Neutralizing human anti-IGFR antibody

FIELD OF THE INVENTION

[0002] The present invention relates to fully human, monoclonal anti-Insulin-like Growth Factor Receptor-I (IGFR1) antibodies as well as methods of using the antibodies and methods of producing the antibodies.

BACKGROUND OF THE INVENTION

[0003] The insulin-like growth factors, also known as somatomedins, include insulin-like growth factor-I (IGF-I) and insulin-like growth factor-II (IGF-II) (Klapper, et al., (1983) Endocrinol. 112:2215 and Rinderknecht, et al., (1978) Febs.Lett. 89:283). These growth factors exert mitogenic activity on various cell types, including tumor cells (Macaulay, (1992) Br. J. Cancer 65:311), by binding to a common receptor named the insulin-like growth factor receptor-1 (IGFR1) (Sepp-Lorenzino, (1998) Breast Cancer Research and Treatment 47:235). Interaction of IGFs with IGFR1 activates the receptor by triggering autophosphorylation of the receptor on tyrosine residues (Butler, et al., (1998) Comparative Biochemistry and Physiology 121:19). Once activated, IGFR1, in turn, phosphorylates intracellular targets to activate cellular signaling pathways. This receptor activation is critical for stimulation of tumor cell growth and survival. Therefore, inhibition of IGFR1 activity represents a valuable potential method to treat or prevent growth of human cancers and other proliferative diseases.

[0004] Several lines of evidence indicate that IGF-I, IGF-II and their receptor IGFR1 are important mediators of the malignant phenotype. Plasma levels of IGF-I have been found to be the strongest predictor of prostate cancer risk (Chan, et al., (1998) Science 279:563) and similar epidemiological studies strongly link plasma IGF-I levels with breast, colon and lung cancer risk.

[0005] Overexpression of Insulin-like Growth Factor Receptor-I has also been demonstrated in several cancer cell lines and tumor tissues. IGFR1 is overexpressed in 40% of all breast cancer cell lines (Pandini, et al., (1999) Cancer Res. 5:1935) and in 15% of lung cancer cell lines. In breast cancer tumor tissue, IGFR1 is overexpressed 6-14 fold and IGFR1 exhibits 2-4 fold higher kinase activity as compared to normal tissue (Webster, et al., (1996) Cancer Res. 56:2781 and Pekonen, et al., (1998) Cancer Res. 48:1343). Ninety percent of colorectal cancer tissue biopsies exhibit elevated IGFR1 levels wherein the extent of IGFR1 expression is correlated with the severity of the disease. Analysis of primary cervical cancer cell cultures and cervical cancer cell lines revealed 3-and 5-fold overexpression of IGFR1, respectively, as compared to normal ectocervical cells (Steller, et al., (1996) Cancer Res. 56:1762). Expression of IGFR1 in synovial sarcoma cells also correlated with an aggressive phenotype (i.e., metastasis and high rate of proliferation; Xie, et al., (1999) Cancer Res. 59:3588).

[0006] Acromegaly, a slowly developing disease, is caused by hypersecretion of growth hormone and IGF-I (Ben-Schlomo, et al., (2001) Endocrin. Metab.Clin. North. Am. 30:565-583). Antagonism of IGFR1 function may be helpful in treating the disease.

[0007] There are several antibodies, which are known in the art, which inhibit the activity of IGFR1. However, these are of relatively low therapeutic value. For example, α-IR3 (Kull, et al., (1983) J. Biol. Chem. 258:6561), 1H7. (Li et al., (1993) Biochem. Biophys. Res. Comm. 196.92-98 and Xiong et al., (1992) Proc. Natl. Acad. Sci., U.S.A. 89:5356-5360; Santa Cruz biotechnology, Inc.; Santa Cruz, Calif.) and MAB391 (R&D Systems; Minneapolis, Minn.) are mouse monoclonal antibodies which interact with IGFR1 and inhibit its activity. Since these are mouse antibodies, their therapeutic utility in humans is limited. When immunocompetent human subjects are administered a dose of mouse antibodies, the subjects produce antibodies against the mouse immunoglobulin sequences. These human anti-mouse antibodies (HAMA) neutralize the therapeutic antibodies and may induce acute toxicity (i.e., a HAMA response).

[0008] One method by which to avert a HAMA response is through the use of fully-human antibodies which lack any foreign (e.g., mouse) amino acid sequences. Although the use of fully-human antibodies is an effective method by which to reduce or prevent human host immune rejection of the therapeutic antibody, rejection of the fully-human antibody can occur. Human rejection of human antibodies may be referred to as a human anti-human antibody response (HAHA response). HAHA response can be mediated by factors such as the presence of rare, low occurrence amino acid sequences in the fully-human antibodies. For this reason, therapeutic antibodies may also be optimized by the inclusion of non-immunogenic or only weakly immunogenic human antibody framework sequences. Preferably, the sequences occur frequently in other human antibodies.

SUMMARY OF THE INVENTION

[0009] The present invention provides fully human anti-human IGFR1 monoclonal antibodies which, preferably, will not induce a HAMA response or will not induce a HAHA response when administered to human subjects and which are useful for treating or preventing diseases which are mediated by IGFR1 (e.g., malignancy).

[0010] The present invention provides a binding composition (e.g., an antibody or antigen-binding fragment thereof) comprising a light chain, wherein the chain comprises the amino acid sequence of the light chain CDR-L1 defined by SEQ ID NO: 8 or 31, the amino acid sequence of the light chain CDR-L2 defined by SEQ ID NO: 9 or 32 and the amino acid sequence of the light chain CDR-L3 defined by SEQ ID NO: 10 or 33. Also provided is a binding composition (e.g., an antibody or antigen-binding fragment thereof) including a heavy chain, wherein the chain includes the amino acid sequence of the heavy chain CDR-H1 defined by SEQ ID NO: 14 or 37, the amino acid sequence of the heavy chain CDR-H2 defined by SEQ ID NO: 15 or 38 and the amino acid sequence of the heavy chain CDR-H3 defined by SEQ ID NO: 16 or 39.

[0011] Preferably, the binding composition (e.g., an antibody or antigen-binding fragment thereof) of the invention comprises a light chain variable region, preferably a mature light chain variable region, which includes amino acids 20-128 of SEQ ID NO: 2, amino acids 21-130 of SEQ ID NO: 25, amino acids 20-128 of SEQ ID NO: 41 or 43 or amino acids 20-128 of SEQ ID NO: 41, 43, 72, 74, 76 or 78 and/or a heavy chain variable region, preferably a mature heavy chain variable region, which includes amino acids 20-137 of SEQ ID NO: 4, amino acids 20-140 of SEQ ID NO: 27, amino acids 20-137 of SEQ ID NO: 45 or amino acids 20-137 of SEQ ID NO: 112.

[0012] Pharmaceutical compositions comprising a binding composition of the present invention and a pharmaceutically acceptable carrier are also provided by the present invention. The binding composition of the invention may also be conjugated to a substance such as polyethylene glycol.

[0013] The present invention also includes a binding composition (e.g., a human antibody or antigen binding fragment thereof) which specifically binds to human IGFR1 comprising a property selected from the group consisting of:

[0014] (a) binds to IGFR1 (e.g., human IGFR1) with a Kd of about 86×10−11 or less;

[0015] (b) Has an off rate (Koff) for IGFR1 (e.g., human IGFR1) of about 6.50×10−5 or smaller;

[0016] (c) Has an on rate (Kon) for IGFR1 (e.g., human IGFR1) of about 0.7×105 or greater;

[0017] (d) Competes with IGF1 for binding to IGFR1 (e.g., human IGFR1);

[0018] (e) Inhibits autophosphorylation (e.g., with an IC50 of 0.10 nM) of IGFR1 (e.g., human IGFR1); and

[0019] (f) Inhibits anchorage-independent growth of a cell expressing IGFR1 (e.g., human IGFR1).

[0020] Preferably, the binding composition comprises all of said properties (a-f). More preferably, the binding composition (e.g., a human antibody or antigen binding fragment thereof) comprises a member selected from:

[0021] (a) a light chain amino acid sequence which comprises CDR-L1 defined by SEQ ID NO: 8, CDR-L2 defined by SEQ ID NO: 9 and CDR-L3 defined by SEQ ID NO: 10;

[0022] (b) a light chain amino acid sequence which comprises CDR-L1 defined by SEQ ID NO: 31, CDR-L2 defined by SEQ ID NO: 32 and CDR-L3 defined by SEQ ID NO: 33;

[0023] (c) a heavy chain amino acid sequence which comprises CDR-H1 defined by SEQ ID NO: 14 or SEQ ID NO: 17, CDR-H2 defined by SEQ ID NO: 15 and CDR-H3 defined by SEQ ID NO: 16; and

[0024] (d) a heavy chain amino acid sequence which comprises CDR-H1 defined by SEQ ID NO: 37 or SEQ ID NO: 70, CDR-H2 defined by SEQ ID NO: 38 and CDR-H3 defined by SEQ ID NO: 39.

