EA044914B1 - WAYS TO IMPROVE ANTI-LIF ANTIBODY TREATMENT RESPONSE IN INDIVIDUALS WITH CANCER - Google Patents

Description

Cross reference to related applications

This application claims priority under the European application serial no.

18382431.7, filed June 18, 2018, and European application serial number 19382131.1, filed February 22, 2019, the contents of which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Leukemia inhibitory factor (LIF) is a member of the interleukin-6 (IL-6) family of cytokines. Based on known physiological activities of LIF, clinical evidence of LIF deficiency, and non-clinical studies of LIF expression in healthy tissues and tumors, LIF inhibition is expected to be well tolerated and result in antitumor efficacy in a range of solid tumors, including but not limited to non-small cell lung cancer (NSCLC), pancreatic cancer, ovarian cancer and glioblastoma multiforme (GBM).

One problem that clinicians face when treating cancer with target-specific therapeutic agents is that not all tumors may respond to a given target-specific therapeutic agent. Even for tumor types known to express LIF or the LIF receptor, there is significant heterogeneity among individual tumors. In addition, not all tumors that express LIF or the LIF receptor may respond equally, so there is a need for effective methods for identifying individuals who may benefit most from treatment with a therapeutic anti-LIF antibody.

Brief description of the invention

The present invention relates to methods of treating cancer in individuals, including administering a therapeutic anti-LIF antibody to those individuals who are likely to respond to said antibody, and methods for determining which individuals are most likely to respond to a therapeutic anti-LIF antibody. Patients with tumors or suffering from cancers that are characterized by expression of LIF or LIF receptor at levels of mRNA or protein exceeding the reference level, as described herein, can be effectively treated with a therapeutic antibody to LIF. In addition, several biomarkers other than LIF biomarkers have been described that can help identify individuals who will benefit from treatment with a therapeutic anti-LIF antibody. These biomarkers, other than LIF biomarkers, can be used alone or in conjunction with an assay that measures LIF or LIF receptor levels. Biomarkers other than LIF biomarkers include immunomodulatory molecules, which indicate an immunosuppressive signature, which includes the presence of immunosuppressive cell types, immunosuppressive cytokines, or immunosuppressive chemokines. It is expected that determination of LIF or LIF receptor levels together with an immunosuppressive signature will increase the predictive power of a method for determining treatment with a therapeutic anti-LIF antibody.

In one aspect, this document describes a method of treating an individual suffering from cancer with a therapeutic leukemia inhibitory factor (LIF) antibody, comprising detecting a level of LIF above a reference level in a biological sample obtained from the individual and administering a therapeutic amount of the antibody. to an individual's LIF if the LIF level is higher than the LIF reference level. In certain embodiments, the anti-LIF therapeutic antibody comprises: an immunoglobulin heavy chain complementarity determining region 1 (VH-CDR1) comprising the amino acid sequence set forth in any of SEQ ID NOs: 1-3; immunoglobulin heavy chain complementarity determining region 2 (VH-CDR2) comprising the amino acid sequence represented by any of SEQ ID NO: 4 or 5; immunoglobulin heavy chain complementarity determining region 3 (VH-CDR3) comprising the amino acid sequence represented by any one of SEQ ID NOs: 6-8; immunoglobulin light chain complementarity determining region 1 (VL-CDR1) comprising the amino acid sequence represented by any of SEQ ID NO: 9 or 10; immunoglobulin light chain complementarity determining region 2 (VL-CDR2) comprising the amino acid sequence represented by any of SEQ ID NO: 11 or 12; and immunoglobulin light chain complementarity determining region 3 (VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the anti-LIF therapeutic antibody comprises an immunoglobulin heavy chain variable region characterized by at least 85, 90, 95 , 97, 98, 99 or 100% identity with SEQ ID NO: 14, 15, 17 or 38, and an immunoglobulin light chain variable region having at least 85, 90, 95, 97, 98, 99 or 100% identity with SEQ ID NO: 18-21. In certain embodiments, the anti-LIF therapeutic antibody comprises an immunoglobulin heavy chain region having at least 85, 90, 95, 97, 98, 99, or 100% identity with SEQ ID NO: 30-33 or 39, and an immunoglobulin light chain region, having at least 85, 90, 95, 97, 98, 99 or 100% identity with SEQ ID NO: 34-37. In certain embodiments, the anti-LIF therapeutic antibody is an IgG antibody comprising two immunoglobulin heavy chains and two immunoglobulin light chains. In certain embodiments, the LIF level is the LIF protein level, and the determination of the level

- 1 044914 involves performing at least one assay that detects LIF protein, or obtaining the results of at least one assay that detects LIF protein. In certain embodiments, the at least one analysis includes an immunohistochemical study. In certain embodiments, the reference level is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 50% of cells staining positive with anti-LIF antibody.

In certain embodiments, the reference level corresponds to an IHC score of 100. In certain embodiments, the reference level corresponds to an IHC score of from about 1 to about 300. In certain embodiments, the reference level corresponds to an IHC score of from about 1 to about 30, from about 1 to about 60, about 1 to about 90, about 1 to about 120, about 1 to about 150, about 1 to about 180, about 1 to about 210, about 1 to about 240, about 1 to about 270, about 1 to about 300, about 30 to about 60, about 30 to about 90, about 30 to about 120, about 30 to about 150, about 30 to about 180, about 30 to about 210, from about 30 to about 240, from about 30 to about 270, from about 30 to about 300, from about 60 to about 90, from about 60 to about 120, from about 60 to about 150, from about 60 to about 180, about 60 to about 210, about 60 to about 240, about 60 to about 270, about 60 to about 300, about 90 to about 120, about 90 to about 150, about 90 to about 180, about 90 to about 210, about 90 to about 240, about 90 to about 270, about 90 to about 300, about 120 to about 150, about 120 to about 180, about 120 to about 210 , from about 120 to about 240, from about 120 to about 270, from about 120 to about 300, from about 150 to about 180, from about 150 to about 210, from about 150 to about 240, from about 150 to about 270, from about 150 to about 300, from about 180 to about 210, from about 180 to about 240, from about 180 to about 270, from about 180 to about 300, from about 210 to about 240, from about 210 to about 270, from about 210 to about 300, about 240 to about 270, about 240 to about 300, or about 270 to about 300. In certain embodiments, the reference level corresponds to an IHC score of about 1, about 30, about 60, about 90, about 120, about 150, about 180, about 210, about 240, about 270, or about 300. In certain embodiments, the reference level corresponds to an IHC score of at least about 1, about 30, about 60, about 90, about 120, about 150, about 180, about 210, about 240, or about 270. In some embodiments, the reference level corresponds to an IHC score of no more than about 30, about 60, about 90, about 120, about 150, about 180, about 210, about 240, about 270, or about 300. In certain embodiments, the reference level is equal to or greater than an IHC score of about 10 to about 100.

In certain embodiments, the at least one assay includes an enzyme-linked immunosorbent assay (ELISA). In certain embodiments, electrochemiluminescence is detected using ELISA. In certain embodiments, the reference level is from about 1 pg/ml to about 10 pg/ml LIF in an undiluted biological sample obtained from an individual. In certain embodiments, the LIF reference level corresponds to the 5th percentile of LIF protein expression in LIF-positive cancers of the same type. In certain embodiments, the LIF reference level corresponds to the 10th percentile of LIF protein expression in LIF-positive cancers of the same type. In certain embodiments, the LIF reference level corresponds to the 5th percentile of LIF protein expression in an exemplary sample of human cancers. In certain embodiments, the LIF reference level corresponds to the 10th percentile of LIF protein expression in an exemplary sample of human cancers. In certain embodiments, the LIF level is a LIF mRNA level, and determining the level includes performing at least one assay that detects LIF mRNA or obtaining the results of at least one assay that detects LIF mRNA. In certain embodiments, the reference level is a level corresponding to

- 2 044914 representing the 5th percentile of LIF mRNA expression in cancers of the same type. In certain embodiments, the reference level is the level corresponding to the 10th percentile of LIF mRNA expression in cancers of the same type. In certain embodiments, the reference level is the level corresponding to the 5th percentile of LIF mRNA expression in an exemplary sample of human cancers. In certain embodiments, the reference level is the level corresponding to the 10th percentile of LIF mRNA expression in an exemplary sample of human cancers. In certain embodiments, the LIF reference level corresponds to the 25th percentile of LIF protein expression in LIF-positive cancers of the same type. In certain embodiments, the LIF reference level corresponds to the 50th percentile of LIF protein expression in LIF-positive cancers of the same type. In certain embodiments, the LIF reference level corresponds to the 25th percentile of LIF protein expression in an exemplary sample of human cancers. In certain embodiments, the LIF reference level corresponds to the 50th percentile of LIF protein expression in an exemplary sample of human cancers. In certain embodiments, the LIF level is a LIF mRNA level, and determining the level includes performing at least one assay that detects LIF mRNA or obtaining the results of at least one assay that detects LIF mRNA. In certain embodiments, the reference level is the level corresponding to the 25th percentile of LIF mRNA expression in cancers of the same type. In certain embodiments, the reference level is the level corresponding to the 50th percentile of LIF mRNA expression in cancers of the same type. In certain embodiments, the reference level is the level corresponding to the 25th percentile of LIF mRNA expression in an exemplary sample of human cancers. In certain embodiments, the reference level is the level corresponding to the 50th percentile of LIF mRNA expression in an exemplary sample of human cancers. In certain embodiments, the LIF level is a LIF DNA level, and determining the level includes performing at least one assay that detects LIF DNA or obtaining the results of at least one assay that detects LIF DNA. In certain embodiments, the at least one assay includes a polymerase chain reaction (PCR). In certain embodiments, the PCR includes quantitative PCR. In certain embodiments, the at least one analysis includes a sequencing reaction. In certain embodiments, the sequencing reaction includes a next generation sequencing reaction. In certain embodiments, the biological sample includes a blood sample. In certain embodiments, the blood sample is blood plasma. In certain embodiments, the blood sample is blood serum. In certain embodiments, the biological sample includes a tissue sample. In certain embodiments, the biological sample is a tumor biopsy. In certain embodiments, the method further includes determining a DNA, mRNA, or protein level of the immunomodulatory molecule that is greater than a reference level of the immunomodulatory molecule. In certain embodiments, the method further includes determining a DNA, mRNA, or protein level of the immunomodulatory molecule that is below a reference level of the immunomodulatory molecule. In certain embodiments, the immunomodulatory molecule is selected from mRNA transcribed from elements of the list consisting of MHCII, CXCL9, CXCL10, CXCR3, PD-L1, CCL7, CCL2, CCL3 and CCL22, or a protein derived from them. In certain embodiments, the method further includes determining a level of a type II macrophage marker (M2) greater than a reference level of DNA, mRNA, or protein of a type II macrophage marker (M2). In certain embodiments, the M2 marker is an mRNA transcribed from the elements of the list consisting of CD206, CD163, PF4, CTSK and ARG1, or a protein derived from them. In certain embodiments, the method further includes determining a level of LIF receptor (LIFR) DNA, mRNA, or protein greater than a reference LIFR level. In certain embodiments, the level of LIFR is detected on an immunomodulatory cell. In certain embodiments, the human cancer is selected from the list consisting of non-small cell lung cancer, ovarian cancer, kidney cancer, bladder cancer, pancreatic cancer, prostate cancer, urogenital tract cancer, gynecological cancer, gastrointestinal cancer, endocrine cancer system, glioblastoma multiforme, breast cancer, melanoma, colorectal cancer, bile duct cancer, cervical cancer, endometrial cancer, squamous cell carcinoma of the head and neck, and combinations thereof. In certain embodiments, the human cancer is selected from the list consisting of non-small cell lung cancer, ovarian cancer, kidney cancer, bladder cancer, and combinations thereof. In certain embodiments, the human cancer is selected from the list consisting of pancreatic cancer, prostate cancer, glioblastoma multiforme, and combinations thereof.

In another aspect, this document describes a method of treating an individual suffering from cancer with a therapeutic anti-leukemia inhibitory factor (LIF) antibody, comprising detecting a level of LIF above a reference level in a biological sample obtained from the individual and administering a therapeutic amount of the antibody. to LIF. In certain embodiments, those

- 3 044914 anti-LIF rapeutic antibody contains: immunoglobulin heavy chain complementarity determining region 1 (VH-CDR1) containing the amino acid sequence represented by any of SEQ ID NO: 1-3; immunoglobulin heavy chain complementarity determining region 2 (VH-CDR2) comprising the amino acid sequence represented by any of SEQ ID NO: 4 or 5; immunoglobulin heavy chain complementarity determining region 3 (VHCDR3) comprising the amino acid sequence represented by any one of SEQ ID NOs: 6-8; immunoglobulin light chain complementarity determining region 1 (VL-CDR1) comprising the amino acid sequence represented by any of SEQ ID NO: 9 or 10; immunoglobulin light chain complementarity determining region 2 (VL-CDR2) comprising the amino acid sequence represented by any of SEQ ID NO: 11 or 12; and immunoglobulin light chain complementarity determining region 3 (VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the anti-LIF therapeutic antibody comprises an immunoglobulin heavy chain variable region characterized by at least 85, 90, 95 , 97, 98, 99 or 100% identity with SEQ ID NO: 14, 15, 17 or 38, and an immunoglobulin light chain variable region having at least 85, 90, 95, 97, 98, 99 or 100% identity with SEQ ID NO: 18-21. In certain embodiments, the anti-LIF therapeutic antibody comprises an immunoglobulin heavy chain region having at least 85, 90, 95, 97, 98, 99, or 100% identity with SEQ ID NO: 30-33 or 39, and an immunoglobulin light chain region, having at least 85, 90, 95, 97, 98, 99 or 100% identity with SEQ ID NO: 34-37. In certain embodiments, the anti-LIF therapeutic antibody is an IgG antibody comprising two immunoglobulin heavy chains and two immunoglobulin light chains. In certain embodiments, the LIF level is a LIF protein level, and determining the level includes performing at least one assay that detects LIF protein or obtaining the results of at least one assay that detects LIF protein. In certain embodiments, the at least one analysis includes an immunohistochemical study. In certain embodiments, the reference level is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 50% of cells staining positive with anti-LIF antibody.

In certain embodiments, the reference level corresponds to an IHC score of 100. In certain embodiments, the reference level corresponds to an IHC score of from about 1 to about 300. In certain embodiments, the reference level corresponds to an IHC score of from about 1 to about 30, from about 1 to about 60, about 1 to about 90, about 1 to about 120, about 1 to about 150, about 1 to about 180, about 1 to about 210, about 1 to about 240, about 1 to about 270, about 1 to about 300, about 30 to about 60, about 30 to about 90, about 30 to about 120, about 30 to about 150, about 30 to about 180, about 30 to about 210, from about 30 to about 240, from about 30 to about 270, from about 30 to about 300, from about 60 to about 90, from about 60 to about 120, from about 60 to about 150, from about 60 to about 180, about 60 to about 210, about 60 to about 240, about 60 to about 270, about 60 to about 300, about 90 to about 120, about 90 to about 150, about 90 to about 180, about 90 to about 210, about 90 to about 240, about 90 to about 270, about 90 to about 300, about 120 to about 150, about 120 to about 180, about 120 to about 210 , from about 120 to about 240, from about 120 to about 270, from about 120 to about 300, from about 150 to about 180, from about 150 to about 210, from about 150 to about 240, from about 150 to about 270, from about 150 to about 300, from about 180 to about 210, from about 180 to about 240, from about 180 to about 270, from about 180 to about 300, from about 210 to about 240, from about 210 to about 270, from about 210 to about 300, about 240 to about 270, about 240 to about 300, or about 270 to about 300. In certain embodiments, the reference level corresponds to an IHC score of about 1, about 30, about 60, about 90, about 120, about 150, about 180, about 210, about 240, about 270, or about 300. In certain embodiments, the reference level corresponds to an IHC score of at least about 1, about 30, about 60, about 90,

- 4 044914 about 120, about 150, about 180, about 210, about 240, or about 270. In some embodiments, the reference level corresponds to an IHC score of no more than about 30, about 60, about 90, about 120, about 150, about 180, approximately 210, approximately 240, approximately 270 or approximately 300.

In certain embodiments, the at least one assay includes an enzyme-linked immunosorbent assay (ELISA). In certain embodiments, electrochemiluminescence is detected using ELISA. In certain embodiments, the reference level is at least about 4 pg/ml LIF in an undiluted biological sample obtained from an individual. In certain embodiments, the LIF reference level corresponds to the 5th percentile of LIF protein expression in LIF-positive cancers of the same type. In certain embodiments, the LIF reference level corresponds to the 10th percentile of LIF protein expression in LIF-positive cancers of the same type. In certain embodiments, the LIF reference level corresponds to the 5th percentile of LIF protein expression in an exemplary sample of human cancers. In certain embodiments, the LIF reference level corresponds to the 10th percentile of LIF protein expression in an exemplary sample of human cancers. In certain embodiments, the LIF level is a LIF mRNA level, and determining the level includes performing at least one assay that detects LIF mRNA or obtaining the results of at least one assay that detects LIF mRNA. In certain embodiments, the reference level is the level corresponding to the 5th percentile of LIF mRNA expression in cancers of the same type. In certain embodiments, the reference level is the level corresponding to the 10th percentile of LIF mRNA expression in cancers of the same type. In certain embodiments, the reference level is the level corresponding to the 5th percentile of LIF mRNA expression in an exemplary sample of human cancers. In certain embodiments, the reference level is the level corresponding to the 10th percentile of LIF mRNA expression in an exemplary sample of human cancers. In certain embodiments, the LIF reference level corresponds to the 25th percentile of LIF protein expression in LIF-positive cancers of the same type. In certain embodiments, the LIF reference level corresponds to the 50th percentile of LIF protein expression in LIF-positive cancers of the same type. In certain embodiments, the LIF reference level corresponds to the 25th percentile of LIF protein expression in an exemplary sample of human cancers. In certain embodiments, the LIF reference level corresponds to the 50th percentile of LIF protein expression in an exemplary sample of human cancers. In certain embodiments, the LIF level is a LIF mRNA level, and determining the level includes performing at least one assay that detects LIF mRNA or obtaining the results of at least one assay that detects LIF mRNA. In certain embodiments, the reference level is the level corresponding to the 25th percentile of LIF mRNA expression in cancers of the same type. In certain embodiments, the reference level is the level corresponding to the 50th percentile of LIF mRNA expression in cancers of the same type. In certain embodiments, the reference level is the level corresponding to the 25th percentile of LIF mRNA expression in an exemplary sample of human cancers. In certain embodiments, the reference level is the level corresponding to the 50th percentile of LIF mRNA expression in an exemplary sample of human cancers. In certain embodiments, the LIF level is a LIF DNA level, and determining the level includes performing at least one assay that detects LIF DNA or obtaining the results of at least one assay that detects LIF DNA. In certain embodiments, the at least one assay comprises a polymerase chain reaction (PCR). In certain embodiments, the PCR includes quantitative PCR. In certain embodiments, the at least one analysis includes a sequencing reaction. In certain embodiments, the sequencing reaction includes a next generation sequencing reaction. In certain embodiments, the biological sample includes a blood sample. In certain embodiments, the blood sample is blood plasma. In certain embodiments, the blood sample is blood serum. In certain embodiments, the biological sample includes a tissue sample. In certain embodiments, the biological sample is a tumor biopsy. In certain embodiments, the method further includes determining a DNA, mRNA, or protein level of the immunomodulatory molecule that is greater than a reference level of the immunomodulatory molecule. In certain embodiments, the method further includes determining a DNA, mRNA, or protein level of the immunomodulatory molecule that is below a reference level of the immunomodulatory molecule. In certain embodiments, the immunomodulatory molecule is selected from mRNA transcribed from elements of the list consisting of MHCII, CXCL9, CXCL10, CXCR3, PD-L1, CCL7, CCL2, CCL3 and CCL22, or a protein derived from them. In certain variants

- 5 044914 implementation of the method further includes determining a level of DNA, mRNA or protein of a type II macrophage marker (M2) exceeding a reference level of a type II macrophage marker (M2). In certain embodiments, the M2 marker is an mRNA transcribed from, or a protein derived from, elements of the list consisting of CD206, CD163, PF4, CTSK and ARG1. In certain embodiments, the method further includes determining a level of LIF receptor (LIFR) DNA, mRNA, or protein greater than a reference LIFR level. In certain embodiments, the level of LIFR is detected on an immunomodulatory cell. In certain embodiments, the human cancer is selected from the list consisting of non-small cell lung cancer, ovarian cancer, kidney cancer, bladder cancer, pancreatic cancer, prostate cancer, urogenital tract cancer, gynecological cancer, gastrointestinal cancer, endocrine cancer system, glioblastoma multiforme, breast cancer, melanoma, colorectal cancer, bile duct cancer, cervical cancer, endometrial cancer, squamous cell carcinoma of the head and neck, and combinations thereof. In certain embodiments, the human cancer is selected from the list consisting of non-small cell lung cancer, ovarian cancer, kidney cancer, bladder cancer, and combinations thereof. In certain embodiments, the human cancer is selected from the list consisting of pancreatic cancer, prostate cancer, glioblastoma multiforme, and combinations thereof.

In another aspect, this document describes a method of treating an individual suffering from cancer with a therapeutic antibody to leukemia inhibitory factor (LIF), comprising determining a level of leukemia inhibitory factor receptor (LIFR) above a reference level in a biological sample obtained from the individual, and administering a therapeutic amount of the anti-LIF antibody to the individual if the LIFR level is higher than the LIFR reference level. In certain embodiments, the level of LIFR is detected on an immunomodulatory cell. In certain embodiments, the anti-LIF therapeutic antibody comprises: an immunoglobulin heavy chain complementarity determining region 1 (VH-CDR1) comprising the amino acid sequence set forth in any of SEQ ID NOs: 1-3; immunoglobulin heavy chain complementarity determining region 2 (VH-CDR2) comprising the amino acid sequence represented by any of SEQ ID NO: 4 or 5; immunoglobulin heavy chain complementarity determining region 3 (VHCDR3) comprising the amino acid sequence represented by any one of SEQ ID NOs: 6-8; immunoglobulin light chain complementarity determining region 1 (VL-CDR1) comprising the amino acid sequence represented by any of SEQ ID NO: 9 or 10; immunoglobulin light chain complementarity determining region 2 (VL-CDR2) comprising the amino acid sequence represented by any of SEQ ID NO: 11 or 12; and immunoglobulin light chain complementarity determining region 3 (VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the anti-LIF therapeutic antibody comprises an immunoglobulin heavy chain variable region characterized by at least 85, 90, 95 , 97, 98, 99 or 100% identity with SEQ ID NO: 14, 15, 17 or 38, and an immunoglobulin light chain variable region having at least 85, 90, 95, 97, 98, 99 or 100% identity with SEQ ID NO: 18-21. In certain embodiments, the anti-LIF therapeutic antibody comprises an immunoglobulin heavy chain region having at least 85, 90, 95, 97, 98, 99, or 100% identity with SEQ ID NO: 30-33 or 39, and an immunoglobulin light chain region, having at least 85, 90, 95, 97, 98, 99 or 100% identity with SEQ ID NO: 34-37. In certain embodiments, the anti-LIF therapeutic antibody is an IgG antibody comprising two immunoglobulin heavy chains and two immunoglobulin light chains. In certain embodiments, the LIFR level is a LIFR protein level, and determining the level includes performing at least one assay that detects LIFR protein or obtaining the results of at least one assay that detects LIFR protein. In certain embodiments, the at least one analysis includes an immunohistochemical study. In certain embodiments, the at least one assay includes an enzyme-linked immunosorbent assay (ELISA). In certain embodiments, electrochemiluminescence is detected using ELISA. In certain embodiments, the at least one analysis includes flow cytometry. In certain embodiments, the LIFR level is a LIFR mRNA level, and determining the level includes performing at least one assay that detects LIFR mRNA or obtaining results from at least one assay that detects LIFR mRNA. In certain embodiments, the LIFR level is a LIFR DNA level, and determining the level includes performing at least one test that detects LIFR DNA or obtaining the results of at least one test that detects LIFR DNA. In certain embodiments, the at least one assay comprises a polymerase chain reaction (PCR). In certain embodiments, the PCR includes quantitative PCR. In certain embodiments, the at least one analysis includes a sequencing reaction. In certain embodiments, the sequencing reaction includes a next generation sequencing reaction. In certain embodiments, biolo

- 6 044914 gical sample includes a blood sample. In certain embodiments, the blood sample is blood plasma. In certain embodiments, the blood sample is blood serum. In certain embodiments, the biological sample includes a tissue sample. In certain embodiments, the biological sample is a tumor biopsy. In certain embodiments, the method further includes determining a DNA, mRNA, or protein level of the immunomodulatory molecule that is greater than a reference level of the immunomodulatory molecule. In certain embodiments, the method further includes determining a DNA, mRNA, or protein level of the immunomodulatory molecule that is below a reference level of the immunomodulatory molecule. In certain embodiments, the immunomodulatory molecule is selected from mRNA transcribed from elements of the list consisting of MHCII, CXCL9, CXCL10, CXCR3, PD-L1, CCL7, CCL2, CCL3 and CCL22, or a protein derived from them. In certain embodiments, the method further includes determining a level of DNA, mRNA, or protein of a type II macrophage marker (M2) greater than a reference level of a type II macrophage marker (M2). In certain embodiments, the M2 marker is an mRNA transcribed from the elements of the list consisting of CD206, CD163, PF4, CTSK and ARG1, or a protein derived from them. In certain embodiments, the method further includes determining a level of LIF DNA, mRNA, or protein that is greater than a reference LIF level. In certain embodiments, the human cancer is selected from the list consisting of non-small cell lung cancer, ovarian cancer, kidney cancer, bladder cancer, pancreatic cancer, prostate cancer, urogenital tract cancer, gynecological cancer, gastrointestinal cancer, endocrine cancer system, glioblastoma multiforme, breast cancer, melanoma, colorectal cancer, bile duct cancer, cervical cancer, endometrial cancer, squamous cell carcinoma of the head and neck, and combinations thereof. In certain embodiments, the human cancer is selected from the list consisting of non-small cell lung cancer, ovarian cancer, kidney cancer, bladder cancer, and combinations thereof. In certain embodiments, the human cancer is selected from the list consisting of pancreatic cancer, prostate cancer, glioblastoma multiforme, and combinations thereof.

