12.1, 2.13.2,
2.14.3, 3.1.1, 4.9.2,4.17.3 or 6.1.1. In another preferred embodiment, the anti-IGF-IR
antibody comprises a light chain comprising an amino acid sequence selected from
SEQ ID NO: 2, 6, 10, 14, 18 or 22, or one or more CDRs from these amino acid
sequences. In another preferred embodiment, the anti-IGF-IR antibody comprises a
heavy chain comprising an amino acid sequence selected from SEQ ID NO: 4, 8, 12,
16, 20 or 24 or one or more CDRs from these amino acid sequences.
Class and Subclass of Anti-IGF-IR Antibodies
The antibody may be an IgG, an IgM, an IgE, an IgA or an IgD
molecule. In a preferred embodiment, the antibody is an IgG and is an IgGl, IgG2,
IgG3 or IgG4 subtype. In a more preferred embodiment, the anti-IGF-IR antibody is
subclass IgG2. In another preferred embodiment, the anti-IGF-IR antibody is the
same class and subclass as antibody 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2,4.17.3 or
6.1.1, which is IgG2.
The class and subclass of anti-IGF-IR antibodies may be determined
by any method known in the art. In general, the class and subclass of an antibody
may be determined using antibodies that are specific for a particular class and
subclass of antibody. Such antibodies are available commercially. The class and
subclass can be determined by ELISA, Western Blot as well as other techniques.
Alternatively, the class and subclass may be determined by sequencing all or a portion
of the constant domains of the heavy and/or light chains of the antibodies, comparing
their amino acid sequences to the known amino acid sequences of various class and
subclasses of immunoglobulins, and determining the class and subclass of the
antibodies.
Species and Molecule Selectivity
In another aspect of the invention, the anti-IGF-IR antibody •
demonstrates both species and molecule selectivity. In one embodiment, the anti-
IGF-IR antibody binds to human, cynomologous or rhesus IGF-JR. In a preferred
embodiment, the anti-IGF-IR antibody does not bind to mouse, rat, guinea pig, dog or
rabbit IGF-IR. In another preferred embodiment, the anti-IGF-IR antibody does not

25
bind to a New World monkey species such as a marmoset. Following the teachings of
the specification, one may determine the species selectivity for the anti-IGF-IR
antibody using methods well known in the art. For instance, one may determine
species selectivity using Western blot, FACS, ELISA or RIA. In a preferred
embodiment, one may determine the species selectivity using Western blot.
In another embodiment, the anti-IGF-IR antibody has a selectivity for
IGF-IR that is at least 50 times greater than its selectivity for insulin receptor. In a
preferred embodiment, the selectivity of the anti-IGF-IR antibody is more than 100
times greater than its selectivity for insulin receptor. In an even more preferred
embodiment, the anti-IGF-IR antibody does not exhibit any appreciable specific
binding to any other protein other than IGF-IR. One may determine the selectivity of
the anti-IGF-IR antibody for IGR-IR using methods well known in the art following
the teachings of the specification. For instance, one may determine the selectivity
using Western blot, FACS, ELISA or RIA. In a preferred embodiment, one may
determine the molecular selectivity using Western blot.
Binding Affinity of Anti-IGF-IR to IGF-IR
In another aspect of the invention, the anti-IGF-IR antibodies bind to
IGF-IR with high affinity. In one embodiment, the anti-IGF-IR antibody binds to
IGF-IR with a Kd of 1 x 10"8 M or less. In a more preferred embodiment, the antibody
binds to IGF-IR with a Kd or 1 x 10"9 M or less. In an even more preferred
embodiment, the antibody binds to IGF-IR with a Kd or 5 x 10"10 M or less. In
another preferred embodiment, the antibody binds to IGF-IR with a Kd or 1 x 10'10 M
or less. In another preferred embodiment, the antibody binds to IGF-IR with
substantially the same Kd as an antibody selected from 2.12.1, 2.13.2, 2.14.3, 3.1.1,
4.9.2, 4.17.3 or 6.1.1. In another preferred embodiment, the antibody binds to IGF-IR
with substantially the same Kd as an antibody that comprises one or more CDRs from
an antibody selected from 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1. In still
another preferred embodiment, the antibody binds to IGF-ER with substantially the
same Kd as an antibody that comprises one of the amino acid sequences selected from
SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24. In another preferred
embodiment, the antibody binds to IGF-IR with substantially the same Kd as an
antibody that comprises one or more CDRs from an antibody that comprises one of

26
the amino acid sequences selected from SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18,
20, 22 or 24.
In another aspect of the invention, the anti-IGF-IR antibody has a low
dissociation rate. In one embodiment, the anti-IGF-ER antibody has an Korrof 1 x
10"1 s'1 or lower. In a preferred embodiment, the Koff-is 5 x 10'5 s'1 or lower. In
another preferred embodiment, the Koff is substantially the same as an antibody
selected from 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1. In another preferred
embodiment, the antibody binds to IGF-IR with substantially the same Koff as an
antibody that comprises one or more CDRs from an antibody selected from 2.12.1,
2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1. In still another preferred embodiment, the
antibody binds to IGF-IR with substantially the same Koff as an antibody that
comprises one of the amino acid sequences selected from SEQ ID NOS: 2, 4, 6, 8, 10,
12,14,16, 18,20, 22 or 24. In another preferred embodiment, the antibody binds to
IGF-IR with substantially the same Koff as an antibody that comprises one or more
CDRs from an antibody that comprises one of the amino acid sequences selected from
SEQ ID NOS: 2,4, 6, 8, 10, 12, 14,16,18, 20, 22.
The binding affinity and dissociation rate of an anti-IGF-IR antibody to
IGF-IR may be determined by any method known in the art. In one embodiment, the
binding affinity can be measured by competitive ELISAs, RIAs or surface plasmon
resonance, such as BIAcore. The dissociation rate can also be measured by surface
plasmon resonance. In a more preferred embodiment, the binding affinity and
dissociation rate is measured by surface plasmon resonance. In an even more
preferred embodiment, the binding affinity and dissociation rate is measured using a
BIAcore. An example of determining binding affinity and dissociation rate is
described below in Example II.
Half-Life of Anti-IGF-IR Antibodies
According to another object of the invention, the anti-IGF-IR antibody
has a half-life of at least one day in vitro or in vivo. In a preferred embodiment, the
antibody or portion thereof has a half-life of at least three days. In a more preferred
embodiment, the antibody or portion thereof has a half-life of four days or longer. In
another embodiment, the antibody or portion thereof has a half-life of eight days or
longer. In another embodiment, the antibody or antigen-binding portion thereof is
derivatized or modified such that it has a longer half-life, as discussed below. In

27
another preferred embodiment, the antibody may contain point mutations to increase
serum half life, such as described WO 00/09560, published February 24,2000.
The antibody half-life may be measured by any means known to one
having ordinary skill in the art. For instance, the antibody half life may be measured
by Western blot, ELISA or R1A over an appropriate period of time. The antibody
half-life may be measured in any appropriate animals, e.g., a monkey, such as a
cynomologous monkey, a primate or a human.
Identification oflGF-IR Epitopes
Recognized by Anti-ICF-IR Antibody
The invention also provides an anti-IGF-IR antibody that binds the
same antigen or epitope as a human anti-IGF-IR antibody. Further, the invention
provides an anti-IGF-IR antibody that cross-competes with a human anti-IGF-IR
antibody. In a preferred embodiment, the human anti-IGF-IR antibody is 2.12.1,
2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1. In another preferred embodiment, the
human anti-IGF-IR comprises one or more CDRs from an antibody selected from
2.12.1,2.13.2, 2.14.3, 3.1.1, 4.9.2,4.17.3 or 6.1.1. In still another preferred
embodiment, the human anti-IGF-IR comprises one of the amino acid sequences
selected from SEQ ID NOS: 2, 4, 6, 8, 10,12, 14, 16,18, 20, 22 or 24. In another
preferred embodiment, the human anti-IGF-IR comprises one or more CDRs from an
antibody that comprises one of the amino acid sequences selected from SEQ ED NOS:
2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22 or 24. In a highly preferred embodiment, the anti-
IGF-IR antibody is another human antibody.
One may determine whether an anti-IGF-IR antibody binds to the same
antigen using a variety of methods known in the art. For instance, one may determine
whether a test anti-IGF-IR antibody binds to the same antigen by using an anti-IGF-
IR antibody to capture an antigen that is known to bind to the anti-IGF-ER antibody,
such as IGF-IR, eluting the antigen from the antibody, and then determining whether
the test antibody will bind to the eluted antigen. One may determine whether an
antibody binds to the same epitope as an anti-IGF-ER antibody by binding the' anti-
IGF-ER antibody to IGF-IR under saturating conditions, and then measuring the
ability of the test antibody to bind to IGF-IR. If the test antibody is able to bind to the
IGF-ER at the same time as the anti-IGF-ER antibody, then the test antibody binds to a
different epitope as the anti-IGF-ER antibody. However, if the test antibody is not

28
able to bind to the IGF-IR at the same time, then the test antibody binds to the same
epitope as the human anti-IGF-IR antibody. This experiment may be performed using
ELISA, RIA or surface plasmon resonance. In a preferred embodiment, the
experiment is performed using surface plasmon resonance. In a more preferred
embodiment, BIAcore is used. One may also determine whether an anti-IGF-IR
antibody cross-competes with an anti-IGF-IR antibody. In a preferred embodiment,
one may determine whether an anti-IGF-IR antibody cross-competes with another by
using the same method that is used to measure whether the anti-IGF-IR antibody is
able to bind to the same epitope as another anti-IGF-IR antibody.
Light and Heavy Chain Usage
The invention also provides an anti-IGF-IR antibody that comprises
variable sequences encoded by a human K gene. In a preferred embodiment, the
variable sequences are encoded by either the VK A27, A3 0 or 012 gene family. In a
preferred embodiment, the variable sequences are encoded by a human VK A30 gene
family. In a more preferred embodiment, the light chain comprises no more than ten
amino acid substitutions from the germline VK A27, A30 or 012, preferably no more
than six amino acid substitutions, and more preferably no more than three amino acid
substitutions. In a preferred embodiment, the amino acid substitutions are
conservative substitutions.
SEQ ID NOS: 2, 6, 10, 14, 18 and 22 provide the amino acid
sequences of the variable regions of six anti-IGF-IR K light chains. SEQ ID NOS: 38,
40 and 42 provide the amino acid sequences of the three germline K light chains from
which the six anti-IGF-IR K light chains are derived. Figs. 1A-1C show the
alignments of the nucleotide sequences of the variable regions of the light chains of
the six anti-IGF-IR antibodies to each other and to the germline sequences from
which they are derived. Following the teachings of this specification, one of ordinary
skill in the art could determine the encoded amino acid sequence of the six anti-IGF-
IR K light chains and the germline K light chains and determine the differences
between the germline sequences and the antibody sequences.
In a preferred embodiment, the VL of the anti-IGF-IR antibody
contains the same amino acid substitutions, relative to the germline amino acid
sequence, as any one or more of the VL of antibodies 2.12.1, 2.13.2, 2.14.3, 3.1.1,
4.9.2, 4.17.3 or 6.1.1. For example, the VL of the anti-IGF-IR antibody may contain

29
one or more amino acid substitutions that are the same as those present in antibody
2.13.2, another amino acid substitution that is the same as that present in antibody
2.14.3, and another amino acid substitution that is the same as antibody 4.9.2. In this
manner, one can mix and match different features of antibody binding in order to
alter, e.g., the affinity of the antibody for IGF-IR or its dissociation rate from the
antigen. In another embodiment, the amino acid substitutions are made in the same
position as those found in any one or more of the VL of antibodies 2.12.1, 2.13.2,
2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1, but conservative amino acid substitutions are
made rather than using the same amino acid. For example, if the amino acid
substitution compared to the germline in one of the antibodies 2.12.1, 2.13.2, 2.14.3,
3.1.1, 4.9.2, 4.17.3 or 6.1.1 is glutamate, one may conservatively substitute aspartate.
Similarly, if the amino acid substitution is serine, one may conservatively substitute
threonine.
In another preferred embodiment, the light chain comprises an amino
acid sequence that is the same as the amino acid sequence of the VL of 2.12.1,2.13.2,
2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1. In another highly preferred embodiment, the light
chain comprises amino acid sequences that are the same as the CDR regions of the
light chain of 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1. In another preferred
embodiment, the light chain comprises an amino acid sequence from at least one CDR
region of the light chain of 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1. In
another preferred embodiment, the light chain comprises amino acid sequences from
CDRs from different light chains. In a more preferred embodiment, the CDRs from
different light chains are obtained from 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or
6.1.1. In another preferred embodiment, the light chain comprises an amino acid
sequence selected from SEQ ED NOS: 2, 6, 10, 14, 18 or 22. In another embodiment,
the light chain comprises an amino acid sequence encoded by a nucleic acid sequence
selected from SEQ ID NOS: 1,5,9, 13, 17 or 21, or a nucleic acid sequence that
encodes an amino acid sequence having 1-10 amino acid insertions, deletions or
substitutions therefrom. Preferably, the amino acid substitutions are conservative
amino acid substitutions. In another embodiment, the antibody or portion thereof
comprises a lambda light chain.
The present invention also provides an anti-IGF-IR antibody or portion
thereof comprises a human heavy chain or a sequence derived from a human heavy

30
chain. In one embodiment, the heavy chain amino acid sequence is derived from a
human VH DP-35, DP-47, DP-70, DP-71 or VIV-4/4.35 gene family. In a preferred
embodiment, the heavy chain amino acid sequence is derived from a human VH DP-
47 gene family. In a more preferred embodiment, the heavy chain comprises no more
than eight amino acid changes from germline VH DP-35, DP-47, DP-70, DP-71 or
VIV-4/4.35, more preferably no more than six amino acid changes, and even more
preferably no more than three amino acid changes.
SEQ ID NOS: 4, 8,12,16,20 and 24 provide the amino acid
sequences of the variable regions of six anti-IGF-IR heavy chains. SEQ ID NOS: 30,
32, 34, 36 and 44 provide the amino acid sequences and SEQ ID NOS: 29, 31, 33, 35
and 43 provide the nucleotide sequences of the germline heavy chains DP-35, DP-47,
DP-70, DP-71 and VIV-4, respectively. Figs. 2A-2D show the alignments of the
amino acid sequences of the variable region of the six anti-IGF-IR antibodies to their
corresponding germline sequences. Following the teachings of this specification, one
of ordinary skill in the art could determine the encoded amino acid sequence of the six
anti-IGF-IR heavy chains and the germline heavy chains and determine the
differences between the germline sequences and the antibody sequences.
In a preferred embodiment, the VH of the anti-IGF-IR antibody
contains the same amino acid substitutions, relative to the germline amino acid
sequence, as anyone or more of the VH of antibodies 2.12.1, 2.13.2,2.14.3, 3.1.1,
4.9.2,4.17.3 or 6.1.1. Similar to what was discussed above, the VH of the anti-IGF-
IR antibody may contain one or more amino acid substitutions that are the same as
those present in antibody 2.13.2, another amino acid substitution that is the same as
that present in antibody 2.14.3, and another amino acid substitution that is the same as
antibody 4.9.2. In this manner, one can mix and match different features of antibody
binding in order to alter, e.g., the affinity of the antibody for IGF-IR or its dissociation
rate from the antigen. In another embodiment, the amino acid substitutions are made
in the same position as those found in any one or more of the VH of antibodies 2.12.1,
2.13.2, 2.14.3, 3.1.1, 4.17.3., 4.9.2 or 6.1.1, but conservative amino acid substitutions
are made rather than using the same amino acid.
In another preferred embodiment, the heavy chain comprises an amino
acid sequence that is the same as the amino acid sequence of the VH of 2.12.1, 2.13.2,
2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1. In another highly preferred embodiment, the

31
heavy chain comprises amino acid sequences that are the same as the CDR regions of
the heavy chain of 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1. In another
preferred embodiment, the heavy chain comprises an amino acid sequence from at
least one CDR region of the heavy chain of 2.12.1, 2.13.2,2.14.3, 3.1.1,4.9.2,4.17.3
or 6.1.1. In another preferred embodiment, the heavy chain comprises amino acid
sequences from CDRs from different heavy chains. In a more preferred embodiment,
the CDRs from different heavy chains are obtained from 2.12.1, 2.13.2,2.14.3, 3.1.1,
4.9.2, 4.17.3 or 6.1.1. In another preferred embodiment, the heavy chain comprises an
amino acid sequence selected from SEQ ID NOS: 4, 8, 12, 16, 20 or 24. In another
embodiment, the heavy chain comprises an amino acid sequence encoded by a nucleic
acid sequence selected from SEQ ID NOS: 3, 7, 11, 15,19 or 23, or a nucleic acid
sequence that encodes an amino acid sequence having 1-10 amino acid insertions,
deletions or substitutions therefrom. In another embodiment, the substitutions are
conservative amino acid substitutions.
Inhibition of IGF-IR Activity by Anti-IGF-IR Antibody
Inhibition of IGF-I Binding to IGF-IR
In another embodiment, the invention provides an ahti-IGF-IR
antibody that inhibits the binding of IGF-I to IGF-IR or the binding of IGF-II to IGF-
IR. In a preferred embodiment, the IGF-IR is human. In another preferred
embodiment, the anti-IGF-IR antibody is a human antibody. In another embodiment,
the antibody or portion thereof inhibits binding between IGF-IR and IGF-I with an
IC50 of no more than 100 nM. In a preferred embodiment, the IC50 is no more than 10
nM. In a more preferred embodiment, the IC50 is no more than 5 nM. The IC50 can
be measured by any method known in the art. Typically, an IC50 can be measured by
ELISA or RIA. In a preferred embodiment, the IC50 is measured by RIA.
In another embodiment, the invention provides an anti-IGF-IR
antibody that prevents activation of the IGF-IR in the presence of IGF-I. In a
preferred embodiment, the anti-IGF-IR antibody inhibits IGF-IR-induced tyrasine
phosphorylation that occurs upon occupancy of the receptor. In another preferred
embodiment, the anti-IGF-IR antibody inhibits downstream cellular events from
occurring. For instance, the anti-IGF-IR can inhibit tyrosine phosphorylation of She
and insulin receptor substrate (IRS) 1 and 2, all of which are normally phosphorylated

32
when cells are treated with IGF-I (Kim et al., J. Biol. Chem. 273: 34543-34550,
1998). One can determine whether an anti-IGF-IR antibody can prevent activation of
IGF-IR in the presence of IGF-1 by determining the levels of autophosphorylation for
IGF-IR, She, IRS-1 or IRS-2 by Western blot or immunopreciptation. In a preferred
embodiment, one would, determine the levels of autophosphorylation of IGF-IR by
Western blot. See, e.g., Example VII.
In another aspect of the invention, the antibody causes the
downregulation of IGF-IR from a cell treated with the antibody. In one embodiment,
the IGF-IR is internalized into the cytoplasm of the cell. After the anti-IGF-IR
antibody binds to IGF-IR, the antibody is internalized, as shown by confocal
microscopy. Without wishing to be bound to any theory, it is believed that the
antibody-IGF-IR complex is internalized into a lysosome and degraded. One may
measure the downregulation of IGF-IR by any method known in the art including
immunoprecipitation, confocal microscopy or Western blot. See, e.g., Example VII.
In a preferred embodiment, the antibody is selected 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2,
or 6.1.1, or comprises a heavy chain, light chain or antigen-binding region thereof.
Activation of IGF-IR by Anti-IGF-IR Antibody
Another aspect of the present invention involves activating anti-IGF-IR
antibodies. An activating antibody differs from an inhibiting antibody because it
amplifies or substitutes for the effects of IGF-I on IGF-IR. In one embodiment, the
activating antibody is able to bind to IGF-IR and cause it to be activated in the
absence of IGF-I. This type of activating antibody is essentially a mimic of IGF-I. In
another embodiment, the activating antibody amplifies the effect of IGF-I on IGF-IR.
This type of antibody does not activate IGF-IR by itself, but rather increases the
activation of IGF-ER in the presence of IGF-I. A mimic anti-IGF-IR antibody may be
easily distinguished from an amplifying anti-IGF-IR antibody by treating cells in vitro
with an antibody in the presence or absence of low levels of IGF-I. If the antibody is
able to cause IGF-IR activation in the absence of IGF-I, e.g., it increases IGF-IR
tyrosine phosphorylation, then the antibody is a mimic antibody. If the antibody
cannot cause IGF-IR activation in the absence of IGF-I but is able to amplify the
amount of IGF-IR activation, then the antibody is an amplifying antibody. In a
preferred embodiment, the activating antibody is 4.17.3. In another preferred
embodiment, the antibody comprises one or more CDRs from 4.17.3. In another

