EA044934B1 - ANTIBODIES TO LIF AND DOSAGE FORMS BASED ON THEM - Google Patents

Description

Cross reference

This application claims priority and benefit from European Application No. 18382327.7, filed May 14, 2018; European Application No. 18382359.0, filed May 25, 2018; European Application No. 19382208.7, filed March 26, 2019; European Application No. 19382331.7, filed May 3, 2019, each of which is incorporated in its entirety.

BACKGROUND OF THE INVENTION

Leukemia inhibitory factor (LIF) is an interleukin-6 (IL-6)-type cytokine that is involved in various biological activities, including inhibition of cell differentiation. Human LIF is a 202 amino acid polypeptide that exerts biological effects through binding to the cell surface LIF receptor (LIFR or CD118), which heterodimerizes with gp130. This leads to the activation of growth-promoting signaling pathways such as the mitogen-activated protein kinase (MAPK) pathway and the Janus-activated kinase (JAK/STAT) pathway. High LIF expression levels and high serum LIF levels have been demonstrated to be associated with poor prognosis in many types of cancer.

Brief Description of the Present Invention

Disclosed herein are anti-LIF antibodies that antagonize or block LIF activity. The anti-LIF antibodies described herein are useful for the treatment of cancer. In particular, certain anti-LIF antibodies, when administered at a therapeutically effective dose, had excellent and unexpected efficacy in reducing tumor volumes in both mouse and non-human primate cancer models. Thus, the present invention provides methods and pharmaceutical compositions for the treatment of cancer using specific antibodies to LIF in certain dosages (both using weight-based doses and using fixed doses).

In another aspect, described herein is a method of treating an individual suffering from cancer, comprising administering to the individual a recombinant antibody that specifically binds leukemia inhibitory factor (LIF) comprising: (a) immunoglobulin heavy chain complementarity determining region 1 (VH-CDR1), containing the amino acid sequence presented under any of SEQ ID NO: 1-3; (b) immunoglobulin heavy chain complementarity determining region 2 (VH-CDR2) comprising the amino acid sequence represented by any of SEQ ID NO: 4 or 5; (c) immunoglobulin heavy chain complementarity determining region 3 (VH-CDR3) comprising the amino acid sequence represented by any one of SEQ ID NOs: 6-8; (d) immunoglobulin light chain complementarity determining region 1 (VLCDR1) comprising the amino acid sequence represented by any of SEQ ID NO: 9 or 10; (e) 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 (5) immunoglobulin light chain complementarity determining region 3 (VL-CDR3) containing the amino acid sequence represented by SEQ ID NO: 13; wherein the recombinant antibody is administered to the individual at a dose of from about 75 to about 2000 milligrams. In certain embodiments, the recombinant antibody binds to glycosylated LIF. In certain embodiments, the recombinant antibody comprises at least one framework region derived from a human antibody framework region. In certain embodiments, the recombinant antibody is humanized. In certain embodiments, the recombinant antibody is deimmunized. In certain embodiments, the recombinant antibody comprises two immunoglobulin heavy chains and two immunoglobulin light chains. In certain embodiments, the recombinant antibody is an IgG antibody. In certain embodiments, the recombinant antibody is a Fab, F(ab) ₂ , single domain antibody, single chain variable fragment (scFv), or nanoantibody. In certain embodiments, the recombinant antibody specifically binds to LIF with a dissociation constant (K _D ) of less than about 200 pmol/L. In certain embodiments, the recombinant antibody specifically binds to LIF with a dissociation constant (K _D ) of less than about 100 picomol/L. In certain embodiments, VH-CDR1 contains the amino acid sequence set forth under SEQ ID NO: 1 (GFTFSHAWMH), VH-CDR2 contains the amino acid sequence set forth under SEQ ID NO: 4 (QIKAKSDDYATYYAESVKG), VH-CDR3 contains the amino acid sequence set forth under SEQ ID NO: 6 (TCWEWDLDF), VL-CDR1 contains the amino acid sequence shown under SEQ ID NO: 9 (RSSQSLLDSDGHTYLN), VL-CDR2 contains the amino acid sequence shown under SEQ ID NO: 11 (SVSNLES) and VLCDR3 contains the amino acid sequence shown under under SEQ ID NO: 13 (MQATHAPPYT). In certain embodiments, VH-CDR1 contains the amino acid sequence set forth under SEQ ID NO: 2 (GFTFSHAW), VH-CDR2 contains the amino acid sequence set forth under SEQ ID NO: 5 (IKAKSDDYAT), VH-CDR3 contains the amino acid sequence set forth under SEQ ID NO: 6 (TCWEWDLDF), VL-CDR1 contains the amino acid sequence presented under SEQ ID NO: 10 (QSLLDSDGHTYLN), VL-CDR2 contains the amino acid sequence presented under SEQ ID NO: 12 (SVS) and VL- CDR3 contains the amino acid sequence shown under SEQ ID NO: 13 (MQATHAPPYT). In certain embodiments, VH-CDR1 contains the amino acid sequence set forth under SEQ ID NO: 3 (HAWMH), VH-CDR2 contains the amino acid sequence set forth under SEQ ID NO: 4 (QIKAKSDDYATYYAESVKG), VH-CDR3 contains the amino acid sequence set forth under SEQ ID NO: 7 (WEWDLDF), VL-CDR1 contains the amino acid sequence presented under SEQ ID NO: 9 (RSSQSLLDSDGHTYLN), VLCDR2 contains the amino acid sequence presented under SEQ ID NO: 11 (SVSNLES) and VL-CDR3 contains the amino acid sequence presented under SEQ ID NO: 13 (MQATHAPPYT). In certain embodiments, the recombinant antibody contains one or more heavy chain framework region 1 (VH-FR1) amino acid sequences that are at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence presented under any of SEQ ID NO: 14-17, an amino acid sequence of heavy chain framework region 2 (VH-FR2) that is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence presented under any of SEQ ID NO: 18 or 19, an amino acid sequence of heavy chain framework region 3 (VH-FR3) that is at least about 80%, 90%, 95%, 97%, 98% or 99% is identical to the amino acid sequence set forth in any of SEQ ID NOs: 20-22, and to the amino acid sequence of heavy chain framework region 4 (VH-FR4) that is at least about 80%, 90%, 95%, 97%, 98% identical or 99% identical to the amino acid sequence presented under any of SEQ ID NO: 23-25. In certain embodiments, the recombinant antibody comprises one or more heavy chain framework region 1 (VH-FR1) amino acid sequences that are identical to the amino acid sequence set forth in any of SEQ ID NOs: 14-17, heavy chain framework region 2 (VH-FR1) amino acid sequence FR2), which is identical to the amino acid sequence presented under any of SEQ ID NO: 18 or 19, the amino acid sequence of heavy chain framework region 3 (VHFR3), which is identical to the amino acid sequence presented under any of SEQ ID NO: 20-22, and the amino acid a heavy chain framework region 4 (VH-FR4) sequence which is identical to the amino acid sequence set forth in any one of SEQ ID NOs: 23-25. In certain embodiments, the recombinant antibody comprises one or more light chain framework region 1 (VL-FR1) amino acid sequences that are at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence presented under any of SEQ ID NO: 26-29, an amino acid sequence of light chain framework region 2 (VL-FR2) that is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence presented under any of SEQ ID NO: 3033, an amino acid sequence of light chain framework region 3 (VL-FR3) that is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid the sequence set forth under any of SEQ ID NOs: 34-37, and an amino acid sequence of light chain framework region 4 (VL-FR4) that is at least about 80%, 90%, 95%, 97%, 98%, or 99 % identical to the amino acid sequence presented under any of SEQ ID NO: 38-40. In certain embodiments, the recombinant antibody comprises one or more light chain framework region 1 (VL-FR1) amino acid sequences that are identical to the amino acid sequence set forth in any of SEQ ID NOs: 26-29, light chain framework region 2 (VL-) amino acid sequence FR2), which is identical to the amino acid sequence presented under any of SEQ ID NO: 30-33, the amino acid sequence of light chain framework region 3 (VL-FR3), which is identical to the amino acid sequence presented under any of SEQ ID NO: 34-37, and an amino acid sequence of light chain framework region 4 (VL-FR4) which is identical to the amino acid sequence set forth in any one of SEQ ID NOs: 38-40. In certain embodiments, the recombinant antibody binds to at least one 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 of SEQ ID NO: 68. In certain embodiments, the recombinant antibody binds to at least five 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: 68. In certain embodiments, the recombinant antibody binds to at least ten of the following residues: A13,114, R15, H16, P17, C18, H19, N20, Q25, Q29, Q32, D120, R123, S127, N128, L130, C131, C134, S135, H138 of SEQ ID NO: 68. In certain embodiments, the recombinant antibody binds to 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: 68. In certain embodiments, cancer includes advanced solid tumor, glioblastoma, stomach cancer, skin cancer, prostate cancer, pancreatic cancer, breast cancer, testicular cancer, thyroid cancer, cancer

- 2 044934 head and neck, liver cancer, kidney cancer, esophageal cancer, ovarian cancer, colon cancer, 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 recombinant antibody is administered as a component of a pharmaceutical formulation, wherein the pharmaceutical formulation comprises the recombinant antibody and further comprises a pharmaceutically acceptable excipient, carrier, or diluent. In certain embodiments, the pharmaceutical composition has a pH value of approximately 6.0. In certain embodiments, the pharmaceutical composition contains about 25 mM histidine, about 6% sucrose, and about 0.01% polysorbate 80, with the recombinant antibody included at a concentration of about 20 mg/ml. In certain embodiments, the recombinant antibody is administered intravenously. In certain embodiments, the recombinant antibody is administered once a week. In certain embodiments, the recombinant antibody is administered approximately once every two weeks. In certain embodiments, the recombinant antibody is administered approximately once every three weeks. In certain embodiments, the recombinant antibody is administered approximately once every four weeks. In certain embodiments, the recombinant antibody is administered at a dose of approximately 75 mg. In certain embodiments, the recombinant antibody is administered at a dose of approximately 225 mg. In certain embodiments, the recombinant antibody is administered at a dose of approximately 750 mg. In certain embodiments, the recombinant antibody is administered at a dose of approximately 1125 mg. In certain embodiments, the recombinant antibody is administered at a dose of approximately 1500 mg. In certain embodiments, the recombinant antibody is administered at a dose of approximately 2000 mg.

In another aspect, described herein is a method of treating an individual suffering from cancer, comprising administering to the individual a recombinant antibody that specifically binds leukemia inhibitory factor (LIF) comprising: (a) an immunoglobulin heavy chain variable region (VH) amino acid sequence, which is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence set forth in any of SEQ ID NO: 41, 42, 44 or 66; and (b) an immunoglobulin light chain variable region (VL) sequence with an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth under any of SEQ IDs NO: 45-48; wherein the recombinant antibody is administered to the individual at a dose of from about 75 to about 2000 mg. In certain embodiments, the VH sequence is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 42; and the VL sequence is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 46. In certain embodiments, the VH sequence is identical to the amino acid sequence set forth in SEQ ID NO: 42; and the VL sequence is identical to the amino acid sequence set forth in SEQ ID NO: 46. In certain embodiments, the recombinant antibody comprises two immunoglobulin heavy chains and two immunoglobulin light chains. In certain embodiments, the recombinant antibody is an IgG antibody. In certain embodiments, the cancer includes advanced solid tumor, glioblastoma, stomach cancer, skin cancer, prostate cancer, pancreatic cancer, breast cancer, testicular cancer, thyroid cancer, head and neck cancer, liver cancer, kidney cancer, esophageal cancer , ovarian cancer, colon cancer, 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 recombinant antibody is administered as a component of a pharmaceutical formulation, wherein the pharmaceutical formulation comprises the recombinant antibody and further comprises a pharmaceutically acceptable excipient, carrier, or diluent. In certain embodiments, the pharmaceutical composition has a pH value of approximately 6.0. In certain embodiments, the pharmaceutical composition contains about 25 mM histidine, about 6% sucrose, and about 0.01% polysorbate 80, with the recombinant antibody included at a concentration of about 20 mg/ml. In certain embodiments, the recombinant antibody is administered intravenously. In certain embodiments, the recombinant antibody is administered approximately once per week. In certain embodiments, the recombinant antibody is administered approximately once every two weeks. In certain embodiments, the recombinant antibody is administered approximately once every three weeks. In certain embodiments, the recombinant antibody is administered approximately once every four weeks. In certain embodiments, the recombinant antibody is administered at a dose of approximately 75 mg. In certain embodiments, the recombinant antibody is administered at a dose of approximately 225 mg. In certain embodiments, the recombinant antibody is administered at a dose of approximately 750 mg. In certain embodiments, the recombinant antibody is administered at a dose of approximately 1125 mg. In certain embodiments, the recombinant antibody is administered at a dose of approximately 1500 mg. In certain embodiments, the recombinant antibody is administered at a dose of approximately 2000 mg.

In another aspect, described herein is a method of treating an individual suffering from cancer, comprising administering to the individual a recombinant antibody that specifically binds leukemia inhibitory factor (LIF), comprising: (a) an immunoglobulin heavy chain sequence with an amino acid sequence that is at least approximately 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence presented under any of SEQ ID NO: 57-60 or 67; and (b) an immunoglobulin light chain sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 61-64 ; wherein the recombinant antibody is administered to the individual at a dose of from about 75 to about 2000 mg. In certain embodiments, the immunoglobulin heavy chain sequence is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 58; and the immunoglobulin light chain sequence is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 62. In certain embodiments, the immunoglobulin heavy chain sequence is identical to the amino acid sequence , presented under SEQ ID NO: 58; and the immunoglobulin light chain sequence is identical to the amino acid sequence set forth in SEQ ID NO: 62. In certain embodiments, the recombinant antibody comprises two immunoglobulin heavy chains and two immunoglobulin light chains. In certain embodiments, the recombinant antibody is an IgG antibody. In certain embodiments, the cancer includes advanced solid tumor, glioblastoma, stomach cancer, skin cancer, prostate cancer, pancreatic cancer, breast cancer, testicular cancer, thyroid cancer, head and neck cancer, liver cancer, kidney cancer, esophageal cancer , ovarian cancer, colon cancer, 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 recombinant antibody is administered as a component of a pharmaceutical formulation, wherein the pharmaceutical formulation comprises the recombinant antibody and further comprises a pharmaceutically acceptable excipient, carrier, or diluent. In certain embodiments, the pharmaceutical composition has a pH value of approximately 6.0. In certain embodiments, the pharmaceutical composition contains about 25 mM histidine, about 6% sucrose, and about 0.01% polysorbate 80, with the recombinant antibody included at a concentration of about 20 mg/ml. In certain embodiments, the recombinant antibody is administered intravenously. In certain embodiments, the recombinant antibody is administered approximately once per week. In certain embodiments, the recombinant antibody is administered approximately once every two weeks. In certain embodiments, the recombinant antibody is administered approximately once every three weeks. In certain embodiments, the recombinant antibody is administered approximately once every four weeks. In certain embodiments, the recombinant antibody is administered at a dose of approximately 75 mg. In certain embodiments, the recombinant antibody is administered at a dose of approximately 225 mg. In certain embodiments, the recombinant antibody is administered at a dose of approximately 750 mg. In certain embodiments, the recombinant antibody is administered at a dose of approximately 1125 mg. In certain embodiments, the recombinant antibody is administered at a dose of approximately 1500 mg. In certain embodiments, the recombinant antibody is administered at a dose of approximately 2000 mg.