In another aspect, this document describes a method of treating an individual suffering from cancer with a therapeutic antibody against leukemia inhibitory factor (LIF), comprising determining a level of leukemia inhibitory factor receptor (LIFR) above a reference level in a biological sample obtained from the individual and administering a therapeutic amount of the anti-LIF antibody to the individual. In certain embodiments, the level of LIFR is detected on an immunomodulatory cell. In certain embodiments, the anti-LIF therapeutic antibody comprises: an immunoglobulin heavy chain complementarity determining region 1 (VH-CDR1) comprising the amino acid sequence set forth in any of SEQ ID NOs: 1-3; immunoglobulin heavy chain complementarity determining region 2 (VH-CDR2) comprising the amino acid sequence represented by any of SEQ ID NO: 4 or 5; immunoglobulin heavy chain complementarity determining region 3 (VH-CDR3) comprising the amino acid sequence represented by any one of SEQ ID NOs: 6-8; immunoglobulin light chain complementarity determining region 1 (VL-CDR1) comprising the amino acid sequence represented by any of SEQ ID NO: 9 or 10; immunoglobulin light chain complementarity determining region 2 (VL-CDR2) comprising the amino acid sequence represented by any of SEQ ID NO: 11 or 12; and immunoglobulin light chain complementarity determining region 3 (VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the anti-LIF therapeutic antibody comprises an immunoglobulin heavy chain variable region characterized by at least 85, 90, 95 , 97, 98, 99 or 100% identity with SEQ ID NO: 14, 15, 17 or 38, and an immunoglobulin light chain variable region having at least 85, 90, 95, 97, 98, 99 or 100% identity with SEQ ID NO: 18-21. In certain embodiments, the anti-LIF therapeutic antibody comprises an immunoglobulin heavy chain region having at least 85, 90, 95, 97, 98, 99, or 100% identity with SEQ ID NO: 30-33 or 39, and an immunoglobulin light chain region, having at least 85, 90, 95, 97, 98, 99 or 100% identity with SEQ ID NO: 34-37. In certain embodiments, the anti-LIF therapeutic antibody is an IgG antibody comprising two immunoglobulin heavy chains and two immunoglobulin light chains. In certain embodiments, the LIFR level is a LIFR protein level, and determining the level includes performing at least one assay that detects LIFR protein or obtaining the results of at least one assay that detects LIFR protein. In certain embodiments, the at least one analysis includes an immunohistochemical study. In certain embodiments, the at least one assay includes an enzyme-linked immunosorbent assay (ELISA). In certain embodiments, electrochemiluminescence is detected using ELISA. In certain embodiments, the at least one analysis includes flow cytometry. In certain embodiments, the LIFR level is the LIFR mRNA level, and determining the level includes conducting

- 7 044914 at least one assay that detects LIFR mRNA, or obtaining the results of at least one assay that detects LIFR mRNA. In certain embodiments, the LIFR level is a LIFR DNA level, and determining the level includes performing at least one test that detects LIFR DNA or obtaining the results of at least one test that detects LIFR DNA. In certain embodiments, the at least one assay includes a polymerase chain reaction (PCR). In certain embodiments, the PCR includes quantitative PCR. In certain embodiments, the at least one analysis includes a sequencing reaction. In certain embodiments, the sequencing reaction includes a next generation sequencing reaction. In certain embodiments, the biological sample includes a blood sample. In certain embodiments, the blood sample is blood plasma. In certain embodiments, the blood sample is blood serum. In certain embodiments, the biological sample includes a tissue sample. In certain embodiments, the biological sample is a tumor biopsy. In certain embodiments, the method further includes determining a DNA, mRNA, or protein level of the immunomodulatory molecule that is greater than a reference level of the immunomodulatory molecule. In certain embodiments, the method further includes determining a DNA, mRNA, or protein level of the immunomodulatory molecule that is below a reference level of the immunomodulatory molecule. In certain embodiments, the immunomodulatory molecule is selected from mRNA transcribed from elements of the list consisting of MHCII, CXCL9, CXCL10, CXCR3, PD-L1, CCL7, CCL2, CCL3 and CCL22, or a protein derived from them. In certain embodiments, the method further includes determining a level of DNA, mRNA, or protein of a type II macrophage marker (M2) greater than a reference level of a type II macrophage marker (M2). In certain embodiments, the M2 marker is an mRNA transcribed from, or a protein derived from, elements of the list consisting of CD206, CD163, PF4, CTSK and ARG1. In certain embodiments, the method further includes determining a level of LIF DNA, mRNA, or protein that is greater than a reference LIF level. In certain embodiments, the human cancer is selected from the list consisting of non-small cell lung cancer, ovarian cancer, kidney cancer, bladder cancer, pancreatic cancer, prostate cancer, urogenital tract cancer, gynecological cancer, gastrointestinal cancer, endocrine cancer system, glioblastoma multiforme, breast cancer, melanoma, colorectal cancer, bile duct cancer, cervical cancer, endometrial cancer, squamous cell carcinoma of the head and neck, and combinations thereof. In certain embodiments, the human cancer is selected from the list consisting of non-small cell lung cancer, ovarian cancer, kidney cancer, bladder cancer, and combinations thereof. In certain embodiments, the human cancer is selected from the list consisting of pancreatic cancer, prostate cancer, glioblastoma multiforme, and combinations thereof.

Brief description of graphic materials

In fig. Figure 1 shows Western blot results demonstrating inhibition of LIF-induced STAT3 phosphorylation by various humanized anti-LIF antibodies.

In fig. 2A and 2B show Western blot results demonstrating inhibition of LIF-induced STAT3 phosphorylation by the humanized and parental 5D8 antibody.

In fig. 3A shows the IC ₅₀ for inhibition of LIF in U-251 cells using the h5D8 antibody.

In fig. 3B shows representative IC ₅₀ determined from the dose-response curve of pSTAT3 inhibition by r5D8 and h5D8 under conditions of stimulation with endogenous LIF. Representative curves are shown (n=1 h5D8, n=2 r5D8).

In fig. 4 shows Western blot results demonstrating inhibition of LIF-induced STAT3 phosphorylation by various monoclonal antibodies described in this disclosure.

In fig. Figure 5 presents the results of immunohistochemical staining and quantification of LIF expression in glioblastoma multiforme (GBM), NSCLC (non-small cell lung carcinoma), ovarian cancer, colorectal cancer tumors, and pancreatic tumors from human patients. Horizontal bars represent mean +/- SEM.

In fig. 6 is a graph showing the results of an experiment conducted in a mouse model of non-small cell lung cancer using the humanized antibody 5D8.

In fig. 7A shows the effect of r5D8 on inhibiting U251 cells in an orthotopic GBM mouse model. Quantification results are shown for day 26.

In fig. 7B shows data from mice inoculated with human U251 GBM cells expressing luciferase and then treated with 100, 200, or 300 μg of h5D8 or vehicle twice weekly. Tumor size was determined by bioluminescence (Xenogen IVIS Spectrum) on day 7. The graph shows individual tumor measurements, with horizontal bars indicating the mean ± SEM. Statistical significance was calculated using the unpaired nonparametric Mann-Whitney U test.

- 8 044914

In fig. 8A shows the effect of r5D8 on inhibiting the growth of ovarian cancer cells in a syngeneic mouse model.

In fig. Figure 8B shows individual tumor measurement values at day 25.

In fig. 8C illustrates that h5D8 exhibits a significant reduction in tumor growth when administered at a dose of 200 μg/mouse twice weekly (p < 0.05). Symbols represent mean + SEM, statistical significance compared with carrier (via unpaired nonparametric Mann-Whitney U test).

In fig. 9A shows the effect of r5D8 on inhibiting the growth of colorectal cancer cells in a syngeneic mouse model.

In fig. Figure 9B shows individual tumor measurement values at day 17.

In fig. 10A shows decreased macrophage infiltration of tumor sites in an orthotopic GBM mouse model with representative imaging and quantification of CCL22+ cells.

In fig. 10B shows decreased macrophage polarization in a human organotypic tissue slice culture model. A representative image (left) and quantification (right) are shown.

In fig. 10C shows decreased macrophage polarization to tumor sites in a syngeneic mouse model of ovarian cancer with representative imaging and quantification of CCL22+ cells.

In fig. 10D shows decreased macrophage infiltration of tumor sites in a syngeneic mouse model of colorectal cancer with representative imaging and quantification of CCL22+ cells.

In fig. 11A shows an increase in the number of non-myeloid effector cells in a syngeneic mouse model of ovarian cancer after treatment with r5D8.

In fig. 11B shows an increase in the number of non-myeloid effector cells in a syngeneic mouse model of colorectal cancer after treatment with r5D8.

In fig. 11C shows a decrease in the percentage of CD4+ TREG cells in the NSCLC mouse cancer model following treatment with r5D8.

In fig. 12 shows data from mice bearing CT26 tumors that were treated twice weekly with PBS (control) or r5D8 administered intraperitoneally in the presence or absence of depleting antibodies to CD4 and CD8. The graph shows individual tumor measurements at day 13 expressed as mean tumor volume + SEM. Statistical differences between groups were determined using the unpaired nonparametric Mann-Whitney U test. R5D8 inhibited the growth of CT26 tumors (*p<0.05). Tumor growth inhibition by r5D8 was significantly reduced in the presence of depleting antibodies to CD4 and CD8 (****p < 0.0001).

In fig. 13A provides an overview of the cocrystal structure of h5D8 Fab in complex with LIF. The gp130 interaction site is mapped to the LIF surface (dark shaded).

In fig. 13B demonstrates detailed interactions between LIF and h5D8 and shows salt-bridge-forming residues and h5D8 residues with buried surface area greater than 100 A ² .

In fig. 14A shows a superposition of five crystal structures of h5D8 Fab and indicates a high degree of similarity, despite the fact that they crystallized under different chemical conditions.

In fig. Figure 14B demonstrates an extensive network of van der Waals interactions mediated by unpaired Cys100. This residue is highly ordered, participates in the formation of HCDR1 and HCDR3 conformations, and is not involved in unwanted rearrangement of disulfide bonds. Distances between residues are shown as dashed lines and labeled.

In fig. 15A demonstrates binding of h5D8 C100 mutants to human LIF by ELISA.

In fig. 15B demonstrates binding of h5D8 C100 mutants to mouse LIF by ELISA.

In fig. 16A demonstrated by Octet that h5D8 does not block the binding between LIF and LIFR. Sequential binding of h5D8 to LIF followed by binding to LIFR.

In fig. 16B and 16C demonstrate ELISA analysis of the binding of LIF/mAb complexes to immobilized LIFR or gp130. Signals of species-specific peroxidase-conjugated anti-IgG antibodies (anti-human for (-) and h5D8, anti-rat for r5d8 and B09), providing detection of the antibody portion of mAb/LIF complexes bound to LIFR (Fig. 16B) or gp130 (Fig. 16C) , immobilized on tablets.

In fig. 17A and 17B demonstrate LIF (FIG. 16A) or LIFR (FIG. 16B) mRNA expression in 72 different human tissues.

In fig. Figure 18 shows mRNA expression levels in different cancer types, stratified into high, medium-high, medium-low, and low levels. Expression data represent LIF transcript levels measured in 7769 samples selected from 22 categories derived from The Cancer Genome Atlas and divided into quartiles across the entire sample.

- 9 044914

In fig. 19A shows the correlation (r ² ) between LIF mRNA levels and CCL7 mRNA levels. Samples for each category were obtained from The Cancer Genome Atlas and the association between LIF and CCL7 was assessed by Pearson correlation.

In fig. 19B shows the correlation (r ² ) between LIF mRNA levels and CCL2 mRNA levels. Samples for each category were obtained from The Cancer Genome Atlas and the association between LIF and CCL2 was assessed by Pearson correlation.

In fig. 19C shows the correlation (r ² ) between LIF mRNA levels and CCL3 mRNA levels. Samples for each category were obtained from The Cancer Genome Atlas and the association between LIF and CCL3 was assessed by Pearson correlation.

In fig. 19D shows the correlation (r ² ) between LIF mRNA levels and CCL22 mRNA levels. Samples for each category were obtained from The Cancer Genome Atlas and the association between LIF and CCL22 was assessed by Pearson correlation.

In fig. 20A shows the correlation (r ² ) between LIF mRNA levels and the expression signature typical of a type II macrophage (M2).

In fig. 20B shows the correlation between the expression levels of LIF and CD163, CD206 and CCL2 in GBM and ovarian cancer. Regression plots between expression levels of LIF and CD163, CD206, CCL2 (in Iog2 RSEM) in tumor cohorts from TCGA in GBM and ovarian (OV) cancer.

In fig. 20C shows IHC correlation of the indicated markers from 20 GBM tumors. Correlations between LIF and CCL2, CD206, CD163 and CXCL9 are shown using R-squared (R ² ) coefficients.

In fig. 20D shows the percentage and mean fluorescence intensity (MFI) of CCL2+ and CXCL9+ in TAMs (CD11b ⁺ Ly6G ^- Ly6C ^- ) from GL261N tumors treated or untreated with anti-LIF antibodies.

In fig. 20E shows the percentage of double positive cells compared to cells positive for TAM markers. Quantification of CXCL9 refers to the total number of cells.

In fig. 20F shows the percentage of CCR2, CXCR3 and LIFR receptors in TAM (CD11b ⁺ Ly6G ^- Ly6C ^- ) and CD8 ⁺ T cell populations (CD3 ⁺ CD8 ⁺ ) determined by flow cytometry. Data are presented as mean ± SEM. Statistical analysis was performed using the Mann-Whitney T test. *P < 0.05.

In fig. 20G shows GL261N tumor growth in CXCL9'/' and CCL2' ^/_ mice or mice treated with the indicated antibodies, with data shown as total flux (photon/s).

In fig. 20H shows a fold increase in the number of tumor-infiltrating CD8 T cells with the indicated treatments. Data represent mean ± SEM. Statistical analyzes were performed using the Mann-Whitney T test. *P < 0.05; **P < 0.01; ***P < 0.001; ****p < 0.0001.

In fig. 20I shows the percentage of double positive cells relative to Iba1 ⁺ cells and the percentage of CXCL9+ cells in organotypic GBM sections (patients 1, 2, 3) incubated with 10 μg/ml anti-LIF antibody for 3 days, relative to the total cells. Data represent the mean of all patients ± SEM. Statistical analyzes were performed using the Mann-Whitney T test. *P < 0.05, **P <0.01; ***P <0.001; ****P < 0.0001.

In fig. Figure 20J shows endpoint M1 and M2 gene transcript levels in CT26 tumors determined by qRT-PCR of whole tumor lysate (n = 8). Data are presented as fold change relative to the mean of IgG1-treated control tumors (Hprt, Tbp, Tfrc).

In fig. 20K shows the results of quantitative determination of the population of pan-myeloid cells (CD11b ⁺ in CD45+ cells), TAM (CD11b ⁺ Ly6C ^nU3kuy F4/80+ in CD45+ cells), MHC II ⁺ TAM (MHC II ⁺ in CD11b ⁺ Ly6C ^nU3kuy F4/80+ cells) and MHC II expression in TAM. Data are shown as mean ± s.e.m. (N values are reported for each experiment), *p < 0.05, ** p < 0.01, *** p < 0.001.

In fig. 21A and B show the level of LIF receptor on primary macrophages differentiated from primary monocytes from 3 different donors, determined by flow cytometry (FIG. 21A), and quantified by interpolation against fluorescent calibration microspheres (FIG. 21B).

In fig. Figure 22 shows an increase in CD206 and CD163 levels in primary _M0 macrophages in response to treatment with LIF (20 nM) for 72 hours.

In fig. Figure 23 shows increased secretion of CCL22 in response to treatment with LIF (20 nM) in _MO macrophages and M1 and M2 macrophages (lower right corner).

In fig. Figure 24 shows LIF receptor expression on tumor-associated macrophages in serous ovarian cancer, stage III-C (two different donors, two left panels), and lung adenocarcinoma, stage III-A (two different donors, two right panels), determined via flow cytometry. Control plots are fluorescent controls minus one (FMO).

- 10 044914

In fig. 25 shows LIF receptor expression on tumor-derived monocyte-derived myeloid-derived suppressor cells (M-MDSC), tumor-derived polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) in serous ovarian cancer, stage

Sh-C (two different donors, two left panels), and lung adenocarcinoma, stage Sh-A (two different donors, two right panels), determined by flow cytometry. Control plots are fluorescence minus one (FMO). Samples gated for CD11b ⁺ CD33+

HLA-DR ^hu3k ™.

Detailed Description of the Invention

In one aspect, this document describes a method of treating an individual suffering from cancer with a therapeutic leukemia inhibitory factor (LIF) antibody, comprising detecting a level of LIF above a reference level in a biological sample obtained from the individual and administering a therapeutic amount of the antibody. to an individual's LIF if the LIF level is higher than the LIF reference level.

In another aspect, this document describes a method of treating an individual suffering from cancer with a therapeutic anti-leukemia inhibitory factor (LIF) antibody, comprising detecting a level of LIF above a reference level in a biological sample obtained from the individual and administering a therapeutic amount of the antibody. to LIF.

In another aspect, this document describes a method of treating an individual suffering from cancer with a therapeutic antibody to leukemia inhibitory factor (LIF), comprising determining a level of leukemia inhibitory factor receptor (LIFR) above a reference level in a biological sample obtained from the individual, and administering a therapeutic amount of the anti-LIF antibody to the individual if the LIFR level is higher than the LIFR reference level.

In another aspect, this document describes a method of treating an individual suffering from cancer with a therapeutic antibody against leukemia inhibitory factor (LIF), comprising determining a level of leukemia inhibitory factor receptor (LIFR) above a reference level in a biological sample obtained from the individual and administering a therapeutic amount of the anti-LIF antibody to the individual.

As used herein, the term individual, patient or subject refers to individuals diagnosed with, suspected of, or at risk of developing a cancer, tumor or neoplasm. In certain embodiments, the individual is a mammal. In certain embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. In certain embodiments, the individual is a human.

As used herein, unless otherwise specified, the term immunomodulatory molecule refers to any molecule, polypeptide or protein that is present either in a tumor or in the tumor microenvironment that modulates or causes modulation of the innate and/or adaptive immune system, including without limitation immunosuppressive chemokines, immunosuppressive cytokines, or immune checkpoint inhibitor molecules. Immunomodulatory molecules can be produced by immunomodulatory cells, tumor/cancerous cells or stromal cells. Immunomodulatory molecules include, but are not limited to, CCL7, CCL2, CCL3, CCL22, MHCII, CXCL9, CXCL10, CXCR3, and PD-L1.

As used herein, unless otherwise noted, the term immunomodulatory cell refers to any cell of the immune system that is capable of producing immunomodulatory factors and includes dendritic cells, macrophages, tumor-associated macrophages, type I macrophages, type II macrophages, myeloid suppressor cells. tumor-derived polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), helper T cells, regulatory T cells, activated T cells, antigen-trained T cells, cytotoxic T cells, etc.

As used herein, the term antibody, unless otherwise specified, includes antigen-binding antibody fragments, i.e. antibody fragments that retain the ability to specifically bind to an antigen that binds to the full-length antibody, such as fragments that retain one or more CDR regions. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; antibodies consisting of only heavy chains, single chain antibody molecules, such as single chain variable fragments (scFv), nanoantibodies, and multispecific antibodies formed from antibody fragments with different specificities, such as bispecific antibodies. In certain embodiments, the antibodies are humanized so as to reduce an individual's immune response to the antibody. For example, antibodies can be chimeric, eg, a non-human variable region with a human constant region, or antibodies grafted with CDRs, eg, a non-human CDR with a constant region and human variable region framework sequences. In certain embodiments, the antibodies are deimmunized after humanization. Deimmunization involves the removal or mutation of one or more T cell epitopes

- 11 044914 in the constant region of the antibody. In certain embodiments, the antibodies described herein are monoclonal. As used herein, the term recombinant antibody is an antibody that contains an amino acid sequence obtained from two different species or two different sources, and includes synthetic molecules, for example, an antibody that contains a non-human CDR region and a human framework or constant region. In certain embodiments, the recombinant antibodies of the present invention are derived from or synthesized from a recombinant DNA molecule. The terms cancer and tumor refer to a physiological condition in mammals that is characterized by impaired cellular growth. Cancer is a class of diseases in which a group of cells exhibit uncontrolled or unwanted growth. Cancer cells can also spread to other places, which can lead to the formation of metastases. Dissemination of cancer cells in the body can occur, for example, through lymph or blood. Uncontrolled growth, invasion and formation of metastases are also called the malignant properties of cancer. These malignant properties distinguish cancer from benign tumors, which generally do not invade or metastasize.

As used herein, the term therapeutic antibody is an antibody that is administered to an individual and is believed to exhibit one or more beneficial effects useful in the treatment of cancer. Therapeutic antibodies of the present invention include antibodies having CDR sequences, immunoglobulin heavy and/or light chain variable regions, or immunoglobulin heavy and light chains completely identical to h5D8, or CDRs that differ from h5D8 but which have similar binding characteristics (epitope, affinity or biological effect) and are capable of exhibiting one or more beneficial effects useful for the treatment of cancer.

As used herein, the term therapeutic amount is a dosage of a therapeutic antibody intended to produce one or more beneficial effects useful for treating cancer.

Certain specific therapeutic amounts are discussed in detail herein.