[0025] The present invention also includes an isolated nucleic acid encoding a peptide selected from:

[0026] (a) amino acids 20-128 of SEQ ID NO: 2;

[0027] (b) amino acids 21-130 of SEQ ID NO: 25;

[0028] (c) amino acids 20-128 of SEQ ID NO: 72;

[0029] (d) amino acids 20-128 of SEQ ID NO: 74;

[0030] (a) amino acids 20-137 of SEQ ID NO: 4;

[0031] (b) amino acids 20-140 of SEQ ID NO: 27;

[0032] (c) amino acids 20-137 of SEQ ID NO: 45;

[0033] (d) amino acids 20-137 of SEQ ID NO: 112;

[0034] (e) amino acids 20-128 of SEQ ID NO: 76; and

[0035] (f) amino acids 20-128 of SEQ ID NO: 78.

[0036] Preferably, the nucleic acid is selected from:

[0037] (a) nucleotides 58-384 of SEQ ID NO: 1;

[0038] (b) nucleotides 61-390 of SEQ ID NO: 24;

[0039] (c) nucleotides 58-384 of SEQ ID NO: 71;

[0040] (d) nucleotides 58-384 of SEQ ID NO: 73.

[0041] (e) nucleotides 58-411 of SEQ ID NO: 3;

[0042] (f) nucleotides 58-420 of SEQ ID NO: 26;

[0043] (g) nucleotides 58-411 of SEQ ID NO: 44;

[0044] (h) nucleotides 58-411 of SEQ ID NO: 111;

[0045] (i) nucleotides 58-384 of SEQ ID NO: 75; and

[0046] (j) nucleotides 58-384 of SEQ ID NO: 77.

[0047] The present invention also provides a recombinant vector comprising any of the foregoing polynucleotides along with a host cell comprising the vector.

[0048] The present invention also comprises a polypeptide selected from:

[0049] (a) amino acids 20-128 of SEQ ID NO: 2;

[0050] (b) amino acids 21-130 of SEQ ID NO: 25;

[0051] (c) amino acids 20-128 of SEQ ID NO: 72;

[0052] (d) amino acids 20-128 of SEQ ID NO: 74;

[0053] (e) amino acids 20-137 of SEQ ID NO: 4;

[0054] (f) amino acids 20-140 of SEQ ID NO: 27;

[0055] (g) amino acids 20-137 of SEQ ID NO: 45;

[0056] (h) amino acids 20-137 of SEQ ID NO: 112;

[0057] (i) amino acids 20-128 of SEQ ID NO: 76; and

[0058] (j) amino acids 20-128 of SEQ ID NO: 78.

[0059] Preferably, the binding composition of the present invention is a human antibody comprising at least one (e.g., 1 or 2) light chain/heavy chain combination selected from:

[0060] a) a light chain variable region comprising amino acids 20-128 of SEQ ID NO: 2 and a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 4; [15H12/19D12 mature LC-15H12/19D12 mature HC]

[0061] b) a light chain variable region comprising amino acids 21-130 of SEQ ID NO: 25 and a heavy chain variable region comprising amino acids 20-140 of SEQ ID NO: 27; [1H3 mature LC-1H3 mature HC]

[0062] c) a light variable region comprising amino acids 20-128 of SEQ ID NO: 72 and a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 45; [mature LCC-mature HCA]

[0063] d) a light variable region comprising amino acids 20-128 of SEQ ID NO: 74 and a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 45; [mature LCD-mature HCA]

[0064] e) a light variable region comprising amino acids 20-128 of SEQ ID NO: 76 and a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 45; [mature LCE-mature HCA]

[0065] f) a light variable region comprising amino acids 20-128 of SEQ ID NO: 78 and a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 45; [mature LCF-mature HCA]

[0066] g) a light variable region comprising amino acids 20-128 of SEQ ID NO: 72 and a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 112; [mature LCC-mature HCB]

[0067] h) a light variable region comprising amino acids 20-128 of SEQ ID NO: 74 and a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 112; [mature LCD-mature HCB]

[0068] i) a light variable region comprising amino acids 20-128 of SEQ ID NO: 76 and a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 112; [mature LCE-mature HCB] and

[0069] j) a light variable region comprising amino acids 20-128 of SEQ ID NO: 78 and a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 112. [mature LCF-mature HCB].

[0070] More preferably, the human antibody is a tetramer comprising two of the foregoing light/heavy chain pairs. Preferably, the human antibody includes mature LCF paired with mature HCA or mature HCB.

[0071] Also provided is a method for making a polypeptide comprising amino acids 20-128 of SEQ ID NO: 2, amino acids 20-137 of SEQ ID NO: 4, amino acids 21-130 of SEQ ID NO: 25, amino acids 20-140 of SEQ ID NO: 27, amino acids 20-128 of SEQ ID NO: 41, 43, 72, 74, 76 or 78, amino acids 20-137 of SEQ ID NO: 45 or amino acids 20-137 of SEQ ID NO: 112 comprising culturing the host cell under conditions in which the polypeptide is produced. Preferably, the polypeptide is also isolated from the host cell.

[0072] The invention also provides a method for treating or preventing a medical condition in a subject which is mediated by elevated expression or activity of Insulin-like Growth Factor Receptor-I or by elevated expression of one or more of its ligands (e.g., IGF-I or IGF-II) comprising administering a binding composition of the invention (e.g., antibody or antigen-binding fragment of the invention) to the subject.

[0073] Preferably, the binding composition comprises a member selected from:

[0074] (a) a light chain amino acid sequence which comprises CDR-L1 defined by SEQ ID NO: 8, CDR-L2 defined by SEQ ID NO: 9 and CDR-L3 defined by SEQ ID NO: 10;

[0075] (b) a light chain amino acid sequence which comprises CDR-L1 defined by SEQ ID NO: 31, CDR-L2 defined by SEQ ID NO: 32 and CDR-L3 defined by SEQ ID NO: 33;

[0076] (c) a heavy chain amino acid sequence which comprises CDR-H1 defined by SEQ ID NO: 14 or SEQ ID NO: 17, CDR-H2 defined by SEQ ID NO: 15 and CDR-H3 defined by SEQ ID NO: 16; and

[0077] (d) a heavy chain amino acid sequence which comprises CDR-H1 defined by SEQ ID NO: 37 or SEQ ID NO: 70, CDR-H2 defined by SEQ ID NO: 38 and CDR-H3 defined by SEQ ID NO: 39.

[0078] The present invention includes any plasmid selected from the group consisting of:

[0079] (i) CMV promoter-15H12/19D12 HCA (γ4)—

[0080] Deposit name: “15H12/19D12 HCA (γ4)”;

[0081] ATCC accession No.______;

[0082] (ii) CMV promoter-15H12/19D12 HCB (γ4)—

[0083] Deposit name: “15H12/19D12 HCB (γ4)”;

[0084] ATCC accession No.______;

[0085] (iii) CMV promoter-15H12/19D12 HCA (γ1)—

[0086] Deposit name: “15H12/19D12 HCA (γ1)”;

[0087] ATCC accession No.______;

[0088] (iv) CMV promoter-15H12/19D12 LCC (κ)—

[0089] Deposit name: “15H12/19D12 LCC (κ)”;

[0090] ATCC accession No.______;

[0091] (v) CMV promoter-15H12/19D12 LCD (κ)—

[0092] Deposit name: “15H12/19D12 LCD (κ)”;

[0093] ATCC accession No.______;

[0094] (vi) CMV promoter-15H12/19D12 LCE (κ)—

[0095] Deposit name: “15H12/19D12 LCE (κ)”;

[0096] ATCC accession No.______; and

[0097] (vii) CMV promoter-15H12/19D12 LCF (κ)—

[0098] Deposit name: “15H12/19D12 LCF (κ)”;

[0099] ATCC accession No.______;

[0100] as well as the nucleic acid inserts of any of the foregoing plasmids. Also included are the nucleic acid portions of the inserts encoding the immunoglobulin variable regions included in the plasmid inserts optionally including the immunoglobulin constant region (i.e., excluding the signal sequence). Also included are any polypeptides encoded by the nucleic acids of any of the foregoing plasmid inserts as well as polypeptides encoding the immunoglobulin variable regions included in any insert optionally including the immunoglobulin constant region (i.e., excluding the signal sequence).

[0101] The above-identified plasmids were deposited, under the Budapest Treaty, on ______ with the American Type Culture Collection (ATCC); 10801 University Boulevard; Manassas, Va. 20110-2209. All restrictions on access to the plasmids deposited in ATCC will be removed upon grant of a patent.

[0102] Preferably, the binding composition is combined with a pharmaceutically acceptable carrier in a pharmaceutical composition. Such medical conditions, as contemplated by the present invention, include acromegaly, ovarian cancer, pancreatic cancer, benign prostatic hyperplasia, breast cancer, prostate cancer, bone cancer, lung cancer, colorectal cancer, cervical cancer, synovial sarcoma, diarrhea associated with metastatic carcinoid, vasoactive intestinal peptide secreting tumors, gigantism, psoriasis, atherosclerosis, smooth muscle restenosis of blood vessels and inappropriate microvascular proliferation.