33
preferred embodiment, the antibody is derived from either or both of the germline
sequences 012 (light chain) and/or D71 (heavy chain).
Inhibition of IGF-IR Tyrosine Phosphorylation, IGF-IR Levels and Tumor Cell
Growth In Vivo by Anti-IGF-IR Antibodies
Another embodiment of the invention provides an anti-IGF-IR
antibody that inhibits IGF-IR tyrosine phosphorylation and receptor levels in vivo. In
one embodiment, administration of anti-IGF-IR antibody to an animal causes a
reduction in IGF-IR phosphotyrosine signal in IGF-IR-expressing tumors. In a
preferred embodiment, the anti-IGF-IR antibody causes a reduction in
phosphotyrosine signal by at least 20%. In a more preferred embodiment, the anti-
IGF-IR antibody causes a decrease in phosphotyrosine signal by at least 60%, more
preferably 50%. In an even more preferred embodiment, the antibody causes a
decrease in phosphotyrosine signal of at least 40%, more preferably 30%, even more
preferably 20%. In a preferred embodiment, the antibody is administered
approximately 24 hours before the levels of tyrosine phosphorylation are measured.
The levels of tyrosine phosphorylation may be measured by any method known in the
art, such as those described infra. See, e.g., Example III and Figure 5. In a preferred
embodiment, the antibody is selected from 2.12.1, 2.13.2, 2.14.3, 3.1.1,4.9.2, or
6.1.1, or comprises a heavy chain, light chain or antigen-binding portion thereof.
In another embodiment, administration of anti-IGF-IR antibody to an
animal causes a reduction in IGF-IR levels in IGF-IR-expressing tumors. In a
preferred embodiment, the anti-IGF-IR antibody causes a reduction in receptor levels
by at least 20% compared to an untreated animal. In a more preferred embodiment,
the anti-IGF-IR antibody causes a decrease in receptor levels to at least 60%, more
preferably 50% of the receptor levels in an untreated animal. In an even more
preferred embodiment, the antibody causes a decrease in receptor levels to at least
40%, more preferably 30%. In a preferred embodiment, the antibody is administered
approximately 24 hours before the IGF-IR levels are measured. The IGF-IR levels
may be measured by any method known in the art, such as those described infra. See,
e.g., Example VIII and Figure 6. In a preferred embodiment, the antibody is selected
from 2.12.1, 2.13.2, 2.14.3, 3.1.1,4.9.2, or 6.1.1, or comprises a heavy chain, light
chain or antigen-binding portion thereof.

34
In another embodiment, an anti-IGF-IR antibody inhibits tumor cell
growth in vivo. The tumor cell may be derived from any cell type including, without
limitation, epidermal, epithelial, endothelial, leukemia, sarcoma, multiple myeloma or
mesodermal cells. Examples of tumor cells include A549 (non-small cell lung
carcinoma) cells, MCF-7 cells, Colo 205 cells, 3T3/IGF-IR cells and A431 cells. In a
preferred embodiment, the antibody inhibits tumor cell growth as compared to the
growth of the tumor in an untreated animal. In a more preferred embodiment, the
antibody inhibits tumor cell growth by 50%. In an even more preferred embodiment,
the antibody inhibits tumor cell growth by 60%, 65%, 70% or 75%. In one
embodiment, the inhibition of tumor cell growth is measured at least 7 days after the
animals have started treatment with the antibody. In a more preferred embodiment,
the inhibition of tumor cell growth is measured at least 14 days after the animals have
started treatment with the antibody. In another preferred embodiment, another
antineoplastic agent is administered to the animal with the anti-IGF-IR antibody. In a
preferred embodiment, the antineoplastic agent is able to further inhibit tumor cell
growth. In an even more preferred embodiment, the antineoplastic agent is
adriamycin, taxol, tamoxifen, 5-fluorodeoxyuridine (5-FU) or CP-358,774. In a
preferred embodiment, the co-administration of an antineoplastic agent and the anti-
IGF-IR antibody inhibits tumor cell growth by at least 50%, more preferably 60%,
65%, 70% or 75%, more preferably 80%, 85% or 90% after a period of 22-24 days.
See, e.g., Fig. 7 and Example IX. In a preferred embodiment, the antibody is selected
from 2.12.1, 2.13.2,2.14.3, 3.1.1, 4.9.2, or 6.1.1, or comprises a heavy chain, light
chain or antigen-binding portion thereof.
Induction of Apoptosis by Anti-IGF-IR Antibodies
Another aspect of the invention provides an anti-IGF-IR antibody that
induces cell death. In one embodiment, the antibody causes apoptosis. The antibody
may induce apoptosis either in vivo or in vitro. In general, tumor cells are more
sensitive to apoptosis than normal cells, such that administration of an anti-IGF-IR
antibody causes apoptosis of a tumor cell preferentially to that of a normal cell. In
another embodiment, the administration of an anti-IGF-IR antibody decreases levels
of an enzyme, akt, which is involved in the phosphatidyl inositol (PI) kinase pathway.
The PI kinase pathway, in turn, is involved in the cell proliferation and prevention of
apoptosis. Thus, inhibition of akt can cause apoptosis. In a more preferred

35
embodiment, the antibody is administered in vivo to cause apoptosis of an IGF-IR-
expressing cell. In a preferred embodiment, the antibody is selected from 2.12.1,
2.13.2, 2.14.3, 3.1.1, 4.9.2, or 6.1.1, or comprises a heavy chain, light chain or
antigen-binding portion thereof.
Methods of Producing Antibodies and Antibody-Producing Cell Lines
Immunization
In one embodiment of the instant invention, human antibodies are
produced by immunizing a non-human animal comprising some or all of the human
immunoglobulin locus with an IGF-IR antigen. In a preferred embodiment, the non-
human animal is a XENOMOUSE™, which is an engineered mouse strain that
comprises large fragments of the human immunoglobulin loci and is deficient in
mouse antibody production. See, e.g., Green et al. Nature Genetics 7:13-21 (1994)
and United States Patents 5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181,
6,091,001, 6,114,598 and 6,130,364. See also WO 91/10741, published July 25,
1991, WO 94/02602, published February 3, 1994, WO 96/34096 and WO 96/33735,
both published October 31, 1996, WO 98/16654, published April 23, 1998, WO
98/24893, published June 11,1998, WO 98/50433, published November 12, 1998,
WO 99/45031, published September 10, 1999, WO 99/53049, published October 21,
1999, WO 00 09560, published February 24, 2000 and WO 00/037504, published
June 29, 2000. The XENOMOUSE ™ produces an adult-like human repertoire of
fully human antibodies, and generates antigen-specific human Mabs. A second
generation XENOMOUSE ™ contains approximately 80% of the human antibody
repertoire through introduction of megabase sized, germline configuration YAC
fragments of the human heavy chain loci and K light chain loci. See Mendez et al.
Nature Genetics 15:146-156 (1997), Green and Jakobovits J. Exp. Med. 188:483-495
(1998), the disclosures of which are hereby incorporated by reference.
The invention also provides a method for making anti-IGF-IR
antibodies from non-human, non-mouse animals by immunizing non-human -
transgenic animals that comprise human immunoglobulin loci. One may produce
such animals using the methods described immediately above. The methods disclosed
in these patents may modified as described in United States Patent 5,994,619. In a

36
preferred embodiment, the non-human animals may be rats, sheep, pigs, goats, cattle
or horses.
In another embodiment, the non-human animal comprising human
immunoglobulin gene loci are animals that have a "minilocus" of human
immunoglobulins. In the minilocus approach, an exogenous Ig locus is mimicked
through the inclusion of individual genes from the Ig locus. Thus, one or more VH
genes, one or more DH genes, one or more JH genes, a mu constant region, and a
second constant region (preferably a gamma constant region) are formed into a
construct for insertion into an animal. This approach is described, inter alia, in U.S.
Patent No. 5,545,807, 5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016,
5,770,429, 5,789,650, 5,814,318, 5,591,669, 5,612,205, 5,721,367, 5,789,215, and
5,643,763, hereby incorporated by reference.
An advantage of the minilocus approach is the rapidity with which
constructs including portions of the Ig locus can be generated and introduced into
animals. However, a potential disadvantage of the minilocus approach is that there
may not be sufficient immunoglobulin diversity to support full B-cell development,
such that there may be lower antibody production.
In order to produce a human anti-IGF-IR antibody, a non-human
animal comprising some or all of the human immunoglobulin loci is immunized with
an IGF-IR antigen and the antibody or the antibody-producing cell is isolated from the
animal. The IGF-IR antigen may be isolated and/or purified IGF-IR and is preferably
a human IGF-IR. In another embodiment, the IGF-IR antigen is a fragment of IGF-
IR, preferably the extracellular domain of IGF-IR. In another embodiment, the IGF-IR
antigen is a fragment that comprises at least one epitope of IGF-ER. In another
embodiment, the IGF-IR antigen is a cell that expresses IGF-IR on its cell surface,
preferably a cell that overexpresses IGF-IR on its cell surface.
Immunization of animals may be done by any method known in the
art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual. New York: Cold
Spring Harbor Press, 1990. Methods for immunizing non-human animals such as
mice, rats, sheep, goats, pigs, cattle and horses are well known in the art. See, e.g.,
Harlow and Lane and United States Patent 5,994,619. In a preferred embodiment, the
IGF-IR antigen is administered with a adjuvant to stimulate the immune response.
Such adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl

37
dipeptides) or ISCOM (immunostimulating complexes). Such adjuvants may protect
the polypeptide from rapid dispersal by sequestering it in a local deposit, or they may
contain substances that stimulate the host to secrete factors that are chemotactic for
macrophages and other components of the immune system. Preferably, if a
polypeptide is being administered, the immunization schedule will involve two or
more administrations of the polypeptide, spread out over several weeks.
Example I provides an protocol for immunizing a XENOMOUSE
with full-length human IGF-IR in phosphate-buffered saline.
Production of Antibodies and Antibody-Producing Cell Lines
After immunization of an animal with an IGF-IR antigen, antibodies
and/or antibody-producing cells may be obtained from the animal. An anti-IGF-IR
antibody-containing serum is obtained from the animal by bleeding or sacrificing the
animal. The serum may be used as it is obtained from the animal, an immunoglobulin
fraction may be obtained from the serum, or the anti-IGF-IR antibodies may be
purified from the serum. Serum or immunoglobulins obtained in this manner are
polyclonal, which are disadvantageous because the amount of antibodies that can be
obtained is limited and the polyclonal antibody has a heterogeneous array of
properties.
In another embodiment, antibody-producing immortalized hybridomas
may be prepared from the immunized animal. After immunization, the animal is
sacrificed and the splenic B cells are fused to immortalized myeloma cells as is well-
known in the art. See, e.g., Harlow and Lane, supra. In a preferred embodiment, the
myeloma cells do not secrete immunoglobulin polypeptides (a non-secretory cell
line). After fusion and antibiotic selection, the hybridomas are screened using IGF-
IR, a portion thereof, or a cell expressing IGF-IR. In a preferred embodiment, the
initial screening is performed using an enzyme-linked immunoassay (ELISA) or a
radioimmunoassay (RIA), preferably an ELISA. An example of ELISA screening is
provided in WO 00/37504, herein incorporated by reference.
In another embodiment, antibody-producing cells may be prepared
from a human who has an autoimmune disorder and who expresses anti-IGF-IR
antibodies. Cells expressing the anti-IGF-IR antibodies may be isolated by isolating
white blood cells and subjecting them to fluorescence-activated cell sorting (FACS)
or by panning on plates coated with IGF-IR or a portion thereof. These cells may be

38
fused with a human non-secretory myeloma to produce human hybridomas expressing
human anti-IGF-IR antibodies. In general, this is a less preferred embodiment
because it is likely that the anti-IGF-IR antibodies will have a low affinity for IGF-IR.
Anti-IGF-IR antibody-producing hybridomas are selected, cloned and
further screened for desirable characteristics, including robust hybridoma growth,
high antibody production and desirable antibody characteristics, as discussed further
below. Hybridomas may be cultured and expanded in vivo in syngeneic animals, in
animals that lack an immune system, e.g., nude mice, or in cell culture in vitro.
Methods of selecting, cloning and expanding hybridomas are well known to those of
ordinary skill in the art.
Preferably, the immunized animal is a non-human animal that
expresses human immunoglobulin genes and the splenic B cells are fused to a
myeloma derived from the same species as the non-human animal. More preferably,
the immunized animal is a XENOMOUSE and the myeloma cell line is a non-
secretory mouse myeloma, such as the myeloma cell line is NSO-bcl2. See, e.g.,
Example I.
In one aspect, the invention provides hybridomas are produced that
produce human anti-IGF-IR antibodies. In a preferred embodiment, the hybridomas
are mouse hybridomas, as described above. In another preferred embodiment, the
hybridomas are produced in a non-human, non-mouse species such as rats, sheep,
pigs, goats, cattle or horses. In another embodiment, the hybridomas are human
hybridomas, in which a human non-secretory myeloma is fused with a human cell
expressing an anti-IGF-IR antibody.
Nucleic Acids, Vectors, Host Cells and
Recombinant Methods of Making Antibodies
Nucleic Acids
Nucleic acid molecules encoding anti-IGF-IR antibodies of the
invention are provided. In one embodiment, the nucleic acid molecule encodes a
heavy and/or light chain of an anti-IGF-IR immunoglobulin. In a preferred
embodiment, a single nucleic acid molecule encodes a heavy chain of an anti-IGF-IR
immunoglobulin and another nucleic acid molecule encodes the light chain of an anti-
IGF-IR immunoglobulin. In a more preferred embodiment, the encoded

39
immunoglobulin is a human immunoglobulin, preferably a human IgG. The encoded
light chain may be a λ chain or a K chain, preferably a K chain.
The nucleic acid molecule encoding the variable region of the light
chain may be derived from the A30, A27 or 012 VK gene. In a preferred
embodiment, the light chain is derived from the A30 VK gene. In another preferred
embodiment, the nucleic acid molecule encoding the light chain comprises the joining
region derived from JKI, JK2 or JK4. In an even more preferred embodiment, the
nucleic acid molecule encoding the light chain contains no more than ten amino acid
changes from the germline A30 VK gene, preferably no more than six amino acid
changes, and even more preferably no more than three amino acid changes.
The invention provides a nucleic acid molecule that encodes a variable
region of the light chain (VL) containing at least three amino acid changes compared
to the germline sequence, wherein the amino acid changes are identical to the amino
acid changes from the germline sequence from the VL of one of the antibodies 2.12.1,
2.13.2, 2.14.3, 3.1.1, 4.9.2,4.17.3 or 6.1.1. The invention also provides a nucleic acid
molecule comprising a nucleic acid sequence that encodes the amino acid sequence of
the variable region of the light chain of 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or
6.1.1. The invention also provides a nucleic acid molecule comprising a nucleic acid
sequence that encodes the amino acid sequence of one or more of the CDRs of any
one of the light chains of 2.12.1, 2.13.2, 2.14.3, 3.1.1,4.9.2, 4.17.3 or 6.1.1. In a
preferred embodiment, the nucleic acid molecule comprises a nucleic acid sequence
that encodes the amino acid sequence of all of the CDRs of any one of the light chains
of 2.12.1, 2.13.2, 2.14.3, 3.1.1,4.9.2,4.17.3 or 6.1.1. In another embodiment, the
nucleic acid molecule comprises a nucleic acid sequence that encodes the amino acid
sequence of one of SEQ ID NOS: 2, 6, 10, 14, 18 or 22 or comprises a nucleic acid
sequence of one of SEQ ID NOS: 1, 5, 9,13, 17 or 21. In another preferred
embodiment, the nucleic acid molecule comprises a nucleic acid sequence that
encodes the amino acid sequence of one or more of the CDRs of any one of SEQ ID
NOS: 2, 6, 10, 14, 18 or 22 or comprises a nucleic acid sequence of one or more of
the CDRs of any one of SEQ ID NOS: 1, 5, 9, 13, 17 or 21. In a more preferred
embodiment, the nucleic acid molecule comprises a nucleic acid sequence that
encodes the amino acid sequence of all of the CDRs of any one of SEQ ID NOS: 2, 6,

40
10, 14, 18 or 22 or comprises a nucleic acid sequence of all the CDRs of any one of
SEQIDNOS: 1,5,9,13, 17 or 21.
The invention also provides a nucleic acid molecules that encodes an
amino acid sequence of a VL that has an amino acid sequence that is at least 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a VL described
above, particularly to a VL that comprises an amino acid sequence of one of SEQ ID
NOS: 2, 6, 10, 14, 18 or 22. The invention also provides a nucleic acid sequence that
is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a
nucleic acid sequence of one of SEQ ID NOS: 1, 5, 9, 13,17 or 21. In another
embodiment, the invention provides a nucleic acid molecule encoding a VL that
hybridizes under highly stringent conditions to a nucleic acid molecule encoding a VL
as described above, particularly a nucleic acid molecule that comprises a nucleic acid
sequence encoding an amino acid sequence of SEQ ID NOS: 2, 6, 10, 14, 18 or 22.
The invention also provides a nucleic acid sequence encoding an VL that hybridizes
under highly stringent conditions to a nucleic acid molecule comprising a nucleic acid
sequence of one of SEQ ID NOS: 1, 5, 9, 13, 17 or 21.
The invention also provides a nucleic acid molecule encoding the
variable region of the heavy chain (VH) is derived from the DP-35, DP-47, DP-71 or
VTV-4/4.35 VH gene, preferably the DP-35 VH gene. In another preferred
embodiment, the nucleic acid molecule encoding the VH comprises the joining region
derived from JH6 or JH5, more preferably JH6. In another preferred embodiment, the
D segment is derived from 3-3, 6-19 or 4-17. In an even more preferred embodiment,
the nucleic acid molecule encoding the VH contains no more than ten amino acid
changes from the germline DP-47 gene, preferably no more than six amino acid
changes, and even more preferably no more than three amino acid changes. In a
highly preferred embodiment, the nucleic acid molecule encoding the VH contains at
least one amino acid change compared to the germline sequence, wherein the amino
acid change is identical to the amino acid change from the germline sequence from
the heavy chain of one of the antibodies 2.12.1, 2.13.2, 2.14.3, 3.1.1,4.9.2,4.17.3 or
6.1.1. In an even more preferred embodiment, the VH contains at least three amino
acid changes compared to the germline sequences, wherein the changes are identical
to those changes from the germline sequence from the VH of one of the antibodies
2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1.

