# Patent

## Title
Biomarkers for pre-selection of patients for anti-IGF1R therapy

## Abstract

The present invention provides methods for identifying patients whose cancers are likely to be responsive to IGF1R inhibitory anti-cancer therapy along with methods for treating such patients. Patients identified by a method of the present invention can be treated with any of several known IGF1R inhibitory agents including antibodies, small molecule inhibitors and anti-sense nucleic acids.

## Background
The present application claims the benefit of U.S. provisional patent application No. 60/633,156; filed Dec. 3, 2004, which is herein incorporated by reference in its entirety.


FIELD OF THE INVENTION
The present invention relates to methods for selecting patients for anti-cancer therapy.
BACKGROUND OF THE INVENTION
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 (IGF1R) (Sepp-Lorenzino, (1998) Breast Cancer Research and Treatment 47:235). Interaction of IGFs with IGF1R activates the receptor by triggering autophosphorylation of the receptor on tyrosine residues (Butler, et al., (1998) Comparative Biochemistry and Physiology 121:19). Once activated, IGF1R, 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 IGF1R activity represents a valuable potential method to treat or prevent growth of human cancers and other proliferative diseases.
Several lines of evidence indicate that IGF-I, IGF-II and their receptor IGF1R 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.
Overexpression of Insulin-like Growth Factor Receptor-1 has also been demonstrated in several cancer cell lines and tumor tissues. IGF1R 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, IGF1R is overexpressed 6-14 fold and IGF1R 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 IGF1R levels wherein the extent of IGF1R 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 IGF1R, respectively, as compared to normal ectocervical cells (Steller, et al., (1996) Cancer Res. 56:1762). Expression of IGF1R 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).
Currently, there are several known anti-cancer therapies that target IGF1R. For example, anti-IGF1R antibodies are owned by Schering Corp (see WO 2003/100008); Pfizer (see WO 2002/53596 or WO 2004/71529); Pierre Fabre medicament (see WO 2003/59951), Pharmacia Corp. (see WO 2004/83248), Immunogen, Inc. (see WO 2003/106621), Hoffman La Roche (see WO 2004/87756) and Imclone Systems Inc. (IMC-A12; see Burtrum et. al Cancer Research 63:8912-8921(2003)). Additionally, Novartis owns a small molecule IGFR inhibitor, NVP-ADW-742 (see WO 2002/92599) as does Biotech Research Ventures PTE Ltd (see WO 2003/39538). Antisense Therapeutics Ltd. also owns an anti-sense therapy that inhibits IGF1R expression, ATL-1101.
Agents that decrease IGF1R function and/or expression are effective in the treatment of some cancer patients. However, it is expected that a portion of cancer patients may not respond to such treatments. Therefore, a need exists in the art for a method to identify specific cancer populations and/or specific cancer patients who are most likely to respond to one or more anti-cancer therapies that target IGF1R.
SUMMARY OF THE INVENTION
The present invention provides, inter alia, a method for treating cancers by pre-selecting patients whose tumors express appreciable levels of IGF-II and/or phosphorylated IRS-1 (insulin receptor substrate-1), thereby increasing the likelihood of a response, in the patient, to therapeutics targeting IGF1R.
The present invention provides a method for treating a tumor in a patient comprising (a) selecting a patient or patient population having a tumor known to express one or more of the following:
(i) IRS-1 phosphorylation on tyrosine 896;
(ii) IRS-1 phosphorylation on tyrosine 612;
(iii) IRS-1 phosphorylation on any tyrosine;
(iv) IGF-II;
(v) IGF1R phosphorylation on any tyrosine; or
(vi) IGF1R; and
(b) administering to said patient a therapeutically effective amount of an IGF1R inhibitory agent.
The present invention comprises a method for treating a tumor in a patient comprising: (a) selecting a patient having a tumor expressing one or more of the following:
(i) IRS-1 phosphorylation on tyrosine 896;
(ii) IRS-1 phosphorylation on tyrosine 612;
(iii) IRS-1 phosphorylation on any tyrosine;
(iv) IGF-II;
(v) IGF1R phosphorylation on any tyrosine; or
(vi) IGF1R; and
(b) administering to said patient a therapeutically effective amount of an IGF1R inhibitory agent. In an embodiment of the invention, the cancer is selected from the group consisting of bladder cancer, Wilm's cancer, bone cancer, prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), colon cancer, rectal cancer, colorectal cancer, endometrial cancer, multiple myeloma, estrogen receptor-positive breast cancer, estrogen receptor-negative breast cancer, cervical cancer, synovial sarcoma, ovarian cancer, pancreatic cancer, neuroblastoma, rhabdomyosarcoma, osteosarcoma and vasoactive intestinal peptide secreting tumors. In an embodiment of the invention, the agent is selected from the group consisting of an isolated antibody or antigen-binding fragment thereof that binds specifically to human IGF1R and is a member selected from the group consisting of: (i) an isolated antibody or antigen-binding fragment thereof that binds specifically to human IGF1R comprising one or more CDRs from a light chain variable region comprising amino acids 20-128 of SEQ ID NO: 8 and/or one or more CDRs from a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 10; (ii) an isolated antibody or antigen-binding fragment thereof comprising one or more CDRs from a heavy chain immunoglobulin comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 19-28, 35-38, 43, 45 or 73-98;

(iii) an isolated antibody or antigen-binding fragment thereof comprising one or more CDRs from a light chain immunoglobulin comprising the amino acid sequence of SEQ ID NO: 10, 12-18, 29-34, 39, 40, 41, 42, 44 or 58-72; and (iv) an isolated single-chain antibody (scfv) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 46-51; or

(v)





or ATL-1101. In an embodiment of the invention, the isolated antibody or antigen-binding fragment thereof comprises: (i) an isolated immunoglobulin heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 19-28, 35-38, 43, 45 and 73-98; (ii) an isolated immunoglobulin light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12-18, 29-34, 39, 40, 41, 42, 44 and 58-72; (iii) an isolated antibody produced by a hybridoma deposited at the American Type Culture Collection under deposit number PTA-2792, PTA-2788, PTA-2790, PTA-2791, PTA-2789 or PTA-2793; (iv) an isolated antibody or antigen-binding fragment thereof that binds specifically to human IGF1R comprising a light chain variable region comprising amino acids 20-128 of SEQ ID NO: 8 and/or a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 10; and/or (v) an isolated antibody comprising an immunoglobulin light chain encoded by the plasmid contained in the cell line deposited at the American Type Culture Collection under deposit number PTA-5220 and an immunoglobulin heavy chain encoded by the plasmid contained in a cell line deposited at the American Type Culture Collection under deposit number PTA-5214 or PTA-5216. In an embodiment of the invention, phosphorylation of tyrosine on IRS-1 or IGF1R is determined by western blot analysis, ELISA or flow cytometry analysis. In an embodiment of the invention, IGF-II expression is determined by western blot analysis, ELISA, quantitative PCR or by northern blot analysis. In an embodiment of the invention, IGF1R expression is determined by western blot analysis or ELISA.

The present invention provides a method for selecting a therapy for a patient or a patient population with a tumor, comprising: (a) determining whether the patient's tumor is known to express one or more of the following:
(i) IRS-1 phosphorylation on tyrosine 896;
(ii) IRS-1 phosphorylation on tyrosine 612;
(iii) IRS-1 phosphorylation on any tyrosine;
(iv) IGF-II;
(v) IGF1R phosphorylation on any tyrosine; or
(vi) IGF1R; and/or
(b) determining whether the patient's tumor expresses one or more of the following:
(i) IRS-1 phosphorylation on tyrosine 896;
(ii) IRS-1 phosphorylation on tyrosine 612;
(iii) IRS-1 phosphorylation on any tyrosine;
(iv) IGF-II;
(v) IGF1R phosphorylation on any tyrosine; or
(vi) IGF1R; and
(c) selecting an IGF1R inhibitory agent as the therapy if the patient's tumor is known to express one or more of (i)-(vi) and/or if the patient's tumor expresses one or more of (i)-(vi). In an embodiment of the invention, the agent is selected from the group consisting of an isolated antibody or antigen-binding fragment thereof that binds specifically to human IGF1R and is a member selected from the group consisting of: (i) an isolated antibody or antigen-binding fragment thereof that binds specifically to human IGF1R comprising one or more CDRs from a light chain variable region comprising amino acids 20-128 of SEQ ID NO: 8 and/or a one or more CDRs from a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 10; (ii) an isolated antibody or antigen-binding fragment thereof comprising one or more CDRs from a heavy chain immunoglobulin comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 19-28, 35-38, 43, 45 or 73-98; (iii) an isolated antibody or antigen-binding fragment thereof comprising one or more CDRs from a light chain immunoglobulin comprising the amino acid sequence of SEQ ID NO: 10, 12-18, 29-34, 39, 40, 41, 42, 44 or 58-72; and (iv) an isolated single-chain antibody (scfv) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 46-51; or




or ATL-1101. In an embodiment of the invention, the isolated antibody or antigen-binding fragment thereof comprises: (i) an isolated immunoglobulin heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 19-28, 35-38, 43, 45 and 73-98; (ii) an isolated immunoglobulin light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12-18, 29-34, 39, 40, 41, 42, 44 and 58-72; (iii) an isolated antibody produced by a hybridoma deposited at the American Type Culture Collection under deposit number PTA-2792, PTA-2788, PTA-2790, PTA-2791, PTA-2789 or PTA-2793; (iv) an isolated antibody or antigen-binding fragment thereof that binds specifically to human IGF1R comprising a light chain variable region comprising amino acids 20-128 of SEQ ID NO: 8 and/or a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 10; and/or (v) an isolated antibody comprising an immunoglobulin light chain encoded by the plasmid contained in the cell line deposited at the American Type Culture Collection under deposit number PTA-5220 and an immunoglobulin heavy chain encoded by the plasmid contained in a cell line deposited at the American Type Culture Collection under deposit number PTA-5214 or PTA-5216.

The present invention also provides a method for advertising an IGF1R inhibitory agent or a pharmaceutically acceptable composition thereof comprising promoting, to a target audience, the use of the agent or pharmaceutical composition thereof for treating a patient or patient population whose tumors express or are known to express one or more of the following:
(i) IRS-1 phosphorylation on tyrosine 896;
(ii) IRS-1 phosphorylation on tyrosine 612;
(iii) IRS-1 phosphorylation on any tyrosine;
(iv) IGF-II;
(v) IGF1R phosphorylation on any tyrosine; or
(vi) IGF1R.
In an embodiment of the invention, the agent is selected from the group consisting of an isolated antibody or antigen-binding fragment thereof that binds specifically to human IGF1R and is a member selected from the group consisting of: (i) an isolated antibody or antigen-binding fragment thereof that binds specifically to human IGF1R comprising one or more CDRs from a light chain variable region comprising amino acids 20-128 of SEQ ID NO: 8 and/or one or more CDRs from a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 10; (ii) an isolated antibody or antigen-binding fragment thereof comprising one or more CDRs from a heavy chain immunoglobulin comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 19-28, 35-38, 43, 45 or 73-98; (iii) an isolated antibody or antigen-binding fragment thereof comprising one or more CDRs from a light chain immunoglobulin comprising the amino acid sequence of SEQ ID NO: 10, 12-18, 29-34, 39, 40, 41, 42, 44 or 58-72; and (iv) an isolated single-chain antibody (scfv) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 46-51; or




or ATL-1101. In an embodiment of the invention, the isolated antibody or antigen-binding fragment thereof comprises: (i) an isolated immunoglobulin heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 19-28, 35-38, 43, 45 and 73-98; (ii) an isolated immunoglobulin light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12-18, 29-34, 39, 40, 41, 42, 44 and 58-72; (iii) an isolated antibody produced by a hybridoma deposited at the American Type Culture Collection under deposit number PTA-2792, PTA-2788, PTA-2790, PTA-2791, PTA-2789 or PTA-2793; (iv) an isolated antibody or antigen-binding fragment thereof that binds specifically to human IGF1R comprising a light chain variable region comprising amino acids 20-128 of SEQ ID NO: 8 and/or a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 10; and/or (v) an isolated antibody comprising an immunoglobulin light chain encoded by the plasmid contained in the cell line deposited at the American Type Culture Collection under deposit number PTA-5220 and an immunoglobulin heavy chain encoded by the plasmid contained in a cell line deposited at the American Type Culture Collection under deposit number PTA-5214 or PTA-5216.