As used herein, the terms treatment or treatment refer to an intervention in a disease state designed to produce one or more beneficial effects. Treatment of cancer/tumor includes methods intended to cause or actually cause the disease to become stable, partial response, complete response, improve progression-free survival, improve overall survival, shrink tumors, slow tumor growth, stop tumor growth or prevent or reduction of metastasis. In certain cases, the therapeutic methods described herein may be used as maintenance treatment after successful treatment or to prevent recurrence or metastasis of a particular tumor or cancer. It is known that not all individuals will respond to the same extent, or at all, to a given administration of a therapeutic antibody, however, even if no response is detected, such individuals are nevertheless considered to have received treatment.

As used herein, the term biomarker is a measurable molecule in an individual, the presence of which is an indicator of the disease state of that individual. Without limitation, biomarkers include, for example, proteins and their post-translational modifications, polypeptides, nucleic acids, DNA, RNA, amino acids, fatty acids, lipids, sterols, carbohydrates or metabolites and metabolic intermediates of amino acids, fatty acids, lipids, sterols and carbohydrates.

As used herein, the term IHC score refers to a parameter used to quantify the expression levels of LIF or LIF receptor in a test sample. the IHC value of a sample is determined by staining the sample with a specific antibody to LIF or LIF receptor using immunohistochemistry. Each tumor cell is assigned an intensity level value ranging from 0 for no staining to 3+ for the most intense staining, 2+ for moderate cell staining, and 1+ for weak cell staining. The IHC score can then be calculated using the following equation:

[1x(% cells 1+)+2x(% cells 2+)+3x(% cells 3+)]

The IHC score can range from 1 to 300. The IHC score in a sample can be used directly to provide an indication of LIF expression levels or can be compared to a reference IHC score to determine whether an individual will respond to treatment with a therapeutic anti-LIF antibody.

As used herein, the term immunohistochemistry, or IHC, refers to a laboratory test using antibodies, affinity molecules, and dyes to test against specific antigens (biomarkers) in a tissue or cell sample. Antibodies can be associated with an enzyme or a fluorescent dye. IHC can be combined with other dyes

- 12 044914 methods that do not involve the use of antibodies, or methods that further detail the structure of a tissue or cell, for example, dyes that stain nuclear or cellular membranes. IHC can be performed on paraffin-embedded, formalin-fixed tissue or biopsies. IHC can also be performed on cells in suspension, with the cells subsequently centrifuged or attached to a microscope slide or coverslip. IHC samples can be suitably analyzed by visible light microscopy or imaging or fluorescence microscopy or imaging. Quantification can be done manually or using a computer program (eg Image J).

As used herein, the term reference level refers to a predetermined criterion used as a standard for evaluating values or data obtained from a sample obtained from an individual. The reference level may be an absolute value; relative value; value with upper and lower limits; range of values; average value; median value; average value; or a value compared to a specific reference or baseline value. The reference level may be based on a sample value obtained from an individual, such as, for example, a value obtained from a sample obtained from a test subject, but at an earlier point in time. The reference level may be based on a large number of samples, for example, from a population of subjects of similar chronological age, gender, disease state, or otherwise combined group, or based on a pool of samples with the inclusion or exclusion of the test sample. The reference level can also be determined using an exemplary number of cancer/tumor samples obtained from different individuals affected by cancer. The reference level can also be determined using biological samples obtained from individuals affected by cancer or a disease other than cancer. These biological samples obtained from individuals affected by cancer or a disease other than cancer may include, for example, tissue biopsies, blood, blood plasma, serum, stool, urine, cerebrospinal fluid, cervical smear, or seminal fluid. An exemplary sample may include measurements of at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more individuals or cancer/tumor biological samples obtained from individuals.

Percentage (%) of sequence identity relative to a polypeptide or antibody reference sequence is the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a polypeptide or antibody reference sequence after sequence alignment and introduction of gaps as necessary to achieve the maximum percentage of sequence identity and without regard to any conservative substitutions as part of sequence identity. Alignment to determine percent amino acid sequence identity can be achieved by various known methods, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR). Suitable parameters for sequence alignment can be determined, including the algorithms needed to achieve maximum alignment over the entire length of the sequences being compared. However, herein, % amino acid sequence identity values are obtained using the ALIGN-2 sequence comparison computer program. The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc., and the source code has been filed with user documentation with the United States Copyright Office, Washington, DC 20559, where it is registered under US Copyright Registration Number TXU510087. The ALIGN-2 program is open source and can be obtained from Genentech, Inc., South San Francisco, California, USA, or can be compiled from source code. The ALIGN-2 program must be compiled for use on the UNIX operating system, including the digital version of UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

Where ALIGN-2 is used to compare amino acid sequences, the % amino acid sequence identity attributable to a given amino acid sequence A to, with or in comparison to a given amino acid sequence B (which can alternatively be paraphrased as: a given amino acid sequence sequence A, which is characterized by or has a certain % amino acid sequence identity with respect to, with or in comparison with a given amino acid sequence B) is calculated as follows: the quotient of X/Y multiplied by 100, where X is the number of amino acid residues taken into account as identical matches by the ALIGN-2 sequence alignment program in alignment A and B of the program, and where Y represents the total number of amino acid residues in B. It should be noted that if the length of the amino acid sequence A is not equal to the length of the amino acid sequence B, then the % identity amino acid sequence identity of A with respect to B will not be equal to the % amino acid sequence identity of B with respect to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are derived as described in the previous paragraph. using the ALIGN-2 computer program.

The term epitope includes any determinant capable of binding to an antigen-binding protein, such as an antibody. An epitope is a region of an antigen that is bound by an antigen binding protein that targets that antigen, and when the antigen is a protein, includes specific amino acids that directly contact the antigen binding protein. Most often, epitopes are located on proteins, but in some cases they can be located on other types of molecules, such as saccharides or lipids. Epitope determinants may include reactive surface moieties of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structure characteristics and/or specific charge characteristics. Typically, antibodies specific for a particular target antigen will preferentially recognize an epitope on the target antigen among a complex array of proteins and/or macromolecules. In certain embodiments, the antibodies disclosed herein recognize a linear sequence of amino acids. In certain embodiments, the antibodies disclosed herein recognize conformational (non-linear) groups of amino acids.

Structural attributes of the antibodies described herein.

The complementarity determining region (CDR) is part of the variable region of an immunoglobulin (antibody) that is primarily responsible for the antigen binding specificity of the antibody. CDR regions vary significantly between antibodies, even if the antibodies specifically bind the same target or epitope. The heavy chain variable region contains three CDR regions, abbreviated as VH-CDR1, VH-CDR2 and VH-CDR3; and the light chain variable region contains three CDR regions, abbreviated as VL-CDR1, VL-CDR2 and VL-CDR3. These CDR regions are sequentially located in the variable region, with CDR1 located closest to the N terminus and CDR3 located closest to the C terminus. Between the CDRs are framework regions that contribute to structure and exhibit much less variability than the CDR regions. The heavy chain variable region contains four framework regions, abbreviated as VHFR1, VH-FR2, VH-FR3 and VH-FR4; and the light chain variable region contains four framework regions, abbreviated as VL-FR1, VL-FR2, VL-FR3 and VL-FR4. Complete full length bivalent antibodies containing two heavy and light chains will contain: 12 CDRs, with three unique heavy chain CDRs and three unique light chain CDRs; 16 FR regions, with four unique heavy chain FR regions and four unique light chain FR regions. In certain embodiments, the antibodies described herein contain at least three heavy chain CDRs. In certain embodiments, the antibodies described herein contain at least three light chain CDRs. In certain embodiments, the antibodies described herein contain at least three heavy chain CDRs and three light chain CDRs. The exact boundaries of the amino acid sequence of the specified CDR or FR can be easily determined using any of a number of well-known schemes, including the schemes described in Kabat et al. (1991), Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (numbering scheme after Kabat); Al-Lazikani et al., (1997) JMB 273,927-948 (Chothia numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), Antibody-antigen interactions: Contact analysis and binding site topography, (Contact numbering scheme); Lefranc MP et al., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains, Dev Comp Immunol, 2003 Jan;27(1):55-77 (IMGT numbering scheme); and Honegger A and Pluckthun A, Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool, J Mol Biol, 2001 Jun 8;309(3):657-70, (Aho numbering scheme). CDRs herein are identified among the variable sequences provided using various numbering systems, using the Kabat, IMGT, Chothia numbering system, or any combination thereof. The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, Kabat's schema is based on structural alignments, whereas Chothia's schema is based on structural information. The numbering for both the Kabat and Chothia schemes is based on the most common sequence lengths of the antibody region, with insertions designated by letters, such as 30a, and deletions occurring in some antibodies. The two schemes place certain insertions and deletions (insertions/deletions) in different positions, resulting in different numberings. The Contact scheme is based on the analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. In certain embodiments, CDRs defined from the variable regions disclosed herein include CDRs defined according to a Chothia, Kabat, IMGT, Contact or Aho scheme, or any combination thereof.

The term variable region or variable domain refers to the heavy or light chain domain of an antibody that is involved in the binding of the antibody to an antigen. The heavy chain and light chain variable domains (VH and VL, respectively) of a native antibody typically have similar structures, with each domain containing four conserved framework regions (FR) and three CDRs (see, for example, Kindt et al. Kuby Immunology , 6th ed, W.H. Freeman and Co., page 91(2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. In addition, antibodies that bind a particular antigen can be isolated using the VH or VL domain of an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively (see, for example, Portolano et al., J. Immunol. 150 :880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)). In certain embodiments, the antibodies described herein contain variable regions of rat origin. In certain embodiments, the antibodies described herein contain CDRs of rat origin. In certain embodiments, the antibodies described herein contain variable regions of murine origin. In certain embodiments, the antibodies described herein contain CDRs of murine origin.

Changes (eg, substitutions) may be made to the CDR, for example, to increase the affinity of the antibody. Such changes can be made to the codons encoding CDRs at a high rate of mutation during somatic maturation (see, for example, Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and the resulting variant can be tested for binding affinity . Affinity maturation (eg, using error polymerase PCR, strand shuffling, CDR randomization, or oligonucleotide-directed mutagenesis) can be used to improve antibody affinity (see, eg, Hoogenboom et al. Methods in Molecular Biology 178:1-37 (2001 )). CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling (see, for example, Cunningham and Wells Science, 244:1081-1085 (1989)). CDR-H3 and CDR-L3 in particular are often targeted. Alternatively or additionally, the crystal structure of the antigen-antibody complex is analyzed to determine the points of contact between the antibody and the antigen. Such contact residues and adjacent residues may be targeted or may be excluded as candidates for replacement. The options can be tested to determine if they have the required properties.

In certain embodiments, the antibodies described herein contain a constant region in addition to a variable region. The heavy chain constant region (CH) contains four domains, abbreviated as _CH1 , CH2, CH3 and CH4, located at the C-terminus of the full heavy chain polypeptide, on the C-terminal side of the variable region. The light chain constant region (CL) is significantly smaller than _CH and is located at the C-terminus of the full light chain polypeptide, on the side of the C-terminus of the variable region. The constant region is highly conserved and contains different isotypes that are associated with slightly different functions and properties. In certain embodiments, the constant region is not required for binding of the antibody to the target antigen. In certain embodiments, antibody constant regions of both the heavy and light chains are not required for antibody binding. In certain embodiments, the antibodies described herein lack one or more light chain constant regions, heavy chain constant regions, or both chains. Most monoclonal antibodies are of the IgG isotype; which is further divided into four subclasses: IgG1, IgG ₂ , IgG ₃ and IgG ₄ . In certain embodiments, the antibodies described herein include any subclass of IgG. In certain embodiments, the IgG subclass includes IgG1. In certain embodiments, the IgG subclass includes IgG ₂ . In certain embodiments, the IgG subclass includes IgG ₃ . In certain embodiments, the IgG subclass includes IgG4.

Antibodies contain a crystallizable fragment region (Fc region), which is responsible for binding to complement and Fc receptors. The Fc region contains the CH2, CH3 and CH4 regions of the antibody molecule. The Fc region of an antibody is responsible for complement activation and antibody-dependent cell-mediated cytotoxicity (ADCC). The Fc region also contributes to the serum half-life of the antibody. In certain embodiments, the Fc region of the therapeutic antibodies described herein contains one or more amino acid substitutions that promote complement-mediated cell lysis. In certain embodiments, the Fc region of the therapeutic antibodies described herein contains one or more amino acid substitutions that promote ADCC. In certain embodiments, the Fc region of the therapeutic antibodies described herein contains one or more amino acid substitutions that reduce the efficiency of complement-mediated cell lysis. In certain embodiments, the Fc region of therapeutic antibodies described herein contains one or more amino acid substitutions that increase the extent of binding of the antibody to the Fc receptor. In certain embodiments, the Fc receptor includes FcyRI (CD64), FcyRIIA (CD32), FcyRIIIA (CD16a), FcyRIIIB (CD16b), or any combination thereof. In certain embodiments, the Fc region of the therapeutic antibodies described herein contains one or more amino acid substitutions that increase the serum half-life of the antibody. In certain embodiments, one or more amino acid substitutions that increase

- 15 044914 half-life of a therapeutic antibody in blood serum, increase the affinity of the antibody for the neonatal Fc receptor (FcRn).

Antibodies useful in clinical settings are often humanized to reduce immunogenicity in human subjects. Humanized antibodies improve the safety and effectiveness of monoclonal antibody therapy. One common method of humanization is to obtain a monoclonal antibody from any suitable animal (eg mouse, rat, hamster) and replace the constant region with a human constant region; Antibodies constructed in this way are called chimeric. Another common method is CDR grafting, in which non-human V-FRs are replaced with human V-FRs. In the CDR grafting method, all residues except the CDR region are of human origin. In certain embodiments, the antibodies described herein are humanized. In certain embodiments, the antibodies described herein are chimeric. In certain embodiments, CDRs are grafted onto the antibodies described herein.

Methods for Determining Treatment with a Therapeutic Anti-LIF Antibody The methods described herein involve treating an individual with a therapeutic anti-LIF antibody if the level of a biomarker exceeds a reference level in a sample obtained from the individual. The sample may include a blood sample, a blood plasma sample, a blood serum sample, a urine sample, a stool sample, or a tissue sample, such as a tissue biopsy from a suspected or known tumor. The biomarker may include LIF, LIF receptor, macrophage type II (M2) cell marker, regulatory T cell marker, activated T cell, antigen trained T cell, cytotoxic T cell, immunosuppressive cytokine, or immunosuppressive chemokine, or phosphorylated STAT3, or any another immunomodulatory molecule. Biomarker levels can be determined using any of the commonly used molecular or cellular biology techniques, such as, but not limited to: quantification of mRIK using semi-quantitative PCR, digital PCR, real-time PCR or RNA sequencing; or protein quantification using Western blotting, flow cytometry, mass cytometry, ELISA, immunofluorescence assay, or homogeneous quantitative protein assays (eg, AlphaLISA®). In certain embodiments, the biomarker is determined through an immunohistochemical study using an antibody specific for the particular biomarker. Immunohistochemical testing can be performed on a biopsy or blood sample obtained from an individual. Using immunohistochemistry, the IHC score for a particular protein can be determined and compared to a reference level or control sample. In addition, protein, mRNA, or DNA levels of biomarker combinations can be determined to guide treatment decisions.

In certain embodiments, the biomarker is LIF. In certain embodiments, an individual treated with antibodies of the present invention has been selected for treatment as having a LIF-positive tumor/LIF-positive cancer. In certain embodiments, the tumor is LIF-positive or produces elevated levels of LIF. In certain embodiments, LIF positivity is determined by comparison with a reference value or established pathological criteria. In certain embodiments, a LIF-positive tumor expresses LIF in an amount that is greater than 2, 3, 5, 10, 100 times or more the amount of LIF that the non-transformed cell from which the tumor originates expresses. In certain embodiments, the tumor has acquired ectopic expression of LIF.

LIF protein levels can be determined quantitatively or semiquantitatively using immunohistochemistry. In certain embodiments, the IHC score for LIF can be calculated in a sample obtained from an individual and if the IHC score is or is greater than about 1, 5, 10, 25, 50, 75, 100, 110, 120, 130, 140, 150, 160 , 170, 180, 190, 200, 210, 220, 230, 240, or 250, then the individual is administered a therapeutic anti-LIF antibody. In certain embodiments, the therapeutic antibody is h5D8 or an antigen binding fragment thereof. In certain embodiments, the sample is a tissue sample or a tissue biopsy sample. In certain embodiments, the percentage of LIF-positive cells can be determined in a sample obtained from an individual, and if the percentage of LIF-positive cells is greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80%, then the individual is administered a therapeutic anti-LIF antibody. In certain embodiments, the therapeutic antibody is h5D8 or an antigen binding fragment thereof. In certain embodiments, the sample is a tissue sample or a tissue biopsy sample. The reference level of the IHC score for LIF is derived from the levels observed in a population of at least N samples. In certain embodiments, N is equal to or greater than 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, or greater. In certain embodiments, the sample includes cancers of a similar type (e.g., defining a reference level for a specific cancer) or all cancers (e.g., defining a reference level for all

- 16 044914 types of cancer). Different cancer types may have different reference levels indicating an increased chance of successful treatment with h5D8, thus the LIF IHC score may be cancer type specific. In certain embodiments, the IHC score for LIF is specific for any or more of non-small cell lung cancer, ovarian cancer, kidney cancer, bladder cancer, pancreatic cancer, prostate cancer, urogenital tract cancer, gynecological cancer, gastrointestinal cancer, cancer endocrine system, glioblastoma multiforme, breast cancer, melanoma, colorectal cancer, bile duct cancer, cervical cancer, endometrial cancer and squamous cell carcinoma of the head and neck.

LIF protein levels can be determined quantitatively or semi-quantitatively using an ELISA-based assay. In certain embodiments, the amount of LIF protein can be determined in a sample obtained from an individual, and if the amount of protein is greater than 1 picogram/milliliter (pg/ml), 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 , 30, 40, 50, 60, 70, 80, 90 pg/ml, then the individual is administered a therapeutic anti-LIF antibody. In certain embodiments, the therapeutic antibody is h5D8 or an antigen binding fragment thereof. In certain embodiments, the amount of LIF protein can be determined in a sample obtained from an individual, and if the amount of protein is greater than 100 pg/mL), 200, 300, 400, 500, 600, 700, 800, 900 pg/mL, 1 nanogram/milliliter (ng /ml), 2, 3, 4, 5, 6, 7, 8, 9, 10 ng/ml, then the individual is administered a therapeutic anti-LIF antibody. In certain embodiments, the therapeutic antibody is h5D8 or an antigen binding fragment thereof. In certain embodiments, the amount of LIF protein can be determined in a sample obtained from an individual, and if the amount of protein is greater than 100 picograms/milliliter (pg/ml), 200, 300, 400, 500, 600, 700, 800, 900 pg/ml, 1 nanogram/milliliter (ng/ml), 2, 3, 4, 5, 6, 7, 8, 9, 10 ng/ml, the individual is administered a therapeutic anti-LIF antibody. In certain embodiments, the therapeutic antibody is h5D8 or an antigen binding fragment thereof. In certain embodiments, the sample is a blood sample, a blood plasma sample, or a blood serum sample. The ELISA reference level is derived from the levels observed in a population of at least N samples. In certain embodiments, N is equal to or greater than 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, or greater. In certain embodiments, the sample includes cancers of a similar type (eg, defining a reference level for a specific cancer) or all cancers (eg, defining a reference level for all cancers). Different cancer types may have different reference levels, indicating an increased chance of successful treatment with h5D8, thus the LIF ELISA reference level may be cancer specific. In certain embodiments, the LIF ELISA reference level is specific for any or more of non-small cell lung cancer, ovarian cancer, kidney cancer, bladder cancer, pancreatic cancer, prostate cancer, urogenital cancer, gynecological cancer, gastrointestinal cancer tract, endocrine system cancer, glioblastoma multiforme, breast cancer, melanoma, colorectal cancer, bile duct cancer, cervical cancer, endometrial cancer and squamous cell carcinoma of the head and neck.

LIF mRNA levels can be determined quantitatively or semiquantitatively using real-time PCR or RNA sequencing. In certain embodiments, the LIF mRNA level can be determined, and if the LIF mRNA level is greater than a level corresponding to 25-, 30-, 35-, 40-, 45-, 50-, 55-, 60-, 65-, 70-, or 75 th percentile, then the individual is administered a therapeutic anti-LIF antibody. In certain embodiments, the therapeutic antibody is h5D8 or an antigen binding fragment thereof. The percentile reference level refers to the mRNA levels observed in a population of at least N samples. In certain embodiments, N is equal to or greater than 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, or greater. In certain embodiments, the sample includes cancers of a similar type (eg, defining a reference level for a specific cancer) or all cancers (eg, defining a reference level for all cancers). Different cancer types may have different reference levels, indicating an increased chance of successful treatment with h5D8, thus the LIF mRNA reference level may be cancer type specific. In certain embodiments, the LIF mRNA reference level is specific for any or more of non-small cell lung cancer, ovarian cancer, kidney cancer, bladder cancer, pancreatic cancer, prostate cancer, urogenital tract cancer, gynecological cancer, gastrointestinal cancer , endocrine cancer, glioblastoma multiforme, breast cancer, melanoma, colorectal cancer, bile duct cancer, cervical cancer, endometrial cancer and squamous cell carcinoma of the head and neck.

LIF signals through binding to the LIF receptor and gp130. In certain embodiments, the antibodies disclosed herein are useful for treating tumors or cancers that express the LIF receptor (CD 118) either directly on the cancer cell or on tumor-associated myeloid cells (eg, macrophages or tumor suppressor cells).

- 17 044914 cells of myeloid origin), stromal cells (cancer-associated fibroblasts) or endothelial cells. Tumor-associated macrophages may be specific immunosuppressive macrophages, such as type II (M2) macrophages.

In certain embodiments, the biomarker is a LIF receptor. In certain embodiments, an individual treated with antibodies of the present invention has been selected for treatment as having a LIF receptor-positive tumor/LIF receptor-positive cancer. In certain embodiments, an individual treated with antibodies of the present invention has been selected for treatment as having LIF receptor-positive infiltrates in tumor sites, as assessed, for example, by IHC, flow cytometry, or mRNA quantitation. Such infiltrates may include immunomodulatory cells such as tumor-associated macrophages, type II macrophages, myeloid-derived suppressor cells, tumor-derived polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC).

In certain embodiments, the antibodies disclosed herein are useful for treating tumors or cancers that express the LIF receptor. A LIF receptor-positive tumor can be determined by histopathological analysis or flow cytometry and in certain embodiments includes a cell that binds an anti-LIF receptor antibody with greater than 2x, 3x, 4x, 5x, 10x or greater potency compared to an isotype control In certain embodiments, the tumor has acquired ectopic expression of the LIF receptor. In a certain embodiment, the cancer cell is a cancer stem cell. In a certain embodiment, a LIF receptor-positive tumor or cancer can be identified by immunohistochemistry using an anti-LIF receptor antibody. In certain embodiments, the level of LIF receptor protein or mRNA is determined in association with one or more cell populations associated with an immunosuppressive response. In certain embodiments, the cell population is myeloid cells, macrophage cells, M2 cells, neutrophils, myeloid-derived suppressor cells, M-MDSC tumor cells, or PMN-MDSC tumor cells.

LIF receptor protein levels can be determined quantitatively or semiquantitatively using immunohistochemistry. The LIF receptor IHC assay may be based on LIF receptor expressed on all cells in the sample, all immune cells in the sample, all myeloid-derived cells in the sample, or all macrophages in the sample. In certain embodiments, the IHC score for the LIF receptor can be calculated in a sample obtained from an individual, and if the IHC score is or is greater than about 1, 5, 10, 25, 50, 75, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250, then the individual is administered a therapeutic anti-LIF antibody. In certain embodiments, the therapeutic antibody is h5D8 or an antigen binding fragment thereof. In certain embodiments, the sample is a tissue sample or a tissue biopsy sample. In certain embodiments, the percentage of LIF receptor positive cells can be determined in a sample obtained from an individual, and if the percentage of LIF receptor positive cells is greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80%, then the individual is administered a therapeutic anti-LIF antibody. In certain embodiments, the therapeutic antibody is h5D8 or an antigen binding fragment thereof. In certain embodiments, the sample is a tissue sample or a tissue biopsy sample. The reference level of the IHC score for the LIF receptor is derived from the levels observed in a population of at least N samples. In certain embodiments, N is equal to or greater than 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, or greater. In certain embodiments, the sample includes cancers of a similar type (eg, defining a reference level for a specific cancer) or all cancers (eg, defining a reference level for all cancers).