[0103] The binding compositions may be administered to a subject, for example, by a parenteral route. Combination therapies comprising administration of a binding composition of the present invention in association with an anti-cancer therapy agent or in association with an anti-cancer therapeutic procedure are also provided.

[0104] A method for producing a fully-human anti-IGFR1 antibody which comprises the steps of immunizing a transgenic non-human animal having a genome comprising a human heavy chain transgene and a human light chain transgene with IGFR1 antigenic polypeptide, preferably amino acids 30-902 of SEQ ID NO: 19 and/or a cell (e.g., HEK293) which expresses IGFR1 on its surface, such that antibodies are produced by B cells of the animal; isolating B. cells of the animal; fusing the B cells with myeloma cells to form immortal, hybridoma cells that secrete human monoclonal antibodies specific for IGFR1; and isolating the human monoclonal antibodies specific for IGFR1 is also provided.

DETAILED DESCRIPTION

[0105] Preferred embodiments of the present invention include a fully human, monoclonal antibody or antigen-binding fragment thereof which specifically recognizes and binds to Insulin-like Growth Factor Receptor-I, preferably amino acids 30-902 of SEQ ID NO: 19. Preferably, the antibody or antigen-binding fragment thereof is 1H3, 15H12, 19D12, 15H12/19D12 LCA, 15H12/19D12 LCB, 15H12/19D12 LCC, 15H12/19D12 LCD, 15H12/19D12 LCE, 15H12/19D12 LCF, 15H12/19D12 HCA or 15H12/19D12 HCB.

[0106] A binding composition or agent refers to a molecule that binds with specificity to IGFR1, e.g., in a ligand-receptor type fashion or an antibody-antigen interaction, e.g., proteins which specifically associate with IGFR1, e.g., in a natural physiologically relevant protein-protein interaction, either covalent or non-covalent. The term “binding composition” is preferably a polypeptide, such as a full antibody or antigen-binding fragment thereof of the present invention (e.g., 15H12/19D12 LCA, 15H12/19D12 LCB, 15H12/19D12 LCC, 15H12/19D12 LCD, 15H12/19D12 LCE, 15H12/19D12 LCF, 15H12/19D12 HCA OR 15H12/19D12 HCB or any peptide set forth, below, in Table 1).

[0107] The antibodies and antigen-binding fragments of the invention may be used to inhibit growth of cells, preferably malignant cells, both in vitro and in vivo. Without being bound by a single theory, the antibodies and antigen-binding fragments of the invention may inhibit cellular growth by inhibiting the interaction between IGFR1 and a ligand for the receptor, such as Insulin-like Growth Factor-I (IGF-I) or Insulin-like Growth Factor-II (IGF-II). The antibodies and antigen-binding fragments may also inhibit IGFR1 autophosphorylation, inhibit anchorage-independent growth of cells (e.g., cancer cells) expressing IGFR1 and inhibit activation of AKT kinase by inducing degradation of IGFR1. Preferably, the antibodies and antigen-binding fragments neutralize the activity of IGFR1 and/or down-regulate IGFR1. The antibodies and antigen-binding fragments may be used to treat or prevent diseases which are mediated by IGFR1. The present invention also provides methods for making the antibodies and antigen-binding fragments of the invention.

[0108] The term “antibody molecule” refers to whole antibodies (e.g., IgG, preferably, IgG1 or IgG4) and fragments, preferably antigen-binding fragments, thereof. Antibody fragments include Fab antibody fragments, F(ab)2 antibody fragments, Fv antibody fragments, single chain Fv antibody fragments and dsFv antibody fragments.

[0109] The terms “IGFR1” “Insulin-like Growth Factor Receptor-I” and “Insulin-like Growth Factor Receptor, type I” are well known in the art. Although IGFR1 may be from any organism, it is preferably from an animal, more preferably from a mammal (e.g., mouse, rat, rabbit, sheep or dog) and most preferably from a human. The nucleotide and amino acid sequence of a typical human IGFR1 precursor has the Genbank Accession No. X04434 or NM—000875 (SEQ ID NO: 19). Cleavage of the precursor (e.g., between amino acids 710 and 711) produces an α-subunit and a β-subunit which associate to form a mature receptor. In preferred embodiments of the invention, amino acids 30-902, from the full length IGFR1 polypeptide are used as an antigen for generation of anti-IGFR1 antibodies.

[0110] The terms “IGF-I” “Insulin-like Growth Factor-I” and “Insulin-like Growth Factor, type I” are also well known in the art. The terms “IGF-II” “Insulin-like Growth Factor-II” and “Insulin-like Growth Factor, type 11” are also well known in the art. Although IGF-I or IGF-II may be from any organism, they are preferably from an animal, more preferably from a mammal (e.g., mouse, rat, rabbit, sheep or dog) and most preferably from a human. The nucleic acid and amino acid sequence of typical, human IGF-I and IGF-II have the Genbank Accession No. XM—052648 (SEQ ID NO: 20) and NM—000612 (SEQ ID NO: 21), respectively. The term “sIGFR1” or “soluble IGFR1” includes any soluble fragment of IGFR1 (e.g., human IGFR1), preferably a fragment from which the receptor trans-membrane region has been deleted, more preferably amino acids 30-902 of SEQ ID NO: 19.

[0111] The amino acid sequence of the variable region of preferred, fully human, monoclonal anti-IGFR1 antibody molecules of the invention (e.g., 1H3, 15H12 and 19D12) along with the nucleotide sequences of nucleic acids which encode the regions are summarized in Table 1. The present invention includes any nucleic acid or polypeptide (e.g., antibody) which comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) of any of the nucleic acids or polypeptides (including mature fragments thereof) set forth, below, in Table 1. Table 1 also includes a summary of the amino acid and nucleotide sequences which correspond to the CDR regions of the antibodies. The amino acid and nucleotide sequences corresponding to the variable region of 15H12 and 19D12 are identical; for this reason, only a single sequence for each variable region or CDR is shown.