The present invention also provides an article of manufacture comprising, packaged together, a pharmaceutical composition comprising an IGF1R inhibitory agent and a pharmaceutically acceptable carrier and a label stating that the agent or pharmaceutical composition is indicated for treating patients having a tumor expressing or known to express one or more of the following:
(i) IRS-1 phosphorylation on tyrosine 896;
(ii) IRS-1 phosphorylation on tyrosine 612;
(iii) IRS-1 phosphorylation on any tyrosine;
(iv) IGF-II;
(v) IGF1R phosphorylation on any tyrosine; or
(vi) IGF1R.
In an embodiment of the invention, the agent is selected from the group consisting of an isolated antibody or antigen-binding fragment thereof that binds specifically to human IGF1R and is a member selected from the group consisting of: (i) an isolated antibody or antigen-binding fragment thereof that binds specifically to human IGF1R comprising one or more CDRs from a light chain variable region comprising amino acids 20-128 of SEQ ID NO: 8 and/or a one or more CDRs from a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 10; (ii) an isolated antibody or antigen-binding fragment thereof comprising one or more CDRs from a heavy chain immunoglobulin comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 19-28, 35-38, 43, 45 or 73-98; (iii) an isolated antibody or antigen-binding fragment thereof comprising one or more CDRs from a light chain immunoglobulin comprising the amino acid sequence of SEQ ID NO: 10, 12-18, 29-34, 39, 40, 41, 42, 44 or 58-72; and (iv) an isolated single-chain antibody (scfv) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 46-51; or




or ATL-1101. In an embodiment of the invention, the isolated antibody or antigen-binding fragment thereof comprises: (i) an isolated immunoglobulin heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 19-28, 35-38, 43, 45 and 73-98; (ii) an isolated immunoglobulin light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12-18, 29-34, 39, 40, 41, 42, 44 and 58-72; (iii) an isolated antibody produced by a hybridoma deposited at the American Type Culture Collection under deposit number PTA-2792, PTA-2788, PTA-2790, PTA-2791, PTA-2789 or PTA-2793; (iv) an isolated antibody or antigen-binding fragment thereof that binds specifically to human IGF1R comprising a light chain variable region comprising amino acids 20-128 of SEQ ID NO: 8 and/or a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 10; and/or (v) an isolated antibody comprising an immunoglobulin light chain encoded by the plasmid contained in the cell line deposited at the American Type Culture Collection under deposit number PTA-5220 and an immunoglobulin heavy chain encoded by the plasmid contained in a cell line deposited at the American Type Culture Collection under deposit number PTA-5214 or PTA-5216.

The present invention further provides a method for manufacturing an IGF1R inhibitory agent or a pharmaceutical composition thereof comprising combining in a package the agent or pharmaceutical composition and a label stating that the agent or pharmaceutical composition is indicated for treating patients having a tumor expressing or known to express one or more of the following:
(i) IRS-1 phosphorylation on tyrosine 896;
(ii) IRS-1 phosphorylation on tyrosine 612;
(iii) IRS-1 phosphorylation on any tyrosine;
(iv) IGF-II;
(v) IGF1R phosphorylation on any tyrosine; or
(vi) IGF1R.
In an embodiment of the invention, the agent is selected from the group consisting of an isolated antibody or antigen-binding fragment thereof that binds specifically to human IGF1R and is a member selected from the group consisting of: (i) an isolated antibody or antigen-binding fragment thereof that binds specifically to human IGF1R comprising one or more CDRs from a light chain variable region comprising amino acids 20-128 of SEQ ID NO: 8 and/or a one or more CDRs from a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 10; (ii) an isolated antibody or antigen-binding fragment thereof comprising one or more CDRs from a heavy chain immunoglobulin comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 19-28, 35-38, 43, 45 or 73-98; (iii) an isolated antibody or antigen-binding fragment thereof comprising one or more CDRs from a light chain immunoglobulin comprising the amino acid sequence of SEQ ID NO: 10, 12-18, 29-34, 39, 40, 41, 42, 44 or 58-72; and (iv) an isolated single-chain antibody (scfv) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 46-51;




or ATL-1101. In an embodiment of the invention, the isolated antibody or antigen-binding fragment thereof comprises: (i) an isolated immunoglobulin heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 19-28, 35-38, 43, 45 and 73-98; (ii) an isolated immunoglobulin light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12-18, 29-34, 39, 40, 41, 42, 44 and 58-72; (iii) an isolated antibody produced by a hybridoma deposited at the American Type Culture Collection under deposit number PTA-2792, PTA-2788, PTA-2790, PTA-2791, PTA-2789 or PTA-2793; (iv) an isolated antibody or antigen-binding fragment thereof that binds specifically to human IGF1R comprising a light chain variable region comprising amino acids 20-128 of SEQ ID NO: 8 and/or a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 10; and/or (v) an isolated antibody comprising an immunoglobulin light chain encoded by the plasmid contained in the cell line deposited at the American Type Culture Collection under deposit number PTA-5220 and an immunoglobulin heavy chain encoded by the plasmid contained in a cell line deposited at the American Type Culture Collection under deposit number PTA-5214 or PTA-5216.

The present invention also provides a method for identifying a patient whose tumor is likely to be responsive to an IGF1R inhibitory agent comprising: (a) determining whether the patient has a tumor known to express one or more of the following:
(i) IRS-1 phosphorylation on tyrosine 896;
(ii) IRS-1 phosphorylation on tyrosine 612;
(iii) IRS-1 phosphorylation on any tyrosine;
(iv) IGF-II;
(v) IGF1R phosphorylation on any tyrosine; or
(vi) IGF1R; and/or
(b) determining whether the patient has a tumor expressing one or more of the following:
(i) IRS-1 phosphorylation on tyrosine 896;
(ii) IRS-1 phosphorylation on tyrosine 612;
(iii) IRS-1 phosphorylation on any tyrosine;
(iv) IGF-II;
(v) IGF1R phosphorylation on any tyrosine; or
(vi) IGF1R.
In an embodiment of the invention, the agent is selected from the group consisting of an isolated antibody or antigen-binding fragment thereof that binds specifically to IGF1R and is a member selected from the group consisting of: (i) an isolated antibody or antigen-binding fragment thereof that binds specifically to human IGF1R comprising one or more CDRs from a light chain variable region comprising amino acids 20-128 of SEQ ID NO: 8 and/or a one or more CDRs from a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 10; (ii) an isolated antibody or antigen-binding fragment thereof comprising one or more CDRs from a heavy chain immunoglobulin comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 19-28, 35-38, 43, 45 or 73-98; (iii) an isolated antibody or antigen-binding fragment thereof comprising one or more CDRs from a light chain immunoglobulin comprising the amino acid sequence of SEQ ID NO: 10, 12-18, 29-34, 39, 40, 41, 42, 44 or 58-72; and (iv) an isolated single-chain antibody (scfv) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 46-51; or (v)




or ATL-1101. In an embodiment of the invention, the isolated antibody or antigen-binding fragment thereof comprises: (i) an isolated immunoglobulin heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 19-28, 35-38, 43, 45 and 73-98; (ii) an isolated immunoglobulin light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12-18, 29-34, 39, 40, 41, 42, 44 and 58-72; (iii) an isolated antibody produced by a hybridoma deposited at the American Type Culture Collection under deposit number PTA-2792, PTA-2788, PTA-2790, PTA-2791, PTA-2789 or PTA-2793; (iv) an isolated antibody or antigen-binding fragment thereof that binds specifically to human IGF1R comprising a light chain variable region comprising amino acids 20-128 of SEQ ID NO: 8 and/or a heavy chain variable region comprising amino acids 20-137 of SEQ ID NO: 10; and/or (v) an isolated antibody comprising an immunoglobulin light chain encoded by the plasmid contained in the cell line deposited at the American Type Culture Collection under deposit number PTA-5220 and an immunoglobulin heavy chain encoded by the plasmid contained in a cell line deposited at the American Type Culture Collection under deposit number PTA-5214 or PTA-5216.




BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. IRS-1 Phosphorylation is Higher in Human Lung Tumor vs. Normal Tissue Samples. Western blot analysis results for normal and tumor tissue samples from four different patients. Lanes marked “T” contained tumor tissue and lanes marked “N” contained normal tissue.
FIG. 2. Antibody 19D12/15H12 LCF/HCA: In Vivo Efficacy. The level of tumor growth inhibition observed in xenograft mice administered antibody 19D12/15H12 LCF/HCA is indicated along with the type of tumor evaluated and the cell line used to establish each tumor.
FIG. 3. In Vivo Efficacy of 19D12/15H12 LCF/HCA Correlates with Sensitivity of IRS-1 Phosphorylation to IGF-I. In the “−” and “+” lanes, the quantity of phosphorylated IRS-1, in each cell line evaluated, is shown in the absence and presence of IGF-I, respectively. The level of in vivo efficacy of 19D12/15H12 LCF/HCA at inhibiting growth of the indicated cell line (see FIG. 2) is also indicated.
FIG. 4. Overexpression of IGF-II mRNA in Human Ovarian Tumor Samples. The normalized level of IGF-II mRNA expression observed in each of the 20 normal ovarian tissue samples and 36 cancerous ovarian tissue samples is shown.
FIG. 5. Overexpression of IGF-II mRNA in Human Colorectal Tumor Samples. The normalized level of IGF-II mRNA expression observed in each of the 36 normal ovarian tissue samples and 36 cancerous colorectal tissue samples is shown.



DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method for treating cancer or for identifying patients whose cancer is likely to be responsive to an IGF1R inhibitory agent. The method is useful, inter alia, for increasing the likelihood that administration of an IGF1R inhibitory anti-cancer therapy to a patient will be efficacious.
The terms “IGF1R”, “IGFR1”, “Insulin-like Growth Factor Receptor-I” and “Insulin-like Growth Factor Receptor, type I” are well known in the art. Although IGF1R 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 IGF1R precursor has the Genbank Accession No. X04434 or NM—000875. 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.
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 II” 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 and NM—000612, respectively.
IGF1R Inhibitory Agents
The term “IGF1R inhibitory agent” includes any substance that decreases the expression, ligand binding, kinase activity or any other biological activity of IGF1R that will elicit a biological or medical response of a tissue, system, subject or patient that is being sought by the administrator (such as a researcher, doctor or veterinarian) which includes any measurable alleviation of the signs, symptoms and/or clinical indicia of cancer (e.g., tumor growth) and/or the prevention, slowing or halting of progression or metastasis of cancer to any degree.
In an embodiment of the invention, an IGF1R inhibitory agent that can be administered to a patient in a method according to the invention is any isolated anti-insulin-like growth factor receptor-1 (IGF1R) antibody or fragment thereof (e.g., monoclonal antibodies (e.g., fully human 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, dsFv antibody fragments, humanized antibodies, chimeric antibodies or anti-idiotypic antibodies) such as any of those disclosed in any of Burtrum et. al Cancer Research 63:8912-8921(2003); in French Patent Applications FR2834990, FR2834991 and FR2834900 and in PCT Application Publication Nos. WO 03/100008; WO 03/59951; WO 04/71529; WO 03/106621; WO 04/83248; WO 04/87756 and WO 02/53596.
In an embodiment of the invention, an IGF1R inhibitory agent that can be administered to a patient in a method according to the invention is an isolated anti-insulin-like growth factor receptor-1 (IGF1R) antibody comprising a mature or unprocessed 19D12/15H12 Light Chain-C, D, E or F and a mature 19D12/15H12 heavy chain-A or B. In an embodiment of the invention, an IGF1R inhibitory agent that can be administered to a patient in a method according to the invention is an isolated antibody that specifically binds to IGF1R that comprises one or more complementarity determining regions (CDRs) of 19D12/15H12 Light Chain-F and/or 19D12/15H12 heavy chain-A (e.g., all 3 light chain CDRs and all 3 heavy chain CDRs).
The amino acid and nucleotide sequences of the 19D12/15H12 antibody chains are shown below. Dotted, underscored type indicates the signal peptide. Solid underscored type indicates the CDRs. Plain type indicates the framework regions. Mature fragments lack the signal peptide.












