Combination of a LIF inhibitor and a platinum-based anti-tumor agent for treating cancer

Abstract

Described herein are methods of treating cancer using a combination of a leukemia inhibitory factor (LIF) binding polypeptide and a platinum-based anti-tumor agent.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of European application serial number 18382432.5 filed on June 18, 2018, the contents of which are hereby incorporated by reference in their entirety. Background Art

[0003] Leukemia inhibitory factor (LIF) is an interleukin 6 (IL-6) type cytokine that participates in a variety of biological activities, including the inhibition of cell differentiation. Human LIF is a 202 amino acid polypeptide that exerts its biological effects by binding to the cell surface LIF receptor (LIFR or CD118) heterodimerized with gp130. This leads to the activation of growth-promoting signaling pathways such as the mitogen-activated protein kinase (MAPK) and Janus-activated kinase (JAK / STAT) pathways. High expression levels and high serum levels of LIF have been shown to be associated with poor prognosis in many types of cancer.

[0004] Platinum-based antitumor agents, also known as platinums, are coordination complexes of platinum that have been shown to be useful for treating a variety of cancers. Mechanistically, platinum-based antitumor agents cause cell death by cross-linking DNA or embedding DNA. This inhibits the repair and synthesis of DNA in cells. Ultimately, these cellular processes are disrupted, leading to the onset of apoptosis. Common platinum-based antitumor agents may include cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatinite, phenathriplatin, picoplatin, seraplatin, lobaplatin or heptaplatin. Summary of the invention

[0005] Methods for treating or preventing cancer, tumors or other neoplasms in individuals are described herein. Methods and compositions of matter include combinations of LIF binding polypeptides and platinum-based anti-tumor agents. These methods can utilize anti-LIF antibodies that antagonize or block LIF activity, as well as platinum-based anti-tumor agents. In particular, these combinations exhibit surprising synergistic effects when compared to anti-LIF antibodies or platinum-based anti-tumor agents alone.

[0006] In one aspect, the present invention describes the use of a leukemia inhibitory factor (LIF) binding polypeptide in combination with a platinum-based anti-tumor agent for treating cancer in an individual. In certain embodiments, the LIF binding polypeptide and the platinum-based anti-tumor agent are administered to the individual in separate formulations. In certain embodiments, the LIF binding polypeptide and the platinum-based anti-tumor agent are administered to the individual in the same formulation. In certain embodiments, the LIF binding polypeptide is administered to the individual before the platinum-based anti-tumor agent is administered to the individual. In certain embodiments, the platinum-based anti-tumor agent is administered to the individual before the LIF binding polypeptide is administered to the individual. In certain embodiments, the platinum-based anti-tumor agent is administered to the individual while the LIF binding polypeptide is administered to the individual. In certain embodiments, the platinum-based anti-tumor agent is administered to the individual. In certain embodiments, the LIF binding polypeptide comprises a fragment of an immunoglobulin variable region or an immunoglobulin heavy chain constant region. In certain embodiments, the LIF binding polypeptide comprises an antibody that specifically binds to LIF. In certain embodiments, the antibody that specifically binds to LIF comprises at least one framework region derived from a human antibody framework region. In certain embodiments, the antibody that specifically binds to LIF is humanized. In certain embodiments, the antibody that specifically binds to LIF is deimmunized. In certain embodiments, the antibody that specifically binds to LIF comprises two heavy immunoglobulin chains and two light immunoglobulin chains. In certain embodiments, the antibody that specifically binds to LIF is an IgG antibody. In certain embodiments, the antibody that specifically binds to LIF is a Fab, F(ab)2, a single domain antibody, a single chain variable fragment (scFv) or a nanobody. In certain embodiments, the antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy chain complementary determining region 1 (VH-CDR1) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 1-3; (b) an immunoglobulin heavy chain complementary determining region 2 (VH-CDR2) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 4 or 5; (c) an immunoglobulin heavy chain complementary determining region 3 (VH-CDR3) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 6-8; (d) an immunoglobulin light chain complementary determining region 1 (VL-CDR1) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 9 or 10; (e) an immunoglobulin light chain complementary determining region 2 (VL-CDR2) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 11 or 12; and (f) an immunoglobulin light chain complementary determining region 3 (VL-CDR3) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 12 or 13. The amino acid sequence described in NO:13.In certain embodiments, the antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy chain variable region (VH) sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 41, 42, 44 or 66; and (b) an immunoglobulin light chain variable region (VL) sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 45-48. 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 antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy chain sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one 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%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 61-64. In certain embodiments, the antibody that specifically binds to LIF has a K of less than about 200 picomolar. D In certain embodiments, the antibody that specifically binds to LIF has a K of less than about 100 picomolar. D In certain embodiments, the platinum-based antitumor agent includes cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatinium tetranitrate, phenoplatin, picoplatin, setraplatin or a combination thereof. In certain embodiments, the platinum-based antitumor agent is cisplatin. In certain embodiments, the cancer includes advanced solid tumors, glioblastoma, gastric 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 cancer or pancreatic adenocarcinoma. In certain embodiments, the cancer is refractory to treatment with a therapeutic amount of LIF-binding polypeptide or a platinum-based antitumor agent administered as a monotherapy.