1TABLE 1Summary of amino acid and nucleotide sequences of the invention.SEQUENCESEQUENCEIDENTIFIERNucleotide sequence encodingSEQ ID NO: 1the 15H12 and 19D12 light chainvariable region-including signalpeptide (15H12/19D12 LC)Amino acid sequence ofSEQ ID NO: 2the 15H12 and 19D12 light chainvariable region-including signalpeptideNucleotide sequence encodingSEQ ID NO: 3the 15H12 and 19D12 heavychain variable region includingsignal peptide (15H12/19D12 HC)Amino acid sequence of theSEQ ID NO: 415H12 and 19D12 heavy chainvariable region including signalpeptideNucleotide sequence encodingSEQ ID NO: 5the 15H12 and 19D12 CDR-L1Nucleotide sequence encodingSEQ ID NO: 6the 15H12 and 19D12 CDR-L2Nucleotide sequence encodingSEQ ID NO: 7the 15H12 and 19D12 CDR-L3Amino acid sequence of theSEQ ID NO: 815H12 and 19D12 CDR-L1Amino acid sequence of theSEQ ID NO: 915H12 and 19D12 CDR-L2Amino acid sequence of theSEQ ID NO: 1015H12 and 19D12 CDR-L3Nucleotide sequence encodingSEQ ID NO: 11the 15H12 and 19D12 CDR-H1Nucleotide sequence encodingSEQ ID NO: 12the 15H12 and 19D12 CDR-H2Nucleotide sequence encodingSEQ ID NO: 13the 15H12 and 19D12 CDR-H3Amino acid sequence of theSEQ ID NO: 1415H12 and 19D12 CDR-H1Amino acid sequence of theSEQ ID NO: 1515H12 and 19D12 CDR-H2Amino acid sequence of theSEQ ID NO: 1615H12 and 19D12 CDR-H3Amino acid sequence of anSEQ ID NO: 17alternative 15H12 and 19D12CDR-H1Nucleotide sequence encodingSEQ ID NO: 18an alternative 15H12 and 19D12CDR-H1Amino acid sequence of Insulin-SEQ ID NO: 19like Growth Factor Receptor-I(IGFR1)Amino acid sequence of Insulin-SEQ ID NO: 20like Growth Factor-I (IGF1)Amino acid sequence of Insulin-SEQ ID NO: 21like Growth Factor-II (IGF2)Nucleotide sequence of PCRSEQ ID NO: 22primerNucleotide sequence of PCRSEQ ID NO: 23primerNucleotide sequence encodingSEQ ID NO: 24the 1H3 light chain variableregion-including signal peptide(1H3 LC)Amino acid sequence of the 1H3SEQ ID NO: 25light chain variable region-including signal peptideNucleotide sequence encodingSEQ ID NO: 26the 1H3 heavy chain variableregion including signal peptide(1H3 HC)Amino acid sequence of the 1H3SEQ ID NO: 27heavy chain variable regionincluding signal peptideNucleotide sequence encodingSEQ ID NO: 28the 1H3 CDR-L1Nucleotide sequence encodingSEQ ID NO: 29the 1H3 CDR-L2Nucleotide sequence encodingSEQ ID NO: 30the 1H3 CDR-L3Amino acid sequence of the 1H3SEQ ID NO: 31CDR-L1Amino acid sequence of the 1H3SEQ ID NO: 32CDR-L2Amino acid sequence of the 1H3SEQ ID NO: 33CDR-L3Nucleotide sequence encodingSEQ ID NO: 34the 1H3 CDR-H1Nucleotide sequence encodingSEQ ID NO: 35the 1H3 CDR-H2Nucleotide sequence encodingSEQ ID NO: 36the 1H3 CDR-H3Amino acid sequence of the 1H3SEQ ID NO: 37CDR-H1Amino acid sequence of the 1H3SEQ ID NO: 38CDR-H2Amino acid sequence of the 1H3SEQ ID NO: 39CDR-H3Nucleotide sequence encodingSEQ ID NO: 40the 15H12/19D12 light chain A(LCA)Amino acid sequence of theSEQ ID NO: 4115H12/19D12 light chain ANucleotide sequence encodingSEQ ID NO: 42the 15H12/19D12 light chain B(LCB)Amino acid sequence of theSEQ ID NO: 4315H12/19D12 light chain BNucleotide sequence encodingSEQ ID NO: 44the 15H12/19D12 heavy chain A(HCA)Amino acid sequence of theSEQ ID NO: 4515H12/19D12 heavy chain ANucleotide sequence encodingSEQ ID NO: 46the 15H12/19D12 light chain Aframework region 1Amino acid sequence of theSEQ ID NO: 4715H12/19D12 light chain Aframework region 1Nucleotide sequence encodingSEQ ID NO: 48the 15H12/19D12 light chain Aframework region 2Amino acid sequence of theSEQ ID NO: 4915H12/19D12 light chain Aframework region 2Nucleotide sequence encodingSEQ ID NO: 50the 15H12/19D12 light chain Aframework region 3Amino acid sequence of theSEQ ID NO: 5115H12/19D12 light chain Aframework region 3Nucleotide sequence encodingSEQ ID NO: 52the 15H12/19D12 light chain Aframework region 4Amino acid sequence of theSEQ ID NO: 5315H12/19D12 light chain Aframework region 4Nucleotide sequence encodingSEQ ID NO: 54the 15H12/19D12 light chain Bframework region 1Amino acid sequence of theSEQ ID NO: 5515H12/19D12 light chain Bframework region 1Nucleotide sequence encodingSEQ ID NO: 56the 15H12/19D12 light chain Bframework region 2Amino acid sequence of theSEQ ID NO: 5715H12/19D12 light chain Bframework region 2Nucleotide sequence encodingSEQ ID NO: 58the 15H12/19D12 light chain Bframework region 3Amino acid sequence of theSEQ ID NO: 5915H12/19D12 light chain Bframework region 3Nucleotide sequence encodingSEQ ID NO: 60the 15H12/19D12 light chain Bframework region 4Amino acid sequence of theSEQ ID NO: 6115H12/19D12 light chain Bframework region 4Nucleotide sequence encodingSEQ ID NO: 62the 15H12/19D12 heavy chain Aframework region 1Amino acid sequence of theSEQ ID NO: 6315H12/19D12 heavy chain Aframework region 1Nucleotide sequence encodingSEQ ID NO: 64the 15H12/19D12 heavy chain Aframework region 2Amino acid sequence of theSEQ ID NO: 6515H12/19D12 heavy chain Aframework region 2Nucleotide sequence encodingSEQ ID NO: 66the 15H12/19D12 heavy chain Aframework region 3Amino acid sequence of theSEQ ID NO: 6715H12/19D12 heavy chain Aframework region 3Nucleotide sequence encodingSEQ ID NO: 68the 15H12/19D12 heavy chain Aframework region 4Amino acid sequence of theSEQ ID NO: 6915H12/19D12 heavy chain Aframework region 4Amino acid sequence of theSEQ ID NO: 70alternative 1H3 CDR-H1Nucleotide sequence encodingSEQ ID NO: 71the 15H12/19D12 light chain C(LCC)Amino acid sequence of theSEQ ID NO: 7215H12/19D12 light chain CNucleotide sequence encodingSEQ ID NO: 73the 15H12/19D12 light chain D(LCD)Amino acid sequence of theSEQ ID NO: 7415H12/19D12 light chain DNucleotide sequence encodingSEQ ID NO: 75the 15H12/19D12 light chain E(LCE)Amino acid sequence of theSEQ ID NO: 7615H12/19D12 light chain ENucleotide sequence encodingSEQ ID NO: 77the 15H12/19D12 light chain F(LCF)Amino acid sequence of theSEQ ID NO: 7815H12/19D12 light chain FNucleotide sequence encodingSEQ ID NO: 79the 15H12/19D12 light chain Cframework region 1Amino acid sequence of theSEQ ID NO: 8015H12/19D12 light chain Cframework region 1Nucleotide sequence encodingSEQ ID NO: 81the 15H12/19D12 light chain Cframework region 2Amino acid sequence of theSEQ ID NO: 8215H12/19D12 light chain Cframework region 2Nucleotide sequence encodingSEQ ID NO: 83the 15H12/19D12 light chain Cframework region 3Amino acid sequence of theSEQ ID NO: 8415H12/19D12 light chain Cframework region 3Nucleotide sequence encodingSEQ ID NO: 85the 15H12/19D12 light chain Cframework region 4Amino acid sequence of theSEQ ID NO: 8615H12/19D12 light chain Cframework region 4Nucleotide sequence encodingSEQ ID NO: 87the 15H12/19D12 light chain Dframework region 1Amino acid sequence of theSEQ ID NO: 8815H12/19D12 light chain Dframework region 1Nucleotide sequence encodingSEQ ID NO: 89the 15H12/19D12 light chain Dframework region 2Amino acid sequence of theSEQ ID NO: 9015H12/19D12 light chain Dframework region 2Nucleotide sequence encodingSEQ ID NO: 91the 15H12/19D12 light chain Dframework region 3Amino acid sequence of theSEQ ID NO: 9215H12/19D12 light chain Dframework region 3Nucleotide sequence encodingSEQ ID NO: 93the 15H12/19D12 light chain Dframework region 4Amino acid sequence of theSEQ ID NO: 9415H12/19D12 light chain Dframework region 4Nucleotide sequence encodingSEQ ID NO: 95the 15H12/19D12 light chain Eframework region 1Amino acid sequence of theSEQ ID NO: 9615H12/19D12 light chain Eframework region 1Nucleotide sequence encodingSEQ ID NO: 97the 15H12/19D12 light chain Eframework region 2Amino acid sequence of theSEQ ID NO: 9815H12/19D12 light chain Eframework region 2Nucleotide sequence encodingSEQ ID NO: 99the 15H12/19D12 light chain Eframework region 3Amino acid sequence of theSEQ ID NO: 10015H12/19D12 light chain Eframework region 3Nucleotide sequence encodingSEQ ID NO: 101the 15H12/19D12 light chain Eframework region 4Amino acid sequence of theSEQ ID NO: 10215H12/19D12 light chain Eframework region 4Nucleotide sequence encodingSEQ ID NO: 103the 15H12/19D12 light chain Fframework region 1Amino acid sequence of theSEQ ID NO: 10415H12/19D12 light chain Fframework region 1Nucleotide sequence encodingSEQ ID NO: 105the 15H12/19D12 light chain Fframework region 2Amino acid sequence of theSEQ ID NO: 10615H12/19D12 light chain Fframework region 2Nucleotide sequence encodingSEQ ID NO: 107the 15H12/19D12 light chain Fframework region 3Amino acid sequence of theSEQ ID NO: 10815H12/19D12 light chain Fframework region 3Nucleotide sequence encodingSEQ ID NO: 109the 15H12/19D12 light chain Fframework region 4Amino acid sequence of theSEQ ID NO: 11015H12/19D12 light chain Fframework region 4Nucleotide sequence encodingSEQ ID NO: 111the 15H12/19D12 heavy chain B(HCB)Amino acid sequence of theSEQ ID NO: 11215H12/19D12 heavy chain BNucleotide sequence encodingSEQ ID NO: 113the 15H12/19D12 heavy chain Bframework region 1Amino acid sequence of theSEQ ID NO: 11415H12/19D12 heavy chain Bframework region 1Nucleotide sequence encodingSEQ ID NO: 115the 15H12/19D12 heavy chain BF framework region 2Amino acid sequence of theSEQ ID NO: 11615H12/19D12 heavy chain Bframework region 2Nucleotide sequence encodingSEQ ID NO: 117the 15H12/19D12 heavy chain Bframework region 3Amino acid sequence of theSEQ ID NO: 11815H12/19D12 heavy chain Bframework region 3Nucleotide sequence encodingSEQ ID NO: 119the 15H12/19D12 heavy chain Bframework region 4Amino acid sequence of theSEQ ID NO: 12015H12/19D12 heavy chain Bframework region 4

[0112] CDR-L1 is the first complementarity determining region (CDR) which occurs in the light chain, CDR-L2 is the second CDR which occurs on the light chain and CDR-L3 is the third CDR which occurs on the light chain.