Modified 19D12/15H12 Light Chain-C (SEQ ID NO: 1)


 








 


(SEQ ID NO: 2)


 








 


Modified 19D12/15H12 Light Chain-D (SEQ ID NO: 3)


 








 


(SEQ ID NO: 4)


 








 


Modified 19D12/15H12 Light Chain-E (SEQ ID NO: 5)


 








 


(SEQ ID NO: 6)


 








 


19D12/15H12 Light Chain-F (LCF; SEQ ID NO: 7)


 








 


(SEQ ID NO: 8)


 








 


19D12/15H12 heavy chain-A (HCA; SEQ ID NO: 9)


 








 


(SEQ ID NO: 10)


 








 


Modified 19D12/15H12 heavy chain-B (SEQ ID NO: 11)


 








 


(SEQ ID NO: 12)


 















Plasmids comprising a CMV promoter operably linked to the 15H12/19D12 LCC, LCD, LCE, LCF or to the 15H12/19D12 HCA or HCB have been deposited at the American Type Culture Collection (ATCC); 10801 University Boulevard; Manassas, Va. 20110-2209 on May 21, 2003. The deposit names and the ATCC accession numbers for the plasmids are set forth below:
(1) CMV promoter-15H12/19D12 HCA (γ4)—
Deposit name: “15H12/19D12 HCA (γ4)”
ATCC accession No.: PTA-5214
(2) CMV promoter-15H12/19D12 HCB (γ4)—
Deposit name: “15H12/19D12 HCB (γ4)”
ATCC accession No.: PTA-5215
(3) CMV promoter-15H12/19D12 HCA (γ1)—
Deposit name: “15H12/19D12 HCA (γ1)”;
ATCC accession No.: PTA-5216
(4) CMV promoter-15H12/19D12 LCC (κ)—
Deposit name: “15H12/19D12 LCC (κ)”;
ATCC accession No.: PTA-5217
(5) CMV promoter-15H12/19D12 LCD (κ)—
Deposit name: “15H12/19D12 LCD (κ)”;
ATCC accession No.: PTA-5218
(6) CMV promoter-15H12/19D12 LCE (κ)—
Deposit name: “15H12/19D12 LCE (κ)”;
ATCC accession No.: PTA-5219
(7) CMV promoter-15H12/19D12 LCF (κ)—
Deposit name: “15H12/19D12 LCF (κ)”;
ATCC accession No.: PTA-5220
All restrictions on access to the plasmids deposited in ATCC will be removed upon grant of a patent. In an embodiment of the present invention, an anti-IGF1R antibody or antigen-binding fragment thereof of the invention comprises any of the CDRs or Ig heavy or light chains or variable regions thereof in any of PTA-5214-PTA-5220. In an embodiment of the invention, the antibody comprises a light chain encoded by the plasmid deposited under number PTA-5220 and a heavy chain encoded by the plasmid deposited under number PTA-5214 or PTA-5216.
In an embodiment, an antibody that binds “specifically” to human IGF1R binds with Kd of about 1.28×10−10 M or less by Biacore measurement or with a Kd of about 2.05×10−12 or less by KinExA measurement.
In an embodiment of the invention, an IGF1R inhibitory agent that can be administered to a patient in a method according to the invention comprises any light chain immunoglobulin and/or a heavy chain immunoglobulin as set forth in Published International Application No. WO 2002/53596 which is herein incorporated by reference in its entirety. For example, in an embodiment, the antibody comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 47 and 51 as set forth in WO 2002/53596 and/or a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 45 and 49 as set forth in WO 2002/53596.
In an embodiment of the invention, an IGF1R inhibitory agent that can be administered to a patient in a method according to the invention comprises any light chain immunoglobulin and/or a heavy chain immunoglobulin as set forth in Published International Application No. WO 2003/59951 which is herein incorporated by reference in its entirety. For example, in an embodiment, the antibody comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 54, 61 and 65 as set forth in WO 2003/59951 and/or a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 69, 75, 79 and 83 as set forth in WO 2003/59951.
In an embodiment of the invention, an IGF1R inhibitory agent that can be administered to a patient in a method according to the invention comprises any light chain immunoglobulin and/or a heavy chain immunoglobulin as set forth in Published International Application No. WO 2004/83248 which is herein incorporated by reference in its entirety. For example, in an embodiment, the antibody comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141 and 143 as set forth in WO 2004/83248 and/or a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140 and 142 as set forth in WO 2004/83248.
In an embodiment of the invention, an IGF1R inhibitory agent that can be administered to a patient in a method according to the invention comprises any light chain immunoglobulin and/or a heavy chain immunoglobulin as set forth in Published International Application No. WO 2003/106621 which is herein incorporated by reference in its entirety. For example, in an embodiment, the antibody comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-12, 58-69, 82-86, 90, 94, 96, 98, as set forth in WO 2003/106621 and/or a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 13, 70-81, 87, 88, 92 as set forth in WO 2003/106621.
In an embodiment of the invention, an IGF1R inhibitory agent that can be administered to a patient in a method according to the invention comprises any light chain immunoglobulin and/or a heavy chain immunoglobulin as set forth in Published International Application No. WO 2004/87756 which is herein incorporated by reference in its entirety. For example, in an embodiment, the antibody comprises a light chain variable region comprising an amino acid sequence of SEQ ID NO: 2 as set forth in WO 2004/87756 and/or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 1 as set forth in WO 2004/87756.
Furthermore, the scope of the present invention comprises any antibody or antibody fragment comprising one or more CDRs and/or framework regions of any of the light chain immunoglobulin or heavy chain immunoglobulins set forth in WO 2002/53596; WO 2003/59951; WO 2004/83248; WO 2003/106621 or WO 2004/87756 as identified by any of the methods set forth in Chothia et al., J. Mol. Biol. 186:651-663 (1985); Novotny and Haber, Proc. Natl. Acad. Sci. USA 82:4592-4596 (1985) or Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., (1987)).
In an embodiment of the invention, anti-IGF1R antibody is produced by a hybridoma that is deposited at the American Type Culture Collection under deposit no. PTA-2792, PTA-2788, PTA-2790, PTA-2791, PTA-2789 or PTA-2793.
In an embodiment of the invention, an anti-IGF1R antibody of the invention comprises an immunoglobulin heavy chain variable region comprising an amino acid sequence selected from the group consisting of:








(SEQ ID NO: 13)










1
grlgqawrsl rlscaasgft fsdyymswir qapgkglewv syisssgstr



51
dyadsvkgrf tisrdnakns lylqmnslra edtavyycvr dgvettfyyy


101
yygmdvwgqg ttvtvssast kgpsvfplap csrstsesta algclvkdyf


151
pepvtvswns galtsgvhtf psca


 








(SEQ ID NO: 14)










1
vqllesgggl vqpggslrls ctasgftfss yamnwvrqap gkglewvsai



51
sgsggttfya dsvkgrftis rdnsrttlyl qmnslraedt avyycakdlg


101
wsdsyyyyyg mdvwgqgttv tvss


 








(SEQ ID NO: 15)










1
gpglvkpset lsltctvsgg sisnyywswi rqpagkglew igriytsgsp



51
nynpslksrv tmsvdtsknq fslklnsvta adtavyycav tifgvviifd


101
ywgqgtlvtv ss


 








(SEQ ID NO: 16)










1
evqllesggg lvqpggslrl scaasgftfs syamswvrqa pgkglewvsa



51
isgsggityy adsvkgrfti srdnskntly lqmnslraed tavyycakdl


101
gygdfyyyyy gmdvwgqgtt vtvss


 








(SEQ ID NO: 17)










1
pglvkpsetl sltctvsggs issyywswir qppgkglewi gyiyysgstn



51
ynpslksrvt isvdtsknqf slklssvtaa dtavyycart ysssfyyygm


101
dvwgqgttvt vss


 








(SEQ ID NO: 18)










1
evqllesggg lvqpggslrl scaasgftfs syamswvrqa pgkglewvsg



51
itgsggstyy adsvkgrfti srdnskntly lqmnslraed tavyycakdp


101
gttvimswfd pwgqgtlvtv ss






In an embodiment of the invention, an anti-IGF1R antibody of the invention comprises an immunoglobulin light chain variable region comprising an amino acid sequence selected from the group consisting of:








(SEQ ID NO: 19)










1
asvgdrvtft crasqdirrd lgwyqqkpgk apkrliyaas rlqsgvpsrf



51
sgsgsgteft ltisslqped fatyyclqhn nyprtfgqgt eveiirtvaa


101
psvfifppsd eqlksgtasv vcllnnfypr eakvqw


 








(SEQ ID NO: 20)










1
diqmtqfpss lsasvgdrvt itcrasqgir ndlgwyqqkp gkapkrliya



51
asrlhrgvps rfsgsgsgte ftltisslqp edfatyyclq hnsypcsfgq


101
gtkleik


 








(SEQ ID NO: 21)










1
sslsasvgdr vtftcrasqd irrdlgwyqq kpgkapkrli yaasrlqsgv



51
psrfsgsgsg teftltissl qpedfatyyc lqhnnyprtf gqgteveiir


 








(SEQ ID NO: 22)










1
diqmtqspss lsasvgdrvt itcrasqgir sdlgwfqqkp gkapkrliya



51
asklhrgvps rfsgsgsgte ftltisrlqp edfatyyclq hnsypltfgg


101
gtkveik


 








(SEQ ID NO: 23)










1
gdrvtitcra sqsistflnw yqqkpgkapk llihvasslq ggvpsrfsgs



51
gsgtdftlti sslqpedfat yycqqsynap ltfgggtkve ik


 








(SEQ ID NO: 24)










1
ratlscrasq svrgrylawy qqkpgqaprl liygassrat gipdrfsgsg



51
sgtdftltis rlepedfavf ycqqygsspr tfgqgtkvei k






In an embodiment of the invention, the anti-IGF1R antibody comprises a light chain immunoglobulin, or a mature fragment thereof (i.e., lacking signal sequence), or variable region thereof, comprising the amino acid sequence of:








(SEQ ID NO: 25)










1
mdmrvpaqll gllllwfpga rcdiqmtqsp sslsasvgdr vtitcrasqg



51
irndlgwyqq kpgkapkrli yaasslgsgv psrfsgsgsg teftltissl


101
qpedfatyyc lqhnsypwtf gqgtkveikr tvaapsvfif ppsdeqlksg


151
tasvvcllnn fypreakvqw kvdnalqsgn sqesvteqds kdstyslsst


201
ltlskadyek hkvyacevth qglsspvtks fnrgec;


 








(SEQ ID NO: 26)










1
mdmrvpaqll gllllwfpga rcdiqmtqsp sslsasvgdr vtftcrasqd



51
irrdlgwyqq kpgkapkrli yaasrlqsgv psrfsgsgsg teftltissl


101
qpedfatyyc lqhnnyprtf gqgteveiir tvaapsvfif ppsdeqlksg


151
tasvvcllnn fypreakvqw kvdnalqsgn sqesvteqds kdstyslsst


201
ltlskadyek hkvyacevth qglsspvtks fnrgec;


 








(SEQ ID NO: 27)










1
mdmrvpaqll gllllwfpga rcdiqmtqsp sslsasvgdr vtitcrasqg



51
irndlgwyqq kpgkapkrli yaasslqsgv psrfsgsgsg teftltissl


101
qpedfatyyc lqhnsypytf gggtkleikr tvaapsvfif ppsdeqlksg


151
tasvvcllnn fypreakvqw kvdnalqsgn sqesvteqds kdstyslsst


201
ltlskadyek hkvyacevth qglsspvtks fnrgec;


or


 








(SEQ ID NO: 28)










1
mdmrvpaqll gllllwfpga rcdiqmtqfp sslsasvgdr vtitcrasqg



51
irndlgwyqq kpgkapkrli yaasrlhrgv psrfsgsgsg teftltissl


101
qpedfatyyc lqhnsypcsf gggtkleikr tvaapsvfif ppsdeqlksg


151
tasvvcllnn fypreakvqw kvdnalqsgn sqesvteqds kdstyslsst


201
ltlskadyek hkvyacevth qglsspvtks fnrgec.






In an embodiment of the invention, the signal sequence is amino acids 1-22 of SEQ ID NOs: 25-28. In an embodiment of the invention, the mature variable region is underscored.