41
In one embodiment, the nucleic acid molecule comprises a nucleic acid
sequence that encodes the amino acid sequence of the VH of 2.12.1, 2.13.2, 2.14.3,
3.1.1, 4.9.2, 4.17.3 or 6.1.1. In another embodiment, the nucleic acid molecule
comprises a nucleic acid sequence that encodes the amino acid sequence of one or
more of the CDRs of the heavy chain of 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or
6.1.1. In a preferred embodiment, the nucleic acid molecule comprises a nucleic acid
sequence that encodes the amino acid sequences of all of the CDRs of the heavy chain
of 2.12.1, 2.13.2, 2.14.3, 3.1.1,4.9.2, 4.17.3 or 6.1.1. In another preferred
embodiment, the nucleic acid molecule comprises a nucleic acid sequence that
encodes the amino acid sequence of one of SEQ ID NOS: 4, 8, 12, 16, 20 or 24 or that
comprises a nucleic acid sequence of one of SEQ ID NOS: 3, 7, 11, 15, 19 or 23. In
another preferred embodiment, the nucleic acid molecule comprises a nucleic acid
sequence that encodes the amino acid sequence of one or more of the CDRs of any
one of SEQ ID NOS: 4, 8, 12, 16, 20 or 24 or comprises a nucleic acid sequence of
oneormoreoftheCDRsofanyoneofSEQIDNOS:3, 7, 11, 15,19 or 23. In a
preferred embodiment, the nucleic acid molecule comprises a nucleic acid sequence
that encodes the amino acid sequences of all of the CDRs of any one of SEQ ID NOS:
4, 8, 12, 16, 20 or 24 or comprises a nucleic acid sequence of all of the CDRs of any
oneofSEQIDNOS:3, 7, 11, 15, 19 or 23.
In another embodiment, the nucleic acid molecule encodes an amino
acid sequence of a VH that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98% or 99% identical to one of the amino acid sequences encoding a VH as described
immediately above, particularly to a VH that comprises an amino acid sequence of
one of SEQ ID NOS: 4, 8,12, 16, 20 or 24. The invention also provides a nucleic
acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or
99% identical to a nucleic acid sequence of one of SEQ ID NOS: 3, 7, 11, 15, 19 or
23. In another embodiment, the nucleic acid molecule encoding a VH is one that
hybridizes under highly stringent conditions to a nucleic acid sequence encoding a
VH as described above, particularly to a VH that comprises an amino acid sequence
of one of SEQ ID NOS: 4, 8, 12, 16, 20 or 24. The invention also provides a nucleic
acid sequence encoding a VH that hybridizes under highly stringent conditions to a
nucleic acid molecule comprising a nucleic acid sequence of one of SEQ ID NOS: 3,
7, 11, 15, 19or23.

42
The nucleic acid molecule encoding either or both of the entire heavy
and light chains of an anti-IGF-IR antibody or the variable regions thereof may be
obtained from any source that produces an anti-IGF-IR antibody. Methods of
isolating mRNA encoding an antibody are well-known in the art. See, e.g., Sambrook
et al. The mRNA may be used to produce cDNA for use in the polymerase chain
reaction (PCR) or cDNA cloning of antibody genes. In one embodiment of the
invention, the nucleic acid molecules may be obtained from a hybridoma that
expresses an anti-IGF-IR antibody, as described above, preferably a hybridoma that
has as one of its fusion partners a transgenic animal cell that expresses human
immunoglobulin genes, such as a XENOMOUSE "*, non-human mouse transgenic
animal or a non-human, non-mouse transgenic animal. In another embodiment, the
hybridoma is derived from a non-human, non-transgenic animal, which may be used,
e.g., for humanized antibodies.
A nucleic acid molecule encoding the entire heavy chain of an anti-
IGF-IR antibody may be constructed by fusing a nucleic acid molecule encoding the
variable domain of a heavy chain or an antigen-binding domain thereof with a
constant domain of a heavy chain. Similarly, a nucleic acid molecule encoding the
light chain of an anti-IGF-IR antibody may be constructed by fusing a nucleic acid
molecule encoding the variable domain of a light chain or an antigen-binding domain
thereof with a constant domain of a light chain. The nucleic acid molecules encoding
the VH and VL chain may be converted to full-length antibody genes by inserting
them into expression vectors already encoding heavy chain constant and light chain
constant regions, respectively, such that the VH segment is operatively linked to the
heavy chain constant region (CH) segment(s) within the vector and the VL segment is
operatively linked to the light chain constant region (CL) segment within the vector.
Alternatively, the nucleic acid molecules encoding the VH or VL chains are converted
into full-length antibody genes by linking, e.g., ligating, the nucleic acid molecule
encoding a VH chain to a nucleic acid molecule encoding a CH chain using standard
molecular biological techniques. The same may be achieved using nucleic acid
molecules encoding VL and CL chains. The sequences of human heavy and light
chain constant region genes are known in the art. See, e.g., Kabat et al., Sequences of
Proteins of Immunological Interest, 5th Ed., NIH Publ. No. 91-3242, 1991. Nucleic
acid molecules encoding the full-length heavy and/or light chains may then be

43
expressed from a cell into which they have been introduced and the anti-IGF-IR
antibody isolated.
In a preferred embodiment, the nucleic acid encoding the variable
region of the heavy chain encodes the amino acid sequence of SEQ ID NOS: 4, 8,12,
16, 20 or 24, and the nucleic acid molecule encoding the variable region of the light
chains encodes the amino acid sequence of SEQ ID NOS: 2, 6, 10, 14, 18 or 22. SEQ
ID NO: 28 depicts the amino acid sequence and SEQ ID NO: 27 depicts the nucleic
acid sequence encoding the constant region of the heavy chain of the anti-IGF-IR
antibodies 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 and 6.1.1. SEQ ID NO: 26
depicts the amino acid sequence and SEQ ID NO: 25 depicts the nucleic acid
sequence encoding the constant region of the light chain of the anti-IGF-IR antibodies
2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 and 6.1.1. Thus, in a preferred embodiment,
the nucleic acid molecule encoding the constant domain of the heavy chain encodes
SEQ ID NO: 28, and the nucleic acid molecule encoding the constant domain of the
light chain encodes SEQ ID NO: 26. In a more preferred embodiment, the nucleic
acid molecule encoding the constant domain of the heavy chain has the nucleic acid
sequence of SEQ ID NO: 27, and the nucleic acid molecule encoding the constant
domain has the nucleic acid sequence of SEQ ID NO: 25.
In another embodiment, a nucleic acid molecule encoding either the
heavy chain of an anti-IGF-IR antibody or an antigen-binding domain thereof, or the
light chain of an anti-IGF-IR antibody or an antigen-binding domain thereof may be
isolated from a non-human, non-mouse animal that expresses human immunoglobulin
genes and has been immunized with an IGF-IR antigen. In other embodiment, the
nucleic acid molecule may be isolated from an anti-IGF-IR antibody-producing cell
derived from a non-transgenic animal or from a human patient who produces anti-
IGF-IR antibodies. Methods of isolating mRNA from the anti-IGF-IR antibody-
producing cells may be isolated by standard techniques, cloned and/or amplified using
PCR and library construction techniques, and screened using standard protocols to
obtain nucleic acid molecules encoding anti-IGF-IR heavy and light chains. .
The nucleic acid molecules may be used to recombinantly express
large quantities of anti-IGF-IR antibodies, as described below. The nucleic acid
molecules may also be used to produce chimeric antibodies, single chain antibodies,
immunoadhesins, diabodies, mutated antibodies and antibody derivatives, as

44
described further below. If the nucleic acid molecules are derived from a non-human,
non-transgenic animal, the nucleic acid molecules may be used for antibody
humanization, also as described below.
In another embodiment, the nucleic acid molecules of the invention
may be used as probes or PCR primers for specific antibody sequences. For instance,
a nucleic acid molecule probe may be used in diagnostic methods or a nucleic acid
molecule PCR primer may be used to amplify regions of DNA that could be used,
inter alia, to isolate nucleic acid sequences for use in producing variable domains of
anti-IGF-IR antibodies. In a preferred embodiment, the nucleic acid molecules are
oligonucleotides. In a more preferred embodiment, the oligonucleotides are from
highly variable regions of the heavy and light chains of the antibody of interest. In an
even more preferred embodiment, the oligonucleotides encode all or a part of one or
moreoftheCDRs.
Vectors
The invention provides vectors comprising the nucleic acid molecules
of the invention that encode the heavy chain or the antigen-binding portion thereof.
The invention also provides vectors comprising the nucleic acid molecules of the
invention that encode the light chain or antigen-binding portion thereof. The
invention also provides vectors comprising nucleic acid molecules encoding fusion
proteins, modified antibodies, antibody fragments, and probes thereof.
To express the antibodies, or antibody portions of the invention, DNAs
encoding partial or full-length light and heavy chains, obtained as described above,
are inserted into expression vectors such that the genes are operatively linked to
transcriptional and translational control sequences. Expression vectors include
plasmids, retroviruses, cosmids, YACs, EBV derived episomes, and the like. The
antibody gene is ligated into a vector such that transcriptional and translational control
sequences within the vector serve their intended function of regulating the
transcription and translation of the antibody gene. The expression vector and
expression control sequences are chosen to be compatible with the expression host
cell used. The antibody light chain gene and the antibody heavy chain gene can be
inserted into separate vector. In a preferred embodiment, both genes are inserted into
the same expression vector. The antibody genes are inserted into the expression
vector by standard methods (e.g., ligation of complementary restriction sites on the

45
antibody gene fragment and vector, or blunt end ligation if no restriction sites are
present).
A convenient vector is one that encodes a functionally complete human
CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so
that any VH or VL sequence can be easily inserted and expressed, as described above.
In such vectors, splicing usually occurs between the splice donor site in the inserted J
region and the splice acceptor site preceding the human C region, and also at the
splice regions that occur within the human CH exons. Polyadenylation and
transcription termination occur at native chromosomal sites downstream of the coding
regions. The recombinant expression vector can also encode a signal peptide that
facilitates secretion of the antibody chain from a host cell. The antibody chain gene
may be cloned into the vector such that the signal peptide is linked in-frame to the
amino terminus of the antibody chain gene. The signal peptide can be an
immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide
from a non-immunoglobulin protein).
In addition to the antibody chain genes, the recombinant expression
vectors of the invention carry regulatory sequences that control the expression of the
antibody chain genes in a host cell. It will be appreciated by those skilled in the art
that the design of the expression vector, including the selection of regulatory
sequences may depend on such factors as the choice of the host cell to be transformed,
the level of expression of protein desired, etc. Preferred regulatory sequences for
mammalian host cell expression include viral elements that direct high levels of
protein expression in mammalian cells, such as promoters and/or enhancers derived
from retroviral LTRs, cytomegalovirus (CMV) (such as the CMV
promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer),
adenovirus, (e.g., the adenovirus major late promoter (AdMLP)), polyoma and strong
mammalian promoters such as native immunoglobulin and actin promoters. For
further description of viral regulatory elements, and sequences thereof, see e.g., U.S.
Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bell et al. and U.S. Pat. No.
4,968,615 by Schaffher et al.
In addition to the antibody chain genes and regulatory sequences, the
recombinant expression vectors of the invention may carry additional sequences, such
as sequences that regulate replication of the vector in host cells (e.g., origins of

46
replication) and selectable marker genes. The selectable marker gene facilitates
selection of host cells into which the vector has been introduced (see e.g., U.S. Pat.
Nos. 4,399.216, 4,634,665 and 5,179,017, all by Axel et al.). For example, typically
the selectable marker gene confers resistance to drugs, such as G418, hygromycin or
methotrexate, on a host cell into which the vector has been introduced. Preferred
selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in
dhfr- host cells with methotrexate selection/amplification) and the neo gene (for G418
selection).
Non-Hybridoma Host Cells and Methods
of Recombinantly Producing Protein
Nucleic acid molecules encoding the heavy chain or an antigen-
binding portion thereof and/or the light chain or an antigen-binding portion thereof of
an anti-IGF-IR antibody, and vectors comprising these nucleic acid molecules, can be
used for transformation of a suitable mammalian host cell. Transformation can be by
any known method for introducing polynucleotides into a host cell. Methods for
introduction of heterologous polynucleotides into mammalian cells are well known in
the art and include dextran-mediated transfection, calcium phosphate precipitation,
polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of
the polynucleotide(s) in liposomes, biolistic injection and direct microinjection of the
DNA into nuclei. In addition, nucleic acid molecules may be introduced into
mammalian cells by viral vectors. Methods of transforming cells are well known in
the art. See, e.g., U.S. Patent Nos. 4,399,216, 4,912,040,4,740,461, and 4,959,455
(which patents are hereby incorporated herein by reference).
Mammalian cell lines available as hosts for expression are well known
in the art and include many immortalized cell lines available from the American Type
Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO)
cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney
cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3
cells, and a number of other cell lines. Mammalian host cells include human,-mouse,
rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Cell lines of particular
preference are selected through determining which cell lines have high expression
levels. Other cell lines that may be used are insect cell lines, such as Sf9 cells,
amphibian cells, bacterial cells, plant cells and fungal cells. When recombinant

47
expression vectors encoding the heavy chain or antigen-binding portion thereof, the
light chain and/or antigen-binding portion thereof are introduced into mammalian host
cells, the antibodies are produced by culturing the host cells for a period of time
sufficient to allow for expression of the antibody in the host cells or, more preferably,
secretion of the antibody into the culture medium in which the host cells are grown.
Antibodies can be recovered from the culture medium using standard protein
purification methods.
Further, expression of antibodies of the invention (or other moieties
therefrom) from production cell lines can be enhanced using a number of known
techniques. For example, the glutamine synthetase gene expression system (the GS
system) is a common approach for enhancing expression under certain conditions.
The GS system is discussed in whole or part in connection with European Patent Nos.
0 216 846, 0 256 055, and 0 323 997 and European Patent Application No.
89303964.4.
It it likely that antibodies expressed by different cell lines or in
transgenic animals will have different glycosylation from each other. However, all
antibodies encoded by the nucleic acid molecules provided herein, or comprising the
amino acid sequences provided herein are part of the instant invention, regardless of
the glycosylation of the antibodies.
Transgenic Animals
The invention also provides transgenic non-human animals comprising
one or more nucleic acid molecules of the invention that may be used to produce
antibodies of the invention. Antibodies can be produced in and recovered from tissue
or bodily fluids, such as milk, blood or urine, of goats, cows, horses, pigs, rats, mice,
rabbits, hamsters or other mammals. See, e.g., U.S. Patent Nos. 5,827,690, 5,756,687,
5,750,172, and 5,741,957. As described above, non-human transgenic animals that
comprise human immunoglobulin loci can be produced by immunizing with IGF-IR
or a portion thereof.
In another embodiment, non-human transgenic animals are produced
by introducing one or more nucleic acid molecules of the invention intr the animal by
standard transgenic techniques. See Hogan, supra. The transgenic cells used for
making the transgenic animal can be embryonic stem cells or somatic cells. The
transgenic non-human organisms can be chimeric, nonchimeric heterozygotes, and

48
nonchimeric homozygotes. See, e.g., Hogan et ai, Manipulating the Mouse Embrvo:
A Laboratory Manual 2ed., Cold Spring Harbor Press (1999); Jackson et al., Mose
Genetics and Transgenics: A Practical Approach. Oxford University Press (2000); and
Pinkert, Trans genie Animal Technology: A Laboratory Handbook, Academic Press
(1999). In another embodiment, the transgenic non-human organisms may have a
targeted disruption and replacement that encodes a heavy chain and/or a light chain of
interest. In a preferred embodiment, the transgenic animals comprise and express
nucleic acid molecules encoding heavy and light chains that bind specifically to IGF-
IR, preferably human IGF-IR. In another embodiment, the transgenic animals
comprise nucleic acid molecules encoding a modified antibody such as a single-chain
antibody, a chimeric antibody or a humanized antibody. The anti-IGF-IR antibodies
may be made in any transgenic animal. In a preferred embodiment, the non-human
animals are mice, rats, sheep, pigs, goats, cattle or horses. The non-human transgenic
animal expresses said encoded polypeptides in blood, milk, urine, saliva, tears, mucus
and other bodily fluids.
Phage Display Libraries
The invention provides a method for producing an anti-IGF-IR
antibody or antigen-binding portion thereof comprising the steps of synthesizing a
library of human antibodies on phage, screening the library with a IGF-IR or a portion
thereof, isolating phage that bind IGF-IR, and obtaining the antibody from the phage.
One method to prepare the library of antibodies comprises the steps of immunizing a
non-human host animal comprising a human immunoglobulin locus with IGF-IR or
an antigenic portion thereof to create an immune response, extracting cells from the
host animal the cells that are responsible for production of antibodies; isolating RNA
from the extracted cells, reverse transcribing the RNA to produce cDNA, amplifying
the cDNA using a primer, and inserting the cDNA into phage display vector such that
antibodies are expressed on the phage. Recombinant anti-IGF-IR antibodies of the
invention may be obtained in this way.
Recombinant anti-IGF-IR human antibodies of the invention in
addition to the anti-IGF-IR antibodies disclosed herein can be isolated by screening of
a recombinant combinatorial antibody library, preferably a scFv phage display library,
prepared using human VL and VH cDNAs prepared from mRNA derived from human
lymphocytes. Methodologies for preparing and screening such libraries are known in

49
the art. There are commercially available kits for generating phage display libraries
(e.g., the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01;
and the Stratagene SurfZAP™ phage display kit, catalog no. 240612). There are also
other methods and reagents that can be used in generating and screening antibody
display libraries (see, e.g., Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCT
Publication No. WO 92/18619; Dower et al. PCT Publication No. WO 91/17271;
Winter et al. PCT Publication No. WO 92/20791; Markland et al. PCT Publication
No. WO 92/15679; Breitling et al. PCT Publication No. WO 93/01288; McCafferty et
al. PCT Publication No. WO 92/01047; Garrard et al. PCT Publication No. WO
92/09690; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum.
Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;
McCafferty et al., Nature (1990) 348:552-554; Griffiths et al. (1993) EMBO J
12:725-734; Hawkins et al. (1992) J. Mol. Biol. 226:889-896; Clackson et al. (1991)
Nature 352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580;
Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc
Acid Res 19:4133-4137; and Barbas et al. (1991) Proc. Natl. Acad. Sci. USA
88:7978-7982.
In a preferred embodiment, to isolate human anti-IGF-IR antibodies
with the desired characteristics, a human anti-IGF-IR antibody as described herein is
first used to select human heavy and light chain sequences having similar binding
activity toward IGF-IR, using the epitope imprinting methods described in
Hoogenboom et al., PCT Publication No. WO 93/06213. The antibody libraries used
in this method are preferably scFv libraries prepared and screened as described in
McCafferty et al., PCT Publication No. WO 92/01047, McCafferty et al., Nature
(1990) 348:552-554; and Griffiths et al., (1993) EMBO J 12:725-734. The scFv
antibody libraries preferably are screened using human IGF-IR as the antigen.
Once initial human VL and VH segments are selected, "mix and
match" experiments, in which different pairs of the initially selected VL and VH
segments are screened for IGF-IR binding, are performed to select preferred VL/VH
pair combinations. Additionally, to further improve the quality of the antibody, the
VL and VH segments of the preferred VL/VH pair(s) can be randomly mutated,
preferably within the CDR3 region of VH and/or VL, in a process analogous to the in
vivo somatic mutation process responsible for affinity maturation of antibodies during