[0007] In another aspect, described herein is the use of an antibody that specifically binds to leukemia inhibitory factor (LIF) in combination with a platinum-based anti-tumor agent for treating cancer in an individual, wherein the LIF-binding antibody comprises: (a) an immunoglobulin heavy chain complementary determining region 1 (VH-CDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 1-3; (b) an immunoglobulin heavy chain complementary determining region 2 (VH-CDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 4 or 5; (c) an immunoglobulin heavy chain complementary determining region 3 (VH-CDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 6-8; (d) an immunoglobulin light chain complementary determining region 1 (VL-CDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 9 or 10; (e) an immunoglobulin light chain complementary determining region 2 (VL-CDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: NO: the amino acid sequence set forth in any one of 11 or 12; and (f) an immunoglobulin light chain complementary determining region 3 (VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO:13.

[0008] In another aspect, described herein is a method for treating an individual with cancer, comprising administering to the individual with cancer an effective amount of: (a) a leukemia inhibitory factor (LIF) binding polypeptide; and (b) a platinum-based anti-tumor agent. In certain embodiments, the method comprises administering to the individual with cancer an effective amount of the LIF binding polypeptide. In certain embodiments, the method comprises administering to the individual with cancer an effective amount of the platinum-based anti-tumor agent. In certain embodiments, the LIF binding polypeptide comprises a fragment of an immunoglobulin variable region or an immunoglobulin heavy chain constant region. In certain embodiments, the LIF binding polypeptide comprises an antibody that specifically binds to LIF. In certain embodiments, the antibody that specifically binds to LIF comprises at least one framework region derived from a human antibody framework region. In certain embodiments, the antibody that specifically binds to LIF is humanized. In certain embodiments, the antibody that specifically binds to LIF is deimmunized. In certain embodiments, the antibody that specifically binds to LIF comprises two immunoglobulin heavy chains and two immunoglobulin light chains. In certain embodiments, the antibody that specifically binds to LIF is an IgG antibody. In certain embodiments, the antibody that specifically binds to LIF is a Fab, F(ab)2, a single domain antibody, a single chain variable fragment (scFv), or a nanobody. In certain embodiments, the antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy chain complementary determining region 1 (VH-CDR1) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 1-3; (b) an immunoglobulin heavy chain complementary determining region 2 (VH-CDR2) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 4 or 5; (c) an immunoglobulin heavy chain complementary determining region 3 (VH-CDR3) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 6-8; (d) an immunoglobulin light chain complementary determining region 1 (VL-CDR1) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 9 or 10; (e) an immunoglobulin light chain complementary determining region 2 (VL-CDR2) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 11 or 12; and (f) an immunoglobulin light chain complementary determining region 3 (VL-CDR3) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 12 or 13. The amino acid sequence described in NO:13.In certain embodiments, the antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy chain variable region (VH) sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 41, 42, 44 or 66; and (b) an immunoglobulin light chain variable region (VL) sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 45-48. In certain embodiments, the VH sequence is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% 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%, 99% or 100% 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 antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy 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 one of SEQ ID NOs: 57-60 or 67; and 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 one of SEQ ID NOs: 61-64. In certain embodiments, the antibody that specifically binds to LIF has a K of less than about 200 picomolar. D In certain embodiments, the antibody that specifically binds to LIF has a K of less than about 100 picomolar. DIn certain embodiments, the platinum-based anti-tumor agent includes cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatinium tetranitrate, phenoplatin, picoplatin, setraplatin or a combination thereof. In certain embodiments, the platinum-based anti-tumor agent is cisplatin. In certain embodiments, the cancer includes advanced solid tumors, glioblastoma, gastric 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 cancer or pancreatic adenocarcinoma. In certain embodiments, the cancer is refractory to treatment with a therapeutic amount of a LIF-binding polypeptide inhibitor. In certain embodiments, the cancer is refractory to treatment with a therapeutic amount of a platinum-based anti-tumor agent. In certain embodiments, the leukemia inhibitory factor (LIF) binding polypeptide and the platinum-based anti-tumor agent are administered separately. In certain embodiments, the LIF binding polypeptide and the platinum-based anti-tumor agent are administered simultaneously. In certain embodiments, the LIF-binding polypeptide and the platinum-based anti-tumor agent are administered as a single composition.