In an embodiment of the invention, the anti-IGF1R antibody comprises a heavy chain immunoglobulin or a mature fragment thereof (i.e., lacking signal sequence), or a variable region thereof, comprising the amino acid sequence of:








(SEQ ID NO: 29)










1
mefglswvfl vaiikgvqcq vqlvesgggl vkpggslrls caasgftfsd



51
yymswirqap gkglewvsyi sssgstiyya dsvkgrftis rdnaknslyl


101
qmnslraedt avyycarvlr flewllyyyy yygmdvwgqg ttvtvssast


151
kgpsvfplap csrstsesta algclvkdyf pepvtvswns galtsgvhtf


201
pavlqssgly slssvvtvps snfgtqtytc nvdhkpsntk vdktverkcc


251
vecppcpapp vagpsvflfp pkpkdtlmis rtpevtcvvv dvshedpevq


301
fnwyvdgvev hnaktkpree qfnstfrvvs vltvvhqdwl ngkeykckvs


351
nkglpapiek tisktkgqpr epqvytlpps reemtknqvs ltclvkgfyp


401
sdiavewesn gqpennyktt ppmldsdgsf flyskltvdk srwqqgnvfs


451
csvmhealhn hytqkslsls pgk;


 








(SEQ ID NO: 30)










1
mefglswvfl vaiikgvqcq aqlvesgggl vkpggslrls caasgftfsd



51
yymswirqap gkglewvsyi sssgstrdya dsvkgrftis rdnaknslyl


101
qmnslraedt avyycvrdgv ettfyyyyyg mdvwgqgttv tvssastkgp


151
svfplapcsr stsestaalg clvkdyfpep vtvswnsgal tsgvhtfpav


201
lqssglysls svvtvpssnf gtqtytcnvd hkpsntkvdk tverkccvec


251
ppcpappvag psvflfppkp kdtlmisrtp evtcvvvdvs hedpevqfnw


301
yvdgvevhna ktkpreeqfn stfrvvsvlt vvhqdwlngk eykckvsnkg


351
lpapiektis ktkgqprepq vytlppsree mtknqvsltc lvkgfypsdi


401
avewesngqp ennykttppm ldsdgsffly skltvdksrw qqgnvfscsv


451
mhealhnhyt qkslslspgk;


 








(SEQ ID NO: 31)










1
mefglswlfl vailkgvqce vqllesgggl vqpggslrls caasgftfss



51
yamswvrqap gkglewvsai sgsggstyya dsvkgrftis rdnskntlyl


101
qmnslraedt avyycakgys sgwyyyyyyg mdvwgqgttv tvssastkgp


151
svfplapcsr stsestaalg clvkdyfpep vtvswnsgal tsgvhtfpav


201
lqssglysls svvtvpssnf gtqtytcnvd hkpsntkvdk tverkccvec


251
ppcpappvag psvflfppkp kdtlmisrtp evtcvvvdvs hedpevqfnw


301
yvdgvevhna ktkpreeqfn stfrvvsvlt vvhqdwlngk eykckvsnkg


351
lpapiektis ktkgqprepq vytlppsree mtknqvsltc lvkgfypsdi


401
avewesngqp ennykttppm ldsdgsffly skltvdksrw qqgnvfscsv


451
mhealhnhyt qkslslspgk;


or


 








(SEQ ID NO: 32)










1
mefglswlfl vailkgvqce vqllesgggl vqpggslrls ctasgftfss



51
yamnwvrqap gkglewvsai sgsggttfya dsvkgrftis rdnsrttlyl


101
qmnslraedt avyycakdlg wsdsyyyyyg mdvwgqgttv tvssastkgp


151
svfplapcsr stsestaalg clvkdyfpep vtvswnsgal tsgvhtfpav


201
lqssglysls svvtvpssnf gtqtytcnvd hkpsntkvdk tverkccvec


251
ppcpappvag psvflfppkp kdtlmisrtp evtcvvvdvs hedpevqfnw


301
yvdgvevhna ktkpreeqfn stfrvvsvlt vvhqdwlngk eykckvsnkg


351
lpapiektis ktkgqprepq vytlppsree mtknqvsltc lvkgfypsdi


401
avewesngqp ennykttppm ldsdgsffly skltvdksrw qqgnvfscsv


451
mhealhnhyt qkslslspgk.






In an embodiment of the invention, the signal sequence is amino acids 1-19 of SEQ ID NOs: 29-32. In an embodiment of the invention, the mature variable region is underscored.

In an embodiment of the invention, the anti-IGF1R antibody comprises a light chain variable region comprising the amino acid sequence of any of SEQ ID NOs: 19-24 paired with a heavy chain variable region comprising an amino acid sequence of any of SEQ ID NOs: 13-18, respectively. In an embodiment of the invention, the anti-IGF1R antibody comprises a mature light chain variable region comprising an amino acid sequence of any of SEQ ID NOs: 25 or 26 paired with a heavy chain variable region comprising an amino acid sequence of any of SEQ ID NOs: 29 or 30. In an embodiment of the invention, the anti-IGF1R antibody comprises a mature light chain variable region comprising an amino acid sequence of any of SEQ ID NOs: 27 or 28 paired with a heavy chain variable region comprising an amino acid sequence of any of SEQ ID NOs: 31 or 32.
In an embodiment of the invention, an anti-IGF1R antibody of the invention comprises an immunoglobulin heavy chain or mature fragment or variable region of 2.12.1 fx (SEQ ID NO: 33) (in an embodiment of the invention, the leader sequence is underscored):









1
mefglswvfl vaiikgvqcq vqlvesgggl vkpggslrls caasgftfsd



 


51
yymswirqap gkglewvsyi sssgstrdya dsvkgrftis rdnaknslyl


 


101
qmnslraedt avyycardgv ettfyyyyyg mdvwgqgttv tvssastkgp


 


151
svfplapcsr stsestaalg clvkdyfpep vtvswnsgal tsgvhtfpav


 


201
lqssglysls svvtvpssnf gtqtytcnvd hkpsntkvdk tverkccvec


 


251
ppcpappvag psvflfppkp kdtlmisrtp evtcvvvdvs hedpevqfnw


 


301
yvdgvevhna ktkpreeqfn stfrvvsvlt vvhqdwlngk eykckvsnkg


 


351
lpapiektis ktkgqprepq vytlppsree mtknqvsltc lvkgfypsdi


 


401
avewesngqp ennykttppm ldsdgsffly skltvdksrw qqgnvfscsv


 


451
mhealhnhyt qkslslspgk






In an embodiment of the invention, an anti-IGF1R antibody of the invention comprises mature immunoglobulin heavy chain variable region 2.12.1 fx (amino acids 20-144 or SEQ ID NO: 33; SEQ ID NO: 34):








q vqlvesgggl vkpggslrls caasgftfsd yymswirqap gkglewvsyi sssgstrdya



 


dsvkgrftis rdnaknslyl qmnslraedt avyycardgv ettfyyyyyg mdvwgqgttv tvss






In an embodiment of the invention, an anti-IGF1R antibody of the invention comprises an immunoglobulin light chain or mature fragment or variable region 2.12.1 fx (SEQ ID NO: 35) (in an embodiment of the invention, the leader sequence is underscored):









1
mdmrvpaqll gllllwfpga rcdiqmtqsp sslsasvgdr vtitcrasqd



 


51
irrdlgwyqq kpgkapkrli yaasrlqsgv psrfsgsgsg teftltissl


 


101
qpedfatyyc lqhnnyprtf gqgtkveikr tvaapsvfif ppsdeqlksg


 


151
tasvvcllnn fypreakvqw kvdnalqsgn sqesvteqds kdstyslsst


 


201
ltlskadyek hkvyacevth qglsspvtks fnrgec






In an embodiment of the invention, an anti-IGF1R antibody of the invention comprises mature immunoglobulin light chain variable region 2.12.1 fx (amino acids 23-130 of SEQ ID NO: 35; SEQ ID NO: 36):








diqmtqsp sslsasvgdr vtitcrasqd irrdlgwyqq kpgkapkrli yaasrlqsgv



 


psrfsgsgsg teftltissl qpedfatyyc lqhnnyprtf gqgtkveikr






In an embodiment of the invention, an anti-IGF1R antibody of the invention comprises a humanized 7C10 immunoglobulin light chain variable region; version 1 (SEQ ID NO: 37):









1
dvvmtqspls lpvtpgepas iscrssqsiv hsngntylqw ylqkpgqspq



 


51
lliykvsnrl ygvpdrfsgs gsgtdftlki srveaedvgv yycfqgshvp


 


101
wtfgqgtkve ik






In an embodiment of the invention, an anti-IGF1R antibody of the invention comprises humanized 7C10 immunoglobulin light chain variable region; version 2 (SEQ ID NO: 38):









1
divmtqspls lpvtpgepas iscrssqsiv hsngntylqw ylqkpgqspq



 


51
lliykvsnrl ygvpdrfsgs gsgtdftlki srveaedvgv yycfqgshvp


 


101
wtfgqgtkve ik






In an embodiment of the invention, an anti-IGF1R antibody of the invention comprises a humanized 7C10 immunoglobulin heavy chain variable region; version 1 (SEQ ID NO: 39):









1
qvqlqesgpg lvkpsetlsl tctvsgysit ggylwnwirq ppgkglewmg



 


51
yisydgtnny kpslkdriti srdtsknqfs lklssvtaad tavyycaryg


 


101
rvffdywgqg tlvtvss






In an embodiment of the invention, an anti-IGF1R antibody of the invention comprises the humanized 7C10 immunoglobulin heavy chain variable region; version 2 (SEQ ID NO: 40):









1
qvqlqesgpg lvkpsetlsl tctvsgysit ggylwnwirq ppgkglewig



 


51
yisydgtnny kpslkdrvti srdtsknqfs lklssvtaad tavyycaryg


 


101
rvffdywgqg tlvtvss






In an embodiment of the invention, an anti-IGF1R antibody of the invention comprises the humanized 7C10 immunoglobulin heavy chain variable region; version 3 (SEQ ID NO: 41):









1
qvqlqesgpg lvkpsetlsl tctvsgysis ggylwnwirq ppgkglewig



 


51
yisydgtnny kpslkdrvti svdtsknqfs lklssvtaad tavyycaryg


 


101
rvffdywgqg tlvtvss






In an embodiment of the invention, an anti-IGF1R antibody of the invention comprises A12 immunoglobulin heavy chain variable region (SEQ ID NO: 42):









1
evqlvqsgae vkkpgssvkv sckasggtfs syaiswvrqa pgqglewmgg



 


51
iipifgtany aqkfqgrvti tadkststay melsslrsed tavyycarap


 


101
lrflewstqd hyyyyymdvw gkgttvtvss






In an embodiment of the invention, an anti-IGF1R antibody of the invention comprises A12 immunoglobulin light chain variable region (SEQ ID NO: 43):









1
sseltqdpav svalgqtvri tcqgdslrsy yaswyqqkpg qapvlviygk



51
nnrpsgipdr fsgsssgnta sltitgaqae deadyycnsr dnsdnrlifg


101
ggtkltvls






or (SEQ ID NO: 105):



















1
sseltqdpav
svalgqtvri
tcqgdslrsy
yatwyqqkpg
qapilviyge



 


51
nkrpsgipdr
fsgsssgnta
sltitgaqae
deadyycksr
dgsgqhlvfg


 


101
ggtkltvlg









In an embodiment of the invention, an anti-IGF1R antibody of the invention comprises 1A immunoglobulin heavy chain variable region (SEQ ID NO: 44):









1
evqlvqsggg lvhpggslrl scagsgftfr nyamywvrqa pgkglewvsa



 


51
igsgggtyya dsvkgrftis rdnaknslyl qmnslraedm avyycarapn


 


101
wgsdafdiwg qgtmvtvss;






optionally including one or more of the following mutations: R30, S30, N31, S31, Y94, H94, D104, E104.