[0113] Similarly, CDR-H1 is the first CDR which occurs on the heavy chain, CDR-H2 is the second CDR which occurs on the heavy chain and CDR-H3 is the third CDR which occurs on the heavy chain.

[0114] FR-L1 is the first framework region of the light chain, FR-L2 is the second framework region of the light chain, FR-L3 is the third framework region of the light chain, FR-L4 is the fourth framework region on the light chain, FR-H1 is the first framework region of the heavy chain, FR-H2 is the second framework region of the heavy chain, FR-H3 is the third framework region of the heavy chain and FR-H4 is the fourth framework region of the heavy chain. These terms and the arrangement of CDRs and FRs on an immunoglobulin chain are well known in the art.

[0115] A mature light chain variable region of the invention, which lacks the signal peptide (i.e., first 19 or 20 residues), is amino acids 20-128 of SEQ ID NO: 2, 41,43, 72, 74, 76 or 78 which is encoded by nucleotides 58-384 of SEQ ID NO: 1, 40, 42, 71, 73, 75, or 77 or amino acids 21-130 of SEQ ID NO: 25 which is encoded by nucleotides 61-390 of SEQ ID NO: 24.

[0116] A mature heavy chain variable region, which lacks the signal peptide (i.e., first 19 residues), is amino acids 20-137 of SEQ ID NO: 4, 45 or 112 which is encoded by nucleotides 58-411 of SEQ ID NO: 3, 44 or 111 or amino acids 20-140 of SEQ ID NO: 27 which is encoded by nucleotides 58-420 of SEQ ID NO: 26.

[0117] In some embodiments the 15H12 and 19D12 CDR-H1 is GFTFSSFAMH (SEQ ID NO: 17) which is encoded by the nucleotide sequence of SEQ ID NO: 18. In some embodiments the 1H3 CDR-H1 is NYAMH (SEQ ID NO: 70).

[0118] The present invention also includes antibodies and antigen-binding fragments which include the framework regions of the antibodies and antigen-binding fragments of the invention. Preferably, FR-L1 is amino acids 20-42 of SEQ ID NO: 2 or amino acids 21-43 of SEQ ID NO: 25; FR-L2 is amino acids 54-68 of SEQ ID NO: 2 or amino acids 55-69 of SEQ ID NO: 25; FR-L3 is amino acids 76-107 of SEQ ID NO: 2 or amino acids 77-108 of SEQ ID NO: 25; FR-L4 is amino acids 117-128 of SEQ ID NO: 2 or amino acids 128-130 of SEQ ID NO: 25; FR-H1 is amino acids 20-44 or 20-49 of SEQ ID NO: 4 or amino acids 20-44 or 20-49 of SEQ ID NO: 27; FR-H2 is amino acids 55-68 of SEQ ID NO: 4 or amino acids 55-68 of SEQ ID NO: 27; FR-H3 is amino acids 85-116 of SEQ ID NO: 4 or amino acids 85-116 of SEQ ID NO: 27 and FR-H4 is amino acids 127-137 of SEQ ID NO: 4 or amino acids 130-140 of SEQ ID NO: 27.

[0119] In preferred embodiments, the antibody molecules of the present invention include FR-L1 defined by amino acids 20-42 of SEQ ID NO: 41 or 43; FR-L2 defined by amino acids 54-68 of SEQ ID NO: 41 or 43; FR-L3 defined by amino acids 76-107 of SEQ ID NO: 41 or 43; and FR-L4 defined by amino acids 117-128 of SEQ ID NO: 41 or 43. Furthermore, preferred embodiments include antibody molecules including FR-H1 defined by amino acids 20-44 of SEQ ID NO: 45; FR-H2 defined by amino acids 55-68 of SEQ ID NO: 45; FR-H3 defined by amino acids 85-116 of SEQ ID NO: 45; and FR-H4 defined by amino acids 127-137 of SEQ ID NO: 45.

[0120] Molecular Biology

[0121] In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein “Sambrook, et al., 1989”); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. (1985)); Transcription And Translation (B. D. Hames & S. J. Higgins, eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel, et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

[0122] A “polynucleotide”, “nucleic acid” or “nucleic acid molecule” may refer to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in single stranded form, double-stranded form or otherwise.

[0123] A “polynucleotide sequence”, “nucleic acid sequence” or “nucleotide sequence” is a series of nucleotide bases (also called “nucleotides”) in a nucleic acid, such as DNA or RNA, and means any chain of two or more nucleotides.

[0124] A “coding sequence” or a sequence “encoding” an expression product, such as a RNA, polypeptide, protein, or enzyme, is a nucleotide sequence that, when expressed, results in production of the product.

[0125] The term “gene” means a DNA sequence that codes for or corresponds to a particular sequence of ribonucleotides or amino acids which comprise all or part of one or more RNA molecules, proteins or enzymes, and may or may not include regulatory DNA sequences, such as promoter sequences, which determine, for example, the conditions under which the gene is expressed. Genes may be transcribed from DNA to RNA which may or may not be translated into an amino acid sequence.

[0126] “Amplification” of DNA as used herein may denote the use of polymerase chain reaction (PCR) to increase the concentration of a particular DNA sequence within a mixture of DNA sequences. For a description of PCR see Saiki, et al., Science (1988) 239: 487. In a specific embodiment, the present invention includes a nucleic acid, which encodes an anti-IGFR1 antibody, an anti-IGFR1 antibody heavy or light chain, an anti-IGFR1 antibody heavy or light chain variable region, an anti-IGFR1 antibody heavy or light chain constant region or anti-IGFR1 antibody CDR (e.g., CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 or CDR-H3) which can be amplified by PCR.

[0127] As used herein, the term “oligonucleotide” refers to a nucleic acid, generally of at least 10 (e.g., 10, 11, 12, 13 or 14), preferably at least 15 (e.g., 15, 16, 17, 18 or 19), and more preferably at least 20 nucleotides (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30), preferably no more than 100 nucleotides (e.g., 40, 50, 60, 70, 80 or 90), that may be hybridizable to a genomic DNA molecule, a cDNA molecule, or an mRNA molecule encoding a gene, mRNA, cDNA, or other nucleic acid of interest. Oligonucleotides can be labeled, e.g., by incorporation of 32P-nucleotides, 3H-nucleotides, 14C-nucleotides, 35S-nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated. In one embodiment, a labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid. In another embodiment, oligonucleotides (one or both of which may be labeled) can be used as PCR primers, either for cloning full length or a fragment of the gene, or to detect the presence of nucleic acids. Generally, oligonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer.

[0128] The sequence of any nucleic acid (e.g., a nucleic acid encoding an IGFR1 gene or a nucleic acid encoding an anti-IGFR1 antibody or a fragment or portion thereof) may be sequenced by any method known in the art (e.g., chemical sequencing or enzymatic sequencing). “Chemical sequencing” of DNA may denote methods such as that of Maxam and Gilbert (1977) (Proc. Natl. Acad. Sci. USA 74:560), in which DNA is randomly cleaved using individual base-specific reactions. “Enzymatic sequencing” of DNA may denote methods such as that of Sanger (Sanger, et al., (1977) Proc. Natl. Acad. Sci. USA 74:5463).

[0129] The nucleic acids herein may be flanked by natural regulatory (expression control) sequences, or may be associated with heterologous sequences, including promoters, internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5′-and 3′-non-coding regions, and the like.

[0130] A “promoter” or “promoter sequence” is a DNA regulatory region capable of binding an RNA polymerase in a cell (e.g., directly or through other promoter-bound proteins or substances) and initiating transcription of a coding sequence. A promoter sequence is, in general, bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at any level. Within the promoter sequence may be found a transcription initiation site (conveniently defined, for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. The promoter may be operably associated with other expression control sequences, including enhancer and repressor sequences or with a nucleic acid of the invention (e.g., SEQ ID NO: 1, 3, 5-7, 11-13, 18, 22-24, 26, 28-30 or 34-36). Promoters which may be used to control gene expression include, but are not limited to, cytomegalovirus (CMV) promoter (U.S. Pat. Nos. 5,385,839 and 5,168,062), the SV40 early promoter region (Benoist, et al., (1981) Nature 290:304-310), the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto, et al., (1980) Cell 22:787-797), the herpes thymidine kinase promoter (Wagner, et al., (1981) Proc. Natl. Acad. Sci. USA 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster, et al., (1982) Nature 296:39-42); prokaryotic expression vectors such as the β-lactamase promoter (Villa-Komaroff, et al., (1978) Proc. Natl. Acad. Sci. USA 75:3727-3731), or the tac promoter (DeBoer, et al., (1983) Proc. Natl. Acad. Sci. USA 80:21-25); see also “Useful proteins from recombinant bacteria” in Scientific American (1980) 242:74-94; and promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter or the alkaline phosphatase promoter.

[0131] A coding sequence is “under the control of”, “functionally associated with” or “operably associated with” transcriptional and translational control sequences in a cell when the sequences direct RNA polymerase mediated transcription of the coding sequence into RNA, preferably mRNA, which then may be trans-RNA spliced (if it contains introns) and, optionally, translated into a protein encoded by the coding sequence.