[0009] In another aspect, a method for treating an individual with cancer is described herein, the method comprising administering an effective amount of a leukemia inhibitory factor (LIF) binding polypeptide to the individual with cancer, wherein a therapeutic amount of a platinum-based anti-tumor agent has been administered to the individual. In certain embodiments, the method inhibits the growth or metastasis of the cancer. In certain embodiments, the LIF binding polypeptide comprises a fragment of an immunoglobulin variable region or an immunoglobulin heavy chain constant region. In certain embodiments, the LIF binding polypeptide comprises an antibody that specifically binds to LIF. In certain embodiments, the LIF binding polypeptide comprises at least one framework region derived from a human immunoglobulin framework region. In certain embodiments, the antibody that specifically binds to LIF is humanized. In certain embodiments, the antibody that specifically binds to LIF is deimmunized. In certain embodiments, the antibody that specifically binds to LIF comprises two immunoglobulin heavy chains and two immunoglobulin light chains. In certain embodiments, the antibody that specifically binds to LIF is an IgG antibody. In certain embodiments, the antibody that specifically binds to LIF is a Fab, F(ab)2, a single domain antibody, a single chain variable fragment (scFv), or a nanobody. In certain embodiments, the antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy chain complementary determining region 1 (VH-CDR1) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 1-3; (b) an immunoglobulin heavy chain complementary determining region 2 (VH-CDR2) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 4 or 5; (c) an immunoglobulin heavy chain complementary determining region 3 (VH-CDR3) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 6-8; (d) an immunoglobulin light chain complementary determining region 1 (VL-CDR1) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 9 or 10; (e) an immunoglobulin light chain complementary determining region 2 (VL-CDR2) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 11 or 12; and (f) an immunoglobulin light chain complementary determining region 3 (VL-CDR3) comprising an amino acid sequence as set forth in SEQ ID NOs: 11 or 12. NO: 13. In certain embodiments, the antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy chain variable region (VH) sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 41, 42, 44 or 66; and (b) an immunoglobulin light chain variable region (VL) sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 45-48.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 antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy chain sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one 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%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 61-64. In certain embodiments, the antibody that specifically binds to LIF has a K of less than about 200 picomolar. D In certain embodiments, the antibody that specifically binds to LIF has a K of less than about 100 picomolar. D In certain embodiments, the platinum-based antitumor agent includes cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenoplatin, picoplatin, setraplatin or a combination thereof. In certain embodiments, the platinum-based antitumor agent is cisplatin. In certain embodiments, the cancer includes advanced solid tumors, glioblastoma, gastric 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 cancer or pancreatic adenocarcinoma. In certain embodiments, the cancer is refractory to treatment with a therapeutic amount of a platinum-based antitumor agent.

