HK1098162B - Antibodies to insulin-like growth factor i receptor - Google Patents

Description

Antibodies to insulin-like growth factor I receptor

The present application is a divisional application with parent application number 01821808.3 (International application number PCT/US01/51113), application date 2001, 12/20, entitled "antibodies to insulin-like growth factor I receptor".

Technical Field

This application claims priority to U.S. provisional application 60/259,927 filed on 5/1/2001.

Technical Field

Insulin-like growth factor (IGF-I) is a 7.5-kD polypeptide that circulates in plasma at very high concentrations and is found in most tissues. IGF-I stimulates differentiation and proliferation of cells and is required for sustained proliferation of most mammalian cell types. These types of cells include human diploid fibroblasts, epithelial cells, smooth muscle cells, T lymphocytes, neural cells, bone marrow cells, chondrocytes, osteoblasts, bone marrow stem cells, and the like. For a review of the types of cells that mediate cell proliferation via IGF-I/IGF-I receptor interactions see Goldring et al, eukar. gene Express, 1: 31-326(1991).

The first step in the transduction pathway leading to IGF-I-stimulated cell proliferation or differentiation is the binding of IGF-I or IGF-II (or insulin at supraphysiological concentrations) to the IGF-I receptor. The IGF-I receptor consists of two types of subunits: alpha subunit (a 130-135kD protein which is all extracellular and plays a role in ligand binding) and beta subunit (a 95-kD transmembrane protein with transmembrane and cytoplasmic domains). IGF-IR belongs to the family of tyrosine kinase growth factor receptors (μ llrich et al, Cell 61: 203-212, 1990) and is structurally similar to the insulin receptor (μ llrich et al, EMBO J.5: 2503-2512, 1986). IGF-IR is initially synthesized as a single chain precursor receptor polypeptide, which is then assembled by glycosylation, proteolytic cleavage and covalent binding into a 460-kD mature heterotetramer containing two alpha-subunits and two beta-subunits. The beta-subunit has tyrosine kinase activity activated by a ligand. This activity is associated with signaling pathways mediating ligand action, including autophosphorylation of the β -subunit and phosphorylation of IGF-IR substrates.

The in vivo serum concentration of IGF-I is dependent on the presence of pituitary Growth Hormone (GH). Although the liver is a major site of GH-dependent IGF-I synthesis. Recent work has shown that most normal tissues also produce IGF-I. IGF-I may also be produced by various neoplastic tissues. IGF-I can therefore act as a regulator of normal or abnormal cell proliferation through 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 that interacts with IGF-IR is regulated by IGFBP. For a review of IGFBP and IGF-I see Grimberg et al, j.cell. physiol.183: 1-9, 2000.

The maintenance of tumor cells by IGF-I and/or IGF-IR in vitro and in vivo has been strongly demonstrated. IGF-IR increases in lung tumors (Kaiser et al, J.cancer Res. Clin Oncol.119: 665. 668, 1993; Moody et al, Life Sciences 52: 1161. 1173, 1993; Maca. mu. ley et al, Cancer Res., 50: 2511. sup. 2517, 1990), breast tumors (Pollak et al, Cancer Lett.38: 223. sup. 230, 1987; Foekens et al, Cancer Res.49: 7002. sup. 7009, 1989; C. mu. llen et al, Cancer Res.49: 7002. sup. 7009, 1990; Aretega et al, J.Clin. invest.84: 1418. sup. 1423, 1989), prostate and colon tumors (RemaoleBennet et al, J.Clin. Endocrinol. Met.75: 609, 1992. Guenth. sup. 102. sup. pol. 102. sup. seq.). Deregulated expression of IGF-I in the prostate epithelium leads to tumor formation 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 stimulates the growth of fibrosarcomas that overexpress IGF-IR (Butler et al, Cancer Res.58: 3021-27, 1998). Furthermore, individuals with elevated levels of IGF-I are at greater risk for common cancer than individuals with low levels of IGF-I within the normal range (Rosen et al, Trends Endocrinol. Metab.10: 136-41, 1999). Many of these types of tumor cells respond to IGF-I with proliferative signals in culture (Nakanishi et al, J.Clin.invest.82: 354-359, 1988; free et al, J.mol.Endocrinol.3: 509-514, 1989), and autocrine or paracrine loops of in vitro proliferation have been deduced (LeRoith et al, Endocrine Revs.16: 143-163, 1995; Yee et al, mol.Endocrinol.3: 509-514, 1989). For a review of the role of IGF-I/IGF-I receptor interactions in the growth of various human tumors see Maca μ lay, br.j. cancer, 65: 311-320, 1992.

Increased IGF-I levels are also associated with several noncancerous pathological conditions including acromegaly and gigantism (Barkan, Cleveland Clin.J.Med.65: 343, 347-. Increased levels of IGF-I may also lead to diabetes or its complications, such as microvascular proliferation (Smith et al, nat. Med.5: 1390-. The reduction in IGF-I levels, particularly when GH serum levels are reduced or when GH insensitivity or resistance is present, is associated with conditions such as short stature (Laron, Paediatr. Dr. mu.gs 1: 155-159, 1999), neuropathy, loss of muscle mass and osteoporosis (Rosen et al, Trends Endocrinol. Metab.10: 136-141, 1999).

It has been demonstrated that interference with IGF-IR causes inhibition of IGF-I-mediated or IGF-II-mediated cell growth using antisense expression vectors or antisense oligonucleotides to IGF-IR RNA (see, e.g., Wraight et al, nat. Biotech.18: 521-526, 2000). The antisense strategy has been successful in inhibiting cell proliferation of several normal cell types and human tumor cell lines. Growth can also be inhibited by 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 vectors expressing antisense RNA to IGF-I RNA (Trojan et al, Science 259: 94-97, 1992). In addition, antibodies to IGF-IR (Aretag et al, Breast Cancer. Res. Treatm., 22: 101-106, 1992; and Kalebic et al, Cancer Res.54: 5531-5534, 1994) and dominant negative mutants of IGF-IR (Prager et 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) are capable of reversing the transformed phenotype, inhibiting tumorigenesis and inducing loss of the metastatic phenotype.

IGF-I also plays an important role in the regulation of apoptosis. Apoptosis, i.e., apoptosis, is associated with a variety of developmental processes including immune and nervous system maturation processes. In addition to roles in developmental processes, apoptosis has been implicated in an important cytoprotective role in preventing tumorigenesis (Williams, Cell 65: 1097-. Inhibition of the apoptosis program by various genetic lesions may lead to the development and progression of malignant tumors.

IGF-I avoids apoptosis due to removal of cytokines in IL-3 dependent hematopoietic cells (Rodriguez-Tarduchy, G. et al, J. Immunol.149: 535. 540, 1992) and also avoids apoptosis due to removal of serum from Rat-1/mycER cells (Harrington, E. et al, EMBO J.13: 3286-. The inhibitory effect of IGF-I on apoptosis plays an important role in the late-committed phase of the cell cycle and also in cells that are blocked by etoposide or thymidine during cell cycle progression. Experimental evidence that fibroblast survival driven by c-myc is dependent on IGF-I suggests that IGF-IR plays an important role in the maintenance of tumor cells, a role distinct from the proliferative effects of IGF-I or IGF-IR, by specifically inhibiting apoptosis. This may be similar to what is believed to be a function exerted by other suppressor-apoptosis genes such as bcl-2 in promoting tumor survival (McDonnell et al, Cell 57: 79-88, 1989; Hockenberry et al, Nature 348: 334-.

The protective effect of IGF-I on apoptosis depends on allowing IGF-IR on cells to interact with IGF-I (Resnicoff et al, Cancer Res. 55: 3739-3741, 1995). Evidence for demonstrating that IGF-IR has the function of inhibiting apoptosis during maintenance of tumor cells can also be provided by the following studies: the quantitative relationship between IGF-IR levels, the extent of apoptosis and tumor incidence in rat syngeneic tumors was determined using IGF-IR antisense oligonucleotides (Rescinoff et al, Cancer Res.55: 3739-3741, 1995). Overexpression of IGF-1R has been found to protect tumor cells in vitro from etoposide-induced apoptosis (Sell et al, Cancer Res.55: 303-24306, 1995), and even more surprisingly, levels of IGF-IR were found to be lower than wild-type levels, thereby causing massive apoptosis of tumor cells in vivo (Resnicoff et al, Cancer Res.55: 2463-2469, 1995).

Possible strategies for inducing apoptosis or for inhibiting cell proliferation associated with increased IGF-I, increased IGF-II and/or increased IGF-IR receptors include inhibiting IGF-I levels or IGF-II levels or avoiding IGF-I binding to IGF-IR. For example, the long-acting somatostatin analog octreotide (octreotide) has been used to reduce IGF synthesis and/or secretion. Soluble IGF-IR has been used to induce apoptosis of tumor cells in vivo and to inhibit tumorigenesis in animal experimental systems (D' Ambrosio et al, cancer Res.56: 4013-20, 1996). In addition, IGF-IR antisense oligonucleotides, peptide analogs of IGF-I, and antibodies to IGF-IR have been used to reduce IGF-I or IGF-IR expression (see above). However, none of these compounds are suitable for long-term administration to human patients. In addition, although IGF-I has been administered to patients to treat short stature, osteoporosis, decrease muscle mass, neuropathy, or diabetes, the combination of IGF-I with IGFBPs often makes treatment with IGF-I very difficult or ineffective.

Thus, since IGF-I and IGF-IR have effects on conditions such as cancer and other proliferative diseases when IGF-I and/or IGF-IR are overexpressed, and little effect on conditions such as short stature and frailty when IGF-I and/or IGF-IR are underexpressed, there is a need to generate antibodies to IGF-IR that inhibit or stimulate IGF-IR. Although the presence of anti-IGF-IR antibodies in certain patients with autoimmune diseases has been reported, these antibodies have not been purified, and have not been shown to be suitable for inhibiting IGF-I activity in 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, Autoimmnity 16: 251, 257, 1993; drexhage et al, nether.j.of med.45: 285-293, 1994. Thus, it would be desirable to have high-affinity human anti-IGF-IR antibodies that can be used for the treatment of human diseases.

Brief description of the drawings

FIGS. 1A-1C show alignments of the nucleotide sequences of the light chain variable regions of six human anti-IGF-IR antibodies with each other and with germline sequences. FIG. 1A shows the alignment of the nucleotide sequences of the light chain variable region (VL) of antibodies 2.12.1(SEQ ID NO: I), 2.13.2(SEQ ID NO: 5), 2.14.3(SEQ ID NO: 9) and 4.9.2(SEQ ID NO: 13) with each other and with the germline V.kappa.A 30 sequence (SEQ ID NO: 39). FIG. IB shows the alignment between the nucleotide sequence of the VL of antibody 4.17.3 (SEQ ID NO: 17) and the germline V κ O12 sequence (SEQ ID NO: 41). FIG. 1C shows the alignment between the nucleotide sequence of the VL of antibody 6.1.1(SEQ ID NO: 21) and the germline V κ A27 sequence (SEQ ID NO: 37). The alignment sequences also represent the CDR regions of the VL of the various antibodies. The consensus sequences of FIGS. 1A-1C are shown in SEQ ID NO: shown at 53-55.

FIGS. 2A-2D show alignments of the nucleotide sequences of the heavy chain variable regions of six human anti-IGF-IR antibodies with each other and with germline sequences. FIG. 2A shows the alignment between the nucleotide sequence of the VH of antibody 2.12.1(SEQ ID NO: 3) and the germline VH DP-35 sequence (SEQ ID NO: 29). FIG. 2B shows the alignment between the nucleotide sequence of the VH of antibody 2.14.3(SEQ ID NO: 11) and the germline VIV-4/4.35 sequence (SEQ ID NO: 43). FIGS. 2C-1 and 2C-2 show the alignment 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) with each other and with the germline VH DP-47 sequence (SEQ ID NO: 31). FIG. 2D shows an alignment between the nucleotide sequence of the VH of antibody 4.17.3 (SEQ ID NO: 19) and the germline VH DP-71 sequence (SEQ ID NO: 35). The alignment also represents the CDR regions of the antibody. The consensus sequences of FIGS. 2A-2D are shown in SEQ ID NO: 56-59.

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 down-regulation on the cell surface (lower panel).

FIG. 5 shows that anti-IGF-IR antibodies 2.13.2 and 4.9.2 attenuate IGF-IR phosphotyrosine signaling in 3T3-IGF-IR tumors.

FIG. 6 shows that anti-IGF-IR antibodies 2.13.2 and 4.9.2 reduce IGF-IR within 3T 3-IGF-IR.

FIG. 7 shows that anti-IGF-IR antibody 2.13.2 inhibits growth of 3T3-IGF-IR tumor in vivo alone (left panel) or in combination with doxorubicin (right panel).

FIG. 8 shows the relationship between serum levels of anti-IGF-IR antibody 2.13.2 and IGF-IR down-regulation in 3T3-IGF-IR tumors.

FIG. 9 shows that multiple doses of anti-IGF-IR antibody 2.13.2 inhibit the growth of 3T3-IGF-IR tumors in vivo alone or in combination with doxorubicin.

FIG. 10 shows that anti-IGF-IR antibody 2.13.2 inhibits the growth of large tumors in vivo in combination with doxorubicin.

FIG. 11 shows that anti-IGF-IR antibody 2.13.2 inhibits Colo205 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 Colo205 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 paclitaxel.

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 doxorubicin (right panel).

Figure 15 shows that multiple doses of anti-IGF-IR antibody 2.13.2 alone or in combination with tamoxifen (tamoxifen) can inhibit MCF-7 tumor growth in vivo.

FIG. 16 shows that multiple doses of anti-IGF-IR antibody 2.13.2 inhibit A431 tumor growth in vivo alone (left panel) or in combination with the epidermal growth factor receptor (EGF-R) tyrosine kinase inhibitor CP-358,774.

FIG. 17 shows the pharmacokinetic evaluation of a single intravenous injection of anti-IGF-IR antibody 2.13.2 in cynomolgus monkeys (Cynomologus monkeys).

FIG. 18 shows that the combination of anti-IGF-IR antibody 2.13.2 with doxorubicin enhances the down-regulation of IGF-IR on 3T3-IGF-IR tumors in vivo.

Figure 19A shows the number of mutations that occurred 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 of the heavy and light chains of antibodies 2.13.2 and 2.12.1 with the germline sequences from which they were obtained. FIG. 19B shows an alignment of the heavy chain amino acid sequence of antibody 2.13.2(SEQ ID NO: 45) with the amino acid sequence of germline sequence DP-47(3-23)/D6-19/JH6 (SEQ ID NO: 46). FIG. 19C shows an alignment between the amino acid sequence of the light chain of antibody 2.13.2(SEQ ID NO: 47) and the amino acid sequence of germline sequence A30/Jk 2(SEQ ID NO: 48). FIG. 19D shows an alignment between the heavy chain amino acid sequence of antibody 2.12.1(SEQ ID NO: 49) and the amino acid sequence of germline sequence DP-35(3-11)/D3-3/JH6 (SEQ ID NO: 50). FIG. 19E shows an alignment between the light chain amino acid sequence of antibody 2.12.1(SEQ ID NO: 51) and the amino acid sequence of germline sequence A30/Jkl (SEQ ID NO: 52). In FIGS. 19B-E, the signal sequences are shown in italics, the CDRs are underlined, the variable regions are in bold, the Framework (FR) mutations are highlighted by placing a plus sign ("+") on the amino acid residue, and the CDR mutations are highlighted by placing an asterisk on the amino acid residue.

Brief description of the invention

The present invention provides isolated antibodies or antigen-binding portions thereof that bind to IGF-IR, preferably antibodies or antigen-binding portions thereof that bind to primate and human IGF-IR, and more preferably antibodies that are human. The invention provides anti-IGF-IR antibodies that inhibit the binding of IGF-I or IGF-II to IGF-IR, and also provides anti-IGF-IR antibodies that activate IGF-IR.

The invention provides pharmaceutical compositions comprising the antibodies and a pharmaceutically acceptable carrier. The pharmaceutical composition may further comprise another ingredient, such as an antineoplastic agent or an imaging agent.

Diagnostic and therapeutic methods are also provided. Diagnostic methods include methods for diagnosing the presence or location of an IGF-IR-expressing tissue using anti-IGF-IR antibodies. The method of treatment comprises administering the antibody to a subject in need thereof, preferably in combination with administration of another therapeutic agent.

The invention provides isolated cell lines, such as hybridomas, that produce anti-IGF-IR antibodies.

The invention also provides nucleic acid molecules encoding the heavy and/or light chains of an anti-IGF-IR antibody, or antigen-binding portions thereof. The invention provides vectors and host cells comprising the nucleic acid molecules and methods for recombinantly producing polypeptides encoded by the nucleic acid molecules.

Also provided are non-human transgenic animals that express the heavy and/or light chains of an anti-IGF-IR antibody or antigen-binding portions thereof. The invention also provides for treating a subject in need of treatment with an effective amount of a nucleic acid molecule encoding the heavy and/or light chain of an anti-IGF-IR antibody or antigen-binding portion thereof

Detailed Description

Definitions and general techniques

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention have the same meaning as commonly understood by one of ordinary skill in the art. Also, unless otherwise required by context, singular terms include the plural and plural terms include the singular. Generally, the terminology and technology associated with 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. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods known in the art and described in various general and more specific references that are cited and discussed in the present specification. See, e.g., Sambrook et al molecular Cloning: a Laboratory Manual (A Laboratory Manual), second edition, Cold spring harbor Laboratory Press, Cold spring harbor, New York (1989) and Ausubel et al, Current Protocols in Mobile μ lar Biology, Greens Publishing Association (Green Publishing Associates) (1992) and Harlow and Lane Antibodies (Harlow and Lane Antibodies): a Laboratory Manual (A Laboratory Manual), Cold spring harbor Laboratory Press, Cold spring harbor, New York (1990), which are incorporated herein by reference. Enzymatic reactions and purification techniques were performed according to the manufacturer's instructions, general methods in the art, or methods described herein. The nomenclature, laboratory procedures, and techniques related to analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly employed in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and administration, and treatment of patients.

Unless otherwise indicated, the following terms are understood to have the following meanings:

the term "polypeptide" includes natural or artificial proteins, protein fragments and polypeptide analogs of a protein sequence. The polypeptide may be monomeric or in multiple specific forms.

The term "isolated protein" or "isolated polypeptide" refers to a protein that is (1) free of naturally associated components with which it coexists in its natural state, (2) free of the same other proteins, (3) expressed by a cell of a different species, or (4) does not occur in nature, in terms of its source. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell in which it is naturally produced is "separated" from the components with which it is naturally associated. Proteins substantially free of naturally associated components can also be provided 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 the sample has a single species of polypeptide. The polypeptide or protein may be in monomeric or multimeric form. A substantially pure polypeptide or protein will generally comprise about 50%, 60, 70%, 80% or 90% W/W of the protein sample, preferably about 95%, more preferably having a purity of 99% or more. Protein purity or homogeneity can be determined by a variety of methods well known in the art, such as polyacrylamide gel electrophoresis of a protein sample followed by detection of a single polypeptide electrophoretic band by staining the gel with a staining agent well known in the art. For specific purposes, higher degrees of separation can be achieved by employing HPLC or other purification techniques well known in the art.

The term "polypeptide fragment" as used herein refers to a polypeptide having an amino-terminal and/or carboxy-terminal deletion, with the remaining amino acid sequence being identical to the corresponding site in the naturally occurring sequence. Fragments are typically at least 5, 6, 8 or 10 amino acids in length, preferably at least 14 amino acids in length, more preferably at least 20 amino acids in length, usually at least 50 amino acids in length, even more preferably at least 70, 80, 90, 100, 150 or 200 amino acids in length.

The term "polypeptide analog" as used herein refers to a polypeptide consisting of a fragment of at least 25 amino acids in length that is substantially identical to a partial amino acid sequence and that has at least one of the following properties: (1) specifically bind to IGF-IR under appropriate binding conditions, (2) block the binding of IGF-I or IGF-II to IGF-IR, or (3) reduce the surface expression of IGF-IR cells or tyrosine phosphorylation in vitro or in vivo. Polypeptide analogs typically have conservative amino acid substitutions (or insertions or deletions) with respect to the naturally occurring sequence. Analogs are generally at least 20 amino acids in length, preferably at least 50, 60, 70, 80, 90, 100, 150 or 200 amino acids in length or longer, and often have the same length as a naturally occurring full-length polypeptide.

Preferred amino acid substitutions are those described below: (1) reduced susceptibility to proteolysis, (2) reduced susceptibility to oxidation, (3) altered binding affinity for formation of protein complexes, (4) altered affinity and (5) other physicochemical or performance properties imparted or altered to such analogs. Analogs can include sequences of various muteins in addition to the naturally occurring peptide sequences. 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 peptide outside the region where intermolecular contacts are formed). Substitutions of a conservative amino acid do not substantially change the structural properties of the parent sequence (e.g., a substituted amino acid does not disrupt the helical structure present in the parent sequence or disrupt other types of secondary structures that determine the properties of the parent sequence). Examples of secondary and tertiary Structures of artificially identified polypeptides are described in "Proteins, Structures and molecular Principles (Proteins, Structures and molecular μ lar Principles)" (creatton, ed., w.h.freeman and Company, new york (1984)); "Protein Structure entry" (c.branden and j.tooze, eds., garland publishing, new york, n.y. (1991)); and Thornton et al, Nature 354 (Nature): 105(1991), each of which is incorporated herein by reference.

Non-peptide analogs are commonly used in the pharmaceutical industry as drugs with properties similar to the template peptide. These types of non-peptide compounds are named "peptidomimetics" (peptide minetics or peptidomimetics). Fauchere, J.adv.Dr. mu.g Res.15: 29 (1986); veber and Freidinger TINS p.392 (1985); and Evans et al, j.med.chem.30: 1229(1987), which is incorporated herein by reference. Such compounds are usually developed with the aid of computer molecular simulations. Peptidomimetics structurally similar to the therapeutic peptides can be employed to provide equivalent therapeutic or prophylactic effects. In general, peptidomimetics are structurally similar to polypeptides of the exemplary nature (paramigm) (i.e., polypeptides having a desired biochemical property or pharmaceutical activity), such as human antibodies, but having one or more moieties optionally selected from the group consisting of-CH₂NH--、--CH₂S--、--CH₂-CH₂-, - -CH- - - - (cis and trans) - -, - -COCH₂--、--CH(OH)CH₂- - -and- -CH₂A bond of SO-, a peptide bond substituted by methods well known in the art. Systematic substitution of one or more amino acids of the consensus sequence with the same type of D-amino acid (e.g., D-lysine in place of L-lysine) can be used to prepare more stable peptides. In addition, restriction peptides containing a consensus sequence or substantially identical consensus sequence variants can be prepared 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 that are capable of forming intramolecular disulfide bonds of cyclized peptides.