50
a natural immune response. This in vitro affinity maturation can be accomplished by
amplifying VH and VL regions using PCR primers complimentary to the VH CDR3
or VL CDR3, respectively, which primers have been "spiked" with a random mixture
of the four nucleotide bases at certain positions such that the resultant PCR products
encode VH and VL segments into which random mutations have been introduced into
the VH and/or VL CDR3 regions. These randomly mutated VH and VL segments can
be rescreened for binding to IGF-IR.
Following screening and isolation of an anti-IGF-IR antibody of the
invention from a recombinant immunoglobulin display library, nucleic acid encoding
the selected antibody can be recovered from the display package (e.g., from the phage
genome) and subcloned into other expression vectors by standard recombinant DNA
techniques. If desired, the nucleic acid can be further manipulated to create other
antibody forms of the invention, as described below. To express a recombinant
human antibody isolated by screening of a combinatorial library, the DNA encoding
the antibody is cloned into a recombinant expression vector and introduced into a
mammalian host cells, as described above.
Class Switching
Another aspect of the instant invention is to provide a mechanism by
which the class of an anti-IGF-IR antibody may be switched with another. In one
aspect of the invention, a nucleic acid molecule encoding VL or VH is isolated using
methods well-known in the art such that it does not include any nucleic acid
sequences encoding CL or CH. The nucleic acid molecule encoding VL or VH are
then operatively linked to a nucleic acid sequence encoding a CL or CH from a
different class of immunoglobulin molecule. This may be achieved using a vector or
nucleic acid molecule that comprises a CL or CH chain, as described above. For
example, an anti-IGF-IR antibody that was originally IgM may be class switched to
an IgG. Further, the class switching may be used to convert one IgG subclass to
another, e.g., from IgGl to IgG2. A preferred method for producing an antibody of
the invention comprising a desired isotypes comprises the steps of isolating a nucleic
acid encoding the heavy chain of an anti-IGF-IR antibody and a nucleic acid encoding
the light chain of an anti-IGF-IR antibody, obtaining the variable region of the heavy
chain, ligating the variable region of the heavy chain with the constant domain of a

51
heavy chain of the desired isotype, expressing the light chain and the ligated heavy
chain in a cell, and collecting the anti-IGF-IR antibody with the desired isotype.
Antibody Derivatives
One may use the nucleic acid molecules described above to generate
antibody derivatives using techniques and methods known to one of ordinary skill in
the art.
Humanized Antibodies
As was discussed above in connection with human antibody
generation, there are advantages to producing antibodies with reduced
immunogenicity. This can be accomplished to some extent using techniques of
humanization and display techniques using appropriate libraries. It will be
appreciated that murine antibodies or antibodies from other species can be humanized
or primatized using techniques well known in the art. See e.g., Winter and Harris
Immunol Today 14:43-46 (1993) and Wright et al. Crit. Reviews in Immunol. 12125-
168 (1992). The antibody of interest may be engineered by recombinant DNA
techniques to substitute the CHI, CH2, CH3, hinge domains, and/or the framework
domain with the corresponding human sequence {see WO 92/02190 and U.S. Patent
Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,792, 5,714,350, and 5,777,085). In a
preferred embodiment, the anti-IGF-IR antibody can be humanized by substituting the
CHI, CH2, CH3, hinge domains, and/or the framework domain with the
corresponding human sequence while maintaining all of the CDRS of the heavy
chain, the light chain or both the heavy and light chains.
Mutated Antibodies
In another embodiment, the nucleic acid molecules, vectors and host
cells may be used to make mutated anti-IGF-IR antibodies. The antibodies may be
mutated in the variable domains of the heavy and/or light chains to alter a binding
property of the antibody. For example, a mutation may be made in one or more of the
CDR regions to increase or decrease the Koff of the antibody for IGF-IR, to increase or
decrease Koff, or to alter the binding specificity of the antibody. Techniques in site-
directed mutagenesis are well-known in the art. See, e.g., Sambrook et al. and
Ausubel et al., supra. In a preferred embodiment, mutations are made at an amino

52
acid residue that is known to be changed compared to germline in a variable region of
an anti-IGF-IR antibody. In a more preferred embodiment, one or more mutations are
made at an amino acid residue that is known to be changed compared to the germline
in a variable region or CDR region of one of the anti-IGF-IR antibodies 2.12.1,
2.13.2, 2.14.3,3.1.1,4.9.2,4.17.3 or 6.1.1. In another embodiment, one or more
mutations are made at an amino acid residue that is known to be changed compared to
the germline in a variable region or CDR region whose amino acid sequence is
presented in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22 or 24, or whose
nucleic acid sequence is presented in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21
or 23. In another embodiment, the nucleic acid molecules are mutated in one or more
of the framework regions. A mutation may be made in a framework region or
constant domain to increase the half-life of the anti-IGF-IR antibody. See, e.g., WO
00/09560, published February 24, 2000, herein incorporated by reference. In one
embodiment, there may be one, three or five point mutations and no more than ten
point mutations. A mutation in a framework region or constant domain may also be
made to alter the immunogenicity of the antibody, to provide a site for covalent or
non-covalent binding to another molecule, or to alter such properties as complement
fixation. Mutations may be made in each of the framework regions, the constant
domain and the variable regions in a single mutated antibody. Alternatively,
mutations may be made in only one of the framework regions, the variable regions or
the constant domain in a single mutated antibody.
In one embodiment, there are no greater than ten amino acid changes
in either the VH or VL regions of the mutated anti-IGF-IR antibody compared to the
anti-IGF-IR antibody prior to mutation. In a more preferred embodiment, there is no
more than five amino acid changes in either the VH or VL regions of the mutated
anti-IGF-IR antibody, more preferably no more than three amino acid changes. In
another embodiment, there are no more than fifteen amino acid changes in the
constant domains, more preferably, no more than ten amino acid changes, even more
preferably, no more than five amino acid changes.
Modified Antibodies
In another embodiment, a fusion antibody or immunoadhesin may be
made which comprises all or a portion of an anti-IGF-IR antibody linked to another
polypeptide. In a preferred embodiment, only the variable regions of the anti-IGF-IR

53
antibody are linked to the polypeptide. In another preferred embodiment, the VH
domain of an anti-IGF-IR antibody are linked to a first polypeptide, while the VL
domain of an anti-IGF-IR antibody are linked to a second polypeptide that associates
with the first polypeptide in a manner in which the VH and VL domains can interact
with one another to form an antibody binding site. In another preferred embodiment,
the VH domain is separated from the VL domain by a linker such that the VH and VL
domains can interact with one another (see below under Single Chain Antibodies).
The VH-linker-VL antibody is then linked to the polypeptide of interest. The fusion
antibody is useful to directing a polypeptide to an IGF-IR-expressing cell or tissue.
The polypeptide may be a therapeutic agent, such as a toxin, growth factor or other
regulatory protein, or may be a diagnostic agent, such as an enzyme that may be
easily visualized, such as horseradish peroxidase. In addition, fusion antibodies can
be created in which two (or more) single-chain antibodies are linked to one another.
This is useful if one wants to create a divalent or polyvalent antibody on a single
polypeptide chain, or if one wants to create a bispecific antibody.
To create a single chain antibody, (scFv) the VH- and VL-encoding
DNA fragments are operatively linked to another fragment encoding a flexible linker,
e.g., encoding the amino acid sequence (Gly4 -Ser)3 (SEQ ID NO: 60), such that the
VH and VL sequences can be expressed as a contiguous single-chain protein, with the
VL and VH regions joined by the flexible linker (see e.g., Bird et al. (1988) Science
242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883;
McCafferty et al., Nature (1990) 348:552-554). The single chain antibody may be
monovalent, if only a single VH and VL are used, bivalent, if two VH and VL are
used, or polyvalent, if more than two VH and VL are used.
In another embodiment, other modified antibodies may be prepared
using anti-IGF-IR-encoding nucleic acid molecules. For instance, "Kappa bodies" (111
et al., Protein Eng 10: 949-57 (1997)), "Minibodies" (Martin et al., EMBO J13: 5303-
9 (1994)), "Diabodies" (Holliger et al., PNAS USA 90: 6444-6448 (1993)), or
"Janusins" (Traunecker et al., EMBO J10: 3655-3659 (1991) and Traunecker et al.
"Janusin: new molecular design for bispecific reagents" Int J Cancer Suppl 7:51-52
(1992)) may be prepared using standard molecular biological techniques following the
teachings of the specification.

54
In another aspect, chimeric and bispecific antibodies can be generated.
A chimeric antibody may be made that comprises CDRs and framework regions from
different antibodies. In a preferred embodiment, the CDRs of the chimeric antibody
comprises all of the CDRs of the variable region of a light chain or heavy chain of an
anti-IGF-IR antibody, while the framework regions are derived from one or more
different antibodies. In a more preferred embodiment, the CDRs of the chimeric
antibody comprise all of the CDRs of the variable regions of the light chain and the
heavy chain of an anti-IGF-IR antibody. The framework regions may be from another
species and may, in a preferred embodiment, be humanized. Alternatively, the
framework regions may be from another human antibody.
A bispecific antibody can be generated that binds specifically to IGF-
IR through one binding domain and to a second molecule through a second binding
domain. The bispecific antibody can be produced through recombinant molecular
biological techniques, or may be physically conjugated together. In addition, a single
chain antibody containing more than one VH and VL may be generated that binds
specifically to IGF-IR and to another molecule. Such bispecific antibodies can be
generated using techniques that are well known for example, in connection with (i)
and (ii) see e.g., Fanger et al. Immunol Methods 4: 72-81 (1994) and Wright and
Harris, supra, and in connection with (iii) see e.g., Traunecker et al. Int. J. Cancer
(Suppl.) 7: 51-52 (1992). In a preferred embodiment, the bispecific antibody binds to
IGF-IR and to another molecule expressed at high level on cancer or tumor cells. In a
more preferred embodiment, the other molecule is erbB2 receptor, VEGF, CD20 or
EGF-R.
In a embodiment, the modified antibodies described above are
prepared using one or more of the variable regions or one or more CDR regions from
one of the antibodies selected from 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1.
In another embodiment, the modified antibodies are prepared using one or more of the
variable regions or one or more CDR regions whose amino ?.cid sequence is presented
in SEQ ID NOS: 2,4, 6, 8, 10,12, 14, 16,18, 20, 22 or 24, or whose nucleic acid
sequence is presented in SEQ ID NOS: 1,3,5,7,9,11, 13, 15, 17, 19, 21 or 23.
Derivatized and Labeled Antibodies
An antibody or antibody portion of the invention can be derivatized or
linked to another molecule (e.g., another peptide or protein). In general, the

55
antibodies or portion thereof is derivatized such that the IGF-DR. binding is not
affected adversely by the derivatization or labeling. Accordingly, the antibodies and
antibody portions of the invention are intended to include both intact and modified
forms of the human anti-IGF-IR antibodies described herein. For example, an
antibody or antibody portion of the invention can be functionally linked (by chemical
coupling, genetic fusion, noncovalent association or otherwise) to one or more other
molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody),
a detection agent, a cytotoxic agent, a pharmaceutical agent, and/or a protein or
peptide that can mediate associate of the antibody or antibody portion with another
molecule (such as a streptavidin core region or a polyhistidine tag).
One type of derivatized antibody is produced by crosslinking two or
more antibodies (of the same type or of different types, e.g., to create bispecific
antibodies). Suitable crosslinkers include those that are heterobifunctional, having two
distinctly reactive groups separated by an appropriate spacer (e.g.,
m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g.,
disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company,
Rockford, 111.
Another type of derivatized antibody is a labeled antibody. Useful
detection agents with which an antibody or antibody portion of the invention may be
derivatized include fluorescent compounds, including fluorescein, fluorescein
isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride,
phycoerythrin, lanthanide phosphors and the like. An antibody may also be labeled
with enzymes that are useful for detection, such as horseradish peroxidase,
P-galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like. When
an antibody is labeled with a detectable enzyme, it is detected by adding additional
reagents that the enzyme uses to produce a reaction product that can be discerned.
For example, when the agent horseradish peroxidase is present, the addition of
hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is
detectable. An antibody may also be labeled with biotin, and detected through
indirect measurement of avidin or streptavidin binding. An antibody may be labeled
with a magnetic agent, such as gadolinium. An antibody may also be labeled with a
predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine
zipper pair sequences, binding sites for secondary antibodies, metal binding domains,

56
epitope tags). In some embodiments, labels are attached by spacer arms of various
lengths to reduce potential steric hindrance.
An anti-IGF-IR antibody may also be labeled with a radiolabeled
amino acid. The radiolabel may be used for both diagnostic and therapeutic purposes.
For instance, the radiolabel may be used to detect IGF-ER-expressing tumors by x-ray
or other diagnostic techniques. Further, the radiolabel may be used therapeutically as
a toxin for cancerous cells or tumors. Examples of labels for polypeptides include,
but are not limited to, the following radioisotopes or radionuclides — 3H, I4C, 15N, 35S,
90Y.99Tc,IIIIn,125I,131I.
An anti-IGF-IR antibody may also be derivatized with a chemical
group such as polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrate
group. These groups may be useful to improve the biological characteristics of the
antibody, e.g., to increase serum half-life or to increase tissue binding.
Pharmaceutical Compositions and Kits
The invention also relates to a pharmaceutical composition for the
treatment of a hyperproliferative disorder in a mammal which comprises a
therapeutically effective amount of a compound of the invention and a
pharmaceutically acceptable carrier. In one embodiment, said pharmaceutical
composition is for the treatment of cancer such as brain, lung, squamous cell, bladder,
gastric, pancreatic, breast, head, neck, renal, kidney, ovarian, prostate, colorectal,
esophageal, gynecological or thyroid cancer. In another embodiment, said
pharmaceutical composition relates to non-cancerous hyperproliferative disorders
such as, without limitation, restenosis after angioplasty and psoriasis. In another
embodiment, the invention relates to pharmaceutical compositions for the treatment of
a mammal that requires activation of IGF-IR, wherein the pharmaceutical
composition comprises a therapeutically effective amount of an activating antibody of
the invention and a pharmaceutically acceptable carrier. Pharmaceutical compositions
comprising activating antibodies may be used to treat animals that lack sufficient IGF-
I or IGF-II, or may be used to treat osteoporosis, frailty or disorders in which the
mammal secretes too little active growth hormone or is unable to respond to growth
hormone.
The anti-IGF-IR antibodies of the invention can be incorporated into
pharmaceutical compositions suitable for administration to a subject. Typically, the

57
pharmaceutical composition comprises an antibody of the invention and a
pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable
carrier" includes any and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the like that are
physiologically compatible. Examples of pharmaceutically acceptable carriers include
one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol
and the like, as well as combinations thereof. In many cases, it will be preferable to
include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol,
or sodium chloride in the composition. Pharmaceutically acceptable substances such
as wetting or minor amounts of auxiliary substances such as wetting or emulsifying
agents, preservatives or buffers, which enhance the shelf life or effectiveness of the
antibody or antibody portion.
The compositions of this invention may be in a variety of forms. These
include, for example, liquid, semi-solid and solid dosage forms, such as liquid
solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets,
pills, powders, liposomes and suppositories. The preferred form depends on the
intended mode of administration and therapeutic application. Typical preferred
compositions are in the form of injectable or infusible solutions, such as compositions
similar to those used for passive immunization of humans with other antibodies. The
preferred mode of administration is parenteral (e.g., intravenous, subcutaneous,
intraperitoneal, intramuscular). In a preferred embodiment, the antibody is
administered by intravenous infusion or injection. In another preferred embodiment,
the antibody is administered by intramuscular or subcutaneous injection.
Therapeutic compositions typically must be sterile and stable under the
conditions of manufacture and storage. The composition can be formulated as a
solution, microemulsion, dispersion, liposome, or other ordered structure suitable to
high drug concentration. Sterile injectable solutions can be prepared by incorporating
the anti-IGF-IR antibody in the required amount in an appropriate solvent with one or
a combination of ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle that contains a basic dispersion medium and the
required other ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the preferred methods of

58
preparation are vacuum drying and freeze-drying that yields a powder of the active
ingredient plus any additional desired ingredient from a previously sterile-filtered
solution thereof. The proper fluidity of a solution can be maintained, for example, by
the use of a coating such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. Prolonged absorption of
injectable compositions can be brought about by including in the composition an
agent that delays absorption, for example, monostearate salts and gelatin.
The antibodies of the present invention can be administered by a
variety of methods known in the art, although for many therapeutic applications, the
preferred route/mode of administration is intraperitoneal, subcutaneous,
intramuscular, intravenous or infusion. As will be appreciated by the skilled artisan,
the route and/or mode of administration will vary depending upon the desired results.
In one embodiment, the antibodies of the present inventon can be administered as a
single dose or may be administered as multiple doses.
In certain embodiments, the active compound may be prepared with a
carrier that will protect the compound against rapid release, such as a controlled
release formulation, including implants, transdermal patches, and microencapsulated
delivery systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters,
and polylactic acid. Many methods for the preparation of such formulations are
patented or generally known to those skilled in the art. See, e.g., Sustained and
Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc.,
New York, 1978.
In certain embodiments, the anti-IGF-IR of the invention may be orally
administered, for example, with an inert diluent or an assimilable edible carrier. The
compound (and other ingredients, if desired) may also be enclosed in a hard or soft
shell gelatin capsule, compressed into tablets, or incorporated directly into the
subject's diet. For oral therapeutic administration, the compounds may be incorporated
with excipients and used in the form of ingestible tablets, buccal tablets, troches,
capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound
of the invention by other than parenteral administration, it may be necessary to coat
the compound with, or co-administer the compound with, a material to prevent its
inactivation.

59
Supplementary active compounds can also be incorporated into the
compositions. In certain embodiments, an anti-IGF-IR of the invention is
coformulated with and/or coadministered with one or more additional therapeutic
agents, such as a chemotherapeutic agent, an antineoplastic agent or an anti-tumor
agent. For example, an anti-IGF-IR antibody may be coformulated and/or
coadministered with one or more additional therapeutic agents. These agents include,
without limitation, antibodies that bind other targets (e.g., antibodies that bind one or
more growth factors or cytokines, their cell surface receptors or IGF-1), IGF-I binding
proteins, antineoplastic agents, chemotherapeutic agents, anti-tumor agents, antisense
oligonucleotides against IGF-IR or IGF-I, peptide analogues that block IGF-IR
activation, soluble IGF-IR, and/or one or more chemical agents that inhibit IGF-I
production or activity, which are known in the art, e.g., octreotide. For a
pharmaceutical composition comprising an activating antibody, the anti-IGF-IR
antibody may be formulated with a factor that increases cell proliferation or prevents
apoptosis. Such factors include growth factors such as IGF-I, and/or analogues of
IGF-I that activate IGF-IR. Such combination therapies may require lower dosages of
the anti-IGF-IR antibody as well as the co-administered agents, thus avoiding possible
toxicities or complications associated with the various monotherapies. In one
embodiment, the antibody and one or more additional therapeutic agent.
The pharmaceutical compositions of the invention may include a
"therapeutically effective amount" or a "prophylactically effective amount" of an
antibody or antibody portion of the invention. A "therapeutically effective amount"
refers to an amount effective, at dosages and for periods of time necessary, to achieve
the desired therapeutic result. A therapeutically effective amount of the antibody or
antibody portion may vary according to factors such as the disease state, age, sex, and
weight of the individual, and the ability of the antibody or antibody portion to elicit a
desired response in the individual. A therapeutically effective amount is also one in
which any toxic cr detrimental effects of the antibody or antibody portion are
outweighed by the therapeutically beneficial effects. A "prophylactically effective
amount" refers to an amount effective, at dosages and for periods of time necessary, to
achieve the desired prophylactic result. Typically, since a prophylactic dose is used in
subjects prior to or at an earlier stage of disease, the prophylactically effective amount
will be less than the therapeutically effective amount.