[0010] In another aspect, a method for treating an individual with cancer is described herein, the method comprising administering an effective amount of a platinum-based antitumor agent to the individual with cancer, wherein a therapeutic amount of a leukemia inhibitory factor (LIF) binding polypeptide has been administered to the individual. In certain embodiments, the method inhibits the growth or metastasis of the cancer. In certain embodiments, the LIF binding polypeptide comprises a fragment of an immunoglobulin variable region or an immunoglobulin heavy chain constant region. In certain embodiments, the LIF binding polypeptide comprises an antibody that specifically binds to LIF. In certain embodiments, the LIF binding polypeptide comprises at least one framework region derived from a human immunoglobulin framework region. In certain embodiments, the antibody that specifically binds to LIF is humanized. In certain embodiments, the antibody that specifically binds to LIF is deimmunized. In certain embodiments, the antibody that specifically binds to LIF comprises two immunoglobulin heavy chains and two immunoglobulin light chains. In certain embodiments, the antibody that specifically binds to LIF is an IgG antibody. In certain embodiments, the antibody that specifically binds to LIF is a Fab, F(ab)2, a single domain antibody, a single chain variable fragment (scFv), or a nanobody. In certain embodiments, the antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy chain complementary determining region 1 (VH-CDR1) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 1-3; (b) an immunoglobulin heavy chain complementary determining region 2 (VH-CDR2) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 4 or 5; (c) an immunoglobulin heavy chain complementary determining region 3 (VH-CDR3) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 6-8; (d) an immunoglobulin light chain complementary determining region 1 (VL-CDR1) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 9 or 10; (e) an immunoglobulin light chain complementary determining region 2 (VL-CDR2) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 11 or 12; and (f) an immunoglobulin light chain complementary determining region 3 (VL-CDR3) comprising an amino acid sequence as set forth in SEQ ID NOs: 11 or 12. NO: 13. In certain embodiments, the antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy chain variable region (VH) sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 41, 42, 44 or 66; and (b) an immunoglobulin light chain variable region (VL) sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 45-48.In certain embodiments, the VH sequence is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% 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%, 99% or 100% 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 antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy chain sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one 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%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 61-64. In certain embodiments, the antibody that specifically binds to LIF has a K of less than about 200 picomolar. D In certain embodiments, the antibody that specifically binds to LIF has a K of less than about 100 picomolar. D In certain embodiments, the platinum-based antitumor agent includes cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenoplatin, picoplatin, setraplatin or a combination thereof. In certain embodiments, the platinum-based antitumor agent is cisplatin. In certain embodiments, the cancer includes advanced solid tumors, glioblastoma, gastric 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 cancer or pancreatic adenocarcinoma. In certain embodiments, the cancer is refractory to treatment with a therapeutic amount of a LIF-binding polypeptide inhibitor.

[0011] In another aspect, the present invention describes a method for treating an individual having cancer, comprising administering to the individual having cancer an effective amount of: (a) an antibody that specifically binds to leukemia inhibitory factor (LIF), comprising: (i) an immunoglobulin heavy chain complementary determining region 1 (VH-CDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 1-3; (ii) an immunoglobulin heavy chain complementary determining region 2 (VH-CDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 4 or 5; (iii) an immunoglobulin heavy chain complementary determining region 3 (VH-CDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 6-8; (iv) an immunoglobulin light chain complementary determining region 1 (VL-CDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 9 or 10; (v) an immunoglobulin light chain complementary determining region 2 (VL-CDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: ID NO: 11 or 12; and (vi) an immunoglobulin light chain complementary determining region 3 (VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13; (b) a platinum-based antitumor agent.