In an embodiment of the invention, an anti-IGF1R antibody of the invention comprises 1A immunoglobulin light chain variable region (SEQ ID NO: 45):









1
diqmtqspss lsasvgdrvt itcrasqgis swlawyqqkp ekapksliya



 


51
asslqsgvps rfsgsgsgtd ftltisslqp edfatyycqq ynsypptfgp


 


101
gtkvdik;






optionally including one or more of the following mutations: P96, 196, P100, Q100, R103, K103, V104, L104, D105, E105

In an embodiment of the invention, an anti-IGF1R antibody of the invention comprises single chain antibody (fv) 8A1 (SEQ ID NO: 46):









1
evqlvqsgae vkkpgeslti sckgpgynff nywigwvrqm pgkglewmgi



 


51
iyptdsdtry spsfqgqvti svdksistay lqwsslkasd tamyycarsi


 


101
rycpggrcys gyygmdvwgq gtmvtvssgg ggsggggsgg ggsseltqdp


 


151
avsvalgqtv ritcqgdslr syyaswyqqk pgqapvlviy gknnrpsgip


 


201
drfsgsssgn tasltitgaq aedeadyycn srdssgnhvv fgggtkltvl


 


251
g






In an embodiment of the invention, an anti-IGF1R antibody of the invention comprises single chain antibody (fv) 9A2 (SEQ ID NO: 47):









1
qvqlvqsgae vrkpgasvkv scktsgytfr nydinwvrqa pgqglewmgr



 


51
isghygntdh aqkfqgrftm tkdtststay melrsltfdd tavyycarsq


 


101
wnvdywgrgt lvtvssgggg sggggsgggg salnfmltqp hsvsespgkt


 


151
vtisctrssg siasnyvqwy qqrpgssptt vifednrrps gvpdrfsgsi


 


201
dtssnsaslt isglktedea dyycqsfdst nlvvfgggtk vtvlg






In an embodiment of the invention, an anti-IGF1R antibody of the invention comprises single chain antibody (fv) 11A4 (SEQ ID NO: 48):









1
evqllesggg lvqpggslrl scaasgftfs syamswvrqa pgkglewvsa



 


51
isgsggstyy adsvkgrfti srdnskntly lqmnslraed tavyycassp


 


101
yssrwysfdp wgqgtmvtvs sggggsgggg sggggsalsy eltqppsvsv


 


151
spgqtatitc sgddlgnkyv swyqqkpgqs pvlviyqdtk rpsgiperfs


 


201
gsnsgniatl tisgtqavde adyycqvwdt gtvvfgggtk ltvlg






In an embodiment of the invention, an anti-IGF1R antibody of the invention comprises single chain antibody (fv) 7A4 (SEQ ID NO: 49):









1
evqlvqsgae vkkpgeslti sckgsgynff nywigwvrqm pgkdlewmgi



 


51
iyptdsdtry spsfqgqvti svdksistay lqwsslkasd tamyycarsi


 


101
rycpggrcys gyygmdvwgq gtmvtvssgg gssggggsgg ggsseltqdp


 


151
avsvalgqtv ritcrgdslr nyyaswyqqk pgqapvlviy gknnrpsgip


 


201
drfsgsssgn tasltitgaq aedeadyycn srdssgnhmv fgggtkltvl


 


251
g






In an embodiment of the invention, an anti-IGF1R antibody of the invention comprises single chain antibody (fv) 11A1 (SEQ ID NO: 50):









1
evqlvesggg vvqpgrslrl scaasgftfs dfamhwvrqi pgkglewlsg



 


51
lrhdgstayy agsvkgrfti srdnsrntvy lqmnslraed tatyycvtgs


 


101
gssgphafpv wgkgtlvtvs sggggsgggg sggggsalsy vltqppsasg


 


151
tpgqrvtisc sgsnsnigty tvnwfqqlpg tapklliysn nqrpsgvpdr


 


201
fsgsksgtsa slaisglqse deadyycaaw ddslngpvfg ggtkvtvlg






In an embodiment of the invention, an anti-IGF1R antibody of the invention comprises single chain antibody (fv) 7A6 (SEQ ID NO: 51)









1
evqlvqsgae vkkpgeslti sckgsgynff nywigwvrqm pgkglewmgi



 


51
iyptdsdtry spsfqgqvti svdksistay lqwsslkasd tamyycarsi


 


101
rycpggrcys gyygmdvwgq gtlvtvssgg ggsggggsgg ggsseltqdp


 


151
avsvalgqtv ritcqgdslr syytnwfqqk pgqapllvvy aknkrpsgip


 


201
drfsgsssgn tasltitgaq aedeadyycn srdssgnhvv fgggtkltvl


 


251
g






In an embodiment of the invention, an anti-IGF1R antibody or an antigen-binding fragment thereof (e.g., a heavy chain or light chain immunoglobulin) of the invention comprises one or more complementarity determing regions (CDR) selected from the group consisting of:











sywmh;
(SEQ ID NO: 52)




 



einpsngrtnynekfkr;
(SEQ ID NO: 53)



 



grpdyygsskwyfdv;
(SEQ ID NO: 54)



 



rssqsivhsnvntyle;
(SEQ ID NO: 55)



 



kvsnrfs;
(SEQ ID NO: 56)



and



 



fqgshvppt.
(SEQ ID NO: 57)






In an embodiment of the invention, an anti-IGF1R antibody or an antigen-binding fragment thereof of the invention comprises a heavy chain immunoglobulin variable region selected from the group consisting of:








(SEQ ID NO: 58)










1
qvqlvqsgae vvkpgasvkl sckasgytft sywmhwvkqr pgqglewige



51
inpsngrtny nqkfqgkatl tvdkssstay mqlssltsed savyyfargr


101
pdyygsskwy fdvwgqgttv tvs;


 








(SEQ ID NO: 59)










1
qvqfqqsgae lvkpgasvkl sckasgytft sylmhwikqr pgrglewigr



51
idpnnvvtkf nekfkskatl tvdkpsstay melssltsed savyycarya


101
ycrpmdywgq gttvtvss;


 








(SEQ ID NO: 60)










1
qvqlqqsgae lvkpgasvkl sckasgytft sywmhwvkqr pgqglewige



51
inpsngrtny nekfkrkatl tvdkssstay mqlssltsed savyyfargr


101
pdyygsskwy fdvwgagttv tvs;


 








(SEQ ID NO: 61)










1
qvqlqqsgae lmkpgasvki sckatgytfs sfwiewvkqr pghglewige



51
ilpgsggthy nekfkgkatf tadkssntay mqlssltsed savyycargh


101
syyfydgdyw gqgtsvtvss;


 








(SEQ ID NO: 62)










1
qvqlqqpgsv lvrpgasvkl sckasgytft sswihwakqr pgqglewige



51
ihpnsgntny nekfkgkatl tvdtssstay vdlssltsed savyycarwr


101
ygspyyfdyw gqgttltvss;


 








(SEQ ID NO: 63)










1
qvqlqqpgae lvkpgasvkl sckasgytft sywmhwvkqr pgrglewigr



51
idpnsggtky nekfkskatl tvdkpsstay mqlssltsed savyycaryd


101
yygssyfdyw gqgttltvss;


 








(SEQ ID NO: 64)










1
qvqlvqsgae vvkpgasvkl sckasgytft sywmhwvkqr pgqglewige



51
inpsngrtny nqkfqgkatl tvdkssstay mqlssltsed savyyfargr


101
pdyygsskwy fdvwgqgttv tvs;


 








(SEQ ID NO: 65)










1
qvqlqqsgae lvkpgasvkl sckasgytft sywmhwvkqr pgqglewige



51
inpsngrtny nekfkrkatl tvdkssstay mqlssltsed savyyfargr


101
pdyygsskwy fdvwgagttv tvss;


 








(SEQ ID NO: 66)










1
qvqlvqsgae vvkpgasvkl sckasgytft sywmhwvkqr pgqglewige



51
inpsngrtny nqkfqgkatl tvdkssstay mqlssltsed savyyfargr


101
pdyygsskwy fdvwgqgttv tvss;


 








(SEQ ID NO: 67)










1
qvqlqqsgae lvkpgasvkl sckasgytft sywmhwvkqr pgrglewigr



51
idpnsggtky nekfkskatl tvdkpsstay mqlssltsed savyycaryd


101
yygssyfdyw gqgttvtvss;


 








(SEQ ID NO: 68)










1
qiqlqqsgpe lvrpgasvki sckasgytft dyyihwvkqr pgeglewigw



51
iypgsgntky nekfkgkatl tvdtssstay mqlssltsed savyfcargg


101
kfamdywgqg tsvtvss;


 








(SEQ ID NO: 69)










1
qvqlqqsgae lvkpgasvkl sckasgytft sywmhwvkqr pgqglewige



51
inpsngrtny nekfkrkatl tvdkssstay mqlssltsed savyyfargr


101
pdyygsskwy fdvwgagttv tvss;


 








(SEQ ID NO: 70)










1
qiqlqqsgpe lvkpgasvki sckasgytft dyyinwmkqk pgqglewigw



51
idpgsgntky nekfkgkatl tvdtssstay mqlssltsed tavyfcarek


101
ttyyyamdyw gqgtsvtvsa;


 








(SEQ ID NO: 71)










1
vqlqqsgael mkpgasvkis ckasgytfsd ywiewvkqrp ghglewigei



51
lpgsgstnyh erfkgkatft adtssstaym qlnsltseds gvyyclhgny


101
dfdgwgqgtt ltvss;








and



 








(SEQ ID NO: 72)










1
qvqllesgae lmkpgasvki sckatgytfs sfwiewvkqr pghglewige



51
ilpgsggthy nekfkgkatf tadkssntay mqlssltsed savyycargh


101
syyfydgdyw gqgtsvtvss;






and/or a light chain immunoglobulin variable region selected from the group consisting of:








(SEQ ID NO: 73)










1
dvlmtqipvs lpvslgdqas iscrssqiiv hnngntylew ylqkpgqspq



51
lliykvsnrf sgvpdrfsgs gsgtdftlki srveaedlgv yycfqgshvp


101
ftfgsgtkle ikr;


 








(SEQ ID NO: 74)










1
dvlmtqtpls lpvslgdpas iscrssqsiv hsnvntylew ylqkpgqspk



51
lliykvsnrf sgvpdrfsgs gagtdftlri srveaedlgi yycfqgshvp


101
ptfgggtkle ikr;


 








(SEQ ID NO: 75)










1
dvlmtqtpls lpvslgdpas iscrssqsiv hsnvntylew ylqkpgqspr



51
lliykvsnrf sgvpdrfsgs gagtdftlri srveaedlgi yycfqgshvp


101
ptfgggtkle ikr;


 








(SEQ ID NO: 76)










1
dvlmtqtpls lpvslgdpas iscrssqsiv hsnvntylew ylqkpgqspk



51
lliykvsnrf sgvpdrfsgs gagtdftlri srveaedlgi yycfqgshvp


101
ptfgggtkle ikr;


 








(SEQ ID NO: 77)










1
dvlmtqtpls lpvslgdpas iscrssqsiv hsnvntylew ylqkpgqspr



51
lliykvsnrf sgvpdrfsgs gagtdftlri srveaedlgi yycfqgshvp


101
ptfgggtkle ikr;


 








(SEQ ID NO: 78)










1
dvlmtqtpls lpvslgdqas iscrssqxiv hsngntylew ylqkpgqspk



51
lliykvsnrf sgvpdrfsgs gsgtdftlki srveaedlgv yycfqgshvp


101
xtfgggtkle ikr;


 








(SEQ ID NO: 79)










1
dvvmtqtpls lpvslgdpas iscrssqsiv hsnvntylew ylqkpgqspk



51
lliykvsnrf sgvpdrfsgs gagtdftlri srveaedlgi yycfqgshvp


101
ptfgggtkle ikr;


 








(SEQ ID NO: 80)










1
dvvmtqtpls lpvslgdpas iscrssqsiv hsnvntylew ylqkpgqspr



51
lliykvsnrf sgvpdrfsgs gagtdftlri srveaedlgi yycfqgshvp


101
ptfgggtkle ikr;


 








(SEQ ID NO: 81)










1
dvlmtqtpls lpvslgdpas iscrssqsiv hsnvntylew ylqkpgqspr



51
lliykvsnrf sgvpdrfsgs gagtdftlri srveaedlgi yycfqgshvp


101
ptfgggtkle ikr;


 








(SEQ ID NO: 82)










1
dvlmtqipvs lpvslgdqas iscrssqiiv hnngntylew ylqkpgqspq



51
lliykvsnrf sgvpdrfsgs gsgtdftlki srveaedlgv yycfqgshvp


101
ftfgsgtkle ikr;


 








(SEQ ID NO: 83)










1
dvlmtqtpls lpvslgdqas iscrfsqsiv hsngntylew ylqksgqspk



51
lliykvsnrf sgvpdrfsgs gsgtdftlki srveaedlgv yycfqgshvp


101
rtfgggtkle ikr;


 








(SEQ ID NO: 84)










1
dvlmtqtpls lpvslgdqas iscrssqsiv hsnvntylew ylqkpgqspk



51
lliykvsnrf sgvpdrfsgs gsgtdftlri srveaedlgi yycfqgshvp


101
ptfgggtkle ikr;


 








(SEQ ID NO: 85)










1
dvvmtqtpls lpvslgdpas iscrssqsiv hsnvntylew ylqkpgqspk



51
lliykvsnrf sgvpdrfsgs gagtdftlri srveaedlgi yycfqgshvp


101
ptfgggtkle ikr;


 








(SEQ ID NO: 86)










1
elvmtqtpls lpvslgdqas iscrssqtiv hsngdtyldw flqkpgqspk



51
lliykvsnrf sgvpdrfsgs gsgtdftlki srveaedlgv yycfqgshvp


101
ptfgggtkle ikr;


 








(SEQ ID NO: 87)










1
dvlmtqtpls lpvslgdpas iscrssqsiv hsnvntylew ylqkpgqspk



51
lliykvsnrf sgvpdrfsgs gagtdftlri srveaedlgi yycfqgshvp


101
ptfgggtkle ikr;


 








(SEQ ID NO: 88)










1
dvvmtqtpls lpvslgdpas iscrssqsiv hsnvntylew ylqkpgqspr



51
lliykvsnrf sgvpdrfsgs gagtdftlri srveaedlgi yycfqgshvp


101
ptfgggtkle ikr;


 








(SEQ ID NO: 89)