In another aspect, this document describes a pharmaceutical composition for use in the treatment of cancer in an individual, wherein the pharmaceutical composition contains a pharmaceutically acceptable excipient, carrier or diluent and a recombinant antibody, wherein the recombinant antibody specifically binds to leukemia inhibitory factor (LIF) and contains: (a) immunoglobulin heavy chain complementarity determining region 1 (VH-CDR1) comprising the amino acid sequence represented by any of SEQ ID NOs: 1-3; (b) immunoglobulin heavy chain complementarity determining region 2 (VH-CDR2) comprising the amino acid sequence represented by any of SEQ ID NO: 4 or 5; (c) immunoglobulin heavy chain complementarity determining region 3 (VH-CDR3) comprising the amino acid sequence represented by any of SEQ ID NOs: 6-8; (d) immunoglobulin light chain complementarity determining region 1 (VL-CDR1) comprising the amino acid sequence represented by any of SEQ ID NO: 9 or 10; (e) 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 (f) immunoglobulin light chain complementarity determining region 3 (VL-CDR3) containing the amino acid sequence represented by

- 4 044934

SEQ ID NO: 13; wherein the recombinant antibody is administered to the individual at a dose of from about 75 to about 2000 mg. In certain embodiments, the pharmaceutical composition contains about 25 mM histidine, about 6% sucrose, and about 0.01% polysorbate 80, with the recombinant antibody included at a concentration of about 20 mg/ml. In certain embodiments, the pharmaceutical composition has a pH value of approximately 6.0. In certain embodiments, the recombinant antibody binds to glycosylated LIF. In certain embodiments, the recombinant antibody comprises at least one framework region derived from a human antibody framework region. In certain embodiments, the recombinant antibody is humanized. In certain embodiments, the recombinant antibody is deimmunized. In certain embodiments, the recombinant antibody comprises two immunoglobulin heavy chains and two immunoglobulin light chains. In certain embodiments, the recombinant antibody is an IgG antibody. In certain embodiments, the recombinant antibody is a Fab, F(ab) ₂ , single domain antibody, single chain variable fragment (scFv), or nanoantibody. In certain embodiments, the recombinant antibody specifically binds to LIF with a dissociation constant (K _D ) of less than about 200 pmol/L. In certain embodiments, the recombinant antibody specifically binds to LIF with a dissociation constant (K _D ) of less than about 100 picomol/L. In certain embodiments, VH-CDR1 contains the amino acid sequence set forth under SEQ ID NO: 1 (GFTFSHAWMH), VH-CDR2 contains the amino acid sequence set forth under SEQ ID NO: 4 (QIKAKSDDYATYYAESVKG), VH-CDR3 contains the amino acid sequence set forth under SEQ ID NO: 6 (TCWEWDLDF), VL-CDR1 contains the amino acid sequence shown under SEQ ID NO: 9 (RSSQSLLDSDGHTYLN), VL-CDR2 contains the amino acid sequence shown under SEQ ID NO: 11 (SVSNLES) and VL-CDR3 contains the amino acid sequence , presented under SEQ ID NO: 13 (MQATHAPPYT). In certain embodiments, VH-CDR1 contains the amino acid sequence presented under SEQ ID NO: 2 (GFTFSHAW), VHCDR2 contains the amino acid sequence presented under SEQ ID NO: 5 (IKAKSDDYAT), VH-CDR3 contains the amino acid sequence presented under SEQ ID NO : 6 (TCWEWDLDF), VL-CDR1 contains the amino acid sequence shown under SEQ ID NO: 10 (QSLLDSDGHTYLN), VL-CDR2 contains the amino acid sequence shown under SEQ ID NO: 12 (SVS) and VL-CDR3 contains the amino acid sequence shown under under SEQ ID NO: 13 (MQATHAPPYT). In certain embodiments, VH-CDR1 contains the amino acid sequence set forth under SEQ ID NO: 3 (HAWMH), VH-CDR2 contains the amino acid sequence set forth under SEQ ID NO: 4 (QIKAKSDDYATYYAESVKG), VH-CDR3 contains the amino acid sequence set forth under SEQ ID NO: 7 (WEWDLDF), VL-CDR1 contains the amino acid sequence shown under SEQ ID NO: 9 (RSSQSLLDSDGHTYLN), VL-CDR2 contains the amino acid sequence shown under SEQ ID NO: 11 (SVSNLES) and VL-CDR3 contains the amino acid sequence , presented under SEQ ID NO: 13 (MQATHAPPYT). In certain embodiments, the recombinant antibody comprises one or more heavy chain framework region 1 (VH-FR1) amino acid sequences that are at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence presented under any of SEQ ID NO: 14-17, an amino acid sequence of heavy chain framework region 2 (VH-FR2) that is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence presented under any of SEQ ID NO: 18 or 19, an amino acid sequence of heavy chain framework region 3 (VH-FR3) that is at least about 80%, 90%, 95%, 97%, 98% or 99% is identical to the amino acid sequence set forth in any of SEQ ID NOs: 20-22, and to the amino acid sequence of heavy chain framework region 4 (VH-FR4) that is at least about 80%, 90%, 95%, 97%, 98% identical or 99% identical to the amino acid sequence presented under any of SEQ ID NO: 23-25. In certain embodiments, the recombinant antibody comprises one or more heavy chain framework region 1 (VH-FR1) amino acid sequences that are identical to the amino acid sequence set forth in any of SEQ ID NOs: 14-17, heavy chain framework region 2 (VH-FR1) amino acid sequence FR2), which is identical to the amino acid sequence presented under any of SEQ ID NO: 18 or 19, the amino acid sequence of heavy chain framework region 3 (VH-FR3), which is identical to the amino acid sequence presented under any of SEQ ID NO: 20-22, and an amino acid sequence of heavy chain framework region 4 (VH-FR4) which is identical to the amino acid sequence set forth in any one of SEQ ID NOs: 23-25. In certain embodiments, the recombinant antibody comprises one or more light chain framework region 1 (VL-FR1) amino acid sequences that are at least about 80%, 90%, 95%, 97%, 98%, or 99% identical

- 5 044934 is identical to the amino acid sequence set forth in any of SEQ ID NOs: 26-29, the amino acid sequence of light chain framework region 2 (VL-FR2), which is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 30-33, the amino acid sequence of light chain framework region 3 (VL-FR3) that is at least about 80%, 90%, 95%, 97 %, 98% or 99% identical to the amino acid sequence set forth under any of SEQ ID NO: 3437, and the amino acid sequence of light chain framework region 4 (VL-FR4) that is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence presented under any of SEQ ID NO: 38-40. In certain embodiments, the recombinant antibody comprises one or more light chain framework region 1 (VL-FR1) amino acid sequences that are identical to the amino acid sequence set forth in any of SEQ ID NOs: 26-29, light chain framework region 2 (VL-) amino acid sequence FR2), which is identical to the amino acid sequence presented under any of SEQ ID NO: 30-33, the amino acid sequence of light chain framework region 3 (VLFR3), which is identical to the amino acid sequence presented under any of SEQ ID NO: 34-37, and the amino acid a light chain framework region 4 (VL-FR4) sequence which is identical to the amino acid sequence set forth in any one of SEQ ID NOs: 38-40. In certain embodiments, the recombinant antibody binds to at least one 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 of SEQ ID NO: 68. In certain embodiments, the recombinant antibody binds to at least five 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: 68. In certain embodiments, the recombinant antibody binds to at least ten 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 of SEQ ID NO: 68. In certain embodiments, the recombinant antibody binds to 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: 68. In certain embodiments, cancer includes advanced solid tumor, glioblastoma, stomach cancer, skin cancer, prostate cancer, pancreatic cancer, breast cancer, testicular cancer, thyroid cancer, head and neck cancer, liver cancer, kidney cancer, esophageal cancer, ovarian cancer, cancer 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 recombinant antibody is administered intravenously. In certain embodiments, the recombinant antibody is administered approximately once per week. In certain embodiments, the recombinant antibody is administered approximately once every two weeks. In certain embodiments, the recombinant antibody is administered approximately once every three weeks. In certain embodiments, the recombinant antibody is administered approximately once every four weeks. In certain embodiments, the recombinant antibody is administered at a dose of approximately 75 mg. In certain embodiments, the recombinant antibody is administered at a dose of approximately 225 mg. In certain embodiments, the recombinant antibody is administered at a dose of approximately 750 mg. In certain embodiments, the recombinant antibody is administered at a dose of approximately 1125 mg. In certain embodiments, the recombinant antibody is administered at a dose of approximately 1500 mg. In certain embodiments, the recombinant antibody is administered at a dose of approximately 2000 mg. In another aspect, this document describes a pharmaceutical composition for use in the treatment of cancer in an individual, wherein the pharmaceutical composition contains a pharmaceutically acceptable excipient, a carrier or diluent, and a recombinant antibody, wherein the recombinant antibody specifically binds to leukemia inhibitory factor (LIF) and contains: (a) an immunoglobulin heavy chain variable region (VH) sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any of SEQ ID NOs : 41, 42, 44 or 66; and (b) an immunoglobulin light chain variable region (VL) sequence with an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth under any of SEQ IDs NO: 4548; wherein the recombinant antibody is administered to the individual at a dose of from about 75 to about 2000 mg. In certain embodiments, the pharmaceutical composition contains about 25 mM histidine, about 6% sucrose, and about 0.01% polysorbate 80, with the recombinant antibody included at a concentration of about 20 mg/ml. In certain embodiments, the pharmaceutical composition has a pH value of approximately 6.0. In certain embodiments, the recombinant antibody binds to glycosylated LIF. In certain embodiments, the VH sequence is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence represented by

- 6 044934 No. under SEQ ID NO: 42; and the VL sequence is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 46. In certain embodiments, the VH sequence is identical to the amino acid sequence set forth in SEQ ID NO: 42; and the VL sequence is identical to the amino acid sequence set forth in SEQ ID NO: 46. In certain embodiments, the recombinant antibody comprises two immunoglobulin heavy chains and two immunoglobulin light chains. In certain embodiments, the recombinant antibody is an IgG antibody. In certain embodiments, the recombinant antibody is a Fab, F(ab) ₂ , single domain antibody, single chain variable fragment (scFv), or nanoantibody. In certain embodiments, the recombinant antibody specifically binds to LIF with a dissociation constant (KD) of less than about 200 pmol/L. In certain embodiments, the recombinant antibody specifically binds to LIF with a dissociation constant (KD) of less than about 100 picomol/L. In certain embodiments, the cancer includes advanced solid tumor, glioblastoma, stomach cancer, skin cancer, prostate cancer, pancreatic cancer, breast cancer, testicular cancer, thyroid cancer, head and neck cancer, liver cancer, kidney cancer, esophageal cancer , ovarian cancer, colon cancer, 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 recombinant antibody is administered intravenously. In certain embodiments, the recombinant antibody is administered approximately once per week. In certain embodiments, the recombinant antibody is administered approximately once every two weeks. In certain embodiments, the recombinant antibody is administered approximately once every three weeks. In certain embodiments, the recombinant antibody is administered approximately once every four weeks. In certain embodiments, the recombinant antibody is administered at a dose of approximately 75 mg. In certain embodiments, the recombinant antibody is administered at a dose of approximately 225 mg. In certain embodiments, the recombinant antibody is administered at a dose of approximately 750 mg. In certain embodiments, the recombinant antibody is administered at a dose of approximately 1125 mg. In certain embodiments, the recombinant antibody is administered at a dose of approximately 1500 mg. In certain embodiments, the recombinant antibody is administered at a dose of approximately 2000 mg.

In another aspect, this document describes a pharmaceutical composition for use in the treatment of cancer in an individual, wherein the pharmaceutical composition contains a pharmaceutically acceptable excipient, carrier or diluent and a recombinant antibody, wherein the recombinant antibody specifically binds to leukemia inhibitory factor (LIF) and contains: ( a) an immunoglobulin heavy chain sequence with an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 57-60 or 67 ; and (b) an immunoglobulin light chain sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 61-64 ; wherein the recombinant antibody is administered to the individual at a dose of from about 75 to about 2000 mg. In certain embodiments, the pharmaceutical composition contains about 25 mM histidine, about 6% sucrose, and about 0.01% polysorbate 80, with the recombinant antibody included at a concentration of about 20 mg/ml. In certain embodiments, the pharmaceutical composition has a pH value of approximately 6.0. In certain embodiments, the recombinant antibody binds to glycosylated LIF. In certain embodiments, the immunoglobulin heavy chain sequence is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 58; and the immunoglobulin light chain sequence is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 62. In certain embodiments, the immunoglobulin heavy chain sequence is identical to the amino acid sequence , presented under SEQ ID NO: 58; and the immunoglobulin light chain sequence is identical to the amino acid sequence set forth in SEQ ID NO: 62. In certain embodiments, the recombinant antibody comprises two immunoglobulin heavy chains and two immunoglobulin light chains. In certain embodiments, the recombinant antibody specifically binds to LIF with a dissociation constant (KD) of less than about 200 pmol/L. In certain embodiments, the recombinant antibody specifically binds to LIF with a dissociation constant (KD) of less than about 100 picomol/L. In certain embodiments, the cancer includes advanced solid tumor, glioblastoma, stomach cancer, skin cancer, prostate cancer, pancreatic cancer, breast cancer, testicular cancer, thyroid cancer, head and neck cancer, liver cancer, kidney cancer, esophageal cancer , ovarian cancer, colon cancer, lung cancer, lymphoma or soft tissue cancer. In certain variants

- 7 044934 implementation cancer includes non-small cell lung cancer, epithelial ovarian carcinoma or pancreatic adenocarcinoma. In certain embodiments, the recombinant antibody is administered intravenously. In certain embodiments, the recombinant antibody is administered approximately once per week. In certain embodiments, the recombinant antibody is administered approximately once every two weeks. In certain embodiments, the recombinant antibody is administered approximately once every three weeks. In certain embodiments, the recombinant antibody is administered approximately once every four weeks. In certain embodiments, the recombinant antibody is administered at a dose of approximately 75 mg. In certain embodiments, the recombinant antibody is administered at a dose of approximately 225 mg. In certain embodiments, the recombinant antibody is administered at a dose of approximately 750 mg. In certain embodiments, the recombinant antibody is administered at a dose of approximately 1125 mg. In certain embodiments, the recombinant antibody is administered at a dose of approximately 1500 mg. In certain embodiments, the recombinant antibody is administered at a dose of approximately 2000 mg.

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 LI-induced STAT3 phosphorylation by various monoclonal antibodies described herein.

In fig. Figure 5 presents the results of immunohistochemical staining and quantification of LIF expression in tumors of glioblastoma multiforme (GBM), NSCLC (non-small cell lung carcinoma), ovarian cancer, and colorectal cancer 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.

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 demonstrates that h5D8 shows a significant reduction in tumor growth when administered at a dose of 200 μg/mouse twice a week (p < 0.05). Symbols represent mean + SEM, statistical significance is compared with the 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 a decrease in macrophage infiltration in a human organotypic tissue section culture model. A representative image (left) and quantification (right) are shown.

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

In fig. 10D shows the reduction in macrophage infiltration of tumor sites in a syngeneic mouse model of colorectal cancer with a representative image and quantitative

- 8 044934 determination 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 the 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. 18A-C show images of a patient at cycle 7 (C7) of treatment with h5D8 (750 mg) at three target lesions - 2 rectus lesions and 1 pouch of Douglas lesion - as well as previous radiation therapy ports. In fig. 18A shows the #1 rectus muscle - the left panel shows the target lesion (1) and the right panel shows the XRT lesion (2), size: 37.8 mm. In fig. 18B shows rectus muscle #2 - left panel shows target lesion (3) and right panel shows XRT lesion (4), size: 24.3 mm. In fig. 18C shows pouch of Douglas - left panel shows target lesion (5) and right panel shows XRT lesion (6), size: 25 mM.

In fig. 19 shows the stabilization of the LIF level in the saturated state relative to the time of the 1st dose for subject 0210-003.

In fig. 20A-C show evidence of modulation of biomarkers that are indicators of the potential mechanism of action of LIF inhibition in tumor biopsies obtained from 0201-003. Data demonstrate results before treatment with h5D8 compared to results during treatment with h5D8. In fig. 20A shows antitumor immunity as percentage (%) change in CD68 frequency; % change in CD8 frequency and % change in Foxp3 frequency. In fig. 20B shows macrophage phenotype characteristics as % change in CD163 frequency; % change in CD206 frequency; and % change in MHCII frequency. In fig. 20C shows the effect of h5D8 treatment on pSTAT3 as % change in the number of nuclei stained for pSTAT3+.

In fig. 21A-C show evidence of modulation of biomarkers that are indicators of the potential mechanism of action of LIF inhibition in a tumor biopsy obtained from subject 0301-003. Data demonstrate results before treatment with h5D8 compared to results during treatment with h5D8. In fig. 21A shows antitumor immunity as percentage (%) change in CD68 frequency; % change in CD8 incidence and %

- 9 044934 changes in the frequency of occurrence of Foxp3. In fig. 21B shows macrophage phenotype characteristics as % change in CD163 frequency; % change in CD205 frequency; and % change in MHCII frequency. In fig. 21C shows the effects of h5D8 treatment on pSTAT3 as % change in the number of nuclei stained for pSTAT3+.

In fig. 22 shows evidence of modulation of biomarkers that are indicators of the potential mechanism of action of LIF inhibition in a tumor biopsy obtained from subject 0301-004. Data show results before treatment with h5D8 compared to results during treatment with h5D8. In fig. 22 shows antitumor immunity as a percentage (%) change in CD68 frequency; % change in CD8 frequency and % change in Foxp3 frequency.

In fig. 23 shows the LIF level stabilization pattern relative to the time of the 1st dose for subject 0201-004.

In fig. 24 shows evidence of modulation of biomarkers that are indicators of the potential mechanism of action of LIF inhibition in a tumor biopsy obtained from subject 0201-004. Data show results before treatment with h5D8 compared to results during treatment with h5D8. In fig. Figure 24 shows the characteristics of the macrophage phenotype as a % change in the frequency of occurrence of CD163; % change in CD205 frequency and % change in MHCII frequency.

In fig. 25 shows the stabilization of the LIF level in the saturated state relative to the time of the 1st dose for subject 0301-002.

In fig. Figure 26 shows the effects of h5D8 in dose cohorts 1–5.

Shown is the geometric mean of plasma h5D8 levels in patients relative to the start of the 1st infusion in dose cohorts 1–5. Additional treatment cycles are indicated (mg per dose every 3 weeks). The number of patients analyzed in each cohort was: cohort 1 n=2, cohort 2 n=1, cohort 3 n=10, cohort 4 n=8 and cohort 5 n=3.

In fig. 27A and B show stabilization of LIF levels in patients after treatment with h5D8 in dose cohorts 1-5 relative to the time of the first infusion. In fig. 27B shows total LIF levels in patients after the first two cycles of h5D8 in dose cohorts 1-4.

Detailed Description of 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 stated.