60
Dosage regimens may be adjusted to provide the optimum desired
response (e.g., a therapeutic or prophylactic response). For example, a single bolus
may be administered, several divided doses may be administered over time or the
dose may be proportionally reduced or increased as indicated by the exigencies of the
therapeutic situation. Pharmaceutical composition comprising the antibody or
comprising a combination therapy comprising the antibody and one or more
additional therapeutic agents may be formulated for single or multiple doses. It is
especially advantageous to formulate parenteral compositions in dosage unit form for
ease of administration and uniformity of dosage. Dosage unit form as used herein
refers to physically discrete units suited as unitary dosages for the mammalian
subjects to be treated; each unit containing a predetermined quantity of active
compound calculated to produce the desired therapeutic effect in association with the
required pharmaceutical carrier. The specification for the dosage unit forms of the
invention are dictated by and directly dependent on (a) the unique characteristics of
the active compound and the particular therapeutic or prophylactic effect to be
achieved, and (b) the limitations inherent in the art of compounding such an active
compound for the treatment of sensitivity in individuals. A particularly useful
formulation is 5 mg/ml anti-IGF-IR antibody in a buffer of 20mM sodium citrate, pH
5.5, 140mM NaCl, and 0.2mg/ml polysorbate 80.
An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of an antibody or antibody portion of the invention
is 0.1-100 mg/kg, more preferably 0.5-50 mg/kg, more preferably 1-20 mg/kg, and
even more preferably 1-10 mg/kg. It is to be noted that dosage values may vary with
the type and severity of the condition to be alleviated. It is to be further understood
that for any particular subject, specific dosage regimens should be adjusted over time
according to the individual need and the professional judgment of the person
administering or supervising the administration of the compositions, and that dosage
ranges set forth herein are exemplary only and are not intended to limit the scope or
practice of the claimed composition. In one embodiment, the therapeutically or
prophylactically effective amount of an antibody or antigen-binding portion thereof is
administered along with one or more additional therapeutic agents.
In another aspect, the invention relates to administration of an anti-
IGF-IR antibody for the treatment of cancer in a dose of less than 300 mg per month.

61
Another aspect of the present invention provides kits comprising the
anti-IGF-IR antibodies and the pharmaceutical compositions comprising these
antibodies. A kit may include, in addition to the antibody or pharmaceutical
composition, diagnostic or therapeutic agents. A kit may also include instructions for
use in a diagnostic or therapeutic method. In a preferred embodiment, the kit includes
the antibody or a pharmaceutical composition thereof and a diagnostic agent that can
be used in a method described below. In another preferred embodiment, the kit
includes the antibody or a pharmaceutical composition thereof and one or more
therapeutic agents, such as an additional antineoplastic agent, anti-tumor agent or
chemotherapeutic agent, that can be used in a method described below.
This invention also relates to pharmaceutical compositions for
inhibiting abnormal cell growth in a mammal which comprise an amount of a
compound of the invention in combination with an amount of a chemotherapeutic
agent, wherein the amounts of the compound, salt, solvate, or prodrug, and of the
chemotherapeutic agent are together effective in inhibiting abnormal cell growth.
Many chemotherapeutic agents are presently known in the art. In one embodiment,
the chemotherapeutic agents is selected from the group consisting of mitotic
inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor
inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival
agents, biological response modifiers, anti-hormones, e.g. anti-androgens, and anti-
angiogenesis agents.
Anti-angiogenesis agents, such as MMP-2 (matrix-metalloproteinase
2) inhibitors, MMP-9 (matrix-metalloproteinase 9) inhibitors, and COX-II
(cyclooxygenase II) inhibitors, can be used in conjunction with a compound of the
invention. Examples of useful COX-II inhibitors include CELEBREX™ (alecoxib),
valdecoxib, and rofecoxib. Examples of useful matrix metalloproteinase inhibitors
are described in WO 96/33172 (published October 24,1996), WO 96/27583
(published March 7, 1996), European Patent Application No. 97304971.1 (filed July
8, 1997), European Patent Application No. 99308617.2 (filed October 29, 1999), WO
98/07697 (published February 26, 1998), WO 98/03516 (published January 29, 1998),
WO 98/34918 (published August 13, 1998), WO 98/34915 (published August 13,
1998), WO 98/33768 (published August 6, 1998), WO 98/30566 (published July 16,
1998), European Patent Publication 606,046 (published July 13, 1994), European

62
Patent Publication 931,788 (published July 28, 1999), WO 90/05719 (published May
31, 1990), WO 99/52910 (published October 21, 1999), WO 99/52889 (published
October 21, 1999), WO 99/29667 (published June 17, 1999), PCT International
Application No. PCT7JJB98/01113 (filed July 21,1998), European Patent Application
No. 99302232.1 (filed March 25,1999), Great Britain patent application number
9912961.1 (filed June 3, 1999), United States Provisional Application No. 60/148,464
(filed August 12, 1999), United States Patent 5,863,949 (issued January 26,1999),
United States Patent 5,861,510 (issued January 19, 1999), and European Patent
Publication 780,386 (published June 25, 1997), all of which are incorporated herein in
their entireties by reference. Preferred MMP inhibitors are those that do not
demonstrate arthralgia. More preferred, are those that selectively inhibit MMP-2
and/or MMP-9 relative to the other matrix-metalloproteinases (i.e. MMP-1, MMP-3,
MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-10, MMP-11, MMP-12, and
MMP-13). Some specific examples of MMP inhibitors useful in the present invention
are AG-3340, RO 32-3555, RS 13-0830, and the compounds recited in the following
list: 3-[[4-(4-fiuoro-phenoxy)-benzenesulfonyl]-(l-hydroxycarbamoyl-cyclopentyl)-
amino]-propionicacid;3-exo-3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-8-oxa-
bicyclo[3.2.1]octane-3-carboxylic acid hydroxyamide; (2R, 3R) l-[4-(2-chloro-4-
fluoro-benzyloxy)-benzenesulfonyl]-3-hydroxy-3-methyl-piperidine-2-carboxylic
acid hydroxyamide; 4-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-tetrahydro-
pyran-4-carboxylic acid hydroxyamide; 3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-
(1 -hydroxycarbamoyl-cyclobutyl)-amino]-propionic acid; 4-[4-(4-chloro-phenoxy)-
benzenesulfonylamino]-tetrahydro-pyran-4-carboxylic acid hydroxyamide; (R) 3-[4-
(4-chloro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-3-carboxylic acid
hydroxyamide; (2R, 3R) l-[4-(4-fiuoro-2-methyl-benzyloxy)-benzenesulfonyl]-3-
hydroxy-3-methyl-piperidine-2-carboxylic acid hydroxyamide; 3-[[4-(4-fluoro-
phenoxy)-benzenesulfonyl]-( 1 -hydroxycarbamoyl-1 -methyl-ethyl)-amino]-propionic
acid; 3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(4-hydroxycarbamoyl-tetrahydro-
pyran-4-yl)-amino]-propionic acid; 3-exo-3-[4-(4-chloro-phenoxy)-
benzenesulfonylamino]-8-oxa-icyclo[3.2. l]octane-3-carboxylic acid hydroxyamide;
3-endo-3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-8-oxa-icyclo[3.2.1]octane-3-
carboxylic acid hydroxyamide; and (R) 3-[4-(4-fluoro-phenoxy)-

63
benzenesulfonylarnino]-tetrahydro-furan-3-carboxylic acid hydroxyamide; and
pharmaceutically acceptable salts and solvates of said compounds.
A compound of the invention can also be used with signal transduction
inhibitors, such as agents that can inhibit EGF-R (epidermal growth factor receptor)
responses, such as EGF-R antibodies, EGF antibodies, and molecules that are EGF-R
inhibitors; VEGF (vascular endothelial growth factor) inhibitors, such as VEGF
receptors and molecules that can inhibit VEGF; and erbB2 receptor inhibitors, such as
organic molecules or antibodies that bind to the erbB2 receptor, for example,
HERCEPTIN™ (Genentech, Inc.). EGF-R inhibitors are described in, for example in
WO 95/19970 (published July 27,1995), WO 98/14451 (published April 9, 1998),
WO 98/02434 (published January 22, 1998), and United States Patent 5,747,498
(issued May 5,1998), and such substances can be used in the present invention as
described herein. EGFR-inhibiting agents include, but are not limited to, the
monoclonal antibodies C225 and anti-EGFR 22Mab (ImClone Systems Incorporated),
ABX-EGF (Abgenix/Cell Genesys), EMD-7200 (Merck KgaA), EMD-5590 (Merck
KgaA), MDX-447/H-477 (Medarex Inc. and Merck KgaA), and the compounds ZD-
1834, ZD-1838 and ZD-1839 (AstraZeneca), PKI-166 (Novartis), PKI-166/CGP-
75166 (Novartis), PTK 787 (Novartis), CP 701 (Cephalon), leflunomide
(Pharmacia/Sugen), CI-1033 (Warner Lambert Parke Davis), CI-1033/PD 183,805
(Warner Lambert Parke Davis), CL-387,785 (Wyeth-Ayerst), BBR-1611 (Boehringer
Mannheim GmbH/Roche), Naamidine A (Bristol Myers Squibb), RC-3940-II
(Pharmacia), BBX-1382 (Boehringer Ingelheim), OLX-103 (Merck & Co.), VRCTC-
310 (Ventech Research), EGF fusion toxin (Seragen Inc.), DAB-389
(Seragen/Lilgand), ZM-252808 (Imperial Cancer Research Fund), RG-50864
(INSERM), LFM-A12 (Parker Hughes Cancer Center), WHI-P97 (Parker Hughes
Cancer Center), GW-282974 (Glaxo), KT-8391 (Kyowa Hakko) and EGF-R Vaccine
(York Medical/Centro de Immunologia Molecular (CIM)). These and other EGF-R-
inhibiting agents can be used in the present invention.
VEGF inhibitors, for example SU-5416 and SU-6668 (Sugen Inc.),
SH-268 (Schering), and NX-1838 (NeXstar) can also be combined with the
compound of the present invention. VEGF inhibitors are described in, for example in
WO 99/24440 (published May 20, 1999), PCT International Application
PCT/IB99/00797 (filed May 3, 1999), in WO 95/21613 (published August 17, 1995),

64
WO 99/61422 (published December 2, 1999), United States Patent 5,834,504 (issued
November 10, 1998), WO 98/50356 (published November 12, 1998), United States
Patent 5,883,113 (issued March 16, 1999), United States Patent 5,886,020 (issued
March 23, 1999), United States Patent 5,792,783 (issued August 11, 1998), WO
99/10349 (published March 4,1999), WO 97/32856 (published September 12, 1997),
WO 97/22596 (published June 26, 1997), WO 98/54093 (published December 3,
1998), WO 98/02438 (published January 22, 1998), WO 99/16755 (published April 8,
1999), and WO 98/02437 (published January 22, 1998), all of which are incorporated
herein in their entireties by reference. Other examples of some specific VEGF
inhibitors useful in the present invention are IM862 (Cytran Inc.); anti-VEGF
monoclonal antibody of Genentech, Inc.; and angiozyme, a synthetic ribozyme from
. Ribozyme and Chiron. These and other VEGF inhibitors can be used in the present
invention as described herein.
ErbB2 receptor inhibitors, such as GW-282974 (Glaxo Wellcome pic),
and the monoclonal antibodies AR-209 (Aronex Pharmaceuticals Inc.) and 2B-1
(Chiron), can furthermore be combined with the compound of the invention, for
example those indicated in WO 98/02434 (published January 22,1998), WO
99/35146 (published July 15,1999), WO 99/35132 (published July 15,1999), WO
98/02437 (published January 22,1998), WO 97/13760 (published April 17, 1997),
WO 95/19970 (published July 27,1995), United States Patent 5,587,458 (issued
December 24, 1996), and United States Patent 5,877,305 (issued March 2, 1999),
which are all hereby incorporated herein in their entireties by reference. ErbB2
receptor inhibitors useful in the present invention are also described in United States
Provisional Application No. 60/117,341, filed January 27, 1999, and in United States
Provisional Application No. 60/117,346, filed January 27, 1999, both of which are
incorporated in their entireties herein by reference. The erbB2 receptor inhibitor
compounds and substance described in the aforementioned PCT applications, U.S.
patents, and U.S. provisional applications, as well as other compounds and substances
that inhibit the erbB2 receptor, can be used with the compound of the present"
invention in accordance with the present invention.
Anti-survival agents include anti-IGF-ER antibodies and anti-integrin
agents, such as anti-integrin antibodies.

65
Diagnostic Methods of Use
The anti-IGF-IR antibodies may be used to detect IGF-IR in a
biological sample in vitro or in vivo. The anti-IGF-IR antibodies may be used in a
conventional immunoassay, including, without limitation, an ELISA, an RIA, FACS,
tissue immunohistochemistry, Western blot or immunoprecipitation. The anti-IGF-IR
antibodies of the invention may be used to detect IGF-IR from humans. In another
embodiment, the anti-IGF-IR antibodies may be used to detect IGF-IR from Old
World primates such as cynomologous and rhesus monkeys, chimpanzees and apes.
The invention provides a method for detecting anti-IGF-IR in a biological sample
comprising contacting a biological sample with an anti-IGF-IR antibody of the
invention and detecting the bound antibody bound to anti-IGF-IR, to detect the IGF-
IR in the biological sample. In one embodiment, the anti-IGF-IR antibody is directly
labeled with a detectable label. In another embodiment, the anti-IGF-IR antibody (the
first antibody) is unlabeled and a second antibody or other molecule that can bind the
anti-IGF-IR antibody is labeled. As is well known to one of skill in the art, a second
antibody is chosen that is able to specifically bind the specific species and class of the
first antibody. For example, if the anti-IGF-ER antibody is a human IgG, then the
secondary antibody may be an anti-human-IgG. Other molecules that can bind to
antibodies include, without limitation, Protein A and Protein G, both of which are
available commercially, e.g., from Pierce Chemical Co.
Suitable labels for the antibody or secondary have been disclosed
supra, and include various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, magnetic agents and radioactive materials. Examples of
suitable enzymes include horseradish peroxidase, alkaline phosphatase,
P-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples of suitable
fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate,
rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an
example of a luminescent material includes luminol; an example of a magnetic agent
includes gadolinium; and examples of suitable radioactive material includel25l, I31I,
35Sor3H.
In an alternative embodiment, IGF-ER can be assayed in a biological
sample by a competition immunoassay utilizing IGF-IR standards labeled with a

66
detectable substance and an unlabeled anti-IGF-IR antibody. In this assay, the
biological sample, the labeled IGF-IR standards and the anti-IGF-IR antibody are
combined and the amount of labeled IGF-IR standard bound to the unlabeled antibody
is determined. The amount of IGF-IR in the biological sample is inversely
proportional to the amount of labeled IGF-IR standard bound to the anti-IGF-IR
antibody.
One may use the immunoassays disclosed above for a number of
purposes. In one embodiment, the anti-IGF-IR antibodies may be used to detect IGF-
IR in cells in cell culture. In a preferred embodiment, the anti-IGF-IR antibodies may
be used to determine the level of tyrosine phosphorylation, tyrosine
autophosphorylation of IGF-IR, and/or the amount of IGF-IR on the cell surface after
treatment of the cells with various compounds. This method can be used to test
compounds that may be used to activate or inhibit IGF-IR. In this method, one
sample of cells is treated with a test compound for a period of time while another
sample is left untreated. If tyrosine autophosphorylation is to be measured, the cells
are lysed and tyrosine phosphorylation of the IGF-IR is measured using an
immunoassay described above or as described in Example III, which uses an ELISA.
If the total level of IGF-IR is to be measured, the cells are lysed and the total IGF-IR
level is measured using one of the immunoassays described above.
A preferred immunoassay for determining IGF-IR tyrosine
phosphorylation or for measuring total IGF-IR levels is an ELISA or Western blot. If
only the cell surface level of IGF-IR is to be measured, the cells are not lysed, and the
cell surface levels of IGF-IR are measured using one of the immunoassays described
above. A preferred immunoassay for determining cell surface levels of IGF-IR
includes the steps of labeling the cell surface proteins with a detectable label, such as
biotin or 125I, immunoprecipitating the IGF-IR with an anti-IGF-IR antibody and then
detecting the labeled IGF-IR. Another preferred immunoassay for determining the
localization of IGF-IR, e.g., cell surface levels, is by using immunobistochemistry.
Methods such as ELISA, RIA, Western blot, immunohistochemistry, cell surface
labeling of integral membrane proteins and immunoprecipitation are well known in
the art. See, e.g., Harlow and Lane, supra. In addition, the immunoassays may be
scaled up for high throughput screening in order to test a large number of compounds
for either activation or inhibition of IGF-IR.

67
The anti-IGF-IR antibodies of the invention may also be used to
determine the levels of IGF-IR in a tissue or in cells derived from the tissue. In a
preferred embodiment, the tissue is a diseased tissue. In a more preferred
embodiment, the tissue is a tumor or a biopsy thereof. In a preferred embodiment of
the method, a tissue or a biopsy thereof is excised from a patient. The tissue or biopsy
is then used in an immunoassay to determine, e.g., IGF-IR levels, cell surface levels
of IGF-IR, levels of tyrosine phosphorylation of IGF-IR, or localization of IGF-IR by
the methods discussed above. The method can be used to determine if a tumor
expresses IGF-IR at a high level.
The above-described diagnostic method can be used to determine
whether a tumor expresses high levels of IGF-IR, which may be indicative that the
tumor will respond well to treatment with anti-IGF-IR antibody. The diagnostic
method may also be used to determine whether a tumor is potentially cancerous, if it
expresses high levels of IGF-IR, or benign, if it expresses low levels of IGF-IR.
Further, the diagnostic method may also be used to determine whether treatment with
anti-IGF-IR antibody (see below) is causing a tumor to express lower levels of IGF-
IR and/or to express lower levels of tyrosine autophosphorylation, and thus can be
used to determine whether the treatment is successful. In general, a method to
determine whether an anti-IGF-IR antibody decreases tyrosine phosphorylation
comprises the steps of measuring the level of tyrosine phosphorylation in a cell or
tissue of interest, incubating the cell or tissue with an anti-IGF-IR antibody or
antigen-binding portion thereof, then re-measuring the level of tyrosine
phosphorylation in the cell or tissue. The tyrosine phosphorylation of IGF-IR or of
another protein(s) may be measured. The diagnostic method may also be used to
determine whether a tissue or cell is not expressing high enough levels of IGF-IR or
high enough levels of activated IGF-IR, which may be the case for individuals with
dwarfism, osteoporosis or diabetes. A diagnosis that levels of IGF-IR or active IGF-
IR are too low could be used for treatment with activating anti-IGF-IR antibodies,
IGF-I or other therapeutic agents for increasing IGF-IR levels or activity.
The antibodies of the present invention may also be used in vivo to
localize tissues and organs that express IGF-IR. In a preferred embodiment, the anti-
IGF-IR antibodies can be used localize IGF-IR-expressing tumors. The advantage of
the anti-IGF-IR antibodies of the present invention is that they will not generate an