An "immunoglobulin" is a tetrameric molecule. In naturally occurring immunoglobulins, each tetramer is composed of two pairs of identical peptide chains, each pair having one "light" (about 25kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain comprises a variable region of about 100 to 110 or more amino acids primarily for recognition of an antigen. The carboxy-terminal portion of each chain defines a constant region that functions primarily as an effector. Human light chains are classified into kappa and lambda light chains. Heavy chains are classified as μ, Δ, γ, a or ε, and thus define the antibody isotype as IgM, IgD, IgG, IgA and IgE, respectively. In both light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, while the heavy chain may also contain a "D" region of more than about 10 amino acids. See generally, basic Immunology chapter 7(Pa μ l, w. editor, 2 nd edition RavenPress, n.y. (1989)) which is incorporated by reference in its entirety for all purposes. Each pair of light/heavy chain variable regions forms an antibody binding site such that a complete immunoglobulin has two binding sites.

Immunoglobulin chains have the same general structure of relatively conserved Framework Regions (FRs) joined by three hypervariable regions, also known as complementarity determining regions or CDRs. The CDRs from both chains in each pair are lined by a framework region to allow binding to a specific epitope. From N-terminus to C-terminus, both light and heavy chains contain the FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 domains. Amino acid assignments for each domain were determined according to Kabat Sequences of Proteins of Immunological Interest (national institutes of Health, Bethesda, Md. (1987and 1991)), or Chothia & Lesk J.mol.biol.196: 901-917 (1987); chothia et al Nature 342: 878-883 (1989).

"antibody" refers to an intact immunoglobulin or an antigen-binding portion thereof that competes with the intact antibody for specific binding. Antigen binding portions can be prepared by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen binding portions specifically include Fab, Fab ', F (ab')₂Fv, dAb and complementarity determining region fragments, single chain antibodies (scFv), chimeric antibodies, diabodies and polypeptides comprising at least a portion of an immunoglobulin sufficient for the polypeptide to have the property of specifically binding to an antigen.

As used herein, antibodies designated, for example, as 2.12.1, 2.13.2, 2.14.3, 4.9.2, 4.17.3, and 6.1.1 are antibodies derived from hybridomas of the same name. For example, antibody 2.12.1 is derived from hybridoma 2.12.1.

The Fab fragment is a monovalent fragment consisting of the VL, VH, CL and CHI domains; f (ab')₂The fragment is a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; the Fd fragment consists of the VH and CH1 domains; the Fv fragment consists of the VL and VH domains of a single arm of an antibody; and dAb fragments (Ward et al, Nature 341: 544-546, 1989) consist of VH domains.

A single chain antibody (scFv) is an antibody in which the VL and VH regions pair to form monovalent molecules linked by a synthetic linker, allowing these molecules to be made into single chain proteins (Bird et al, Science 242: 423 Acad. Sci. 1988 and Huston et al, Proc. Natl. Acad. Sci. USA 85: 5879-. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide single chain, but use a linker that is too short to allow pairing between the two domains on the same chain, thereby allowing the domains to pair with complementary domains on other chains to create two antigen binding sites (see, e.g., Holliger, P. et al, Proc. Natl. Acad. Sci. USA 90: 6444-. One or more CDRs may be introduced covalently or noncovalently into the molecule to make it an immunoadhesin. Immunoadhesins can incorporate CDRs as part of a long polypeptide chain, can covalently link CDRs to another polypeptide chain, or can non-covalently incorporate CDRs. The CDRs allow 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 the same or different from each other. For example, a naturally occurring immunoglobulin has two identical binding sites, a single chain antibody or Fab fragment has one binding site, and a "bispecific" or "bifunctional" antibody has two different binding sites.

An "isolated antibody" is an antibody that (1) does not bind to a naturally occurring binding composition, including other naturally occurring binding antibodies, with which it coexists in its native state, (2) does not contain other proteins of the same species, (3) is expressed by a cell of a different species, or (4) does not occur in nature. Examples of isolated antibodies include anti-IGF-IR antibodies purified by IGF-IR affinity, anti-IGF-IR antibodies synthesized in vitro by hybridomas or other cell lines, and human anti-IGF-IR antibodies obtained from transgenic mice.

The term "human antibody" includes all antibodies having one or more variable and constant regions derived from human immunoglobulin sequences. In a preferred embodiment, all variable and constant regions are derived from human immunoglobulin sequences (a fully human antibody). These antibodies can be prepared in a variety of ways as described below.

A humanized antibody is an antibody derived from a non-human species in which certain amino acids in the framework and constant regions of the heavy and light chains are mutated so as to avoid or eliminate an immune response in humans. Alternatively, humanized antibodies can be prepared by fusing together the constant regions of a human antibody and the variable regions of a non-human antibody. Examples of how to prepare humanized antibodies are found in US6054297, US5886152 and US 5877293.

The term "chimeric antibody" refers to an antibody that contains one or more regions derived from one antibody and one or more regions derived from one or more other antibodies. In a preferred embodiment, one or more of the CDRs are obtained 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, CDRs from more than one human anti-IGF-IR antibody are mixed together to match a chimeric antibody. For example, a chimeric antibody may contain one light chain CDR1 derived from a first human anti-IGF-IR antibody, the CDR1 may bind to light chain CDR2 and CDR3 derived from a second human anti-IGF-IR antibody, and the heavy chain-derived CDRs may be derived from a third human anti-IGF-IR antibody. Furthermore, the framework regions may be derived from one and the same anti-IGF-IR antibody, from one or more different antibodies, such as human antibodies, or from humanized antibodies.

A "neutralizing antibody" or "inhibitory antibody" is an antibody that inhibits the binding of IGF-IR to IGF-I, wherein excess 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 about 40%, more preferably 60%, even more preferably 80%, or more preferably 85%. The reduction in binding capacity can be determined by methods known to those of ordinary skill in the art, for example, by in vitro competitive binding assays. An example of determining a reduction in the amount of binding of IGF-I to IGF-IR is provided in example IV below.

An "activating antibody" is an antibody that activates IGF-IR by at least about 20% when added to a cell, tissue or organism that expresses IGF-IR. In a preferred embodiment, the antibody activates IGF-IR activity by at least 40%, more preferably by 60%, even more preferably by 80%, or even more preferably by 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 determined by determining the amount of tyrosine autophosphorylation of IGF-IR.

Fragments or analogs of the antibodies can be prepared without difficulty by those skilled in the art following the techniques described herein. The preferred amino-and carboxy-termini of the fragments or analogs are present near the boundaries of the functional domains. Structural or functional domains can be determined by comparison of a library of nucleotide and/or amino acid sequences with a public or appropriate database. Preferably, computerized comparison methods are employed to determine sequence motifs or predicted protein configuration domains present in other proteins of known structure and/or function. Methods for determining the sequence of a protein that folds into a known three-dimensional structure are known. Bowie et al, Science 253: 164(1991).

The term "Surface plasmon resonance (Surface plasmon resonance)" as used herein refers to an optical phenomenon capable of analyzing biospecific interactions in real time by detecting changes in protein concentration within a biosensing matrix, for example, using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, n.j.). For further explanation 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 Johnson, b.et al (1991) anal. biochem.198: 268-277.

Noun "K_off"refers to the dissociation rate constant for separating an antibody from an antibody/antigen complex.

Noun "K_d"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 are typically composed of chemically active surface groups of molecules such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics as well as specific charged characteristics. Antibodies are said to bind specifically to antigens with dissociation constants < 1uM, preferably < 100nM and most preferably < 10 nM.

As used herein, twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (second edition, by E.S. Golub and D.R. Gren, eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. Stereoisomers of twenty conventional amino acids (e.g., D-amino acids), unnatural amino acids such as α -, α -disubstituted amino acids, N-alkyl amino acids, lactic acid and other unconventional amino acids may also be suitable components of the polypeptides of the invention. Examples of unconventional amino acids include: 4-hydroxyproline, gamma-carboxyglutamic acid,. epsilon. -N, N, N-trimethyllysine,. epsilon. -N-acetyl lysine, O-phosphoserine, N-acetyl serine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, s-N-methylarginine and other similar amino and imino acids (e.g., 4-hydroxyproline). In the polypeptide annotation used herein, the left direction is the amino terminal direction and the right direction is the carboxy terminal direction, according to standard usage and convention.

The term "polynucleotide" as referred to herein means a polymeric form of nucleotides, ribonucleotides or deoxyribonucleotides or an altered form of each nucleotide of at least 10 bases in length. The term includes single-stranded and double-stranded forms of DNA.

The term "isolated polynucleotide" as used herein refers to a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, which "isolated polynucleotide" (1) does not bind to all or a portion of the polynucleotide to which the "isolated polynucleotide" naturally occurs, (2) is operably linked to the polynucleotide to which the "isolated polynucleotide" naturally occurs, or (3) does not naturally occur as part of a longer sequence, depending on the source.

The term "oligonucleotide" as referred to herein includes naturally occurring and altered nucleotides linked together by naturally occurring and non-naturally occurring oligonucleotide linkages. Oligonucleotides are a subset of polynucleotides, typically 200 bases or less in length. Preferably the oligonucleotide is 10 to 60 bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Although oligonucleotides may be double-stranded, e.g., used to construct gene mutants, oligonucleotides are typically single-stranded, e.g., used as probes. The oligonucleotide of the invention may be either a sense or antisense oligonucleotide.

The term "naturally occurring nucleotide" as referred to herein includes deoxyribonucleotides and ribonucleotides. The term "altered nucleotide" referred to herein includes nucleotides having altered or substituted sugar groups and the like. The term "oligonucleotide linkage" as referred to herein includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphororaniladate, phosphoramidate, and the like. See, for example, LaPlanche et al, nucleic acids res.14: 9081 (1986); stec et al, J.Am.chem.Soc.106: 6077 (1984); stein et al, nucleic acids res.16: 3209 (1988); zon et al, Anti-Cancer Dr μ g Design 6: 539 (1991); zon et al, Oligonucleotides and antigens: a Practical Approach, pp.87-108(F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); stec et al, US 5151510; uhlmann and Peyman Chemical Reviews 90: 543(1990), the entire contents of which are incorporated herein by reference. The oligonucleotide may also contain a label for detection, if desired.

"operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that regulate the gene of interest in trans or remotely. The term "expression control sequence" as referred to herein refers to a polynucleotide sequence necessary for expression and processing of a coding sequence to which it is ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; RNA efficient processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that increase translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and, if desired, sequences that promote secretion of the protein. The nature of such control sequences varies from host organism to host organism; in prokaryotes, such control sequences typically include a promoter, a ribosome binding site, and a transcription termination sequence; in eukaryotes, such control sequences typically include promoters and transcription termination sequences. Term(s) for

"control sequences" are intended to include, at a minimum, all components necessary for expression and processing, but may also include additional components whose presence is advantageous, e.g., leader sequences and fusion partner sequences.

The term "vector" as used herein refers to a nucleic acid molecule capable of transferring another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can 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). By introduction into a host cell, other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell and thereby 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 defined 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" and "vector" may be used interchangeably, and plasmid is the most common form of vector. However, the invention is intended to include 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 refers to a cell into which a recombinant expression vector has been introduced. It is understood that the term refers not only to the particular experimental cell but also to a daughter cell of such a cell. Because certain modifications may occur in the progeny due to mutation or to 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" as referred to herein refers to binding that is detectable and specific. According to the present invention, polynucleotides, oligonucleotides and fragments thereof selectively hybridize to nucleic acid strands under hybridization and wash conditions that minimize the amount of detectable binding to non-specific nucleic acids. "high stringency" or "highly stringent" conditions are used to obtain selective hybridization conditions known in the art and discussed herein. An example of "high stringency" or "highly stringent" conditions is one such method: a method of incubating a polynucleotide with another polynucleotide, wherein a polynucleotide can be immobilized on a solid surface such as a membrane, maintained in a hybridization buffer of 6 XSSPE or SSC, 50% formamide, 5 XDenhardt's reagent, 0.5% SDS, 100. mu.g/ml denatured salmon sperm DNA fragment at a hybridization temperature of 42 ℃ for 12-16 hours, and then washed twice with a washing buffer consisting of 1 XSSC, 0.5% SDS at 55 ℃. See also Sambrook et al, supra, pages 9.50-9.55.

The term "percent sequence identity" as used in reference to nucleic acid sequences refers to the residues in the two sequences that are the same when aligned for maximum correspondence. The length of the sequence identity comparison may extend at least about 9 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 many different algorithms in the art that can be used to determine nucleotide sequence identity. For example, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, a program in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wisconsin. FASTA, including for example the programs FASTA2 and FASTA3, provides alignments and percent sequence identity for the regions of maximum overlap between query and search sequences (Pearson, Methods enzymol.183: 63-98 (1990); Pearson, Methods mol.biol.132: 185-219 (2000); Pearson, Methods enzvmol.266: 227-258 (1996); Pearson, J.mol.biol.276: 71-84 (1998); incorporated herein by reference). Unless otherwise specified, default parameters for a particular program or algorithm are used. For example, percent sequence identity between nucleic acid sequences can be determined using FASTA (word size 6 and NOPAM coefficient of scoring matrix) with default parameters or using Gap with default parameters provided by GCGV region 6.1, which is incorporated herein by reference.

Unless otherwise indicated, reference to a nucleic acid sequence shall include its complement. Thus, reference to a nucleic acid sequence having a particular sequence should be understood to include its complementary strand having a complementary sequence.

In the field of molecular biology, researchers use the terms "percent sequence identity", "percent sequence similarity", and "percent sequence homology" interchangeably. In the present application, these terms have the same meaning only with respect to nucleic acid sequences.

The terms "substantially similar" or "sequence substantially similar" in reference to a nucleic acid or fragment thereof refer to a nucleotide sequence identity of at least about 85%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% nucleotide bases as determined by any known algorithm of sequence identity, such as FASTA, BLAST or Gap, as described above, when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions.

The term "substantial identity" as applied to polypeptides means that two peptide sequences have 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 when optimally aligned, for example by the GAP or BESTFIT program using default GAP weights. Preferably, the different residue positions differ due to conservative amino acid substitutions. "conservative amino acid substitution" refers to a substitution in which one amino acid residue is replaced with another amino acid residue having a side chain (R group) of similar chemical nature (e.g., charge or hydrophobicity). Typically, conservative amino acid substitutions do not substantially alter the function of the protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage of sequence identity or degree of similarity may be adjusted up to correct for the conservative nature of the substitution. Means for making such adjustments are well known to those skilled in the art. See, e.g., Pearson, Methods mol. biol. 24: 307-31(1994), which is incorporated herein by reference. Examples of groups of amino acids with chemically similar side chains include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxy side chain: serine and threonine; 3) amide-containing side chain: asparagine and glutamine; 4) aromatic side chain: phenylalanine, tyrosine and tryptophan; 5) basic side chain: lysine, arginine and histidine; and 6) the sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid and asparagine-glutamine.

Alternatively, conservative substitutions are described in Gonnet et al, Science 256: 1443-45(1992), which is incorporated herein by reference, has any change in the PAM250 log-likelihood matrix (log-likehood matrix) that has a positive value. A "moderately conservative" substitution is any change in the PAM250 log-likelihood matrix that has a non-negative value.

Sequence similarity, also known as sequence identity, of polypeptides is typically determined using sequence analysis software. Protein analysis software compares similar sequences using a determination of the degree of similarity due to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG includes programs such as "Gap" and "Bestfit" which, under default parameters, can be used to determine sequence homology or sequence identity between closely related polypeptides, such as between homologous polypeptides or wild-type proteins derived from different species of organisms and muteins thereof. See, e.g., GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1 with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity for regions of greatest overlap between query and search sequences (Pearson (1990); Pearson (2000)). Another preferred algorithm when comparing the sequences of the invention to a database containing a variety of sequences from different organisms is the computer program BLAST, in particular blastp or tblastn, using default parameters. See, e.g., Altsch μ l et al, j.mol.biol.215: 403-; altsch. mu.l et al, Nucleic Acids Res.25: 3389-402 (1997); incorporated herein by reference.

The length of polypeptide sequences over which homology comparisons are made is generally 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 35 residues. When searching a database comprising sequences derived from a plurality of different organisms, it is preferred to perform a comparison of the amino acid sequences.

As used herein, the term "label" or "labeled" refers to the incorporation of another molecule into an antibody. In one embodiment, the label is a detectable label, such as incorporation of a radiolabeled amino acid or attachment of a biotin moiety to the polypeptide, which can be detected by labeled avidin (e.g., streptavidin containing a fluorescent label or having enzymatic activity, which can be measured by optical or colorimetric methods). In another embodiment, the label may be of therapeutic nature, such as a drug conjugate or toxin. Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Of labels for polypeptidesExamples include, but are not limited to, the following: a radioisotope or radionuclide (e.g.,³H，¹⁴C，¹⁵N，³⁵S，⁹⁰Y，⁹⁹Tc，¹¹¹In，¹²⁵I，¹³¹I) fluorescent markers (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic markers (e.g., horseradish peroxidase,. beta. -galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotin groups, predetermined polypeptide epitopes recognized by a second reporter (e.g., leucine zipper sequence, binding site for a second antibody, metal binding region, epitope tail), magnetic agents such as gadolinium chelate, toxins such as pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthrax dione (diychloroxy anthralin dione), mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine (procaine), tetracaine (tetracaine), lidocaine (lidocaine), propranolol (propranolol) and puromycin and analogues or homologues thereof.

In certain embodiments, attachment of the labels is performed by spacer arms of various lengths to reduce potential steric hindrance.

The term "agent" as used herein means a compound, a mixture of compounds, a biological macromolecule, or an extract made from biological material. The term "agent or drug" as used herein refers to a compound or composition that, when properly administered to a patient, is capable of producing a desired therapeutic effect. Other chemical terms herein are used according to conventional usage in the art, as exemplified by the McGralv-Hill chemical glossary dictionary (McGralv-Hill dictionary of chemical terms) (Parker, S., Ed., McGraw-Hill, San Francisco (1985)), which is incorporated herein by reference).

The term "antineoplastic agent" as used herein refers to a drug having functional properties that inhibit the development or progression of a tumor, particularly a malignant (cancerous) lesion such as a carcinoma, sarcoma, lymphoma or leukemia, in a human. Inhibition of metastasis is often a property of antineoplastic agents.

The term "patient" includes human and veterinary subjects.

Human anti-IGF-IR antibodies and characterization thereof

Human antibodies avoid some of the problems created by antibodies having mouse or rat variable and/or constant regions. The presence of such mouse or rat derived sequences can lead to rapid clearance of the antibody or can elicit an immune response in the patient to the antibody. Thus, in one embodiment, the invention provides humanized anti-IGF-IR antibodies. In a preferred embodiment, the invention provides a fully human anti-IGF IR antibody by introducing human immunoglobulin genes into a rodent such that the rodent produces a fully human antibody. More preferred are fully human anti-human IGF-IR antibodies. Intact human anti-IGF-IR antibodies are expected to minimize the inherent immunogenic and allergic responses to mouse or mouse-derived monoclonal antibodies (mabs), thereby increasing the efficacy and safety of the administered antibodies. The use of fully human antibodies is expected to provide substantial advantages in the treatment of chronic and recurrent human diseases, such as inflammation and cancer, which may require repeated administration of antibodies. 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 has a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18 or 22, or one or more CDRs derived from these amino acid sequences. In another preferred embodiment, the anti-IGF-IR antibody has a heavy chain variable region comprising a sequence selected from the group consisting of seq id NOs: 4, 8, 12, 16, 20 or 24, or one or more heavy chains derived from the CDRs of these amino acid sequences.

Classes and subclasses of anti-IGF-IR antibodies

The antibody may be an IgG, IgM, IgE, IgA or IgD molecule. In a preferred embodiment, the antibody is an IgG and is of the IgG1, IgG2, IgG3 or IgG4 subtype. In a more preferred embodiment, the anti-IGF-IR antibody is subclass IgG 2. In another preferred embodiment, the anti-IGF-IR antibody is of the same class or 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 IgG 2.

The class and subclass of anti-IGF-IR antibodies can be determined by methods known in the art. In general, the class and subclass of an antibody can be determined using antibodies specific for a particular class and subclass of antibody. Such antibodies are commercially available. The class and subclass can be determined by ELISA, Western blot, and other techniques. Alternatively, class and subclass can be determined by sequencing the heavy and/or light constant regions of an antibody, comparing its amino acid sequence to known amino acid sequences of immunoglobulins of different classes and subclasses, and determining the class and subclass of the antibody.

Selectivity of species and molecules

According to another aspect of the invention, the anti-IGF-IR antibodies demonstrate species and molecular selectivity. In one embodiment, the anti-IGF-IR antibody binds to human, cynomolgus or rhesus IGF-IR.. 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 bind to new world breeds of monkeys, such as marmoset (marmoset). The species selectivity of anti-IGF-IR antibodies may be determined using methods known in the art in accordance with the teachings of the present specification. Species selectivity can be determined, for example, by Western blotting, FACS, ELISA or RIA. In a preferred embodiment, species selectivity can be determined by Western blotting.

In another embodiment, the anti-IGF-IR antibody is selective for IGF-IR at least 50-fold greater than for the insulin receptor. In a preferred embodiment, the anti-IGF-IR antibody is at least 100-fold more selective for IGF-IR than for insulin receptor. In a more preferred embodiment, the anti-IGF-IR antibody does not have any appreciable specific binding to proteins other than IGF-IR. The selectivity of anti-IGF-IR antibodies for IGR-IR can be determined using methods known in the art and in accordance with the teachings of the present specification. For example, selectivity can be determined by Western blotting, FACS, ELISA or RIA. In a preferred embodiment, the selectivity of the molecule can be determined by Western blotting.

Binding affinity of anti-IGF-IR to IGF-IR

According to another aspect of the invention, the anti-IGF-IR antibody binds to IGF-IR with high affinity. In one embodiment, the anti-IGF-IR antibody binds to K of IGF-IR_dIs 1 × 10^-8M or less. In a preferred embodiment, the antibody binds to K of IGF-IR_dIs 1 × 10^-9M or less. In a more preferred embodiment, the antibody binds to K of IGF-IR_dIs 5x10^-10M or less. In another preferred embodiment, the antibody binds to K of IGF-IR_dIs 1 × 10^-10M or less. In another preferred embodiment, the antibody binds to K of IGF-IR_dK of 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_dAre substantially the same. In another preferred embodiment, the antibody binds to K of IGF-IR_dK of an antibody comprising one or more CDRs selected from the group consisting of 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3, or 6.1.1 antibodies_dAre substantially the same. In a more preferred embodiment, the antibody binds to K of IGF-IR_dAnd a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24, or a pharmaceutically acceptable salt thereof_dAre substantially the same. In a preferred embodimentIn this embodiment, the antibody binds to K of IGF-IR_dK with antibodies containing one or more CDRs_dSubstantially the same, wherein the CDRs are derived from a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24.

According to another aspect of the invention, the anti-IGF-IR antibody has a very low off-rate. In one embodiment, the anti-IGF-IR antibody has a K_offIs 1 × 10^-4s^-1Or lower. In a preferred embodiment, K_offIs 1 × 10^-5s^-1Or lower. In another preferred embodiment, K_offSubstantially identical to 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_offSubstantially identical to an antibody comprising one or more CDRs derived 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 a more preferred embodiment, the antibody binds to IGF-IR_offAnd a polypeptide containing a nucleotide sequence selected from SEQ ID

ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24, or a pharmaceutically acceptable salt thereof. In another preferred embodiment, the antibody binds to IGF-IR_offSubstantially identical to an antibody comprising one or more CDRs wherein said CDRs are derived from a polypeptide comprising a sequence selected from the group consisting of 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 can be determined by any method known in the art. In one embodiment, the binding affinity is determined by competitive ELISA, RIA or surface plasmon resonance, such as BIAcore. The dissociation rate can also be determined by surface plasmon resonance. In a more preferred embodiment, the binding affinity and dissociation rate is determined by surface plasmon resonance. In an even more preferred embodiment, binding affinity and dissociation rate is determined by BIAcore. An example of determining binding affinity and dissociation rate is described in example II below.