Different cancer types may have different reference levels indicating an increased chance of successful treatment with h5D8, thus the LIF receptor IHC score may be cancer specific. In certain embodiments, the LIF receptor IHC score is specific for any or more of non-small cell lung cancer, ovarian cancer, kidney cancer, bladder cancer, pancreatic cancer, prostate cancer, urogenital cancer, gynecological cancer, gastrointestinal cancer tract, endocrine system cancer, glioblastoma multiforme, breast cancer, melanoma, colorectal cancer, bile duct cancer, cervical cancer, endometrial cancer and squamous cell carcinoma of the head and neck.

LIF receptor protein levels can be determined quantitatively or semiquantitatively using a flow cytometry-based assay. In certain embodiments, the level of LIF receptor protein can be determined in a sample obtained from an individual and if the amount of protein is greater than 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x compared to a control antibody (e.g. , isotype control), then the individual is administered a therapeutic anti-LIF antibody. In certain embodiments, the therapeutic antibody is h5D8 or an antigen binding fragment thereof. In certain embodiments, the sample is a blood sample, a blood plasma sample, or a blood serum sample. The flow cytometry reference level is derived from the levels observed in a population of at least N samples. In certain embodiments, N is equal to or greater than 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, or greater. In certain embodiments, the sample includes cancers of a similar type (eg, defining a reference level for a specific cancer) or all cancers (eg, defining a reference level for all cancers). Different cancer types may have different reference levels indicating an increased chance of treatment success with h5D8, thus the flow cytometry LIF reference may be cancer specific. In certain embodiments, the flow cytometry LIF reference level is specific for any or more of non-small cell lung cancer, ovarian cancer, kidney cancer, bladder cancer, pancreatic cancer, prostate cancer, urogenital cancer, gynecological cancer, gastrointestinal cancer intestinal tract, endocrine system cancer, glioblastoma multiforme, breast cancer, melanoma, colorectal cancer, bile duct cancer, cervical cancer, endometrial cancer and squamous cell carcinoma of the head and neck.

LIF receptor mRNA levels can be determined quantitatively or semiquantitatively using real-time PCR or RNA sequencing. In certain embodiments, the LIF receptor mRNA level can be determined and if the LIF receptor mRNA level is greater than a level corresponding to 25-, 30-, 35-, 40-, 45-, 50-, 55-, 60-, 65-, 70-, or 75th percentile, then the individual is administered a therapeutic anti-LIF antibody. In certain embodiments, the therapeutic antibody is h5D8 or an antigen binding fragment thereof. The percentile reference level refers to the mRNA levels observed in a population of at least N samples. In certain embodiments, N is equal to or greater than 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, or greater. In certain embodiments, the sample includes cancers of a similar type (eg, defining a reference level for a specific cancer) or all cancers (eg, defining a reference level for all cancers). Different cancer types may have different reference levels, indicating an increased chance of successful treatment with h5D8, thus the LIF mRNA reference level may be cancer type specific. In certain embodiments, the LIF mRNA reference level is specific for any or more of non-small cell lung cancer, ovarian cancer, kidney cancer, bladder cancer, pancreatic cancer, prostate cancer, urogenital tract cancer, gynecological cancer, gastrointestinal cancer , endocrine cancer, glioblastoma multiforme, breast cancer, melanoma, colorectal cancer, bile duct cancer, cervical cancer, endometrial cancer and squamous cell carcinoma of the head and neck.

Additional biomarkers described herein and useful in methods of treating an individual with a therapeutic anti-LIF antibody include immunosuppressive biomarkers. This paper demonstrates that LIF and the LIF receptor are important for signal transduction in various types of immunomodulatory cells and thus immunosuppressive biomarkers can serve as indicators of the success of potential treatments. These biomarkers can be used alone or combined with determination of LIF and LIF receptor levels. In certain embodiments, if the protein, mRNA, or DNA level of an immunosuppressive biomarker exceeds a reference level, then the individual is administered a therapeutic anti-LIF antibody. In certain embodiments, the therapeutic antibody is h5D8 or an antigen binding fragment thereof. In certain embodiments, if the protein, mRNA, or DNA level of the immunosuppressive biomarker exceeds the reference level, and the LIF level exceeds the reference level, then the individual is administered a therapeutic anti-LIF antibody. In certain embodiments, the therapeutic antibody is h5D8 or an antigen binding fragment thereof. In certain embodiments, if the protein, mRNA, or DNA level of the immunosuppressive biomarker exceeds the reference level, and the LIF receptor level exceeds the reference level, then the individual is administered a therapeutic anti-LIF antibody. In certain embodiments, the therapeutic antibody is h5D8 or an antigen binding fragment thereof.

In certain embodiments, a combination of all three of LIF, a LIF receptor, and an immunosuppressive biomarker can be used to select an individual for treatment. Important immunomodulatory and immunosuppressive biomarkers of the present invention include biomarkers that are associated with regulatory T cells, activated T cells, antigen-trained T cells, cytotoxic T cells, and their corresponding functional activity products, including chemokines and cytokines released by macrophages, associated with a tumor or present in the tumor microenvironment; markers of myeloid suppressor cells

- 19 044914 origin or markers of macrophages, including M2 macrophages. In certain embodiments, the biomarker is an immunomodulatory molecule, such as a costimulatory molecule, antigen presenting molecule, cytokine, or chemokine, that acts on regulatory T cells. In certain embodiments, the costimulatory molecule, antigen presenting molecule, cytokine, or chemokine that acts on regulatory T cells is selected from the list consisting of MHCII, CXCL9, CXCL10, CXCR3, PD-L1, CCL7, CCL2, CCL3, and CCL22. In certain embodiments, the antigen presenting molecule that acts on regulatory T cells is MHCII. In certain embodiments, the cytokine or chemokine that acts on regulatory T cells is CXCL9. In certain embodiments, the cytokine or chemokine that acts on regulatory T cells is CXCL10. In certain embodiments, the cytokine or chemokine that acts on regulatory T cells is CXCR3. In certain embodiments, the co-stimulatory molecule that acts on regulatory T cells is PD-L1. In certain embodiments, the cytokine or chemokine that acts on regulatory T cells is CCL7. In certain embodiments, the cytokine or chemokine that acts on regulatory T cells is CCL2. In certain embodiments, the cytokine or chemokine that acts on regulatory T cells is CCL3. In certain embodiments, the cytokine or chemokine that acts on regulatory T cells is CCL22. In certain embodiments, a patient is selected for treatment if MHCII levels are below a reference level. In certain embodiments, if the MHCII level is below 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x compared to a control antibody (eg, an isotype control), then the individual is administered a therapeutic anti-LIF antibody. In certain embodiments, a patient is selected for treatment if CXCL9 levels are below a reference level. In certain embodiments, if the level of CXCL9 is below 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x compared to a control antibody (eg, an isotype control), then the individual is administered a therapeutic anti-LIF antibody. In certain embodiments, a patient is selected for treatment if CXCL10 concentration levels are below the reference level. In certain embodiments, if the level of CXCL10 is below 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x compared to a control antibody (eg, an isotype control), then the individual is administered a therapeutic anti-LIF antibody. In certain embodiments, a patient is selected for treatment if CXCR3 concentration levels are below a reference level. In certain embodiments, if the level of CXCR3 is below 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x compared to a control antibody (eg, an isotype control), then the individual is administered a therapeutic anti-LIF antibody. In certain embodiments, a patient is selected for treatment if PD-L1 concentration levels are below a reference level. In certain embodiments, if the PD-L1 level is below 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x compared to a control antibody (e.g., an isotype control), then the individual is administered a therapeutic antibody to LIF. In certain embodiments, a patient is selected for treatment if CCL7 levels exceed a reference level. In certain embodiments, if the level of CCL7 exceeds 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x compared to a control antibody (eg, an isotype control), then the individual is administered a therapeutic anti-LIF antibody. In certain embodiments, a patient is selected for treatment if CCL2 levels exceed a reference level. In certain embodiments, if the CCL2 level is greater than 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x compared to a control antibody (eg, an isotype control), then the individual is administered a therapeutic anti-LIF antibody. In certain embodiments, a patient is selected for treatment if CCL3 levels exceed a reference level. In certain embodiments, if the CCL3 level is greater than 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x compared to a control antibody (eg, an isotype control), then the individual is administered a therapeutic anti-LIF antibody. In certain embodiments, a patient is selected for treatment if CCL22 levels exceed a reference level. In certain embodiments, if the level of CCL22 is greater than 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x compared to a control antibody (eg, an isotype control), then the individual is administered a therapeutic anti-LIF antibody. In certain embodiments, the immunosuppressive biomarker is an M2 macrophage cell marker. In certain embodiments, the M2 macrophage cell marker is selected from the list consisting of CD206, CD163, PF4, CTSK, and ARG1. In certain embodiments, the M2 macrophage cell marker is CD206. In certain embodiments, the M2 macrophage cell marker is CD163. In certain embodiments, the M2 macrophage cell marker is PF4. In certain embodiments, the M2 macrophage cell marker is CTSK. In certain embodiments, the M2 macrophage cell marker is ARG1. In certain embodiments, a patient is selected for treatment if CD206 levels exceed a reference level. In certain embodiments, if the CD206 level is greater than 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x compared to a control antibody (e.g., an isotype control), then

- 20 044914 a therapeutic antibody to LIF is administered to an individual. In certain embodiments, a patient is selected for treatment if CD163 levels exceed a reference level. In certain embodiments, if the CD163 level is greater than 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x compared to a control antibody (eg, an isotype control), then the individual is administered a therapeutic anti-LIF antibody. In certain embodiments, a patient is selected for treatment if PF4 levels exceed a reference level. In certain embodiments, if the PF4 level is greater than 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x compared to a control antibody (eg, an isotype control), then the individual is administered a therapeutic anti-LIF antibody. In certain embodiments, a patient is selected for treatment if CTSK levels exceed a reference level. In certain embodiments, if the CTSK level is greater than 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x compared to a control antibody (eg, an isotype control), then the individual is administered a therapeutic anti-LIF antibody. In certain embodiments, a patient is selected for treatment if ARG1 levels exceed a reference level. In certain embodiments, if the level of ARG1 exceeds 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x compared to a control antibody (eg, an isotype control), then the individual is administered a therapeutic anti-LIF antibody. Protein levels of immunosuppressive biomarkers can be determined by Western blotting, ELISA, flow cytometry, or IHC; mRNA levels of immunosuppressive biomarkers can be determined using qPCR or RNA sequencing. In certain embodiments, the therapeutic antibody is h5D8 or an antigen binding fragment thereof. In certain embodiments, if the level of an immunosuppressive biomarker described herein exceeds a reference level, the patient is selected for treatment with a therapeutic anti-LIF antibody. In certain embodiments, if the level of the immunosuppressive biomarker exceeds the reference level, and the LIF level exceeds the reference level, then the individual is administered a therapeutic anti-LIF antibody. In certain embodiments, if the level of the immunosuppressive biomarker exceeds the reference level, and the level of the LIF receptor exceeds the reference level, then the individual is administered a therapeutic anti-LIF antibody. In certain embodiments, a combination of all three of LIF, a LIF receptor, and an immunosuppressive biomarker can be used to select an individual for treatment.

Additional biomarkers described herein and useful in methods for determining whether an individual has been treated with a therapeutic antibody to LIF include markers of LIF signaling. In certain embodiments, the LIF signaling marker is phosphorylated STAT3. In certain embodiments, a patient is selected for treatment if phosphorylated STAT3 levels exceed a reference level. In certain embodiments, if the level of pSTAT3 is greater than 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x compared to a control antibody (eg, an isotype control), then the individual is administered a therapeutic anti-LIF antibody. In certain embodiments, the therapeutic antibody is h5D8 or an antigen binding fragment thereof.

Therapeutic antibodies to LIF.

The 5D8 antibody described herein was obtained from rats immunized with DNA encoding human LIF. The original version of the rat antibody is designated r5D8, the humanized version is designated h5D8.

5D8.

The antibodies described herein were obtained from rats immunized with DNA encoding human LIF. One such antibody (5D8) has been cloned and sequenced and contains a CDR (using a combination of Kabat and LMGT CDR numbering methods) with the following amino acid sequences: VH-CDR1 corresponding to SEQ ID NO: 1 (GFTFSHAWMH), VHCDR2 corresponding to SEQ ID NO : 4(QLKAKSDDYATYYAESVKG), VH-CDR3 corresponding to SEQ ID NO: 6 (TCWEWDLDF), VL-CDR1 corresponding to SEQ ID NO: 9 (RSSQSLLDSDGHTYLN), VL-CDR2 corresponding to SEQ ID NO: 11 (SVSNLES), and VL -CDR3 corresponding to SEQ ID NO: 13 (MQATHAPPYT). This antibody was humanized by grafting with a CDR, and its humanized variant is designated h5D8. The V _H and V _L regions are provided under SEQ ID NOs: 15 and 19.

In certain embodiments, described herein is a therapeutic antibody that specifically binds LIF containing a VH-CDR1 that is at least 80% or 90% identical to the sequence set forth at SEQ ID NO: 1 (GFTFSHAWMH), VH-CDR2, which is at least 80%, 90% or 95% identical to the sequence presented under SEQ ID NO: 4 (QLKAKSDDYATYYAESVKG), and VH-CDR3 is at least 80% or 90% identical to the sequence presented under SEQ ID NO: 6 ( TCWEWDLDF). In certain embodiments, described herein is a therapeutic antibody that specifically binds LIF containing VL-CDR1 that is at least 80% or 90% identical to the sequence set forth at SEQ ID NO: 9 (RSSQSLLDSDGHTYLN), VL-CDR2, which is at least 80% identical to the sequence presented under SEQ ID NO: 11 (SVSNLES), and VL-CDR3 is at least 80 or 90% identical to the sequence presented under SEQ ID NO: 13

- 21 044914 (MQATHAPPYT). In certain embodiments, disclosed herein is a therapeutic antibody that specifically binds LIF comprising VH-CDR1 set forth under SEQ ID NO: 1 (GFTFSHAWMH), VH-CDR2 set forth under SEQ ID NO: 4 (QIKAKSDDYATYYAESVKG), VH-CDR3 , presented under SEQ ID NO: 6 (TCWEWDLDF), VL-CDR1, presented under SEQ ID NO: 9 (RSSQSLLDSDGHTYLN), VL-CDR2, presented under SEQ ID NO: 11 SVSNLES), and VL-CDR3, presented under SEQ ID NO: 13 (MQATHAPPYT). Certain conservative amino acid substitutions are provided in the CDR amino acid sequences of the present invention. In certain embodiments, the antibody contains CDRs that differ from the amino acid sequence represented by any of SEQ ID NOs: 1, 4, 6, 9, 11 and 13 by 1, 2, 3 or 4 amino acids. In certain embodiments, the antibody contains CDRs that differ from the amino acid sequence represented by any of SEQ ID NOs: 1, 4, 6, 9, 11, and 13 by 1, 2, 3, or 4 amino acids and do not affect binding affinity by more than than 10%, 20% or 30%. In certain embodiments, antibodies that specifically bind LIF contain one or more human heavy chain framework regions.

In certain embodiments, described herein is a therapeutic antibody that specifically binds LIF comprising a VH-CDR1 amino acid sequence that is at least 80% or 90% identical to the sequence set forth in SEQ ID NO: 1 (GFTFSHAWMH), the VH amino acid sequence -CDR2, which is at least 80, 90 or 95% identical to the sequence presented under SEQ ID NO: 4 (QIKAKSDDYATYYAESVKG), and the amino acid sequence of VH-CDR3, which is at least 80% or 90% identical to the sequence presented under SEQ ID NO: 8 (TSWEWDLDF). In certain embodiments, described herein is a therapeutic antibody that specifically binds LIF comprising a VL-CDR1 amino acid sequence that is at least 80% or 90% identical to the sequence set forth at SEQ ID NO: 9 (RSSQSLLDSDGHTYLN), the VL amino acid sequence -CDR2, which is at least 80% identical to the sequence presented under SEQ ID NO: 11 (SVSNLES), and the amino acid sequence of VLCDR3, which is at least 80% or 90% identical to the sequence presented under SEQ ID NO: 13 ( MQATHAPPYT). In certain embodiments, described herein is a therapeutic antibody that specifically binds LIF comprising the amino acid sequence VH-CDR1 set forth in SEQ ID NO: 1 (GFTFSHAWMH), the amino acid sequence VH-CDR2 set forth in SEQ ID NO: 4 (QIKAKSDDYATYYAESVKG) , amino acid sequence of VH-CDR3 presented under SEQ ID NO: 8 (TSWEWDLDF), amino acid sequence of VL-CDR1 presented under SEQ ID NO: 9 (RSSQSLLDSDGHTYLN), amino acid sequence of VL-CDR2 presented under SEQ ID NO: 11 (SVSNLES ), and the amino acid sequence of VL-CDR3 shown under SEQ ID NO: 13 (MQATHAPPYT). Certain conservative amino acid substitutions are provided in the CDR amino acid sequences of the present invention. In certain embodiments, the antibody contains CDRs that differ from the amino acid sequence represented by any of SEQ ID NOs: 1, 4, 8, 9, 11 and 13 by 1, 2, 3 or 4 amino acids. In certain embodiments, the antibody contains CDRs that differ from the amino acid sequence represented by any of SEQ ID NOs: 1.4, 8, 9, 11, and 13 by 1, 2, 3, or 4 amino acids and do not affect binding affinity by more than than 10, 20 or 30%.

In certain embodiments, disclosed herein is a therapeutic antibody that specifically binds LIF comprising a humanized heavy chain variable region comprising an amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98 % or approximately 99% identical to the amino acid sequence set forth in any of SEQ ID NO: 14, 15, or 17. In certain embodiments, described herein is a therapeutic antibody that specifically binds LIF containing a humanized heavy chain variable region comprising an amino acid sequence, presented under any of SEQ ID NOs: 14, 15 and 17. In certain embodiments, described herein is a therapeutic antibody that specifically binds LIF comprising a humanized light chain variable region comprising an amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence presented under any of SEQ ID NO: 18-21. In certain embodiments, described herein is a therapeutic antibody that specifically binds LIF comprising a humanized light chain variable region comprising the amino acid sequence set forth in any of SEQ ID NOs: 18-21. In certain embodiments, the antibody specifically binds human LIF.

In certain embodiments, disclosed herein is a therapeutic antibody that specifically binds LIF comprising a humanized severe

- 22 044914 chain containing an amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98% or about 99% identical to the amino acid sequence presented under SEQ ID NO:15; and a humanized light chain variable region comprising an amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 19 In certain embodiments, disclosed herein is a therapeutic antibody that specifically binds LIF comprising a humanized heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 15; and a humanized light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 19.

In certain embodiments, disclosed herein is a therapeutic antibody that specifically binds LIF comprising a humanized heavy chain variable region comprising an amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98 % or approximately 99% identical to the amino acid sequence presented under SEQ ID NO: 38; and a humanized light chain variable region comprising an amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 19 In certain embodiments, described herein is a therapeutic antibody that specifically binds LIF comprising a humanized heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 38; and a humanized light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 19.

In certain embodiments, disclosed herein is a therapeutic antibody that specifically binds LIF comprising a humanized heavy chain comprising an amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or approximately 99% identical to the amino acid sequence presented under any of SEQ ID NO: 30-33; and a humanized light chain comprising an amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in any of SEQ ID NO: 34 -37. In certain embodiments, described herein is a therapeutic antibody that specifically binds LIF comprising a humanized heavy chain comprising the amino acid sequence represented by any of SEQ ID NOs: 30-33; and a humanized light chain comprising the amino acid sequence represented by any one of SEQ ID NOs: 34-37.

In certain embodiments, disclosed herein is a therapeutic antibody that specifically binds LIF comprising a humanized heavy chain comprising an amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or approximately 99% identical to the amino acid sequence presented under SEQ ID NO: 31; and a humanized light chain comprising an amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 35. B In certain embodiments, a therapeutic antibody is disclosed herein that specifically binds LIF comprising a humanized heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 31; and a humanized heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 35. In certain embodiments, a therapeutic antibody that specifically binds LIF is described herein comprising a humanized heavy chain comprising an amino acid sequence that is at least about 80% , about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence presented under SEQ ID NO:39; and a humanized light chain comprising an amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 35. B In certain embodiments, a therapeutic antibody is disclosed herein that specifically binds LIF comprising a humanized heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 39; and a humanized light chain comprising the amino acid sequence set forth in SEQ ID NO: 35.

In certain embodiments, a recombinant antibody is described herein,

- 23 044914 which specifically binds leukemia inhibitory factor (LIF) containing heavy chain complementarity determining region 1 (VH-CDR1) containing the amino acid sequence represented by SEQ ID NO: 3; heavy chain complementarity determining region 2 (VHCDR2) containing the amino acid sequence shown under SEQ ID NO: 4; heavy chain complementarity determining region 3 (VH-CDR3) containing the amino acid sequence shown under SEQ ID NO: 7; light chain complementarity determining region 1 (VL-CDR1) containing the amino acid sequence shown under SEQ ID NO: 9; and light chain complementarity determining region 2 (VL-CDR2) containing the amino acid sequence shown under SEQ ID NO: 11; and light chain complementarity determining region 3 (VL-CDR3) containing the amino acid sequence set forth in SEQ ID NO: 13.

In certain embodiments, described herein is a recombinant antibody that specifically binds leukemia inhibitory factor (LIF) containing heavy chain complementarity determining region 1 (VH-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 2; heavy chain complementarity determining region 2 (VHCDR2) containing the amino acid sequence shown under SEQ ID NO: 5; heavy chain complementarity determining region 3 (VH-CDR3) containing the amino acid sequence shown under SEQ ID NO: 6; light chain complementarity determining region 1 (VL-CDR1) containing the amino acid sequence shown under SEQ ID NO: 10; and light chain complementarity determining region 2 (VL-CDR2) containing the amino acid sequence shown under SEQ ID NO: 12; and light chain complementarity determining region 3 (VL-CDR3) containing the amino acid sequence set forth in SEQ ID NO: 13. Certain conservative amino acid substitutions are provided in the CDR amino acid sequences of the present invention. In certain embodiments, the antibody contains CDRs that differ from the amino acid sequence represented by any of SEQ ID NOs: 2, 5, 6, 10, 12 and 13 by 1, 2, 3 or 4 amino acids. In certain embodiments, the antibody contains CDRs that differ from the amino acid sequence represented by any of SEQ ID NOs: 2, 5, 6, 10, 12, and 13 by 1, 2, 3, or 4 amino acids and do not affect binding affinity by more than than 10, 20 or 30%.

In certain embodiments, described herein is a recombinant antibody that specifically binds leukemia inhibitory factor (LIF) containing heavy chain complementarity determining region 1 (VH-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 3; heavy chain complementarity determining region 2 (VHCDR2) containing the amino acid sequence shown under SEQ ID NO: 4; heavy chain complementarity determining region 3 (VH-CDR3) containing the amino acid sequence shown under SEQ ID NO: 7; light chain complementarity determining region 1 (VL-CDR1) containing the amino acid sequence shown under SEQ ID NO: 9; and light chain complementarity determining region 2 (VL-CDR2) containing the amino acid sequence shown under SEQ ID NO: 11; and light chain complementarity determining region 3 (VL-CDR3) containing the amino acid sequence set forth in SEQ ID NO: 13. Certain conservative amino acid substitutions are provided in the CDR amino acid sequences of the present invention. In certain embodiments, the antibody contains CDRs that differ from the amino acid sequence represented by any of SEQ ID NOs: 3, 4, 7, 9, 11 and 13 by 1, 2, 3 or 4 amino acids. In certain embodiments, the antibody contains CDRs that differ from the amino acid sequence set forth in any of SEQ ID NOs: 3, 4, 7, 9, 11, and 13 by 1, 2, 3, or 4 amino acids and do not affect binding affinity by more than than 10, 20 or 30%.