In another aspect, described herein is a method of treating an individual suffering from cancer, comprising administering to the individual a recombinant antibody that specifically binds leukemia inhibitory factor (LIF) comprising: (a) immunoglobulin heavy chain complementarity determining region 1 (VH-CDR1), containing the amino acid sequence presented under any of SEQ ID NO: 1-3; (b) immunoglobulin heavy chain complementarity determining region 2 (VH-CDR2) comprising the amino acid sequence represented by any of SEQ ID NO: 4 or 5; (c) immunoglobulin heavy chain complementarity determining region 3 (VH-CDR3) comprising the amino acid sequence represented by any of SEQ ID NOs: 6-8; (d) immunoglobulin light chain complementarity determining region 1 (VLCDR1) comprising the amino acid sequence represented by any of SEQ ID NO: 9 or 10; (e) 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 (5) immunoglobulin light chain complementarity determining region 3 (VL-CDR3) containing the amino acid sequence represented by SEQ ID NO: 13; wherein the recombinant antibody is administered to the individual at a dose of from about 75 to about 2000 mg.

In another aspect, described herein is a method of treating an individual suffering from cancer, comprising administering to the individual a recombinant antibody that specifically binds leukemia inhibitory factor (LIF) comprising: (a) an immunoglobulin heavy chain variable region (VH) amino acid sequence, which is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence presented under any of SEQ ID NO: 41, 42, 44 or 66; and (b) an immunoglobulin light chain variable region (VL) sequence with an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth under any of SEQ IDs NO: 45-48; wherein the recombinant antibody is administered to the individual at a dose of from about 75 to about 2000 mg. In another aspect, this document describes a method of treating an individual suffering from cancer, comprising administering to the individual a recombinant antibody that specifically binds leukemia inhibitory factor (LIF) comprising: (a) an immunoglobulin heavy chain sequence with an amino acid sequence that at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence presented under any of SEQ ID NO: 57-60 or 67; and (b) an immunoglobulin light chain sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 61-64 ; wherein the recombinant antibody is administered to the individual at a dose of from about 75 to about 2000 mg.

In another aspect, this document describes a pharmaceutical composition for use in the treatment of cancer in an individual, wherein the pharmaceutical composition contains a pharmaceutically acceptable excipient, carrier or diluent and a recombinant antibody, wherein the recombinant antibody specifically binds to leukemia inhibitory factor (LIF) and contains: (a) immunoglobulin heavy chain complementarity determining region 1 (VH-CDR1) comprising the amino acid sequence represented by any of SEQ ID NOs: 1-3; (b) immunoglobulin heavy chain complementarity determining region 2 (VH-CDR2) comprising the amino acid sequence represented by any of SEQ ID NO: 4 or 5; (c) immunoglobulin heavy chain complementarity determining region 3 (VH-CDR3) comprising the amino acid sequence represented by any one of SEQ ID NOs: 6-8; (d) immunoglobulin light chain complementarity determining region 1 (VL-CDR1) comprising the amino acid sequence represented by any of SEQ ID NO: 9 or 10; (e) 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 (f) immunoglobulin light chain complementarity determining region 3 (VL-CDR3) containing the amino acid sequence represented by SEQ ID NO: 13; wherein the recombinant antibody is administered to the individual at a dose of from about 75 to about 2000 milligrams. In another aspect, this document describes a pharmaceutical composition for use in the treatment of cancer in an individual, wherein the pharmaceutical composition contains a pharmaceutically acceptable excipient, carrier or diluent and a recombinant antibody, wherein the recombinant antibody specifically binds to leukemia inhibitory factor (LIF) and contains: ( a) an immunoglobulin heavy chain variable region (VH) sequence with an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any of SEQ ID NO: 41, 42, 44 or 66; and (b) an immunoglobulin light chain variable region (VL) sequence with an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth under any of SEQ IDs NO: 45-48; wherein the recombinant antibody is administered to the individual at a dose of from about 75 to about 2000 mg.

In another aspect, this document describes a pharmaceutical composition for use in the treatment of cancer in an individual, wherein the pharmaceutical composition contains a pharmaceutically acceptable excipient, carrier or diluent and a recombinant antibody, wherein the recombinant antibody specifically binds to leukemia inhibitory factor (LIF) and contains: ( a) an immunoglobulin heavy chain sequence with an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 57-60 or 67 ; and (b) an immunoglobulin light chain sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 61-64 ; wherein the recombinant antibody is administered to the individual at a dose of from about 75 to about 2000 mg. As used herein, unless otherwise indicated, the expression approximately refers to an amount that is within at least 10% of that stated.

As used herein, the terms individual, subject and patient are used interchangeably and include people who have been diagnosed or suspected of having a tumor, cancer or other neoplasm. As used herein, the term treat or treatment refers to interventions in a normal or pathological condition of an individual, designed or intended to alleviate at least one sign or symptom associated with the specified normal or pathological condition. The concept of treating or treating cancer as described herein refers to interventions designed to induce a complete response, a partial response, slow the progression of the cancer or tumor being treated, reduce tumor size or tumor burden, or slow tumor growth or tumor burden. Treatment also refers to interventions aimed at reducing the metastasis or grade of cancer or tumor. One skilled in the art will appreciate that given the heterogeneous population of individuals suffering from a disease, not all individuals will respond equally or to any extent to a given treatment. However, these individuals are considered to be undergoing treatment. Unsuccessful treatment usually leads to progression of the disease and the need for additional treatment with another therapeutic agent. In certain aspects, the antibodies and methods described herein can be used to maintain remission of a cancer or prevent the recurrence of the same cancer or another cancer related to the cancer being treated.

As used herein, the term immune checkpoint inhibitor refers to a drug that inhibits a biological molecule (immune checkpoint molecule) produced by the body that negatively regulates the antitumor/anticancer activity of T cells in the body. The immune checkpoint molecule can be produced by a tumor, an immune cell in the tumor microenvironment, or an immune cell existing not in the tumor microenvironment but in the bloodstream or lymphatic system. Immune checkpoint molecules include, but are not limited to, PD-1, PDL-1, PDL-2, CTLA4, TIM-3, LAG-3, VISTA, SIGLEC7, PVR, TIGIT, IDO, KIR, A2AR, B7-NZ, B7H4, and NOX2.

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 in the constant region of an 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.

Percentage (%) sequence identity to a reference polypeptide or antibody sequence is the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a reference polypeptide or antibody sequence after sequence alignment and the introduction of gaps as necessary to achieve the maximum percentage of sequence identity and without considering 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.

- 12 044934

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 obtained as described in the previous paragraph using a computer program ALIGN-2.

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. Structural Attributes of 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 VH-FR1, 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 divalent 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 (Kabat numbering scheme); Al-Lazikani et al., (1997) JMB 273,927-948 (Chotia numbering scheme); MacCallum et al., J. Mol. Biol. 262:132-145 (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, Chotia 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 alignment, whereas Chotia's schema is based on structural information. The numbering for both the Kabat and Chotia schemes is based on the most common sequence lengths of the antibody region, with insertions designated by letters, such as 30a, and deletions appearing in some antibodies. Two

- 13 044934 schemes place certain insertions and deletions (insertions/deletions) in different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is in many respects similar to the Chotia numbering scheme. In certain embodiments, CDRs may be determined using any combination of IMGT, Chotia, Kabat, Contact, and Aho methods.

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 mutation rate 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 _CH 1, _CH 2, 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 substantially smaller than CH and is located at the C-terminus of the full light chain polypeptide, adjacent to 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, IgG2, IgG3 and IgG4. 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 IgG2. In certain embodiments, the IgG subclass includes IgG3. 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 C _H 2, C _H 3 and C _H 4 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 antibodies described herein contains one or more amino acid substitutions that promote complement-mediated cell lysis. In certain embodiments, the Fc region of the antibodies described herein contains one or more amino acid substitutions that contribute to ADCC. In certain embodiments, the Fc region of the antibodies described herein contains one or more amino acid substitutions that reduce complement-mediated cell lysis. In certain embodiments, the Fc region of the antibodies described herein contains one or more amino acid substitutions that increase 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 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 the serum half-life of the antibody increase the affinity of the antibody for the neonatal Fc receptor (FcRn).

In certain embodiments, the antibodies of the present invention are variants that have some, but not all, effector functions, making them a desirable candidate for applications in which the in vivo half-life of the antibody is important but some effector functions (such as complement activation and ADCC) are unnecessary or harmful. In vitro and/or in vivo cytotoxicity assays can be performed to confirm the reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcyR binding ability (thus likely lacking ADCC activity) but retains FcRn binding ability. Non-limiting examples of in vitro assays to evaluate the ADCC activity of a molecule of interest are described in US Pat. Nos. 5,500,362 and 5,821,337. Alternatively, non-radioactive analytical techniques (eg, the ACTI™ and CytoTox 96® non-radioactive cytotoxicity assays) can be used. Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs), monocytes, macrophages, and natural killer (NK) cells.

Antibodies may have an increased half-life and improved binding to the neonatal Fc receptor (FcRn) (see, for example, US 2005/0014934). Such antibodies may contain an Fc region with one or more substitutions therein that enhance binding of the Fc region to FcRn, and include antibodies with substitutions at one or more residues of the Fc region: 238, 256, 265, 272, 286, 303, 305, 307 , 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434 according to the EU numbering system (see, for example, US Patent No. 7371826). Other examples of Fc region variants are also discussed (see, for example, Duncan & Winter, Nature 322:738-40 (1988); US Pat. Nos. 5,648,260 and 5,624,821; and WO 94/29351).

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, the antibodies described herein are grafted onto CDRs.

Humanization usually reduces or has little effect on the overall affinity of the antibody. This document describes antibodies that unexpectedly have greater affinity for their target after humanization. In certain embodiments, humanization increases the affinity of the antibody by 10%. In certain embodiments, humanization increases the affinity of the antibody by 25%. In certain embodiments, humanization increases the affinity of the antibody by 35%. In certain embodiments, humanization increases the affinity of the antibody by 50%. In certain embodiments, humanization increases the affinity of the antibody by 60%. In certain embodiments, humanization increases the affinity of the antibody by 75%. In certain embodiments, humanization increases the affinity of the antibody by 100%. Accordingly, affinity is measured using surface plasmon resonance (SPR). In certain embodiments, affinity is measured using glycosylated human LIF. In certain embodiments, glycosylated human LIF has been immobilized on the surface of an SPR chip. In certain embodiments, the antibody binds to a K _D of less than about 300 nanomol/L, 200 nanomol/L, 150 nanomol/L, 125 nanomol/L, 100 nanomol/L, 90 nanomol/L, 80 nanomol/L, 70 nanomol /L, 60 nanomol/L, 50 nanomol/L, 40 nanomol/L or less. Antibodies of the Present Invention The antibodies described herein were obtained from rats immunized with DNA encoding human LIF.

In certain embodiments, described herein is an antibody (5D8) that specifically binds LIF containing the VH-CDR1 region represented by any of SEQ ID

- 15 044934

NO: 1-3, VH-CDR2, presented under any of SEQ ID NO: 4 or 5, and VH-CDR3, presented under any of SEQ ID NO: 6-8. In certain embodiments, described herein is an antibody that specifically binds LIF comprising the VL-CDR1 region represented by any of SEQ ID NO: 9 or 10, VL-CDR2 represented by SEQ ID NO: 11 or 12, and VL- CDR3, shown under SEQ ID NO: 13. In certain embodiments, an antibody that specifically binds LIF is described herein, containing the VH-CDR1 region, presented under any of SEQ ID NO: 1-3, VH-CDR2, presented under any of SEQ ID NO: 4 or 5, and VH-CDR3, represented by any of SEQ ID NO: 6-8, VL-CDR1, presented by any of SEQ ID NO: 9 or 10, VL-CDR2, presented by SEQ ID NO: 11 or 12, and VL-CDR3 provided under SEQ ID NO: 13. In certain embodiments, the antibody specifically binds to human LIF.

In certain embodiments, an antibody that specifically binds LIF comprises one or more human heavy chain framework regions comprising a VH-FR1 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99 % identical to the amino acid sequence set forth under any of SEQ ID NO: 1417, an amino acid sequence of VH-FR2 that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth under by any of SEQ ID NO: 18 or 19, an amino acid sequence of VH-FR3 that is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence set forth in any of SEQ ID NO : 20-22, or an amino acid sequence of a VHFR4 region that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 23-25. In certain embodiments, one or more human heavy chain framework regions comprise a VH-FR1 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 15, an amino acid sequence of VH-FR2 that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 19, the amino acid sequence of VH-FR3 that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 20, and the amino acid sequence of VH-FR4 that is at least about 80 %, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, an antibody that specifically binds LIF comprises one or more human light chain framework regions containing an amino acid sequence of VL-FR1 that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 26-29, the amino acid sequence of VL-FR2 that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 30-33, a VL-FR3 amino acid sequence that is at least is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 34-37, or to the VL-FR4 amino acid sequence that is at least about 80% identical , 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence presented under any of SEQ ID NO: 38-40. In certain embodiments, one or more human light chain framework regions comprise a VL-FR1 amino acid sequence that is at least about 80%, about 90%, or about 95% identical to the amino acid sequence set forth in SEQ ID NO: 27, the VL amino acid sequence -FR2 that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 31 to the amino acid sequence of VL-FR3 that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 35, and the amino acid sequence of VL-FR4, which is at least about 80%, 90%, 95% identical , 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 38. In certain embodiments, one or more human heavy chain framework regions and one or more human light chain framework regions comprise the amino acid sequence VH-FR1 that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 15, the amino acid sequence of VH-FR2, which is at least about 80% identical, 90 %, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 19, the amino acid sequence of VH-FR3, which is at least about 80%, 90%, 95%, 97%, 98 % or 99% identical to the amino acid sequence set forth in SEQ ID NO: 20 to the amino acid sequence of VH-FR4 that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth under SEQ ID NO: 24, amino acid sequence

- 16 044934 body of VL-FR1 that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 27 to the amino acid sequence of VL-FR2, which is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 31 to the amino acid sequence of VL-FR3, which is at least about 80% identical, 90 %, 95%, 97%, 98% or 99% identical to the amino acid sequence presented under SEQ ID NO: 35, and the amino acid sequence of VL-FR4, which is at least 80%, 90%, 95%, 97%, 98 % or 99% identical to the amino acid sequence set forth in SEQ ID NO: 38. In certain embodiments, an antibody that specifically binds LIF comprises one or more human heavy chain framework regions comprising: an amino acid sequence of VH-FR1 that is identical to the amino acid sequence presented under any of SEQ ID NO: 14-17, the amino acid sequence of VH-FR2, which is identical to the amino acid sequence presented under any of SEQ ID NO: 18 or 19, the amino acid sequence of VH-FR3, which is identical to the amino acid sequence presented under any of SEQ ID NO: 20-22, or an amino acid sequence of the VH-FR4 region that is identical to the amino acid sequence presented under any of SEQ ID NO: 23-25. In certain embodiments, one or more human heavy chain framework regions comprise a VH-FR1 amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO: 15, a VH-FR2 amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO: 19 , an amino acid sequence of VH-FR3 that is identical to the amino acid sequence set forth in SEQ ID NO: 20, and an amino acid sequence of VH-FR4 that is identical to the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, an antibody that specifically binds LIF , contains one or more human light chain framework regions containing a VL-FR1 amino acid sequence that is identical to the amino acid sequence set forth under any of SEQ ID NOs: 26-29, a VL-FR2 amino acid sequence that is identical to the amino acid sequence set forth under any of SEQ ID NO: 30-33, an amino acid sequence of VL-FR3 that is identical to the amino acid sequence presented under any of SEQ ID NO: 34-37, or an amino acid sequence of VL-FR4 that is identical to the amino acid sequence presented under any of SEQ ID NO: : 38-40. In certain embodiments, one or more human light chain framework regions comprise a VL-FR1 amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO: 27, a VL-FR2 amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO: 31 , an amino acid sequence of VL-FR3 that is identical to the amino acid sequence set forth in SEQ ID NO: 35, and an amino acid sequence of VL-FR4 that is identical to the amino acid sequence set forth in SEQ ID NO: 38. In certain embodiments, one or more human framework regions heavy chain and one or more human light chain framework regions contain an amino acid sequence of VH-FR1 that is identical to the amino acid sequence set forth in SEQ ID NO: 15, an amino acid sequence of VH-FR2 that is identical to the amino acid sequence set forth in SEQ ID NO: 19, the amino acid sequence of VH-FR3, which is identical to the amino acid sequence presented under SEQ ID NO: 20, the amino acid sequence of VH-FR4, which is identical to the amino acid sequence presented under SEQ ID NO: 24, the amino acid sequence of VL-FR1, which is identical to the amino acid sequence presented under SEQ ID NO: 27, the amino acid sequence of VL-FR2, which is identical to the amino acid sequence presented under SEQ ID NO: 31, the amino acid sequence of VL-FR3, which is identical to the amino acid sequence presented under SEQ ID NO: 35, and the amino acid sequence of VL- FR4, which is identical to the amino acid sequence presented under SEQ ID NO: 38. In certain embodiments, the antibody specifically binds human LIF. 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 IMGT 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 (QIKAKSDDYATYYAESVKG), 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.