68
immune response upon administration. The method comprises the steps of
administering an anti-IGF-IR antibody or a pharmaceutical composition thereof to a
patient in need of such a diagnostic test and subjecting the patient to imaging analysis
determine the location of the IGF-IR-expressing tissues. Imaging analysis is well
known in the medical art, and includes, without limitation, x-ray analysis, magnetic
resonance imaging (MRI) or computed tomography (CE). In another embodiment of
the method, a biopsy is obtained from the patient to determine whether the tissue of
interest expresses IGF-IR rather than subjecting the patient to imaging analysis. In a
preferred embodiment, the anti-IGF-IR antibodies may be labeled with a detectable
agent that can be imaged in a patient. For example, the antibody may be labeled with
a contrast agent, such as barium, which can be used for x-ray analysis, or a magnetic
contrast agent, such as a gadolinium chelate, which can be used for MRI or CE.
Other labeling agents include, without limitation, radioisotopes, such as 99Tc. In
another embodiment, the anti-IGF-IR antibody will be unlabeled and will be imaged
by administering a second antibody or other molecule that is detectable and that can
bind the anti-IGF-IR antibody.
Therapeutic Methods of Use
In another embodiment, the invention provides a method for inhibiting
IGF-IR activity by administering an anti-IGF-IR antibody to a patient in need thereof.
Any of the types of antibodies described herein may be used therapeutically. In a
preferred embodiment, the anti-IGF-IR antibody is a human, chimeric or humanized
antibody. In another preferred embodiment, the IGF-IR is human and the patient is a
human patient. Alternatively, the patient may be a mammal that expresses an IGF-IR
that the anti-IGF-IR antibody cross-reacts with. The antibody may be administered to
a non-human mammal expressing an IGF-IR with which the antibody cross-reacts
(i.e. a primate, or a cynomologous or rhesus monkey) for veterinary purposes or as an
animal model of human disease. Such animal models may be useful for evaluating
the therapeutic efficacy of antibodies of this invention.
As used herein, the term "a disorder in which IGF-IR activity is
detrimental" is intended to include diseases and other disorders in which the presence
of high levels of IGF-IR in a subject suffering from the disorder has been shown to be
or is suspected of being either responsible for the pathophysiology of the disorder or a
factor that contributes to a worsening of the disorder. Accordingly, a disorder in

69
which high levels of IGF-IR activity is detrimental is a disorder in which inhibition of
IGF-IR activity is expected to alleviate the symptoms and/or progression of the
disorder. Such disorders may be evidenced, for example, by an increase in the levels
of IGF-ER on the cell surface or in increased tyrosine autophosphorylation of IGF-IR
in the affected cells or tissues of a subject suffering from the disorder. The increase in
IGF-IR levels may be detected, for example, using an anti-IGF-IR antibody as
described above.
In a preferred embodiment, an anti-IGF-IR antibody may be
administered to a patient who has an IGF-IR-expressing tumor. A tumor may be a
solid tumor or may be a non-solid tumor, such as a lymphoma. In a more preferred
embodiment, an anti-IGF-IR antibody may be administered to a patient who has an
IGF-IR-expressing tumor that is cancerous. In an even more preferred embodiment,
the anti-IGF-IR antibody is administered to a patient who has a tumor of the lung,
breast, prostate or colon. In a highly preferred embodiment, the method causes the
tumor not to increase in weight or volume or to decrease in weight or volume. In
another embodiment, the method causes the IGF-IR on the tumor to be internalized.
In a preferred embodiment, the antibody is selected from 2.12.1, 2.13.2, 2.14.3, 3.1.1,
4.9.2, or 6.1.1, or comprises a heavy chain, light chain or antigen-binding region
thereof.
In another preferred embodiment, an anti-IGF-IR antibody may be
administered to a patient who expresses inappropriately high levels of IGF-1. It is
known in the art that high-level expression of IGF-I can lead to a variety of common
cancers. In a more preferred embodiment, the anti-IGF-IR antibody is administered to
a patient with prostate cancer, glioma or fibrosarcoma. In an even more preferred
embodiment, the method causes the cancer to stop proliferating abnormally, or not to
increase in weight or volume or to decrease in weight or volume.
In one embodiment, said method relates to the treatment of cancer such
as brain, squamous cell, bladder, gastric, pancreatic, breast, head, neck, esophageal,
prostate, colorectal, lung, renal, kidney, ovarian, gynecological or thyroid cancer.
Patients that can be treated with a compounds of the invention according to the
methods of this invention include, for example, patients that have been diagnosed as
having lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head
and neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal

70
cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer,
gynecologic tumors (e.g., uterine sarcomas, carcinoma of the fallopian tubes,
carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina or
carcinoma of the vulva), Hodgkin's disease, cancer of the esophagus, cancer of the
small intestine, cancer of the endocrine system (e.g., cancer of the thyroid,
parathyroid or adrenal glands), sarcomas of soft tissues, cancer of the urethra, cancer
of the penis, prostate cancer, chronic or acute leukemia, solid tumors of childhood,
lymphocytic lymphomas, cancer of the bladder, cancer of the kidney or ureter (e.g.,
renal cell carcinoma, carcinoma of the renal pelvis), or neoplasms of the central
nervous system (e.g., primary CNS lymphoma, spinal axis tumors, brain stem gliomas
or pituitary adenomas).
The antibody may be administered once, but more preferably is
administered multiple times. The antibody may be administered from three times
daily to once every six months. The administering may be on a schedule such as three
times daily, twice daily, once daily, once every two days, once every three days, once
weekly, once every two weeks, once every month, once every two months, once every
three months and once every six months. The antibody may be administered via an
oral, mucosal, buccal, intranasal, inhalable, intravenous, subcutaneous, intramuscular,
parenteral, intratumor or topical route. The antibody may be administered at a site
distant from the site of the tumor. The antibody may also be administered
continuously via a minipump. The antibody may be administered once, at least twice
or for at least the period of time until the condition is treated, palliated or cured. The
antibody generally will be administered for as long as the tumor is present provided
that the antibody causes the tumor or cancer to stop growing or to decrease in weight
or volume. The antibody will generally be administered as part of a pharmaceutical
composition as described supra. The dosage of antibody will generally be in the
range of 0.1-100 mg/kg, more preferably 0.5-50 mg/kg, more preferably 1-20 mg/kg,
and even more preferably 1-10 mg/kg. The serum concentration of the antibody may
be measured by any method known in the art. See, e.g., Example XVII below. The
antibody may also be administered prophylactically in order to prevent a cancer or
tumor from occurring. This may be especially useful in patients that have a "high
normal" level of IGF-1 because these patients have been shown to have a higher risk
of developing common cancers. See Rosen et al., supra.

71
In another aspect, the anti-IGF-IR antibody may be co-administered
with other therapeutic agents, such as antineoplastic drugs or molecules, to a patient
who has a hyperproliferative disorder, such as cancer or a tumor. In one aspect, the
invention relates to a method for the treatment of the hyperproliferative disorder in a
mammal comprising administering to said mammal a therapeutically effective amount
of a compound of the invention in combination with an anti-tumor agent selected
from the group consisting of, but not limited to, mitotic inhibitors, alkylating agents,
anti-metabolites, intercalating agents, growth factor inhibitors, cell cycle inhibitors,
enzymes, topoisomerase inhibitors, biological response modifiers, anti-hormones,
kinase inhibitors, matrix metalloprotease inhibitors, genetic therapeutics and anti-
androgens. In a more preferred embodiment, the antibody may be administered with
an antineoplastic agent, such as adriamycin or taxol. In another preferred
embodiment, the antibody or combination therapy is administered along with
radiotherapy, chemotherapy, photodynamic therapy, surgery or other immunotherapy.
In yet another preferred embodiment, the antibody will be administered with another
antibody. For example, the anti-IGF-IR antibody may be administered with an
antibody or other agent that is known to inhibit tumor or cancer cell proliferation, e.g.,
an antibody or agent that inhibits erbB2 receptor, EGF-R, CD20 or VEGF.
Co-administration of the antibody with an additional therapeutic agent
(combination therapy) encompasses administering a pharmaceutical composition
comprising the anti-IGF-IR antibody and the additional therapeutic agent and
administering two or more separate pharmaceutical compositions, one comprising the
anti-IGF-IR antibody and the other(s) comprising the additional therapeutic agent(s).
Further, although co-administration or combination therapy generally means that the
antibody and additional therapeutic agents are administered at the same time as one
another, it also encompasses instances in which the antibody and additional
therapeutic agents are administered at different times. For instance, the antibody may
be administered once every three days, while the additional therapeutic agent is
administered once daily. Alternatively, the antibody may be administered prior to or
subsequent to treatment of the disorder with the additional therapeutic agent.
Similarly, administration of the anti-IGF-IR antibody may be administered prior to or
subsequent to other therapy, such as radiotherapy, chemotherapy, photodynamic
therapy, surgery or other immunotherapy

72
The antibody and one or more additional therapeutic agents (the
combination therapy) may be administered once, twice or at least the period of time
until the condition is treated, palliated or cured. Preferably, the combination therapy
is administered multiple times. The combination therapy may be administered from
three times daily to once every six months. The administering may be on a schedule
such as three times daily, twice daily, once daily, once every two days, once every
three days, once weekly, once every two weeks, once every month, once every two
months, once every three months and once every six months, or may be administered
continuously via a minipump. The combination therapy may be administered via an
oral, mucosal, buccal, intranasal, inhalable, intravenous, subcutaneous, intramuscular,
parenteral, intratumor or topical route. The combination therapy may be administered
at a site distant from the site of the tumor. The combination therapy generally will be
administered for as long as the tumor is present provided that the antibody causes the
tumor or cancer to stop growing or to decrease in weight or volume.
In a still further embodiment, the anti-IGF-IR antibody is labeled with
a radiolabel, an immunotoxin or a toxin, or is a fusion protein comprising a toxic
peptide. The anti-IGF-IR antibody or anti-IGF-IR antibody fusion protein directs the
radiolabel, immunotoxin, toxin or toxic peptide to the IGF-IR-expressing tumor or
cancer cell. In a preferred embodiment, the radiolabel, immunotoxin, toxin or toxic
peptide is internalized after the anti-IGF-IR antibody binds to the IGF-IR on the
surface of the tumor or cancer cell.
In another aspect, the anti-IGF-IR antibody may be used
therapeutically to induce apoptosis of specific cells in a patient in need thereof. In
many cases, the cells targeted for apoptosis are cancerous or tumor cells. Thus, in a
preferred embodiment, the invention provides a method of inducing apoptosis by
administering a therapeutically effective amount of an anti-IGF-IR antibody to a
patient in need thereof. In a preferred embodiment, the antibody is selected from
2.12.1, 2.13.2, 2.14.3, 3.1.1,4.9.2, or 6.1.1, or comprises a heavy chain, light chain or
antigen-binding region thereof.
In another aspect, the anti-IGF-IR antibody may be used to treat
noncancerous states in which high levels of IGF-1 and/or IGF-IR have been associated
with the noncancerous state or disease. In one embodiment, the method comprises the
step of administering an anti-IGF-ER antibody to a patient who has a noncancerous

73
pathological state caused or exacerbated by high levels of IGF-I and/or IGF-ER levels
or activity. In a preferred embodiment, the noncancerous pathological state is
acromegaly, gigantism, psoriasis, atherosclerosis, smooth muscle restenosis of blood
vessels or inappropriate microvascular proliferation, such as that found as a
complication of diabetes, especially of the eye. In a more preferred embodiment, the
anti-IGF-IR antibody slows the progress of the noncancerous pathological state. In a
more preferred embodiment, the anti-IGF-IR antibody stops or reverses, at least in
part, the noncancerous pathological state.
In another aspect, the invention provides a method of administering an
activating anti-IGF-IR antibody to a patient in need thereof. In one embodiment, the
activating antibody or pharmaceutical composition is administered to a patient in need
thereof in an amount effective to increase IGF-IR activity. In a more preferred
embodiment, the activating antibody is able to restore normal IGF-IR activity. In
another preferred embodiment, the activating antibody may be administered to a
patient who has small stature, neuropathy, a decrease in muscle mass or osteoporosis.
In another preferred embodiment, the activating antibody may be administered with
one or more other factors that increase cell proliferation, prevent apoptosis or increase
IGF-IR activity. Such factors include growth factors such as IGF-I, and/or analogues
of IGF-I that activate IGF-IR. In a preferred embodiment, the antibody is selected
from 4.17.3, or comprises a heavy chain, light chain or antigen-binding portion
thereof.
Gene Therapy
The nucleic acid molecules of the instant invention may be
administered to a patient in need thereof via gene therapy. The therapy may be either
in vivo or ex vivo. In a preferred embodiment, nucleic acid molecules encoding both a
heavy chain and a light chain are administered to a patient. In a more preferred
embodiment, the nucleic acid molecules are administered such that they are stably
integrated into the chromosome of B cells because these cells are specialized for
producing antibodies. In a preferred embodiment, precursor B cells are transfected or
infected ex vivo and re-transplanted into a patient in need thereof. In another
embodiment, precursor B cells or other cells are infected in vivo using a virus known
to infect the cell type of interest. Typical vectors used for gene therapy include
liposomes, plasmids, or viral vectors, such as retroviruses, adenoviruses and adeno-

74
associated viruses. After infection either in vivo or ex vivo, levels of antibody
expression may be monitored by taking a sample from the treated patient and using
any immunoassay known in the art and discussed herein.
In a preferred embodiment, the gene therapy method comprises the
steps of administering an effective amount of an isolated nucleic acid molecule
encoding the heavy chain or the antigen-binding portion thereof of the human
antibody or portion thereof and expressing the nucleic acid molecule. In another
embodiment, the gene therapy method comprises the steps of administering an
effective amount of an isolated nucleic acid molecule encoding the light chain or the
antigen-binding portion thereof of the human antibody or portion thereof and
expressing the nucleic acid molecule. In a more preferred method, the gene therapy
method comprises the steps of administering an effective amount of an isolated
nucleic acid molecule encoding the heavy chain or the antigen-binding portion thereof
of the human antibody or portion thereof and an effective amount of an isolated
nucleic acid molecule encoding the light chain or the antigen-binding portion thereof
of the human antibody or portion thereof and expressing the nucleic acid molecules.
The gene therapy method may also comprise the step of administering another anti-
cancer agent, such as taxol, tamoxifen, 5-FU, adriamycin or CP-358,774.
In order that this invention may be better understood, the following
examples are set forth. These examples are for purposes of illustration only and are
not to be construed as limiting the scope of the invention in any manner.
EXAMPLE I: Generation of Hvbridomas Producing Anti-IGF-IR Antibody
Antibodies of the invention were prepared, selected, and assayed as
follows:
Immunization and hybridoma generation
Eight to ten week old XENOMICE ™ were immunized
intraperitoneally or in their hind footpads with either the extracellular domain of
human IGF-IR (10 ug/dose/mouse), or with 3T3-IGF-IR or 300.19-IGF-IR cells,
which are two transfected cell lines that express human IGF-IR on their plasma
membranes (10 x 106 cells/dose/mouse). This dose was repeated five to seven times
over a three to eight week period. Four days before fusion, the mice received a final
injection of the extracellular domain of human IGF-IR in PBS. Spleen and lymph

75
node lymphocytes from immunized mice were fused with the non-secretory myeloma
P3-X63-Ag8.653 cell line and were subjected to HAT selection as previously
described (Galfre and Milstein, Methods Enzymol. 73:3-46, 1981). A panel of
hybridomas all secreting IGF-ER specific human IgG2k antibodies were recovered.
Seven hybridomas producing monoclonal antibodies specific for IGF-IR were
selected for further study and were designated 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2,
4.17.3 and 6.1.1.
Hybridomas 2.12.1, 2.13.2, 2.14.3, 3.1.1,4.9.2 and 4.17.3 were
deposited in the American Type Culture Collection (ATCC), 10801 University
Boulevard, Manassas, VA 20110-2209, on December 12, 2000 with the following
deposit numbers:

Hvbndoma Deposit No.
2.12.1 PTA-2792
2.13.2 PTA-2788
2.14.3 PTA-2790
3.1.1 PTA-2791
4.9.2 PTA-2789
4.17.3 PTA-2793
EXAMPLE II: Determination of Affinity Constants (Kd) of Fully
Human Anti-IGF-IR Monoclonal Antibodies by BIAcore
We performed affinity measures of purified antibodies by surface
plasmon resonance using the BIAcore 3000 instrument, following the manufacturer's
protocols.
Protocol 1
To perform kinetic analyses, protein-A was immobilized on the
sensorchip surfaces of the BIAcore. The sensorchip was then used to capture the anti-
IGF-IR antibodies of the present invention. Different concentrations of the
extracellular domain of IGF-IR were injected on the sensorchip and the binding and
dissociation kinetics of the interactions between the anti-IGF-IR antibodies and the
extracellular domain of IGF-IR were analyzed. The data were evaluated with global
fit Langmuir 1:1, using baseline drift models available on the BIAevaluation software
provided by BIAcore.

76
Protocol 2
BIAcore measurements were performed essentially as described by
Fagerstam et al. "Detection of antigen-antibody interactions by surface plasmon
resonance. Applications to epitope mapping." J. Mol. Recog. 3: 208-214. (1990).
Table I lists affinity measurements for representative anti-IGF-IR
antibodies of the present invention:
Table I

The kinetic analyses indicates that the antibodies prepared in
accordance with the invention possess high affinities and strong binding constants for
the extracellular domain of IGF-IR.
EXAMPLE III: Antibody-mediated Inhibition of
IGF-I-induced Phosphorylation of IGF-IR
We performed ELISA experiments in order to determine whether the
antibodies of this invention were able to block IGF-I-mediated activation of IGF-IR.
IGF-I-mediated activation of IGF-IR was detected by increased receptor-associated
tyrosine phosphorylation.
ELISA Plate Preparation
We prepared ELISA capture plates by adding 100 ul blocking buffer
(3% bovine serum albumin [BSA] in Tris-buffered saline [TBS]) to each well of a
ReactiBind Protein G-coated 96-well plates (Pierce) and incubated the plates with

77
shaking for 30 minutes at room temperature. We diluted rabbit pan-specific SC-713
anti-IGF-IR antibody (Santa Cruz) in blocking buffer to a concentration of 5 ug/ml
and added 100 ul diluted antibody to each well. We incubated the plates with shaking
for 60-90 minutes at room temperature. We then washed the plates five times with
wash buffer (TBS + 0.1% Tween 20) and gently blotted the remaining buffer out onto
paper towels. These plates were not allowed to dry out prior to the addition of lysate.
Preparation of Lysate from IGF-IR-expressing Cells
We placed IGF-IR-transfected NIH-3T3 cells (5xl04/ml) in 100 ul of
growth media (DMEM high glucose media supplemented with L-glutamine (0.29
mg/ml), 10% heat-inactivated FBS, and 500 ug/ml each of geneticin, penicillin and
streptomycin) in 96-well U-bottom plates. We incubated the plates at 37°C, 5% CO2
overnight to allow the cells to attach. We decanted the media from the plates and
replaced it with 100 ul fresh growth media per well. For testing, we diluted the
potential anti-IGF-IR antibodies to five times the desired final concentration in
growth media and added 25 JJ.1 per well. All samples were performed in triplicate.
We then incubated the plates at 37°C for one hour. We stimulated the cells with 25
ul/well of 600 ng/ml IGF-1 (prepared in growth media) and incubate the plates at
room temperature for 10 minutes. We then decanted the media by inverting the plates
and blotting gently onto paper towels and lysed the adherent cells by adding 50 ul of
lysis buffer (50 mM HEPES, pH 7.4, 10 mM EDTA, 150 mM NaCl, 1.5 mM MgCl2)
1.6 mM NaVO4,1% Triton X-100,1% glycerol supplemented immediately before use
with one EDTA-free protease inhibitor tablet [Roche Molecular Sciences] per 50 ml)
and shaking for 5 minutes at room temperature. We added 200 ul dilution buffer (50
mM HEPES, pH 7.4,1.6 mM NaVO4) to each well and mixed by pipetting up and
down. We transferred 100 ul of lysate from each well to each well of the ELISA
capture plate prepared as described above and incubated with gentle shaking for two
hours at room temperature.
ELISA with Anti-tyrosine-phosphate (pTYR) Antibodies
We removed the cell lysate by inverting the plates, washed the plates
five times with wash buffer and blotted on paper towels. We added 100 ul per well
pTYR-specific antibody (HRP-PY54) diluted in blocking buffer to a concentration of