Half-life of anti-IGF-IR antibodies

According to another object of the invention, the half-life of the anti-IGF-IR antibody is at least one day in vitro or in vivo. In a preferred embodiment, the antibody or fragment thereof has a half-life of at least three days. In a more preferred embodiment, the antibody or fragment thereof has a half-life of four days or more. In another embodiment, the antibody or fragment thereof has a half-life of eight days or more. In another embodiment, the antibody, or antigen-binding portion thereof, is derivatized or altered to have a longer half-life, as described below. In another preferred embodiment, the antibody may contain point mutations to increase serum half-life, such as described in WO 00/09560 published 24/2/2000.

The half-life of an antibody can be determined by any method known to one of ordinary skill in the art. For example, the half-life of the antibody can be determined by Western blotting, ELISA or RIA over a suitably long period of time. The half-life of the antibody can be determined in an appropriate animal, e.g., a monkey, such as a cynomolgus monkey, a primate or human.

Identification of IGF-IR epitopes recognized by anti-IGF-IR antibodies

The invention also provides anti-IGF-IR antibodies that bind to the same antigen or epitope as human anti-IGF-IR antibodies. Furthermore, the present invention provides anti-IGF-IR antibodies that cross-compete with human anti-IGF-IR antibodies. 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 antibody comprises one or more CDRs from an antibody selected from the group consisting of 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 and 6.1.1. In another more preferred embodiment, the human anti-IGF-IR antibody comprises a heavy chain variable region selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24. In another preferred embodiment, the human anti-IGF-IR antibody comprises one or more CDRs derived from a polypeptide comprising a sequence selected from the group consisting of seq id 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.

Whether an anti-IGF-IR antibody binds to the same antigen can be determined using a variety of methods known in the art. For example, it can be determined whether an anti-IGF-IR antibody binds to the same antigen by capturing an antigen known to bind to an anti-IGF-IR antibody, such as IGF-IR, with the anti-IGF-IR antibody, eluting the antigen from the antibody, and then determining whether the tested antibody binds to the eluted antigen. Whether an antibody binds to the same epitope as an anti-IGF-IR antibody can be determined by binding the anti-IGF-IR antibody to IGF-IR under saturating conditions and then assaying the ability of the tested antibody to bind to IGF-IR. If the tested antibody is capable of binding to IGF-IR at the same time as the anti-IGF-IR antibody, then the tested antibody binds to a different epitope than the anti-IGF-IR antibody. However, if the tested antibody is not able to bind to IGF-IR at the same time, the tested antibody binds to the same epitope as the human anti-IGF-IR antibody. The experiment can be performed by ELISA, RIA or surface plasmon resonance. In a preferred embodiment, the experiment is performed using epicytoplasmic group resonance. In a more preferred embodiment, BIAcore is used. It can also be determined whether the anti-IGF-IR antibody cross-competes with the anti-IGF-IR antibody. In a preferred embodiment, it can be determined whether an anti-IGF-IR antibody cross-competes with another anti-IGF-IR antibody by using the same method as it is determined whether an anti-IGF-IR antibody is capable of binding the same epitope as another anti-IGF-IR antibody.

Hydroxyl chain and heavy chain usage

The invention also provides anti-IGF-IR antibodies that contain variable sequences encoded by the human kappa gene. In a preferred embodiment, the variable sequence is encoded by the vka 27, a30 or O12 gene family. In a preferred embodiment, the variable sequence is encoded by the human vka 30 gene family. In a more preferred embodiment, the light chain comprises no more than 10 amino acid substitutions by germline vka 27, a30 or O12, preferably no more than 6 amino acid substitutions, more preferably no more than 3 amino acid substitutions. In a preferred embodiment, the amino acid substitutions are conservative substitutions.

SEQ ID NO: 2, 6, 10, 14, 18 and 22 provide the variable region amino acid sequences of six anti-IGF-IR kappa light chains. SEQ ID NO: 38, 40 and 42 provide the variable region amino acid sequences of three germline kappa light chains from which the six anti-IGF-IR kappa light chains were derived. FIGS. 1A-1C show an alignment of the nucleotide sequences of the light chain variable regions of the six anti-IGF-IR antibodies with each other and with the germline sequences from which they were derived. In light of the teachings of the present specification, one of ordinary skill in the art will be able to determine the amino acid sequences of the six encoded anti-IGF-IR kappa light chains and germline kappa light chains, and will also be able to determine the differences between germline and 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 VLs 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 an anti-IGF-IR antibody may contain one or more amino acid substitutions identical to the substitutions present in antibody 2.13.2, another amino acid substitution identical to the substitutions present in antibody 2.14.3, and another amino acid substitution identical to the substitutions present in antibody 4.9.2. In this way, the different characteristics of antibody binding can be combined and matched together in order to alter, for example, the affinity of the antibody for IGF-IR and the dissociation rate from the antigen. In another embodiment, amino acid substitutions are made at the same positions as found in any one or more of the VLs 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 do not employ the same amino acids. For example, aspartic acid can be conservatively converted if the amino acid substitution made in one of antibodies 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3, or 6.1.1 is glutamic acid, relative to the germline. Similarly, if an amino acid substitution is made with serine, then threonine can be conservatively substituted.

In another preferred embodiment, the light chain comprises an amino acid sequence identical to the amino acid sequence of 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 the same amino acid sequence 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 derived from at least one light chain CDR region 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 derived from CDRs of different light chains. In a more preferred embodiment, the CDRs derived from the 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 a sequence selected from SEQ ID NOs: 2, 6, 10, 14, 18 or 22. In another embodiment, the light chain comprises a heavy chain consisting of a sequence selected from SEQ ID NOs: 1, 5, 9, 13, 17 or 21 or an amino acid sequence encoded by a nucleotide sequence encoding an amino acid sequence having 1-10 amino acid insertions, deletions or substitutions. Preferably, the amino acid substitution is a conservative amino acid substitution. In another embodiment, the antibody or fragment thereof comprises a lambda light chain.

The invention also provides anti-IGF-IR antibodies, or portions thereof, comprising a human heavy chain or sequences derived from a human heavy chain. In one embodiment, the heavy chain amino acid sequence is derived from human V_HDP-35, DP-47, DP-70, DP-71 or VIV-4/4.35 gene families. In a preferred embodiment, the heavy chain amino acid sequence is derived from human V_HDP47 gene family. In a more preferred embodiment of the process according to the invention,the heavy chain contains no more than 8 germline V_HAmino acid changes of DP-35, DP-47, DP-70, DP-71 or VIV-4/4.35 gene, more preferably no more than 6 amino acid changes, and even more preferably no more than 3 amino acid changes.

SEQ ID NO: 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 NO: 30, 32, 34, 36 and 44 provide the amino acid sequences of germline heavy chains DP-35, DP-47, DP-70, DP-71 and VIV-4, respectively, and the amino acid sequences of SEQ ID NOs: 29, 31, 33, 35 and 43 provide the nucleotide sequences of germline heavy chains DP-35, DP-47, DP-70, DP-71 and VIV-4, respectively. FIGS. 2A-2D show alignments between the amino acid sequences of the variable regions of the six anti-IGF-IR antibodies and their germline sequences. In light of the teachings of the present specification, one of ordinary skill in the art will be able to determine the amino acid sequences of the six encoded anti-IGF-IR heavy chains and the germline heavy chain, and will also be able to determine the differences between the germline sequence and the antibody sequence.

In a preferred embodiment, the VH of the anti-IGF-IR antibody contains the same amino acid substitution relative to the germline amino acid sequence as any one 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. Similarly to the above, the VH of the anti-IGF-IR antibody may contain one or more amino acid substitutions identical to the substitutions present in antibody 2.13.2, another amino acid substitution identical to the substitutions present in antibody 2.14.3, and another amino acid substitution identical to the substitutions present in antibody 4.9.2. In this way, the different characteristics of antibody binding can be combined and matched together in order to alter, for example, the affinity of the antibody for IGF-IR and the dissociation rate from the antigen. In another embodiment, amino acid substitutions are made at the same positions as found in any one or more of the VH's 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 do not employ the same amino acids.

In another preferred embodiment, the heavy chain comprises an amino acid sequence identical to 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 very preferred embodiment, the heavy chain comprises the same amino acid sequence 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 derived from at least one CDR region in 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 chains comprise amino acid sequences derived from the CDRs of different heavy chains. In a more preferred embodiment, the CDRs derived from the 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 a sequence selected from SEQ ID NOs: 4, 8, 12, 16, 20 or 24. In another embodiment, the heavy chain comprises a heavy chain consisting of a sequence selected from SEQ ID NOs: 3, 7, 11, 15, 19 or 23 or an amino acid sequence encoded by a nucleotide sequence encoding an amino acid sequence having 1-10 amino acid insertions, deletions or substitutions. In another embodiment, the substitution is a conservative amino acid substitution.

Inhibition of IGF-IR activity by anti-IGF-IR antibodies

Inhibiting IGF-I binding to IGF-IR

In another embodiment, the invention provides anti-IGF-IR antibodies that inhibit the binding of IGF-I to IGF-IR or 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 fragment thereof inhibits the binding of IGF-IR and IGF-I with an IC of no more than 100nM₅₀. In a preferred embodiment, the IC₅₀Not more than 10 nM. In a more preferred embodiment, the IC₅₀Not more than 5 nM. The IC₅₀Can be determined by methods known in the art. Typically, ICs₅₀Can be determined by ELISA or RIA. In a preferred embodiment, the IC₅₀Can be determined by RIA.

In another embodiment, the invention provides anti-IGF-IR antibodies that prevent IGF-IR activation in the presence of IGF-I. In a preferred embodiment, the anti-IGF-IR antibody inhibits IGF-IR-induced tyrosine phosphorylation at the receptor. In another preferred embodiment, the anti-IGF-IR antibody inhibits the progression of a downstream cellular event. For example, anti-IGF-IR inhibits tyrosine phosphorylation of Shc and Insulin Receptor Substrates (IRS)1 and 2, all of which are normally phosphorylated when cells are treated with IGF-I (Kim et al, J.biol.chem.273: 34543-. Whether an anti-IGF-IR antibody inhibits IGF-IR activity in the presence of IGF-1 can be determined by measuring the level of autophosphorylation of IGF-IR, Shc, IRS-1 or IRS-2 by Western blotting or immunoprecipitation. In a preferred embodiment, the level of autophosphorylation of IGF-IR can be determined by Western blotting. See, e.g., example VII.

According to another aspect of the invention, the antibody causes down-regulation of IGF-IR in antibody-treated cells. In another embodiment, IGF-IR is internalized into the cytoplasm of the cell. When the anti-IGF-IR antibody binds to IGF-IR, the antibody is internalized as shown by focused microscopy. Without wishing to be bound by any theory, it is believed that the antibody-IGF-IR complex is internalized into lysosomes and degraded. The down-regulation of IGF-IR can be determined by methods known in the art, including immunoprecipitation, focused microscopy, or Western blotting. See, e.g., example VII. 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 contains a heavy chain, light chain or antigen binding region thereof.

Activation of IGF-IR by anti-IGF-IR antibodies

Another aspect of the invention relates to activating anti-IGF-IR antibodies. Activating antibodies differ from inhibiting antibodies in that activation enhances or replaces the effects of IGF-I on IGF-IR. In one embodiment, the activating antibody is capable of binding to IGF-IR and activating it in the absence of IGF-I. Such activating antibodies are essentially a mimetic of IGF-1. In another embodiment, the activating antibody enhances the effect of IGF-I on IGF-IR. Such antibodies do not themselves activate IGF-IR, but do enhance IGF-IR activation in the presence of IGF-1. The mimetic anti-IGF-IR antibodies can be readily distinguished from the amplified anti-IGF-IR antibodies by treating the cells in vitro with the antibodies in the presence or absence of low concentrations of IGF-1. An antibody is a mimetic antibody if it is capable of causing activation of IGF-IR in the absence of IGF-1, e.g., enhances tyrosine phosphorylation of IGF-IR. An antibody is an amplifying antibody if it does not cause IGF-IR activation in the absence of IGF-1 but does amplify the amount of IGF-IR activation. In a preferred embodiment, the activating antibody is 4.17.3. In another preferred embodiment, the antibody contains one or more CDRs derived from 4.17.3. In another preferred embodiment, the antibody is derived from one 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 anti-IGF-IR antibodies that inhibit IGF-IR tyrosine phosphorylation and receptor levels in vivo. In one embodiment, administration of the anti-IGF-IR antibody to an animal results in attenuation of IGF-IR phosphotyrosine signaling within the GF-IR-expressing tumor. In a preferred embodiment, the anti-IGF-IR antibody reduces phosphotyrosine signaling by at least 20%. In a more preferred embodiment, the anti-IGF-IR antibody reduces phosphotyrosine signaling by at least 60%, more preferably by 50%. In a more preferred embodiment, the antibody reduces phosphotyrosine signaling by at least 40%, more preferably by 30%, even more preferably by 20%. In a preferred embodiment, the antibody is administered about 24 hours prior to the determination of tyrosine phosphorylation. The level of tyrosine phosphorylation can be determined by any method known in the art, such as described below. 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, a light chain or an antigen-binding fragment thereof.

In another embodiment, administration of the anti-IGF-IR antibody to an animal causes a decrease in the level of IGF-IR in an IGF-IR-expressing tumor. In a preferred embodiment, the anti-IGF-IR antibody reduces receptor levels by at least 20% compared to untreated animals. In a more preferred embodiment, the anti-IGF-IR antibody reduces receptor levels to at least 60%, more preferably 50%, of the receptor levels in the untreated animal. In an even more preferred embodiment, the antibody reduces the receptor level to at least 40%, more preferably 30%. In a preferred embodiment, the antibody is administered about 24 hours prior to the determination of IGF-IR levels. IGF-IR levels can be determined by any method known in the art, such as the methods described below. 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, a light chain or an antigen-binding fragment thereof.

In another embodiment, the anti-IGF-IR antibody inhibits the growth of tumor cells in vivo. The tumor cells can be derived from any cell type including, but not limited to, epidermal cells, epithelial cells, endothelial cells, leukemia cells, sarcoma cells, multiple myeloma, or mesodermal cells. Examples of tumor cells include A549 (non-small cell lung cancer) cells, MCF-7 cells, Colo205 cells, 3T3/IGF-IR cells and A431 cells. In a preferred embodiment, the antibody inhibits the growth of tumor cells as compared to the growth of tumor cells in an untreated animal. In a more preferred embodiment, the antibody inhibits the growth of tumor cells by 50%. In an even more preferred embodiment, the antibody inhibits the growth of tumor cells by 60%, 65%, 70% or 75%. In one embodiment, inhibition of tumor cell growth is determined at least 7 days after the initial treatment of the animal with the antibody. In a more preferred embodiment, the inhibition of tumor cell growth is determined at least 14 days after the start of treatment of the animal with the antibody. In another preferred embodiment, another anti-neoplastic agent is administered to the animal in combination with an anti-IGF-IR antibody. In a preferred embodiment, the antineoplastic agent is capable of further inhibiting the growth of a tumor. In an even more preferred embodiment, the antineoplastic agent is doxorubicin, paclitaxel, tamoxifen, 5-fluorodeoxyuridine (5-FU) or CP-358, 774. In a preferred embodiment, the tumor cell growth is inhibited by at least 50%, more preferably 60%, 65%, 70% or 75%, more preferably 80%, 85% or 90% 22-24 days after co-administration of the anti-tumor agent and the anti-IGF-IR antibody. See, e.g., fig. 7and 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, a light chain or an antigen-binding fragment thereof.

Apoptosis induced by anti-IGF-IR antibodies

One aspect of the invention provides anti-IGF-IR antibodies that induce cell death. In one embodiment, the antibody causes apoptosis. The antibodies can induce apoptosis in vivo or in vitro. In general, tumor cells are more sensitive to apoptosis than normal cells, and thus administration of an anti-IGF-IR antibody preferentially causes apoptosis of tumor cells over normal cells. In another embodiment, administration of an anti-IGF-IR antibody reduces the level of the enzyme, akt, involved in the Phosphatidylinositol (PI) kinase pathway. The PI kinase pathway is in turn involved in the propagation of cells and the prevention of apoptosis. Thus, inhibition of akt can cause apoptosis. In a more preferred embodiment, the antibody is administered in vivo so as to cause apoptosis of the IGF-IR-expressing cells. 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, a light chain or an antigen-binding fragment thereof.

Methods for producing antibodies and antibody-producing cell lines

Immunization

In one embodiment of the invention, human antibodies are prepared by immunizing a non-human animal with an IGF-IR antigen, which contains part or all of the human immunoglobulin locus. In a preferred embodiment, the non-human animal is XENOMOUSE^TMWhich is a kind of person containingImmunoglobulin lociAnd mouse antibody production is deficient. See, e.g., Green et al Nature Genetics 7: 13-21(1994) and US patents 5916771, 5939598, 5985615, 5998209, 6075181, 6091001, 6114598 and 6,130,364. See also WO 91/10741 published on month 7and 25 of 1991, WO 94/02602 published on month 2 and 3 of 1994, WO 96/34096 and WO 96/33735 published on month 10 and 31 of 1996, WO 98/16654 published on month 4 and 23 of 1998, WO 98/24893 published on month 6 and 11 of 1998, WO 98/50433 published on month 11 and 12 of 1998, WO 99/45031 published on month 9 and 10 of 1999, WO99/53049 published on month 21 of 1999, WO 0009560 published on month 2 and 24 of 2000, and WO 00/037504 published on month 6 and 29 of 2000. XENOMOUSE^TMHuman repertoires of mature forms of fully human antibodies are generated and antigen-specific human mabs are generated. Second generation Xenomouses by introduction of megabase-sized germline-structured YAC fragments of the human heavy and kappa light chain loci^TMContains about 80% of human antibody components. See Mendez et al Nature Genetics 15: 146-: 483-495(1998), the entire disclosure of which is incorporated herein by reference.

The invention also provides methods for producing anti-IGF-IR antibodies from non-human, non-mouse animals by immunizing non-human transgenic animals containing human immunoglobulin loci. Such animals can be produced using the methods described directly above. The methods disclosed in these patents can be improved as disclosed in US 5994619. In a preferred embodiment, the non-human animal may be a rat, sheep, pig, goat, cow or horse.

In another embodiment, the non-human animal containing a human immunoglobulin locus is an animal having a "minilocus" (minilocus) of a human immunoglobulin. In the minilocus approach, the foreign Ig locus is mimicked by the inclusion of individual genes derived from the Ig locus. Thus, one or more V_HGene, one or more D_HGene, J or J_HThe gene, a mu constant region and a second constant region (preferably a gamma constant region) form a construct suitable for insertion into an animal. Such methods are described in U.S. patent nos. 5545807, 5545806, 5625825, 5625126, 563425, 5661016, 5770429, 5789650, 5814318, 5591669, 5612205, 5721367, 5789215 and 5643763, which are incorporated herein by reference.

The advantage of the minilocus approach is that constructs containing a portion of the Ig locus can be rapidly prepared and introduced into animals. However, a potential disadvantage of the small locus approach is that there is insufficient immunoglobulin diversity to support the development of intact B cells, and thus antibody production may be low.

To produce human anti-IGF-IR antibodies, a non-human animal containing some or all of the human immunoglobulin loci is immunized with an IGF-IR antigen and the antibodies or antibody-producing cells are isolated from the animal. The IGF-IR antigen may be isolated and/or purified IGF-IR, and is preferably human IGF-IR. In another embodiment, the IGF-IR antigen is an IGF-IR fragment, preferably the extracellular domain of IGF-IR. In another embodiment, the IGF-IR antigen is a fragment containing at least one epitope of IGF-IR. In another embodiment, the IGF-IR antigen is a cell that expresses IGF-IR on the surface of a cell, preferably a cell that overexpresses IGF-IR on the surface of a cell.

Immunization of animals may be carried out according to 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 known in the art. See, for example, Harlow and Lane and U.S. patent US 5994619. In a preferred embodiment, the IGF-IR antigen is administered with an adjuvant to elicit an immune response. Such adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptide) or ISCOM (immune stimulating complex). Such adjuvants may protect the polypeptide from rapid dispersion by sequestering it by local deposition, 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 the polypeptide is administered, the immunization program will comprise two or more polypeptide administrations over a time span of several weeks.

Example I provides immunization of XENOMOUSE with human full-length IGF-IR dissolved in phosphate buffer^TMThe scheme (2).

Preparation of antibodies and antibody-producing cell lines

After immunizing an animal with IGF-IR antigen, antibodies and/or antibody-producing cells can be obtained from the animal. Serum containing anti-IGF-IR antibodies is obtained from animals by exsanguination or killing the animal. Serum obtained from an animal may be used, immunoglobulin fractions may be obtained from the serum or anti-IGF-IR antibodies may be purified from the serum. The serum or immunoglobulin obtained in this way is polyclonal, which has the disadvantage that the amount of antibody obtained is very limited and that the polyclonal antibodies have a heterogeneous character.

In another embodiment, an antibody-producing immortal hybridoma can be prepared from the immunized animal. Following immunization, the animals are sacrificed and the B cells of the spleen are fused to immortal myeloma cells, as is 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-secreting cell line). After fusion and antibiotic selection, hybridomas are screened using IGF-IR, partial fragments thereof or IGF-IR expressing cells. In a preferred embodiment, the initial screening is performed by enzyme linked immunosorbent assay (ELISA) or Radioimmunoassay (RIA), preferably by ELISA. An example of an ELISA screen is provided by WO00/37504, incorporated herein by reference.

In another embodiment, the antibody-producing cells are prepared in vivo from a human that has an autoimmune deficiency and expresses an anti-IGF-IR antibody. Cells expressing anti-IGF-IR antibodies can be isolated by isolating white blood cells and subjecting these cells to fluorescence-activated cell sorting (FACS) or by scanning a plate coated with IGF-IR or a partial fragment thereof. These cells can be fused with human non-secretory myelomas to produce hybridomas expressing human anti-IGF-IR antibodies. In general, this is a less preferred embodiment, since the affinity of anti-IGF-IR antibodies to IGF-IR appears to be low.

Hybridomas producing anti-IGF-IR antibodies are selected, cloned, and further screened for desirable properties, including healthy growth of the hybridomas, high antibody production, and desirable antibody properties, as discussed further below. Hybridomas can be cultured and expanded in vivo in syngeneic animals, animals lacking the immune system, such as nude mice, or can be cultured and expanded in vitro in cell culture. Methods of selecting, cloning and expanding hybridomas are well known to those of ordinary skill in the art.

Preferably, the animal being immunized is a non-human animal expressing human immunoglobulin genes, and the B cells of the spleen are fused to a myeloma derived from the same animal species as the non-human animal. More preferably, the immunized animal is XeNOMOUS^TMAnd the myeloma cell line is a non-secretory mouse myeloma, such as NSO-bcl 2.