In certain embodiments, disclosed herein is a therapeutic antibody that specifically binds LIF comprising a humanized heavy chain comprising an amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or approximately 99% identical to the amino acid sequence presented under any of SEQ ID NO: 22-25; and a humanized light chain comprising an amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in any of SEQ ID NO: 26 -29. In certain embodiments, described herein is a therapeutic antibody that specifically binds LIF comprising a humanized heavy chain comprising the amino acid sequence represented by any of SEQ ID NOs: 22-25; and a humanized light chain comprising the amino acid sequence represented by any one of SEQ ID NOs: 26-29.

In certain embodiments, described herein is a therapeutic antibody that specifically binds LIF containing a humanized heavy chain containing amino

- 24044914 an amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 23; and a humanized light chain comprising an amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 27. B In certain embodiments, a therapeutic antibody is disclosed herein that specifically binds LIF comprising a humanized heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 23; and a humanized light chain comprising the amino acid sequence set forth in any one of SEQ ID NO: 27.

In certain embodiments, disclosed herein is a therapeutic antibody that specifically binds LIF comprising a humanized heavy chain comprising an amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or approximately 99% identical to the amino acid sequence presented under SEQ ID NO: 39; and a humanized light chain comprising an amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 27. B In certain embodiments, a therapeutic antibody is disclosed herein that specifically binds LIF comprising a humanized heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 39; and a humanized light chain comprising the amino acid sequence set forth in any one of SEQ ID NO: 27.

Epitopes Bound by Therapeutically Useful Anti-LIF Antibodies Disclosed herein is a unique epitope of human LIF that, when bound, inhibits the biological activity of LIF (eg, STAT3 phosphorylation), and suppresses tumor growth in vivo and produces a therapeutic effect. The therapeutic antibody of the present invention may be a therapeutic antibody that does not contain a CDR from h5D8, but binds to the same or similar epitope (amino acid residues) as h5D8. A similar epitope is an epitope that binds within the binding region of the specified epitope. The epitope described herein consists of two discontinuous stretches of amino acids (residue 13 to residue 32 and residue 120 to residue 138 of human LIF) that are present in two different topological domains (alphahelices A and C) of the human LIF protein. This bonding is a combination of weak (van der Waals forces), moderate (hydrogen bonds) and strong (salt bridge) interactions. In certain embodiments, the contact residue is a residue on LIF that forms a hydrogen bond with a residue on the anti-LIF antibody. In certain embodiments, the contact residue is a residue on LIF that forms a salt bridge with a residue on an anti-LIF antibody. In certain embodiments, the contact residue is a residue on LIF that interacts via van der Waals forces with a residue on the anti-LIF antibody and is within at least 5, 4, or 3 angstroms of distance from it. The therapeutic antibody may bind this epitope, bind a lesser portion of this epitope, or overlap with this epitope and can be used for the assay described herein.

In certain embodiments, a therapeutic antibody described herein is an isolated antibody that binds any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen or twenty of the following residues: A13, 114, R15, H16, P17, C18, H19, N20, Q25, Q29, Q32, D120, R123, S127, N128, L130, C131, C134, S135 or H138 from SEQ ID NO: 40. In certain embodiments, an isolated antibody is described herein that binds all of the following residues: A13, I14, R15, H16, P17, C18, H19, N20, Q25, Q29, Q32, D120, R123, S127, N128, L130, C131, C134, S135, or H138 from SEQ ID NO: 40. In certain embodiments, an isolated antibody is described herein that binds all of the following residues: A13, 114, R15, H16, P17, C18 , H19, N20, Q25, Q29, Q32, D120, R123, S127, N128, L130, C131, C134, S135, or H138 of SEQ ID NO: 40. In certain embodiments, the antibody binds only those residues that are involved in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only those residues that are involved in strong interactions with the antibody. In a certain embodiment, the antibody interacts with helices A and C in LIF. In a certain embodiment, the antibody blocks the interaction of LIF with gp130.

Indications for use.

In certain embodiments, therapeutic antibodies disclosed herein inhibit LIF signaling in cells. In certain embodiments, the IC ₅₀ value for biological inhibition by the antibody under serum starvation conditions in U-251 cells is less than or equal to about 100, 75, 50, 40, 30, 20, 10, 5, or 1 nmol/L. In certain variants

- 25 044914 implementation of the IC ₅₀ value for biological inhibition by antibody under serum starvation conditions in U-251 cells is less than or equal to approximately 900, 800, 700, 600, 500, 400, 300, 200 or

100 nmol/l.

In certain embodiments, antibodies useful for treating cancer or tumor are disclosed herein. In certain embodiments, cancer includes tumors of the breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head, neck, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testicle and liver. In certain embodiments, tumors that can be treated with antibodies of the present invention include adenoma, adenocarcinoma, angiosarcoma, astrocytoma, epithelial carcinoma, germinoma, glioblastoma, glioma, hemangioendothelioma, hemangiosarcoma, hematoma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma and/or teratoma. In certain embodiments, the tumor/cancer is selected from the group consisting of acral lentiginous melanoma, actinic keratosis, adenocarcinoma, adenoid cystic carcinoma, adenomas, adenosarcoma, adenosquamous carcinoma, astrocytic tumors, Bartholin's gland carcinoma, basal cell carcinoma, bronchial gland carcinoma, capillary carcinoma cynoid , carcinomas, carcinosarcoma, cholangiocarcinoma, chondrosarcoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymal sarcoma, Ewing sarcoma, focal nodular hyperplasia, gastrinoma, germline cell tumors, glioblastoma, glu kagonomas, hemangioblastomas, hemangioendotheliomas , hemangiomas, hepatic adenomas, hepatic adenomatosis, hepatocellular carcinoma, insulinitis, intraepithelial neoplasm, intraepithelial squamous cell neoplasm, invasive squamous cell carcinoma, large cell carcinoma, liposarcoma, lung carcinoma, lymphoblastic leukemia, lymphocytic leukemia, leiomyosarcoma, melanoma, malignant melanoma, malignant mesothelial tumor, schwannoma, medulloblastoma, medulloepithelioma, mesothelioma, mucoepidermoid carcinoma, myeloid leukemia, neuroblastoma, neuroepithelial adenocarcinoma, nodular melanoma, osteosarcoma, ovarian carcinoma, papillary serous adenocarcinoma, pituitary tumors, plasmacytoma, pseudosarcoma, predilection carcinoma static gland, pulmonary blastoma, renal cell carcinoma, retinoblastoma , rhabdomyosarcoma, sarcoma, serous carcinoma, squamous cell carcinoma, small cell carcinoma, soft tissue carcinoma, somatostatin-secreting tumor, squamous cell carcinoma, undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, vaginal/vulvar carcinoma, tumor secreting vasoactive intestinal poly peptide (VIPpomes), and Wilms tumors. In certain embodiments, the tumor/cancer to be treated with one or more antibodies of the present invention includes brain cancer, head and neck cancer, colorectal carcinoma, acute myeloid leukemia, pre-B cell acute lymphoblastic leukemia, bladder cancer, astrocytoma, preferably grade II, III or IV astrocytoma, glioblastoma, glioblastoma multiforme, small cell cancer and non-small cell cancer, preferably non-small cell lung cancer, lung adenocarcinoma, metastatic melanoma, androgen-independent metastatic prostate cancer, androgen-dependent metastatic prostate cancer, adenocarcinoma prostate and breast cancer, preferably ductal breast cancer and/or breast carcinoma. In certain embodiments, the cancer to be treated with the antibodies of the present invention includes glioblastoma. In certain embodiments, the cancer to be treated with one or more antibodies of the present invention includes pancreatic cancer. In certain embodiments, the cancer to be treated with one or more antibodies of the present invention includes ovarian cancer. In certain embodiments, the cancer to be treated with one or more antibodies of the present invention includes lung cancer. In certain embodiments, the cancer to be treated with one or more antibodies of the present invention includes prostate cancer. In certain embodiments, the cancer to be treated with one or more antibodies of the present invention includes colon cancer. In certain embodiments, the cancer being treated includes glioblastoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, or lung cancer. In a certain embodiment, the cancer is refractory to other treatment. In a certain embodiment, the cancer being treated is recurrent. In a certain embodiment, the cancer is recurrent/refractory glioblastoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, urogenital cancer, gynecological cancer, gastrointestinal cancer, endocrine cancer, or lung cancer. In certain embodiments, the cancer includes advanced solid tumor, glioblastoma, stomach cancer, skin cancer, prostate cancer, pancreatic cancer, breast cancer, testicular cancer, thyroid cancer, colorectal cancer, bile duct cancer, cervical cancer, endometrial cancer , head and neck cancer, liver cancer, kidney cancer, esophageal cancer, ovarian cancer, cancer

- 26 044914 colon, lung cancer, lymphoma or soft tissue cancer. In certain embodiments, the cancer includes non-small cell lung cancer, epithelial ovarian carcinoma, or pancreatic adenocarcinoma. In certain embodiments, the cancer includes an advanced solid tumor.

Therapeutic methods.

In certain embodiments, therapeutic antibodies can be administered by any route suitable for administering pharmaceutical compositions containing antibodies, such as, for example, subcutaneous, intraperitoneal, intravenous, intramuscular, intratumoral, or intracerebral, etc. In certain embodiments, the antibodies are administered intravenously. In certain embodiments, the antibodies are administered according to a suitable dosing schedule, for example, once a week, twice a week, once a month, twice a month, etc. In certain embodiments, the antibodies are administered once every three weeks. Antibodies can be administered in any therapeutically effective amount. In certain embodiments, a therapeutically acceptable amount is from about 0.1 mg/kg to about 50 mg/kg. In certain embodiments, a therapeutically acceptable amount is from about 1 mg/kg to about 40 mg/kg. In certain embodiments, a therapeutically acceptable amount is from about 5 mg/kg to about 30 mg/kg. The therapeutic antibody can be administered at a fixed dose regardless of the weight or weight of the individual to whom the h5D8 antibody is administered. The h5D8 antibody may be administered at a fixed dose regardless of the weight or weight of the individual to whom the therapeutic antibody is administered, as long as the individual's weight is at least about 37.5 kg. The fixed dose of therapeutic antibody to be administered may range from about 75 mg to about 2000 mg. The fixed dose of therapeutic antibody to be administered may be from about 225 mg to about 2000 mg, from about 750 mg to about 2000 mg, from about 1125 mg to about 2000 mg, or from about 1500 mg to about 2000 mg. The fixed dose of therapeutic antibody to be administered may be approximately 75 mg. The fixed dose of therapeutic antibody to be administered may be approximately 225 mg. The fixed dose of therapeutic antibody to be administered may be approximately 750 mg. The fixed dose of therapeutic antibody to be administered may be approximately 1125 mg. The fixed dose of therapeutic antibody to be administered may be approximately 1500 mg. The fixed dose of therapeutic antibody to be administered may be approximately 2000 mg.

Other dosages of the therapeutic antibody are contemplated. The fixed dose of therapeutic antibody to be administered may be approximately 50, 100, 150, 175, 200, 250, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650 , 675, 700, 725, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 1100, 1150, 1175, 1200, 1225, 1250, 1275, 1300, 1325 , 1350, 1375, 1400, 1425, 1450, 1475, 1525, 1550, 1575, 1600, 1625, 1650, 1675, 1700, 1725, 1750, 1775, 1800, 1825, 1850, 1875, 1900, 1925, 1950, 1975 , 2025, 2050, 2075 or 2100 mg. Any of these doses can be administered once a week, once every two weeks, once every three weeks, or once every four weeks.

The therapeutic antibody can be administered in a dose based on the weight or body weight of the individual to whom the therapeutic antibody is administered. The body weight-adjusted dose of therapeutic antibody to be administered may range from about 1 mg/kg to about 25 mg/kg. The body weight-adjusted dose of therapeutic antibody to be administered may be from about 3 mg/kg to about 25 mg/kg, from about 10 mg/kg to about 25 mg/kg, from about 15 mg/kg to about 25 mg/kg, or from about 20 mg/kg to about 25 mg/kg. The body weight-adjusted dose of h5D8 to be administered may be approximately 1 mg/kg. The body weight-adjusted dose of therapeutic antibody to be administered may be approximately 3 mg/kg. The body weight-adjusted dose of therapeutic antibody to be administered may be approximately 10 mg/kg. The body weight-adjusted dose of therapeutic antibody to be administered may be approximately 15 mg/kg. The body weight-adjusted dose of therapeutic antibody to be administered may be approximately 20 mg/kg. The body weight-adjusted dose of therapeutic antibody to be administered may be approximately 25 mg/kg.

Any of the doses detailed herein can be administered intravenously over a period of at least about 60 minutes; however, this period may vary somewhat depending on the conditions relating to each individual administration.

Pharmaceutically acceptable excipients, carriers and diluents.

In certain embodiments, the antibodies of the present invention are administered as a suspension in a sterile solution. In certain embodiments, the solution contains a physiologically acceptable concentration of salt (eg, NaCl). In certain embodiments, the solution contains from about 0.6 to 1.2% NaCl. In certain embodiments, the solution contains from about 0.7 to 1.1% NaCl. In certain embodiments, the solution contains from about 0.8 to 1.0% NaCl. In certain embodiments, the highly concentrated antibody stock solution can be diluted in approximately 0.9% NaCl. In certain embodiments, the solution contains approximately 0.9% NaCl. In certain embodiments, the solution further contains one or more of the following components: buffers, for example, acetate, citrate, histidine, succinate, phosphate, bicarbonate and hydroxymethylaminomethane (Tris); surfactants such as polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), polysorbate and poloxamer 188; polyol/disaccharide/polysaccharides, for example glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose and dextran 40; amino acids such as histidine, glycine or arginine; antioxidants, for example ascorbic acid, methionine; and chelating agents such as EGTA or EGTA. In certain embodiments, the antibodies of the present invention are transported/stored in lyophilized form and reconstituted prior to administration. In certain embodiments, the lyophilized antibody formulations contain a bulking agent such as mannitol, sorbitol, sucrose, trehalose, and dextran 40. In a certain embodiment, the anti-LIF antibodies of the present invention can be transported and stored as a concentrated stock solution to be diluted at the site use for treatment. In certain embodiments, the stock solution contains about 25 mM histidine, about 6% sucrose, about 0.01% polysorbate, and about 20 mg/ml anti-LIF antibody. In certain embodiments, the pH of the solution is approximately 6.0. In certain embodiments, the formulation administered to the individual is an aqueous solution containing about 25 mM histidine, about 6% sucrose, about 0.01% polysorbate 80, and about 20 mg/ml h5D8 antibody. In certain embodiments, the pH of the solution is approximately 6.0.

Examples

The following illustrative examples represent embodiments of the compositions and methods described herein and are not intended to be limiting in any way.

Example 1. Preparation of rat antibodies specific to LIF.

The cDNA encoding amino acids 23–202 of human LIF was cloned into expression plasmids (Aldevron GmbH, Freiburg, Germany). Groups of laboratory rats (Wistar) were immunized by intradermal injection of DNA-coated gold particles using a hand-held particle bombardment device (gene gun). Cell surface expression of transiently transfected HEK cells was confirmed using tag antibodies that recognize a tag attached to the N terminus of the LIF protein. Serum samples were collected after serial immunizations and tested by flow cytometry on HEK cells transiently transfected with the above-mentioned expression plasmids. Antibody-producing cells were isolated and fused with mouse myeloma cells (Ag8) according to standard procedures. Hybridomas producing LIF-specific antibodies were identified by flow cytometry screening as described above. Hybridoma positive cell pellets were prepared using Cellular RNA Stabilizer and Protectant (RNAlater, Cat# AM7020, ThermoFisher Scientific) and further processed for antibody variable domain sequencing.

Example 2. Preparation of mouse antibodies specific to LIF.

The cDNA encoding amino acids 23–202 of human LIF was cloned into expression plasmids (Aldevron GmbH, Freiburg, Germany). Groups of laboratory mice (NMRI) were immunized by intradermal injection of DNA-coated gold particles using a hand-held particle bombardment device (gene gun). Cell surface expression of transiently transfected HEK cells was confirmed using tag antibodies that recognize a tag attached to the N terminus of the LIF protein. Serum samples were collected after serial immunizations and tested by flow cytometry on HEK cells transiently transfected with the above-mentioned expression plasmids. Antibody-producing cells were isolated and fused with mouse myeloma cells (Ag8) according to standard procedures. Hybridomas producing LIF-specific antibodies were identified by flow cytometry screening as described above. Hybridoma positive cell pellets were prepared using Cellular RNA Stabilizer and Protectant (RNAlater, Cat# AM7020, ThermoFisher Scientific) and further processed for antibody variable domain sequencing.

Example 3: Humanization of rat LIF-specific antibodies.

One clone (5D8), obtained from rat immunization, was selected for subsequent humanization. Humanization was performed using standard CDR grafting methods. The heavy chain and light chain regions were cloned from the 5D8 hybridoma using standard molecular cloning techniques and sequenced by the Sanger method. A BLAST search of the human heavy chain and light chain variable sequences was then performed, and 4 sequences from each were selected as acceptor scaffolds for humanization. These acceptor scaffolds were deimmunized to remove T cell response epitopes. CDR1, CDR2 and CDR3 of the heavy chain and light chain of 5D8 were cloned into 4 different heavy chain acceptor scaffolds (H1-H4) and 4 different light chain acceptor scaffolds (L1-L4). All 16 different antibodies were then tested for: expression in CHO-S cells (Selexis); inhibition of LIF-induced STAT3 phosphorylation; and binding affinity by surface plasmon resonance (SPR). The experimental data are summarized in table. 1.

________Table 1. Summary of humanization data 5D8_________

Combination of heavy chain and light chain Inhibition of LIF induced by pSTAT3 according to fig. 1 Affinity by SPR, K _D1 (pm) Expression (µg/ml) H0L0 +++ 133±46 393 H1L1 - n. P. 627 H1L2 +++ 55±23 260 H1L3 +++ 54±31 70 H1L4 - n. P. 560 H2L1 - n. P. 369 H2L2 +++ 52±22 392 H2L3 ++ 136±19 185 H2L4 - n. P. 78 H3L1 n. P. n. P. No expression H3L2 n. P. n. P. No expression H3L3 n. P. n. P. No expression H3L4 n. P. n. P. No expression H4L1 - n. P. 259 H4L2 ++ 913±308 308 H4L3 + 252 H4L4 - n. P. 186

n. n. - did not check; H0L0 is a chimeric antibody with the complete rat heavy and light chain variable regions.

Expression characteristics of transfected cells were compared in Erlenmeyer flasks (seeding 3x10 ⁵ cells/ml, culture volume 200 ml) during fed-batch culture after 10 days of cell culture. At this point, the cells were collected and the secreted antibody was purified using a protein A column and then quantified. Expression of all humanized antibodies was observed except those containing the H3 heavy chain. The H2 and L2 variable regions performed well compared to other variable regions (SEQ ID NO: 15 and SEQ ID NO: 19).

Inhibition of LIF-induced phosphorylation of STAT3 at tyrosine 705 was determined by Western blotting. U251 glioma cells were seeded in 6-well plates at a density of 100,000 cells/well. Cells were cultured in complete medium for 24 hours before any treatment, and then cells were serum starved for 8 hours. Cells were then incubated overnight with the indicated antibodies at a concentration of 10 μg/ml. After treatment, proteins were isolated using radioimmunoprecipitation assay (RIPA) lysis buffer containing phosphatase and protease inhibitors, quantified (BCA protein assay, Thermo Fisher Scientific), and used in Western blotting. For Western blotting, membranes were blocked for 1 h in 5% nonfat dry milk -TBST and incubated with primary antibody overnight (p-STAT3, cat. no. 9145, Cell Signaling or STAT3, cat. no. 9132, Cell Signaling) or 30 min (β-actin peroxidase, catalog no. A3854, Sigma-Aldrich). The membranes were then washed in TBST, incubated with secondary antibody, and washed again. Proteins were detected by chemiluminescence (SuperSignal Substrate, catalog no. 34076, Thermo Fisher Scientific). These results are shown in Fig. 1. The darker the pSTAT3 band, the less inhibition. Inhibition was high in lanes designated 5D8 (non-humanized rat antibody), A (H0L0), C (H1L2), D (H1L3), and G (H2L2); inhibition was moderate in H (H2L3), O (H4L2) and P (H4L3); inhibition was absent in B (H1L1), E (H1L4), F (H2L1), I (H2L4), N (H4L1) and Q (H4L4).

Next, antibodies that showed inhibition of LIF-induced STAT3 phosphorylation.

- 29 044914 was analyzed by SPR to determine binding affinity. Briefly, the binding of humanized antibodies A (H0L0), C (H1L2), D (H1L3) and G (H2L2), H (H2L3) and O (H4L2) to amino-linked hLIF was detected in an assay using the Biacore™ 2002 instrument. Kinetic constants and affinity indicators were determined by mathematical approximation of sensorgrams (Langmuir interaction model [A+B=AB]) among all sensorgrams obtained on all surfaces of the sensor chip at six ligand concentrations. The best fit curves (minimum Chi2 value) for each concentration were used to calculate kinetic constants and affinity indices. See table. 1.

Since divalent antibodies were used as analytes in the experimental model, the sensograms with the best approximation were also analyzed based on the divalent analyte fitting model [A+B=AB; AB+B=AB2] to gain a more detailed understanding of the mechanism of target binding by humanized antibodies. Analysis of kinetic sensorgrams using the divalent analyte approximation model [A+B=AB; AB+B=AB2] confirmed the ranking of mAb samples by relative affinity.

Humanized 5D8 containing H2 and L2 was selected for more detailed analysis due to its high binding affinity and high yield from batch culture.

Example 4: Humanization of clone 5D8 improves binding to LIF.

Clone H2L2 (h5D8) was selected for further analysis and binding to the parent rat 5D8 (r5D8) and mouse clone 1B2 was compared by SPR. Antibody 1B2 is a previously described murine anti-LIF antibody deposited with Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM ACC3054) and included for comparison purposes. Recombinant human LI isolated from E. coli and HEK-293 cells, respectively, was used as ligands. LIF from human or E. coli was covalently bound to the surface of Biacore optical sensor chips using amine immobilization chemistry, and binding affinities were calculated based on kinetic constants.

Materials and methods.

Human LIF from E. coli was obtained from Millipore, catalog reference LIF 1010; human LIF from HEK-293 cells was obtained from ACRO Biosystems, catalog reference LIF-H521b. LIF was immobilized on sensor chips using the Biacore Amine Coupling Kit (BR-1000-50; GE-Healthcare, Uppsala, Sweden). Samples were analyzed on a Biacore™ 2002 instrument using CM5 optical sensor chips (BR-1000-12; GE-Healthcare, Uppsala, Sweden). Biacore HBS-EP buffer was used during the runs (BR-1001-88; GE-Healthcare, Uppsala, Sweden). Kinetic analysis of binding sensorgrams was performed using the BIAevaluation 4.1 program. Kinetic constants and affinity indicators were determined by mathematical approximation of sensorgrams (Langmuir interaction model [A+B=AB]) among all sensorgrams obtained on all surfaces of the sensor chip at increasing analyte concentrations. Sensograms were also analyzed based on the approximation model for the sensogram of a divalent analyte [A+B=AB; AB+B=AB ₂ ], including component analysis to assess the contribution of the divalent analyte to the specific Langmuir affinities of the antibody for the target (eg, contribution to avidity). To calculate kinetic constants and affinity indices, best fit curves (minimum Chi ² value) for each concentration were used. A summary of the affinity experiment data is shown in Table. 2 (human LIF obtained in E. coli) and table. 3 (human LIF produced in HEK 293 cells).

table 2

Improved binding 5D8 after humanization K _D [pm] hLIF (E. coli) Langmuir sensogram approximation 1:1 Divalent analyte approximation Mouse antibody 1B2 400±210 1500±200 r5D8 (rat) 130±30 780±130 h5D8 (humanized) 26±14 82±25

- 30 044914

Table 3

Improved 5D8 binding after humanization K _D [pm] hLIF (HEK 293) Langmuir sensogram approximation 1:1 Divalent analyte approximation Mouse antibody 1B2 320±150 3900±900 r5D8 (rat) 135±100 410±360 h5D8 (humanized) 13±6 63±30

A 1:1 Langmuir sensogram fit model from this series of experiments shows that the humanized antibody 5D8 (h5D8) bound to human LIF with an affinity that was ∼10-25-fold greater than that of murine 1B2 and r5D8.