78
0.2 μg/ml and incubated the plates with shaking for 30 minutes at room temperature.
We then washed these plates five times with wash buffer and blotted on paper towels.
We detected binding of the HRP-PY54 antibody by adding 100 ul per
well of TMB peroxidase substrate solution (Kirkegaard & Perry) and incubating with
shaking as the color developed (approximately 2-10 minutes). We stopped the color
development reaction by adding 100 ul per well of TMB stop solution (Kirkegaard &
Perry). We then shook the plates for 10 seconds at room temperature to mix the
solution and quantitated by measurement at OD45onm-
Table II and Figure 4 show the results of this experiment performed
with several antibodies of the invention. The results of this experiment demonstrate
the ability of the antibodies of this invention to block IGF-I-mediated activation of
IGF-IR as shown by increased receptor-associated tyrosine phosphorylation.
Furthermore, these results can be used to quantify the relative potency of the
antibodies of this invention.
Table II

MonoclonalAntibody IC50(Hg/ml)
2.12.1 0.172
2.13.2 0.0812
2.14.3 0.325
4.9.2 0.0324
EXAMPLE IV: Antibodv-mediated Blocking of IGF-I/IGF-IR Binding
We performed ELISA experiments to quantitate the ability of the
antibodies of the invention to inhibit IGF-I binding to IGF-IR in a cell-based assay.
We plated IGF-IR-transfected NIH-3T3 cells (5xlO4/ml) in 100 ul of DMEM high
glucose media supplemented with L-glutamine (0.29 mg/ml), 10% heat-inactivated
FBS, and 500 ug/ml each of geneticin, penicillin and streptomycin in 96-well.U-
bottom plates. We then incubated the plates at 37°C, 5% CO2 overnight to allow cells
to attach. We then decanted the media from the plates and replaced it with 100 ul
fresh media per well. For testing, we diluted antibodies in assay media (DMEM high
glucose media supplemented with L-glutamine, 10% heat-inactivated FBS, 200 jig/ml

79
BSA and 500 ug/ml each of geneticin, penicillin and streptomycin) to the desired final
concentration and added 50 ul per well. All samples were performed in triplicate.
We then incubated the plates at 37°C for ten minutes. We diluted [I25I]-IGF-I to a
concentration of 1 uCi/ml is assay media and added 50 ul per well of the plate. As a
control for background radioactivity, we added cold IGF-I to a final concentration of
100 ng/ml. We incubated the plates for 10 minutes at 37°C, decanted the media by
blotting gently onto paper towels and washed twice with assay media. We then lysed
the cells by adding 50 p.1 0.1 N NaOH, 0.1% SDS and shaking the plates for five
minutes at room temperature. We then transferred the samples to a scintillation plate,
added 150 ul OptiPhase Supermix and read the signal on a Wallac Micro-Beta
counter.
Table III and Figure 3 show the results of this experiment performed
with three representative antibodies of the invention. This experiment demonstrated
that antibodies of the invention specifically inhibit binding of [125I]-IGF-I to cells
overexpressing IGF-IR.
Table in

Monoclonal Antibody IC50
2.12.1 0.45 ug/ml
2.13.2 0.18 ng/ml
4.9.2 0.1 ug/ml
EXAMPLE V: Epitope Mapping Studies
Having demonstrated that the antibodies of the invention recognize
IGF-IR, we performed epitope mapping studies with several antibodies of the
invention. We focused these experiments particularly on the 2.12.1, 2.13.2, 2.14.3,
and 4.9.2 antibodies.
We conducted BIAcore competition studies to determine whether the
antibodies of this invention bind to the same or distinct site on the IGF-IR molecule.
We bound the extracellular domain (ECD) of IGF-IR to a BIAcore sensorchip as
described above in Example II. We bound a first antibody of the invention to this
sensorchip-bound IGF-IR under saturating conditions. We then measured the ability
of subsequent secondary antibodies of the invention to compete with the primary

80
antibody for binding to IGF-IR. This technique enabled us to assign the antibodies of
this invention to different binding groups.
We performed this experiment with antibodies 2.12.1, 2.13.2,2.14.3,
and 4.9.2. We observed that 2.13.2 and 4.9.2 compete for the same site on the
extracellular domain of IGF-IR. The other antibodies, 2.12.1 and 2.14.3, bind to sites
on IGF-IR that are different from both each other and from the site bound by 2.13.2
and 4.9.2.
EXAMPLE VI: Species Crossreactivitv of the Antibodies of the Invention
In order to determine the species crossreactivity of the antibodies of
the invention, we performed several experiments including immunoprecipitation,
antibody-mediating blocking of IGF-I-induced receptor phosphorylation and FACS
analysis.
To perform immunoprecipitation experiments, we plated cells in
DMEM high glucose media supplemented with L-glutamine (0.29 mg/ml), 10% heat-
inactivated FBS, and 500 ug/ml each of geneticin, penicillin and streptomycin to 50%
confluence in T25 flasks. We then added 100 uJ of an antibody of the invention in
Hank's buffered saline solution (HBSS; Gibco BRL) at a concentration of 1 ug/ml.
We incubated the plates for 30 minutes at 37°C in an incubator and then stimulated
the cells with IGF-I at 100 ng/ml for 10 minutes at room temperature. We lysed the
cells in RJPA buffer (Harlow and Lane, supra) and immunoprecipitated IGF-IR with
2 ng of pan-specific SC-713 anti-IGF-ER antibody (Santa Cruz) plus protein A
agarose beads for 1 hour at 4°C. We pelleted the beads and wash three times with
PBS/T (PBS + 0.1% Tween-20) and then boiled the beads in 40 ul Laemmli buffer
containing 5% pME.
The samples prepared as described above were then analyzed by
Western blot. We loaded 12 ul of each sample per lane on 4-10% gradient Novex™
gels run with IX MES buffer (Novex™). Gels were run at 150V for 1 hour or at 200V
for approximately 30 minutes. We thei. transferred the gel to a membrane in Novex™
transfer buffer with 10% methanol either overnight at 100mA or for 1-1.5 hours at
250mA. We then allowed the membrane to dry completely and blocked at room
temperature with TBS (Tris-buffered saline pH 8.0) containing Superblock (Pierce
Chemical Co.). We added the IGF-IR blotting antibody SC713 (Santa Cruz) to detect
immunoprecipitated IGF-IR.

81
This experiment was performed with antibodies of the invention,
particularly 2.12.1,2.13.2,4.17.3 and 4.9.2, on cells from a variety of animals. We
found that antibodies 2.12.1, 2.13.2 and 4.9.2 were able to bind human, but not
canine, guinea pig, rabbit or IGF-IR. Further, these antibodies were able to bind
COS7 and Rhesus IGF-IR, both derived from old world monkeys, but not IGF-IR
from the marmoset, which is a new world monkey. These experiments indicate that
the antibodies are highly specific.
Antibody-mediated Blocking of
JGF-I/IGF-IR Binding in Non-human Primates
Following our observation that the antibodies of the invention
recognize IGF-IR from old world monkeys, we also tested their ability to block IGF-
I/IGF-IR binding in cells derived from these old world monkeys. We plated cells in
DMEM high glucose media supplemented with L-glutamine, 10% heat-inactivated
FBS, and 500 ug/ml each of geneticin, penicillin and streptomycin to 50% confluence
in T25 flasks. We then added an antibody of the invention, or media without antibody
as a control, and stimulated the cells with IGF-I at 100 ng/ml for 10 minutes at room
temperature. After stimulation, we lysed the cells and immunoprecipitated IGF-IR
with pan-specific IGF-IR antibody SC713 as described above. We then performed
Western blot analysis as described above using HRP-PY54 antibody to detect
phosphorylated tyrosine in the activated IGF-IR.
We observed that antibodies of this invention, in particular 2.13.2 and
4.9.2 could block IGF-I-induced phosphorylation of IGF-IR in both COS7 and Rhesus
cells. The IC50 for the observed inhibition was 0.02 ug/ml and 0.005 ug/ml for COS7
and Rhesus IGF-IR, respectively.
Determination of Cross-species Affinity
of Antibodies of the Invention
We performed FACS analysis to determine the affinity of the
antibodies of the invention for IGF-IR from other animals, particularly the old world
monkeys described above. We incubated aliquots of human and monkey cells (5xl05)
for 1 hour on ice with increasing concentrations of biotinylated anti-IGF-IR
antibodies of the invention or with a biotinylated anti-keyhole limpet hemocyanin
(KLH) antibody (Abgenix) as a negative control. We then incubated the samples for
30 minutes on ice with steptavidin-conjugated RPE (phycoerythrin). We measured

82
binding by flow cytometry and analyzed the histograms of fluorescence intensity
(FI2-H) versus cell number (Counts) using CellQuest software. We calculated
binding (Kd) for each antibody from graphs of mean fluorescence intensity versus
antibody concentration. In most experiments, we measured binding in cultured
human MCF-7 cells and either rhesus or cynomologous tissue culture cells. We
controlled for depletion of the antibody by measuring binding over a range of cell
concentrations.
We performed the aforementioned FACS analysis to test the ability of
antibodies of the invention, particularly 2.13.2 and 4.9.2, to bind human, rhesus and
cynomologous cells. We observed a half maximal binding (Kd of 0.1 ug/ml for all
cell lines tested.
EXAMPLE VII: IGF-I Receptor Downregulation
We performed blocking experiments essentially as described above in
Example IV up to the addition of [l25I]-labeled IGF-I. At this point, we boiled the
cells in 40 \il Laemmli buffer containing 50% me. We then analyzed the samples by
western blot analysis as described above in Example VI and probed the blots with
both pan-specific IGF-IR antibody SC713 to quantify the levels of IGF-IR and HRP-
PY54 antibody to monitor the levels of phosphorylated tyrosine in the activated IGF-
IR.
As observed previously (Example III), we observed blockage of IGF-I-
induced phosphorylation of IGF-IR following the treatment of cells with an antibody
of this invention (Figure 4). Further, we observed that this blockage of IGF-I-induced
phosphorylation was followed by downregulation of the IGF-IR in these cells. See,
e.g., Fig. 4. IGF-IR levels were maximally reduced 16 hours after stimulation with
IGF-I in the presence of an antibody of the invention.
EXAMPLE VIII: Effects of the Antibodies of the Invention on IGF-IR in vivo
We determined whether the effects of the antibodies of the invention
on IGF-IR as described in the previous examples would occur in vivo. We induced
tumors in athymic mice according to published methods (V.A. Pollack et al.,
"Inhibition of epidermal growth factor receptor-associated tyrosine phosphorylation in
human carcinomas with CP-358,774: Dynamics of receptor inhibition in situ and
antitumor effects in athymic mice," J. Pharmacol. Exp. Ther. 291:739-748 (1999).

Briefly, we injected IGF-IR-transfected NIH-3T3 cells (5xlO6) subcutaneously into 3-
4 week-old athymic (nu/nu) mice with 0.2 ml of Matrigel preparation. We then
injected mice with an antibody of the invention intraperitoneally after established (i.e.
approximately 400 mm3) tumors formed.
After 24 hours, we extracted the tumors, homogenized them and
determined the level of IGF-IR. To determine IGF-IR levels, we diluted the SC-713
antibody in Blocking buffer to a final concentration of ug/ml and added 100 ul to each
well of a Reacti-Bind Goat anti-rabbit (GAR) coated plate (Pierce). We incubated the
plates at room temperature for 1 hour with shaking and then washed the plates five
times with wash buffer. We then weighed tumor samples that had been prepared as
described above and homogenized them in lysis buffer (1 ml/100 mg). We diluted
12.5 ul of tumor extract with lysis buffer to a final volume of 100 ul and added this to
each well of a 96-well plate. We incubated the plates at room temperature with
shaking for 1-2 hours and then washed the plates five times with Wash buffer. We
then added 100 ul HRP-PY54 or biotinylated anti-IGF-IR antibody in Blocking buffer
to each well and incubated at room temperature with shaking for 30 minutes. We then
washed the plates five times with wash buffer and developed the plates. We
developed the plates probed with HRP-PY54 by adding 100 ul of the TMB microwell
substrate per well and stopped color development with the addition 100 ul 0.9 M
H2SO4. We then quantitated the signal by shaking for 10 seconds and measuring
OD450nm- The signal was normalized to total protein. We developed plates probed
with anti-IGF-IR antibody by adding 100 ul of streptavidin-HRP diluted in Blocking
buffer to each well, incubating at room temperature with shaking for 30 minutes and
then continuing as described for HRP-PY54.
We observed that intraperitoneal injection of an antibody of this
invention, particularly 2.13.2 and 4.9.2, resulted in inhibition of IGF-IR activity as
measured by a decrease of [both IGF-IR phosphotyrosine (phosphorylated IGF-IR)
and] total IGF-IR protein (Figure 6). In addition, we also observed a decrease in IGF-
IR phosphotyrosine (phosphorylated IGF-IR) (Figure 5). Without wishingto be bound
by any theory, the decreased levels of IGF-IR phosphotyrosine may be due to the
decreased levels of IGF-IR protein in vivo after treatment with the antibody or may be
due to a combination of decreased levels of IGF-IR protein and a decrease in tyrosine
phosphorylation on the IGF-IR that is present due to blocking of activation by ligand

84
(e.g., IGF-I or IGF-II). Furthermore, this inhibition was responsive to the dose of
antibody injected (Figure 6). These data demonstrate that the antibodies of the
invention are able to target the IGF-IR in vivo in a manner analogous to what we
observed in vitro.
EXAMPLE IX: Growth Inhibition (TGD of 3T3/IGF-IR Cell Tumors
We tested whether anti-IGF-IR antibodies of the invention would
function to inhibit tumor growth. We induced tumors as described above (Example
VIII) and when established, palpable tumors formed (i.e. 250 mm3, within 6-9 days),
we treated the mice with a single, 0.20 ml dose of antibody by intraperitoneal
injection. We measured tumor size by Vernier calipers across two diameters every
third day and calculated the volume using the formula (length x [width]2)/2 using
methods established by Geran, et al., "Protocols for screening chemical agents and
natural products against animal tumors and other biological systems," Cancer
Chemother. Rep. 3:1-104.
When we performed this analysis with an antibody of the invention, we
found that a single treatment with antibody 2.13.2 alone inhibited the growth of IGF-
IR-transfected NIH-3T3 cell-induced tumors (Figure 7, left panel). Furthermore, in
combination studies with a single dose of 7.5 mg/kg intravenously-supplied
adriamycin, we observed that administration of a single dose of 2.13.2 enhanced the
effectiveness of adriamycin, a known inhibitor of tumor growth. The combination of
adriamycin with an antibody of the invention, 2.13.2, demonstrated a growth delay of
7 days versus treatment with the antibody or adriamycin alone (Figure 7, right panel).
EXAMPLE X: Relationship of Antibody Levels to IGF-IR Downregulation
Tumors were induced in nude mice as described in Example VIII. The
mice were then treated with 125 ug of 2.13.2 by intraperitoneal injuction, as described
in Example VIII. Tumors were extracted and IGF-IR levels were measured by ELISA
as described in Example VIII. Figure 8 shows the serum 2.13.2 antibody levels and
IGF-IR receptor levels over time. The experiment demonstrates that the IGF-IR is
down-regulated by the antibody and that the degree of IGF-IR inhibition is dose
proportional to the serum concentration of the antibody.

83
EXAMPLE XI: Growth Inhibition of 3T3/IGF-IR Tumors with Multiple Dosing of
Antibody in Combination with Adriamvcin
Tumors were induced in nude mice as described in Example IX. Mice
with established subcutaneous tumors of approximately 250 mm3 were treated on
days 1, 8, 15 and 22 with various amounts of 2.13.2 antibody (i.p.) or 7.5 mg/kg
adriamycin (i.v.), either as single agents or in combination, as described in Example
IX. Figure 9 shows the tumor size in relation to the various treatments over time.
The experiment demonstrates that treatment with an anti-IGF-IR antibody given once
every seven days inhibits tumor cell growth and enhances inhibition of tumor cell
growth in combination with adriamycin, a known tumor inhibitor.
EXAMPLE XII: Growth Inhibition of Large Tumors
Tumors were induced in nude mice as described in Example EX. Mice
with large established subcutaneous tumors of slightly less than 2000 mm3 were
treated on days 1 and 8 with various amounts of 2.13.2 antibody (i.p.) or 7.5 mg/kg
adriamycin (i.v.), either as single agents or in combination, as described in Example
IX. Figure 10 shows the tumor size in relation to the various treatments over time.
Control, antibody alone and adriamycin alone animal groups were terminated at day
5, when the tumor size exceeded 2000 mm3. The experiment demonstrates that
treatment with an anti-IGF-IR antibody in combination with adriamycin is highly
efficacious against large tumors when multiple doses are given.
EXAMPLE XIII: Growth Inhibition of Colorectal Cell Tumors
Tumors were induced in nude mice as described in Example IX except
that Colo 205 cells (ATCC CCL 222) were used. Colo 205 cells are human colorectal
adenocarcinoma cells. Mice with established subcutaneous tumors of approximately
250 mm3 were treated with various amounts of 2.13.2 antibody (i.p.) or with 100
mg/kg 5-fluorodeoxyuridine (5-FU, i.v.), either as single agents or in combination, as
described in Example IX. Figure 11 shows the tumor size in relation to the various
treatments over time. The experiment demonstrates that treatment with an anti-IGF-IR
antibody given once inhibits human colorectal cancer cell growth when provided as a
single agent and enhances the effectiveness of 5-FU, a known tumor inhibitor.
Mice with established Colo 205 tumors were treated on days 1, 8, 15
and 22 with 500 ug 2.13.2 (i.p.), 100 mg/kg 5-FU (i.v.) or a combination thereof.