See, for example, example I.

In one aspect, the invention provides hybridomas that produce human anti-IGF-IR antibodies. In a preferred embodiment, the hybridoma is a mouse hybridoma as described above. In another preferred embodiment, the hybridoma is produced from a non-human, non-mouse breed such as rat, sheep, pig, goat, cow, or horse. In another embodiment, the hybridoma is a human hybridoma in which a human non-secretory myeloma is fused to human cells expressing an anti-IGF-IR antibody.

Nucleic acids, vectors, host cells and recombinant methods for producing antibodies

Nucleic acids

Nucleic acid molecules encoding the anti-IGF-IR antibodies of the invention are provided. In one embodiment, the nucleic acid molecule encodes the heavy and/or light chain of an anti-IGF-IR immunoglobulin. In a preferred embodiment, a single nucleic acid molecule encodes the 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 immunoglobulin is a human immunoglobulin, preferably a human IgG. The encoded light chain may be a lambda chain or a kappa chain, preferably a kappa chain.

The nucleic acid molecule encoding the variable region of the light chain may be derived from the a30, a27 or 012 vk genes. In a preferred embodiment, the light chain is obtained from the a30V kappa gene. In another preferred embodiment, the nucleic acid molecule encoding a light chain comprises a junction region derived from jk 1, jk2 or jk 4. 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 A30V kappa gene, preferably no more than six amino acid changes, even more preferably no more than three amino acid changes,

the invention provides nucleic acid molecules encoding a light chain variable region (VL) having at least three amino acid changes from a germline sequence, wherein the amino acid changes are the same as those of a germline sequence derived 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 nucleic acid molecules comprising a nucleic acid sequence encoding the amino acid sequence of the light chain variable region 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 nucleic acid molecules comprising a nucleic acid sequence encoding the amino acid sequence of one or more 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 encoding the amino acid sequences of all 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 nucleotide sequence encoding SEQ ID NO: 2, 6, 10, 14, 18 or 22 or a nucleic acid sequence comprising one of the amino acid sequences of SEQ ID NO: 1, 5, 9, 13, 17 or 21. In another preferred embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding SEQ ID NO: 2, 6, 10, 14, 18 or 22 or a nucleic acid sequence comprising one or more 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 nucleotide sequence encoding SEQ ID NO: 2, 6, 10, 14, 18 or 22 or a nucleic acid sequence comprising the amino acid sequences of all the CDRs of any one of SEQ ID NOs: 1, 5, 9, 13, 17 or 21, or a pharmaceutically acceptable salt thereof.

The invention also provides nucleic acid molecules encoding amino acid sequences of VLs having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequences of the above VL, in particular comprising the amino acid sequence of SEQ ID NO: 2, 6, 10, 14, 18 or 22, or a pharmaceutically acceptable salt thereof. The invention also provides a polypeptide at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1, 5, 9, 13, 17 or 21, or a pharmaceutically acceptable salt thereof. In another embodiment, the invention provides nucleic acid molecules encoding a VL that hybridizes under highly stringent conditions to a nucleic acid molecule encoding a VL as described above, particularly nucleic acid molecules comprising a nucleotide sequence encoding a VL comprising a nucleotide sequence as set forth in SEQ ID NO: 2, 6, 10, 14, 18 or 22. The invention also provides nucleic acid sequences encoding a VL that hybridizes under highly stringent conditions to a nucleic acid sequence comprising SEQ ID NO: 1, 5, 9, 13, 17 or 21.

The invention also provides nucleic acid molecules encoding the heavy chain (VH) variable region derived from the DP-35, DP-47, DP-71 or Vit-4/4.35VH gene, preferably the DP-35VH gene. In a preferred embodiment, the nucleic acid molecule encoding VH comprises a junction region from JH6 or JH5, more preferably JH 6. In another preferred embodiment, the D fragment is derived from 3-3, 6-19 or 4-17. In an even more preferred embodiment, the nucleic acid molecule encoding a 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 a VH contains at least one amino acid change compared to the germline sequence, wherein the amino acid change is identical to an amino acid change of the germline sequence of the heavy chain derived from 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 sequence, wherein the changes are identical to those of the germline sequence of the VH derived from 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 one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding 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 encoding the amino acid sequence of one or more 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 encoding the amino acid sequences of all 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 encoding seq id NO: 4, 8, 12, 16, 20 or 24 or a nucleic acid sequence comprising the amino 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 nucleotide sequence encoding SEQ ID NO: 4, 8, 12, 16, 20 or 24 or a nucleic acid sequence comprising the amino acid sequence of one or more CDRs of any one of SEQ ID NOs: 3, 7, 11, 15, 19 or 23. In a preferred embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding SEQ ID NO: 4, 8, 12, 16, 20 or 24 or a nucleic acid sequence comprising the amino acid sequences of all the CDRs of any one of SEQ ID NOs: 3, 7, 11, 15, 19 or 23.

In another embodiment, the nucleic acid molecule encodes an amino acid sequence of a VH having an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to one of the amino acid sequences encoding the aforementioned VH, particularly a VH comprising the amino acid sequence of SEQ ID NO: 4, 8, 12, 16, 20 or 24. The invention also provides a polypeptide at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3, 7, 11, 15, 19 or 23, or a pharmaceutically acceptable salt thereof. In another embodiment, the nucleic acid molecule encoding a VH is a nucleic acid molecule that hybridizes under highly stringent conditions to a nucleic acid molecule encoding a VH as described above, in particular to a nucleic acid molecule comprising a nucleotide sequence encoding a VH as set forth in SEQ ID NO: 4, 8, 12, 16, 20 or 24, or a pharmaceutically acceptable salt thereof. The invention also provides a nucleic acid molecule encoding a VH that hybridizes under highly stringent conditions to a nucleic acid molecule comprising SEQ ID NO: 3, 7, 11, 15, 19 or 23, or a nucleic acid molecule thereof.

Encoding one or both of the intact heavy and light chains of an anti-IGF-IR antibodyThe nucleic acid molecules of the variable regions thereof may be obtained from any source that produces anti-IGF-IR antibodies. Methods for isolating mRNA encoding antibodies are known in the art. See, e.g., Sambrook et al. The mRNA can be used to prepare cDNA for Polymerase Chain Reaction (PCR) or antibody gene cDNA cloning. In one embodiment of the invention, the nucleic acid molecule may be obtained from a hybridoma expressing an anti-IGF-IR antibody as described above, preferably a hybridoma having a transgenic animal cell expressing human immunoglobulin genes as one of its fusion partners, such as XENOMOUSE^TMA non-human mouse transgenic animal or a non-human, non-mouse transgenic animal. In another embodiment, the hybridoma is obtained from a non-human, non-transgenic animal, which is used, for example, for a humanized antibody.

Nucleic acid molecules encoding the entire heavy chain of an anti-IGF-IR antibody may be constructed by fusing a nucleic acid molecule encoding the variable region of the heavy chain or an antigen-binding region thereof to the constant region of the 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 region of the light chain, or an antigen-binding region thereof, to the constant region of the light chain. Nucleic acid molecules encoding VH and VL can be converted to full-length antibody genes by inserting them into expression vectors that already encode heavy chain constant regions and light chain constant regions, respectively, such that the VH fragment is operably linked to a heavy chain constant region (CH) fragment in the vector and the VL fragment is operably linked to a light chain constant region (CL) fragment in the vector. Alternatively, nucleic acid molecules encoding a VH or VL chain are converted to full-length antibody genes by linking nucleic acid molecules encoding a VH chain to nucleic acid molecules encoding a CH chain using standard molecular biology techniques. The same effect can be achieved by 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, 5 th edition, NIH publication No. 91-3242, 1991. The nucleic acid molecules encoding the full-length heavy and/or light chains may be introduced into a cell for expression and isolation of the anti-IGF-IR antibody.

In a preferred embodiment, the nucleic acid molecule encoding the heavy chain variable region encodes the amino acid sequence of SEQ ID NO: 4, 8, 12, 16, 20 or 24, and the nucleic acid molecule encoding the light chain variable region encodes the amino acid sequence of SEQ ID NO: 2, 6, 10, 14, 18 or 22. SEQ ID NO: 28 describes the amino acid sequence of the heavy chain constant region of antibodies 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 and 6.1.1 and SEQ ID NO: 27 describes nucleic acid sequences encoding the heavy chain constant regions of 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 describes the amino acid sequence of 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 and SEQ ID NO: 25 describes nucleic acid sequences encoding the light chain constant regions of 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 heavy chain constant region encodes the amino acid sequence of SEQ ID NO: 28, and the nucleic acid molecule encoding the light chain constant region encodes SEQ ID NO: 26. in a more preferred embodiment, the nucleic acid molecule encoding the heavy chain constant region has the amino acid sequence of SEQ ID NO: 27, and the nucleic acid molecule encoding the constant region has the sequence set forth in SEQ ID NO: 25.

In another embodiment, the nucleic acid molecule encoding the heavy chain or antigen-binding region thereof, or the light chain or antigen-binding region thereof, of an anti-IGF-IR antibody may be isolated from a non-human, non-mouse animal that expresses human immunoglobulin genes and is immunized with an IGF-IR antigen. In another embodiment, the nucleic acid molecule can be isolated from an anti-IGF-IR antibody-producing cell obtained from a non-transgenic animal or from a patient producing an anti-IGF-IR antibody. mRNA can be isolated from anti-IGF-IR antibody-producing cells by standard techniques, cloned and/or amplified using PCR and library construction techniques, and screened using standard protocols to obtain nucleic acid molecules encoding the heavy and light chains of an anti-IGF-IR antibody.

The nucleic acid molecules can 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, mutant antibodies and antibody derivatives, as described further below. Also, as described below, the nucleic acid molecule can be used to humanize an antibody if it is obtained from a non-human, non-transgenic animal.

In another embodiment, the nucleic acid molecules of the invention can be used as probes or PCR primers for specific antibody sequences. For example, nucleic acid molecule probes may be used in diagnostic methods or nucleic acid molecule PCR primers may be used to amplify regions of DNA that may be used to isolate nucleic acid sequences for use in the production of anti-IGF-IR antibody variable regions. In a preferred embodiment, the nucleic acid molecule is an oligonucleotide. In a more preferred embodiment, the oligonucleotides are derived from the hypervariable regions of the heavy and light chains of the antibody of interest. In an even more preferred embodiment, the oligonucleotide encodes all or part of one or more CDRs.

Carrier

The invention provides vectors comprising a nucleic acid molecule of the invention encoding a heavy chain or antigen-binding fragment thereof. The invention also provides vectors comprising a nucleic acid molecule of the invention encoding a light chain or an antigen-binding fragment thereof. The invention also provides vectors containing nucleic acid molecules encoding the fusion proteins, altered antibodies, antibody fragments, and probes thereof.

To express the antibody or antibody fragment of the present invention, the DNA encoding partial or full-length light and heavy chains obtained according to the above-described method is inserted into an expression vector so that the gene is operably linked to a transcriptional or translational control sequence. Expression vectors include plasmids, retroviruses, cosmids, YACs, derivative episomes of EBV, and the like. The antibody gene is ligated into a vector such that transcriptional and translational control sequences in the vector serve the intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene may be inserted into separate vectors. In a preferred embodiment, the two genes are inserted into the same expression vector. The antibody gene is inserted into an expression vector by standard methods (e.g., complementary restriction sites on the antibody gene fragment are ligated to the in vivo, or blunt-ended if no restriction sites are present).

A suitable vector is one which encodes a functionally complete human CH or CL immunoglobulin sequence with appropriate processing restriction sites to enable any VH or VL sequence to be readily inserted and expressed as described above. In such vectors, splicing is typically performed between the splice donor site in the inserted J region and the splice acceptor site in front of the human C region, and also in the splice region present within the human CH exon. Polyadenylation and transcription termination occur at natural chromosomal sites downstream of the coding region. The recombinant expression vector may also encode a signal peptide that facilitates secretion of the antibody chain from the 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 may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin).

In addition to the antibody chain gene, the recombinant expression vector of the present invention carries a regulatory sequence that controls the expression of the antibody chain gene in a host cell. It will be appreciated by those skilled in the art that the design of vectors for expression, including the choice of regulatory sequences, will depend on such factors as the choice of host cell to be transformed, the level of expression of the desired protein, and the like. Preferred regulatory sequences for expression in mammalian host cells include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers from retroviral LTRs, Cytomegalovirus (CMV), such as the CMV promoter/enhancer, simian virus 40(SV40), such as the SV40 promoter/enhancer, adenoviruses, e.g., the adenovirus major late promoter (AdMLP), polyoma, and mammalian strong promoters such as the native immunoglobulin and actin promoters. Further description of viral regulatory elements and their sequences is found, for example, in U.S. Pat. No. 5,5168062 to Stinski, U.S. Pat. No. 5,4510245 to Bell et al, and U.S. Pat. No. 4,4968615 to Schaffner.

In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may contain additional sequences, such as sequences that regulate replication of the vector in a host cell (e.g., initiation of replication) and a selectable marker gene. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4399216, 4634665 and 5,179,017, all by Axel et al). For example, the selectable marker gene typically confers resistance to drugs such as G418, hygromycin or methotrexate on the host cell into which the vector is introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for DHFR host cells with methotrexate selectivity/amplification) and the neo gene (for G418 selection).

Non-hybridoma host cells and methods for recombinant production of proteins

Nucleic acid molecules encoding the heavy and/or light chains of anti-IGF-IR antibodies or antigen-binding fragments thereof, as well as vectors containing these nucleic acid molecules, may be used to transform suitable mammalian host cells. Transformation may be carried out by introducing the polynucleotide into a host cell by any known method. Methods for introducing heterologous polynucleotides into mammalian cells are known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide with liposomes, guided missile (biolistic) injection of DNA and direct microinjection into the nucleus. Alternatively, the nucleic acid molecule may be introduced into a mammalian cell by a viral vector. Methods for transforming cells are known in the art. See, for example, U.S. Pat. Nos. 4399216, 4912040, 4740461 and 4959455 (which are incorporated herein by reference).

Mammalian cell lines as expression hosts are known in the art and include many immortalized cells obtained from the American Type Culture Collection (ATCC). These cells include 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., HepG2), a549 cells, 3T3 cells, and some other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells. Particularly preferred cell lines are selected by 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 a recombinant expression vector encoding the heavy chain or antigen-binding fragment thereof, the light chain and/or an antigen-binding fragment thereof is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient for expression of the antibody in the host cell, or more preferably, for secretion of the antibody into the medium in which the host cell is cultured. The antibody is recovered from the medium using standard protein purification methods.

Moreover, expression of the antibodies of the invention (or other portions derived from the antibodies) by the producer cell line can be enhanced using a variety of known techniques. For example, the glutamine synthetase gene expression system (GS system) is a common means of enhancing expression under certain conditions. The GS system is discussed in whole or in part in european patents EP0216846, EP0256055 and EP0323997 and european patent application 89303964.4.

Antibodies expressed by different cell lines or transgenic animals appear to have different glycosylation from one another. However, antibodies encoded by the nucleic acid molecules provided herein, or antibodies containing the amino acid sequences provided herein, are part of the invention, regardless of the glycosylation of the antibody.

Transgenic animals

The invention also provides a composition containing one or more of the inventionThe invention is a non-human transgenic animal for producing a nucleic acid molecule of an antibody of the invention. Antibodies can be produced and recovered in and from tissues or body fluids such as milk, blood or urine of goats, cows, horses, pigs, rats, mice, rabbits, hamsters or other mammals. See, for example, US patents 5827690, 5756687, 5750172 and 5,741,957. As described above, the human being is includedImmunoglobulin lociThe non-human transgenic animal of (a) can be prepared by immunization with IGF-IR or a fragment thereof.

In another embodiment, a non-human transgenic animal is prepared by introducing one or more nucleic acid molecules of the invention into the animal using standard transgenic techniques. See Hogan, supra. The transgenic cell used to prepare the transgenic animal may be an embryonic stem cell or a somatic cell. The non-human transgenic organism may be chimeric, non-chimeric hybrid and non-chimeric homozygote. See for example Hogan et al,Manipμlating the MouseEmbrvo：A Laboratorv Manual2ed., cold spring harbor Press (1999); the result of Jackson et al,Mose Genetics and Transaenics：A Practical Approachoxford university press (2000); and a (d) a (n) kert,Transgenic Animal Technology：ALaboratory Handbook，academic Press (1999). In another embodiment, the non-human transgenic organism may have target cleavage and substitution encoding the desired heavy and/or light chain. In a preferred embodiment, the transgenic animal contains and expresses nucleic acid molecules encoding heavy and light chains that specifically bind to IGF-IR, preferably human IGF-IR. In another embodiment, the transgenic animal contains a nucleic acid molecule encoding an altered antibody, such as a single chain antibody, a chimeric antibody, or a humanized antibody. anti-IGF-IR antibodies can be prepared in any transgenic animal. In a preferred embodiment, the non-human animal is a mouse, rat, sheep, pig, goat, cow, or horse. The non-human transgenic animal expresses the murine encoded polypeptide in blood, milk, urine, saliva, tears, mucus, and other body fluids.

Phage display libraries

The present invention provides a method for producing an anti-IGF-IR antibody or antigen-binding fragment thereof, comprising the steps of: synthesizing a phage library of human antibodies, screening the library with IGF-IR or a fragment thereof, isolating phage that bind IGF-IR and obtaining said antibodies from within the phage. One method of preparing an antibody library comprises the steps of: immunizing humans with IGF-IR or antigen binding fragments thereofImmunoglobulin lociThereby generating an immune response, extracting cells capable of producing antibodies from the host animal; isolating RNA from the extracted cells, reverse transcribing the RNA to prepare cDNA, amplifying the cDNA using primers, and inserting the cDNA into a phage display vector to express an antibody in the phage. Recombinant anti-IGF-IR antibodies of the invention may be obtained in this manner.

In addition to the anti-IGF-IR antibodies disclosed herein, recombinant human anti-IGF-IR antibodies of the invention may also be isolated by screening recombinant combinatorial antibody libraries, preferably scFv phage display libraries, prepared using human VL and VH cDNAs made from mRNA derived from human lymphocytes. Methods for preparing and screening such libraries are known in the art. There are commercially available kits for preparing phage display libraries (e.g., Pharmacia recombinant phage antibody System, catalog No. 27-9400-01; and Stratagene SurfZAp^TMPhage display kit, catalog No. 240612). There are also other methods and reagents that can be used to prepare and screen antibody display libraries (see, e.g., U.S. Pat. No. 5,220,09 to Ladner et al; PCT publication WO 92/18619 to Kang et al; PCT publication WO 91/17271 to Dower et al; PCT publication WO 92/20791 to Winter et al; PCT publication WO 92/15679 to Markland et al; PCT publication WO 93/01288 to Breitling et al; PCT publication WO92/01047 to McCafferty et al; PCT publication WO 92/09690 to Garrrard et al; Fuchs et al (1991) Bio/Technology 9: 1370-hs et al, (1993) EMBO J12: 725-; hawkins et al, (1992) J.mol.biol.226: 889-896; clackson et al, (1991) Nature 352: 624-; 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 desired properties, human heavy and light chain sequences having similar binding activity to IGF-IR are first selected using the human anti-IGF-IR antibodies described herein according to the epitope imprinting method described in PCT publication WO 93/06213 to Hoogenboom et al. The antibody library used in the method is preferably a scFv library which is prepared according to PCT publication WO92/01047 to McCafferty et al, Nature (1990) 348: 552 and 554; and Griffiths et al, (1993) EMBO J12: 725-734 for preparation and screening. The scFv antibody library is preferably screened against human IGF-IR as an antigen.

Once the initial VL and VH fragments are screened out, a "mix and compare" experiment is performed to select the preferred VL/VH pairing conjugates in which IGF-IR is combined to screen out different pairs of initially selected VL and VH fragments. In addition, to further improve antibody quality, preferred VL/VH paired VL and VH fragments may be randomly mutated in a process similar to the in vivo somatic mutation process that results in maturation of antibody avidity during the natural immune response, preferably within the CDR3 regions of the VL and/or VH. This in vitro affinity maturation can be accomplished by amplifying the VH and VL regions using PCR primers complementary to VHCDR3 or VL CDR3, respectively, which primers are "spiked" with a random mixture of four nucleotide bases at certain positions such that the resulting PCR product encodes VH and VL fragments in which random mutations are introduced into the VH and/or VL CDR3 regions. These randomly mutated VH and VL fragments can be screened for binding to IGF-IR again.

After screening and isolation of the anti-IGF-IR antibodies of the invention from the recombinant immunoglobulin display library, the nucleic acid encoding the selection antibody may 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 may be further processed to produce other forms of antibodies of the invention, as described below. To express the antibody isolated by screening the combinatorial library, DNA encoding the antibody is cloned into a recombinant expression vector and then introduced into the mammalian cells described above.

Type conversion

Another aspect of the invention is to provide a mechanism for the conversion of anti-IGF-IR antibodies to another. According to one aspect of the invention, a nucleic acid molecule encoding a VL or VH is isolated such that the nucleic acid molecule does not contain any nucleic acid sequence encoding a CL or CH, using methods known in the art. The nucleic acid molecule encoding VL or VH is then operably linked to a nucleic acid molecule encoding CL or CH derived from a different type of immunoglobulin molecule. As described above, this can be achieved by using vectors or nucleic acid molecules containing CL or CH chains. For example, an anti-IGF-IR antibody that is initially an IgM may be transformed to an IgG. Furthermore, class switching can be used to convert one IgG subtype to another, for example from IgGI to IgG 2. One preferred method for producing antibodies of the invention comprising an isotype of interest comprises the steps of: isolating nucleic acid encoding the heavy chain of the anti-IGF-IR antibody and nucleic acid encoding the light chain of the anti-IGF-IR antibody to obtain the variable region of the heavy chain, linking the variable region of the heavy chain to the constant region of a heavy chain of the desired isotype, expressing the light chain and the linked heavy chain in a cell and collecting the anti-IGF-IR antibody of the desired isotype.

Antibody derivatives

Antibody derivatives can be prepared using the above-described nucleic acid molecules by techniques and methods known to those of ordinary skill in the art.