1
dvlmtqtpvs lsvslgdqas iscrssqsiv hstgntylew ylqkpgqspk



51
lliykisnrf sgvpdrfsgs gsgtdftlki srveaedlgv yycfqashap


101
rtfgggtkle ikr;


 








(SEQ ID NO: 90)










1
dvlmtqtpls lpvslgdqas isckssqsiv hssgntyfew ylqkpgqspk



51
lliykvsnrf sgvpdrfsgs gsgtdftlki srveaedlgv yycfqgship


101
ftfgsgtkle ikr;


 








(SEQ ID NO: 91)










1
dieltqtpls lpvslgdqas iscrssqsiv hsngntylew ylqkpgqspk



51
lliykvsnrf sgvpdrfsgs gsgtdftlki srveaedlgv yycfqgshvp


101
ytfgggtkle ikr;


 








(SEQ ID NO: 92)










1
dvlmtqtpls lpvslgdqas iscrssqsiv hsnvntylew ylqkpgqspk



51
lliykvsnrf sgvpdrfsgs gsgtdftlri srveaedlgi yycfqgshvp


101
ptfgggtkle ikr;


 








(SEQ ID NO: 93)










1
dvvmtqtpls lpvslgdpas iscrssqsiv hsnvntylew ylqkpgqspr



51
lliykvsnrf sgvpdrfsgs gagtdftlri srveaedlgi yycfqgshvp


101
ptfgggtkle ikr;


 








(SEQ ID NO: 94)










1
dvlmtqtpls lpvslgdqas iscrssqsiv hsnvntylew ylqkpgqspk



51
lliykvsnrf sgvpdrfsgs gsgtdftlri srveaedlgi yycfqgshvp


101
ptfgggtkle ikr;


 








(SEQ ID NO: 95)










1
dvvmtqtpls lpvslgdpas iscrssqsiv hsnvntylew ylqkpgqspk



51
lliykvsnrf sgvpdrfsgs gagtdftlri srveaedlgi yycfqgshvp


101
ptfgggtkle ikr;


 








(SEQ ID NO: 96)










1
dvlmtqtpls lpvslgdqas iscrsnqtil lsdgdtylew ylqkpgqspk



51
lliykvsnrf sgvpdrfsgs gsgtdftlki srveaedlgv yycfqgshvp


101
ptfgggtkle ikr;


 








(SEQ ID NO: 97)










1
dvlmtqtpls lpvslgdqas iscrssqtiv hsngntylew ylqkpgqspk



51
lliykvtnrf sgvpdrfsgs gsgtdftlki srveaedlgv yycfqgthap


101
ytfgggtkle ikr;








and



 








(SEQ ID NO: 98)










1
dvlmtqtpls lpvslgdqas iscrssqsiv hsngntylew ylqkpgqspk



51
lliysissrf sgvpdrfsgs gsgtdftlki srvqaedlgv yycfqgshvp


101
ytfgggtkle ikr.






The scope of the present invention includes methods wherein a patient is administered an anti-insulin-like growth factor receptor-1 (IGF1R) antibody wherein the variable region of the antibody is linked to any immunoglobulin constant region. In an embodiment, the light chain variable region is linked to a κ chain constant region. In an embodiment, the heavy chain variable region is linked to a γ1, γ2, γ3 or γ4 chain constant region. Any of the immunoglobulin variable regions set forth herein, in embodiments of the invention, can be linked to any of the foregoing constant regions.
In an embodiment of the invention, an IGF1R inhibitory agent that can be administered to a patient in a method according to the invention is AEW-541 (NVP-AEW-541; NVP-AEW-541-NX-7):




(Novartis; East Hanover, N.J.; see WO 2002/92599);





(WO 2003/39538).

In an embodiment of the invention, an IGF1R inhibitory agent that can be administered to a patient in a method according to the invention is any IGF1R anti-sense nucleic acid. For example, in an embodiment, the anti-sense IGF1R nucleic acid is ATL-1101 (Antisense Therapeutics Ltd; Australia). In an embodiment, the IGF1R anti-sense nucleic acid comprises any of the following nucleotide sequences: 5′-ATCTCTCCGCTTCCTTTC-3′ (SEQ ID NO: 99), 5′-ATCTCTCCGCTTCCTTTC-3′ (SEQ ID NO: 100), 5′-ATCTCTCCGCTTCCTTTC-3′ (SEQ ID NO: 101) or any IGF1R antisense nucleic acid set forth in any of US Published Patent Application No. US20030096769; Published International Application No. WO 2003/100059; Fogarty et al., Antisense Nucleic Acid Drug Dev. 2002 December; 12(6):369-77; White et al., J Invest Dermatol. 2002 June; 118(6):1003-7; White et al., Antisense Nucleic Acid Drug Dev. 2000 June; 10(3):195-203; or Wraight et al., Nat Biotechnol. 2000 May; 18(5):521-6.
In an embodiment of the invention, an IGF1R inhibitory agent that can be administered to a patient in a method according to the invention is an anti-IGF-I or II antibody; for example, any antibody disclosed in WO 2003/93317 or EP00492552.
The scope of the present invention includes any kinase inhibitor compound set forth in published international applications WO 2004/030627 or WO 2004/030625. In an embodiment, the kinase inhibitor is (±)-4-[2-(3-chloro-4-fluoro-phenyl)-2-hydroxy-ethylamino]-3-[6-(imidazol-1-yl)-4-methyl-1H-benzimidazol-2-yl]-1H-pyridin-2-one:




(optionally in combination with paclitaxel or with cetuximab).