The h5D8 antibody was then tested against several LIF species using SPR. SPR binding kinetics of h5D8 was tested for recombinant LIF analytes obtained from different species and expression systems: human LIF (E. coli, HEK293 cells); mouse LIF (B. coli cells, CHO); rat LIF (E. coli); Cynomolgus macaque LIF (yeast, HEK293 cells).

Materials and methods.

The h5D8 antibody was immobilized on the surface of the sensor chip by non-covalent Fc-specific capture. Recombinant Ig(Fc)-specific S. aureus A/G protein was used as a capture agent to provide sterically uniform and flexible antibody presentation to LIF analytes. Sources of LIF analytes are as follows: human human LIF (from E. coP; reference in Millipore LIF catalog 1050); human LIF (from HEK cells, ACRO Biosystems LIF-H521); mouse LIF (E.coP; Millipore cat. no. NF-LIF2010); mouse LIF (from CHO cells; Reprokine catalog no. RCP09056); monkey LIF (Kingfisher Biotech yeast cat# RP1074Y); Monkey LIF produced in HEK-293 cells. Overall, h5D8 exhibited binding to multiple species of LIF. A summary of the data from the affinity experiment is shown in Table. 4.

Table 4

Broad species reactivity of humanized 5D8 Langmuir sensogram approximation 1:1 Analyst Average value of K _a (1/M s) [10 ⁵ ] Average value K _d (1/s) [ΙΟ' ⁵ ] Average value K _D [pm] Human LIF (E. coll) 8.5 ±0.7 7.2 ± 0.7 86 ±9 Human LIF (HEK-293) 5.5 ± 0.02 3.1 ±0.7 56 ± 13 Mouse LIF (E. coli) 21.4 ±3.7 5.7 ± 1.0 27 ±6 Mouse LIF (CHO cells) 6.5 ± 0.7 1.1 ±0.3 17 ± 4 LIF cynomolgus monkey (yeast) 6.3 ± 0.8 5.4±0.7 89 ± 10 LIF cynomolgus macaque (NEK-293) 2.4 ± 0.2 3.3± 0.3 134 ±6

Example 5 Humanized clone 5D8 inhibits LIF-induced STATS phosphorylation in vitro.

To determine the biological activity of h5D8, humanized and parental versions were tested in a cell culture model of LIF activation. In fig. Figure 2A shows that the humanized clone exhibited increased inhibition of STAT3 (Tyr 705) phosphorylation when a glioma cell line was incubated with human LIF. In fig. 2B shows an experiment with the same model as in FIG. 2A, repeated with different dilutions of h5D8 antibody.

Ways.

U251 glioma cells were seeded in 6-well plates at a density of 150,000 cells/well. Cells were cultured in complete medium for 24 h before any treatment. Cells were then treated overnight by incubation with r5D8 anti-LIF antibody or h5D8 anti-LIF antibody at a concentration of 10 μg/ml or left untreated (control cells).

- 31 044914

After treatment, proteins were isolated using radioimmunoprecipitation assay (RIPA) lysis buffer containing phosphatase and protease inhibitors, quantified (BCAprotein assay, Thermo Fisher Scientific), and used in Western blotting. For Western blotting, membranes were blocked for 1 h in 5% skim milk - TB ST and incubated with primary antibody overnight (p-STAT3, cat. no. 9145, Cell Signaling or STAT3, cat. no. 9132, Cell Signaling) or 30 minutes (β-actin peroxidase, catalog no. A3854, Sigma-Aldrich). Membranes were then washed in TBST, incubated with secondary antibody if necessary, and washed again. Proteins were detected by chemiluminescence (SuperSignal Substrate, catalog no. 34076, Thermo Fisher Scientific).

Example 6: 1C ₅₀ value when treated with h5D8 antibody on endogenous LIF levels in U-251 cells.

It was also determined that for biological inhibition of h5D8 under conditions of serum starvation in U-251 cells, 1C ₅₀ is only 490 picomol/l (Fig. 3A). See representative results in FIG. FOR and ZV and in table. 5.

Table 5

Origin of the cell line Cell line name Treatment 1C ₅₀ (nM) IC ₉ o (nM) Inhibition JAK (%) Endogenous LIF condition n=l n=2 Average value SD Average value Average meaning G.B.M. U251 h5D8 0.78 0.54 0.66 0.12 4.1 84% r5D8 1.6 1.5 1.4 0.15 8.5 86% 1.2 1.4

Ways.

U-251 cells were seeded at a rate of 600,000 cells per 6-cm dish (per condition). Cells were treated with h5D8 at the appropriate concentration (titer) overnight at 37°C under serum starvation conditions (0.1% FBS). As a positive control for pSTAT3, recombinant LIF (R&D no. 7734-LF/CF) was used to stimulate cells at 1.79 nM for 10 min at 37°C. JAK inhibitor I (Calbiochem #420099) was used as a negative control for pSTAT3 at 1 μM for 30 min at 37°C. Cells were then collected on ice to obtain lysates according to the protocols of the Meso Scale Discovery Multi-Spot Assay System Total STAT3 (p/n K150SND-2) and Phospho-STAT3 (Tyr705) (p/n K150SVD-2) kits for measurement protein levels determined using MSD Meso Sector S600.

Example 7 Additional antibodies that specifically bind to human human LIF.

Other rat antibody clones (10G7 and 6B5) have been identified that specifically bind human LIF, and a summary of their binding characteristics is given in Table 1 below. 6, clone 1B2 serves for comparison.

Ways.

Real-time binding kinetic analysis was performed for anti-LIF mAbs 1B2, 10G7, and 6B5 immobilized on the surface of optical sensor chips.

CM5, using recombinant LIF target proteins [human LIF (E. coli); Millipore Cat. No. LIF 1010 and human LIF (HEK293 cells); ACRO Biosystems Cat. No. LIF-H521b] as analytes.

Kinetic constants and affinity indices were obtained by mathematical approximation of the sensogram using the 1:1 Langmuir binding model using global algorithms (simultaneous approximation of sets of sensograms), as well as single curve fitting algorithms. The reliability of global approximations was assessed using k _obs analysis.

-32044914

Table 6

Measuring the affinity of additional antibodies to LIF Langmuir sensogram approximation 1:1 Analyst Clone Average value of K _a (1/Ms) Average Ka value (1/s) Average value K _D [nM] Human LIF (E coli) 1B2 1.1 ±0.4E5 1.1 ±0.ЗЭ-3 9.7 ± 1.4 Human LIF (NEK-293) 1B2 2.0 ± 0.04E6 1.4±0.2E-3 0.7 ±0.03 Human LIF (E. coll) 10G7 7.9 ± 5.8E4 6.0 ± 2.ЗЭ-4 12.6 ±9.5 Human LIF (NEK-293) 10G7 3.6 ± 1.75E5 3.1 ±0.5E-4 1D ±0.6 Human LIF (E. coli) 6B5 And. And. And. And. And. And. Human LIF (NEK-293) 6B5 3.6 ± 1.7E5 3.1 ±0.5E-4 62 ±6

Example 8 Additional anti-LIF antibodies inhibit LIF-induced STATS phosphorylation in vitro.

Additional clones were tested for their ability to inhibit LIF-induced STAT3 phosphorylation in cell culture. As shown in FIG. 4, clones 10G7 and the previously described r5D8 showed high inhibition of LIF-induced STAT3 phosphorylation compared to clone 1B2. Polyclonal antisera to LIF (item) were included as a positive control. Although 6B5 did not show inhibition, this may be explained by the possible lack of binding of 6B5 to non-glycosylated LIF, which was used in this experiment.

Ways.

Glioma cells obtained from the patient were seeded in 6-well plates at a density of 150,000 cells/well. Cells were cultured in GBM medium, which consisted of Neurobasal medium (Life Technologies) supplemented with B27 (Life Technologies), penicillin/streptomycin, and growth factors (20 ng/ml EGF and 20 ng/ml FGF-2 [PeproTech]) for 24 h before any processing. The next day, cells were treated with recombinant LIF produced in E. coli, or with a mixture of recombinant LIF with the indicated antibodies for 15 min (final concentration 10 μg/ml for antibodies and 20 ng/ml recombinant LIF), or not treated . After treatment, proteins were isolated using radioimmunoprecipitation assay (RIPA) lysis buffer containing phosphatase and protease inhibitors, quantified (BCA-protein assay, Thermo Fisher Scientific), and used in Western blotting. For Western blotting, membranes were blocked for 1 h in 5% skim milk - TBST and incubated with primary antibody overnight (pSTAT3, cat. no. 9145, Cell Signaling) or 30 min (β-actin peroxidase, cat. no. A3854 , Sigma-Aldrich). Membranes were then washed in TBST, incubated with secondary antibody if necessary, and washed again. Proteins were detected by chemiluminescence (SuperSignal Substrate, catalog no. 34076, Thermo Fisher Scientific).

Example 9 LIF is highly overexpressed in several tumor types.

Immunohistochemical analysis was performed on several types of human tumors to determine the extent of LIF expression. In fig. Figure 5 shows that LIF is highly expressed in glioblastoma multiforme (GBM), non-small cell lung cancer (NSCLC), ovarian cancer, colorectal cancer (CRC) and pancreatic tumors.

Example 10 Humanized clone h5D8 inhibits tumor growth in a mouse model of non-small cell lung carcinoma.

To determine the ability of the humanized clone 5D8 to inhibit LIF-positive cancer in vivo, this antibody was tested in a mouse model of non-small cell lung carcinoma (NSCLC). In fig. Figure 6 shows a reduction in tumor growth in mice treated with this antibody compared to the vehicle negative control.

Ways.

Non-small cell lung cancer (NSCLC) cell line KLN205 with high levels of LIF

- 33 044914 was infected with lentivirus for stable expression of the firefly luciferase gene for the purpose of monitoring bioluminescence in vivo. To establish a mouse model of 5x105 non-small cell lung cancer (NSCLC) cells, KLN205 was orthotopically implanted into the left lung of immunocompetent syngeneic DBA/2 mice at 8 weeks of age via intercostal puncture. Mice were treated with vehicle control or with 15 or 30 mg/kg h5D8 antibody intraperitoneally twice a week and tumor growth was monitored by bioluminescence. To visualize bioluminescence, mice were intraperitoneally injected with 0.2 ml of 15 mg/ml D-luciferin under 12% isoflurane inhalation anesthesia. Bioluminescence signals were monitored using an IVIS 2000 series system (Xenogen Corp., Alameda, CA, USA), consisting of a highly sensitive cooled CCD camera. Living Image software (Xenogen Corp.) was used to collate the imaging data and integrate common bioluminescence signals in each boxed area. Data were analyzed using total photon flux (photons/second) in regions of interest (ROI). The results demonstrate that treatment with the h5D8 antibody promotes tumor regression. Data are presented as mean ± SEM.

Example 11: h5D8 inhibits tumor growth in a mouse model of glioblastoma multiforme.

In an orthotopic GBM tumor model using the human luciferase-expressing U251 cell line, r5D8 significantly reduced tumor volumes in mice treated with 300 μg of r5D8 and h5D8 by intraperitoneal injection twice weekly. The results of this study are shown in Fig. 7A (quantitative determination on day 26 after treatment). This experiment was also conducted using mice that were treated with 200 μg or 300 μg of humanized h5D8, and the results showed a statistically significant reduction in tumor after 7 days of treatment.

Ways.

U251 cells stably expressing luciferase were collected, washed in PBS, centrifuged at 400g for 5 min, resuspended in PBS, and counted using an automated cytometer (Countess, Invitrogen). Cells were kept on ice to maintain optimal viability. Mice were anesthetized by intraperitoneal administration of ketamine (Ketolar50®)/xylacine (Rompun®) (75 and 10 mg/kg, respectively). Each mouse was carefully placed in a stereotaxic device and immobilized. Hair was removed from the scalp with depilatory cream, and the scalp was cut with a scalpel to expose the skull. Using a drill, a small incision was carefully made at the coordinates 1.8 mm lateral and 1 mm anterior to the junction of the lambdoid and sagittal sutures. 5 μl of cells were inoculated using a 30G Hamilton syringe into the right striatum at a depth of 2.5 mm. The head incision was closed with tissue adhesive Hystoacryl (Braun) and mice were administered the subcutaneous analgesic meloxicam (Metacam®) (1 mg/kg). The final number of cells implanted in each mouse was 3x105.

Mice were treated twice weekly with intraperitoneal administration of h5D8. Treatment began on day 0 immediately after tumor cell inoculation. Mice received a total of 2 doses of h5D8 or vehicle control.

Body weight and tumor volume. Body weight was measured twice a week, and tumor growth was quantified by bioluminescence on day 7 (Xenogen IVIS Spectrum). To quantify bioluminescence activity in vivo, mice were anesthetized with isofluorane and injected intraperitoneally with the substrate luciferin (PerkinElmer) (167 μg/kg).

Tumor size, determined by bioluminescence (Xenogen IVIS Spectrum), was assessed on day 7. Individual tumor scores and mean ± SEM for each treatment group were calculated. Statistical significance was determined using the unpaired nonparametric Mann-Whitney U test.

Example 12: h5D8 inhibits tumor growth in a mouse model of ovarian cancer.

The efficacy of r5D8 was assessed in two other syngeneic tumor models. In an orthotopic ID8 ovarian tumor model, r5D8 antibody, administered 300 μg intraperitoneally twice weekly, significantly suppressed tumor growth as measured by abdominal volume (FIGS. 8A and 8B). The results in Fig. 8C show that h5D8 antibody also reduced tumor volume at doses of 200 μg and above.

Ways.

ID8 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Gibco, Invitrogen), 40 U/ml penicillin, and 40 μg/ml streptomycin (PenStrep) (Gibco, Invitrogen ) and 0.25 μg/ml plasmocin (Invivogen).

ID8 cells were harvested, washed in PBS, centrifuged at 400 g for 5 min, and resuspended in PBS. Cells were kept on ice to maintain optimal viability, and 200 μl of cell suspension was injected intraperitoneally using a 27-gauge needle. The final number of cells implanted into mice was 5x106.

Mice were treated twice weekly with i.p. h5D8 at different doses as indicated. Body weight was measured twice a week, and tumor progression was monitored by measuring abdominal girth using a caliper (Fisher Scientific).

Example 13: r5D8 inhibits tumor growth in a mouse model of colorectal cancer.

In mice bearing subcutaneous CT26 colon tumors, r5D8 antibody (administered 300 μg intraperitoneally twice weekly) significantly suppressed tumor growth (Figures 9A and 9B).

Ways.

CT26 cells were cultured in Roswell Park Memorial Institute medium (RPMI [Gibco, Invitrogen]) supplemented with 10% fetal bovine serum (FBS), 40 U/ml penicillin and 40 μg/ml streptomycin (PenStrep), and 0.25 μg/ml plasmocin .

CT26 cells (8x105) were trypsinized, washed with PBS, centrifuged at 400 g for 5 min and resuspended in 100 μl PBS. Cells were kept on ice to avoid cell death. CT26 cells were administered to mice by subcutaneous injection using a 27G needle.

300 μg of r5D8 or vehicle control was administered to mice by intraperitoneal injection twice weekly starting on day 3 after CT26 cell implantation.

Body weight and tumor volume were measured three times a week. Tumor volume was measured with a caliper (Fisher Scientific).

Example 14: r5D8 reduces inflammatory infiltration in tumor models.

In the U251 orthotopic GBM model, the expression of CCL22, a marker of M2-polarized macrophages, was significantly reduced in r5D8-treated tumors, as shown in Fig. 10A. This result was also confirmed in a physiologically relevant organotypic tissue slice culture model using r5D8, in which three patient samples showed a significant reduction in the expression of CCL22 and CD206 (MRC1) (also a marker of M2 macrophages) after treatment, as shown in FIG. 10V (compare MRC1 and CCL22 in the top row, control, and in the bottom row, with processing). In addition, r5D8 also reduced the number of CCL22+ M2 macrophages in syngeneic ID8 (Fig. 10C) and CT26 (Fig. 10D) tumors from immunocompetent mice.

Example 15: r5D8 increases the number of non-myeloid effector cells.

To investigate additional immune mechanisms, the effects of r5D8 on T cells and other non-myeloid effector immune cells in the tumor microenvironment were assessed. In a syngeneic ID8 ovarian orthotopic tumor model, treatment with r5D8 resulted in increased numbers of intratumoral NK cells and increased total and activated CD4+ and CD8+ T cells, as shown in FIG. 11A. Similarly, in a syngeneic CT26 colon tumor model, the r5D8 antibody increased the number of intratumoral NK cells, increased the number of CD4+ and CD8+ T cells, and tended to decrease the number of CD4 ⁺ CD25 ⁺ FoxP3 ⁺ T regulatory cells, as shown in Fig. 11B. A trend toward decreased numbers of CD4 ⁺ CD25 ⁺ FoxP3 ⁺ T regulatory cells was also observed in the syngeneic KLN205 orthotopic tumor model after treatment with r5D8, as shown in FIG. 11C. Consistent with the requirement for T cells to mediate efficacy, depletion of CD4+ and CD8+ T cells in the CT26 model inhibited the antitumor efficacy of r5D8, as shown in FIG. 12.

Methods for T cell depletion.

CT26 cells were cultured in RPMI culture medium (Gibco, Invitrogen) supplemented with 10% fetal bovine serum (FBS [Gibco, Invitrogen]), 40 U/ml penicillin and 40 μg/ml streptomycin (PenStrep [Gibco, Invitrogen]) and 0. 25 μg/ml plasmocin (Invivogen). CT26 cells (5x105) were collected, washed with PBS, centrifuged at 400 g for 5 min and resuspended in 100 μl PBS. Cells were kept on ice to avoid cell death. CT26 cells were administered to mice in both flanks by subcutaneous injection using a 27G syringe. Mice were treated twice weekly with r5D8 intraperitoneal administration as specified in the study design. Vehicle control (PBS), rat r5D8 and/or anti-CD4, and anti-CD8 were administered to mice by intraperitoneal injection (IP) twice weekly as specified in the study design. All antibody treatments were used simultaneously.

Example 16 Crystal structure of h5D8 complexed with human LIF.

The crystal structure of h5D8 was solved to 3.1 Å resolution to determine the epitope on LIF to which h5D8 bound and to identify the residues of h5D8 that are involved in binding. The cocrystal structure revealed that the N-terminal loop of LIF is located centrally between the h5D8 light and heavy chain variable regions (Fig. 13A). In addition, h5D8 interacts with helix residues A and C of LIF to form a discontinuous conformational epitope. The binding is due to several salt bridges, H-bonds and van der Waals interactions (Table 7, Fig. 13B). The h5D8 epitope on LIF spans the region of interaction with gp130. See Boulanger, M.J., Bankovich, A.J., Kortemme, T., Baker, D. & Garcia, K.C. Convergent mechanisms for recognition of divergent cytokines by the shared signaling receptor gp130. Molecular cell 12, 577-589 (2003). The results are summarized below in the table. 7 and shown in FIG. 13.

- 35 044914

Table 7

Summary of the X-ray crystal structure of h5D8 complexed with human LIF LIF residue (epitope) Type interactions Remnant h5D8 (paratope, numbering according to Kabat) A1a13 V.D.W. L-Tyr49, L-Asn53 Pe14-O HB L-Ser50-OG Not V.D.W. L-His30, L-Tyr32, L-Tyr49, L-Ser50 H-Trp97 Argl5-NE S.B. L-Glu55-OE1, L-Glu55-OE2 Argl5-NH1 S.B. L-Glu55-OE1, L-Glu55-OE2 Argl5-NH2 S.B. L-Glu55-OE1, L-Glu55-OE2 Argl5-O HB L-Asn34-ND2 Argl5 V.D.W. L-Asn34, L-Leu46, L-Tyr49, L-Glu55, L- Ser56 H-Glu96, H-Trp97, H-Asp98, H-Leu99, H- AsplOl Hisl6-NE2 S.B. H-Aspl01-OD2 Hisl6 V.D.W. L-Tyr32, L-Asn34, L-Met89 H-Trp95, H-Glu96, H-Trp97, H-Aspl01 Prol7 V.D.W. L-Tyr32, L-Ala91 H-Trp97 Cysl8 V.D.W. L-Tyr32 H-Trp33, H-Trp97 Hisl9-NE2 S.B. H-Glu96-OE1, H-Glu96-OE2 Hisl9 V.D.W. H-His31, H-Trp33, H-Glu96 Asn20-OD1 HB H-Lys52-NZ Asn20-ND2 HB H-Asp53-OD1 Asn20 V.D.W. H-Trp33, H-Lys52, H-Asp53 Gln25-NE2 HB H-Asp53-OD2 Gln25 V.D.W. H-His31, H-Ser52C, H-Asp53 Gln29 V.D.W. H-His31 Gln32 V.D.W. H-Lys52B Aspl20-OD2 HB H-Ser30-OG Aspl20 V.D.W. H-Thr28, H-Ser30 Argl23-NE HB H-Thr28-OG Argl23 V.D.W. H-Thr28 Glyl24 V.D.W. H-His31 Leul25 V.D.W. H-His31 Serl27-OG HB H-Asp98-OD2 Serl27-O HB H-Trp97-NE1 Serl27 V.D.W. H-His31, H-Trp97, H-Asp98 Asnl28-OD1 HB H-His31-NE2 Asnl28 V.D.W. H-His31 Leul30 V.D.W. H-Trp97 Cysl31 V.D.W. H-Trp97 Cysl34 V.D.W. H-Trp97 Serl35-O HB L-His30-NE2 Serl35 V.D.W. L-His30 Hisl38 V.D.W. L-His30

- 36 044914

VDW, van der Waals forces, low-energy coupling; HB, hydrogen bond (medium energy bond); SB, salt bridge (high energy bond).

Ways.

LIF was transiently expressed in HEK 293S (Gnt I ^−/− ) cells and purified using Ni-NTA affinity chromatography followed by gel filtration chromatography in 20 mM Tris, pH 8.0, and 150 mM NaCl. Recombinant Fab h5D8 was transiently expressed in HEK 293F cells and purified using KappaSelect affinity chromatography followed by cation exchange chromatography. Purified h5D8 Fab and LIF were mixed at a molar ratio of 1:2.5 and incubated at room temperature for 30 min before deglycosylation using EndoH. Gel filtration chromatography was then used to purify the complex. The complex was concentrated to 20 mg/mL and prepared for crystallization tests using dilute matrix screening tests. Crystals formed at 4°C under conditions containing 19% (v/v) isopropanol, 19% (w/v) PEG 4000, 5% (v/v) glycerol, 0.095 M sodium citrate, pH 5.6. The crystal was diffracted with a resolution of 3.1 A at the 08ID-1 radiation channel of the Canadian Light Source (CLS) synchrotron. Data were collected, processed and scaled using XDS according to Kabsch et al. Xds. Acta crystallographica. Section D, Biological crystallography 66, 125-132 (2010). Structures were determined by molecular replacement using Phaser according to McCoy et al. Phaser crystallographic software. J Appl Crystallogr 40, 658-674 (2007). Performed several iterations of model building and refinement using Coot and phenix.refine until the structures approached acceptable R _work and R _free . See Emsley et al. Features and development of Coot. Acta crystallographica. Section D, Biological crystallography 66, 486-501 (2010) and Adams et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta crystallographica. Section D, Biological crystallography 66, 213-221 (2010) respectively. Figures were drawn in PyMOL (PyMOL molecular graphics system, version 2.0, Schrodinger, LLC).