Humanized antibodies

The method for producing a human antibody as described above has an advantage of producing an antibody with reduced immunogenicity. This can be achieved to some extent by immunological techniques and display techniques using appropriate libraries. It is understood that murine antibodies or antibodies derived from other species may be humanized or primatized using techniques known in the art. See, e.g., Winter and harrisim Today 14: 43-46(1993) and Wright et al, crit. reviews in Immunol.12125-168 (1992). Antibodies of interest are engineered by recombinant DNA techniques using the corresponding human sequences in place of CH1, CH2, CH3, hinge and/or framework regions (see WO 92/02190 and US patents 5530101, 5585089, 5693761, 5693792, 5714350 and 5777085). In a preferred embodiment, the anti-IGF-IR antibody is humanized by replacing CH1, CH2, CH3, the hinge region and/or the framework regions with corresponding human sequences while retaining all CDRs of the heavy chain, the light chain or both the heavy and light chains

Mutant antibodies

In another embodiment, the nucleic acid molecules, vectors and host cells may be used to prepare mutant anti-IGF-IR antibodies. The antibodies may be mutated in the variable regions of the heavy and/or light chains in order to alter the binding properties of the antibody. For example, mutations can be made in one or more CDR regions to increase or decrease the K of the antibody to IGF-IR_dIncreasing or decreasing K_offOr altering the binding characteristics of the antibody. Site-directed mutagenesis techniques are known in the art. See, e.g., Sambrook et al, and Ausubel et al, supra. In a preferred embodiment, amino acid residues within the variable region of the anti-IGF-IR antibody that are known to be altered from germline are mutated. In a more preferred embodiment, one or more mutations are made to amino acid residues within the variable or CDR regions 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 that are known to be altered from germline. In another embodiment, the amino acid sequence is as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 orThe nucleic acid sequence is shown as SEQ ID NO: 1, 3, 5,7, 9, 11, 13, 15, 17, 19, 21 or 23, and one or more mutations at amino acid residues within the variable or CDR regions known to have been altered relative to the germline. In another embodiment, the nucleic acid molecule is mutated in one or more framework regions. Mutations are made in the framework or constant regions to extend the half-life of the anti-IGF-IR antibody. See, for example, WO 00/09560 published at 2/24/2000, which is incorporated herein by reference. In another embodiment, there may be one, three or five point mutations and no more than ten point mutations. Mutations may be made in the framework or constant regions to alter the immunogenicity of the antibody, to provide sites 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, constant and variable regions within a single mutant antibody. Or may be mutated in only one of the framework, variable or constant regions within a single mutated antibody.

In one embodiment, there are no more than ten amino acid changes in the VH or VL region of the mutated anti-IGF-IR antibody relative to the pre-mutated anti-IGF-IR antibody. In a more preferred embodiment, there are no more than five amino acid changes, more preferably no more than three amino acid changes in the VH or VL region of the mutated anti-IGF-IR antibody. In another embodiment, there are no more than fifteen amino acid changes in the constant region, more preferably no more than ten amino acid changes, and even more preferably no more than five amino acid changes.

Altered antibodies

In another embodiment, a fusion antibody or immunoadhesin may be prepared comprising an intact anti-IGF-IR antibody or fragment thereof linked to another polypeptide. In a preferred embodiment, only the variable region of the anti-IGF-IR antibody is linked to the polypeptide. In another preferred embodiment, the VH domain of an anti-IGF-IR antibody is linked to a first polypeptide and the VL domain of an anti-IGF-IR antibody is linked to a second polypeptide which binds to the first polypeptide in a manner such that the VH and VL domains are capable of interacting to form an antibody binding site. In another preferred embodiment, the VH region is separated from the VL region by a linker to allow interaction between the VH and VL regions (see single chain antibodies below). The VH-linker-VL antibody is then linked to the antibody of interest. The fusion antibody is used to target the polypeptide to IGF-IR-expressing cells or tissues. 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 can be easily visualized, such as horseradish peroxidase. In addition, the fusion antibody may be made in a form in which two (or more) single-chain antibodies are linked to each other. This can be done if it is desired to prepare a bivalent or multivalent antibody in the form of a single polypeptide chain, or if it is desired to prepare a bispecific antibody.

For the preparation of single chain antibodies (scFv), VH-and VL-encoding DNA fragments are operably linked to another fragment encoding a flexible linker, e.g., a fragment encoding the amino acid sequence (Gly4-Ser)₃(SEQ ID NO: 60) such that the VH and VL sequences are expressed as a continuous single-chain protein with VL and VH domains linked by a 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 one VH and VL is used, bivalent if two VH and VL are used, or multivalent if more than two VH and VL are used.

In another embodiment, other altered antibodies can be prepared using anti-IGF-IR-encoding nucleic acid molecules. For example, "kappa bodies" (Ill et al, Protein Eng 10: 949-57(1997), "bodies (Minibody)" (Martin et al, EMBO J13: 5303-9(1994), "diplodies" (Holliger et al, PNAS USA 90: 6444-.

Alternatively, chimeric and bispecific antibodies can be prepared. Chimeric antibodies can be prepared containing CDRs and framework regions derived from different antibodies. In a preferred embodiment, the CDRs of the chimeric antibody comprise all the CDRs of the light or heavy chain variable region of the anti-IGF-IR antibody, and the framework regions are derived from one or more different antibodies. In a more preferred embodiment, the CDRs of the chimeric antibody comprise all the CDRs of the light or heavy chain variable region of an anti-IGF-IR antibody. The framework regions may be derived from another variety and, in a preferred embodiment, may be humanized. Alternatively, the framework region may be derived from another human antibody.

Bispecific antibodies can be prepared that specifically bind to IGF-IR via one binding domain and to a second molecule via a second binding domain. The bispecific antibodies can be produced by recombinant molecular biotechnology or can be physically conjugated together. Alternatively, single chain antibodies containing more than one VH and VL and which specifically bind IGF-IR and another molecule may be prepared. Such bispecific antibodies can be prepared using known techniques, e.g., in conjunction with (i) and (ii) see, e.g., Fanger et al, Immunol Methods 4: 72-81(1994), and Wright and Harris, as described above, and in conjunction (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 another molecule that is expressed at high levels on cancer or tumor cells. In a more preferred embodiment, the other molecule is the erbB2 receptor, VEGF, CD20 or EGF-R.

In one embodiment, the above-described altered antibody is prepared using one or more variable regions or one or more CDR regions of 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 embodiment, the altered antibody is a monoclonal antibody that utilizes a polypeptide whose amino acid sequence is set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 or a nucleic acid sequence thereof as set forth in SEQ ID NO: 1, 3, 5,7, 9, 11, 13, 15, 17, 19, 21 or 23, or one or more CDR regions.

Derivatized and labeled antibodies

The antibody or antibody fragment of the invention may be derivatized or linked to another molecule (e.g., another polypeptide or protein). Typically, the antibody or fragment thereof is derivatized to protect IGF-IR binding from adverse effects of derivatization or labeling. Thus, the antibodies or fragments thereof of the invention are designed to contain both intact and altered forms of the human anti-IGF-IR-antibodies described herein. For example, an antibody or fragment thereof of the invention can be functionally linked (by chemical coupling, genetic fusion, non-covalent binding, etc.) to one or more other molecular entities such as another antibody (e.g., a bispecific antibody or diabody), a detection agent, a cytotoxic agent, an agent, and/or a protein or peptide capable of mediating binding of the antibody or fragment thereof to another molecule, such as a streptavidin nuclear region or a polyhistidine tag.

One type of derivatized antibody is produced by cross-linking two or more antibodies (same or different types of antibodies, e.g., to create a bispecific antibody). Suitable crosslinking agents include those that are hetero-bifunctional (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homo-bifunctional (e.g., disuccinimidyl suberate) having two different reactive groups separated by a suitable spacer. Such cross-linking agents are available from Pierce chemical Company, Rockford, III.

Another type of derivatized antibody is a labeled antibody. Useful detection reagents that can be derivatized with the antibodies or antibody fragments of the invention include fluorescent compounds including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. The antibody may also be labeled with an enzyme for detection such as horseradish peroxidase, beta-galactosidase, luciferin bacillus, alkaline phosphatase, glucose oxidase, or the like. When the antibody is labeled with a detectable enzyme, the antibody is detected by adding additional reagents that are used by the enzyme to produce a product that can be distinguished. For example, when the reagent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine results in the production of a detectable colored reaction product. Antibodies can also be labeled with biotin and detected by indirect determination of avidin or streptavidin binding. The antibody may be labelled with a magnetic agent such as gadolinium. The antibody can also be labeled with a predetermined polypeptide epitope recognized by a second reporter molecule (e.g., leucine zipper pair sequence, a second antibody binding site, a metal binding region, an epitope tail). In some embodiments, the markers are connected by spacer arms of different lengths in order to reduce potential steric hindrance.

The anti-IGF-IR antibody may also be labeled with a radiolabeled amino acid. The radiolabel may be used for diagnostic or therapeutic purposes. For example, IGF-IR-expressing tumors can be detected by X-ray or other diagnostic techniques using the radiolabel. Furthermore, the radiolabel may be used therapeutically as a toxin to cancer cells or tumours. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionuclides-³H，¹⁴C，¹⁵N，³⁵S，⁹⁰Y， ⁹⁹Tc，¹¹¹In，¹²⁵I，¹³¹I。

The anti-IGF-IR antibodies may also be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl groups, or sugar groups. These groups can be used to improve the biological properties of the antibody, for example to increase serum half-life or to increase tissue binding.

Pharmaceutical composition kit

The invention also relates to pharmaceutical compositions for treating hyperproliferative disorders in a mammal comprising a therapeutically effective amount of a compound of the present invention and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition is used to treat cancer such as brain, lung, squamous cell, bladder, stomach, pancreas, breast, head, neck, kidney, ovary, prostate, colorectal, esophageal, gynecological, or thyroid cancer. In another embodiment, the pharmaceutical composition relates to non-cancerous hyperproliferative disorders such as, but not limited to, restenosis and psoriasis after angioplasty. In another embodiment, the invention relates to a pharmaceutical composition for treating a mammal in need of activating IGF-IR, wherein the composition comprises a therapeutically effective amount of an activating antibody of the invention and a pharmaceutically acceptable carrier. Pharmaceutical compositions containing the activating antibody may be used to treat animals lacking sufficient IGF-I or IGF-II, or may be used to treat osteoporosis, frailty, or conditions in which the mammal has too little or no response to growth hormone secretion.

The anti-IGF-IR antibodies of the present invention can be combined into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition contains an antibody of the invention and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all agents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible. Examples of pharmaceutically acceptable carriers include a wide variety of water, saline, phosphate buffer, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it is preferred to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable substances such as wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers can extend the shelf life or effectiveness of the antibody or antibody fragment.

The compositions of the present 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 dosage form depends on the mode of administration and the therapeutic use. 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. Whether or not 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 a more 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 compositions may be formulated as solutions, microemulsions, dispersions, liposomes or other ordered structures suitable for high drug concentrations. Sterile injectable solutions can be prepared by the next step: the desired amount of anti-IGF-IR antibody is combined with the desired amount of one or a combination of the above components in a suitable solvent and then filter sterilized. Generally, dispersions can be prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. For sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a solution which has been sterile-filtered. The desired fluidity of the 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 the injectable compositions can be brought about by including in the composition a substance which delays absorption, for example, monostearate salts and gelatin.

Although the preferred route/mode of administration for many therapeutic uses is intraperitoneal, subcutaneous, intramuscular, intravenous or infusion administration, the antibodies of the invention may be administered by a variety of methods known in the art. As will be appreciated by those skilled in the art, the route/mode of administration will vary with the desired result. In one embodiment, the antibodies of the invention can be administered in a single dose or in multiple doses.

In certain embodiments, the active compounds can be formulated with carriers that will prevent rapid release of the compound, such as in controlled release formulations including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Various methods for preparing such formulations are patented or known to those skilled in the art. See, e.g., Sustained and controlledRelease Dr μ g Delivery Systems, J.R. Robinson, eds., Marcel Dekker, Inc., New York, 1978.

In certain embodiments, the anti-IGF-IR of the invention may be administered orally, for example with an inert diluent or an assimilable edible carrier. The compounds (and other ingredients, if desired) may also be encapsulated in a hard or soft shelled gelatin capsule, compressed into a tablet or incorporated directly into the diet of the subject. For oral therapeutic administration, the compounds may be combined with excipients and used in the form of an ingestible tablet, buccal tablet, lozenge, capsule, elixir, suspension, syrup, wafer, or the like. In order to administer the compounds of the present invention by means other than parenteral administration, it is necessary to coat the compounds with or co-administer the compounds with substances that avoid their inactivation.

Supplementary active compounds may also be combined into the composition. In certain embodiments, the anti-IGF-IR of the present invention can be formulated and/or administered with one or more additional therapeutic agents, such as chemotherapeutic agents, antineoplastic agents, or antineoplastic agents. For example, the anti-IGF-IR antibody can be formulated and/or administered with one or more additional therapeutic agents. Such agents include, but are not limited to, antibodies that bind to other targets (e.g., antibodies that bind to one or more growth factors or cytokines, their cell surface receptors, or IGF-I), IGF-I-binding proteins, antineoplastic agents, chemotherapeutic agents, antineoplastic agents, anti-IGF-IR or IGF-I antisense oligonucleotides, peptide analogs that inhibit IGF-IR activation, soluble IGF-IR and/or one or more chemical agents known in the art that inhibit IGF-I production or activity, such as octreotide (octreotide). To prepare a pharmaceutical composition containing the activated antibody, the anti-IGF-IR antibody may be formulated with factors that enhance cell proliferation or prevent apoptosis. Such factors include growth factors, such as IGF-I, and/or IGF-I analogs that activate IGF-IR. This combination therapy requires low doses of anti-IGF-IR antibodies and co-administered agents, thus avoiding possible toxicity or complications associated with various monotherapies. In one embodiment, the antibody and one or more additional therapeutic agents.

The pharmaceutical compositions of the invention may contain a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antibody fragment of the invention. "therapeutically effective amount" means an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. The therapeutically effective amount of the antibody or antibody fragment may vary with such factors as the disease state, age, sex and weight of the individual, the ability of the antibody or antibody fragment to elicit a desired response in the individual. A therapeutically effective amount is also one in which the beneficial effects of the treatment outweigh the toxic or deleterious effects of the antibody or antibody fragment. By "prophylactically effective amount" is meant an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, the prophylactically effective amount will be lower than the therapeutically effective amount due to the prophylactic dose used prior to or early in the disease.

The dosage regimen may be adjusted to provide the optimal result desired (e.g., a therapeutic or prophylactic result). For example, a bolus may be administered in a single dose, several divided doses may be administered over a period of time, or may be proportionally reduced or increased depending on the urgency of the treatment. The pharmaceutical composition containing the antibody or the pharmaceutical composition containing a combination comprising the antibody and one or more additional therapeutic agents may be formulated as a single or multiple dose. It is particularly advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of administration. Unit dosage forms, as used herein, refer to physically discrete units suitable 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 unit dosage form of the present invention depends on (a) the unique properties of the active compounds and the particular therapeutic or prophylactic effect to be achieved and (b) the inherent limitations in the art of such active compounds with respect to the sensitivity of individual therapy. A particularly useful formulation is 5mg/ml anti-IGF-IR antibody in a buffer of 20mM sodium citrate, pH5.5, 140mM NaCI and 0.2mg/ml Tween 80(polysorbate 80).

An exemplary, non-limiting therapeutically or prophylactically effective amount of an antibody or antibody fragment of the invention ranges from 0.1 to 100mg/kg, more preferably from 0.5 to 50mg/kg, more preferably from 1 to 20mg/kg, and even more preferably from 1 to 10 mg/kg. It should be noted that the dosage values may vary with the type and severity of the condition to be alleviated. It is further understood that the specific dosage regimen for a particular subject should be adjusted over time according to the individual needs and the professional judgment of the individual in administering and monitoring the dosage of the composition and that the dosage ranges given herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions. In one embodiment, a therapeutically effective amount or a prophylactically effective amount of the antibody or antigen-binding fragment thereof is administered with one or more additional therapeutic agents.

In another aspect, the invention relates to the administration of an anti-IGF-IR antibody at a dose of less than 300mg per month for the treatment of cancer.

In another aspect of the invention, kits containing anti-IGF-IR antibodies and pharmaceutical compositions containing these antibodies are provided. The kit contains, in addition to the antibody or composition, a diagnostic or therapeutic agent. The kit may also contain instructions for use in a diagnostic or therapeutic method. In a preferred embodiment, the kit contains the antibody or pharmaceutical composition thereof and a diagnostic reagent that can be used in the methods described below. In another preferred embodiment, the kit contains the antibody or pharmaceutical composition thereof and one or more therapeutic agents that can be used in the methods described below, such as additional antineoplastic agents, or chemotherapeutic agents.

The present invention also relates to a pharmaceutical composition for inhibiting abnormal cell growth in a mammal, the pharmaceutical composition comprising an amount of a compound of the present invention and an amount of a chemotherapeutic agent, wherein the amount of the compound, salt, solvate or prodrug, and the amount of the chemotherapeutic agent act together in inhibiting abnormal cell growth. Many chemotherapeutic agents are currently known in the art. In one embodiment, the chemotherapeutic agent is selected from the group consisting of mitotic inhibitors, alkylating agents, anti-metabolites, intercalating agents, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormones, such as anti-androgens, and anti-angiogenic agents.

Anti-angiogenic agents such as MMP-2 (matrix-metalloproteinase 2) inhibitors, MMP-9 (matrix-metalloproteinase 9) inhibitors, and COX-II (cyclooxygenase II) inhibitors may be combined with the compounds of the present invention. Examples of useful COX-II inhibitors include CELEBREX^TM(alecoxib), valdecoxib and rofecoxib. Examples of useful matrix-metalloprotease inhibitors are described in WO 96/33172 (published at 24.10.1996), WO96/27583 (published at 7.3.1996), European patent application 97304971.1 (filed at 8.7.1997), European patent application 99308617.2 (filed at 29.10.1999), WO 98/07697 (published at 26.2.1998), WO 98/03516 (published at 29.1.1998), WO 98/34918 (published at 13.8.1998), WO 98/34915 (published at 13.8.13.1998), WO 98/33768 (published at 6.8.1998), WO 98/30566 (published at 16.7.1998), European patent applicationsPublication 606,046 (published on 13/7/1994), European patent publication 931,788 (published on 28/7/1999), WO 90/05719 (published on 31/5/1990), WO 99/52910 (published on 21/10/1999), WO 99/52889 (published on 21/10/1999), WO 99/29667 (published on 17/6/1999), PCT international patent application 98/01113 (filed on 21/7/1998), European patent application 99302232.1 (filed on 25/3/1999), Notepad patent application 9912961.1 (filed on 3/1999), US provisional application 60/148,464 (filed on 12/8/1999), US patent US5863949 (granted on 26/1/1999), US patent US5861510 (filed on 19/1/1999), and European patent application 780,386 (published on 25/6/1997), All documents are incorporated herein by reference in their entirety. Preferred MMP inhibitors are those which do not exhibit arthralgia. More preferred are those inhibitors which selectively inhibit MMP-2 and/or MMP-9 relative to 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 that are useful in the present invention are AG-3340, RO32-3555, RS13-0830 and the following compounds: 3- [ [4- (4-fluoro-phenoxy) -benzenesulfonyl group]- (1-hydroxycarbamoyl-cyclopentyl) amino]-propionic acid; 3-exo-3- [4- (4-fluoro-phenoxy) -benzenesulfonylamino]-8-oxa-bicyclo [3.2.1]Octane-3-carboxylic acid hydroxyamide; (2R, 3R)1- [4- (2-chloro-4-fluoro-benzyloxy) -benzenesulfonyl]-3-hydroxy-3-methyl-piperidine-2-carboxylic acid hydroxyamide; 4- [4- (4-fluoro-phenoxy) -benzenesulfonylamino group]-tetrahydro-pyran-4-carboxylic acid hydroxyamide; 3- [ [4- (4-fluoro-phenoxy) -benzenesulfonyl group]- (1-hydroxycarbamoyl-cyclobutyl) -amino]-propionic acid; 4- [4- (4-chloro-phenoxy) -benzenesulfonylamino group]-tetrahydro-pyran-4-carboxylic acid hydroxyamide; (R)3- [4 (4-chloro-phenoxy) -benzenesulfonylamino group]-tetrahydro-pyran-3-carboxylic acid hydroxyamide; (2R, 3R)1- [4- (4-fluoro-2-methyl-benzyloxy) -benzenesulfonyl]-3-hydroxy-3-methyl-piperidine-2-carboxylic acid hydroxyamide; 3- [ [4- (4-fluoro-phenoxy) -benzenesulfonyl group]- (1-hydroxycarbamoyl-1-methyl-ethyl) -amino]-propionic acid; 3- [ [4- (4-fluoro-phenoxy) -benzenesulfonyl group]- (4-hydroxycarbamoyl-tetrahydro-pyran-4-yl) -amino]-propionic acid; 3-exo-3- [4- (4-chloro-phenoxy-benzenesulfonyl) -benzeneAmino) -8-oxa-bicyclo [3.2.1]Octane-3-carboxylic acid hydroxyamide; 3-endo-3- [4- (4-fluoro-phenoxy) -benzenesulfonylamino]-8-oxa-bicyclo [3.2.1]Octane-3-carboxylic acid hydroxyamide; and (R)3- [4- (4-fluoro-phenoxy) benzenesulfonylamino group]-tetrahydro-furan-3 carboxylic acid hydroxyamide; and pharmaceutically acceptable salts and solvents thereof.

The compounds of the present invention may also be used with the following inhibitors: signal transduction inhibitors such as agents capable of inhibiting EGF-R (epidermal growth factor receptor) responses such as EGF-R antibodies, EGF antibodies, and EGF-R inhibitor molecules; VEGF (vascular endothelial growth factor) inhibitors, such as VEGF receptors and molecules capable of inhibiting VEGF; and erbB2 receptor inhibitors, such as organic molecules or antibodies capable of binding to erbB2 receptor, e.g. HERCEPTIN^TM(Genentech, Inc.). EGF-R inhibitors are described, for example, in WO95/19970 (published about 27 days 7 in 1995), WO 98/14451 (published 9 days 4/1998), WO 98/02434 (published 22 days 1/1998), and U.S. Pat. No. 5/1998) in U.S. Pat. No. 5,47498, and such substances may be used in the invention described herein. EGFR-inhibitors include, but are not limited to, monoclonal antibody 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 (Metarex Inc. and Merck KgaA), and compounds ZD1834, ZD-1838 and ZD-1839(AstraZeneca), PKI-166(Novartis), PKI-166/CGP75166(Novartis), PTK 787(Novartis), CP701(Cephalon), leflunomide (Pharmacia/S. mu. gen), CI-1033(Warner Lambert Parvis), CI-1033/PD 183,805(Warner Lambert Parkheir 785, Bridger-1031 (Szerie-Biotech), Roche-1033/Warner-183,805 (Warner Lambourne, Warner-Parthror, Szerie-R1, Szerie-R-1611 (Szerie) OLX-103 (Merck)&Co.), VRCTC310(Ventech Research), EGF fusion toxin (Seragen Inc.), DAB-389(Seragen/Lilgand), ZM-252808(Imperial Research Fund), RG-50864(INSERM), LFM-A12(Parker H. mu.ghes Cancer Center), WHI-P97(Parker H. mu.ghes Cancer Center), GW-282974(Glaxo), KT-8391(Kyowa Hakko) and EGF-R vaccine (York Medical/Central de Imm)unilogia Molec μ lar (CIM)). These and other EGF-inhibitors may be used in the present invention.