In an embodiment of the invention, the IGR1R inhibitory agent is a soluble fragment of IGF1R (e.g., amino acids 30-902 of IGF1R) or siRNA (small interfering RNA) against IGF-1R.
In an embodiment, IGF1R comprises the amino acid sequence set forth under Genbank Accession No.: XM—052648 or NM—000612.
The present invention also includes embodiments wherein the patient receives both an IGF1R inhibitory agent in association with one or more other anti-cancer agents, including, but not limited to paclitaxel, thalidomide, docetaxel, gefitinib, temozolomide, lonafarnib, tipifarnib, letrozole, doxorubicin, cis-platin, oxaliplatin, camptothecan, topotecan, etoposide, vincristine, vinblastine, raloxifene, gemcitabine, retinoic acid, tamoxifen, trastuzumab, cetuximab or octreotide; or anti-cancer therapeutic procedures including, but not limited to, surgical tumorectomy or anti-cancer radiation therapy. The present invention further includes embodiment wherein two or more IGF1R inhibitory agents are administered in association with one another.
The term “in association” indicates that the components of the combinations of the invention can be formulated into a single composition for simultaneous delivery or formulated separately into two or more compositions (e.g., a kit). Furthermore, each component of a combination of the invention can be administered to a subject at a different time than when the other component is administered; for example, each administration may be given non-simultaneously at several intervals over a given period of time. Moreover, the separate components may be administered to a subject by the same or by a different route (e.g., orally, intravenously, intratumorally).
Generation of Antibodies
Any suitable method can be used to elicit an antibody with the desired biologic properties to inhibit IGF1R. It is desirable to prepare monoclonal antibodies (mAbs) from various mammalian hosts, such as mice, rodents, primates, humans, etc. Description of techniques for preparing such monoclonal antibodies may be found in, e.g., Stites, et al. (eds.) BASIC AND CLINICAL IMMUNOLOGY (4th ed.) Lange Medical Publications, Los Altos, Calif., and references cited therein; Harlow and Lane (1988) ANTIBODIES: A LABORATORY MANUAL CSH Press; Goding (1986) MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.) Academic Press, New York, N.Y. Thus, monoclonal antibodies may be obtained by a variety of techniques familiar to researchers skilled in the art. Typically, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell. See Kohler and Milstein (1976) Eur. J. Immunol. 6:511-519. Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods known in the art. See, e.g., Doyle, et al. (eds. 1994 and periodic supplements) CELL AND TISSUE CULTURE: LABORATORY PROCEDURES, John Wiley and Sons, New York, N.Y. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, one may isolate DNA sequences which encode a monoclonal antibody or a binding fragment thereof by screening a DNA library from human B cells according, e.g., to the general protocol outlined by Huse, et al. (1989) Science 246:1275-1281. Modified antibodies can be generated, for example, by introducing mutations in DNA encoding an immunoglobulin chain, for example, by use of conventional recombinant biological techniques.
Other suitable techniques involve selection of libraries of antibodies in phage or similar vectors. See, e.g., Huse et al., Science 246:1275-1281 (1989); and Ward et al., Nature 341:544-546 (1989). The polypeptides and antibodies of the present invention may be used with or without modification, including chimeric or humanized antibodies. Frequently, the polypeptides and antibodies will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulins may be produced, see Cabilly U.S. Pat. No. 4,816,567; and Queen et al. (1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033; or made in transgenic mice, see Mendez et al. (1997) Nature Genetics 15:146-156. Further methods for producing chimeric, humanized and human antibodies are well known in the art. See, e.g., U.S. Pat. No. 5,530,101, issued to Queen et al, U.S. Pat. No. 5,225,539, issued to Winter et al, U.S. Pat. No. 4,816,397 issued to Boss et al, all of which are incorporated by reference in their entirety.
Tumor Analysis
The methods of the present method comprise determining whether tumor cells comprising one or more of the following characteristics:
(i) IRS-1 phosphorylation on tyrosine 896;
(ii) IRS-1 phosphorylation on tyrosine 612;
(iii) IRS-1 phosphorylation on any tyrosine;
(iv) IGF-II expression;
(v) IGF1R phosphorylation on any tyrosine; or
(vi) expression of IGF1R.
Tumor cells can be assayed to determine whether any of these characteristics are present by any of several methods commonly known in the art. In an embodiment, IRS-1 or IGF1R tyrosine phosphorylation can be determine by western blot analysis with an anti-phosphotyrosine antibody. For example, anti-phosphotyrosine antibodies PY20, PT66 and P-Try-100 are commercially available from PerkinElmer Life and Analytical Sciences, Inc. (Boston, Mass.); and anti-phosphotyrosine antibody 4G10 is commercially available from Upstate Cell Signaling Solutions (Waltham, Mass.). Western blot analysis is a conventional method that is well known in the art. In an embodiment, IRS-1 or IGF1R tyrosine phosphorylation can be determine by an Enzyme linked immunosorbent assay (ELISA) or immunoprecipitation. In an embodiment, expression of IGF1R or IGF-II by tumor cells can, similarly, be determined by western blot analysis, immunoprecipitation or by ELISA. Any of several anti-IGF1R antibodies known in the art, for example, as described herein, can be used.
Many references are available to provide guidance in applying the above techniques (Kohler et al., Hybridoma Techniques (Cold Spring Harbor Laboratory, New York, 1980); Tijssen, Practice and Theory of Enzyme Immunoassays (Elsevier, Amsterdam, 1985); Campbell, Monoclonal Antibody Technology (Elsevier, Amsterdam, 1984); Hurrell, Monoclonal Hybridoma Antibodies: Techniques and Applications (CRC Press, Boca Raton, Fla., 1982); Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987)).
In an embodiment of the invention, IGF-II expression by a tumor cell can be determined by IGF-II RNA detection. In an embodiment of the invention, IGF-II RNA is determined by northern blot analysis. Northern blot analysis is a conventional technique well known in the art and is described, for example, in Molecular Cloning, a Laboratory Manual, second edition, 1989, Sambrook, Fritch, Maniatis, Cold Spring Harbor Press, 10 Skyline Drive, Plainview, N.Y. 11803-2500.
Dosage
In an embodiment, an IGF1R inhibitory agent is administered to a patient at a “therapeutically effective dosage” or “therapeutically effective amount” which preferably inhibits a disease or condition (e.g., tumor growth) to any extent—preferably by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80%-100% relative to untreated subjects. In an embodiment of the invention, the term “therapeutically effective amount” or “therapeutically effective dosage” means that amount or dosage of an IGF1R inhibitory agent (e.g., an anti-IGF1R antibody or antigen-binding fragment thereof) that will elicit a biological or medical response of a tissue, system, subject or host that is being sought by the administrator (such as a researcher, doctor or veterinarian) which includes any measurable alleviation of the signs, symptoms and/or clinical indicia of cancer (e.g., tumor growth) and/or the prevention, slowing or halting of progression or metastasis of cancer to any degree. The ability of an IGF1R inhibitory agent to inhibit cancer can be evaluated in an animal model system predictive of efficacy in human tumors. Alternatively, efficacy can be evaluated by examining the ability of a treatment of the invention or any component thereof to inhibit tumor cell growth in vitro by assays well-known to the skilled practitioner. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.
A clinician may use any of several methods known in the art to measure the effectiveness of a particular dosage scheme of an IGF1R inhibitory agent. For example, tumor size can be determined in a non-invasive route, such as by X-ray, positron emission tomography (PET) scan, computed tomography (CT) scan or magnetic resonance imaging (MRI).
A cancer or a tumor cell is “responsive” to an IGF1R inhibitory agent if the agent can provide any measurable alleviation of the signs, symptoms and/or clinical indicia of cancer (e.g., tumor growth) and/or the prevention, slowing or halting of progression or metastasis of cancer to any degree.
Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a dose may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of an IGF1R inhibitory agent employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. The effectiveness of a given dose or treatment regimen of IGF1R inhibitory agent can be determined, for example, by determining whether a tumor being treated in the subject shrinks or ceases to grow.
In an embodiment of the invention, administration of IGF1R inhibitory agent is by injection proximal to the site of the target (e.g., tumor). In an embodiment, a therapeutically effective daily dose of IGF1R inhibitory agent or pharmaceutical composition thereof is administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day. In an embodiment, a “therapeutically effective” dosage of any anti-IGFR antibody (e.g., 19D12/15H12 LCF/HCA) is in the range of about 3 mg/kg (body weight) to about 20 mg/kg (e.g., 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg or 20 mg/kg) per day. In an embodiment, a “therapeutically effective dosage” of a chemotherapeutic agent (e.g., an IGF1R inhibitory agent) is whenever possible as set forth in the Physicians' Desk Reference 2003 (Thomson Healthcare; 57th edition (Nov. 1, 2002)) which is herein incorporated by reference. For example, in an embodiment of the invention, a therapeutically effective dosage of NVP-ADW-742 is about 1 mg/kg/day to about 50 mg/kg/day (e.g., 5 mg/kg/day, 10 mg/kg/day, 15 mg/kg/day, 20 mg/kg/day, 25 mg/kg/day, 30 mg/kg/day, 35 mg/kg/day, 40 mg/kg/day, 45 mg/kg/day).
Therapeutic Methods and Administration
An IGF1R inhibitory agent can be used to inhibit or reduce the growth or proliferation of any cell, such as a malignant cell, either in vitro (e.g., in cell culture) or in vivo (e.g., within the body of a subject suffering from a disease mediated by elevated expression or activity of IGF1R or by elevated expression of its ligand (e.g., IGF-I or IGF-II)). Such inhibition or reduction of growth or proliferation of a cell can be achieved by contacting the cell with the IGF1R inhibitory agent.
In an embodiment, an IGF1R inhibitory agent is for treating cancer in a patient that is characterized by one or more of the following characteristics: IRS-1 phosphorylation on tyrosine 896; (ii) IRS-1 phosphorylation on tyrosine 612; (iii) IRS-1 phosphorylation on any tyrosine; (iv) IGF-II expression; (v) IGF1R phosphorylation on any tyrosine; or (vi) expression of IGF1R. For example, in an embodiment, the cancer is bladder cancer, Wilm's cancer, bone cancer, prostate cancer, lung cancer, endometrial cancer, multiple myeloma, non-small cell lung cancer (NSCLC), colon cancer, rectal cancer, colorectal cancer, breast cancer (estrogen receptor + or estrogen receptor −), cervical cancer, synovial sarcoma, ovarian cancer, pancreatic cancer, neuroblastoma, rhabdomyosarcoma, osteosarcoma, diarrhea associated with metastatic carcinoid or vasoactive intestinal peptide secreting tumor (e.g., VIPoma or Werner-Morrison syndrome).
In an embodiment, it is in initially determined if a prospective patient to be treated with an IGF1R inhibitory agent suffers from a variety of cancer that is commonly known to exhibit one of the following characteristics: (i) IRS-1 phosphorylation on tyrosine 896; (ii) IRS-1 phosphorylation on tyrosine 612; (iii) IRS-1 phosphorylation on any tyrosine; (iv) IGF-II expression; (v) IGF1R phosphorylation on any tyrosine; or (vi) expression of IGF1R. If the patient is determined to suffer from a cancer known to be characterized by one or more of the 6 characteristics set forth above, the patient is selected for treatment with an IGF1R inhibitory agent. A tumor type may be known to comprise any of the listed characteristics, for example, if such is established in scientific literature (e.g., periodic journals or textbooks) or if such is commonly known in the art by practitioners of ordinary skill or if such a characteristic has ever been observed in one or more patients or tumors, or if such can reasonably be inferred from experimental data (e.g., in vitro or in vivo data including animal data).
In an embodiment of the invention, a prospective patient's individual tumor is analyzed and it is determined whether the tumor exhibits one of more of the 6 characteristics: (i) IRS-1 phosphorylation on tyrosine 896; (ii) IRS-1 phosphorylation on tyrosine 612; (iii) IRS-1 phosphorylation on any tyrosine; (iv) IGF-II expression; (v) IGF1R phosphorylation on any tyrosine; or (vi) expression of IGF1R. In this embodiment, if the patient's tumor is determined to be characterized by one or more of the 6 characteristics set forth above, the patient is selected for treatment with an IGF1R inhibitory agent. In an embodiment, it is first determined whether the patient's tumor expresses the characteristic (i) IRS-1 phosphorylation on tyrosine 896 or (ii) IRS-1 phosphorylation on tyrosine 612; then, if such a characteristic is identified, it is determined whether the tumor comprises the characteristic (iv) IGF-II expression; if the patient's tumor is determined to express characteristic (i) or (ii) and characteristic (iv), then the patient is selected for treatment with an IGF1R inhibitory agent.
The cells from a particular patient's tumor can be obtained surgically, for example, by surgical biopsy. For example, a tumor biopsy can be taken by endoscopic biopsy, excisional or incisional biopsy or fine needle aspiration (FNA) biopsy.
The term “patient” or “subject” includes any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit) and most preferably a human.
As stated above, in an embodiment of the invention, where possible, an IGF1R inhibitory agent is administered to a subject in accordance with the Physicians' Desk Reference 2003 (Thomson Healthcare; 57th edition (Nov. 1, 2002)) or as set forth herein.
An IGF1R inhibitory agent can be administered by an invasive route such as by injection (see above). Administration by a non-invasive route (e.g., orally; for example, in a pill, capsule or tablet) is also within the scope of the present invention. In an embodiment of the invention, an anti-IGF1R antibody (e.g., 15H12/19D12 LCF/HCA), or pharmaceutical composition thereof, is administered intravenously, subcutaneously, intramuscularly, intraarterially or intratumorally.
An IGF1R inhibitory agent can be administered with medical devices known in the art. For example, a pharmaceutical composition of the invention can be administered by injection with a hypodermic needle.
The pharmaceutical compositions of the invention may also be administered with a needleless hypodermic injection device; such as the devices disclosed in U.S. Pat. No. 6,620,135; 6,096,002; 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556.
Examples of well-known implants and modules for administering pharmaceutical compositions include: U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments. Many other such implants, delivery systems, and modules are well known to those skilled in the art.
Pharmaceutical Compositions
An IGF1R inhibitory agent can be incorporated into a pharmaceutical composition, along with a pharmaceutically acceptable carrier, suitable for administration to a subject in vivo. The scope of the present invention includes pharmaceutical compositions which are suitable to be administered to a subject by any route including, for example, oral, ocular, topical, pulmonary (inhalation), intratumoral injection, intravenous injection, subcutaneous injection or intramuscular injection.
For general information concerning formulations, see, e.g., Gilman, et al., (eds.) (1990), The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; A. Gennaro (ed.), Remington's Pharmaceutical Sciences, 18th Edition, (1990), Mack Publishing Co., Easton, Pa.; Avis, et al., (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications Dekker, New York; Lieberman, et al., (eds.) (1990) Pharmaceutical Dosage Forms: Tablets Dekker, New York; and Lieberman, et al., (eds.) (1990), Pharmaceutical Dosage Forms: Disperse Systems Dekker, New York, Kenneth A. Walters (ed.) (2002) Dermatological and Transdermal Formulations (Drugs and the Pharmaceutical Sciences), Vol 119, Marcel Dekker.
Pharmaceutically acceptable carriers are conventional and very well known in the art. Examples include aqueous and nonaqueous carriers, stabilizers, antioxidants, solvents, dispersion media, coatings, antimicrobial agents, buffers, serum proteins, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for injection into a subject's body.
Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
Examples of pharmaceutically-acceptable antioxidants include: water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; and oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
Prevention of the presence of microorganisms may be ensured both by sterilization procedures, and by the inclusion of various antimicrobial agents such as EDTA, EGTA, paraben, chlorobutanol, phenol sorbic acid, and the like.
Suitable buffers which may be included in the pharmaceutical compositions of the invention include L-histidine based buffers, phosphate based buffers (e.g., phosphate buffered saline, pH≅7), sorbate based buffers or glycine-based buffers.
Serum proteins which may be included in the pharmaceutical compositions of the invention may include human serum albumin.
Isotonic agents, such as sugars (e.g., sucrose), ethanol, polyalcohols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, mannitol or sorbitol), sodium citrate or sodium chloride (e.g., buffered saline) may also be included in the pharmaceutical compositions of the invention. In an embodiment of the invention, the sugar, for example, glucose or sucrose is present at a high concentration (e.g., about 10-100 mg/ml, e.g., 50 mg/ml, 60 mg/ml or 70 mg/ml).
Prolonged absorption of an injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and/or gelatin.
Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils.
Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art.
Sterile injectable solutions comprising an anti-IGF1R antibody can be prepared by incorporating the antibody or antigen-binding fragment thereof in the required amount in an appropriate solvent, optionally with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the antibody into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional, desired ingredient from a previously sterile-filtered solution thereof.
In an embodiment of the invention, an anti-IGF1R antibody of the invention is in a pharmaceutical formulation comprising a therapeutically effective amount of said antibody, a buffer and sucrose. For example, in an embodiment of the invention, the buffer is any one of phosphate buffer, citrate buffer, histidine buffer, glycine buffer or acetate buffer. The pharmaceutical formulation can be within any suitable pH range. In an embodiment of the invention, the pH is 5.0, 5.5, 6.0, 7.5, or between about 5.5 and about 6 or between about 5 and about 7.
An IGF1R inhibitory agent including an anti-IGF1R antibody or antigen-binding fragment thereof can be orally administered. Pharmaceutical compositions for oral administration may contain, in addition to the binding composition, additives such as starch (e.g., potato, maize or wheat starch or cellulose), starch derivatives (e.g., microcrystalline cellulose or silica), sugars (e.g., lactose), talc, stearate, magnesium carbonate or calcium phosphate. In order to ensure that oral compositions comprising an antibody or antigen-binding fragment of the invention are well tolerated by the patient's digestive system, mucus formers or resins may be included. It may also be desirable to improve tolerance by formulating the antibody or antigen-binding fragment in a capsule which is insoluble in the gastric juices. An exemplary pharmaceutical composition of this invention in the form of a capsule is prepared by filling a standard two-piece hard gelatin capsule with the antibody or antigen-binding fragment of the invention in powdered form, lactose, talc and magnesium stearate. Oral administration of immunoglobulins has been described (Foster, et al., (2001) Cochrane Database System rev. 3:CD001816)
An IGF1R inhibitory agent may also be included in a pharmaceutical composition for topical administration. Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site where treatment is required, such as liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear or nose.
Drops may comprise sterile aqueous or oily solutions or suspensions and may be prepared by dissolving an IGF1R inhibitory agent in a suitable aqueous solution of a bactericidal and/or fungicidal agent and/or any other suitable preservative, and preferably including a surface active agent. The resulting solution may then be clarified by filtration.
Lotions according to the present invention include those suitable for application to the skin or eye. An eye lotion may comprise a sterile, aqueous solution optionally containing a bactericide and may be prepared by methods similar to those for the preparation of drops. Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a moisturizer such as glycerol or an oil such as castor oil or arachis oil.
Creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingredient for external application. They may be made by mixing an IGF1R inhibitory agent in finely-divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with the aid of suitable machinery, with a greasy or non-greasy basis. The basis may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil; wool fat or its derivatives, or a fatty acid such as stearic or oleic acid together with an alcohol such as propylene glycol or macrogels. The formulation may incorporate any suitable surface active agent such as an anionic, cationic or non-ionic surface active such as sorbitan esters or polyoxyethylene derivatives thereof. Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.
An IGF1R inhibitory agent may also be administered by inhalation. A suitable pharmaceutical composition for inhalation may be an aerosol. An exemplary pharmaceutical composition for inhalation of an antibody or antigen-binding fragment of the invention may include: an aerosol container with a capacity of 15-20 ml comprising the antibody or antigen-binding fragment of the invention, a lubricating agent, such as polysorbate 85 or oleic acid, dispersed in a propellant, such as freon, preferably in a combination of 1,2-dichlorotetrafluoroethane and difluorochloromethane. Preferably, the composition is in an appropriate aerosol container adapted for either intranasal or oral inhalation administration.
Kits and Articles of Manufacture
Kits and articles of manufacture of the present invention include an IGF1R inhibitory agent, preferably combined with a pharmaceutically acceptable carrier, in a pharmaceutical formulation, more preferably in a pharmaceutical dosage form such as a pill, a powder, an injectable liquid, a tablet, dispersible granules, a capsule, a cachet or a suppository. See for example, Gilman et al. (eds.) (1990), The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences, supra, Easton, Pa.; Avis et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications Dekker, New York; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets Dekker, New York; and Lieberman et al. (eds.) (1990), Pharmaceutical Dosage Forms: Disperse Systems Dekker, New York.
The kits and articles of manufacture of the present invention also include information, for example in the form of a package insert or label, indicating that the target of the IGF1R inhibitory agent is IGF1R. The term “target” indicates that the agent reduces or inhibits ligand binding, kinase activity, expression or any other biological activity of IGF1R in any way. The insert or label may take any form, such as paper or on electronic media such as a magnetically recorded medium (e.g., floppy disk) or a CD-ROM.
The label or insert may also include other information concerning the pharmaceutical compositions and dosage forms in the kit or article of manufacture. Generally, such information aids patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely. For example, the following information regarding the IGF1R inhibitory agent may be supplied in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, how supplied, proper storage conditions, references and patent information.
EXAMPLES
This section is intended to further describe the present invention and should not be construed to further limit the invention. Any composition or method set forth herein comprises part of the present invention.
In this example, the level of phosphorylation of IRS-1 in human lung tumor tissue was compared to that of normal tissue samples and found to be higher in tumor cells than in normal cells. Also, the in vivo efficacy of the anti-IGF1R antibody 19D12/15H12 LCF/HCA was correlated with the ability of the IGF-1 to cause IRS-1 phosphorylation. In addition, the level of IGF-II mRNA expression was evaluated in 56 different normal and cancerous ovarian and colorectal tissue samples and found to be high in several samples of tumor tissue.
Tumor lysate preparation. Tumor tissues were first weighed and pulverized into fine powder with a pre-chilled pulverizer on dry ice. Tumor powders were transferred into a tube, and 4.5× volume of the buffer A (i.e., 450 ul buffer A per 100 mg tissue) was added. The samples were sonicated for 30 seconds, 0.5× volume of buffer B (i.e., add 50 ul buffer B per 100 mg tissue powder) was added, and samples were spun for 13,000 rpm for 20 min at 4° C. after incubation on ice for 30 min. Supernatants were collected and protein concentrations of the lysates were determined by Bio-Rad assay.
Buffer A: 50 mM Hepes, pH 7.4, 150 mM NaCl, 5% Glycerol, 1.5 mM MgCl2, 2 mM Sodium Vanadate, 5 mM NaF, Protease inhibitors (2× concentrated C complete EDTA-free from Roche-cat #. 1 873 580), Phosphatase inhibitor Cocktail 1 (Sigma p2850), Phosphatase inhibitor Cocktail 2 (Sigma p5726).