Example 17 h5D8 is highly specific for LIF.

The goal was to test the binding of h5D8 to other members of the LIF family to determine binding specificity. Using the Octet96 assay, h5D8 binding to human LIF is approximately 100-fold greater than binding to LIF, the highest homology member of the IL-6 family, oncostatin M (OSM), when both proteins are produced in E. coli. When both proteins are produced in a mammalian system, h5D8 does not exhibit binding to OSM. The data is summarized in table. 8.

Table 8

Summary of h5D8 Cytokine Affinities Using the Octet Assay KD[M] kon [1/M s] kdis[1/c] h5D8 + huLIF (E. coli) 4.3E-10 +/-2.0E-11 HERE+05 +/-HERE+OZ l.3E-04 +/- 5.8E-06 h5D8 + huLIF (from mammalian system) l.3E-09 +/-7.2E-11 l,2E+05 +/-l,3E+03 l.5E-04 +/- 8.5E-06 h5D8 + huOSM (E. coli) 3.6E-08 +/- l.4E-09 8.5E+04 +/-HERE+OZ HERE-OZ +/-4.1E-O5 h5D8 + huOSM (from mammalian system) C.o. C.o. C.o. h5D8 + huIL-6 (E. coli) C.o. C.o. C.o.

S. o. - there is no binding.

Ways.

Binding experiments using Octet: Reagents were used and prepared according to the manufacturer's instructions. The baseline kinetic parameter determination experiment was performed using Octet Data Acquisition software version 9.0.0.26 as follows: Sensor/program setup: i) equilibration (60 s); ii) loading (15 s); w) initial level (60 s); iv) binding (180 s) and v) dissociation (600 s).

Affinity of h5D8 for cytokines according to Octet: Baseline kinetic parameter determination experiment was performed using Octet Data Acquisition software version 9.0.0.26 as follows: Amine Reactive 2ndGeneration Biosensors (AR2G) were hydrated in water for at least 15 min. Conjugation at the amino group of h5D8 with biosensors was carried out in accordance with ForteBio Technical Note 26 (see references) using the Amine Coupling Second Generation Kit. The immersion steps were performed at 30°C, 1000 rpm as follows: i) 60 s equilibration in water; ii) 300 s activation in 20 mM ECD, 10 mM sulfo-NHS in water; iii) 600 s immobilization of 10 μg/ml h5D8 in 10 mM sodium acetate, pH 6.0; iv) 300 with extinction in 1M

- 37 044914 ethanolamine, pH 8.5; v) 120 s at baseline in water. Kinetic parameter experiments were then performed with the following dip and readout steps at 30°C, 1000 rpm: vi) 60 s baseline in 1X kinetic buffer; vii) 180 s binding of appropriate serial dilutions of cytokine in 1X kinetic buffer; viii) 300 s dissociation in 1X kinetic buffer; ix) three regeneration/neutralization cycles alternating with 10 mM glycine, pH 2.0 and 1X kinetic buffer, respectively (5 s each for 3 cycles). After regeneration, the biosensors were reused for subsequent binding assays.

Recombinant human LIF produced in mammalian cells was obtained from ACROBiosystems (LIF-H521b); recombinant human OSM produced in mammalian cells was obtained from R&D (8475-OM/CF); and recombinant human OSM produced in E. coli cells were obtained from R&D (295-OM-050/CF).

Example 18: Crystal structure of fab h5D8.

Five crystal structures of Fab h5D8 were determined under a wide range of chemical conditions. The high resolutions of these structures indicate that the conformations of the CDR residues involve little flexibility and are very similar in different chemical environments. A unique feature of this antibody is the presence of a non-canonical cysteine at position 100 of the heavy chain variable region. Structural analysis shows that the cysteine is unpaired and largely inaccessible to solvent.

Fab H5D8 was prepared by papain digestion of its IgG followed by purification using standard affinity, ion exchange, and size exclusion chromatography techniques. The crystals were obtained using vapor phase diffusion methods, and they allowed the determination of five crystal structures with a resolution of 1.65 to 2.0 A. All structures were solved and assigned to the same crystallographic space group and with similar unit cell sizes (P212121 , a~53.8 A, b-66.5 A, c~ 143.3 A) despite crystallization conditions in a range of five different pH levels: 5.6, 6.0, 6.5, 7.5 and 8 ,5. Thus, these crystal structures allow comparison of the 3D arrangement of h5D8 Fabs without obstruction by crystal packing artifacts and under a wide range of chemical conditions.

Electron density was detected for all complementarity determining region (CDR) residues, which were subsequently modeled. It is noteworthy that LCDR1 and HCDR2 adopted extended conformations that, together with the shallow regions of LCDR3 and HCDR3, formed a binding cavity in the center of the paratope (Fig. 14A). These five structures were very similar across all residues, with all-atom RMSDs ranging from 0.197 to 0.327 Å (Figure 14A). These results indicate that CDR residue conformations are maintained in a variety of chemical environments, including pH levels from 5.6 to 8.5 and ionic strengths from 150 mM to 1 M. Electrostatic surface analysis of the h5D8 paratope showed that positively and negatively charged regions contributed equally to the hydrophilic properties, with no predominant hydrophobic regions. h5D8 has the unusual feature of a non-canonical cysteine at the base of HCDR3 (Cys100). In all five structures, this free cysteine is ordered and does not cause rearrangement of disulfide bonds. In addition, it is not modified by the addition of Cys (cysteinylation) or glutathione (glutathiolation) and interacts due to van der Waals forces (distances 3.5-4.3 A) with the atoms of the main and side chains Leu4, Phe27, Trp33, Met34 , Glu102 and Leu105 heavy chain (Fig. 14B). Finally, Cys100 is a predominantly buried structural residue that appears to be involved in mediating the conformation of CDR1 and HCDR3. Thus, it is unlikely to exhibit reactivity toward other cysteines, as observed by the homogeneous arrangement of this region in the five crystal structures.

Ways.

H5D8-1 IgG was obtained from Catalent Biologies and formulated in 25 mM histidine, 6% sucrose, 0.01% polysorbate 80 at pH 6.0. Active buffer exchange of the formulated IgG with PBS buffer was performed using a MWCO 10K concentrator (Millipore) before digestion with 1:100 μg papain (Sigma) for 1 hour at 37°C in PBS, 1.25 mM EDTA, 10 mM cysteine. Papain-digested IgG was passed through a protein A column (GE Healthcare) using an AKTA Start chromatography system (GE Healthcare). The eluate from the Protein A column, which contained Fab h5D8, was removed and buffer exchanged to 20 mM sodium acetate, pH 5.6, using a 10K MWCO concentrator (Millipore). The resulting sample was loaded onto a Mono S cation exchange column (GE Healthcare) using an AKTA Pure chromatography system (GE Healthcare). Elution with a 1M potassium chloride gradient resulted in a predominant Fab h5D8 peak, which was recovered, concentrated, and purified to uniform size using a Superdex 200 Increase gel filtration column (GE Healthcare) in 20 mM Tris-HCl, 150 mM sodium chloride at pH 8 ,0. The high purity of Fab h5D8 was confirmed by SDS-PAGE under reducing and non-reducing conditions.

Purified Fab h5D8 was concentrated to 25 mg/ml using a 10K MWCO concentrator (Milli

- 38 044914 pore). An Oryx 4 dispenser (Douglas Instruments) was used to model vapor-phase diffusion crystallization experiments with commercial sparse matrix screening assays with 96 conditions JCSG TOP96 (Rigaku reagents) and MCSG-1 (Anatrace) at 20°C. Crystals were obtained and collected after four days under the following five crystallization conditions: 1) 0.085 M sodium citrate, 25.5% (w/v) PEG 4000, 0.17 M ammonium acetate, 15% (v/v) glycerol , pH 5.6; 2) 0.1M MES, 20% (w/v) PEG 6000, 1M lithium chloride, pH 6.0; 3) 0.1M MES, 20% (w/v) PEG 4000, 0.6M sodium chloride, pH 6.5; 4) 0.085M HEPES sodium, 17% (w/v) PEG 4000, 8.5% (v/v) 2-propanol, 15% (v/v) glycerol, pH 7.5; and 5) 0.08 Mtris, 24% (w/v) PEG 4000, 0.16 M magnesium chloride, 20% (v/v) glycerol, pH 8.5. Before flash freezing in liquid nitrogen, 5-15% (v/v) glycerol or 10% (v/v) ethylene glycol was added to the crystal-containing mother liquors as needed. The crystals were exposed to X-ray synchrotron radiation at channel 23-ID-D of the Advanced Photon Source synchrotron (Chicago, Illinois, USA) and diffraction patterns were recorded on a Pilatus3 6M detector. Data were processed using XDS and structures were determined by molecular displacement using Phaser. Refinement was carried out in PHENIX with iterative model building in Coot. The figures were built in PyMOL. All software was available in SBGrid.

Example 19 Mutations of cysteine 100 in h5D8 preserve binding.

Analysis of h5D8 revealed a free cysteine residue at position 100 (C100) in the heavy chain variable region. H5D8 variants were generated by replacing C100 with each natural amino acid to determine binding characteristics to and affinity for human and murine LIF. Binding was characterized by ELISA and Octet assay. The results are summarized in table. 9. EC50 curves for ELISA are shown in FIG. 15 (Fig. 15A, human LIF, and Fig. 15B, mouse LIF).

Table 9

Summary of affinity values determined by Octet assay and EC50 values determined by ELISA Mutation Affinity/Λ^ (M) EC50 binding (nM) Human LIE Mouse LIE Human LIE Mouse LIE syuo <1.0E-12±2.252E-11 9.946E-11 ± 8.272E-12 0.09878 0.1605 C100S 8.311E-10±5.886E-11 2.793E-09± 5.925E-11 n.d. n.d. C100Q 3.87E-09 ± 1.55E-10 2.84E-09 ± 4.85E-11 10.18 26.33 C100N 5.59E-09 ± 1.01E-10 6.68E-09 ± 9.8E-11 13.18 45.87 C100E 2.67E-09 ± 4.64E-11 4.1E-09 ± 7.56E-11 7,179 25.3 C100D 2.02E-09 ± 8.08E-11 6.49E-09 ± 7D6E-11 11.89 22.88 suot 4.36E-10±2.1E-11 1.02E-09± 1.77E-11 5.575 8,753 C100G 2.49E-09 ± 4.2E-11 3,ZZE-09±5.42E-11 21.94 40.17 S100R 2.74E-10 ± 2.97E-10 <1.0E-12 ± 7.64E-10 34.44 101.9 C100 A <1.0E-12±2.713E-11 <1.0E-12± 1.512E-11 0.6705 0.9532 C100V <1.0E-12± 1.805E-11 <1.0E-12 ± 8.086E-12 0.2785 0.3647 C100L <1.0E-12± 1.963E-11 1.998E-10± 1.055E-11 0.454 0.547 S1001 <1.0E-12± 1.424E-11 3.361E-11 ± 7.545E-12 0.299 0.3916 sumptuously 1D55E-09 ± 3,400E-11 2.676E-09±2.449E-11 0.7852 1,563 C100F 4.376E-09± 1D27E-10 1.147E-08±9.099E-11 8,932 21.53 C100Y 1.444E-08± 1D59E-09 2.514E-08 ± 2.047E-09 n.d. n.d. C100W 2.508E-08 ± 7.036E-09 4.819E-08 ± 4.388E-09 n.d. n.d. suon 1.304E-10± 1.416E-10 4.284E-09 ± 1.231E-10 8,254 n.d. syuk 7.477E-08 ± 1.581E-09 6.053E-08 ± 2.589E-09 n.d. n.d. C100R 1.455E-07 ± 6.964E-09 5D42E-08 ± 3.247E-09 n.d. n.d.

Ways.

ELISA: Binding of h5D8 C100 variants to human and mouse LIF was determined by ELISA. Recombinant human or mouse LIF protein was added to Maxisorp 384-well plates at a concentration of 1 μg/ml and left overnight at 4°C. The plates were blocked using 1× blocking buffer for 2 h at room temperature. Titers of each h5D8 C100 variant were added and allowed to bind for 1 h at room temperature. Tablets

- 39 044914 washed three times with PBS+0.05% Tween-20. HRP-conjugated anti-human IgG antibodies were added and allowed to bind for 30 min at room temperature.

The plates were washed three times with PBS+0.05% Tween-20 and developed with 1x TMB substrate. The reaction was stopped with 1 M HCl and the absorbance was measured at 450 nm. Figure plotting and nonlinear regression analysis were performed using Graphpad Prism.

Octet RED96: The affinity of h5D8 C100 variants for human and mouse LIF was determined by BLI using the Octet RED96 system. C100 h5D8 variants were loaded onto human Fc biosensors at a concentration of 7.5 μg/ml after a 30-second baseline in 1x kinetic buffer. Human or mouse LIF protein titers were coupled to loaded biosensors for 90 seconds and allowed to dissociate in 1× kinetic buffer for 300 seconds. KD was calculated using data analysis software using a 1:1 global fitting model.

Example 20: h5D8 blocks LIF binding to gp130 in vitro.

To determine whether h5D8 prevents LIF from binding to LIFR, a molecular binding assay was performed using the Octet RED 96 platform. H5D8 was loaded onto ANS biosensors by capture with anti-human Fc antibody. The biosensors were then immersed in LIF and binding was observed as expected (Figure 16A, middle third). Subsequently, the biosensors were immersed in different concentrations of LIFR. Dose-dependent binding was observed (Fig. 16A, right third). A control experiment demonstrated that this binding was LIF-specific (not shown) and was not due to nonspecific interaction of LIFR with h5D8 or with the biosensors.

To further characterize the binding of h5D8 and LIF, a series of ELISA binding experiments were performed. H5D8 and LIF were preincubated and then plated on plates coated with either recombinant human (hLIFR) or gp130. The lack of binding between the h5D8/LIF complex and the coating substrate may indicate that h5D8 somehow disrupts the binding of LIF to the receptor. In addition, control antibodies that either do not bind LIF (isotype control, indicated (-)) or bind LIF at known binding sites were also used (B09 does not compete with either gp130 or LIFR for LIF binding; r5D8 is the rat parental version h5D8). The ELISA results demonstrated that the h5D8/LIF complex was able to bind hLIFR (as was the r5D8/LIF complex), indicating that these antibodies did not interfere with LIF/LIFR binding (Fig. 16A). Conversely, the h5D8/LIF complex (and the r5D8/LIF complex) was unable to bind recombinant human gp130 (Fig. 16B). This indicates that the gp130 binding site on LIF is affected when LIF is associated with h5D8.

Example 21. Expression of LIF and LIFR in human tissues.

Quantitative real-time PCR was performed on a variety of different human tissue types to determine the expression levels of LIF and LIFR. The average expression levels shown in FIG. 17A and 17B are presented in copies per 100 ng of total RNA. Most tissues expressed at least 100 copies per 100 ng of total RNA. LIF mRNA expression was highest in human adipose tissue (mesentery of the ileum [1]), blood vessel tissue (choroid plexus [6] and mesenteric vessels [8]), and umbilical cord tissue [68]; and the lowest in brain tissues (cortex [20] and substantia nigra [28]). LIFR mRNA expression was highest in human adipose tissue (meso-ileum [1]), blood vessel tissue (pulmonary vasculature [9]), brain tissue [11–28], and thyroid tissue [66]; and the lowest in PBMC [31]. LIF and LIFR mRNA expression levels in cynomolgus macaque tissues were similar to those observed in human tissues, where LIF expression was high in adipose tissue and LIFR expression was high in adipose tissue and low in PBMC (data not shown).

Fabric numbering in Fig. 17A and 17B are as follows: 1 - adipose tissue (ileal mesentery); 2 - adrenal gland; 3 - bladder; 4 - bladder (triangle); 5 - blood vessel (cerebral: middle cerebral artery); 6 - blood vessel (choroid plexus); 7 - blood vessel (coronary artery); 8 - blood vessel (mesenteric (large intestine)); 9 - blood vessel (pulmonary); 10 - blood vessel (renal); 11 - brain (amygdala); 12 - brain (caudate nucleus); 13 - brain (cerebellum); 14 brain - (cortex: anterior cingulate gyrus); 15 brain (cortex: posterior cingulate cortex); 16 - brain (cortex: lateral frontal region); 17 - brain (cortex: medial frontal region); 18 - brain (cortex: occipital region); 19 - brain (cortex: parietal region); 20 - brain (cortex: temporal region); 21 - brain (dorsal raphe nucleus); 22 - brain (hippocampus); 23 - brain (hypothalamus: anterior); 24 - brain (hypothalamus: posterior); 25 - brain (blue spot); 26 - brain (medulla oblongata); 27 - brain (nucleus accumbens); 28 - brain (substantia nigra); 29 - mammary gland; 30 - cecum; 31 - peripheral blood mononuclear cells (PBMC); 32 - colon; 33 - dorsal root ganglia (DRG); 34 - duodenum; 35 - fallopian tube; 36 gallbladder; 37 - heart (left atrium); 38 - heart (left ventricle); 39 - ileum; 40 - jejunum; 41 - kidney (bark); 42 - kidney (medullary substance); 43 - kidney (pelvis); 44 - liver (parenchyma); 45 - liver (bronchus: primary); 46 - liver (bronchus: tertiary); 47 - lung (parenchyma); 48

- 40 044914

- lymph node (tonsil); 49 - muscle (skeletal); 50 - esophagus; 51 - ovary; 52 - pancreas; 53 - pineal gland; 54 - pituitary gland; 55 - placenta; 56 - prostate gland; 57 - rectum; 58 - skin (foreskin); 69 - spinal cord; 60 - spleen (parenchyma); 61 - stomach (antrum); 62 - stomach (body); 63 - stomach (bottom); 64 - stomach (pyloric canal); 65 - testicle; 66 - thyroid gland; 67 - trachea; 68 - umbilical cord; 69 - ureter; 70 - uterus (cervix); 71 - uterus (myometrium); 72 - vas deferens.

Example 22. Dose selection, stepwise dose escalation and fixed dose administration.

Anti-LIF antibody dose selection, dose escalation, and fixed dose administration are described below. Mice and cynomolgus monkeys were used to evaluate the safety of h5D8.

No treatment-related adverse effects were observed in 4-week GLP toxicity studies in mice and monkeys receiving a weekly IV dose of no more than 100 mg/kg. Thus, the highest non-hazardous toxic dose (HNSTD) is >100 mg/kg, and the no observed adverse effect level (NOAEL) was set at 100 mg/kg IV for both species under study conditions. The dosage was scaled to establish the human equivalent dose (HED). A scaling approach based on body surface area (BSA) was adapted to estimate HED. Based on these GLP toxicology studies, the maximum recommended starting dose (MRSD) was calculated as follows:

0.81 mg/kg IV HED from murine NOAEL with a 10-fold safety factor;

>10 mg/kg IV based on 1/10 of the toxic toxic dose in mice;

3.2 mg/kg HED IV from NOAEL cynomolgus monkey with a 10-fold safety factor;

> 16.7 mg/kg IV based on 1/6 HNSTD.

Based on the results of toxicology studies and using a conservative approach to the advanced cancer patient population in the phase 1 study, an MRSD of 1 mg/kg (or fixed dose of 75 mg) IV was supported using these data.

The pharmacologically active dose (PAD) was also taken into account when establishing MRSD. Based on the pharmacological, PK and LIF stabilization data in pharmacological mouse models available to date, the following approach was used to evaluate PAD. Based on the dose response in the U251 xenograft mouse model, a dose of approximately 300 mcg i/p twice weekly was adopted as the optimal effective dose; this dose level was associated with a pre-last dose trough serum level of approximately 230 mcg/mL. Evidence was obtained that maximum stabilization of serum LIF levels in this model was achieved at this 300 μg dose, which was also supported by data on stabilization of serum LIF levels in a GLP toxicity study in mice at doses of 10, 30, and 100 mg. /kg. Using a PK model based on a 2-compartment model extrapolated to PK data from monkeys and scaled to humans, it was determined that a clinical dose of 1500 mg every 3 weeks would produce a C _{min im alna} value of approximately 500 mcg/mL. Likewise, a minimum effective dose of 20 μg twice weekly in this U251 xenograft mouse model was associated with a pre-last dose trough serum level of approximately 20 μg/mL; There was evidence that this 20 mcg dose achieved only approximately 50% of the maximum stabilization of serum LIF levels, which was supported by evidence of minimal stabilization of LIF levels at a dose of 0.5 mg/kg IV in a PK tolerance study in mice. A clinical dose of 75 mg every 3 weeks will provide a C _min im _alna value of approximately 25 mcg/mL. Additional PK-PD data (stabilization of LIF levels) available from syngeneic mouse models supported the PAD obtained in the U251 xenograft mouse model.

Thus, based on both toxicological data in mice and monkeys and data on the minimum effective dose in a murine xenograft model, a starting dose of 75 mg IV was considered appropriate. The maximum clinical dose of 1500 to 2000 mg has been supported by toxicological data. A fixed-dose approach was appropriate based on observations of a linear PK in animal models coupled with the absence of adverse outcomes associated with the study drug.

Example 23: LIF expression in various types of cancer.

Establishing treatment based on LIF levels regardless of cancer type would be a feasible approach if there was heterogeneity in LIF expression across different cancer types. In fig. 18 shows that certain cancers have a higher incidence of high LIF mRNA levels. Even among cancers that have a relatively high incidence of low LIF expression, there is a subset of individuals who will benefit from treatment with an anti-LIF antibody.

Way.

RNA sequencing data were obtained from the Cancer Genome Atlas repository for 7769 samples in 22 categories. Expression of LIF transcripts was classified into high, medium-high, medium-low, and low based on the upper, upper-middle, lower-middle, and lower quartiles of expression.

LIF calculated for all samples.

Example 24 LIF expression correlates with regulatory T cell chemokines in various cancers.

Inhibition of LIF has previously been shown to have the effect of reducing the abundance of immunosuppressive macrophage populations (eg, M2 macrophages) in murine and human ex vivo models (Figures 10A-10D) and reducing the abundance of regulatory CD4+ T cells in a mouse model of NSCLC (Figure 11C ), and infiltration. Thus, cancers that are characterized by high levels of both LIF and regulatory T cell chemokines or myeloid cell or M2 macrophage secreted chemokines may identify a subset of individuals with a high likelihood of responding to treatment with an anti-LIF antibody. Regulatory T cells are immunosuppressive CD4+ cells, and M2 macrophages are macrophages that maintain an anti-inflammatory immunosuppressive environment in tissues, compared to M1 macrophages that maintain a pro-inflammatory environment. In fig. 19A-19D show the correlation of LIF mRNA expression and the mRNA of various regulatory T cell chemokines, namely CCL7 (FIG. 19A), CCL2 (FIG. 19B), CCL3 (FIG. 19A), and CCL22 (FIG. 19A). Likewise, in FIG. 20A shows the correlation between LIF mRNA expression and M2 macrophage signature mRNA. This provides a basis for separating a population of LIF-expressing individuals (even those with low or moderate-low levels of LIF) as those who will potentially respond to a therapeutic anti-LIF antibody if an immunosuppressive signature is present (e.g., based on expression of M2 markers, regulatory T cell chemokines or regulatory T cell markers).

Significant positive correlations between LIF and CCL2, CD163 and CD206 were observed in both human GBM and ovarian cancer TCGA datasets (Figure 20B). No correlation was observed between LIF and CXCL9 (data not shown), but relatively low levels of CXCL9 mRNA were detected in tumors. These results were confirmed at the protein level by analyzing a cohort of 20 GBM patients and immunohistochemically analyzing the tumors for LIF, CXCL9, CCL2, CD163 and CD206. A strong positive correlation was observed between LIF and CCL2, CD 163 and CD206 (Fig. 20C). CXCL9 was expressed in isolated clusters of cells, which explains the low levels of CXCL9 mRNA present in tumors. Of note, CXCL9 showed an inverse correlation with LIF in human GBM (Fig. 20C).