[0012] In another aspect, a kit is described herein, comprising: (a) a leukemia inhibitory factor (LIF) binding polypeptide; and (b) a platinum-based antitumor agent. In certain embodiments, the LIF binding polypeptide comprises a fragment of an immunoglobulin variable region or an immunoglobulin heavy chain constant region. In certain embodiments, the LIF binding polypeptide comprises an antibody that specifically binds to LIF. In certain embodiments, the LIF binding polypeptide comprises at least one framework region derived from a human immunoglobulin framework region. In certain embodiments, the antibody that specifically binds to LIF is humanized. In certain embodiments, the antibody that specifically binds to LIF is deimmunized. In certain embodiments, the antibody that specifically binds to LIF comprises two immunoglobulin heavy chains and two immunoglobulin light chains. In certain embodiments, the antibody that specifically binds to LIF is an IgG antibody. In certain embodiments, the antibody that specifically binds to LIF is a Fab, F(ab)2, a single domain antibody, a single chain variable fragment (scFv) or a nanobody. In certain embodiments, the antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy chain complementary determining region 1 (VH-CDR1) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 1-3; (b) an immunoglobulin heavy chain complementary determining region 2 (VH-CDR2) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 4 or 5; (c) an immunoglobulin heavy chain complementary determining region 3 (VH-CDR3) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 6-8; (d) an immunoglobulin light chain complementary determining region 1 (VL-CDR1) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 9 or 10; (e) an immunoglobulin light chain complementary determining region 2 (VL-CDR2) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 11 or 12; and (f) an immunoglobulin light chain complementary determining region 3 (VL-CDR3) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 12 or 13. NO: 13. In certain embodiments, the antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy chain variable region (VH) sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 41, 42, 44 or 66; and (b) an immunoglobulin light chain variable region (VL) sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 45-48.In certain embodiments, the VH sequence is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% 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%, 99% or 100% 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 antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy chain sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one 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%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 61-64. In certain embodiments, the antibody that specifically binds to LIF has a K of less than about 200 picomolar. D In certain embodiments, the antibody that specifically binds to LIF has a K of less than about 100 picomolar. D In certain embodiments, the platinum-based anti-tumor agent includes cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenaplatin, picoplatin, setraplatin or a combination thereof. In certain embodiments, the platinum-based anti-tumor agent is cisplatin. In certain embodiments, the kit further includes a pharmaceutically acceptable excipient, carrier or diluent.

[0013] In another aspect, a composition is described herein, comprising: (a) a leukemia inhibitory factor (LIF) binding polypeptide; and (b) a platinum-based antitumor agent. In certain embodiments, the LIF binding polypeptide comprises a fragment of an immunoglobulin variable region or an immunoglobulin heavy chain constant region. In certain embodiments, the LIF binding polypeptide comprises an antibody that specifically binds to LIF. In certain embodiments, the LIF binding polypeptide comprises at least one framework region derived from a human immunoglobulin framework region. In certain embodiments, the antibody that specifically binds to LIF is humanized. In certain embodiments, the antibody that specifically binds to LIF is deimmunized. In certain embodiments, the antibody that specifically binds to LIF comprises two immunoglobulin heavy chains and two immunoglobulin light chains. In certain embodiments, the antibody that specifically binds to LIF is an IgG antibody. In certain embodiments, the antibody that specifically binds to LIF is a Fab, F(ab)2, a single domain antibody, a single chain variable fragment (scFv) or a nanobody. In certain embodiments, the antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy chain complementary determining region 1 (VH-CDR1) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 1-3; (b) an immunoglobulin heavy chain complementary determining region 2 (VH-CDR2) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 4 or 5; (c) an immunoglobulin heavy chain complementary determining region 3 (VH-CDR3) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 6-8; (d) an immunoglobulin light chain complementary determining region 1 (VL-CDR1) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 9 or 10; (e) an immunoglobulin light chain complementary determining region 2 (VL-CDR2) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 11 or 12; and (f) an immunoglobulin light chain complementary determining region 3 (VL-CDR3) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 12 or 13. NO: 13. In certain embodiments, the antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy chain variable region (VH) sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 41, 42, 44 or 66; and (b) an immunoglobulin light chain variable region (VL) sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 45-48.In certain embodiments, the VH sequence is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% 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%, 99% or 100% 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 antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy chain sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one 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%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 61-64. In certain embodiments, the antibody that specifically binds to LIF has a K of less than about 200 picomolar. D In certain embodiments, the antibody that specifically binds to LIF has a K of less than about 100 picomolar. D In certain embodiments, the platinum-based antitumor agent includes cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenaplatin, picoplatin, setraplatin or a combination thereof. In certain embodiments, the platinum-based antitumor agent is cisplatin. In certain embodiments, the composition further includes a pharmaceutically acceptable excipient, carrier or diluent. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Depicted is a Western blot showing the inhibition of LIF-induced STAT3 phosphorylation by different anti-LIF humanized antibodies.