In certain embodiments, described herein is an antibody that specifically binds LIF comprising a VH-CDR1 region that is at least 80 or 90% identical

- 17 044934 is identical to the sequence presented under 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 (QIKAKSDDYATYYAESVKG), and VH -CDR3, which is at least 80 or 90% identical to the sequence presented under SEQ ID NO: 6 (TCWEWDLDF). In certain embodiments, disclosed herein is an antibody that specifically binds LIF comprising a VL-CDR1 region 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 (MQATHAPPYT). In certain embodiments, disclosed herein is an antibody that specifically binds LIF comprising the region 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 comprise one or more human heavy chain framework regions comprising: a VH-FR1 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 14-17, an amino acid sequence of VH-FR2 that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence presented under any of SEQ ID NOs: 18 or 19, an amino acid sequence of VH-FR3 that is at least about 90% identical to the amino acid sequence presented under any of SEQ ID NOs: 20-22, or the amino acid sequence of the VH-FR4 region that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 23-25. In certain embodiments, one or more human heavy chain framework regions comprise a VH-FR1 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 15, an amino acid sequence of VH-FR2 that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 19, the amino acid sequence of VH-FR3 that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 20, and the amino acid sequence of VH-FR4 that is at least about 80 %, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, an antibody that specifically binds LIF comprises one or more human light chain framework regions containing : an amino acid sequence of VL-FR1 that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 26-29, amino acid sequence of VL- FR2 that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 30-33, the amino acid sequence of VL-FR3 that is at least is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 34-37, or the amino acid sequence of VL-FR4 that is at least about 80 %, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence presented under any of SEQ ID NO: 38-40. In certain embodiments, one or more human light chain framework regions comprise a VL-FR1 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 27, an amino acid sequence of VL-FR2 that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 31, the amino acid sequence of VL-FR3 that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 35, and the amino acid sequence of VL-FR4 that is at least about 80 %, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 38. In certain embodiments, one or more human framework regions

- 18 044934 heavy chain and one or more human light chain framework regions contain all of a VH-FR1 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence, presented under SEQ ID NO: 15, the amino acid sequence of VH-FR2, which is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence presented under SEQ ID NO: 19, amino acid a VH-FR3 sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 20, a VH-FR4 amino acid sequence that is at least is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO: 24 to the amino acid sequence of VL-FR1 that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth SEQ ID NO: 27, an amino acid sequence of VL-FR2 that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 31, an amino acid sequence VL-FR3 that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 35, and the amino acid sequence of VL-FR4 that is at least is about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 38. In certain embodiments, the antibody specifically binds human LIF. In certain embodiments, an antibody that specifically binds LIF comprises one or more human heavy chain frameworks containing a VH-FR1 amino acid sequence that is identical to the amino acid sequence set forth in any of SEQ ID NOs: 14-17, the VH-FR2 amino acid sequence , which is identical to the amino acid sequence presented under any of SEQ ID NO: 18 or 19, the amino acid sequence of VH-FR3, which is identical to the amino acid sequence presented under any of SEQ ID NO: 20-22, or the amino acid sequence of the VHFR4 region, which is identical to the amino acid sequence the sequence presented under any of SEQ ID NO: 23-25. In certain embodiments, one or more human heavy chain framework regions comprise a VH-FR1 amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO: 15, a VH-FR2 amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO: 19 , an amino acid sequence of VH-FR3 that is identical to the amino acid sequence set forth in SEQ ID NO: 20, and an amino acid sequence of VH-FR4 that is identical to the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, an antibody that specifically binds LIF , contains one or more human light chain framework regions containing an amino acid sequence of VLFR1 that is identical to the amino acid sequence presented under any of SEQ ID NO: 26-29, an amino acid sequence of VL-FR2 that is identical to the amino acid sequence presented under any of SEQ ID NO: 30-33, an amino acid sequence of VL-FR3 that is identical to the amino acid sequence presented under any of SEQ ID NOs: 34-37, or an amino acid sequence of VL-FR4 that is identical to the amino acid sequence presented under any of SEQ ID NO: 38 -40. In certain embodiments, one or more human light chain framework regions comprise a VL-FR1 amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO: 27, a VL-FR2 amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO: 31 , an amino acid sequence of VL-FR3 that is identical to the amino acid sequence set forth in SEQ ID NO: 35, and an amino acid sequence of VL-FR4 that is identical to the amino acid sequence set forth in SEQ ID NO: 38. In certain embodiments, one or more human framework regions heavy chain and one or more human light chain framework regions contain an amino acid sequence of VH-FR1 that is identical to the amino acid sequence set forth in SEQ ID NO: 15, an amino acid sequence of VH-FR2 that is identical to the amino acid sequence set forth in SEQ ID NO: 19, the amino acid sequence of VH-FR3, which is identical to the amino acid sequence presented under SEQ ID NO: 20, the amino acid sequence of VH-FR4, which is identical to the amino acid sequence presented under SEQ ID NO: 24, the amino acid sequence of VL-FR1, which is identical to the amino acid sequence presented under SEQ ID NO: 27, the amino acid sequence of VL-FR2, which is identical to the amino acid sequence presented under SEQ ID NO: 31, the amino acid sequence of VL-FR3, which is identical to the amino acid sequence presented under SEQ ID NO: 35, and the amino acid sequence of VL- FR4, which is identical to the amino acid sequence presented under SEQ ID NO: 38. In certain embodiments, the antibody specifically binds human LIF.

- 19 044934

In certain embodiments, described herein is an 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-CDR2 amino acid sequence , 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 an 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-CDR2 amino acid sequence , which is at least 80% identical to the sequence presented under SEQ ID NO: 11 (SVSNLES), and the amino acid sequence of VL-CDR3, which is at least 80 or 90% identical to the sequence presented under SEQ ID NO: 13 (MQATHAPPYT ). In certain embodiments, disclosed herein is an antibody that specifically binds LIF, comprising the amino acid sequence of VH-CDR1 set forth in SEQ ID NO: 1 (GFTFSHAWMH), the amino acid sequence of 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 set forth in 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, antibodies that specifically bind LIF comprise one or more human heavy chain framework regions comprising: a VH-FR1 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 14-17, an amino acid sequence of VH-FR2 that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence presented under any of SEQ ID NOs: 18 or 19, an amino acid sequence of VH-FR3 that is at least about 90% identical to the amino acid sequence presented under any of SEQ ID NOs: 20-22, or the amino acid sequence of the VH-FR4 region that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 23-25. In certain embodiments, one or more human heavy chain framework regions comprise a VH-FR1 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 15, an amino acid sequence of VH-FR2 that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 19, the amino acid sequence of VH-FR3 that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 20, and the amino acid sequence of VH-FR4 that is at least about 80 %, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, an antibody that specifically binds LIF comprises one or more human light chain framework regions containing a VL-FR1 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 26-29, VL-FR2 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 30-33, a VL-FR3 amino acid sequence that is at least is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 34-37, or to the VLFR4 amino acid sequence that is at least about 80% identical, 90 %, 95%, 97%, 98% or 99% identical to the amino acid sequence presented under any of SEQ ID NO: 38-40. In certain embodiments, one or more human light chain framework regions comprise a VL-FR1 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 27, the amino acid sequence of VL-FR2, which is at least about 80%, 90%,

- 20 044934

95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 31, the amino acid sequence of VL-FR3, which is at least about 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence presented under SEQ ID NO: 35, and an amino acid sequence of VL-FR4 that is at least 90% identical to the amino acid sequence presented under SEQ ID NO: 38. In certain embodiments, one or more human framework regions heavy chain and one or more human light chain framework regions contain all of a VH-FR1 amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 15, an amino acid sequence of VH-FR2 that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 19, an amino acid sequence of VH- FR3 that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 20 to the amino acid sequence of VH-FR4 that is at least about 90 % identical to the amino acid sequence set forth in SEQ ID NO: 24, to an amino acid sequence of VL-FR1 that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth under SEQ ID NO: 27, an amino acid sequence of VL-FR2 that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 31, the amino acid sequence of VL-FR3 that is at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 35, and the amino acid sequence of VL-FR4 that is at least about 80 %, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 38. In certain embodiments, the antibody specifically binds human LIF. In certain embodiments, an antibody that specifically binds LIF comprises one or more human heavy chain framework regions comprising: a VH-FR1 amino acid sequence that is identical to the amino acid sequence set forth in any of SEQ ID NOs: 14-17, a VH-FR1 amino acid sequence FR2, which is identical to the amino acid sequence presented under any of SEQ ID NO: 18 or 19, the amino acid sequence of VH-FR3, which is identical to the amino acid sequence presented under any of SEQ ID NO: 20-22, or the amino acid sequence of the VH-FR4 region, which is identical to the amino acid sequence presented under any of SEQ ID NO: 23-25. In certain embodiments, one or more human heavy chain framework regions comprise a VH-FR1 amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO: 15, a VH-FR2 amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO: 19 , an amino acid sequence of VH-FR3 that is identical to the amino acid sequence set forth in SEQ ID NO: 20, and an amino acid sequence of VH-FR4 that is identical to the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, an antibody that specifically binds LIF , contains one or more human light chain framework regions containing: a VL-FR1 amino acid sequence that is identical to the amino acid sequence presented under any of SEQ ID NO: 26-29, a VL-FR2 amino acid sequence that is identical to the amino acid sequence presented under any from SEQ ID NOs: 30-33, an amino acid sequence of VL-FR3 that is identical to the amino acid sequence presented under any of SEQ ID NOs: 34-37, or an amino acid sequence of VL-FR4 that is identical to the amino acid sequence presented under any of SEQ ID NO: 38-40. In certain embodiments, one or more human light chain framework regions comprise a VL-FR1 amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO: 27, a VL-FR2 amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO: 31 , an amino acid sequence of VL-FR3 that is identical to the amino acid sequence set forth in SEQ ID NO: 35, and an amino acid sequence of VL-FR4 that is identical to the amino acid sequence set forth in SEQ ID NO: 38. In certain embodiments, one or more human framework regions heavy chain and one or more human light chain framework regions contain an amino acid sequence of VH-FR1 that is identical to the amino acid sequence set forth in SEQ ID NO: 15, an amino acid sequence of VH-FR2 that is identical to the amino acid sequence set forth in SEQ ID NO: 19, the amino acid sequence of VH-FR3, which is identical to the amino acid sequence presented under SEQ ID NO: 20, the amino acid sequence of VH-FR4, which is identical to the amino acid sequence presented under SEQ ID NO: 24, the amino acid sequence of VL-FR1, which is identical to the amino acid sequence presented under SEQ ID NO: 27, amino acid

- 21 044934 a VL-FR2 sequence that is identical to the amino acid sequence presented under SEQ ID NO: 31, an amino acid sequence of VL-FR3 that is identical to the amino acid sequence presented under SEQ ID NO: 35, and a VLFR4 amino acid sequence that is identical to the amino acid sequence, presented under SEQ ID NO: 38. In certain embodiments, the antibody specifically binds human LIF.

In certain embodiments, disclosed herein is an 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 NOs: 41, 42, and 44. In certain embodiments, this document describes an antibody that specifically binds LIF containing a humanized heavy chain variable region comprising the amino acid sequence set forth in any of SEQ ID NOs: 41, 42, and 44. In certain embodiments, disclosed herein is an 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: 45-48. In certain embodiments, this document discloses an 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: 45-48. In certain embodiments, the antibody specifically binds human LIF. In certain embodiments, disclosed herein is an 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: 42; 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: 46 In certain embodiments, disclosed herein is an antibody that specifically binds LIF comprising a humanized heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 42; and a humanized heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 46.

In certain embodiments, disclosed herein is an 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: 66; 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: 46 In certain embodiments, disclosed herein is an antibody that specifically binds LIF comprising a humanized heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 66; and a humanized heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 46.

In certain embodiments, disclosed herein is an 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 about 99% identical to the amino acid sequence presented under any of SEQ ID NO: 57-60; 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: 61 -64. In certain embodiments, described herein is an antibody that specifically binds LIF comprising a humanized heavy chain comprising the amino acid sequence represented by any of SEQ ID NOs: 57-60; and a humanized light chain comprising the amino acid sequence set forth in any one of SEQ ID NO: 6164.

In certain embodiments, disclosed herein is an antibody that specifically binds LIF containing a humanized heavy chain containing an amino acid

- 22044934 a 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: 58; 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: 62. B In certain embodiments, this document discloses an antibody that specifically binds LIF comprising a humanized heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 58; and a humanized heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 62. In certain embodiments, an 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 set forth in SEQ ID NO: 67; 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: 62. B In certain embodiments, this document discloses an antibody that specifically binds LIF comprising a humanized heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 67; and a humanized heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 62.

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.

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 defining complementarity about

- 23 044934 light chain region 3 (VL-CDR3) containing the amino acid sequence represented by 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 an 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 about 99% identical to the amino acid sequence presented under any of SEQ ID NO: 49-52; 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: 53 -56. In certain embodiments, described herein is an antibody that specifically binds LIF comprising a humanized heavy chain comprising the amino acid sequence represented by any of SEQ ID NOs: 49-52; and a humanized light chain comprising the amino acid sequence set forth in any one of SEQ ID NO: 5356.

In certain embodiments, disclosed herein is an 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 about 99% identical to the amino acid sequence presented under SEQ ID NO: 50; 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: 54. B In certain embodiments, described herein is an antibody that specifically binds LIF comprising a humanized heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 50; and a humanized light chain comprising the amino acid sequence set forth in any one of SEQ ID NO: 54.

Epitopes Bound by Therapeutically Useful Anti-LIF Antibodies Described 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. 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 (alpha helices 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. In certain embodiments, an isolated antibody is described herein 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, I14, R15, H16, P17, C18, H19, N20, Q25, Q29, Q32, D120, R123, S127, N128, L130, C131, C134, S135 or H138 from SEQ ID NO : 68. 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: 68. 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 of SEQ ID NO: 68. 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. In certain embodiments, described herein is an antibody comprising a CDR with an amino acid sequence

- 24 044934 activity, represented by any of SEQ ID NO: 1, 4, 6, 9, 11 and 13, which relates to 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, I14, R15, H16, P17, C18, H19, N20, Q25, Q29, Q32, D120, R123, S127, N128 , L130, C131, C134, S135, or H138 from SEQ ID NO: 68. In certain embodiments, described herein is an antibody comprising a CDR with an amino acid sequence represented by any of SEQ ID NO: 1, 4, 6, 9, 11 and 13, which binds to 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: 68. 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 certain embodiments, disclosed herein is an antibody comprising a CDR with the amino acid sequence represented by any of SEQ ID NOs: 1, 4, 8, 9, 11, and 13 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, I14, R15, H16, P17, C18, H19, N20, Q25, Q29, Q32, D120, R123, S127, N128, L130, C131, C134, S135, or H138 of SEQ ID NO: 68. In certain embodiments, an antibody comprising a CDR with an amino acid sequence represented by any of the SEQ IDs is disclosed herein. NO: 1, 4, 8, 9, 11 and 13, which binds to 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: 68. 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 certain embodiments, disclosed herein is an antibody comprising CDRs that differ from the amino acid sequence set forth in any of SEQ ID NOs: 1, 4, 6, 9, 11, and 13 at 1, 2, 3, 4, or 5 amino acids, and which 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, I14 , R15, H16, P17, C18, H19, N20, Q25, Q29, Q32, D120, R123, S127, N128, L130, C131, C134, S135, or H138 of SEQ ID NO: 68. In certain embodiments herein describes an antibody containing CDRs that differ from the amino acid sequence presented under any of SEQ ID NO: 1, 4, 6, 9, 11 and 13 at 1, 2, 3, 4 or 5 amino acids, and which binds to 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: 68. In certain embodiments implementation, 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 certain embodiments, disclosed herein is an antibody comprising CDRs that differ from the amino acid sequence set forth in any of SEQ ID NOs: 1, 4, 8, 9, 11, and 13 at 1, 2, 3, 4, or 5 amino acids, and which 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, I14 , R15, H16, P17, C18, H19, N20, Q25, Q29, Q32, D120, R123, S127, N128, L130, C131, C134, S135, or H138 of SEQ ID NO: 68. In certain embodiments herein describes an antibody containing CDRs that differ from the amino acid sequence presented under any of SEQ ID NO: 1, 4, 8, 9, 11 and 13 at 1, 2, 3, 4 or 5 amino acids, and bind to 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: 68. 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 certain embodiments, described herein is an antibody that specifically binds LIF comprising a humanized heavy chain variable region 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:42; and an amino acid sequence of the humanized light chain variable region 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: 46, and which connects any one, two, three, four, five, six, seven, eight, nine, ten,

- 25 044934 eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen or twenty of the following balances: 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: 68. In certain embodiments, described herein is an antibody that specifically binds LIF comprising a humanized heavy chain variable region amino acid sequence that 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: 42; and an amino acid sequence of the humanized light chain variable region 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: 46, and which 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 : 68. 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 certain embodiments, described herein is an antibody that specifically binds LIF comprising a humanized heavy chain variable region 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: 66; and an amino acid sequence of the humanized light chain variable region 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: 46, and which 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, I14, R15, H16, P17, C18, H19, N20, Q25, Q29, Q32, D120, R123, S127, N128, L130, C131, C134, S135, or H138 of SEQ ID NO: 68. In certain embodiments, the implementation is described herein an antibody that specifically binds LIF comprising a humanized heavy chain variable region 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 below SEQ ID NO: 66; and an amino acid sequence of the humanized light chain variable region 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: 46, and which 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 : 68. 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. Indications for Use In certain embodiments, the antibodies described 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 nanomol/L. 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 900, 800, 700, 600, 500, 400, 300, 200, or 100 nmol/L.

In certain embodiments, h5D8 and h5D8 dosages disclosed herein are useful for treating tumors and cancers that express 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-fold, 3-fold, 5-fold, 10-fold, 100-fold, or greater than 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. A LIF-positive tumor can be determined histologically using, for example, immunohistochemical analysis using an anti-LIF antibody; through commonly used molecular biology techniques, such as, for example, quantification of mRNA by real-time PCR or RNA sequencing; or protein quantification, e.g.