Buffer B: Buffer A plus 10% Triton-100

Cell culture lysate preparation. Cells were washed in PBS once, lysed in buffer containing 50 mM Hepes, pH7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 2 mM Na3VO4 and protease inhibitor cocktail (CompleteTM, Roche Diagnostics GmbH; Mannheim, Germany). Samples were spun for 13,000 rpm for 10 min at 4° C. after incubation on ice for 30 min. Supernatants were collected and protein concentrations of the lysates were determined by Bio-Rad assay.
Western analysis. Equal amounts of cell or tumor lysates were separated on a SDS-PAGE, transferred to nitrocellulose filters, probed with desired antibodies, and visualized by ECL (Amersham; Piscataway, N.J.). Antibodies for detecting IGFR and IRS-1 were from Santa Cruz Biotechnology (Santa Cruz, Calif.). Antibodies against phospho-IRS1[pY896] and phospho-IRS1[pY612] were from Biosource (Camarillo, Calif.).
IGF-II protein measurement. Cells from various cell lines were seeded in T-175 plates in medium containing 10% FBS. After cells were attached, medium was changed to serum free medium. Medium was collected, all debris was spun down, and the supernatants were lyophilized. Cells on the plates were trypsinized and counted. Water was added to each lyophilized supernatant sample (1 ml/2×107 cells). IGF-II was measured using the IGF-II ELISA kit from DSL (DSL-10-2600). IGF-II amounts were determined by the standard curve and reported as nanogram IGF-II per 1×106 cells.
IGF-II mRNA measurement. RNAs were made from tumor samples and cDNAs were synthesized. Expression of IGF-II was analyzed on 20 ng of cDNA sample in a Fluorogenic 5′-nuclease PCR assay with specific probes and primers using the ABI Prism 7700 Sequence Detection System (Applied Biosystems; Foster City, Calif.). CT numbers were normalized by determining Ubiquitin (reference gene) mRNA expression in all samples.











IGF2/forward:





AGGAGCTCGAGGCGTTCAG
(SEQ ID NO: 102)



 



IGF2/reverse:



GTCTTGGGTGGGTAGAGCAATC
(SEQ ID NO: 103)



 



probe:



AGGCCAAACGTCACCGTCCCC
(SEQ ID NO: 104)






Xenograft models in mice. Four to five million human tumor cells (H322, H838, A2780, ES2, MCF7, SW-527, SK-N-AS, SK-N-MC) in Matrigel were inoculated subcutaneously into each nude mouse. When the tumor size reached at least  ̃50 mm3, 19D12/15H12 LCF/HCA treatment was initiated and continued with dosing two times per week. 19D12/15H12 LCF/HCA was injected into each nude mouse, intraperitoneally, at 0.004 mg/mouse, 0.02 mg/mouse, 0.1 mg/mouse or 0.5 mg/mouse. Tumor volumes were measured by Labcat.
IRS-1 phosphorylation level in human lung cancer and normal tissue samples. Twelve pairs of samples of normal and cancerous human lung cancerous tissue samples were obtained from patients. Cell lysates were prepared from the tissue samples and subjected to western blot analysis, staining with anti-phospho-IRS1[pY896] as described above. Total IRS-1 was also measured by staining with an anti-IRS antibody.
The western blot data generated in these experiments is set forth in FIG. 1. In 6 out of the 12 sample pairs evaluated (50%), greater phospho-IRS-1 levels were observed in tumor tissue samples than in the corresponding normal tissue sample.
Similar results were observed when the level of IRS-1 phosphorylation was measured in normal and cancerous colorectal tissue samples. The colorectal tissue samples were evaluated essentially identically to that way the lung tissue samples were evaluated.
Correlation of In vivo efficacy of 19D12/15H12 LCF/HCA with IRS-1 phosphorylation. To evaluate in vivo efficacy of 19D12/15H12 LCF/HCA antibody, nude mice bearing human tumor xenografts were administered the antibody or an isotype control, and tumor volume was evaluated over time as described above.
To evaluate IRS-1 phosphorylation in tumor cell lines, cell lines were grown in the presence of absence of 100 ng/ml IGF-I. Cell lysates of A2780, ES2, H322, H838 and SK-N-AS cells were then prepared and evaluate-d by western blot analysis as describe above.
The results of the in vivo efficacy experiments are set forth in FIG. 2. The 19D12/15H12 LCF/HCA antibody was found to be effective at inhibiting the growth of several types of tumors in vivo (e.g., non-small cell lung cancer, ovarian cancer, breast cancer, neuroblastoma).
The results of the experiments measuring basal and IGF-I stimulated IRS-1 phosphorylation in tumor cells are set forth in FIG. 3. The A2780, H322 and SK-N-AS cell lines evaluated exhibited the greatest basal and IGF-I stimulated IRS-1 phosphorylation.
The cell lines that were most sensitive, in vivo, to growth inhibition by 19D12/15H12 LCF/HCA (FIG. 2) were those that showed the greatest basal and IGF-I stimulated IRS-1 phosphorylation (FIG. 3).
IGF-II mRNA expression level in ovarian and colorectal tumor samples. Normal and cancerous ovarian and colorectal tissue samples were obtained from multiple cancer patients. The level of IGF-II mRNA expression was evaluated, by Taqman analysis, as described above. The level of IGF-II mRNA expression of each ovarian tissue sample is set forth in FIG. 4 and the level of IGF-II mRNA expression in each colorectal tissue sample is set forth in FIG. 5. In these experiments, 20% of ovarian tumor samples were found to overexpress IGF-II mRNA as compared to normal ovarian tissue samples. Fifty three percent of colorectal samples were found to overexpress IGF-II mRNA as compared with adjacent, normal colorectal samples.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
Patents, patent applications, Genbank Accession Numbers and publications are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties.

## Claims

1. A method for treating a non-small cell lung tumor, an ovarian tumor, a neuroblastoma tumor or an osteosarcoma tumor in a mammalian patient wherein growth or survival of the tumor is mediated by IGF1R expression or activity said method comprising:
(a) selecting a mammalian patient having a non-small cell lung tumor, an ovarian tumor, a neuroblastoma tumor or an osteosarcoma tumor expressing phosphorylated IRS-1 tyrosine 896 or phosphorylated IRS-1 tyrosine 612; and
(b) administering to said mammalian patient a therapeutically effective amount of an isolated antibody that binds specifically to IGF1R comprising a light chain immunoglobulin variable domain comprising the amino acid sequence: EIVLTQSPGT LSVSPGERATLSCRASQSIGSSLHWYQQKPGQAPRLLIKYASQSLSGIPD RFSGSGSGTDFTLTISRLEPEDFAVYYCHQSSRLPHTFGQGTKVEIKRT (amino acids 20-128 of SEQ ID NO: 8); and a heavy chain immunoglobulin variable domain comprising the amino acid sequence: EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSFAMHWVRQAPGKGLEWISVIDTRGATYYA DSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARLGNFYYGMDVWGQGTTVTVSS (amino acids 20-137 of SEQ ID NO: 10); wherein said antibody inhibits growth or survival of said tumor.

2. The method of claim 1 wherein the tumor is an osteosarcoma tumor.

3. The method of claim 2 wherein the antibody is a monoclonal antibody and the patient is a human.

4. The method of claim 2 wherein the antibody is a fully human monoclonal antibody and the patient is a human.

5. The method of claim 3 wherein said light chain immunoglobulin variable domain is conjugated to an immunoglobulin kappa constant domain and said heavy chain immunoglobulin variable domain is conjugated to an immunoglobulin gamma 1 constant domain.

6. The method of claim 4 wherein said light chain immunoglobulin variable domain is conjugated to an immunoglobulin kappa constant domain and said heavy chain immunoglobulin variable domain is conjugated to an immunoglobulin gamma 1 constant domain.

7. The method of claim 3 wherein said patient is administered the monoclonal antibody in association with one or more additional anti-cancer agents.

8. The method of claim 7 wherein the patient is administered said monoclonal antibody in association with one or more additional anti-cancer agents selected from the group consisting of: paclitaxel, thalidomide, docetaxel, gefitinib, temozolomide, lonafarnib, tipifarnib, letrozole, doxorubicin, cis-platin, oxaliplatin, camptothecin, topotecan, etoposide, vincristine, vinblastine, raloxifene, gemcitabine, retinoic acid, tamoxifen, trastuzumab, cetuximab and octreotide.

9. The method of claim 4 wherein said patient is administered the monoclonal antibody in association with one or more additional anti-cancer agents.

10. The method of claim 9 wherein the patient is administered said monoclonal antibody in association with one or more additional anti-cancer agents selected from the group consisting of: paclitaxel, thalidomide, docetaxel, gefitinib, temozolomide, lonafarnib, tipifarnib, letrozole, doxorubicin, cis-platin, oxaliplatin, camptothecin, topotecan, etoposide, vincristine, vinblastine, raloxifene, gemcitabine, retinoic acid, tamoxifen, trastuzumab, cetuximab and octreotide.

11. The method of claim 8 wherein the additional anti-cancer agent is paclitaxel.

12. The method of claim 8 wherein the additional anti-cancer agent is docetaxel.

13. The method of claim 8 wherein the additional anti-cancer agent is gefitinib.

14. The method of claim 8 wherein the additional anti-cancer agent is cis-platin.

15. The method of claim 8 wherein the additional anti-cancer agent is oxaliplatin.

16. The method of claim 8 wherein the additional anti-cancer agent is octreotide.