[0132] The terms “express” and “expression” mean allowing or causing the information in a gene, RNA or DNA sequence to become manifest; for example, producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene. A DNA sequence is expressed in or by a cell to form an “expression product” such as an RNA (e.g., mRNA) or a protein (e.g., antibody 1H3, 15H12 or 19D12 or a fragment thereof). The expression product itself may also be said to be “expressed” by the cell.

[0133] The terms “vector”, “cloning vector” and “expression vector” mean the vehicle (e.g., a plasmid) by which a DNA or RNA sequence can be introduced into a host cell, so as to transform the host and, optionally, promote expression and/or replication of the introduced sequence.

[0134] The term “transfection” or “transformation” means the introduction of a nucleic acid into a cell. These terms may refer to the introduction of a nucleic acid encoding an anti-IGFR1 antibody or fragment thereof into a cell. The introduced gene or sequence may be called a “clone”. A host cell that receives the introduced DNA or RNA has been “transformed” and is a “transformant” or a “clone”. The DNA or RNA introduced to a host cell can come from any source, including cells of the same genus or species as the host cell, or cells of a different genus or species.

[0135] The term “host cell” means any cell of any organism that is selected, modified, transfected, transformed, grown, or used or manipulated in any way, for the production of a substance by the cell, for example the expression or replication, by the cell, of a gene, a DNA or RNA sequence, a protein or an enzyme.

[0136] The term “expression system” means a host cell and compatible vector which, under suitable conditions, can express a protein or nucleic acid which is carried by the vector and introduced to the host cell. Common expression systems include E. coli host cells and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells and vectors. In a specific embodiment, IGFR1 or an antibody and antigen-binding fragment of the invention may be expressed in human embryonic kidney cells (HEK293). Other suitable cells include CHO (chinese hamster ovary) cells, HeLa cells and NIH 3T3 cells and NSO cells (non-Ig-producing murine myeloma cell line). Nucleic acids encoding an antibody or antigen-binding fragment of the invention, sIGFR1 or IGFR1 may be expressed at high levels in an E.coli/T7 expression system as disclosed in U.S. Pat. Nos. 4,952,496, 5,693,489 and 5,869,320 and in Davanloo, P., et al., (1984) Proc. Nati. Acad. Sci. USA 81, 2035-2039; Studier, F. W., et al., (1986) J. Mol. Biol. 189: 113-130; Rosenberg, A. H., et al., (1987) Gene 56: 125-135; and Dunn, J. J., et al., (1988) Gene 68: 259 which are herein incorporated by reference.

[0137] The present invention contemplates any superficial or slight modification to the amino acid or nucleotide sequences which correspond to the antibodies or antigen-binding fragments of the invention. In particular, the present invention contemplates sequence conservative variants of the nucleic acids which encode the antibodies or antigen-binding fragments of the invention. “Sequence-conservative variants” of a polynucleotide sequence are those in which a change of one or more nucleotides in a given codon results in no alteration in the amino acid encoded at that position. Function-conservative variants of the antibodies of the invention are also contemplated by the present invention. “Function-conservative variants” are those in which one or more amino acid residues in a protein or enzyme have been changed without altering the overall conformation and function of the polypeptide, including, but, by no means, limited to, replacement of an amino acid with one having similar properties. Amino acids with similar properties are well known in the art. For example, polar/hydrophilic amino acids which may be interchangeable include asparagine, glutamine, serine, cysteine, threonine, lysine, arginine, histidine, aspartic acid and glutamic acid; nonpolar/hydrophobic amino acids which may be interchangeable include glycine, alanine, valine, leucine, isoleucine, proline, tyrosine, phenylalanine, tryptophan and methionine; acidic amino acids which may be interchangeable include aspartic acid and glutamic acid and basic amino acids which may be interchangeable include histidine, lysine and arginine.

[0138] The present invention includes anti-IGFR1 antibodies and fragments thereof which are encoded by nucleic acids as described in Table 1 as well as nucleic acids which hybridize thereto. Preferably, the nucleic acids hybridize under low stringency conditions, more preferably under moderate stringency conditions and most preferably under high stringency conditions and, preferably, exhibit IGFR1 binding activity. A nucleic acid molecule is “hybridizable” to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook, et al., supra). The conditions of temperature and ionic strength determine the “stringency” of the hybridization. Typical low stringency hybridization conditions may be 55° C., 5×SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5×SSC, 0.5% SDS. Typical, moderate stringency hybridization conditions are similar to the low stringency conditions except the hybridization is carried out in 40% formamide, with 5× or 6×SSC. High stringency hybridization conditions are similar to low stringency conditions except the hybridization conditions are carried out in 50% formamide, 5× or 6×SSC and, optionally, at a higher temperature (e.g., 57° C., 59° C., 60° C., 62° C., 63° C. or 68° C.). In general, SSC is 0.15M NaC1 and 0.015M Na-citrate. Hybridization requires that the two nucleic acids contain complementary sequences, although, depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the higher the stringency under which the nucleic acids may hybridize. For hybrids of greater than 100 nucleotides in length, equations for calculating the melting temperature have been derived (see Sambrook, et al., supra, 9.50-9.51). For hybridization with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook, et al., supra, 11.7-11.8).

[0139] Also included in the present invention are nucleic acids comprising nucleotide sequences and polypeptides comprising amino acid sequences which are at least about 70% identical, preferably at least about 80% identical, more preferably at least about 90% identical and most preferably at least about 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to the reference nucleotide and amino acid sequences of Table 1 when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences. Polypeptides comprising amino acid sequences which are at least about 70% similar, preferably at least about 80% similar, more preferably at least about 90% similar and most preferably at least about 95% similar (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to the reference amino acid sequences of Table 1 (e.g., SEQ ID NOs. 2 (e.g., amino acids 20-128), 4 (e.g., amino acids 20-137), 8-10, 14-16, 17, 25 (e.g., amino acids 21-130), 27 (e.g., amino acids 20-140), 31-33 or 37-39) when the comparison is performed with a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences, are also included in the present invention.

[0140] Sequence identity refers to exact matches between the nucleotides or amino acids of two sequences which are being compared. Sequence similarity refers to both exact matches between the amino acids of two polypeptides which are being compared in addition to matches between nonidentical, biochemically related amino acids. Biochemically related amino acids which share similar properties and may be interchangeable are discussed above.

[0141] The following references regarding the BLAST algorithm are herein incorporated by reference: BLAST ALGORITHMS: Altschul, S. F., et al., (1990) J. Mol. Biol. 215:403-410; Gish, W., et al., (1993) Nature Genet. 3:266-272; Madden, T. L., et al., (1996) Meth. Enzymol. 266:131-141; Altschul, S. F., et al., (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J., et al., (1997) Genome Res. 7:649-656; Wootton, J. C., et al., (1993) Comput. Chem. 17:149-163; Hancock, J. M. et al., (1994) Comput. Appl. Biosci. 10:67-70; ALIGNMENT SCORING SYSTEMS: Dayhoff, M. O., et al., “A model of evolutionary change in proteins.” in Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3. M. O. Dayhoff (ed.), pp. 345-352, Natl. Biomed. Res. Found., Washington, D.C.; Schwartz, R. M., et al., “Matrices for detecting distant relationships.” in Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3.” M. O. Dayhoff (ed.), pp. 353-358, Natl. Biomed. Res. Found., Washington, D.C.; Altschul, S. F., (1991) J. Mol. Biol. 219:555-565; States, D. J., et al., (1991) Methods 3:66-70; Henikoff, S., et al., (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919; Altschul, S. F., et al., (1993) J. Mol. Evol. 36:290-300; ALIGNMENT STATISTICS: Karlin, S., et al., (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268; Karlin, S., et al., (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877; Dembo, A., et al., (1994) Ann. Prob. 22:2022-2039; and Altschul, S. F. “Evaluating the statistical significance of multiple distinct local alignments.” in Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), (1997) pp. 1-14, Plenum, N.Y.

[0142] Antibody Structure

[0143] In general, the basic antibody structural unit is known to comprise a tetramer. Each tetramer includes two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain may include a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain may define a constant region primarily responsible for effector function. Typically, human light chains are classified as kappa and lambda light chains. Furthermore, human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes).

[0144] The variable regions of each light/heavy chain pair may form the antibody binding site. Thus, in general, an intact IgG antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are, in general, the same.

[0145] Normally, the chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are usually aligned by the framework regions, enabling binding to a specific epitope. In general, from N-terminal to C-terminal, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is, generally, in accordance with the definitions of Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883. The present invention provides antibodies or antigen-binding fragments of the invention comprising CDRs and FRs from the light and heavy chains of 1H3 , 15H12 and 19D12 (e.g., 15H12/19D12 LCA, 15H12/19D12 LCB, 15H12/19D12 HCA, SEQ ID NOs: 2, 4, 25, 27, 41, 43 and 45) as defined by Kabat and Chothia (see above references).