VEGF inhibitors, such as SU-5416 and SU-6668 (S. mu. gen Inc.), SH-268 (Sobering), and NX-1838 (NeXBtar) may also be used in combination with the compounds of the present invention. VEGF inhibitors are described, for example, in WO 99/24440 (published 20/5/1999), PCT International application PCT/IB99/00797 (filed 3/5/1999), WO95/21613 (published 17/8/1995), WO 99/61422 (published 2/12/1999), US5834504 (granted 10/11/19998), WO 98/50356 (published 12/11/1998), US5883113 (granted 16/3/1999), US5886020 (granted 23/1999), US5792783 (granted 11/1998), WO 99/10349 (published 4/1999), WO 97/32856 (published 12/1997), WO 97/22596 (published 26/6/1997), WO 98/54093 (published 3/1998), WO 98/02438 (published 22/1998), WO 3522/1998), WO 5886020 (published 12/9/4/1999), WO 97/22596 (published 26/1998), WO 99/16755 (published on 8.4.1999), and WO 98/02437 (published on 22.1.1998), all of which are incorporated herein by reference in their entirety. Other examples of certain specific VEGF that are used in the present invention are IM862(Cytran Inc.); anti-VEGF monoclonal antibodies of Genentech, inc; and vascular enzyme (angiozyme), a synthetic Ribozyme derived from Ribozyme and Chiron. These and other VEGF inhibitors may be used in the invention described herein.

ErbB2 receptor inhibitors, such as GW-282974(Glaxo Wellcome plc), and monoclonal antibodies AR-209(Aronex Pharmaceuticals Inc.) and 2B-1(Chiron), may be further combined with the compounds of the present invention, such as those described in WO 98/02434 (published 22/1/1998), WO 99/35146 (published 15/7/1999), WO99/35132 (published 15/7/1999), WO 98/02437 (published 22/1/1997), WO 97/13760 (published 17/1997), WO95/19970 (published 27/7/1995), US587458 (published 24/12/1996), and US5877305 (published 2/3/1999), which are all incorporated herein by reference. ErbB2 receptor inhibitors useful in the present invention are also described in U.S. provisional application 60/117,341 filed on 27.1.1999 and in U.S. provisional application 60/117,346 filed on 27.1.1999, both of which are incorporated herein by reference in their entirety. In accordance with the present invention, the erbB2 receptor inhibitor compounds and the substances described in the above-mentioned PCT applications, U.S. patents, and U.S. provisional applications, as well as other compounds and substances that inhibit the erbB2 receptor, may be used with the compounds of the present invention.

Anti-survival agents include anti-IGF-IR antibodies and anti-integrin agents, such as anti-integrin antibodies.

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 conventional immunoassays including, but not limited to, ELISA, RIA, FACS, tissue immunohistochemistry, Western blotting, or immunoprecipitation. The anti-IGF-IR antibodies of the invention may be used to detect IGF-IR in humans. In another embodiment, the anti-IGF-IR antibodies can be used to detect IGF-IR in the Old World (Old World) primates, such as rhesus macaques and rhesus macaques, chimpanzees and apes. The invention provides methods for detecting anti-IGF-IR in a biological sample, comprising contacting the biological sample with an anti-IGF-IR antibody of the invention and detecting the bound antibody that binds to the anti-IGF-IR to detect IGF-IR in the biological sample. In one embodiment, the anti-IGF-IR antibody is directly labeled with a detection label. In another embodiment, the anti-IGF-IR antibody (the primary antibody) is not labeled and the secondary antibody or other molecule capable of binding to the anti-IGF-IR antibody is labeled. It is well known to those skilled in the art to select a second antibody that is capable of specifically binding to a particular species and type of first antibody. For example, if the anti-IGF-IR antibody is human IgG, the second antibody may be an anti-human-IgG. Other molecules capable of binding to antibodies include, but are not limited to, protein a and protein G, both of which are commercially available, for example, from Pierce Chemical Co.

Suitable labels for the antibody or second antibody have been disclosed above and include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, magnetic agents and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent substances include 7-hydroxycoumarin, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; examples of luminescent substances include luminol; examples of the magnetic agent include gadolinium; and examples of suitable radioactive materials include¹²⁵I，¹³¹I，³⁵S or³H。

In another embodiment, the IGF-IR in a biological sample may be analyzed by a competitive immunoassay using an IGF-IR standard labeled with a test agent and an unlabeled IGF-IR antibody. In this assay method, a biological sample, a labeled IGF-IR standard and an anti-IGF-IR antibody are mixed together 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 bound to the anti-IGF-IR antibody.

The immunoassay method described above may be used for some purposes. In one embodiment, the anti-IGF-IR antibody 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, and/or the amount of IGF-IR on the cell surface following treatment of cells with various compounds. The method can be used to test compounds that can be used to activate or inhibit IGF-IR. In this method, a cell sample is treated with a test compound for a period of time while another sample is not treated. If tyrosine autophosphorylation is to be determined, the cells are lysed and tyrosine phosphorylation of IGF-IR is determined using the immunoassay described above or in example III, which utilizes an ELISA. If the total level of IGF-IR is to be determined, the cells are lysed and the total level of IGF-IR is determined using one of the immunoassays described above.

A preferred immunoassay method for determining tyrosine phosphorylation of IGF-IR or for determining total levels of IGF-IR is ELISA or Western blotting. If the cell surface level of IGF-IR is measured alone, cell lysis is not required and the cell surface level of IGF-IR can be measured using one of the immunoassays described above. A preferred immunoassay method for determining cell surface levels of IGF-IR comprises the steps of: with a detectable label, e.g. biotin or¹²⁵I labeling cell surface proteins, immunoprecipitating IGF-IR with an anti-IGF-IR antibody, and detecting the labeled IGF-IR. Another preferred immunoassay method for determining the localization of IGF-IR, e.g., cell surface levels, is by using immunohistochemistry. Methods such as ELISA, RIA, Western blot, immunohistochemistry, cell surface labeling of membrane-integrated proteins and immunoprecipitation are known in the art. See, e.g., Harlow and Lane, supra. In addition, the immunoassay can be scaled up to high throughput screening levels (high throughput screening) in order to detect large amounts of compounds for activating or inhibiting IGF-IR.

The anti-IGF-IR antibodies of the invention may also be used to determine the level of IGF-IR in a tissue or in cells derived from the tissue. In a preferred embodiment, the tissue is diseased tissue. In a more preferred embodiment, the tissue is tumor tissue or a biopsy thereof. In a preferred embodiment of the method the tissue or biopsy thereof is excised from the patient. The tissue or biopsy thereof is then used in an immunoassay to determine, for example, the level of IGF-IR, the cell surface level of IGF-IR, the tyrosine phosphorylation level of IGF-IR or the location of IGF-IR by the methods described above. The method can be used to determine whether tumors express IGF-IR at high levels.

The diagnostic methods described above can be used to determine whether a tumor expresses high levels of IGF-IR, which can be an indication that the tumor responds well to treatment with anti-IGF-IR antibodies. The diagnostic method may also be used to determine whether a tumor is likely cancerous if high levels of IGF-IR are expressed, or benign if low levels of IGF-IR are expressed. Furthermore, the diagnostic method can also be used to determine whether a treatment with an anti-IGF-IR antibody (see below) results in a tumor expressing low levels of IGF-IR and/or exhibiting low levels of tyrosine autophosphorylation, and can thus be used to determine whether the treatment is effective. Generally, the method for determining whether an anti-IGF-IR antibody reduces tyrosine phosphorylation comprises the steps of: measuring the level of tyrosine phosphorylation in the cell or tissue of interest, culturing the cell or tissue with an anti-IGF-IR antibody or antigen-binding fragment thereof, and then measuring the level of tyrosine phosphorylation in the cell or tissue again. Tyrosine phosphorylation of IGF-IR or another protein can be determined. The diagnostic method may also be used to determine whether tissues or cells do not express sufficiently high levels of IGF-IR or sufficiently high levels of activated IGF-IR, as is the case in individuals with short stature, osteoporosis or diabetes. Diagnosis of too low levels of IGF-IR or active IGF-IR may be used to increase IGF-IR levels or activity using an activated anti-IGF-IR antibody, IGF-I or other therapeutic agent.

The antibodies of the invention may also be used to localize IGF-IR expressing tissues and organs in vivo. In a preferred embodiment, the anti-IGF-IR may be used to localize IGF-IR-expressing tumors. The anti-IGF-IR antibodies of the present invention are advantageous in that they do not generate an immune response upon administration. The method comprises the following steps: the anti-IGF-IR antibody or pharmaceutical composition thereof is administered to a patient in need of such a diagnostic test and the patient is subjected to image analysis to determine the location of the IGF-IR-expressing tissue. Image analysis is known in the medical field and includes, but is not limited to, X-ray analysis, Magnetic Resonance Imaging (MRI) or computed tomography (CE). In one embodiment of the method, a biopsy is obtained from the patient to determine whether the tissue of interest expresses IGF-IR without mapping the patientAnd (4) image analysis. In a preferred embodiment, the anti-IGF-IR antibody is labeled with a detection agent that is capable of being visualized in the patient. For example, the antibody may be labelled with a contrast agent such as barium, which may be used for X-ray analysis, or a magnetic contrast agent such as gadolinium chelate, which may be used for MRI or CE. Other labels include, but are not limited to, radioisotopes such as⁹⁹Tc. In another embodiment, the anti-IGF-IR antibody is not labeled and image analysis is performed by applying a secondary antibody or other molecule that is detectable and capable of binding to the anti-IGF-IR antibody.

Application of therapeutic methods

In another embodiment, the invention provides a method of 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 expressing IGF-IR to which an anti-IGF-IR antibody cross-reacts. The antibodies can be administered to a non-human mammal (i.e., primate or cynomolgus or rhesus monkey) expressing IGF-IR to which the antibodies cross-react, for veterinary purposes or as an animal model of human disease. Such animal models can be used to evaluate the therapeutic efficacy of the antibodies of the invention.

The term "a condition in which IGF-IR activity is detrimental" as used herein is intended to include diseases in which the presence of high levels of IGF-IR in a patient has been shown to be or may contribute to the physiopathological cause of the condition or to a factor which worsens the condition. Thus, a condition in which high levels of IGF-IR activity are detrimental is one in which inhibition of IGF-IR activity is expected to alleviate the symptoms and/or progression of the disease. Such a condition may be evidenced, for example, by an increase in the level of IGF-IR at the cell surface or an enhancement of tyrosine phosphorylation in affected cells or tissues of a subject suffering from the condition. An increase in IGF-IR levels may be detected, for example, by using the anti-IGF-IR antibodies described above.

In a preferred embodiment, the anti-IGF-IR antibody is administered to a patient having an IGF-IR-expressing tumor. The tumor may be a solid tumor or may be a non-solid tumor, such as a lymphoma. In a more preferred embodiment, the anti-IGF-IR antibody may be administered to a patient having an IGF-IR-expressing tumor that is cancerous. In an even more preferred embodiment, the anti-IGF-IR antibody can be administered to a patient having a lung tumor, breast tumor, prostate tumor, or colon tumor. In a highly preferred embodiment, the method results in the tumor no longer increasing in weight or volume or decreasing in weight and volume. In another embodiment, the method internalizes IGF-IR on the tumor. 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 fragment thereof.

In another preferred embodiment, the anti-IGF-IR antibody may be administered to a patient that does not properly express high levels of IGF-I. In the art, it is known that high levels of expression of IGF-I can lead to a variety of conventional cancers. In a more preferred embodiment, the anti-IGF-IR antibody is administered to a prostate cancer, glioma or fibrosarcoma patient. In an even more preferred embodiment, the method results in the cancer ceasing abnormal proliferation, or no further increase in weight or volume or a decrease in weight or volume.

In one embodiment, the method relates to the treatment of cancer such as brain, squamous cell, bladder, gastric, pancreatic, breast, head, neck, oesophageal, prostate, colorectal, lung, kidney, ovarian gynaecological or thyroid cancer. Patients treatable with a compound of the invention according to the methods of the invention include, for example, patients diagnosed with lung, bone, pancreatic, skin, head and neck, cutaneous or intraocular melanoma, uterine, ovarian, rectal, cancer of the anal region, stomach, colon, breast, gynecological (e.g., uterine sarcoma, fallopian tube, endometrial, cervical, vaginal or vulval), hodgkin's disease, esophageal, small bowel, cancer of the endocrine system (e.g., thyroid, parathyroid or adrenal), soft tissue sarcoma, urinary tract, penis, prostate, chronic or acute leukemia, solid tumors of childhood, lymphocytic lymphomas, bladder, renal or ureteral (e.g., renal cell, renal pelvis), or central nervous system (e.g., primary CNS lymphoma, pancreatic cancer, skin cancer, head and neck cancer, melanoma of the skin or eyes, carcinoma of the uterus, carcinoma of the ovary, carcinoma of the rectum, carcinoma of the endocrine system (e.g., thyroid, parathyroid, adrenal gland, or central nervous, Spinal axis (Spinal axis) tumors, brain stem glioma, or pituitary gland tumors).

The antibody may be administered once, but more preferably multiple administrations are carried out. The antibody may be administered from three times a day to once every six months. The administration can be according to a regimen such as three times daily, twice daily, once every two days, once every three days, once weekly, once every two weeks, once monthly, once every two months, once every three months, and once every six months. The antibody may be administered by oral, mucosal, buccal, intranasal, inhalation, intravenous, subcutaneous, intramuscular, parenteral, intratumoral, or topical routes of administration. The antibody may be administered at a location remote from the tumor site. The antibody may also be administered continuously by a micro-pump. The antibody may be administered once, at least twice, or at least for a period of time until the condition is treated, alleviated, or cured. Antibodies are generally administered as soon as a tumor appears, if they can stop the growth of the tumor or cancer or reduce the weight or volume of the tumor or cancer. The antibody is typically administered as part of a pharmaceutical composition as described above. The dosage range of the antibody is usually 0.1-100mg/kg, more preferably 0.5-50mg/kg, more preferably 1-20mg/kg, and even more preferably 1-10 mg/kg. The serum concentration of the antibody can be determined by methods known in the art. See, for example, example XVII below. To avoid the development of cancer or tumors, the antibody may also be administered prophylactically. This is particularly applicable to patients with elevated levels of IGF-I, since these patients have proven to be a high risk group with common cancers. See Rosen et al, supra.

In another aspect, the anti-IGF-IR antibody may be co-administered with other therapeutic agents, such as anti-tumor drugs or molecules, to a patient suffering from a hyperproliferative disorder, such as cancer or a tumor. In one aspect, the invention relates to a method for treating a hyperproliferative disorder in a mammal, comprising administering to said mammal a therapeutically effective amount of a compound of the present invention with an anti-neoplastic agent selected from, 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 metalloproteinase inhibitors, genetic therapeutic agents, and anti-androgens. In a more preferred embodiment, the antibody may be administered with an anti-neoplastic agent such as doxorubicin or paclitaxel. In another preferred embodiment, the antibody or combination therapy may be administered in conjunction with radiation therapy, chemotherapy, photodynamic therapy, surgery or other immunotherapy. In another preferred embodiment, the antibody may be administered with another antibody. For example, the anti-IGF-IR antibody may be administered with an antibody or other drug known to inhibit tumor or cancer cell proliferation, such as an antibody or drug that inhibits erbB2 receptor, EGF-R, CD20 or VEGF.

Co-administration of the antibody with an additional therapeutic agent (combination therapy) includes administration of a pharmaceutical composition containing an anti-IGF-IR antibody and an additional therapeutic agent, and administration of two or more separate pharmaceutical compositions, one containing an anti-IGF-IR antibody and the other containing an additional therapeutic agent. Moreover, while co-administration or combination therapy generally means that the antibody and additional therapeutic agent are administered concurrently with each other, it also includes situations where the antibody and additional therapeutic agent are not administered concurrently. For example, the antibody may be administered once every three days, while the additional therapeutic agent may be administered once a day. Or the antibody may be administered before or after treatment of the disorder with the additional therapeutic agent. Similarly, the antibody may be administered before or after other therapies such as radiotherapy, chemotherapy, photodynamic therapy, surgery or other immunotherapy.

The antibody and one or more additional therapeutic agents (combination therapy) may be administered once, twice, or at least for a period of time until the condition is treated, alleviated, or cured. Preferably the combination therapy is administered multiple times. The combination therapy may be administered from three times a day to once every six months. The administration may be performed according to a regimen such as three times daily, twice daily, once every two days, once every three days, once weekly, once every two weeks, once monthly, once every two months, once every three months, and once every six months, or may be performed continuously by a micro pump. The combination therapy may be administered by oral, mucosal, buccal, intranasal, inhalation, intravenous, subcutaneous, intramuscular, parenteral, intratumoral or topical routes of administration. The combination therapy may be administered at a location remote from the tumor site. If the antibody is capable of stopping the growth of the tumor or cancer or reducing the weight or volume of the tumor or cancer, the combination therapy will generally be performed as soon as the tumor appears.

In another embodiment, the anti-IGF-IR antibody is labeled with a radiolabel, immunotoxin or toxin, or is a fusion protein containing a toxic peptide. The anti-IGF-IR antibody or anti-IGF-IR antibody fusion protein localizes the radiolabel, immunotoxin, toxin, or toxic peptide to an IGF-IR-expressing tumor or cancer cell. In a preferred embodiment, the radiolabel, immunotoxin, toxin or toxic peptide is internalized when the anti-IGF-IR antibody binds to IGF-IR on the surface of a tumor or cancer cell.

In another aspect, the anti-IGF-IR antibody is used therapeutically to induce apoptosis in specific cells in a patient in need of treatment. In many cases, the cell targeted for apoptosis is a cancer cell or a tumor cell. Thus, in a preferred embodiment, the invention provides a method of inducing apoptosis by administering to a patient in need thereof a therapeutically effective amount of an anti-IGF-IR antibody. 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 fragment thereof.

In another aspect, the anti-IGF-IR antibodies may be used to treat non-cancerous conditions in which high levels of IGF-I and/or IGF-IR are associated with a non-cancerous condition or disease. In one embodiment, the method comprises the steps of: administering an anti-IGF-IR antibody to a patient having a non-cancerous pathological condition, wherein said non-cancerous pathological condition is caused or exacerbated by high levels of IGF-I and/or IGF-IR levels or activity. In a preferred embodiment, the non-cancerous pathological condition is acromegaly, gigantism, psoriasis, atherosclerosis, such as found as a complication of diabetes, which is restenosis of vascular smooth muscle of the eye or inappropriate proliferation of microvessels. In a more preferred embodiment, the anti-IGF-IR antibody slows the development of non-cancerous pathological conditions. In a more preferred embodiment, the anti-IGF-IR antibody at least partially terminates or reverses non-cancerous pathological symptoms.

In another aspect, the invention provides methods of administering an activating anti-IGF-IR antibody to a patient in need of treatment. In one embodiment, the activating antibody or pharmaceutical composition is administered to a patient in need of treatment in an amount effective to enhance IGF-IR activity. In a more preferred embodiment, the activating antibody is capable of restoring normal IGF-IR activity. In another preferred embodiment, the activating antibody may be administered to a patient with short stature, neuropathy, decreased muscle mass, or osteoporosis. In another preferred embodiment, the activating antibody may be administered with one or more other factors in order to enhance cell proliferation, avoid apoptosis or enhance IGF-IR activity. Such factors include growth factors such as IGF-I and/or IGF-I analogs capable of activating IGF-IR. In another preferred embodiment, the antibody is selected from 4.17.3, or contains a heavy chain, light chain or antigen binding fragment thereof.

Gene therapy

The nucleic acid molecules of the invention may be administered to a patient in need of treatment by gene therapy. The therapy is performed in vivo or ex vivo. In a preferred embodiment, a nucleic acid molecule encoding both the heavy chain and the light chain is administered. In a more preferred embodiment, the nucleic acid molecules are administered so that they are stably integrated into the chromosome of the B cells, since these cells are specialized for the production of antibodies. In a preferred embodiment, precursor B cells are transfected or infected ex vivo and then transplanted into a patient in need of treatment. In another embodiment, precursor B cells or other cells are infected in vivo with a virus known to infect the desired cell type. Typical vectors for gene therapy include liposomes, plasmids or viral vectors such as retroviruses, adenoviruses or adeno-associated viruses. Following infection in vivo or ex vivo, the expression level of the antibody can be monitored by sampling from the treated patient and using immunoassays known in the art and described herein.

In a preferred embodiment, the gene therapy comprises the steps of: administering and expressing an effective amount of an isolated nucleic acid molecule encoding a heavy chain of a human antibody or fragment thereof or an antigen-binding fragment thereof. In another embodiment, the gene therapy comprises the steps of: administering and expressing an effective amount of an isolated nucleic acid molecule encoding a light chain of a human antibody or fragment thereof or an antigen-binding fragment thereof. In a more preferred embodiment, the gene therapy comprises the steps of: administering and expressing an effective amount of an isolated nucleic acid molecule encoding a heavy chain of a human antibody or fragment thereof or an antigen-binding fragment thereof and an effective amount of an isolated nucleic acid molecule encoding a light chain of a human antibody or fragment thereof or an antigen-binding fragment thereof. The gene therapy may also include the administration of another anti-cancer drug, such as paclitaxel, tamoxifen, 5-FU, doxorubicin, or CP-358,774.

In order that the invention may be better understood, the following examples are set forth. These examples are illustrative only and should not be construed as limiting the scope of the invention in any way.

Example 1: generation of anti-IGF-IR antibody producing hybridomas

The antibodies of the invention were prepared, selected and assayed as follows:

immunization and hybridoma production

The extracellular domain of human IGF-IR (10. mu.g/dose/mouse) or 3T3-IGF-IR or 300.19-IGF-IR cells (10X 10)⁶Cells/dose/mouse) on eight to ten weeks old xenomics^TMImmunization is carried out intraperitoneally or in hindpaw pads, where the two cells are two cell lines transfected and expressing human IGF-IR on their plasma membranes. This dose was repeated five to seven times in three to eight weeks. Four days prior to fusion, the mice were injected with the last injection of the extracellular domain of human IGF-IR in PBS. Lymphocytes from the spleen and lymph nodes of immunized mice were fused with non-secreting myeloma P3-X63-Ag8.653 cells and HAT selected as described previously (Galfre and Milstein, Methods enzymol.73: 3-46, 1981). A panel of hybridomas secreting IGF-IR specific human IgG2 kappa antibodies was recovered. Seven hybridomas producing monoclonal antibodies specific for IGF-IR were selected for further study and 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 at 12.12.12.2000 at the American Type Culture Collection (ATCC), 10801 Universal Bo μ levard, Manassas, VA 20110-:

hybridoma cell Accession number

2.12.1P TA-2792

2.13.2P TA-2788

2.14.3P TA-2790

3.1.1P TA-2791

4.9.2P TA-2789

4.17.3P TA-2793

Example II: determination of the affinity constant P (Kd) of the human intact anti-IGF-IRD monoclonal antibody by BIAcore

Determination of affinity of purified antibodies by surface plasmon resonance using a BIAcore 3000 instrument and following the manufacturer's instructions

Scheme 1

For kinetic analysis, protein-A was immobilized on the sensor chip surface of BIAcore. The anti-IGF-IR antibodies of the invention are then captured with the sensor chip. Extracellular regions of IGF-IR were injected at different concentrations onto the sensor chip and the binding and dissociation kinetics of the interaction between the anti-IGF-IR antibody and the extracellular regions of IGF-IR were analyzed. The data were evaluated using a baseline drift model in the BIA evaluation software provided by BIAcore, with a Universal fitting Langmuir (globalfit Langmuir) 1: 1.

Scheme 2

Essentially according to Fagerstat et al, "Detection of anti-inflammatory interactions by surface plasma response. 208-.