[0015] Figure 2A and 2B Depicted is a Western blot showing inhibition of LIF-induced STAT3 phosphorylation by humanized and parental 5D8 antibodies.

[0016] Figure 3A The IC of h5D8 antibody for inhibition of LIF in U-251 cells is shown. 50 .

[0017] Figure 3B Representative ICs of r5D8 and h5D8 inhibition of pSTAT3 under endogenous LIF stimulation conditions are shown. 50Dose response curves. Shown are representative curves (n=1h5D8, n=2r5D8).

[0018] Figure 4 Depicted are Western blots showing inhibition of LIF-induced STAT3 phosphorylation by various monoclonal antibodies described in the present disclosure.

[0019] Figure 5 Depicted are immunohistochemical staining and quantification of LIF expression in glioblastoma multiforme (GBM), NSCLC (non-small cell lung cancer), ovarian cancer, colorectal cancer, and pancreatic tumors from human patients. Bars represent mean + / - standard error of the mean (SEM).

[0020] Fig. 6A is a diagram showing experiments performed in a non-small cell lung cancer mouse model using humanized 5D8 antibody.

[0021] Figure 6B is a diagram showing an experiment performed in a non-small cell lung cancer mouse model using the r5D8 antibody.

[0022] Fig. 7A Shown is the inhibitory effect of r5D8 on U251 cells in an orthotopic mouse model of GBM. Quantification at day 26 is shown.

[0023] Figure 7B Data from mice are shown, and the mice were inoculated with human U251 GBM cells expressing luciferase, and then treated with 100, 200 or 300 μg of h5D8 or vehicle twice a week. Tumor size was determined by bioluminescence (Xenogen IVIS spectrum) on the 7th day. The figure shows individual tumor measurements, and horizontal bars represent mean ± SEM. Statistical significance was calculated using unpaired nonparametric Mann-Whitney U test.

[0024] Fig. 8A The inhibitory effect of r5D8 on ovarian cancer cell growth in a syngeneic mouse model is shown.

[0025] Figure 8B Individual tumor measurements at day 25 are shown.

[0026] Figure 8C It is demonstrated that h5D8 showed a significant reduction in tumor growth when administered at 200 μg / mouse twice weekly (p<0.05). Symbols are mean+SEM, statistical significance compared to vehicle (using unpaired nonparametric Mann-Whitney U test).

[0027] Fig.9AThe inhibitory effect of r5D8 on colorectal cancer cell growth in a syngeneic mouse model is shown.

[0028] Fig. 9B Individual tumor measurements at day 17 are shown.

[0029] Fig. 10A Shown is the reduction of macrophage infiltration into tumor sites in an orthotopic mouse model of GBM, with representative images and quantification of CCL22+ cells.

[0030] Fig. 10B Shown is a reduction in macrophage infiltration in a human organotypic tissue slice culture model.

[0031] Fig. 10C Shown is a reduction in macrophage infiltration into tumor sites in a syngeneic mouse model of ovarian cancer, with representative images and quantification of CCL22+ cells.

[0032] Fig. 10D Shown is a reduction in macrophage infiltration into tumor sites in a syngeneic mouse model of colorectal cancer, with representative images and quantification of CCL22+ cells.

[0033] Fig.10E The inflammatory phenotype of tumor-associated macrophages (TAMs) harvested from tumors treated with h5D8 (15 mg / kg, 2QW) at day 25 (endpoint) is shown. In treated tumors, TAMs polarize toward an M1 pro-inflammatory phenotype. Statistical significance was determined by an unpaired t-test.

[0034] Fig.10F Shown are gene expression data of monocytes cultured with conditioned media from LIF knockdown cells.

[0035] Fig.11A Shown is an increase in non-myeloid effector cells in a syngeneic mouse model of ovarian cancer following treatment with r5D8.