- 26 044934 by Western blotting, flow cytometry, ELISA or homogeneous quantitative protein assay (eg AlphaLISA®). In certain embodiments, the antibodies can be used to treat patients who have been diagnosed with cancer. In certain embodiments, the cancer includes one or more cancer stem cells or is one or more cancer stem cells.

In certain embodiments, h5D8 and dosages of h5D8 disclosed herein are useful for treating tumors in cancers that express the LIF receptor (CD118). 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 is a cancer stem cell. In a certain embodiment, a LIF-positive tumor or cancer can be determined by immunohistochemical analysis using an anti-LIF antibody and an anti-LIF antibody. In a certain embodiment, a LIF-positive tumor is determined by immunohistochemical (IHC) analysis of the level of LIF expression in the top 10%, 20%, 30%, 40%, or top 50% of tumors.

The h5D8 and dosages of h5D8 described here affect many outcomes. In a certain embodiment, the antibodies described herein can reduce the presence of M2 macrophages in tumors by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in the tumor. model compared to a control antibody (eg, isotype control). M2 macrophages can be identified by CCL22 and/or CD206 staining of sections in IHC or by flow cytometry of tumor-infiltrating immune or myeloid cells. In a certain embodiment, the antibodies described herein can reduce the binding of LIF to gp130 in tumors by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a control antibody (eg, isotype control). In a certain embodiment, antibodies described herein can reduce lIf signaling by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in LIF-sensitive cell line compared to a control antibody (eg, isotype control). Using Western blotting, LIF signaling can be measured, for example, by phosphorylation of STAT3 (a downstream target of the LIF signaling pathway). The antibodies herein are also highly specific for LIF compared to other cytokines that are members of the IL-6 family. In certain embodiments, the antibodies bind human LIF with an affinity that is about 10x, about 50x, or about 100x greater than that of any other IL-6 family cytokine. In certain embodiments, anti-LIF antibodies do not bind to other IL-6 family cytokines that are produced in the mammalian system. In certain embodiments, the antibodies do not bind to oncostatin M that is produced in the mammalian system.

The h5D8 and dosages of h5D8 described herein are useful in methods of providing improvements in levels of biomarkers that are predictive of tumor or cancer burden. Tumor markers such as carbohydrate antigen-125 (CA125), carbohydrate antigen 19-9 (CA 19-9) and carcinoembryonic antigen (CEA) are positive prognostic indicators. Typically, low levels of these antigens correlate with treatment success. In certain embodiments, h5D8 reduces serum levels of CEA, CA 19-9, CA125, or a combination thereof when administered at a dose of approximately 1125 or 1500 approximately every 2, 3, or 4 weeks, including stepwise dose increases during this period. In certain embodiments, the tumor-associated marker is CA 19-9, CA-125, CEA, or a combination thereof. In certain embodiments, the tumor-associated marker is CEA. In certain embodiments, the tumor associated marker is CA-125. In certain embodiments, the tumor-associated marker is CA 19-9. Levels of CA 19-9, CA-125, CEA, or a combination thereof can be reduced by approximately 10%, 20%, 25%, 30%, 35%, 40%, or 50% as a result of the treatment described herein. H5D8 and the dosages of h5D8 described herein may also be used to improve other prognostic indicators of tumor/cancer burden or disease impact. In certain embodiments, the prognostic indicator is US Eastern Cooperative Oncology Group (ECOG) status. ECOG status is set on a scale from 0 to 5 as follows: 0 - fully active, able to perform all activities without restrictions, as before the disease; 1 - limited in physically strenuous activities, but ambulates and can do light or sedentary work, such as light housework, office work; 2 - ambulatory and able to independently care for themselves, but unable to perform any work activities; approximately more than 50% of waking hours or less; 3 is only able to take care of himself to a limited extent; confined to bed or chair for more than

- 27 044934

50% of waking time; 4 - completely disabled; cannot perform self-care; completely confined to a bed or chair; 5 - dead. In certain embodiments, the methods and dosages provide a reduction in the ECOG score of 1, 2, 3, or 4. In certain embodiments, the individual's ECOG status is reduced to 0, 1, 2, or 3. In certain embodiments, the individual's ECOG status is not increased by for at least 3, 6, 9 or 12 months. In certain embodiments, an individual's ECOG status is reduced to 0 or 1 upon administration of h5D8 at a dose of approximately 1125 or 1500 approximately every 2, 3, or 4 weeks, including stepwise dose increases during this period. This improvement or delay in progression will be visible after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more treatments.

In certain embodiments, treatment with h5D8 results in disease stabilization. In certain embodiments, h5D8 and the dosages of h5D8 described herein result in disease stabilization for at least 3, 6, 12, 15, 18, 24, 30, or 36 months during treatment. In certain embodiments, the antibodies and dosages described herein result in disease stabilization for at least 3, 6, 12, 15, 18, 24, 30, or 36 months during treatment with h5d8 administered at a dose of approximately 1125 or 1500 approximately every 2, 3 or 4 weeks, including gradual dose increases during this period. In certain embodiments, the antibodies and dosages described herein result in disease stabilization for at least 12 months during treatment with h5d8 administered at a dose of approximately 1125 or 1500 approximately every 2, 3, or 4 weeks, including stepwise increases doses during this period. This delay in progression will be visible after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more treatments.

In certain embodiments, h5D8 and dosages of h5D8 described herein are useful for treating cancer or a tumor. In certain embodiments, h5D8 is useful for treating cancer that is resistant to at least one other treatment. In certain embodiments, the treatment is a chemotherapy or immunotherapy treatment (eg, immune checkpoint inhibitor therapy). In certain embodiments, the immunotherapy targets PD-1, PDL-1, PDL-2, or any combination thereof. In certain embodiments, the immunotherapy is directed to the poliovirus receptor (PVR), TIGIT, or any combination thereof. 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 carcinoma and non-small cell carcinoma, preferably

- 28 044934 non-small cell lung cancer, lung adenocarcinoma, metastatic melanoma, androgen-independent metastatic prostate cancer, androgen-dependent metastatic prostate cancer, prostate adenocarcinoma 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 relapsed/refractory glioblastoma, pancreatic cancer, ovarian cancer, colon cancer, prostate 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, head and neck cancer, liver cancer, kidney cancer, esophageal cancer , ovarian cancer, colon cancer, 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. In certain embodiments, the cancer includes appendiceal cancer, rectal cancer, metastatic myxoid liposarcoma, and paraganglioma.

Therapeutic methods

In certain embodiments, 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, such as once a week, twice a week, once a month, twice a month, etc. In certain embodiments, the antibodies are administered approximately 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 to about 50 mg/kg. In certain embodiments, the therapeutically acceptable amount is from about 1 to about 40 mg/kg. In certain embodiments, the therapeutically acceptable amount is from about 5 to about 30 mg/kg. The h5D8 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 can be administered at a fixed dose regardless of the weight or weight of the individual to whom the h5D8 antibody is administered, provided that the individual weighs at least about 37.5 kg. The fixed dose of h5D8 to be administered may range from about 75 mg to about 2000 mg. The fixed dose of h5D8 to be administered may be from about 225 to about 2000 mg, from about 750 to about 2000 mg, from about 1125 to about 2000 mg, or from about 1500 to about 2000 mg. A fixed dose of h5D8 for administration may be approximately 75 mg. A fixed dose of h5D8 for administration may be approximately 225 mg. A fixed dose of h5D8 for administration may be approximately 750 mg. A fixed dose of h5D8 for administration may be approximately 1125 mg. A fixed dose of h5D8 for administration may be approximately 1500 mg. A fixed dose of h5D8 for administration may be approximately 2000 mg. Other dosages of h5D8 are also expected. The fixed dose of h5D8 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, 1 275, 1300, 1325, 1350, 1375, 1400, 1425, 1450, 1475, 1525, 1550, 1575, 1600, 1625, 1650, 1675, 1700, 1725, 1750, 1775, 1800, 1825, 1850, 1 875, 1900, 1925, 1950, 1975, 2025, 2050, 2075 or 2100 mg. Any of these doses can be administered approximately once per week, approximately once every two weeks, approximately once every three weeks, or approximately once every four weeks.

The fixed dose of h5D8 to be administered may range from about 75 to about 2000 mg approximately once per week. The fixed dose of h5D8 to be administered can range from

- 29 044934 approximately 75 to approximately 1500 mg approximately once a week. The fixed dose of h5D8 to be administered may be from about 225 to about 1500 mg, from about 750 to about 1500 mg, from about 1125 to about 1500 mg about once per week. A fixed dose of h5D8 for administration may be approximately 75 mg approximately once per week. A fixed dose of h5D8 for administration may be approximately 225 mg approximately once per week. A fixed dose of h5D8 for administration may be approximately 750 mg approximately once per week. A fixed dose of h5D8 for administration may be approximately 1125 mg approximately once per week. A fixed dose of h5D8 for administration may be approximately 1500 mg approximately once per week. A fixed dose of h5D8 for administration may be approximately 2000 mg approximately once per week.

The fixed dose of h5D8 to be administered may range from about 75 to about 2000 mg approximately once every two weeks. The fixed dose of h5D8 to be administered may range from about 75 to about 1500 mg approximately once every two weeks. The fixed dose of h5D8 to be administered may be from about 225 to about 1500 mg, from about 750 to about 1500 mg, from about 1125 to about 1500 mg about once every two weeks. A fixed dose of h5D8 for administration may be approximately 75 mg approximately once every two weeks. A fixed dose of h5D8 for administration may be approximately 225 mg approximately once every two weeks. A fixed dose of h5D8 for administration may be approximately 750 mg approximately once every two weeks. A fixed dose of h5D8 for administration may be approximately 1125 mg approximately once every two weeks. A fixed dose of h5D8 for administration may be approximately 1500 mg approximately once every two weeks. A fixed dose of h5D8 to administer may be approximately 2000 milligrams approximately once every two weeks.

The fixed dose of h5D8 to be administered may range from about 75 to about 2000 mg approximately once every three weeks. The fixed dose of h5D8 to be administered may range from about 75 to about 1500 mg approximately once every three weeks. The fixed dose of h5D8 to be administered may be from about 225 to about 1500 mg, from about 750 to about 1500 mg, from about 1125 to about 1500 mg about once every three weeks. A fixed dose of h5D8 for administration may be approximately 75 mg approximately once every three weeks. A fixed dose of h5D8 for administration may be approximately 225 mg approximately once every three weeks. A fixed dose of h5D8 for administration may be approximately 750 mg approximately once every three weeks. A fixed dose of h5D8 for administration may be approximately 1125 mg approximately once every three weeks. A fixed dose of h5D8 for administration may be approximately 1500 mg approximately once every three weeks. A fixed dose of h5D8 to administer may be approximately 2000 mg approximately once every three weeks.

The fixed dose of h5D8 to be administered may range from about 75 to about 2000 mg approximately once every four weeks. The fixed dose of h5D8 to be administered may range from about 75 to about 1500 mg approximately once every four weeks. The fixed dose of h5D8 to be administered may be from about 225 to about 1500 mg, from about 750 to about 1500 mg, from about 1125 to about 1500 mg about once every four weeks. A fixed dose of h5D8 for administration may be approximately 75 mg approximately once every four weeks. A fixed dose of h5D8 for administration may be approximately 225 mg approximately once every four weeks. A fixed dose of h5D8 for administration may be approximately 750 mg approximately once every four weeks. A fixed dose of h5D8 for administration may be approximately 1125 mg approximately once every four weeks. A fixed dose of h5D8 for administration may be approximately 1500 mg approximately once every four weeks. A fixed dose of h5D8 to administer may be approximately 2000 mg approximately once every four weeks. The h5D8 antibody can be administered in a dose based on the weight or body weight of the individual to whom the h5D8 antibody is administered. The body weight-adjusted dose of h5D8 to be administered may range from about 1 mg/kg to about 25 mg/kg. The body weight-adjusted dose of h5D8 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 approximately 20 mg/kg to approximately 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 h5D8 for administration may be approximately 3 mg/kg. The body weight-adjusted dose of h5D8 for administration may be approximately 10 mg/kg. The body weight-adjusted dose of h5D8 for administration may be approximately 15 mg/kg. The body weight-adjusted dose of h5D8 for administration may be approximately 20 mg/kg. The body weight-adjusted dose of h5D8 for administration may be approximately 25 mg/kg. The body weight-adjusted dose of h5D8 to be administered may range from about 1

- 30 044934 mg/kg to approximately 25 mg/kg approximately once every three weeks. The body weight-adjusted dose of h5D8 to be administered may be from about 3 mg/kg to about 25 mg/kg, from about 10 mg/kg to about 20 mg/kg, from about 15 mg/kg to about 25 mg/kg, or from approximately 20 mg/kg to approximately 25 mg/kg approximately once a week, every two, three or four weeks.

Other body weight-adjusted doses of h5D8 are also available. The body weight-adjusted dose of h5D8 may be approximately 2 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 21 mg/kg, 22 mg/kg, 23 mg/kg, 24 mg/kg, 26 mg/kg, 27 mg/kg, 28 mg/kg, 29 mg/kg, or 30 mg/kg. Any of these doses can be administered once per week, approximately once every two weeks, approximately once every three weeks, or approximately once every four weeks.

A body weight-adjusted dose of h5D8 for administration may be approximately 1 mg/kg once weekly. A body weight-adjusted dose of h5D8 for administration may be approximately 3 mg/kg once weekly. A body weight-adjusted dose of h5D8 for administration may be approximately 10 mg/kg once weekly. A body weight-adjusted dose of h5D8 for administration may be approximately 15 mg/kg once weekly. A body weight-adjusted dose of h5D8 for administration may be approximately 20 mg/kg once weekly. A weight-adjusted dose of h5D8 for administration may be approximately 25 mg/kg once weekly

The body weight-adjusted dose of h5D8 for administration may be approximately 1 mg/kg approximately once every two weeks. The body weight-adjusted dose of h5D8 for administration may be approximately 3 mg/kg approximately once every two weeks. A body weight-adjusted dose of h5D8 for administration may be approximately 10 mg/kg approximately once every two weeks. A body weight-adjusted dose of h5D8 for administration may be approximately 15 mg/kg approximately once every two weeks. A body weight-adjusted dose of h5D8 for administration may be approximately 20 mg/kg approximately once every two weeks. A body weight-adjusted dose of h5D8 for administration may be approximately 25 mg/kg approximately once every two weeks.

The body weight-adjusted dose of h5D8 for administration may be approximately 1 mg/kg approximately once every three weeks. The body weight-adjusted dose of h5D8 for administration may be approximately 3 mg/kg approximately once every three weeks. The body weight-adjusted dose of h5D8 for administration may be approximately 10 mg/kg approximately once every three weeks. A body weight-adjusted dose of h5D8 for administration may be approximately 15 mg/kg approximately once every three weeks. A body weight-adjusted dose of h5D8 for administration may be approximately 20 mg/kg approximately once every three weeks. A body weight-adjusted dose of h5D8 for administration may be approximately 25 mg/kg approximately once every three weeks.

The body weight-adjusted dose of h5D8 for administration may be approximately 1 mg/kg approximately once every four weeks. The body weight-adjusted dose of h5D8 for administration may be approximately 3 mg/kg approximately once every four weeks. The body weight-adjusted dose of h5D8 for administration may be approximately 10 mg/kg approximately once every four weeks. The body weight-adjusted dose of h5D8 for administration may be approximately 15 mg/kg approximately once every four weeks. The body weight-adjusted dose of h5D8 for administration may be approximately 20 mg/kg approximately once every four weeks. The body weight-adjusted dose of h5D8 for administration may be approximately 25 mg/kg approximately once every four weeks.

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.

The dosages described herein may result in a serum/plasma half-life of h5D8 of about 15 to about 20 days. In certain embodiments, the dosages result in a serum half-life of about 16 to 19 days. In certain embodiments, the dosages result in a serum half-life of about 17 to 18 days. In certain embodiments, dosages of h5D8 administered at 750 milligrams, 1125 milligrams, or 1500 milligrams result in a serum half-life of approximately 17 or 18 days.

A dosage of any of the amounts described herein can be formulated into a composition for the treatment of a cancer/tumor described herein. 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; polyols/disaccharides/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.

The h5D8 antibody described herein may be included in a kit containing a vial filled with a sterile solution containing the h5D8 antibody at a concentration of about 20 mg/ml, about 25 mM histidine, about 6% sucrose, and about 0.01% polysorbate 80. The vial may be a disposable glass vial. A single-use glass vial may be filled with approximately 10 milliliters of h5D8 antibody at a concentration of approximately 20 mg/ml, approximately 25 mM histidine, approximately 6% sucrose, and approximately 0.01% polysorbate 80. In certain embodiments, the pH of the solution is approximately 6.0. The h5D8 antibody described herein may be included in a kit containing a vial filled with a lyophilized composition containing the h5D8 antibody, which when reconstituted in an appropriate amount of sterile diluent provides a concentration of approximately 20 mg/ml h5D8 antibody, approximately 25 mM histidine, about 6% sucrose and about 0.01% polysorbate 80. The vial may be a disposable glass vial. The antibodies described herein can be administered or prepared or diluted for administration in a variety of ways depending on the dosage level that is ultimately to be delivered to the patient. This can be done, for example, to optimize the pharmaceutical properties of the dosage for the patient, for example to reduce the amount of suspended particles. H5D8 can be prepared at a concentration of approximately 8 mg/mL regardless of the final dose delivered to the patient. In certain embodiments, h5D8 can be formulated at a concentration of no more than about 10, 9, 8, 7, 6, 5, or 4 mg/ml. In certain embodiments, h5D8 can be formulated at a concentration of greater than about 1, 2, 3, 4, 5, 6, or 7 mg/ml.