[0146] Antibody Molecules

[0147] The term “antibody molecule” includes, but is not limited to, antibodies and fragments, preferably antigen-binding fragments, thereof. The term includes monoclonal antibodies, polyclonal antibodies, bispecific antibodies, Fab antibody fragments, F(ab)2 antibody fragments, Fv antibody fragments (e.g., VH or VL), single chain Fv antibody fragments and dsFv antibody fragments. Furthermore, the antibody molecules of the invention may be fully human antibodies or chimeric antibodies. Preferably, the antibody molecules are monoclonal, fully human antibodies; more preferably, the antibody molecules are 1H3, 15H12 or 19D12. Preferably, the antibody molecules include one or more of the variable regions and CDRs whose amino acid and nucleotide sequences are set forth in Table 1.

[0148] The present invention includes any antibody molecule comprising a CDR selected from:

2RASQSIGSSLH;(SEQ ID NO: 8)YASQSLS;(SEQ ID NO: 9)HQSSRLPHT;(SEQ ID NO: 10)SFAMH(SEQ ID NO: 14)GFTFSSFAMH;(SEQ ID NO: 17)VIDTRGATYYADSVKG;(SEQ ID NO: 15)LGNFYYGMDV;(SEQ ID NO: 16)RASQSVSSFLA;(SEQ ID NO: 31)DASNRAP;(SEQ ID NO: 32)QQRSNWPRWT;(SEQ ID NO: 33)GFTFSNYAMH;(SEQ ID NO: 37)AIGAGGDTYYADSVKG; and(SEQ ID NO: 38)GRHRNWYYYNKDY;(SEQ ID NO: 39)NYANH(SEQ ID NO: 70)

[0149] The scope of the present invention includes antibody variable regions of the present invention (e.g., any variable region, mature or unprocessed, indicated in Table 1) linked to any immunoglobulin constant region. If a light chain variable region is linked to a constant region, preferably it is a κ chain. If a heavy chain variable region is linked to a constant region, preferably it is a γ1, γ2, γ3 or γ4 constant region, more preferably, γ1, γ2 or γ4 and even more preferably γ1 or γ4.

[0150] The anti-IGFR1 antibody molecules of the invention preferably recognize human IGFR1, preferably sIGFR1; however, the present invention includes antibody molecules which recognize IGFR1 from different species, preferably mammals (e.g., mouse, rat, rabbit, sheep or dog). The present invention also includes anti-IGFR1 antibodies or fragments thereof which are complexed with IGFR1 or any fragment thereof (e.g., amino acids 30-902 of SEQ ID NO: 19) or with any cell which is expressing IGFR1 or any portion or fragment thereof on the cell surface (e.g., HEK293 cells stably transformed with human IGFR1 or MCF7 (e.g., ATCC Cell Line No. HTB-22)). Such complexes may be made by contacting the antibody or antibody fragment with IGFR1 or the IGFR1 fragment.

[0151] In a preferred embodiment, fully-human monoclonal antibodies directed against IGFR1 are generated using transgenic mice carrying parts of the human immune system rather than the mouse system. These transgenic mice, which may be referred to, herein, as “HuMAb” mice, contain a human immunoglobulin gene miniloci that encodes unrearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous μ and κ chain loci (Lonberg, N., et al., (1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit reduced expression of mouse IgM or κ, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgGκ monoclonal antibodies (Lonberg, N., et al., (1994), supra; reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N., et al., (1995) Intern. Rev. Immunol. 13:65-93, and Harding, F., et al., (1995) Ann. N. Y Acad. Sci 764:536-546). The preparation of HuMab mice is commonly known in the art and is described, for example, in Taylor, L., et al., (1992) Nucleic Acids Research 20:6287-6295; Chen, J., et al., (1993) International Immunology 5: 647-656; Tuaillon, et al., (1993) Proc. Natl. Acad. Sci USA 90:3720-3724; Choi, et al., (1993) Nature Genetics 4:117-123; Chen, J., et al., (1993)EMBO J. 12: 821-830; Tuaillon, et al., (1994) J Immunol. 152:2912-2920; Lonberg, et al., (1994) Nature 368(6474): 856-859; Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Taylor, L., et al., (1994) International Immunology 6: 579-591; Lonberg, N., et al., (1995) Intern. Rev. Immunol. Vol. 13: 65-93; Harding, F., et al., (1995) Ann. N.Y Acad. Sci 764:536-546; Fishwild, D., et al., (1996) Nature Biotechnology 14: 845-851 and Harding, et al., (1995) Annals NY Acad. Sci. 764:536-546; the contents of all of which are hereby incorporated by reference in their entirety. See further, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; 5,770,429 and 5,545,807; and International Patent Application Publication Nos. WO 98/24884; WO 94/25585; WO 93/12227; WO 92/22645 and WO 92/03918 the disclosures of all of which are hereby incorporated by reference in their entity.

[0152] To generate fully human, monoclonal antibodies to IGFR1, HuMab mice can be immunized with an antigenic IGFR1 polypeptide, preferably amino acids 30-902 of SEQ ID NO: 19, as described by Lonberg, N., et al., (1994) Nature 368(6474): 856-859; Fishwild, D., et al., (1996) Nature Biotechnology 14: 845-851 and WO 98/24884. Preferably, the mice will be 6-16 weeks of age upon the first immunization. For example, a purified preparation of IGFR1 or sIGFR1 can be used to immunize the HuMab mice intraperitoneally. The mice can also be immunized with whole HEK293 cells which are stably transformed or transfected with an IGFR1 gene. An “antigenic IGFR1 polypeptide” may refer to an IGFR1 polypeptide of any fragment thereof, preferably amino acids 30-902 of SEQ ID NO: 19, which elicits an anti-IGFR1 immune response, preferably in HuMab mice.

[0153] In general, HuMAb transgenic mice respond well when initially immunized intraperitoneally (IP) with antigen in complete Freund's adjuvant, followed by every other week IP immunizations (usually, up to a total of 6) with antigen in incomplete Freund's adjuvant. Mice can be immunized, first, with cells expressing IGFR1 (e.g., stably transformed HEK293 cells), then with a soluble fragment of IGFR1 (e.g., amino acids 30-902 of SEQ ID NO: 19) and continually receive alternating immunizations with the two antigens. The immune response can be monitored over the course of the immunization protocol with plasma samples being obtained by retroorbital bleeds. The plasma can be screened for the presence of anti-IGFR1 antibodies, for example by ELISA, and mice with sufficient titers of immunoglobulin can be used for fusions. Mice can be boosted intravenously with antigen 3 days before sacrifice and removal of the spleen. It is expected that 2-3 fusions for each antigen may need to be performed. Several mice can be immunized for each antigen. For example, a total of twelve HuMAb mice of the HC07 and HC012 strains can be immunized.

[0154] Hybridoma cells which produce the monoclonal, fully human anti-IGFR1 antibodies may be produced by methods which are commonly known in the art. These methods include, but are not limited to, the hybridoma technique originally developed by Kohler, et al., (1975) (Nature 256:495-497), as well as the trioma technique (Hering, et al., (1988) Biomed. Biochim. Acta. 47:211-216 and Hagiwara, et al., (1993) Hum. Antibod. Hybridomas 4:15), the human B-cell hybridoma technique (Kozbor, et al., (1983) Immunology Today 4:72 and Cote, et al., (1983) Proc. Natl. Acad. Sci. U.S.A 80:2026-2030), and the EBV-hybridoma technique (Cole, et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985). Preferably, mouse splenocytes are isolated and fused with PEG to a mouse myeloma cell line based upon standard protocols. The resulting hybridomas may then be screened for the production of antigen-specific antibodies. For example, single cell suspensions of splenic lymphocytes from immunized mice may by fused to one-sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma cells (ATCC, CRL 1580) with 50% PEG. Cells may be plated at approximately 2×105 cells/mL in a flat bottom microtiter plate, followed by a two week incubation in selective medium containing 20% fetal Clone Serum, 18% “653” conditioned media, 5% origen (IGEN), 4 mM L-glutamine, 1 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50 mg/ml gentamycin and 1× HAT (Sigma; the HAT is added 24 hours after the fusion). After two weeks, cells may be cultured in medium in which the HAT is replaced with HT. Individual wells may then be screened by ELISA for human anti-IGFR1 monoclonal IgG antibodies. Once extensive hybridoma growth occurs, medium can be observed usually after 10-14 days. The antibody secreting hybridomas may be replated, screened again, and if still positive for human IgG, anti-IGFR1 monoclonal antibodies, can be subcloned at least twice by limiting dilution. The stable subclones may then be cultured in vitro to generate small amounts of antibody in tissue culture medium for characterization.

[0155] The anti-IGFR antibody molecules of the present invention may also be produced recombinantly (e.g., in an E.coli/T7 expression system as discussed above). In this embodiment, nucleic acids encoding the antibody molecules of the invention (e.g., VH or VL) may be inserted into a pET-based plasmid and expressed in the E.coli/T7 system. There are several methods by which to produce recombinant antibodies which are known in the art. One example of a method for recombinant production of antibodies is disclosed in U.S. Pat. No. 4,816,567 which is herein incorporated by reference. Transformation can be by any known method for introducing polynucleotides into a host cell. Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, biolistic injection and direct microinjection of the DNA into nuclei. In addition, nucleic acid molecules may be introduced into mammalian cells by viral vectors. Methods of transforming cells are well known in the art. See, for example, U.S. Pat. Nos. 4,399,216; 4,912,040; 4,740,461 and 4,959,455.