86
Figure 12 shows the tumor size in relation to the various treatments over time. The
experiment demonstrates that treatment with an anti-IGF-IR antibody given once
every seven days inhibits human colorectal cancer cell growth and enhances the
effectiveness of 5-FU.
"EXAMPLE XIV: Growth Inhibition of Breast Cancer Cell Tumors
Nude mice as described in Example VIII were implanted with
biodegradable estrogen pellets (0.72 mg 17-P-estradiol/pellet, 60 day release;
Innovative Research of America). After 48 hours, tumors were induced in nude mice
essentially as described in Example IX except that MCF-7 cells (ATCC HTB-22)
were used. MCF-7 cells are estrogen-dependent human breast carcinoma cells. Mice
with established subcutaneous rumors of approximately 250 mm3 were treated with 50
Mg 2.13.2 antibody (i.p.) on days 1,4, 7, 10, 13, 16, 19 and 22 (q3dx7) or with 6.25
mg/kg taxol (i.p.) on days 1, 2, 3,4, 5 (qldx5), either as single agents or in
combination, essentially as described in Example IX. Figure 13 shows the tumor size
in relation to the various treatments over time. The experiment demonstrates that
treatment with an anti-IGF-IR antibody by itself inhibits human breast cancer cell
growth when administered once every three days and also enhances the effectiveness
of taxol, a known breast cancer inhibitor, when given in combination.
Mice having established tumors from MCF-7 cells as described
immediately above were treated on day 1 with various amounts of 2.13.2 antibody
(i.p.) alone or with 3.75 mg/kg adriamycin (i.v.), essentially as described in Example
IX. Figure 14 shows the tumor size in relation to the various treatments over time.
The experiment demonstrates that a single treatment with an anti-IGF-IR antibody by
itself inhibits human breast cancer cell growth and enhances the effectiveness of
adriamycin, a known tumor inhibitor.
Mice having established tumors from MCF-7 cells as described
immediately above were treated with 250 μg 2.13.2 antibody (i.p.) on days 1, 8, 15
and 23 or with a biodegradable tamoxifen pellet (25 mg/pellet, free base, 60 day
release, Innovative Research of America), either as single agents or in combination,
essentially as described in Example IX. The tamoxifen pellet was implanted on day 1
after the tumor was established. Figure 15 shows the tumor size in relation to the
various treatments over time. The experiment demonstrates that treatment with an

88
anti-IGF-IR antibody administered once every seven days inhibits human breast
cancer growth by itself and enhances the effectiveness of tamoxifen, a known tumor
inhibitor.
EXAMPLE XVI: Growth Inhibition of Epidermoid Carcinoma Cell Tumors
Tumors were induced in nude mice essentially as described in Example
IX except that A431 cells (ATCC CRL 1555) were used. A431 cells are human
epidermoid carcinoma cells that overexpress EGFR. Mice with established
subcutaneous tumors of approximately 250 mm3 were treated on days 1, 8,15, 22 and
29 with 500 pg 2.13.2 antibody (i.p.) or were treated once daily for 27 days with 10
mg/kg CP-358,774 given orally (p.o.), either as single agents or in combination, as
described in Example DC. CP-358,774 is described in United States Patent 5,747,498
and Moyer et al., Cancer Research 57: 4838-4848 (1997), herein incorporated by
reference. Figure 16 shows the tumor size in relation to the various treatments over
time. The experiment demonstrates that treatment with an anti-IGF-IR antibody
enhances the effectiveness of CP-358,774, a known EGF-R tyrosine kinase inhibitor,
for inhibiting the growth of a human epidermoid carcinoma tumor.
EXAMPLE XVII: Pharmacokinetics of Anti-IGF-IR Antibodies in vivo
To evaluate the pharmacokinetics of the anti-IGF-IR antibodies,
cynomolgus monkeys were injected intravenously with 3, 30 or 100 mg/kg of 2.13.2
antibody in an acetate buffer. Serum was collected from the monkeys at various time
points and anti-IGF-IR antibody concentrations in the monkeys were determined for a
period of up to ten weeks levels. To quantitate functional serum antibody levels, the
extracellular domain of the human IGF-IR (IGF-I-sR, R&D Systems, Catalog #
391GR) was bound to 96-weIl plates. Monkey serum (diluted between 1:100 and
1:15,000) was added to the assay plates so that each sample would be within the linear
range of the standard curve and incubated under conditions in which any anti-IGF-IR
antibody would bind to IGF-I-sR. After washing the plates, a labeled anti-human IgG
antibody was added to the plates and incubated under conditions in which the anti-
human IgG antibody would bind to the anti-IGF-IR antibody. The plates were then
washed and developed, and a control standard curve and linear regression fits used to
quantitate the amount of anti-IGF-IR antibodies. Figure 17 shows the concentration

88
of 2.13.2 in serum over time. The experiment demonstrates that the half-life of the
anti-IGF-IR antibody is 4.6 to 7.7 days and has a volume distribution of 74-105
mL/kg. Further, the experiment demonstrates that the amounts given are dose-
proportional in the monkey, which indicates that the anti-IGF-IR antibody has
saturated any available IGF-IR binding sites in the body even at the lowest dose of 3
mg/kg.
EXAMPLE XVIII: Combination Therapy of Anti-IGF-IR Antibody and Adriamvcin
Downregulates IGF-IR in vivo
Tumors were induced in nude mice as described in Example IX. Mice
with established subcutaneous tumors of approximately 400 mm3 were treated with a
single injection of 250 [ig 2.13.2 antibody (i.p.) or with 7.5 mg/kg adriamycin (i.v.),
. either as single agents or in combination, as described in Example EX. 72 hours after
administration of the agents, tumors were extracted as described in Example VIII, and
equal amounts of the tumor extracts were subjected to sodium dodecyl phosphate
polyacrylamide gel electrophoresis (SDS PAGE) and western blot analysis using the
anti-IGF-IR antibody SC-713 (Santa Cruz). Figure 18 shows the amounts of IGF-IR
in tumor cells in control animals (first three lanes of each panel), in animals treated
with antibody alone (top panel), in animals treated with adriamycin alone (middle
panel) and in animals treated with antibody and adriamycin (lower panel). Each lane
represents equal amounts of protein from individual tumors from individual mice.
The experiment demonstrates that treatment with adriamycin alone has little effect on
IGF-IR levels and that treatment with antibody alone shows some decrease in IGF-IR
levels. Surprisingly, treatment with adriamycin and antibody together shows a
dramatic decrease in IGF-IR levels, demonstrating that adriamycin and antibody
greatly downregulate IGF-IR levels.
All publications and patent applications cited in this specification are
herein incorporated by reference as if each individual publication or patent application
were specifically and individually indicated to be incorporated by reference. Although
the foregoing invention has been described in some detail by way of illustration and
example for purposes of clarity of understanding, it will be readily apparent to those
of ordinary skill in the art in light of the teachings of this invention that certain
changes and modifications may be made thereto without departing from the spirit or
scope of the appended claims.

WE CLAIM:
1. A monoclonal antibody or an antigen-binding portion thereof that specifically binds to
insulin-like growth factor I receptor (IGF-IR), wherein said antibody comprises a heavy chain
and a light chain; and wherein said antibody comprises the CDR1, CDR2, and CDR3 amino
acid sequences of a variable domain selected from the group consisting of:
a) the light chain variable domain of antibody 2.12.1 (ATCC Deposit No. PTA-2792);
b) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 2;
c) the heavy chain variable domain of antibody 2.12.1;
d) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 4;
and
e) the heavy and light chain variable domains of antibody 2.12.1.
2. The antibody or antigen-binding portion as claimed in claim 1, wherein the variable
domain of said light chain comprises no more than ten amino acid changes from the amino acid
sequence encoded by a germline VK A30 gene.
3. The antibody or antigen-binding portion as claimed in claim 2, wherein the light chain
variable domain comprises the amino acid sequence of SEQ ID NO: 2.
4. The antibody or antigen-binding portion as claimed in claim 1, wherein the variable
domain of said heavy chain comprises no more than eight amino acid changes from the amino
acid sequence encoded by a germline VH DP35 gene.
5. The antibody or antigen-binding portion as claimed in claim 4, wherein the heavy chain
variable domain comprises the amino acid sequence of SEQ ID NO: 4.
6. A monoclonal antibody or an antigen-binding portion thereof that specifically binds to
IGF-IR, wherein said antibody comprises the heavy chain CDR1, CDR2, and CDR3 amino acid
sequences and the light chain CDR1, CDR2, and CDR3 amino acid sequences of antibody
2.12.1 (ATCC Deposit No. PTA-2792).

7. The antibody or antigen-binding portion as claimed in claim 6, wherein said antibody
comprises the heavy chain variable domain and the light chain variable domain of antibody
2.12.1 (ATCC Deposit No. PTA-2792).
8. The antibody as claimed in claim 1, wherein said antibody comprises heavy chain and
light chain amino acid sequences selected from the group consisting of:

a) the amino acid sequence of the heavy chain and the amino acid sequence of the light
chain of 2.12.1 (ATCC Deposit No. PTA-2792), respectively; and
b) the amino acid sequence of SEQ ID NO: 49 without the signal sequence and the amino
acid sequence of SEQ ID NO: 51 without the signal sequence, respectively.

9. A monoclonal antibody that is antibody 2.12.1 (ATCC Deposit No. PTA-2792) or an
antibody with the same amino acid sequences as antibody 2.12.1.
10. The monoclonal antibody as claimed in claim 1, wherein said heavy chain amino acid
sequence comprises the CDR1, CDR2 and CDR3 amino acid sequences in SEQ ID NO: 4 and
wherein said light chain amino acid sequence comprises the CDR1, CDR2 and CDR3 amino
acid sequences in SEQ ID NO: 2.
11. The monoclonal antibody as claimed in claim 10, wherein the heavy chain amino acid
sequence comprises the variable domain amino acid sequence in SEQ ID NO: 4 or said
sequence with up to five conservative amino acid substitutions, and the light chain amino acid
sequence comprises the variable domain amino acid sequence in SEQ ID NO: 2 or said
sequence with up to five amino acid substitutions.
12. The monoclonal antibody as claimed in claim 11, wherein the heavy chain amino acid
sequence comprises the variable domain amino acid sequence in SEQ ID NO: 4 and the light
chain amino acid sequence comprises the variable domain amino acid sequence in SEQ ID NO:
2.
13. A monoclonal antibody or an antigen-binding portion thereof, wherein the heavy chain
of said antibody comprises SEQ ID NO: 49 without the signal sequence and the light chain of
said antibody comprises SEQ ID NO: 51 without the signal sequence.

14. The antibody or antigen-binding portion as claimed in any one of claims 1-13, that is
a) an immunoglobulin G (IgG), an IgM, an IgE, an IgA or an IgD molecule, or is derived
therefrom; or
b) an Fab fragment, an F(ab')2 fragment, an Fv fragment, a single chain antibody, a human
antibody, a humanized antibody, a chimeric antibody or a bispecific antibody.
15. The antibody or antigen-binding portion as claimed in any one of claims 1-14, wherein
the antibody or portion has at least one property selected from the group consisting of:
a) does not bind to mouse, rat, dog or rabbit IGF-IR;
b) binds to cynomologous or rhesus IGF-IR but not to marmoset IGF-IR;
c) inhibits the binding of IGF-I or IGF-II to IGF-IR;
d) has a selectivity for IGF-IR that is at least 50 times greater than its selectivity for insulin
receptor;
e) inhibits tumor growth in vivo;
f) causes IGF-IR disappearance from the cell surface when incubated with a cell
expressing IGF-IR;
g) inhibits IGF-IR-induced tyrosine phosphorylation;
h) binds to IGF-IR with a KD of 7.37 x 10-9 M or less; and
i) has a Koff for IGF-IR of 10-4 s-1 or less.
16. The antibody or antigen-binding portion as claimed in claim 15, wherein the antibody or
portion inhibits binding of IGF-I or IGF-II to IGF-IR, binds to IGF-IR with a KD of 7.37 x 10-9
M or less and has a selectivity for IGF-IR that is at least 50 times greater than its selectivity for
insulin receptor.
17. The antibody or antigen-binding portion as claimed in claim 15, wherein the antibody or
portion comprises all of said properties.
18. The antibody or antigen-binding portion as claimed in any one of claims 1-17, wherein
said antibody or portion inhibits binding between IGF-IR and IGF-I or IGF-II with an IC50 of
less than 100 nM.

19. The antibody or antigen-binding portion as claimed in claim 1, wherein the antibody or
portion cross-competes for binding to IGF-IR with antibody 2.12.1 (ATCC Deposit No. PTA-
2792) or binds to the same epitope of IGF-IR as antibody 2.12.1.
20. The antibody or antigen-binding portion as claimed in claim 19, wherein the antibody or
portion cross-competes for binding to IGF-IR with antibody 2.12.1 (ATCC Deposit No. PTA-
2792) and binds to the same epitope of IGF-IR as antibody 2.12.1.
21. A pharmaceutical composition comprising the antibody or antigen-binding portion as
claimed in any one of claims 1-20, and a pharmaceutically acceptable carrier.
22. The pharmaceutical composition as claimed in claim 21, further comprising an
antineoplastic, chemotherapeutic, anti-angiogenic, or anti-tumor agent.
23. A process for making an antibody as claimed in any one of claims 1-20, comprising the
steps of:

a) immunizing a non-human mammal with an immunogen comprising IGF-IR, wherein the
mammal is capable of expressing human antibodies in its B cells;
b) isolating B cells from the mammal;
c) screening said B cells, or cell lines derived therefrom, to identify a cell line that
produces antibodies that bind to IGF-IR;
d) culturing the cell line that expresses antibodies that bind to IGF-IR; and
e) isolating antibodies that bind to IGF-IR from the cell line.

24. A hybridoma cell line that produces the antibody or antigen-binding portion, as claimed
in any one of claims 1-20.
25. The hybridoma cell line as claimed in claim 24 that produces:

a) antibody 2.12.1 (ATCC Deposit No. PTA-2792), or an antibody that has the same
amino acid sequences as antibody 2.12.1; or
b) an antigen-binding portion of the antibody of (a).

26. A medicament suitable for diagnosing the presence or location of an IGF-IR-expressing
tumor in a subject in need of such diagnostic, wherein said medicament comprises the antibody
or antigen-binding portion, as claimed in any one of claims 1-20.
27. A medicament suitable for treating cancer in a human, wherein said medicament
comprises the antibody or antigen-binding portion, as claimed in any one of claims 1-20.
28. The medicament as claimed in claim 27, wherein said medicament is suitable for
administration with an anti-neoplastic, anti-tumor, anti-angiogenic or chemotherapeutic agent.
29. An isolated nucleic acid molecule that comprises a nucleic acid sequence that encodes
the heavy chain or an antigen-binding portion thereof, a nucleic acid sequence that encodes the
light chain or an antigen-binding portion thereof, or both, of the antibody, as claimed in any one
of claims 1-20.
30. The isolated nucleic acid molecule as claimed in claim 29, wherein the nucleic acid
molecule comprises a nucleic acid sequence selected from the group consisting of:

a) a nucleic acid sequence encoding the heavy chain or an antigen-binding portion thereof
of an antibody comprising the three CDRs from the heavy chain of antibody 2.12.1
(ATCC Deposit No. PTA-2792);
b) a nucleic acid sequence encoding the amino acid sequence of the heavy chain or an
antigen-binding portion thereof of antibody 2.12.1;
c) a nucleic acid sequence encoding the light chain or an antigen-binding portion thereof of
an antibody comprising the three CDRs from the light chain of antibody 2.12.1;
d) a nucleic acid sequence encoding the amino acid sequence of the light chain or an
antigen-binding portion thereof of antibody 2.12.1;
e) a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2 or 4; and
f) the nucleic acid sequence of SEQ ID NO: 1 or 3;
wherein said nucleic acid molecule optionally comprises a nucleic acid sequence encoding the
amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 26.

31. A vector comprising a nucleic acid sequence that encodes the heavy chain or an antigen-
binding portion thereof, a nucleic acid sequence that encodes the light chain or an antigen-
binding portion thereof, or both, of the antibody or portion as claimed in any one of claims 1-
20, wherein the vector optionally comprises expression control sequence(s) operably linked to
the nucleic acid sequences.
32. A method of making an anti-IGF-IR antibody or an antigen-binding portion thereof,
comprising culturing the hybridoma cell line as claimed in claim 24, under suitable conditions
and recovering said antibody or portion.
33. A medicament comprising:

a) an isolated nucleic acid molecule encoding the heavy chain or an antigen-binding
portion thereof of the antibody as claimed in any one of claims 1-20;
b) an isolated nucleic acid molecule encoding the light chain or an antigen-binding portion
thereof of the antibody according to any one of claims 1-20; or
c) isolated nucleic acid molecules encoding the light chain and the heavy chain or
antigen-binding portions thereof of the antibody according to any one of claims 1-20.

The present invention relates to antibodies and antigen-binding protions thereof that specifically bind to insulin-like
growth factor I receptor (IGF-IR), which is preferably human IGF-IR. The invention also relates to human anti-IGF-IR antibodies,
including chimeric, bispecific, derivatized, single chain antibodies or portions of fusion proteins. The invention also relates to isolated heavy and light chain immunoglobulin molecules derived from anti-IGF-IR antibodies and nucleic acid molecules encoding
such momlecules. The present invention also relates to methods of making anti-IGF-IR antibodies, pharmaceutical compositions
comprising these antibodies and methods of using the antibodies and compositions thereof for diagnosis and treatment. The invention
also provides gene therapy methods using nucleic acid molecules encoding the heavy and/or light immunoglobulin molecules that
comprise the human anti-IGF-IR antibodies. The invention also relates to gene therapy methods and transgenic animals comprising
nucleic acid molecules of the present invention.


Documents:

02634-kolnp-2007-abstract.pdf

02634-kolnp-2007-claims.pdf

02634-kolnp-2007-correspondence others.pdf

02634-kolnp-2007-description complete.pdf

02634-kolnp-2007-drawings.pdf

02634-kolnp-2007-form 1.pdf

02634-kolnp-2007-form 2.pdf

02634-kolnp-2007-form 3.pdf

02634-kolnp-2007-form 5.pdf

02634-kolnp-2007-gpa.pdf

02634-kolnp-2007-sequence listing.pdf

2634-KOLNP-2007-(29-03-2012)-CORRESPONDENCE.pdf

2634-KOLNP-2007-(29-03-2012)-FORM-27.pdf

2634-KOLNP-2007-(29-03-2012)-PA-CERTIFIED COPIES.pdf

2634-KOLNP-2007-ABSTRACT 1.1.pdf

2634-kolnp-2007-amanded claims.pdf

2634-KOLNP-2007-ASSIGNMENT 1.1.pdf

2634-KOLNP-2007-ASSIGNMENT.pdf

2634-KOLNP-2007-CLAIMS.pdf

2634-KOLNP-2007-CORRESPONDENCE 1.2.pdf

2634-kolnp-2007-correspondence 1.4.pdf

2634-KOLNP-2007-CORRESPONDENCE OTHERS 1.1.pdf

2634-KOLNP-2007-CORRESPONDENCE-1.3.pdf

2634-KOLNP-2007-DESCRIPTION (COMPLETE) 1.1.pdf

2634-kolnp-2007-description (complete) 1.2.pdf

2634-KOLNP-2007-EXAMINATION REPORT REPLY RECIEVED 1.1.pdf

2634-kolnp-2007-form 1-1.1.pdf

2634-KOLNP-2007-FORM 1.pdf

2634-KOLNP-2007-FORM 13.pdf

2634-KOLNP-2007-FORM 18.pdf

2634-KOLNP-2007-FORM 2 1.1.pdf

2634-kolnp-2007-form 2-1.2.pdf

2634-KOLNP-2007-FORM 3 1.1.pdf

2634-KOLNP-2007-FORM 3.pdf

2634-KOLNP-2007-FORM 5.pdf

2634-KOLNP-2007-FORM 6.pdf

2634-KOLNP-2007-FORM-27.pdf

2634-KOLNP-2007-OTHERS 1.1.pdf

2634-kolnp-2007-others 1.2.pdf

2634-KOLNP-2007-PA.pdf

2634-KOLNP-2007-PCT PRIORITY DOCUMENT NOTIFICATION.pdf

2634-KOLNP-2007-PETITION UNDER RULE 137-1.1.pdf

2634-kolnp-2007-petition under rule 137-1.2.pdf

2634-KOLNP-2007-PETITION UNDER RULE 137.pdf

2634-KOLNP-2007-REPLY TO EXAMINATION REPORT.pdf


Patent Number 250011
Indian Patent Application Number 2634/KOLNP/2007
PG Journal Number 48/2011
Publication Date 02-Dec-2011
Grant Date 28-Nov-2011
Date of Filing 13-Jul-2007
Name of Patentee AMGEN FREMONT INC
Applicant Address 6701 KAISER DRIVE, FREMONT CALIFORNIA 94555
Inventors:
# Inventor's Name Inventor's Address
1 COHEN BRUCE D 41 CARDINAL ROAD, EAST LYME, CT 06333
2 MILLER PENELOPE E 56 HANCOCK DRIVE, MYSTIC, CT 06355
3 MOYER JAMES D. 5 JEFFERSON DRIVE, EAST LYME, CT 06333
4 CORVALAN JOSE R 125 WILLIAMS LANE, FOSTER CITY, CA 94404
5 GALLO MICHAEL 2650 COLWOOD DRIVE, NORTH VANCOUVER, BRITISH COLUMBIA, V79 2R1
6 BEEBE JEAN 383 FORSYTH ROAD, SALEM, CT 06420
PCT International Classification Number A61K39/395
PCT International Application Number PCT/US2001/051113
PCT International Filing date 2001-12-20
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/259,927 2001-01-05 U.S.A.