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.

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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 immunization series 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 immunization of rats, 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 via surface plasmon resonance (SPR). Data from these experiments are summarized in table. 1.

Table 1. 5D8 Humanization Summary

Combination of heavy chain and light chain Inhibition of LIF-induced pSTAT3 according to FIG. 1 Affinity by SPR, K _D i (pm) Expression (µg/ml) H0L0 +++ 133±46 393 H1L1 - n. P. 627 H1L2 +++ 55±23 260 H1L3 +++ 54±31 70 H1L4 - N/A 560 H2L1 - N/A 369 H2L2 +++ 52±22 392 H2L3 ++ 136±19 185 H2L4 - n. P. 78 H3L1 n. P. n. P. No expression H3L2 n. And. n. P. No expression H3L3 n. And. n. P. No expression H3L4 n. And. n. P. No expression H4L1 - n. P. 259 H4L2 ++ 913±308 308 H4L3 + 252 H4L4 - n. P. 186 And. p. = not checked; H0L0 = chimeric antibody with complete rat heavy and light chain variable regions

The 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 except those containing the H3 heavy chain was observed. The H2 and L2 variable regions performed well compared to other variable regions (SEQ ID NO: 42 and SEQ ID

NO: 46).

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 Kit, 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).

Antibodies that exhibited inhibition of LIF-induced STAT3 phosphorylation were then 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. To calculate kinetic constants and affinity indices, best-fitting curves (minimum Chi2 value) were used for each concentration (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 = AB ₂ ] 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.

We selected clone H2L2 (h5D8) for further analysis and compared binding to parental rat 5D8 (r5D8) and mouse clone 1B2 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 LIF isolated from E. coli and HEK-293 cells, respectively, were 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 indices 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. The sensograms were also analyzed based on the approximation model for the sensogram of a divalent analyte [A + B = AB; AB + B = AB2], 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 indicators, the best fit curves (minimum Chi ² value) for each concentration were used. A summary of these affinity experiments is shown in Table. 2 (human LIF obtained in E. colt) and table. 3 (human LIF produced in HEK cells

293).

Table 2. Improved 5D8 binding 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

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±1OO 410±360 h5D8 (humanized) 13±6 63±30

A 1:1 Langmuir sensogram fit model from this series of experiments shows that humanized antibody 5D8 (h5D8) bound to human LIF with an affinity that was ~1025-fold greater than that of mouse 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 (E. 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 LIF (from E. coli; reference in Millipore LIF catalog 1050); human LIF (from HEK cells, ACRO Biosystems LIF-H521); mouse LIF (E. coli; 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 this affinity experiment is shown in Table. 4.

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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 of Ka (1/s) [10' 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 STAT3 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. 2A shows that the humanized clone exhibited increased levels of inhibition of STAT3 (Tyr 705) phosphorylation when the 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.

Methods

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).

After treatment, proteins were isolated using radioimmunoprecipitation assay (RIPA) lysis buffer containing phosphatase and protease inhibitors, quantified (BCA Protein Assay Kit, 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 (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). 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: IC ₅₀ value of h5D8 antibody treatment on endogenous LIF levels in U-251 cells

We also determined that for biological inhibition of h5D8 under serum starvation conditions in U-251 cells, the IC ₅₀ was only 490 pmol/L (Figure 3A). See representative results in FIG. 3A and 3B and in table. 5.

Table 5

Tissue cell line Cell line name Treatment 1C ₅₀ (nM) 1C ₉₀ (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

Methods

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 No. 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 LIF.

Other rat antibody clones (10G7 and 6B5) have been identified that specifically bind human LIF, and their binding characteristics are summarized in Table 1 below. 6, clone 1B2 served as a reference sample.

Methods

Real-time kinetic binding analysis was performed for anti-LIF mAbs 1B2, 10G7, and 6B5 immobilized on the surface of CM5 optical sensor chips 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 a 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 ko _bs analysis.

Table 6. Affinities of additional antibodies to LIF

Langmuir sensogram approximation 1:1

Analyst Clone Average value of K _a (1/Ms) Average value _Kd (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 (HEK293) 1B2 2.0 ± 0.04E6 1.4±0.2E-3 0.7 ±0.03 Human LIF (E. coli) 10G7 7.9 ± 5.8E4 6.0 ± 2.ЗЭ-4 12.6 ±9.5 Human LIF (HEK293) 10G7 3.6 ± 1.75E5 3.1 ±0.5E-4 1D ±0.6 Human LIF (E coli) 6B5 n. P. n. P. n. P. Human LIF (HEK293) 6B5 3.6 ± 1.7E5 3.1±0.5E-4 62 ±6

Example 8 Additional anti-LIF antibodies inhibit LIF-induced STAT3 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 (positive) were included as a positive control, while 6B5 showed no inhibition, this may be explained by the possible lack of binding of 6B5 to non-glycosylated LIF, which was used in this experiment.

Methods

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 Kit, 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 (p-STAT3, cat. no. 9145, Cell Signaling) or 30 min (β-actin peroxidase, cat. no. catalog 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).

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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. As shown in FIG. 5, LIF is highly expressed in glioblastoma multiforme (GBM), non-small cell lung cancer (NSCLC), ovarian cancer, and colorectal cancer (CRC).

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.

Methods

The non-small cell lung cancer (NSCLC) cell line KLN205 with high levels of LIF was infected with lentivirus to stably express the firefly luciferase gene to monitor 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 weekly, 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 (i/p) 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.

Methods

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 (Rompύn®) (75 mg/kg 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 injected intraperitoneally with h5D8 twice a week. 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.

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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 IP 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.

Methods

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 5x10 ⁶ .

Mice were administered h5D8 twice a week i/p 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 i.p. twice weekly) significantly suppressed tumor growth (Figures 9A and 9B).

Methods

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 (IP) 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 ⁺ 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.

T cell depletion methods

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 administered r5D8 intraperitoneally twice weekly 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 twice.

- 39,044,934 per week as stated in the study design. All antibody treatments were used simultaneously. Example 16 Crystal Structure of h5D8 in Complex with Human LIF The crystal structure of h5D8 was solved to 3.1 angstrom 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.

Table 7. Summary of X-ray crystal structure of h5D8 complexed with human LIF

LIF balance Type Remnant h5D8 (paratope, numbering according to (epitope) interactions Kabatu) 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 L-Asn34, L-Leu46, L-Tyr49, L-Glu55, L-Ser56 Argl5 V.D.W. H-Glu96, H-Trp97, H-Asp98, H-Leu99, H-Aspl01 Hisl6-NE2 S.B. H-Aspl01-OD2 Hisl6 L-Tyr32, L-Asn34, L-Met89 V.D.W. H-Trp95, H-Glu96, H-Trp97, H-Aspl01 Prol7 V.D.W. L-Tyr32, L-Ala91

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N-Tgr97 Cysl8 V.D.W. L-Tyr32 N-TgrZZ, N-Tgr97 Hisl9-NE2 S.B. H-Glu96-OE1, H-Glu96-OE2 Hisl9 V.D.W. H-His31, H-Tgr33, H-Glu96 Asn20-OD1 NV H-Lys52-NZ Asn20-ND2 NV H-Asp53-OD1 Asn20 V.D.W. H-Tgr33, H-Lys52, H-Asp53 Gln25-NE2 NV 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 NV H-Ser30-OG Aspl20 V.D.W. H-Thr28, H-Ser30 Argl23-NE NV H-Thr28-OG Argl23 V.D.W. H-Thr28 Glyl24 V.D.W. H-His31 Leul25 V.D.W. H-His31 Serl27-OG NV H-Asp98-OD2 Serl27-O NV H-Trp97-NE1 Serl27 V.D.W. H-His31, H-Trp97, H-Asp98 Asnl28-OD1 NV 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 NV L-His30-NE2 Serl35 V.D.W. L-His30 Hisl38 V.D.W. L-His30 VDW, van der Waals forces, low-energy coupling; HB, hydrogen bond (medium energy bond); SB, salt bridge (high energy bond)

Methods

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 of 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, 125132 (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).

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Example 17: h5D8 is highly specific for LIF

The present inventors attempted 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 Determined 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 ZDE-OZ +/-4DE-05 h5D8 + huOSM (from a mammalian system) C. o. C. o. C. o. h5D8 + huIL-6 (E. coli) C. 0. C. 0. C. 0. C.o. = no binding

Methods

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); iii) baseline (60 s); iv) binding (180 s) and v) dissociation (600 s) Affinity of h5D8 for cytokines according to Octet: the experiment to determine the basic kinetic parameters was carried out using Octet Data Acquisition software version 9.0.0.26 as follows: Amine Reactive 2nd Generation 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 s quench in 1 M ethanolamine, pH 8.5; v) 120 s at baseline in water. Then experiments were carried out to determine the kinetic parameters with the following stages of immersion and readout at 30°C, 1000 rpm; vi) 60 s at 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 diffusion methods, and they made it possible to determine

- 42 044934 five crystal structures with a resolution from 1.65 to 2.0 A. All structures were deciphered 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 ranging from 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 conformations of CDR residues 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 revealed that positively and negatively charged regions contributed equally to hydrophilic properties without 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 our five crystal structures.

Methods

H5D8-1 IgG was obtained from Catalent Biologics 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 h 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 ACTa 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 ACT Pure chromatography system (GE Healthcare). Elution with a gradient of 1 M potassium chloride resulted in a predominant peak of Fab h5D8, 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 h5D8 Fab was concentrated to 25 mg/ml using a 10K MWCO concentrator (Millipore). 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) 0.1 M MES, 20% (w/v) PEG 6000, 1 M lithium chloride, pH 6.0; 3) 0.1 M MES, 20% (w/v) PEG 4000, 0.6 M sodium chloride, pH 6.5; 4) 0.085 M 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 Cysteine 100 Mutations in h5D8 Retain 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 naturally occurring 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. EC ₅₀ curves for ELISA are shown in FIG. 15 (Fig. 15A, human LIF, and Fig. 15B, mouse LIF).

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Table 9. Summary of Affinity Measures Determined by Analysis . Octet, and EU ₅₀ values determined by ELISA.

Mutation Affinity/Λ^ (M) EC50 binding (nM) Human LIF Mouse LIF Human LIF Mouse LIF C100 <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 4DE-09 ± 7.56E-11 7,179 25.3 C100D 2.02E-09 ± 8.08E-11 6.49E-09 ± 7D6E-11 11.89 22.88 C100T 4.36E-10 ± 2DE-11 1.02E-09 ± 1.77E-11 5.575 8,753 C100G 2.49E-09 ± 4.2E-11 3.33E-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 S100M 1D55E-09 ± 3,400E-11 2.676E-09 ± 2.449E-11 0.7852 1,563 C100F 4.376E-09 ± 1D27E-10 1D47E-08 ± 9.099E-11 8,932 21.53 C100Y 1D44E-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. S100K 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.

Methods

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. The plates were 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 bound to the loaded biosensors for 90 s and allowed to dissociate in 1× kinetic buffer for 300 s. 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 were also used that either do not bind LIF (isotype control, indicated (-)) or bind LIF at known binding sites (B09 does not compete with either

- 44 044934 gp130, nor with LIFR for LIF binding; r5D8 is the rat original version of 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 vessels [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 FIG. 17B is 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 gyrus); 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 (PBMCs); 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 - 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); 59 - 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. Therefore, 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 population in the phase 1 study, MRSD was confirmed with these data. component 1 mg/kg (or fixed dose 75 mg) i.v.

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The pharmacologically active dose (PAD) was also taken into account when establishing MRSD. Based on pharmacological, PK and LIF level 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 _trough value of approximately 500 μg/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 produce a _Cmax 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: Phase 1 dose escalation and dose expansion study for h5D8.

To establish the safety profile and appropriate dose of h5D8 as monotherapy against various types of cancer, a phase 1 clinical trial was initiated. The primary objectives were: 1) to evaluate the safety and tolerability of h5D8 in patients with advanced solid tumors; 2) determine the recommended dose for h5D8 monotherapy; and 3) evaluate the preliminary antitumor activity of h5D8 as measured by overall response rate (ORR) according to RECIST 1.1 criteria. Secondary objectives were to: characterize the PK and immunogenicity of h5D8; and 2) evaluate efficacy parameters in patients with advanced solid tumors, including disease control rate (DCR) and progression-free survival (PFS) using RECIST 1.1. The research objectives were to: a) examine the relationship between the pharmacokinetics, pharmacodynamics, and exposure of h5D8 with patient safety and antitumor activity; b) evaluate whether high tumor LIF expression correlates with the antitumor activity of h5D8; c) characterize the pharmacodynamic effects of h5D8 in the periphery and tumor and d) characterize the effect of h5D8 treatment on investigative biomarkers. The study was designed as an open-label phase 1 study and included patients with advanced solid tumor. The study was conducted and is being conducted using a 3 + 3 accelerated dose titration design with fixed doses of h5D8 administered intravenously every 3 weeks (in cohorts with doses of 75 mg, 225 mg, 750 mg, 1125 mg and 1500 mg).

According to the design, antitumor response was assessed using the Response Evaluation Criteria in Solid Tumors (RECIST) guideline 1.1. According to the design, assessments were performed at baseline and every 6 weeks for the first 6 months, and then every 12 weeks until disease progression was confirmed or the patient was withdrawn from the study. According to the design, adverse events were graded according to the Common Terminology Criteria for Adverse Events (CTCAE) version 4.03 and assessed continuously during the study and for 30 days after the last course of treatment.

Forty-one patients were enrolled and dosed. Patient demographics are shown in Table. 10.

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Table 10

Median age (range) 64.5 (36-78) Gender, N (%) Male 20 (50%) Female 20 (50%) Race, N (%) Caucasian 33 (82.5%) Negroid 4 (10%) Mongoloid 2 (5%) Other 1 (2.5%) Tumor type, N (%) Pancreas 13 (31.7%) Ovarian 5 (12.2%) Colorectal 3 (7.3%) Prostate 3 (7.3%) Appendix 2 (4.9%) Cholangiocarcinoma 3 (4.9%) Nasopharynx 4 (4.9%) NSCLC 5 (4.9%) Rectum 6 (4.9%) Squamous 7 (4.9%) Adenoid cystic carcinoma (ACC) 1 (2.4%) Melanoma 1 (2.4%) Metastatic myxoid liposarcoma 1 (2.4%) Paraganglioma 1 (2.4%) Uterine cancer 1 (2.4%)

For the trial, dose-limiting toxicities (DLTs) were defined as 1) effects observed within 21 days after cycle 1, day 1, as assessed by the primary reviewer in consultation with the data review committee (DRC) as possibly related to h5D8; 2) any drug-related adverse event (AE) > grade 3. DLT did not include AEs with a clear alternative explanation and predefined, self-limiting grade 3 AEs, including: 1) fatigue, nausea, vomiting, or diarrhea that resolves to <grade 2 within 72 hours with appropriate drug therapy; 2) temporary (lasting <72 hours) biochemical abnormalities of grade 3, which are considered clinically insignificant; 3) grade 3 neutropenia lasting <72 hours; 4) thrombocytopenia grade 3 without clinically significant bleeding. The DRC may consider as a DLT a drug-related AE of any grade that delays the onset of cycle 2, day 1 by >14 days. Summary of safety data for cohorts 1–5 to date are shown in Table. eleven.

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Table 11. Safety Summary

Cohort 1 Cohort 2 Cohort 3 Cohort 4 Cohort 5 Total Number of subjects with adverse events (AE) 2 (N=2) 1 (N=l) 10 (N= 10) 8 (N=10) 6 (N=ll) 27 (79.4%) (N = 34) Number of subjects with AE > grade 3 2(U) 0 6(11) 1(1) 1(1) 10 (23) (29.4%) Number of subjects with h5D8-related AEs: 0 1(2) 4(8) 3(9) 3(5) 11 (24) (32.4%) SAE 8 0 8 0 2 18 Related to h5D8 SAE 0 0 0 0 0 0 Deadly AE 1 0 1 0 0 2 Infusion-related AEs 0 1 1 0 0 2 DLT for C1 (21 days) 0 0 0 0 0 0 Postponed DLT 0 0 0 0 0 0 AE leading to interruption of h5D8 reception 0 0 1 1 0 2 AEs leading to early termination of h5D8 administration 0 0 1 0 0 1 AEs leading to changes in h5D8 dose 0 0 0 0 0 0

No dose-limiting toxic effects or tolerability issues were observed at any dose. Overall, the data indicate that h58 is safe and well tolerated at all doses tested.

Example 24: Single case studies of patients treated with h5D8.