[0156] Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and a number of other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Cell lines of particular preference are selected through determining which cell lines have high expression levels. Other cell lines that may be used are insect cell lines, such as Sf9. cells, amphibian cells, bacterial cells, plant cells and fungal cells. When recombinant expression vectors encoding the heavy chain or antigen-binding portion thereof, the light chain and/or antigen-binding portion thereof are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, 5 secretion of the antibody into the culture medium in which the host cells are grown.

[0157] Antibodies can be recovered from the culture medium using standard protein purification methods. Further, expression of antibodies of the invention (or other moieties therefrom) from production cell lines can be enhanced using a number of known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions. The GS system is discussed in whole or part in connection with European Patent Nos. 0 216 846, 0 256 055, and 0 323 997 and European Patent Application No. 89303964.4.

[0158] It is likely that antibodies expressed by different cell lines or in transgenic animals will have different glycosylation from each other. However, all antibodies encoded by the nucleic acid molecules provided herein, or comprising the amino acid sequences provided herein are part of the instant invention, regardless of the glycosylation of the antibodies.

[0159] “Koff” refers to the off-rate constant for dissociation of the antibody from an antibody/antigen complex.

[0160] “Kon” refers to the rate at which the antibody associates with the antigen.

[0161] “Kd” refers to the dissociation constant of a particular antibody/antigen interaction. Kd=Koff/Kon.

[0162] The term “monoclonal antibody,” as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Monoclonal antibodies are advantageous in that they may be synthesized by a hybridoma culture, essentially uncontaminated by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being amongst a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. As mentioned above, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler, et al., (1975) Nature 256: 495.

[0163] A polyclonal antibody is an antibody which was produced among or in the presence of one or more other, non-identical antibodies. In general, polyclonal antibodies are produced from a B-lymphocyte in the presence of several other B-lymphocytes which produced non-identical antibodies. Usually, polyclonal antibodies are obtained directly from an immunized animal.

[0164] A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab′ fragments. See, e.g., Songsivilai, et al., (1990) Clin. Exp. Immunol. 79: 315-321, Kostelny, et al., (1992) J Immunol. 148:1547-1553. In addition, bispecific antibodies may be formed as “diabodies” (Holliger, et al., (1993) PNAS USA 90:6444-6448) or as “Janusins” (Traunecker, et al., (1991) EMBO J. 10:3655-3659 and Traunecker, et al., (1992) Int. J. Cancer Suppl. 7:51-52).

[0165] The term “fully human antibody” refers to an antibody which comprises human immunoglobulin protein sequences only. A fully human antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell or in a hybridoma derived from a mouse cell. Similarly, “mouse antibody” refers to an antibody which comprises mouse immunoglobulin sequences only.

[0166] The present invention includes “chimeric antibodies”—an antibody which comprises a variable region of the present invention fused or chimerized with an antibody region (e.g., constant region) from another, non-human species (e.g., mouse, horse, rabbit, dog, cow, chicken). These antibodies may be used to modulate the expression or activity of IGFR1 in the non-human species.

[0167] “Single-chain Fv” or “sFv” antibody fragments have the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. Techniques described for the production of single chain antibodies (U.S. Pat. Nos. 5,476,786; 5,132,405 and 4,946,778) can be adapted to produce anti-IGFR1-specific single chain antibodies. For a review of sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, N.Y., pp. 269-315 (1994).

[0168] “Disulfide stabilized Fv fragments” and “dsFv” refer to antibody molecules comprising a variable heavy chain (VH) and a variable light chain (VL) which are linked by a disulfide bridge.

[0169] Antibody fragments within the scope of the present invention also include F(ab)2 fragments which may be produced by enzymatic cleavage of an IgG by, for example, pepsin. Fab fragments may be produced by, for example, reduction of F(ab)2 with dithiothreitol or mercaptoethylamine. A Fab fragment is a VL-CL chain appended to a VH-CH1 chain by a disulfide bridge. A F(ab)2 fragment is two Fab fragments which, in turn, are appended by two disulfide bridges. The Fab portion of an F(ab)2 molecule includes a portion of the Fc region between which disulfide bridges are located.

[0170] An FV fragment is a VL or VH region.

[0171] Depending on the amino acid sequences of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are at least five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgG-1, IgG-2, IgG-3 and IgG-4; IgA-1 and IgA-2.

[0172] The anti-IGFR1 antibody molecules of the invention may also be conjugated to a chemical moiety. The chemical moiety may be, inter alia, a polymer, a radionuclide or a cytotoxic factor. Preferably the chemical moiety is a polymer which increases the half-life of the antibody molecule in the body of a subject. Suitable polymers include, but are not limited to, polyethylene glycol (PEG) (e.g., PEG with a molecular weight of 2 kDa, 5 kDa, 10 kDa, 12 kDa, 20 kDa, 30 kDa or 40 kDa), dextran and monomethoxypolyethylene glycol (mPEG). Lee, et al., (1999) (Bioconj. Chem. 10:973-981) discloses PEG conjugated single-chain antibodies. Wen, et al., (2001) (Bioconj. Chem. 12:545-553) disclose conjugating antibodies with PEG which is attached to a radiometal chelator (diethylenetriaminpentaacetic acid (DTPA)).

[0173] The antibodies and antibody fragments of the invention may also be conjugated with labels such as 99Tc, 90Y, 111In, 32P, 14C, 125I, 3H, 131I, 11C,15O, 13N, 18F, 35S, 51Cr, 57To, 226Ra, 60Co, 59Fe, 57Se, 152Eu, 67CU, 217Ci, 211At, 212Pb, 47Sc, 109Pd, 234Th, and 40K, 157Gd, 55Mn, 52Tr and 56Fe.

[0174] The antibodies and antibody fragments of the invention may also be conjugated with fluorescent or chemilluminescent labels, including fluorophores such as rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanate, phycoerythrin, phycocyanin, allophycocyanin, o-phthaladehyde, fluorescamine, 152Eu, dansyl, umbelliferone, luciferin, luminal label, isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridimium salt label, an oxalate ester label, an aequorin label, 2,3-dihydrophthalazinediones, biotin/avidin, spin labels and stable free radicals.

[0175] The antibody molecules may also be conjugated to a cytotoxic factor such as diptheria toxin, Pseudomonas aeruginosa exotoxin A chain , ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins and compounds (e.g., fatty acids), dianthin proteins, Phytoiacca americana proteins PAPI, PAPII, and PAP-S, momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, mitogellin, restrictocin, phenomycin, and enomycin.

[0176] Any method known in the art for conjugating the antibody molecules of the invention to the various moieties may be employed, including those methods described by Hunter, et al., (1962) Nature 144:945; David, et al., (1974) Biochemistry 13:1014; Pain, et al., (1981) J. Immunol. Meth. 40:219; and Nygren, J., (1982) Histochem. and Cytochem. 30:407. Methods for conjugating antibodies are conventional and very well known in the art.

Modified Antibody Molecules

[0177] The present invention includes antibodies and antigen-binding fragments (e.g., fully-human antibodies, SFv, dsFv, Fv, chimeric antibodies) comprising a light chain of SEQ ID NOs: 41, 43, 72, 74, 76 or 78 (15H12/19D12 LCA, LCB, LCC, LCD, LCE or LCF); preferably amino acids 20-128 of SEQ ID NOs: 41, 43, 72, 74, 76 or 78 (mature 15H12/19D12 LCA, LCB, LCC, LCD, LCE or LCF). The present invention also includes antibody molecules including the heavy chain of SEQ ID NO: 45 or 112 (15H12/19D12 HCA, HCB); preferably amino acids 20-137 of SEQ ID NO: 45 or 112 (mature 15H12/19D12 HCA, HCB).

[0178] The 15H12/19D12 LCA, LCB, LCC, LCD, LCE and LCF may be dimerized with any other immunoglobulin heavy chain, preferably an immunoglobulin heavy chain of the present invention. Likewise, 15H12/19D12 HCA or HCB may be dimerized with any light chain, preferably a light chain of the present invention. For example, 15H12/19D12 HCA or HCB may be dimerized with 15H12/19D12 LCC, LCD, LCE or LCF.

[0179] Antibodies and antigen-binding fragments comprising 15H12/19D12 LCA, 15H12/19D12 LCB, 15H12/19D12 LCC, 15H12/19D12 LCD, 15H12/19D12 LCE, 15H12/19D12 LCF, 15H12/19D12 HCA or 15H12/19D12 HCB or anyfragment thereof exhibit minimal immunogenicity in a human subject; thereby, leading to a low incidence of HAHA response when administered to a human subject.

[0180] Preferred antibody chains are shown below. Dotted underscored type encodes the signal peptide. Solid underscored type encodes the CDRs. Plain type encodes the framework regions. Most preferably, the antibody chains are mature fragments which lack the signal peptide.

[0181] Modified 19D12/15H12 Light Chain-C (SEQ ID NO: 71)