CXCL9 and CCL2 were chemokines critical for tumor infiltration of CD8 T cells and recruitment of TAMs and Tregs, respectively. Confirmed regulation of CXCL9 and CCL2 to neutralize LIF in TAMs (CD11b ⁺ Ly6GLy6C) (Fig. 20D) Immunostaining and isolation of TAMs demonstrated that CXCL9, CCL2, CD206 and CD163 were mainly expressed in TAMs (Fig. 20E), and treatment with anti-LIF antibody (h5D8) regulated their expression (Fig. 20D, 20E). CXCR3 (CXCL9 receptor), CCR2 (CCL2 receptor), and LIFR were expressed in TAMs and CD8+ T cells (Figure 20F).

CXCL9 and CCL2 knockout mouse models (CXCL9'/', CCL2'/') were used to test the significance of CXCL9 and CCL2 regulation for the oncogenic function of LIF. Tumors in these mouse models were treated with blocking antibodies to CXCL9 and CCL2. Notably, the antitumor response to LIF inhibition was attenuated in CXCL9' ^/ ' mice, but not in CCL?'/' mice (Figure 20G). Likewise, neutralizing antibody to CXCL9, but not antibody to CCL2, attenuated the anti-cancer response to anti-LIF (h5D8) (Fig. 20G). These results demonstrated that the major mediator of anti-LIF (h5D8) response was CXCL9. As expected, blockade of CXCL9 reduced tumor infiltration by CD8+ T cells in response to anti-LIF (h5D8) (Fig. 20H).

To confirm that LIF regulates tumor infiltration by immune cells through CXCL9 repression in tumors from actual cancer patients, organotypic tissue cultures were established from GBM samples recently obtained from patients. These organotypic models allow for the short-term culture of tumor sections that preserve the tissue architecture and stroma (including immune cells) of the patient's tumor. Organotypic tissue cultures obtained from 3 patients whose tumor cells expressed high levels of LIF (Fig. 20I). All 3 cultures had high levels of TAM infiltration, detected by the Iba1 marker, and the majority of TAMs expressed CCL2, CD163 and CD206. Notably, 3-day treatment of an organotypic culture with a neutralizing anti-LIF antibody reduced the expression of CCL2, CD163 and CD206 and increased the expression of CXCL9 (FIG. 201).

Similar to the above observation in the case of human macrophages treated with anti-LIF antibody (h5D8), it was observed that treatment with anti-LIF antibody (h5D8) induced a decrease in the expression of M2 markers CD206 and CD163 in CT26 tumors (Fig. 20J). In contrast, treatment with anti-LIF antibody (h5D8) induced increased expression of M1 immunostimulatory markers, including CXCL9, CXCL10, and PD-L1 (Figure 20J). These results were further extended by also examining TAM phenotypes in MC38 tumors treated with anti-LIF antibody (h5D8). While treated tumors showed no difference in the overall frequency of total myeloid populations or TAM populations, treatment with anti-LIF antibody (h5D8) induced increased expression in both the portion of MHCII-expressing TAMs and increased overall MHCII expression levels (Fig. .20K). A side-by-side comparison of M1/M2 bias in the CT26 and MC38 models was not possible because MC38 TAMs did not express CD206, a key marker used for phenotyping M2 macrophages in the CT26 model. Together, these data demonstrate that anti-LIF antibody treatment suppresses tumor growth in two independent preclinical tumor models. The analysis demonstrates that LIF antibody treatment affected TAM phenotypes but not total TAM numbers or total myeloid cell numbers, suggesting that the observed efficacy is likely due in part to reprogramming TAMs to favor antitumor immunity.

Ways.

RNA sequencing data were obtained from the Cancer Genome Atlas repository. Based on Pearson's correlation, the association between the expression of LIF and various regulatory T cell chemokines (CCL7, CCL2, CCL3, and CCL22) was calculated for tumor samples from cancers of the gallbladder, brain, breast, colon, head and neck, kidney, lung, melanoma, ovarian, pancreatic, prostate and uterine cancer. Based on Pearson correlation, the association between LIF expression and the transcriptional signature representing M2 macrophages was calculated for tumor samples from gallbladder, brain, breast, colon, head and neck, kidney, lung, melanoma, ovarian, pancreatic, prostate and uterus.

Cells were lysed for mRNA extraction (RNeasy Mini or Micro Kit, Qiagen), retrotranscribed (BioRad's iScript Reverse Supermix for mRNA), and qRT-PCR was performed using Taqman probes from Applied Biosystems according to the manufacturer's recommendations. For paraffin-embedded sections, RNA was obtained using the High Pure FFPET RNA isolation kit (Roche) and following the manufacturer's instructions. Reactions were performed on a CFX384 TouchTM Real-Time PCR Detection System (Bio-Rad) and results were expressed as fold change calculated by Ct method compared to control. Mouse or human ACTB or GAPDH were used as internal controls for normalization.

RNA was analyzed on the Affymetrix microarray platform using Mouse Gene 2.1 ST. It was then normalized based on the Robust-Microarray Average (RMA). Bayesian linear regression with paired samples using the limma Bioconductor package identified that genes were differentially expressed in mice treated with anti-LIF antibody.

In mouse experiments, nuclei were counterstained using DAPI and images were acquired using a NIKON Eclipse Ti confocal laser scanning microscope. Immunofluorescence quantification was performed using ImageJ by counting all or up to 100 cells positive for CD 11b, Iba1 or CD3 in 2-3 different fields for each mouse, 3-5 mice/group, and calculating the percentage of cells positive for CCL2 , CD206 and CD163 as part of populations positive for Iba1 (for the GL261N model) or CD68/CD11b (for the ID8 model). For CXCL9, the percentage of cells surrounded by a given cytokine signal in the total cell population was calculated. In the case of organotypic sections, 3–4 fields of each patient were quantified (n = 3). For organotypic tissue immunofluorescence, five different z-axis images for each condition were processed using Fiji-Image J software. For CD8+ T cells, the percentage of CD8 T cells in the entire population was calculated. Data in the graphs are presented as mean ± SEM.

Antibodies for immunofluorescence analysis: human/mouse CCL2 (Novus Biologicals; 1:200), human/mouse CD11b (AbCam; 1:2000), human/mouse Ibal (Wako; 1:1000), mouse CD68 (AbCam; 1:200 ), human/mouse CD206 (Abcam; 1:500), mouse CD163 (Abcam; 1:200), CXCL9 (mouse: Novus Biologicals, 1:200; human: Thermo Fischer Scientific; 1:200) and human CD8 (DAKO ; 1:200).

Human GBM samples were obtained from the University Hospital and the Val de Brona Teaching Hospital. The clinical protocol was approved by the Institutional Review Board and the Val de Brona Clinical Hospital (CEIC) with informed consent obtained from all subjects.

Organotypic cultures of GBM slices were prepared as follows. After resection, surgical specimens were cut with a scalpel into rectangular blocks 5–10 mm long and 1–2 mm wide and individually transferred into 0.4 μm membrane culture inserts (Millipore) in 6-well plates. Before placing the inserts into 6-well plates, 1.2 ml of Neurobasal medium (Life Technologies) supplemented with B27 (Life Technologies), penicillin/streptomycin (Life Technologies) and growth factors (20 ng/ml EGF and 20 ng/ml) was added to each well. ml FGF-2) (PeproTech). Cultures were stored at 37°C, constant humidity, 95% air and 5% CO ² . After one day, sections were treated with rat anti-mouse/human LIF blocking antibody (h5D8) (designated anti-LIF) (laboratory-produced) or its corresponding normal IgG (10 μg/ml) for 3 days. For CXCL9 blocking studies, a neutralizing mouse monoclonal antibody to human CXCL9 (R&D Systems) was added to the culture at a concentration of 1.5 μg/ml. In some cases, 0.1 ng/ml human rIFNy (R&D Systems) was added for 24 hours. Paral

- 43 044914 In line with this, peripheral blood mononuclear cells (PBMCs) were obtained from whole blood of the same patient by density gradient centrifuge separation using Lymphosep (Biowest). PBMCs were cryopreserved in RPMI supplemented with 10% inactivated FBS and 10% DMSO before use. For immune cell infiltration assays, control or LIF inhibition sections were immersed in Matrigel (Corning) followed by the addition of 1 x 10 ⁶ PBMCs to a 24-well plate in complete RPMI medium. In addition, supernatants were collected, organotypic sections were extracted from Matrigel, and then processed for immunofluorescence (IF) analysis and flow cytometry. Under some conditions, PBMCs were resuspended in PBS at a concentration of 10 cells/ml and incubated for 20 min with 5 μM Cell Trace CFSE (Invitrogen). After incubation, cells were washed using RPMI and added to sections embedded in Matrigel. After 24 h, invasion of fluorescent PBMCs into Matrigel was assessed microscopically by counting migrating cells in five different areas for each condition.

Microslides were deparaffinized and hydrated. Antigen retrieval was performed using Citrate Antigen Retrieval Solution ( _DAKO ) pH 6 or 9 for 10 min, 10% peroxidase ( _H2O2 ) and blocking solution (2% BSA) for 1 h at room temperature. The detection system used was En Vision FLEX+ (DAKO) according to the manufacturer's instructions, followed by hematoxylin counterstaining, dehydration, and mounting media (DPX). Quantification of LIF, CCL2, CD163, CD206, and CXCL9 staining in GBM tumors from patients was expressed as an H-score (3 x percentage of strong staining + 2 x percentage of moderate staining + percentage of weak staining) ranging from 0 to 300. Quantification of p -STAT3, Ki67, CC3, and CD8 were performed using ImageJ, counting the total number of cells in three different fields per mouse, five mice/group, and calculating the percentage of positive cells. Data in the graphs are presented as mean ± SEM.

Antibodies for immunohistochemical studies: human LIF (Atlas; 1:200), mouse LIF (AbCam; 1:200), mouse p-STAT3 (Cell Signaling; 1:50), mouse Ki67 (AbCam; 1:200), mouse cleaved caspase 3 (CC3) (Cell Signaling; 1:500), mouse CD8 (Bioss; 1:200), human/mouse CCL2 (Novus Biologicals, 1:200), human CXCL9 (Thermo Fischer Scientific; 1:100) and human CD163 (Leica Novacastra; 1:200).

Example 25 LIF induces type II (M2) macrophage polarization.

Dual stratification by LIF immunosuppressive signature is a potentially reliable way to identify individuals particularly likely to respond to anti-LIF antibody therapy. Additional evidence supporting this is shown in FIG. 21A and 21B, 22 and 23. In FIG. 21A and 21B show that primary human macrophages have increased LIF receptor expression after culturing monocytes for 7 days with 50 ng/ml M-CSF. Shown are cells obtained from three different individuals. In fig. 22 and 23 further show that primary human macrophages have increased expression of the macrophage surface markers CD206 and CD 163 (FIG. 22) and secretion of CCL22 (FIG. 23) after 72 hours of culture with LIF. Notably, CCL22 secretion is much more stable in polarized M2 macrophages (Fig. 23, bottom right). These data provide a mechanistic link between LIF expression and the presence of an immunosuppressive signature, indicating that a dual LIF immunosuppressive signature to guide patient treatment can be applied across all cancer types and tissues.

Way.

Macrophages were differentiated from human peripheral CD14+ monocytes by culturing in RPMI-1640 medium with 10% heat-inactivated FBS, penicillin/streptomycin, and 50 ng/ml M-CSF for 7 days. In some experiments, this was followed by cultivation in the same medium supplemented with 20 nM LIF or PBS (for control), or 100 ng/ml LPS and 25 ng/ml IFNy (for M1 polarization) or 20 ng/ml each of IL- 4, IL-10, and TGFP (for M2 polarization) for additional time periods indicated.

Example 26. Expression of the LIF receptor on myeloid cells in various types of cancer.

Further support for the prospect of a dual immunosuppressive signature of LIF being important for tumor growth and cancer survival is that human tumor-associated macrophages do express the LIF receptor. In fig. 24 and 25 show that macrophages isolated from 3 of 4 separated ovarian and lung cancer tumor cells express the LIF receptor (FIG. 24). In addition, the LIF receptor is expressed on the cell surface of tumor-associated myeloid-derived suppressor cells, both monocytic myeloid-derived suppressor cells and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) (Fig. 25).

Although preferred embodiments of the present invention have been shown and described herein, those skilled in the art will appreciate that such embodiments are provided by way of example only. Numerous variations, changes and substitutions will be apparent to those skilled in the art without departing from the present invention.

- 44 044914

It should be understood that various alternatives to the embodiments of the present invention described herein may be used in the practice of the present invention.

All publications, patent applications, issued patents and other documents referred to herein are incorporated herein by reference to the same extent as if each individual publication, patent application, issued patent or other document had been specifically and separately identified as incorporated herein by reference in its entirety. Definitions that are contained in the text incorporated by reference are excluded to the extent that they conflict with the definitions in the present invention.

Unless otherwise defined, all technical terminology used herein has the same meaning as would be commonly understood by one skilled in the art to which the present invention relates. As used in the present description and the accompanying claims, the singular form includes the defined entities in the plural, unless the context clearly indicates otherwise. Any reference to or herein is intended to cover and/or unless otherwise noted.

As used herein, unless otherwise indicated, the term approximately refers to an amount that is within at least 10% of what is claimed.

Sequences

SEQ ID NO Subsequence 1 GFTFSHAWMH 2 GFTFSHAW 3 HAWMH 4 QIKAKSDDYATYYAESVKG 5 IKAKSDDYAT 6 TCWEWDLDF 7 WEWDLDF 8 TSWEWDLDF 9 RSSQSLLDSDGHTYLN 10 QSLLDSDGHTY AND SVSNLES 12 SVS 13 MQATHAPPYT 14 EVQLVESGGGLVKPGGSLKLSCAASGFTFSHAWMHWVRQAPGKGLEWVAQ IKAKSDDYATYYAESVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTCW EWDLDF WGQGTL VT VS S

- 45 044914

15 QVQLQESGGGLVKPGGSLRLSCAASGFTFSHAWMHWVRQAPGKGLEWVG QIKAKSDDYATYYAESVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTC WEWDLDF WGQGTM VT VS S 16 EVQLVESGGGVVQPGRSLRLSCAASGFTFSHAWMHWVRQAPGKGLEWVAQ IKAKSDDYATYYAESVKGRFSISRDNAKNSLYLQMNSLRVEDTVVYYCTCW EWDLDF WGQGTT VT VS S 17 EVQLMESGGGLVKPGGSLRLSCATSGFTFSHAWMHWVRQAPGKGLEWVGQ IKAKSDDYATYYAESVKGRFTISRDDSKSTLFLQMNNLKTEDTAVYYCTCW EWDLDF WGQGTLVT VS S 18 DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGHTYLNWFQQRPGQSPRRLI YSVSNLESGVPDRFSSGSG TDFTLKISRVEAEDVGLYYCMQATHAPPYTFGQGTKLEIK 19 DIVMTQTPLSSPVTLGQPASISCRSSQSLLDSDGHTYLNWLQQRPGQPPRLLIY SVSNLESGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQATHAPPYTFGQ GTKLEIK 20 DIVMTQTPLSLSVTPGQPASISCRSSQSLLDSDGHTYLNWLLQKPGQPPQLLIY SVSNLESGVPNRFSGSGSGTDFTLKISRVEAEDVGLYYCMQATHAPPYTFGG GTKVEIK 21 DVVMTQSPLSQPVTLGQPASISCRSSQSLLDSDGHTYLNWLQQRPGQSPRRLI YSVSNLESGVPDRFNGSGSGTDFTLSISRVEAEDVGVYYCMQATHAPPYTFG QGTKVEIK 22 MGWTLVFLFLLSVTAGVHSEVQLVESGGGLVKPGGSLKLSCAASGTFFSHA WMHWVRQAPGKGLEWVAQIKAKSDDYATYYAESVKGRFTISRDDSKNTLY LQMNSLKTEDTAVYYCTCWEWDLDFWGQGTLVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 23 MGWTLVFLFLLSVTAGVHSQVQLQESGGGLVKPGGSLRLSCAASGTFFSHA WMHWVRQAPGKGLEWVGQIKAKSDDYATYYAESVKGRFTISRDDSKNTLY

- 46 044914

LQMNSLKTEDTAVYYCTCWEWDLDFWGQGTMVTVSSASTKGPSVFPLAPS SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 24 MGWTLVFLFLLSVTAGVHSEVQLVESGGGVVQPGRSLRLSCAASGTFFSHA WMHWVRQAPGKGLEWVAQIKAKSDDYATYYAESVKGRFSISRDNAKNSLY LQMNSLRVEDTVVYYCTCWEWDLDFWGQGTTVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 25 MGWTLVFLFLLSVTAGVHSEVQLMESGGGLVKPGGSLRLSCATSGFTFSHA WMHWVRQAPGKGLEWVGQIKAKSDDYATYYAESVKGRFTISRDDSKSTLF LQMNNLKTEDTAVYYCTCWEWDLDFWGQGTLVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 26 MVSSAQFLGLLLLCFQGTRCDVVMTQSPLSLPVTLGQPASISCRSSQSLLDSD GHTYLNWFQQRPGQSPRRLIYSVSNLESGVPDRFSGSGSGTDFTLKISRVEAE DVGLYYCMQATHAPPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLS SP VTKSFNRGEC

- 47 044914

27 MVSSAQFLGLLLLCFQGTRCDIVMTQTPLSSPVTLGQPASISCRSSQSLLDSDG HTYLNWLQQRPGQPPRLLIYSVSNLESGVPDRFSGSGAGTDFTLKISRVEAED VGVYYCMQATHAPPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVV CLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLS SP VTKSFNRGEC 28 MVSSAQFLGLLLLCFQGTRCDIVMTQTPLSLSVTPGQPASISCRSSQSLLDSDG HTYLNWLLQKPGQPPQLLIYSVSNLESGVPNRFSGSGSGTDFTLKISRVEAED VGLYYCMQATHAPPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVV CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLS SP VTKSFNRGEC 29 MVSSAQFLGLLLLCFQGTRCDVVMTQSPLSQPVTLGQPASISCRSSQSLLDSD GHTYLNWLQQRPGQ SPRRLIYS VSNLESGVPDRF SSGSGSGTDFTLSISRVEAE DVGVYYCMQATHAPPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSK AD YEKHKVYACEVTHQGLS SP VTKSFNRGEC thirty EVQLVESGGGLVKPGGSLKLSCAASGFTFSHAWMHWVRQAPGKGLEWVAQ IKAKSDDYATYYAESVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTCW EWDLDFWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 31 QVQLQESGGGLVKPGGSLRLSCAASGFTFSHAWMHWVRQAPGKGLEWVG QIKAKSDDYATYYAESVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTC WEWLDFWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEP VT VS WNSGALTS GVHTFP AVLQ S SGL YSLS SWT VP S S SLGTQT YICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL T VDKSRWQQGNVF SC S VMHEALHNHYTQKSLSLSPGK

- 48 044914

32 EVQLVESGGGVVQPGRSLRLSCAASGFTFSHAWMHWVRQAPGKGLEWVAQ IKAKSDDYATYYAESVKGRFSISRDNAKNSLYLQMNSLRVEDTVVYYCTCW EWDLDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 33 EVQLMESGGGLVKPGGSLRLSCATSGFTFSHAWMHWVRQAPGKGLEWVGQ IKAKSDDYATYYAESVKGRFTISRDDSKSTLFLQMNNLKTEDTAVYYCTCW EWDLDFWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 34 DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGHTYLNWFQQRPGQSPRRLI YSVSNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGLYYCMQATHAPPYTFG QGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSS PVTKSFNRGEC 35 DIVMTQTPLSSPVTLGQPASISCRSSQSLLDSDGHTYLNWLQQRPGQPPRLLIY SVSNLESGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQATHAPPYTFGQ GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSP VTKSFNRGEC 36 DIVMTQTPLSLSVTPGQPASISCRSSQSLLDSDGHTYLNWLLQKPGQPPQLLIY SVSNLESGVPNRFSGSGSGTDFTLKISRVEAEDVGLYYCMQATHAPPYTFGG GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSP VTKSFNRGEC

-

Claims (3)

37 DVVMTQSPLSQPVTLGQPASISCRSSQSLLDSDGHTYLNWLQQRPGQSPRRLI YSVSNLESGVPDRFSGSGSGTDFTLSISRVEAEDVGVYYCMQATHAPPYTFG QGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSS PVTKSFNRGEC 38 QVQLQESGGGLVKPGGSLRLSCAASGFTFSHAWMHWVRQAPGKGLEWVG QIKAKSDDYATYYAESVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTS WEWDLDF WGQGTM VT VS S 39 QVQLQESGGGLVKPGGSLRLSCAASGFTFSHAWMHWVRQAPGKGLEWVG QIKAKSDDYATYYAESVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTS WEWLDFWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVT VSWNSGALTSGVHTFP AVLQS SGLYSLS S VVT VPS S SLGTQT YICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM TKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVF SC S VMHEALHNHYTQKSLSLSPGK 40 SPLPITPVNATCAIRHPCHNNLMNQIRSQLAQLNGSANALFILYYTAQGEPFP NNLDKLCGPNVTDFPPFHANGTEKAKLVELYRIVVYLGTSLGNITRDQKILNP SALSLHSKLNATADILRGLLSNVLCRLCSKYHVGHVDVTYGPDTSGKDVFQ KKKLGCQLLGKYKQIIAVLAQAF 41 EQLEESVGDLVKPGASLTLTCTASGFSFSGLYYMCWVRQAPGKGLEWIACI WTGSTDSTYYATWAKGRFTISKTSSTTVTLQMTSLTVADTATYFCARGGGV PGDGYALWGPGTL VT VS S 42 GTELVMTQTPASVSEPVGGTVTINCQASEDISSNLVWYQQKSGQPPKLLIYD ASMLASGVPSRFKGSGSGTQFTLTISDLECADGATYYCQSYYVASSSYFVNG FGGGTEVV CLAIM 1. A method of treating an individual suffering from cancer with a therapeutic antibody to leukemia inhibitory factor (LIF), comprising determining that a level of LIF is greater than a reference level in a biological sample obtained from the individual, further comprising determining that a protein level of an immunomodulatory molecule is greater than a reference level of an immunomodulatory molecule, wherein the immunomodulatory molecule is selected from CCL7, CCL2, CCL3 and CCL22, and administering a therapeutic amount of an anti-LIF antibody to the individual if the LIF level is above the LIF reference level and the protein level of the immunomodulatory molecule is above the immunomodulatory molecule reference level. 2. The method according to claim 1, where the therapeutic antibody to LIF contains: a) immunoglobulin heavy chain complementarity determining region 1 (VH-CDR1) containing the amino acid sequence represented by SEQ ID NO: 2 (GFTFSHAW); b) immunoglobulin heavy chain complementarity determining region 2 (VH-CDR2) containing the amino acid sequence represented by SEQ ID NO: 5 (IKAKSDDYAT); c) immunoglobulin heavy chain complementarity determining region 3 (VH-CDR3) containing the amino acid sequence represented by SEQ ID NO: 6 (TCWEWDLDF); d) immunoglobulin light chain complementarity determining region 1 (VL-CDR1) containing the amino acid sequence represented by SEQ ID NO: 10 (QSLLDSDGHTY); e) immunoglobulin light chain complementarity determining region 2 (VL-CDR2) containing the amino acid sequence represented by SEQ ID NO: 12 (SVS); And f) immunoglobulin light chain complementarity determining region 3 (VL-CDR3) containing the amino acid sequence represented by SEQ ID NO: 13 (MQATHAPPYT). 3. The method of claim 2, wherein the anti-LIF therapeutic antibody comprises an immunoglobulin heavy chain variable region having at least 85, 90, 95, 97, 98, 99 or 100% identity -