Table I lists avidity measurements for a typical anti-IGF-IR antibody of the invention:

TABLE I

Monoclonal antibodies	Kd(M) scheme 1	Kd(M) scheme 2
			2.12.1	7.37×10-9
2.13.2	3.5×10-9	1.53×10-9
			2.14.3	6.41×10-10
3.1.1	1.15×10-9
			4.9.2	6.84×10-10	4.27×10-10
4.17.3	1.3×10-8
			6.1.1	5.65×10-10

Kinetic analysis showed that the antibodies prepared according to the invention have a high affinity and a strong binding constant for the extracellular domain of IGF-IR.

Example III: antibody-mediated inhibition of IGF-I-induced IGF-IR phosphorylation

To determine whether the antibodies of the invention are capable of blocking IGF-I-mediated IGF-IR activation reactions, we performed ELISA experiments. IGF-I-mediated IGF-IR activation is detected by enhanced receptor-related tyrosine phosphorylation.

Preparation of ELISA plates

We prepared ELISA capture plates by adding 100. mu.l of blocking buffer (Tris-buffered saline [ TBS ] containing 3% bovine serum albumin [ BSA ]) to each well of a protein G-coated reactionbind 96-well plate (Pierce) and incubated the plates at room temperature for 30 min with shaking. The rabbit full-specific SC-713 anti-IGF-IR antibody (Santa Cruz) was diluted to a concentration of 5. mu.g/ml with blocking buffer and 100. mu.l of the diluted antibody was added to each well. The plates were incubated at room temperature for 60-90 minutes with shaking. The plates were then washed 5 times with wash buffer (TBS + 0.1% Tween 20) and the remaining buffer was gently blotted dry with a paper towel. The plates could not be completely dried before the addition of the lysate.

Preparation of lysates from IGF-IR-expressing cells

Transfecting IGF-IRNIH-3T3 cells (5X 10)⁴Ml) was placed in 100. mu.l of a medium (DMEM high glucose content medium to which L-glutamine (0.29mg/ml), 10% heat-inactivated FBS and 500. mu.g/ml of each of geneticin, penicillin and streptomycin) in a 96-well U-shaped bottom plate. 5% CO at 37 ℃₂The plates were then incubated overnight to allow the cells to attach. The medium was decanted from the plate and 100. mu.l of fresh medium was added to each well. To perform the assay, the potential anti-IGF-IR antibody was diluted five times with medium and 25. mu.l was added to each well. All samples were run in triplicate. The plates were then incubated at 37 ℃ for 1 hour. Cells were stimulated with 25. mu.l/well of 600ng/ml IGF-1 (prepared with medium) and plates were incubated for 10 minutes at room temperature. The medium was decanted by inverting the plate and blotted gently onto paper towels, and lysed by adding 50. mu.l of lysis buffer (50mM HEPES, pH7.4, 10mM EDTA, 150mM NaCl, 1.5mM MgCl)₂，1.6mM NaVO₄1% Triton X-100, 1% glycerol and 50. mu.l each of a tablet of EDTA-free protease inhibitor [ RocheMolec. mu. lar Sciences ]]Added just prior to use) and shaken at room temperature for 5 minutes to lyse the attached cells. Mu.l of dilution buffer (50mM HEPES, pH7.4, 1.6mM NaVO) was added to each well₄) And mixing was performed by sucking up and sucking down. 100 μ l of lysate in each well was transferred to each well of an ELISA capture plate prepared as described above and incubated for 2 hours at room temperature with gentle shaking.

ELISA Using anti-tyrosine-phosphate (pTYR) antibody

The cell lysate was removed by inverting the plate, washing the plate 5 times with wash buffer and blotting onto paper towels. Mu.l of pTYR-specific antibody (HRP-PY54) diluted to 0.2. mu.g/ml with blocking buffer was added to each well and the plate was incubated at room temperature for 30 minutes with shaking. The plates were then washed 5 times with wash buffer and blotted onto paper towels.

By adding 100. mu.l of TMB peroxidase substrate solution per well(Kirkegaard&Perry) and incubated with shaking to develop color (approximately 1-10 minutes) to detect binding of HRP-PY54 antibody. Add 100. mu.l TMB stop solution (Kirkegaard) per well&Perry) to terminate the chromogenic reaction. The plates were then shaken for 10 seconds at room temperature to mix and pass the solution at OD_450nmMeasured for quantitative analysis.

Table II and FIG. 4 show the results of experiments with several antibodies of the invention. The results of this experiment demonstrate the ability of the antibodies of the invention to block IGF-I-mediated IGF-IR activation, as evidenced by enhanced receptor-related tyrosine phosphorylation. Moreover, these results can be used to quantify the relative potency of the antibodies of the invention.

TABLE II

Monoclonal antibodies	IC50(μg/ml)
		2.12.1	0.172
2.13.2	0.0812
		2.14.3	0.325
4.9.2	0.0324

Example IV: blocking IGF-I/IGF-IR binding by antibody-mediation

ELISA assays were performed in cell-based assays to quantify the ability of the antibodies of the invention to inhibit IGF-I binding to IGF-I. IGF-IR-transfected NIH-3T3 cells (5X 10)⁴Ml) was placed in 100. mu.l of DMEM high glucose medium supplemented with L-glutamine (0.29mg/ml), 10% heat-inactivated FBS and 500. mu.g/ml of each of geneticin, penicillin and streptomycin in a 96-well U-shaped bottom plate. Then 5% CO at 37 ℃₂The plates were then incubated overnight to allow the cells to attach. The medium was decanted from the plate and 100. mu.l of fresh medium was added to each well. For the assay, the antibody was diluted to the desired final concentration with assay medium (DMEM high glucose content medium supplemented with L-glutamine (0.29mg/ml), 10% heat inactivated BSA and 200. mu.g/ml of each of geneticin, penicillin and streptomycin) and 50. mu.l was added to each well. All samples were run in triplicate. The plates were then incubated at 37 ℃ for 10 minutes. Using the measurement medium¹²⁵I]IGF-I was diluted to 1. mu. Ci/ml and 50. mu.l was added to each well of the plate. As a control for background radioactivity, cooled IGF-I was added until a final concentration of 100ng/ml was reached. The plates were incubated at 37 ℃ for 10 minutes, the medium was decanted by gently blotting onto a paper towel, and rinsed twice with assay medium. Cells were lysed by adding 50 μ 10.1N NaOH, 0.1% SDS and shaking the plate for 5 minutes at room temperature. The samples were then transferred to scintillation plates, 150. mu.l of OptiPhaseSupermix was added and the signal on the Wallac Micro-Beta technologist was read.

Table III and FIG. 3 show the results of experiments with three exemplary antibodies of the present invention. This experiment shows that the antibodies of the invention specifically inhibit the binding of [125I ] -IGF-I to cells overexpressing IGF-IR.

TABLE III

Monoclonal antibodies	IC50
		2.12.1	0.45μg/ml
2.13.2	0.18μg/ml
		4.9.2	0.1μg/ml

Example V: study of epitope mapping

Having shown that the antibodies of the present invention are capable of recognizing IGF-IR, we have conducted epitope mapping studies on several antibodies of the present invention. We particularly focused the experiment on the 2.12.1, 2.13.2, 2.14.3 and 4.9.2 antibodies.

We performed BIAcore competition experiments to determine whether the antibodies of the invention bind to the same or different sites on IGF-IR molecules. The extracellular domain (ECD) of IGF-IR was bound to the BIAcore sensor chip described in example II above. The first antibody of the invention is bound to the sensor chip-bound IGF-IR under saturating conditions. Subsequent second antibodies of the invention are then assayed for their ability to compete with the first antibody for binding to IGF-IR. This technique enables us to separate the antibodies of the invention into different binding types.

We performed this experiment on antibodies 2.12.1, 2.13.2, 2.14.3 and 4.9.2. As a result, it was found that 2.13.2 and 4.9.2 compete for the same site on the extracellular domain of IGF-IR. Other antibodies 2.12.1 and 2.14.3 bind to sites on the extracellular domain of IGF-IR that are not identical to each other and are different from the sites to which 2.13.2 and 4.9.2 bind.

Example VI: species Cross-reactivity of antibodies of the invention

To determine the species cross-reactivity of the antibodies of the invention, we performed several experiments, including immunoprecipitation, antibody-mediated blockade of receptor phosphorylation induced by IGF-I, and FACS analysis.

For immunoprecipitation experiments, we placed the cells in T25 shake flasks in DMEM high glucose content medium supplemented with L-glutamine (0.29mg/ml), 10% heat-inactivated FBS, and 500 μ g/ml of each of geneticin, penicillin, and streptomycin until 50% confluence was reached. Then, 100. mu.l of the antibody of the present invention in a 1. mu.g/ml concentration of Hank buffer salt solution (HBSS; Gibco BRL) was added. The plates were incubated at 37 ℃ for 30 minutes in an incubator, and the cells were then stimulated with 100ng/ml IGF-I for 10 minutes at room temperature. The cells were lysed in RIPA buffer (Harlow and Lane, supra) and IGF-IR was immunoprecipitated at 4 ℃ with 2. mu.g of full-specific SC-713 anti-IGF-IR antibody (Santa Cruz) plus protein A agarose beads. The beads were pelleted and washed three times with PBS/T (PBS + 0.1% Tween-20) and then boiled in 40. mu.l Laemmli buffer containing 5% β ME.

Samples prepared as described above were analyzed by Western blotting. Load 12. mu.l of each sample to a 4-10% gradient of Novex^TMIn the lanes on the gel and in IXMES buffer (Novex)^TM) And (4) performing middle-stage operation. Run at 150V for 1 hour or 200V for about 30 minutes. Then by using 10% methanol at 100mA overnight or at 25Transfer of the gel to Novex at 0mM for 1-1.5 hours^TMOn the membrane in transfer buffer. The membrane was then dried thoroughly and blocked with TBS (Tris-buffered saline solution pH 8.0) containing Superlock (Pierce chemical Co.) at room temperature. The immunoprecipitated IGF-IR was detected by addition of the IGF-IR blotting antibody SC713(Santa Cruz).

Such experiments were performed on cells derived from various mammals using the antibodies of the invention, particularly 2.12.1, 2.13.2, 4.17.3 and 4.9.2. As a result, it was found that the antibodies 2.12.1, 2.13.2 and 4.9.2 were able to bind to human but not to canine, guinea pig, rabbit IGF-IR. Furthermore, these antibodies were able to bind both COS 7and rhesus IGF-IR obtained from all old world monkeys, but not IGF-IR obtained from marmosets belonging to new world monkeys. These experiments indicate that the antibodies are highly specific.

Antibody-mediated blockade of IGF-I/IGF-IR binding in non-human primates

After observing that the antibodies of the invention recognize IGF-IR derived from old world monkeys, we also tested their ability to block IGFI/IGF-IR binding in monkeys derived from these old world monkeys. The cells were placed to 50% confluence in DMEM high glucose medium supplemented with L-glutamine, 10% heat-inactivated FBS and 500. mu.g/ml each of geneticin, penicillin and streptomycin in T25 shake flasks. The antibody of the invention was then added, or the antibody-free medium was used as a control, and the cells were stimulated with 100ng/ml IGF-I for 10 minutes at room temperature. Following stimulation, cells were lysed and IGF-IR immunoprecipitated with the full-specific IGF-IR antibody SC713 as described above. Western blot analysis was then performed as described above using the HRP-PY54 antibody to detect phosphorylated tyrosine in activated IGF-IR.

We observed that the antibodies of the invention, in particular 2.13.2 and 4.9.2, were able to block IGF-I-induced phosphorylation of IGF-IR in COS 7and rhesus cells. IC against the inhibitory effect observed for COS 7and rhesus IGF-IR₅₀Are respectively 0.02. mu.g/ml and0.005μg/ml。

determination of Cross-species affinity of antibodies of the invention

FACS analysis was performed to determine the affinity of the antibodies of the invention for IGF-IR from other animals, especially the old world monkeys described above. Human and monkey cells (5X 10) were treated with increasing concentrations of biotinylated anti-IGF-IR antibodies of the invention or biotinylated anti-Keyhole Limpet Hemocyanin (KLH) antibody (Abgenix) as a negative control⁵) An aliquot of (a) was ice-cooled for 1 hour. The sample was then ice-cooled with streptavidin-conjugated RPE (phycoerythrin) for 30 minutes. Binding was determined by flow cytometry and histograms of fluorescence intensity (F12-H) versus cell number (counts) were analyzed using CellQuest software. The binding capacity (K) of each antibody was calculated from a plot of mean fluorescence intensity versus antibody concentration_d). In most experiments, binding was determined in cultured human MCF-7 cells and in tissue culture cells of rhesus monkey or macaque. Antibody consumption is controlled by measuring binding capacity over a range of cell concentrations.

The FACS analysis described above was performed to test the ability of the antibodies of the invention, in particular 2.13.2 and 4.9.2, to bind to human, rhesus and cynomolgus monkey cells. We found that all cell lines tested had a half maximal binding force (Kd) of 0.1. mu.g/ml.

Example VII: down-regulation of IGF-I receptor

A blocking experiment was conducted essentially as in example IV above until the addition of [ 2 ]¹²⁵I]-labeled IGF-I. At this time the cells were boiled in 40. mu.l Laemmli buffer containing 50% ME. The samples were then analyzed by Western blotting as described in example VI above and the blots were detected by quantitative analysis of IGF-IR levels using the full-specific IGF-IR antibody SC713 and monitoring of phosphorylated tyrosine levels in activated IGF-IR using HRP-PY 54.

As observed above (example III), we observed that IGF-I-induced phosphorylation of IGF-IR was blocked after treatment with the antibodies of the invention (FIG. 4). Furthermore, we have found that IGF-IR in these cells is down-regulated after the IGF-I-induced phosphorylation is blocked. See, for example, fig. 4. IGF-IR levels were maximally reduced 16 hours after stimulation with IGF-I in the presence of the antibodies of the invention.

Example VIII: in vivo Effect of the antibodies of the invention on IGF-IR

We determined whether the effect of the antibodies of the invention on IGF-IR described in the above examples would occur in vivo. Tumors were induced in athymic mice according to published methods (V.A. Pollack et al, "Inhibition of epitopic growth factor receptor-associated phosphorinone human carcinomaes with CP-358, 774: Dynamics of receptor in situ and antibodies in therapy," J.Pharmacol. Exp.Ther.291: 739-748 (1999)).

Briefly, IGF-IR-transfected NIH-3T3 cells (5X 10)⁶) Injected subcutaneously with 0.2ml of Matrigel formulation into 3-4 week old athymic (nu/nu) mice. Then at the tumor (i.e. about 400 mm)³) After formation, mice were injected intraperitoneally with the antibodies of the present invention.

After 24 hours, the tumors were removed, homogenized and assayed for IGF-IR levels. To determine the level of IGF-IR, SC-713 antibody was diluted to a final concentration of μ g/ml with blocking buffer and 100 μ l was added to each well of a reaction-Bind goat anti-rabbit (GAR) -coated plate (Pierce). The plates were incubated at room temperature for 1 hour with shaking and then washed 5 times with wash buffer. The tumor samples prepared as described above were then weighed and homogenized in lysis buffer (1ml/100 mg). 12.5. mu.l of the tumor extract was diluted with lysis buffer to a final volume of 100. mu.l and added to each well of a 96-well plate. The plates were incubated at room temperature for 1-2 hours with shaking, then washedBuffer washes 5 times. Then 100. mu.l of HRP-PY54 or biotinylated anti-IGF-IR antibody in blocking buffer was added to each well and incubated at room temperature for 30 minutes with shaking. The plates were then washed 5 times with wash buffer and developed. Plates detected with HRP-PY54 were developed by adding 100. mu.l of TMB microwell substrate per well, followed by adding 100. mu.l of 0.9MH₂SO₄To terminate the color reaction. Then by shaking for 10 seconds and determining the OD_450nmThe signal was quantitatively analyzed. The signal was normalized to total protein. The plates detected by anti-IGF-IR antibody were developed by adding 100. mu.l of streptavidin diluted in blocking buffer to each well, incubating at room temperature for 30 minutes with shaking, and then proceeding as for HRP-PY 54.

As a reduction in the measured IGF-IR phosphotyrosine (phosphorylated IGF-IR) and total IGF-IR protein occurred, we found that intraperitoneal injection of the antibodies of the invention, especially 2.13.2 and 4.9.2, resulted in inhibition of IGF-IR activity (FIG. 6). We also found that IGF-IR phosphotyrosine (phosphorylated IGF-IR) was also reduced (FIG. 5). Without wishing to be bound by any theory, the decrease in the level of phosphotyrosine in IGF-IR may be due to a decrease in the level of IGF-IR protein in vivo following treatment with the antibody or may be due to a simultaneous decrease in the level of IGF-IR protein and tyrosine phosphorylation of IGF-IR, which is present due to blocking of ligand (e.g., IGF-I or IGF-II) activation. Moreover, the inhibition corresponded to the dose of injected antibody (fig. 6). These data indicate that the antibodies of the invention are able to target IGF-IR in vivo in a manner similar to what we observed in vitro.

Example IX: growth Inhibition (TGI) of 3T3/IGF-IR cell tumors

We tested whether the anti-IGF-IR antibodies of the invention act to inhibit growth. Tumors were induced as described above (example VIII) once a defined, palpable tumor (i.e., 250 mm) had formed³Within 6-9 days), 0 is used.Mice were treated by intraperitoneal injection with a single dose of 20ml of antibody. The tumors were sized by measuring two diameters of the tumors with a vernier caliper every three days, according to the method established by Geran et al, "Protocols for screening chemical agents and natural products against tumors and other biological systems,"Cancer Chemother.Rep.3: 1-104, using the formula (length x width]²) The tumor volume was calculated as/2.

When this assay was performed with the antibodies of the invention, it was found that a single treatment with only antibody 2.13.2 inhibited the growth of IGF-IR-transfected NIH-3T3 cell-induced tumors (fig. 7, left). Moreover, in a combined study with a single intravenous infusion of doxorubicin at a single dose of 7.5mg/kg, we observed that administration of a single dose of 2.13.2 enhanced the efficacy of doxorubicin, a known tumor growth inhibitor. The use of doxorubicin in combination with antibody 2.13.2 of the invention demonstrated a delay in growth of 7 days relative to the growth of antibody or doxorubicin alone (figure 7, right).

Example X: relationship of antibody levels to IGF-IR Down-Regulation

Tumors were induced in nude mice as described in example VIII. The mice were then treated by intraperitoneal injection of 125 μ g of 2.13.2 as described in example VIII. Tumors were removed as described in example VIII and IGF-IR levels determined by ELISA. FIG. 8 shows the change over time of serum 2.13.2 antibody levels and IGF-IR receptor levels. This experiment shows that IGF-IR is down-regulated by the antibody and that the extent of IGF-IR inhibition is dose-proportional to the serum concentration of the antibody.

Example XI: inhibition of 3T3/IGF-IR tumor growth by multiple combinations of antibodies with doxorubicin

Nude mice according to the description of example IXThe tumor is induced internally. Treatment of the body with different amounts of 2.13.2 antibody (i.p.) or 7.5mg/kg doxorubicin (i.v.) resulted in about 250mm formation in vivo on days 1, 8, 15 and 22 as described in example IX, either in single dose or in combination³The subcutaneous tumor of (a). Figure 9 shows the tumor size versus time for different treatment methods. This experiment shows that treatment with anti-IGF-IR antibody once every 7 days inhibited tumor cell growth and enhanced tumor cell growth inhibition by the combination with doxorubicin, a known tumor suppressor.

Example XII: inhibition of large tumor growth

Tumors were induced in nude mice as described in example IX. On days 1 and 8, treatment with different amounts of either 2.13.2 antibody (i.p.) or 7.5mg/kg doxorubicin (i.v.) alone or in combination resulted in slightly less than 2000mm in vivo as described in example IX³Subcutaneous large tumor mice. FIG. 10 shows the tumor size versus time for different treatments. The experiments in the control group of animals administered with antibody only and doxorubicin only were terminated on day 5, when the tumor size exceeded 2000mm³. This experiment shows that treatment with doxorubicin, multiple administrations of anti-IGF-IR antibody, has a strong potency against large tumors.

Example XIII: growth inhibition of colorectal cell tumors

Tumors were induced in nude mice as described in example IX, except that Colo205 cells (ATCC CCL 222) were used. Colo205 cells are human colorectal adenocarcinoma cells. Treatment of the body with varying amounts of either 2.13.2 antibody (i.p.) or 100mg/kg of 5-fluorodeoxyuridine (i.v.) resulted in about 250mm formation in vivo, either in single dose or in combination, as described in example IX³The subcutaneous tumor of (a). FIG. 11 shows the tumor size and the time course of the different treatmentsThe relationship (2) of (c). This experiment shows that treatment with anti-IGF-IR antibody once in a single dose inhibits the growth of human colorectal cancer cells and enhances the efficacy of 5-FU, a known tumor suppressor.

On days 1, 8, 15 and 22, Colo205 tumor-forming mice were treated with 500 μ g2.13.2(i.p.), 100mg/kg 5-FU (i.v.), or a combination of both. Figure 12 shows the tumor size versus time for different treatment methods. This experiment shows that treatment with anti-IGF-IR antibody once every 7 days inhibited the growth of human colorectal cancer cells and enhanced the efficacy of 5-FU.

Example XIV: growth inhibition of breast cancer cell tumors

The nude mice were implanted with biodegradable estrogen pellets (0.72mg 17-beta estradiol/pellet, 60 days released; Innovative Research of america) as described in example VIII. After 48 hours, tumors were induced in nude mice essentially as described in example IX, except MCF-7 cells (ATCC HTB-22) were used. MCF-7 cells are human estrogen-dependent breast cancer cells. Substantially as described in example IX, treatment with 50 μ g of the 2.13.2 antibody (i.p.) at days 1, 4, 7, 10, 13, 16, 19 and 22 (q3dx7), or 6.25mg/kg paclitaxel at days 1, 2, 3, 4, 5 (q1dx5) resulted in about 250mm formation in vivo, either in single dose or in combination³The subcutaneous tumor of (a). Figure 13 shows the tumor size versus time for different treatment methods. This experiment shows that the treatment method with anti-IGF-IR antibody administered once every 3 days alone inhibits the growth of human breast cancer cells and enhances the efficacy of paclitaxel when combined with paclitaxel, which is known to be a breast cancer inhibitor.

Mice bearing tumors formed from MCF-7 cells as described above were treated on day 1 with varying amounts of 2.13.2 antibody (i.p.) alone or in combination with 3.75mg/kg of doxorubicin (i.v.) essentially as described in example IX. Figure 14 shows the tumor size versus time for different treatment methods. This experiment shows that treatment with anti-IGF-IR alone inhibited the growth of human breast cancer cells and enhanced the efficacy of doxorubicin, a known tumor suppressor.

On days 1, 8, 15 and 23, mice bearing tumors formed from MCF-7 cells as described above were treated with 250 μ g of 2.13.2 antibody (i.p.) or biodegradable tamoxifen pellets (25 mg/pellet, free base, release for 60 days, Innovative Research of America) in single dose or combination as described in example IX. Tamoxifen pellets were implanted on day 1 after tumor formation. Figure 15 shows the tumor size versus time for different treatment methods. This experiment shows that treatment with anti-IGF-IR alone administered once every 7 days inhibited the growth of human breast cancer cells and enhanced the efficacy of tamoxifen, a known tumor suppressor.