Subject 0106-002 was a 68-year-old Caucasian woman with stage IV pancreatic cancer who was intensively pretreated with 4 lines of prior systemic anticancer therapy against metastatic disease. The subject was initially diagnosed with stage II/III moderate to poorly differentiated pancreatic ductal adenocarcinoma. The subject was treated with neoadjuvant FLOFIRINOX for approximately 167 days and achieved a partial response. The subject underwent radical laparoscopic distal pancreatectomy and splenectomy followed by the adjuvant drug gemcitabine for approximately 7 months when the subject developed recurrent progressive disease in the pancreatic bed. The subject was treated with gemcitabine and abraxane for approximately 2 months, with the best response being a partial response followed by disease progression in the pancreatic bed and peritoneal lymph nodes. The subject then received 5FU and Onivid (liposomal irinotecan) for approximately 2 weeks, which was discontinued due to toxicity. The subject was treated for approximately 2 months with an experimental Wnt inhibitor (from Samumed) and had disease progression with malignant lymphadenopathy above and below the diaphragm. The subject received a pyrimidine nucleoside metabolism inhibitor (Fujiflim) for approximately 4 months, with the best response being a partial response. The subject was confirmed to have progression as well as elevated CA19-9 by imaging and was enrolled in the h5D8 study. The results of her previous treatments prior to the h5D8 trial are summarized in Table 1. 13 (PR=partial response; PD=progressive disease; SD=stable disease).

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Table 12

Palliative anticancer therapy Duration of therapy up to P.D. Best answer CA19-9 level at the beginning of treatment Level CA19-9 with best response Level CA19-9 in PD Gemcitabine/Abrax en 2 months PR 63 UNK 97 Wnt inhibitor 2 months P.D. 1088 N.A. 4195 Pyr imide inhibitor of NEW nucleosides 4 months PR 4195 1249 1658

The subject received her first dose of h5D8 at 1125 mg and the most recent dose was on cycle 9 (C9) (1500 mg) approximately 165 days later. At baseline, the subject had no clinically significant laboratory abnormalities; US Eastern Cooperative Oncology Group (ECOG) status = 1; elevated CA19-9 level was 1658. Two lymph nodes (one thoracic and one abdominal) were identified as target lesions, and abdominal lymphadenopathy was identified as a non-target lesion. On cycle 3 day 1 (C3D1), her ECOG status improved to 0, her CA19-9 level dropped to 1069, and the overall RECIST response was stable disease. Traditionally, the CA19-9 level has been a reliable prognostic marker of the disease. According to the doctor, there was no longer any need to use oxycodone for abdominal pain. The subject received cycle 6, and after approximately 2 weeks, the patient's analgesic requirements increased and the subject was hospitalized away from the trial site. The subject has now returned to baseline, requiring narcotic analgesia twice daily. The subject underwent follow-up scans and was found to be stable. Her CA19-9 levels increased during cycle 5, but returned to below baseline levels on cycle 9 and below the initial decrease on cycle 3. The results are shown in Table 1. 13. U

No treatment-related adverse events were observed in the subject. C9 Tumor Original Table 13 C7 nw C5 markers SA level level 1658 1069 1224 1397 1045.8 19-9 (units/ml) Target lesion Original NW C5 C7 C9 by RECIST Rib cage level 15 mm 17 mm 14 mm 14 mm 13 mm Abdominal 19 mm 19 mm 20 mm 18 mm 15 mm cavity Amount LD 34 mm 36 mm 34 mm 32 mm 28 mm Non-target lesion Abdominal Present Not CR, not Not CR, not Not CR, not Not CR, not cavity P.D. P.D. P.D. P.D. New hearth N.A. No No No No General answer N.A. Stabilization; Stabilization; Stabilization Stabilization;

diseases diseases diseases diseases

Subject 0102-001 was a 78-year-old Caucasian woman with stage IV uterine leiomyosarcoma who was pretreated with 6 lines of systemic anticancer therapy. The subject was initially diagnosed with T1B uterine leiomyosarcoma and underwent radical TAH/BSO. Local recurrence occurred approximately 3 years after the initial diagnosis, for which surgical resection was performed; chemoradiotherapy was declined. Approximately 2 years later, the subject relapsed and was treated with 4 courses of gemcitabine and docetaxel, achieving a mixed response. The subject then received 3 cycles of Doxil, with the best response being progressive disease. This was followed by 2 months of vinorelbine with the best response being PD. Trabectidine was administered for approximately 4 months with mixed response and discontinued due to toxicity. Subject was then treated with DTIC for 4 months with PD as best

- 49 044934 answers. As a result of undergoing radiation therapy (XRT) applied to the major pelvic masses, the patient received a total of 3750 cGy in 10 servings over approximately days and then received Votrient for approximately 1 month with PD as the best response. The results of her previous courses of treatment are presented in Table. 14.

Table 14

Palliative anticancer therapy Duration of therapy until PD Best answer Gemcitabine/docetaxel 3 months Mixed Doxorubicin 2 months P.D. Vinorelbine 2 months P.D. Trabectidin D/C for toxicity Mixed DTIC 4 months P.D. Votrient 2 months P.D.

At baseline, the subject had no clinically significant laboratory abnormalities; ECOG=1 and elevated CA-125 level was 13. Subject received her first dose of 750 mg h5D8 and her CA-125 level decreased to 9 by C3. When assessed at C5, her CA-125 level was 8, and the overall RECIST response was stable disease.

Based on her baseline scans, one (1) pulmonary lesion, 1 liver lesion, 2 rectus muscle lesions, and 1 pouch of Douglas lesion were identified as target lesions, and a peritoneal lesion was identified as a non-target lesion. When assessed at C7, her CA-125 level was 9, and the subject had an overall RECIST response of stable disease. Images from C7 3 target lesions and ports for previous radiation therapy are shown in FIG. 18A-C. And the above results are shown in table. 15.

Table 15

Tumor markers Baseline NW C5 C7 EOT CA125 level (units/ml) 13 9 8 9 10 Target lesion according to RECIST Baseline NW C5 C7 EOT Lung 13.4 mm 17.7 mm 15.7 mm 20.0 mm 27.1 mm Liver 14.9 mm 14.5 mm 17.7 mm 18.1 mm 22.6 mm Rectus muscle, M No. 1 39.1 mm 37.6 mm 38.0 mm 37.8 mm 40.1 mm Rectus muscle, M No. 2 31.9 mm 26.6 mm 22.7 mm 24.3 mm 28.5 mm Douglas space 36.9 mm 28.3 mm 24.9 mm 25.0 mm 25.0 mm Amount LD 136.2 mm 124.7 mm 119 mm 125.2 mm 143.3 mm Non-target lesion Peritoneal nodule Comfort present Not CR/not P.D. Not CR/not P.D. Not CR/not P.D. NE New hearth N.A. No No No No General answer N.A. Stabilization of the disease Stabilization of the disease Stabilization of the disease Progressing her disease

Subject 0101-001 was a 50-year-old woman with stage IV KRAS+ colorectal carcinoma receiving first-line systemic anticancer therapy for metastatic disease. The subject was initially diagnosed with stage III moderately differentiated colorectal adenocarcinoma (T3N2M0). The subject underwent radical robotic resection of the inferior anterior portion followed by 5FU and FOLFOX adjuvant for approximately 7 months. Approximately 2 years later, the subject relapsed with pulmonary metastases and chose watchful waiting and did not receive palliative systemic therapy for stage IV disease. Approximately 1 year after relapse, lung metastases continued to progress. The subject refused first-line chemotherapy against mCRC and chose the h5D8 clinical trial as first-line therapy.

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The subject received her first dose of h5D8 at 225 mg and her most recent dose was at C6 (750 mg) approximately 4 months later. At baseline, the subject had no clinically significant laboratory abnormalities; ECOG status = 0 and elevated tumor marker level (CEA) was 11.8. Two (2) right lower lobe lung lesions were identified as target lesions and 2 non-target lesions (ovary and bone) were identified. When assessed at C3, the response was stable disease and CEA rose to 11.8. When assessed at C5, the subject had stable disease followed by progression on post-treatment radiological imaging at C6. The results are shown in table. 16.

Table 16

Tumor markers Baseline NW C5 EOT CEA 10.5 11.8 17.9 20.1 Target lesion according to RECIST Baseline NW C5 EOT Lung, No. 1 12.8 mm 13.9 mm 15.7 mm 18.6 mm Lung, No. 2 11.7 mm 12.1 mm 12.7 mm 13.5 mm Amount LD 24.5 mm 26 mm 28.4 mm 32.1 mm Non-target lesion Bone Present Not CR, not PD Not CR, not PD Not indicated Ovary Present Not CR, not PD Not CR, not PD Not indicated New hearth N.A. No No No General answer N.A. Stabilization of the disease Stabilization of the disease Progressive disease

Subject 0106-005 was a 75-year-old Caucasian woman with fallopian tube carcinoma. The subject underwent radical BSO FNA, LN excision and tumor mass removal followed by adjuvant carboplatin and Taxol. Approximately 5 years later, the subject relapsed with malignant adenopathy and received palliative radiotherapy (55 cGy) to the LN at the aortic bifurcation. The subject had LN progression and received palliative radiotherapy (60 cGy) to the periaortic LNs. The subject developed lung metastases and was treated with an experimental BET inhibitor for approximately 1 month, which was discontinued due to toxicity, after which she received an experimental anti-PD-1 antibody for approximately 4 months, with best response being stabilization of the disease. The subject was then treated with an experimental TIGIT inhibitor for approximately 200 days, with the best response being stable disease. After progression, the subject was treated with an experimental monoclonal antibody for approximately 4 months, with the best response being stable disease. The subject was confirmed to have progressed and was enrolled in the h5D8 study. The results of her previous courses of treatment are presented in Table. 17.

Table 17

Palliative cancer therapy Duration of therapy until PD Best answer Antibodies to PDL-1 16 months SD TIGIT inhibitor 7 months SD Pyrimidine nucleoside inhibitor 4 months SD

Subject received her first dose of 750 mg h5D8; at C4, 2 months later, the h5D8 cycle dose was increased to 1125 mg. Her last dose (C6) was administered approximately 44 days after this dose increase. Approximately 1 month after treatment, the subject had progression on imaging and had her EOT visit. At baseline, the subject had no clinically significant laboratory abnormalities and her ECOG score = 1.

The subject had no expression of tumor markers in the blood. Two lung lesions (one in the right upper lobe and one in the left upper chest) were selected as target lesions, and multiple pulmonary nodules were selected as non-target lesions. The subject experienced no treatment-related adverse events. The results of these tests are shown in table. 18.

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Table 18

Target lesion Baseline Cycle 3 Cycle 5 EOT RUL 20 mm 20 mm 22 mm 28 mm Left upper chest opening 36 mm 40 mm 41 mm 44 mm Amount LD 56 mm 60 mm 63 mm 72 mm Non-target lesion Pulmonary nodules Present Not CR, not PD Not CR, not PD Not CR, not PD New hearth N.A. No No No General answer N.A. Stabilization of the disease Stabilization of the disease Progressive disease

Subject 0301-002 was a 66-year-old Caucasian woman who was diagnosed with ovarian cancer. The subject was treated with neoadjuvant carboplatin and Taxol, followed by radical hysterectomy, BSO, and omentectomy after approximately 4 months and additional carboplatin/Taxol for an additional approximately 3 months. Approximately 8 months later, the subject relapsed and received palliative carboplatin/Taxol chemotherapy for approximately 6 months; There is no information about the best answer. Approximately 3 months after this chemotherapy, the subject progressed on imaging and was treated with doxorubicin for approximately 7 months, with the best response being stable disease; approximately 4 months later, the subject began to progress again. The subject then received taxol alone for approximately 3 months, with best response being progressive disease. The subject was treated for approximately 6 months with gemcitabine and carboplatin, with the best response being stable disease. Approximately 3 months after treatment, the subject progressed on imaging and was enrolled in the h5D8 study. The results of her previous courses of treatment are presented in Table. 19.

Table 19

Palliative cancer therapy Duration of therapy until PD Best answer Carboplatin/Taxol 9 months UNKNOWN Doxorubicin 11 months SD Takeol 2-3 months P.D. Carboplatin/gemcitabine 5 months NOT EVALUABLE

Subject received her first dose of 1500 mg h5D8. Her most recent dose on C7 was approximately 5 months late. At baseline, the subject had no clinically significant laboratory abnormalities and her ECOG score = 0. At baseline, her CA-125 level was 412.3. Three lesions were selected as target lesions (1 pancreatic implant, 1 subcostal soft tissue, and 1 pleural node) and 3 nontarget lesions were identified (liver, peritoneum, and retroperitoneal lymph nodes). The subject experienced no treatment-related adverse events. The results of its testing are shown in table. 20.

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Table 20

Tumor markers Baseline Cycle 3 Cycle 5 Cycle 7 CA level 125 412.3 Not done 1071.7 1613.6 Target lesion Baseline Cycle 3 Cycle 5 Cycle 7 Right pleural 24 mm 25 mm 25 mm 26 mm node ST mass in the subcostal 51 mm 52 mm 52 mm 61 mm region Implant in 32 mm 30 mm 32 mm 57 mm pancreas gland Amount LD Non-target lesion 107 mm 107 mm 109 mm 144 Segment V Present Not CR, not PD Not CR, not PD Not CR, not PD liver In the peritoneum Present Not CR, not PD Not CR, not PD Not CR, not PD Retroperitoneal Present Not CR, not PD Not CR, not PD Not CR, not PD LN New hearth N.A. No No No General answer N.A. Stabilization Stabilization Progressive diseases diseases disease

Subject 0102-004 was a 65-year-old Caucasian woman who was diagnosed with head and neck cancer. The subject underwent radical total glossectomy followed by adjuvant radiation therapy (6000 cGy) one month later. Approximately 10 months later, the subject relapsed with lung metastases, which were treated with radiation (5000 cGy) over an approximately 1-week course. Progression of lung disease and new bone metastases were observed at 8 months post-irradiation, and the subject was treated with ipilimumab and nivolumab for approximately 1 year and 45 days, with the best response being stable disease. The subject progressed on imaging and was enrolled in the h5D8 study. The results of her previous treatment are presented in Table. 21.

Table 21

Palliative cancer therapy Duration of therapy until PD Best answer Ipilimumab/nivolumab 13 months SD

The subject received her first dose of h5D8 at 1500 mg and her most recent dose at C7 was approximately 155 days later. At baseline, the subject had no clinically significant laboratory abnormalities and her ECOG score was 1. The subject had no serum expression of minor tumor markers. Four lesions were selected as target lesions (2 lung lesions and 2 pleural lesions) and two bone lesions were identified as non-target lesions. The results are shown in table. 22.

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Table 22

Target Original Cycle 3 Cycle 5 Cycle 7 hearth level RLL 37 mm 35.4 mm 36.3 mm 39.4 mm RUL 37.8 mm 36.8 mm 37.2 mm 40.8 mm Pleura 13.3 mm 13.6 mm 13.9 mm 14.3 mm Pleura 37.3 mm 37.5 mm 38.4 38.6 mm Amount LD 125.4 mm 123.3 mm 125.8 133.1 Non-target hearth Bone Present Not CR, not PD Not CR, not PD Not CR, not PD textile Bone Present Not CR, not PD Not CR, not PD Not CR, not PD textile New hearth N.A. No No No General N.A. Stabilization Stabilization Stabilization answer diseases diseases diseases

Subject 0201-003 was a 57-year-old Caucasian male diagnosed with myxoid liposarcoma. The subject was treated with neoadjuvant adriamycin and ifosfamide for approximately 4 months and neoadjuvant radiation therapy (5000 cGy) to the right posterior calf for approximately 35 days. The subject then underwent radical wide resection of the right posterior tibia, excision of the right posterior tibial nerve and popliteal, anterior and posterior tibial and peroneal vessels. The subject relapsed with pleural disease and malignant lymphadenopathy in the chest. The subject was treated with gemcitabine and taxotere for approximately 41 days, with best response being progressive disease. The subject was then treated with dacarbazine for 4 months, with the best response being a partial response. After 22 days, the subject progressed on imaging and was enrolled in the h5D8 study. The results of his previous courses of treatment are presented in Table. 23.

Table 23

Palliative cancer therapy Duration of therapy until PD Best answer G emcitabine/taxotere 1 month P.D. DTIC 4 months PR

The subject received his first dose of h5D8 at 1125 mg and his most recent dose was (C6) (1500 mg) approximately 136 days later. At baseline, the subject had no clinically significant laboratory abnormalities and an ECOG score of 1. The subject had no peripheral blood tumor markers. Two pleural tumor lesions were selected as target lesions and three non-target lesions were selected (1 pleural tumor mass and 2 LNs). The results are shown in table. 24. The subject experienced no treatment-related adverse events.

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Table 24

Target lesion Baseline Cycle 3 Cycle 5 Right lower pleural tumor mass 110 mm 106 mm 103 mm Right upper pleural tumor mass 56 mm 61 mm 64 mm Amount LD 166 mm 167 mm 167 mm Non-target lesion Right pleural tumor mass Present Not CR, not PD Not CR, not PD Right supradiaphragmatic LN Present Not CR, not PD Not CR, not PD Right paratracheal LN Present Not CR, not PD Not CR, not PD New hearth N.A. No No General answer N.A. Stabilization of the disease Stabilization of the disease

Example 25: Biomarkers Indicative of Positive Response to Treatment with h5D8.

Subject 0201-003 was a 57-year-old Caucasian male diagnosed with myeloid liposarcoma. The results of his current treatment regimen are presented in Table. 23. When assessing the subject at C5 of treatment with h5D8 after 12 weeks, there was no increase in RECIST criteria for his pulmonary metastatic lesion size (167 mm) (See Table 24). The subject is completing this treatment regimen (+14 weeks) and the best response recorded was stable disease. Biopsies were collected from the metastatic site of the lung to determine biomarkers for treatment with h5D8. At the time of biopsy collection, the subject showed signs of stabilization of LIF levels in the saturated state. Stabilization of LIF levels in subject 0201-003 is shown in FIG. 19. In biopsies taken during treatment, biomarkers of antitumor immunity were observed compared with the stage before treatment with h5D8. The results are presented in Fig. 20A-C. The results show an increase in the infiltration of CD8-positive T cells, as well as an increase in tumor-associated macrophage (TAM) populations. See fig. 20A. TAM cells showed an immunostimulatory phenotype (MHCII+). See fig. 20V. A decrease in the number of pSTAT3+ nuclei was also observed. See fig. 20C.