Example XVI: growth inhibition of epidermoid carcinoma cell tumors

Tumors were induced in nude mice essentially as described in example IX, except A431 cells (ATCC CRL 1555) were used. A431 cells are human epidermoid cancer cells that overexpress EGFR. Approximately 250mm was formed in vivo by treatment with 500 μ g2.13.2 antibody (i.p.) in single dose or combination on days 1, 8, 15, 22 and 29 as described in example IX³Or by once daily oral administration of 10mg/kg CP-358,774(p.o.) for 27 days. CP-358,774 is described in U.S. Pat. No. 5,47498 and Moyer et al, Cancer Research 57: 4838-4848(1997), incorporated herein by reference. FIG. 16 shows the tumor size versus time for different treatments. This experiment shows that treatment with anti-IGF-I enhances the efficacy of CP-358,774, a known EGF-R tyrosine kinase inhibitor, for inhibiting the growth of human epidermoid carcinoma tumors.

Example XVII: pharmacokinetics of in vivo anti-IGF-IR antibodies

To evaluate the pharmacokinetics of the anti-IGF-IR antibodies, macaques were injected intravenously with 3, 30 or 100mg/kg of 2.13.2 antibody in acetate buffer. Sera were collected from the monkeys at different time points and the anti-IGF-IR antibody concentration in monkeys was determined for 10 weeks. To quantify the levels of functional serum antibodies, the extracellular domain of human IGF-IR (IGF-I-sR, R & D Systems, Catalog #391GR) was ligated into 96-well plates. Monkey serum (diluted 1: 100 to 1: 15,000) was added to the assay plates so that each sample fell within the linear range of the standard curve, and the plates were incubated under conditions in which anti-IGF-IR antibodies were able to bind IGF-I-sR. After washing the plate, labeled anti-human IgG antibody is added to the plate and incubated under conditions in which anti-human IgG antibody is able to bind to anti-IGF-IR antibody. The plates were then washed and developed and the amount of anti-IGF-IR antibody was quantitatively analyzed using a control standard curve and linear regression fit. FIG. 17 shows the change in serum concentration of 2.13.2 with time. This experiment shows that the half-life of the anti-IGF-IR antibody is 4.6 to 7.7 days and its volume distribution is 74-105 mL/kg. Furthermore, this experiment demonstrated that the amount in monkeys was dose-proportional, indicating that the anti-IGF-IR antibody saturates any possible IGF-IR binding site in vivo even at the lowest dose of 3 mg/kg.

Example XVIII: combination therapy of anti-IGF-IR antibodies and doxorubicin down-regulates IGF-IR in vivo

Tumors were induced in nude mice as described in example IX. A single injection of 250. mu.g of either 2.13.2 antibody (i.p.) or 7.5mg/kg doxorubicin (i.v.) in vivo resulted in approximately 400mm formation in a single dose or combination as described in example IX³The subcutaneous tumor of (a). 72 hours after administration of the agent, tumors were removed as described in example VIII and equal amounts of tumor extracts were subjected to sodium dodecyl phosphate polyacrylamide gel electrophoresis (SDS PAGE) and anti-IGF-IR antibody SC-713(Santa Cruz)Western blot analysis. FIG. 18 shows IGF-IR content in tumor cells in control animals (first three lanes of each gel plate), animals treated with antibody only (top panel), animals treated with doxorubicin only (middle panel), and animals treated with antibody and doxorubicin (bottom panel). Each lane represents an equal amount of protein derived from an individual tumor of a mouse individual. This experiment demonstrates that doxorubicin treatment alone had little effect on IGF-IR levels, whereas antibody treatment alone showed some reduction in IGF-IR levels. Surprisingly, treatment with both doxorubicin and antibody significantly reduced IGF-IR levels, indicating that doxorubicin and antibody significantly down-regulated IGF-IR levels.

All publications and patent publications cited in this specification are herein incorporated by reference as if each individual publication or patent publication 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.

Sequence listing

<110>ABGENIX，INC.

PFIZER，INC.

<120> antibodies against insulin-like growth factor I receptor

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Ile Arg Arg Asp Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

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Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser

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Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn

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Asn Tyr Pro Arg Thr Phe Gly Gln Gly Thr Glu Val Glu Ile Ile Arg

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Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

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Phe Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr

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Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

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Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ser Cys Ala

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35 40 45

Tyr Ala Ala Ser Arg Leu His Arg Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Cys

85 90 95

Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

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Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser

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Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Thr Thr Leu Tyr Leu

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Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

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20 25 30

Gly Lys Ala Pro Lys Arg Leu Ile Tyr Ala Ala Ser Arg Leu Gln Ser

35 40 45

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Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

65 70 75 80

Leu Gln His Asn Asn Tyr Pro Arg Thr Phe Gly Gln Gly Thr Glu Val

85 90 95

Glu Ile Ile Arg

100

<210>11

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<212>DNA

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85 90 95

Ile Ile Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

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<210>13

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<212>DNA

<213> human

<400>13

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20 25 30

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35 40 45

Tyr Ala Ala Ser Lys Leu His Arg Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>15

<211>376

<212>DNA

<213> human

<400>15

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<210>16

<211>125

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Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ile Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Leu Gly Tyr Gly Asp Phe Tyr Tyr Tyr Tyr Tyr Gly Met

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210>17

<211>279

<212>DNA

<213> human

<400>17

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tcagcagtct gcaacctgaa gattttgcaa cttactactg tcaacagagt tacaatgccc 240

cactcacttt cggcggaggg accaaggtgg agatcaaac 279

<210>18

<211>92

<212>PRT

<213> human

<400>18

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Thr

1 5 10 15

Phe Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

20 25 30

Ile His Val Ala Ser Ser Leu Gln Gly Gly Val Pro Ser Arg Phe Ser

35 40 45

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

50 55 60

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Asn Ala Pro

65 70 75 80

Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

85 90

<210>19

<211>341

<212>DNA

<213> human

<400>19

cccaggactg gtgaagcctt cggagaccct gtccctcacc tgcactgtct ctggtggctc 60

catcagtagt tactactgga gttggatccg gcagccccca gggaagggac tggagtggat 120

tgggtatatc tattacagtg ggagcaccaa ctacaacccc tccctcaaga gtcgagtcac 180

catatcagta gacacgtcca agaaccagtt ctccctgaag ctgagttctg tgaccgctgc 240

ggacacggcc gtgtattact gtgccaggac gtatagcagt tcgttctact actacggtat 300

ggacgtctgg ggccaaggga ccacggtcac cgtctcctca g 341

<210>20

<211>113

<212>PRT

<213> human

<400>20

Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val

1 5 10 15

Ser Gly Gly Ser Ile Ser Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro

20 25 30

Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser

35 40 45

Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp

50 55 60

Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala

65 70 75 80

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Tyr Ser Ser Ser Phe Tyr

85 90 95

Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

100 105 110

Ser

<210>21

<211>274

<212>DNA

<213> human

<220>

<221> modified base

<222>(240)

<223> a, c, t, g, other or unknown

<400>21

agagccaccc tctcctgtag ggccagtcag agtgttcgcg gcaggtactt agcctggtac 60

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ggcatcccag acaggttcag tggcagtggg tctgggacag acttcactct caccatcagc 180

agactggagc ctgaagattt tgcagtgttt tactgtcagc agtatggtag ttcacctcgn 240

acgttcggcc aagggaccaa ggtggaaatc aaac 274

<210>22

<211>91

<212>PRT

<213> human

<400>22

Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg Gly Arg Tyr

1 5 10 15

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

20 25 30

Tyr Gly Ala Ser SerArg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly

35 40 45

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro

50 55 60

Glu Asp Phe Ala Val Phe Tyr Cys Gln Gln Tyr Gly Ser Ser Pro Arg

65 70 75 80

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

85 90

<210>23

<211>367

<212>DNA

<213> human

<400>23

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcaggt attactggga gtggtggtag tacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagatcca 300

gggactacgg tgattatgag ttggttcgac ccctggggcc agggaaccct ggtcaccgtc 360

tcctcag 367

<210>24

<211>122

<212>PRT

<213> human

<400>24

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Thr Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Pro Gly Thr Thr Val Ile Met Ser Trp Phe Asp Pro Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>25

<211>320

<212>DNA

<213> human

<400>25

gaactgtggc tgcaccatct gtcttcatct tcccgccatc tgatgagcag ttgaaatctg 60

gaactgcctc tgttgtgtgc ctgctgaata acttctatcc cagagaggcc aaagtacagt 120

ggaaggtgga taacgccctc caatcgggta actcccagga gagtgtcaca gagcaggaca 180

gcaaggacag cacctacagc ctcagcagca ccctgacgct gagcaaagca gactacgaga 240

aacacaaagt ctacgcctgc gaagtcaccc atcagggcct gagctcgccc gtcacaaaga 300

gcttcaacag gggagagtgt 320

<210>26

<211>106

<212>PRT

<213> human

<400>26

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

1 5 10 15

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

20 25 30

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

35 40 45

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

50 55 60

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

65 70 75 80

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

85 90 95

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210>27

<211>978

<212>DNA

<213> human

<400>27

gcctccacca agggcccatc ggtcttcccc ctggcgccct gctccaggag cacctccgag 60

agcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120

tggaactcag gcgctctgac cagcggcgtg cacaccttcc cagctgtcct acagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcaacttcgg cacccagacc 240

tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagac agttgagcgc 300

aaatgttgtg tcgagtgccc accgtgccca gcaccacctg tggcaggacc gtcagtcttc 360

ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacgtgc 420

gtggtggtgg acgtgagcca cgaagacccc gaggtccagt tcaactggta cgtggacggc 480

gtggaggtgc ataatgccaa gacaaagcca cgggaggagc agttcaacag cacgttccgt 540

gtggtcagcg tcctcaccgt tgtgcaccag gactggctga acggcaagga gtacaagtgc 600

aaggtctcca acaaaggcct cccagccccc atcgagaaaa ccatctccaa aaccaaaggg 660

cagccccgag aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 720

caggtcagcc tgacctgcct ggtcaaaggc ttctacccca gcgacatcgc cgtggagtgg 780

gagagcaatg ggcagccgga gaacaactac aagaccacac ctcccatgct ggactccgac 840

ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac 900

gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 960

tccctgtctc cgggtaaa 978

<210>28

<211>326

<212>PRT

<213> human

<400>28

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro Gly Lys

325

<210>29

<211>296

<212>DNA

<213> human

<400>29

caggtgcagc tggtggagtc tgggggaggc ttggtcaagc ctggagggtc cctgagactc 60

tcctgtgcag cctctggatt caccttcagt gactactaca tgagctggat ccgccaggct 120

ccagggaagg ggctggagtg ggtttcatac attagtagta gtggtagtac catatactac 180

gcagactctg tgaagggccg attcaccatc tccagggaca acgccaagaa ctcactgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagaga 296

<210>30

<211>98

<212>PRT

<213> human

<400>30

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>31

<211>296

<212>DNA

<213> human

<400>31

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaaga 296

<210>32

<211>98

<212>PRT

<213> human

<400>32

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys

<210>33

<211>296

<212>DNA

<213> human

<400>33

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggggac cctgtccctc 60

acctgcgctg tctctggtgg ctccatcagc agtagtaact ggtggagttg ggtccgccag 120

cccccaggga aggggctgga gtggattggg gaaatctatc atagtgggag caccaactac 180

aacccgtccc tcaagagtcg agtcaccata tcagtagaca agtccaagaa ccagttctcc 240

ctgaagctga gctctgtgac cgccgcggac acggccgtgt attactgtgc gagaga 296

<210>34

<211>98

<212>PRT

<213> human

<400>34

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gly

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Gly Ser Ile Ser Ser Ser

20 25 30

Asn Trp Trp Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Glu Ile Tyr His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>35

<211>293

<212>DNA

<213> human

<400>35

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagt agttactact ggagctggat ccggcagccc 120

ccagggaagg gactggagtg gattgggtat atctattaca gtgggagcac caactacaac 180

ccctccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240

aagctgagct ctgtgaccgc tgcggacacg gccgtgtatt actgtgcgag aga 293

<210>36

<211>97

<212>PRT

<213> human

<400>36

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg

<210>37

<211>290

<212>DNA

<213> human

<400>37

gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120

cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180

gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240

cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcacctcc 290

<210>38

<211>96

<212>PRT

<213> human

<400>38

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

<210>39

<211>288

<212>DNA

<213> human

<220>

<221> modified base

<222>(288)

<223> a, c, t, g, other or unknown

<400>39

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 120

gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtctacag cataatagtt accctccn 288

<210>40

<211>96

<212>PRT

<213> human

<400>40

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Pro

85 90 95

<210>41

<211>288

<212>DNA

<213> human

<400>41

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta cccctcch 288

<210>42

<211>96

<212>PRT

<213> human

<400>42

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro

85 90 95

<210>43

<211>293

<212>DNA

<213> human

<400>43

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagt agttactact ggagctggat ccggcagccc 120

gccgggaagg gactggagtg gattgggcgt atctatacca gtgggagcac caactacaac 180

ccctccctca agagtcgagt caccatgtca gtagacacgt ccaagaacca gttctccctg 240

aagctgagct ctgtgaccgc cgcggacacg gccgtgtatt actgtgcgag aga 293

<210>44

<211>97

<212>PRT

<213> human

<400>44

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Ala Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Tyr Thr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg

<210>45

<211>470

<212>PRT

<213> human

<400>45

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe

35 40 45

Ser Ser Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Thr Thr Phe Tyr Ala

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Thr

85 90 95

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Lys Asp Leu Gly Trp Ser Asp Ser Tyr Tyr Tyr Tyr

115 120 125

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

130 135 140

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

145 150 155 160

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

165 170 175

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

180 185 190

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

195 200 205

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

210 215 220

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

225 230 235 240

Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

245 250 255

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

260 265 270

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

275 280 285

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

290 295 300

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

305 310 315 320

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

325 330 335

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

340 345 350

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

355 360 365

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

370 375 380

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

385 390 395 400

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

405 410 415

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

420 425 430

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

435 440 445

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

450 455 460

Ser Leu Ser Pro Gly Lys

465 470

<210>46

<211>470

<212>PRT

<213> human

<400>46

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

85 90 95

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Lys Gly Tyr Ser Ser Gly Trp Tyr Tyr Tyr Tyr Tyr

115 120 125

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

130 135 140

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

145 150 155 160

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

165 170 175

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

180 185 190

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

195 200 205

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

210 215 220

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

225 230 235 240

Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

245 250 255

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

260 265 270

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

275 280 285

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

290 295 300

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

305 310 315 320

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

325 330 335

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

340 345 350

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

355 360 365

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

370 375 380

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

385 390 395 400

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

405 410 415

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

420 425 430

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

435 440 445

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

450 455 460

Ser Leu Ser Pro Gly Lys

465 470

<210>47

<211>236

<212>PRT

<213> human

<400>47

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Phe Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

35 40 45

Gln Gly Ile Arg Asn Asp Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Arg Leu Ile Tyr Ala Ala Ser Arg Leu His Arg Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln

100 105 110

His Asn Ser Tyr Pro Cys Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210>48

<211>236

<212>PRT

<213> human

<400>48

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

35 40 45

Gln Gly Ile Arg Asn Asp Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Arg Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln

100 105 110

His Asn Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210>49

<211>470

<212>PRT

<213> human

<400>49

Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Ile Lys Gly

1 5 10 15

Val Gln Cys Gln Ala Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Asp Tyr Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ser Tyr Ile Ser Ser Ser Gly Ser Thr Arg Asp Tyr Ala

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn

85 90 95

Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Val Arg Asp Gly Val Glu Thr Thr Phe Tyr Tyr Tyr Tyr

115 120 125

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

130 135 140

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

145 150 155 160

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

165 170 175

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

180 185 190

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

195 200 205

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

210 215 220

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

225 230 235 240

Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

245 250 255

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

260 265 270

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

275 280 285

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

290 295 300

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

305 310 315 320

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

325 330 335

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

340 345 350

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

355 360 365

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

370 375 380

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

385 390 395 400

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

405 410 415

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

420 425 430

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

435 440 445

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

450 455 460

Ser Leu Ser Pro Gly Lys

465 470

<210>50

<211>473

<212>PRT

<213> human

<400>50

Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Ile Lys Gly

1 5 10 15

Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Asp Tyr Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn

85 90 95

Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Val Leu Arg Phe Leu Glu Trp Leu Leu Tyr Tyr

115 120 125

Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr

130 135 140

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

145 150 155 160

Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val

165 170 175

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

180 185 190

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

195 200 205

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly

210 215 220

Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys

225 230 235 240

Val Asp Lys Thr Val GluArg Lys Cys Cys Val Glu Cys Pro Pro Cys

245 250 255

Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

260 265 270

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

275 280 285

Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

290 295 300

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

305 310 315 320

Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His

325 330 335

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

340 345 350

Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln

355 360 365

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

370 375 380

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

385 390 395 400

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

405 410 415

Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu

420 425 430

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

435 440 445

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

450 455 460

Lys Ser Leu Ser Leu Ser Pro Gly Lys

465 470

<210>51

<211>236

<212>PRT

<213> human

<400>51

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Phe Thr Cys Arg Ala Ser

35 40 45

Gln Asp Ile Arg Arg Asp Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Arg Leu Ile Tyr Ala Ala Ser Arg Leu Gln Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln

100 l05 110

His Asn Asn Tyr Pro Arg Thr Phe Gly Gln Gly Thr Glu Val Glu Ile

115 120 125

Ile Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210>52

<211>236

<212>PRT

<213> human

<400>52

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

35 40 45

Gln Gly Ile Arg Asn Asp Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Arg Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln

100 105 110

His Asn Ser Tyr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210>53

<211>326

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: consensus sequences

<220>

<221> modified base

<222>(289)

<223> a, c, t, g, other or unknown

<400>53

gacatccaga tgacccagty tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

wtcacttgcc gggcaagtca ggrcattaga mrtgatttag gctggtwtca gcagaaacca 120

gggaaagcyc ctaagcgcct gatctatgct gcatccmrwt trcammgwgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcmg cctgcagcct 240

gaagattttg caacttatta ctgtytacar cataatartt aycckybsns kttyggcsrr 300

gggaccrags tggaratcaw acgaac 326

<210>54

<211>322

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: consensus sequences

<400>54

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgyaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagy asctwtttaa attggtatca gcagaaacca 120

gggaaagccc ctaarctcct gatcyatgyt gcatccagtt trcaargtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacartr ccccayychc tttcggcgga 300

gggaccaagg tggagatcaa ac 322

<210>55

<211>325

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: consensus sequences

<220>

<221> modified base

<222>(291)

<223> a, c, t, g, other or unknown

<400>55

gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgya gggccagtca gagtgttmgc rgcagstact tagcctggta ccagcagaaa 120

cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180

gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240

cctgaagatt ttgcagtgtw ttactgtcag cagtatggta gytcacctcs nacgttcggc 300

caagggacca aggtggaaat caaac 325

<210>56

<211>376

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: consensus sequences

<400>56

caggtgcagc tggtggagtc tgggggaggc ttggtcaagc ctggagggtc cctgagactc 60

tcctgtgcag cctctggatt cacyttcagt gactactaya tgagctggat ccgccaggct 120

ccagggaagg ggctggartg ggtttcatac attagtagta gtggtagtac cakakactac 180

gcagactctg tgaagggccc attcaccatc tccagggaca acgccaagaa ctcactgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgy gagagatgga 300

gtggaaacta ctttttacta ctactactac ggtatggacg tctggggcca agggaccacg 360

gtcaccgtct cctcag 376

<210>57

<211>358

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: consensus sequences

<220>

<221> modified base

<222>(337)

<223> a, c, t, g, other or unknown

<400>57

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagt arttactact ggagctggat ccggcagccc 120

gccgggaagg gactggagtg gattgggcgt atctatacca gtgggagcmc caactacaac 180

ccctccctca agagtcgagt caccatgtca gtagacacgt ccaagaacca gttctccctg 240

aagctgarct ctgtgaccgc cgcggacacg gccgtgtatt actgtgcggt aacgattttt 300

ggagtggtta ttatctttga ctactggggc cagrganccc tggtcaccgt ctcctcag 358

<210>58

<211>418

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: consensus sequences

<400>58

caggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtrcag cctctggatt cacctttagc agctatgcca tgarctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagst attastggka gtggtggtab yacatwctac 180

gcagactccg tgaagggccc gttcaccatc tccagagaca attccargam cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagatctk 300

ggctrsksyg actyttacta ctactactac ggtatggacg tctggggcca agggacyacg 360

gtgattatga gttggttcga cccctggggc cagggaaccc tggtcaccgt ctcctcag 418

<210>59

<211>364

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: consensus sequences

<400>59

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagt agttactact ggagytggat ccggcagccc 120

ccagggaagg gactggagtg gattgggtat atctattaca gtgggagcac caactacaac 180

ccctccctca agagtcgact caccatatca gtagacacgt ccaagaacca gttctccctg 240

aagctgagyt ctgtgaccgc tgcggacacg gccgtgtatt actgtgccag gacgtatagc 300

agttcgttct actactacgg tatggacgtc tggggccaag ggaccacggt caccgtctcc 360

tcag 364

<210>60

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: Gly-Ser linker

<400>60

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

Claims (6)

1. A human, humanized or chimeric monoclonal antibody or 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, wherein the variable region of the heavy chain and the variable region of the light chain are the heavy chain variable region and the light chain variable region, respectively, of monoclonal antibody 4.9.2 produced by the hybridoma of ATCC deposit No. PTA-2789.

2. The antibody or antigen-binding portion of claim 1, wherein the antibody or portion has at least one property selected from the group consisting of:

i) (ii) IGF-IR that does not bind guinea pigs, dogs, or rabbits;

ii) IGF-IR binding to rhesus monkey or rhesus monkey but not to marmoset;

iii) inhibits binding of IGF-I to IGF-IR;

iv) inhibiting tumor growth in vivo;

v) causing the disappearance of IGF-IR from the cell surface when incubated with cells expressing IGF-IR;

vi) inhibits IGF-IR-induced tyrosine phosphorylation; and

vii) at 8x10^-9K of M or less_dBinding to IGF-IR.

3. The antibody or antigen-binding portion of claim 2, wherein the antibody or portion thereof has all of the properties described.

4. The antibody or antigen-binding portion of any one of claims 1-3, wherein the antibody or portion has an IC of less than 100nM₅₀Inhibiting the binding between IGF-IR and IGF-I.

5. A human, humanized or chimeric monoclonal antibody or 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, wherein the variable region of the heavy chain and the variable region of the light chain are the heavy chain variable region and the light chain variable region, respectively, of monoclonal antibody 4.9.2 produced by the hybridoma of ATCC deposit No. PTA-2789;

wherein the antibody or portion has at least one property selected from the group consisting of:

i) cross-competes with antibody 4.9.2 for binding to IGF-IR;

ii) binds to the same epitope of IGF-IR as antibody 4.9.2;

iii) binds to the same antigen as that bound by antibody 4.9.2;

iv) with a K substantially identical to that of antibody 4.9.2_dBinding to IGF-IR; and

v) binds to IGF-IR with substantially the same off-rate as antibody 4.9.2.

6. The antibody or antigen-binding portion of claim 5, wherein the antibody or portion has all of the properties described.