Subject 0301-003 was a 47-year-old woman with stage IV retroperitoneal paraganglioma. The results of her treatment courses before treatment with h5D8 are presented in Table. 25.

Table 25

Palliative cancer therapy Duration of therapy until PD Best answer Cyclophosphamide/vincristine/dacarbazine 2 years PR Selective RET inhibitor 5 months SD Octreotide 4 months SD

Her evaluation at C3 of treatment with h5D8 after 6 weeks showed stable CEA levels (0.5 ng/mL) and stable disease according to RECIST criteria for metastatic target sites in the lungs and liver. The patient remains on this treatment regimen (+11 weeks) and the best response recorded was stable disease. Biopsies were collected from the metastatic liver site to determine biomarkers for treatment with h5D8. At the moment, data on stabilization of the LIF level have not been processed. In biopsies taken during treatment, biomarkers of antitumor immunity were observed compared with the stage before treatment with h5D8. The results are presented in Fig. 21A-S. From the results, an increase in CD8-positive T cell infiltration was evident, see FIG. 21A, and a decrease in TAM polarization towards suppressive phenotypes (CD163+; CD206+). See fig. 21B. A decrease in the number of pSTAT+ nuclei was also observed. See fig. 21C.

Subject 0301-004 was a 74-year-old man with stage IV pancreatic adenocarcinoma. Prior to treatment with h5D8, the subject completed two courses of treatment. The results of his previous treatment are presented in Table. 26.

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Table 26

Palliative cancer therapy Duration of therapy until PD Best answer Gemcitabine-NAB, paclitaxel 3 months PR FOLFOX 2 months P.D.

Subject 0301-004 had a very aggressive form of cancer that was virtually immediately negative with two first-line therapies given at the time of tumor onset, including immediate onset of advanced disease state on FOLFOX. Subject undergoes this treatment regimen using h5D8 (6 weeks). A biopsy specimen was collected from the metastatic area of the liver. At the moment, data on stabilization of the LIF level have not been processed. In biopsies taken during treatment, biomarkers of antitumor immunity were observed compared with the stage before treatment with h5D8. The results are shown in Fig. 22. Based on immunohistochemical analysis, the subject was characterized as LIF ^low . Despite the rapid progression of the disease with a highly aggressive chemotherapy regimen, an increase in CD8+ T cell populations was observed in the tumor microenvironment (see Fig. 22). Moderate effects were observed on macrophage populations (data not shown). And no differences were observed in pSTAT3+ nuclei (data not shown).

Subject 0201-004 was a 66-year-old man who was diagnosed with stage IV melanoma. The subject received one course of treatment prior to the h5D8 trial. Best response was not evaluable and results not shown (nivolumab; 4 months). Unsuccessful treatment with nivolumab indicated profound immune suppression by the tumor. The subject is completing this treatment regimen (6 weeks) and the best response recorded was progressive disease. A biopsy specimen was collected from the metastatic skin area. At the time of biopsy collection, the subject had generally elevated LIF levels on the stabilization assay, with no evidence of stabilization of LIF levels in the saturated state. The results of the LIF level stabilization assay are shown in FIG. 23. In biopsies taken during treatment, biomarkers of antitumor immunity were observed compared with the stage before treatment with h5D8. The results are shown in Fig. 24. Based on immunohistochemical analysis, the subject was characterized as LIF ^high . Macrophage analysis was limited to the tumor microenvironment (TME) and showed little change in T cell activity (data not shown). Increased polarization of macrophage phenotypes towards MHCII+ was observed (see Fig. 24). Preliminary results from rapid pathology examination indicated that the number of pSTAT3+ nuclei decreased in the sample during treatment.

Subject 0301-002 was a 66-year-old Caucasian woman who was diagnosed with ovarian cancer. The results of her current treatment regimen are presented in Table. 19. When assessing a subject at C5 of treatment with h5D8 at 12 weeks, CA19-9 levels showed an increase (412 to 1072 U/mL), but her target lesions did not show a significant increase according to RECIST criteria (107 to 109 mm) (see table 20). The subject is completing this treatment regimen (+16 weeks) and the best response recorded was stable disease. Biopsies were collected from the metastatic lymph node site to determine biomarkers for treatment with h5D8. At the time of biopsy collection, the subject showed signs of stabilization of LIF levels in the saturated state. Stabilization of LIF levels in subject 0201-003 is shown in FIG. 25. Biomarkers of antitumor immunity were observed in biopsies taken during treatment compared with pretreatment with h5D8. There were no observed changes in tumor immunological biomarkers (data not shown). The subject was classified as LIF ^low based on immunohistochemical analysis.

A summary of the above test results to identify biomarkers for effective treatment using h5D8 is presented in Table. 27. Effects of h5D8 treatment on enhancing the antitumor immune response, TAM modulation, and LIF-mediated signaling are evident across several parameters. (NC=no change; NE=no effect).

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Table 27

Tumor type Biopsy site Dose (mg) Statu with CD8 infiltration Deviations eTAM pSTAT 3 Paraganglioma Liver 112 5 +11 weeks + -in M2 - Myxoid liposarcoma Lung 112 5 +14 weeks ++ ++ to Ml NC Pancreas Liver 112 5 6 weeks + NC NC Melanoma Leather 112 5 6 weeks - +/- - Ovarian Lymph node 150 0 +16 weeks - NE NE

Example 26 PK/PD values of h5D8 in hD5-administered subjects.

To determine the pharmacokinetics of the h5D8 antibody in humans, the study measured serum levels of h5D8 in treated patients. Briefly, quantification of h5D8 antibody levels in human serum samples was performed using a sandwich immunoassay. h5D8 antibody in patient serum samples was captured on rhuLIF (recombinant human LIF) coated MSD plates and detected using sulfo-labeled anti-human IgG (sulfo-anti-h5d8-Fab2-IgG). The signal from the sulfo group was measured using an MSD S600 reader and quantified using the h5D8 standard curve. From the results shown in FIG. 26, it can be seen that h5D8 exhibited standard pharmacokinetics with an estimated half-life of approximately 17 days. In addition, the results show that h5D8 exhibits a linear PK in the dosage range of 750-1500 mg Q3W, with saturation of target-mediated drug distribution occurring at doses greater than 225 mg.

To determine whether the target LIF was bound to the h5D8 antibody, a capture ELISA assay was used to determine total LIF levels after treatment. The plasma half-life of LIF is relatively short and is increased by binding through h5D8, resulting in increased plasma levels of LIF. Thus, an increase in LIF levels in the periphery indicates target binding. In fig. 27A-B show total LIF levels over time in several patients after receiving IV h5D8. Overall, these data show saturation of targets after three cycles of drug administration at non-saturating dose levels (cohorts 2-3) (see FIG. 27B).

Total LIF levels in patient serum samples were quantified using a sandwich immunoassay. Briefly, MSD plates were spotted with anti-LIF capture antibody A4 (rabbit monoclonal). After incubation with patient serum samples, the bound LIF/h5D8 complex was detected using the sulfo-labeled anti-LIF antibody 7C3 PB001. Signals from sulfo groups were measured using an MSD S600 reader, and LIF/h5D8 levels in patient serum were quantified using a rhLIF/h5D8 standard curve.

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. 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.

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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 eleven SVSNLES 12 svs 13 MQATHAPPYT 14 EVQLVESGGGLVKPGGSLKLSCAAS 15 QVQLQESGGGLVKPGGSLRLSCAAS 16 EVQLVESGGGVVQPGRSLRLSCAAS 17 EVQLMESGGGLVKPGGSLRLSCATS 18 WVRQAPGKGLEWVA 19 WVRQAPGKGLEWVG 20 RFTISRDDSKNTLYLQMNSLKTEDTAVYYC 21 RFSISRDNAKNSLYLQMNSLRVEDTVVYYC 22 RFTISRDDSKSTLFLQMNNLKTEDTAVYYC 23 WGQGTLVTVSS 24 WGQGTMVTVSS 25 WGQGTTVTVSS 26 DVVMTQSPLSLPVTLGQPASISC 27 DIVMTQTPLSSPTVTLGQPASISC 28 DIVMTQTPLSLSVTPGQPASISC 29 DVVMTQSPLSQPVTLGQPASISC

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thirty WFQQRPGQSPRRLIY 31 WLQQRPGQPPRLLIY 32 WLLQKPGQPPQLLIY 33 WLQQRPGQSPRRLIY 34 GVPDRF SSGSGSGTDFTLKISRVEAEDVGLYYC 35 GVPDRF SSGSGAGTDFTLKISRVEAEDVGVYYC 36 GVPNRF SSGSGSGTDFTLKISRVEAEDVGLYYC 37 GVPDRFNGSGSGTDFTLSISRVEAEDVGVYYC 38 FGQGTKLEIK 39 FGGGTKVEIK 40 FGQGTKVEIK 41 EVQLVESGGGLVKPGGSLKLSCAASGFTFSHAWMHWVRQAPGKGLEWVAQ IKAKSDDYATYYAESVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTCW EWDLDF WGQGTL VT VS S 42 QVQLQESGGGLVKPGGSLRLSCAASGFTFSHAWMHWVRQAPGKGLEWVG QIKAKSDDYATYYAESVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTC WEWDLDF WGQGTM VT VS S 43 EVQLVESGGGVVQPGRSLRLSCAASGFTFSHAWMHWVRQAPGKGLEWVAQ IKAKSDDYATYYAESVKGRFSISRDNAKNSLYLQMNSLRVEDTVVYYCTCW EWDLDF WGQGTTVT VS S 44 EVQLMESGGGLVKPGGSLRLSCATSGFTFSHAWMHWVRQAPGKGLEWVGQ IKAKSDDYATYYAESVKGRFTISRDDSKSTLFLQMNNLKTEDTAVYYCTCW EWDLDF WGQGTL VT VS S 45 DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGHTYLNWFQQRPGQSPRRLI YSVSNLESGVPDRFSSGSG TDFTLKISRVEAEDVGLYYCMQATHAPPYTFGQGTKLEIK 46 DIVMTQTPLSSPVTLGQPASISCRSSQSLLDSDGHTYLNWLQQRPGQPPRLLIY SVSNLESGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQATHAPPYTFGQ GTKLEIK 47 DIVMTQTPLSLSVTPGQPASISCRSSQSLLDSDGHTYLNWLLQKPGQPPQLLIY SVSNLESGVPNRFSGSGSGTDFTLKISRVEAEDVGLYYCMQATHAPPYTFGG GTKVEIK

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48 DVVMTQSPLSQPVTLGQPASISCRSSQSLLDSDGHTYLNWLQQRPGQSPRRLI YSVSNLESGVPDRFNGSGSGTDFTLSISRVEAEDVGVYYCMQATHAPPYTFG QGTKVEIK 49 MGWTLVFLFLLSVTAGVHSEVQLVESGGGLVKPGGSLKLSCAASGTFFSHA WMHWVRQAPGKGLEWVAQIKAKSDDYATYYAESVKGRFTISRDDSKNTLY LQMNSLKTEDTAVYYCTCWEWDLDFWGQGTLVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 50 MGWTLVFLFLLSVTAGVHSQVQLQESGGGLVKPGGSLRLSCAASGTFFSHA WMHWVRQAPGKGLEWVGQIKAKSDDYATYYAESVKGRFTISRDDSKNTLY LQMNSLKTEDTAVYYCTCWEWDLDFWGQGTMVTVSSASTKGPSVFPLAPS SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 51 MGWTLVFLFLLSVTAGVHSEVQLVESGGGVVQPGRSLRLSCAASGTFFSHA WMHWVRQAPGKGLEWVAQIKAKSDDYATYYAESVKGRFSISRDNAKNSLY LQMNSLRVEDTVVYYCTCWEWDLDFWGQGTTVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK

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52 MGWTLVFLFLLSVTAGVHSEVQLMESGGGLVKPGGSLRLSCATSGFTFSHA WMHWVRQAPGKGLEWVGQIKAKSDDYATYYAESVKGRFTISRDDSKSTLF LQMNNLKTEDTAVYYCTCWEWDLDFWGQGTLVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 53 MVSSAQFLGLLLLCFQGTRCDVVMTQSPLSLPVTLGQPASISCRSSQSLLDSD GHTYLNWFQQRPGQSPRRLIYSVSNLESGVPDRFSGSGSGTDFTLKISRVEAE DVGLYYCMQATHAPPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLS SP VTKSFNRGEC 54 MVSSAQFLGLLLLCFQGTRCDIVMTQTPLSSPVTLGQPASISCRSSQSLLDSDG HTYLNWLQQRPGQPPRLLIYSVSNLESGVPDRFSGSGAGTDFTLKISRVEAED VGVYYCMQATHAPPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVV CLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLS SP VTKSFNRGEC 55 MVSSAQFLGLLLLCFQGTRCDIVMTQTPLSLSVTPGQPASISCRSSQSLLDSDG HTYLNWLLQKPGQPPQLLIYSVSNLESGVPNRFSGSGSGTDFTLKISRVEAED VGLYYCMQATHAPPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVV CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLS SP VTKSFNRGEC 56 MVSSAQFLGLLLLCFQGTRCDVVMTQSPLSQPVTLGQPASISCRSSQSLLDSD GHTYLNWLQQRPGQSPRRLIYSVSNLESGVPDRFSGSGSGTDFTLSISRVEAE DVGVYYCMQATHAPPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLS SP VTKSFNRGEC 57 EVQLVESGGGLVKPGGSLKLSCAASGFTFSHAWMHWVRQAPGKGLEWVAQ IKAKSDDYATYYAESVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTCW EWDLDFWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE

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PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 58 QVQLQESGGGLVKPGGSLRLSCAASGFTFSHAWMHWVRQAPGKGLEWVG QIKAKSDDYATYYAESVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTC WEWLDFWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEP VT VS WNSGALTS GVHTFP AVLQ S SGL YSLS SWT VP S S SLGTQT YICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVF SC S VMHEALHNHYTQKSLSLSPGK 59 EVQLVESGGGVVQPGRSLRLSCAASGFTFSHAWMHWVRQAPGKGLEWVAQ IKAKSDDYATYYAESVKGRFSISRDNAKNSLYLQMNSLRVEDTVVYYCTCW EWDLDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 60 EVQLMESGGGLVKPGGSLRLSCATSGFTFSHAWMHWVRQAPGKGLEWVGQ IKAKSDDYATYYAESVKGRFTISRDDSKSTLFLQMNNLKTEDTAVYYCTCW EWDLDFWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

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Claims (3)

61 DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGHTYLNWFQQRPGQSPRRLI YSVSNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGLYYCMQATHAPPYTFG QGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSS R VTKSFNRGEC 62 DIVMTQTPLSSPVTLGQPASISCRSSQSLLDSDGHTYLNWLQQRPGQPPRLLIY SVSNLESGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQATHAPPYTFGQ GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSP VTKSFNRGEC 63 DIVMTQTPLSLSVTPGQPASISCRSSQSLLDSDGHTYLNWLLQKPGQPPQLLIY SVSNLESGVPNRFSGSGSGTDFTLKISRVEAEDVGLYYCMQATHAPPYTFGG GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSP VTKSFNRGEC 64 DVVMTQSPLSQPVTLGQPASISCRSSQSLLDSDGHTYLNWLQQRPGQSPRRLI YSVSNLESGVPDRFSGSGSGTDFTLSISRVEAEDVGVYYCMQATHAPPYTFG QGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSS P VTKSFNRGEC 65 BLANK SAMPLE 66 QVQLQESGGGLVKPGGSLRLSCAASGFTFSHAWMHWVRQAPGKGLEWVG QIKAKSDDYATYYAESVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTS WEWDLDF WGQGTM VT VS S 67 QVQLQESGGGLVKPGGSLRLSCAASGFTFSHAWMHWVRQAPGKGLEWVG QIKAKSDDYATYYAESVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTS WEWLDFWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEP VT VS WNSGALTS GVHTFP AVLQ S SGL YSLS SWT VP S S SLGTQT YICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM TKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVF SC S VMHEALHNHYTQKSLSLSPGK 68 SPLPITPVNATCAIRHPCHNNLMNQIRSQLAQLNGSANALFILYYTAQGEPFP NNLDKLCGPNVTDFPPFHANGTEKAKLVELYRIVVYLGTSLGNITRDQKILNP SALSLHSKLNATADILRGLLSNVLCRLCSKYHVGHVDVTYGPDTSGKDVFQ KKKLGCQLLGKYKQIIAVLAQAF CLAIM 1. A method of treating an individual suffering from cancer, comprising administering to the individual a recombinant antibody that specifically binds leukemia inhibitory factor (LIF), comprising: 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 1) immunoglobulin light chain complementarity determining region 3 (VL-CDR3) containing the amino acid sequence represented by SEQ ID NO: 13 (MQATHAPPYT); and wherein the recombinant antibody is administered to the individual at a dose of approximately 1500 mg. 2. The method according to claim 1, where the recombinant antibody is humanized. 3. The method according to any one of claims 1 or 2, where the recombinant antibody contains two heavy chains im-