[0036] Fig. 11B Shown is an increase in non-myeloid effector cells in a syngeneic mouse model of colorectal cancer following treatment with r5D8.

[0037] Fig. 11C Figure 3 shows the expression of CD4+ T cells in a NSCLC cancer mouse model after treatment with r5D8. REG The percentage of cells decreased.

[0038] Fig.12Data for mice bearing CT26 tumors treated with PBS (control) or r5D8 intraperitoneally twice a week in the presence or absence of anti-CD4 and anti-CD8 depletion antibodies are shown. The figure shows individual tumor measurements at d13, expressed as mean tumor volume + SEM. Statistical differences between groups were determined by unpaired nonparametric Mann-Whitney U test. R5D8 inhibited the growth of CT26 tumors (*p < 0.05). In the presence of anti-CD4 and anti-CD8 depletion antibodies, the growth inhibitory effect of r5D8 on tumors was significantly reduced (****p < 0.0001).

[0039] Fig.13A An overview of the co-crystal structure of h5D8 Fab in complex with LIF is shown. The gp130 interaction site is mapped on the surface of LIF (dark shading).

[0040] Fig. 13B The detailed interaction between LIF and h5D8 is presented, showing the residues forming salt bridges and the buried surface area is larger than of h5D8 residues.

[0041] Fig.14A A superposition of five h5D8 Fab crystal structures is shown and indicates a high degree of similarity despite crystallization under different chemical conditions.

[0042] Fig. 14B An extensive network of van der Waals interactions mediated by unpaired Cys100 is shown. This residue is well ordered, participates in shaping the conformations of HCDR1 and HCDR3, and does not participate in undesired disulfide mispairing. Distances between residues are shown and labeled with dashed lines.

[0043] Fig.15A Binding of h5D8 C100 mutant to human LIF was demonstrated by ELISA.

[0044] Fig. 15B Binding of h5D8 C100 mutant to mouse LIF was demonstrated by ELISA.

[0045] Fig.16A h5D8 does not block the binding between LIF and LIFR as demonstrated by Octet. h5D8 binds sequentially to LIF followed by LIFR.

[0046] Fig. 16B and 16CELISA analysis of LIF / mAb complexes bound to immobilized LIFR or gp130 is shown. Signals from species-specific peroxidase-conjugated anti-IgG antibodies (anti-human antibodies for (-) and h5D8, and anti-rat antibodies for r5d8 and B09) detected binding to immobilized LIFR ( Fig. 16B ) or gp130( Fig. 16C ) The antibody portion of the mAb / LIF complex of the coated plate.

[0047] Fig.17A and 17B The expression of LIF in 72 different human tissues was demonstrated. Fig.17A ) or LIFR ( Fig. 17B )'s mRNA expression.

[0048] Fig.18 Data from mice with subcutaneously implanted CT26 tumors treated with h5D8 and cisplatin are shown.The graph shows the time course of tumor growth in terms of mean tumor volume for vehicle, control IgG1, h5D8 alone, cisplatin alone, and h5D8+cisplatin combination groups.

[0049] Fig.19A and 19B Shown are the results from the treatment with h5D8 and doxorubicin ( Fig.19A ) or h5D8 and paclitaxel ( Fig.19B ) Tumor volume data over time for mice bearing subcutaneously implanted CT26 tumors treated with .

[0050] Fig. 20 Data from mice with intradermally implanted CT26 tumors treated with h5D8 and cisplatin are shown.The graph shows the time course of tumor growth in terms of mean tumor volume for vehicle, control IgG1, h5D8 alone, cisplatin alone, and h5D8+cisplatin combination groups. DETAILED DESCRIPTION

[0051] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. As used in this specification and the appended claims, the singular forms "a / an" and "the" include plural references unless the context clearly indicates otherwise. Unless otherwise indicated, any reference to "or" herein is intended to encompass "and / or".

[0052] In one aspect, described herein is the use of a leukemia inhibitory factor (LIF) binding polypeptide in combination with a platinum-based anti-tumor agent for treating cancer in an individual.

[0053] In another aspect, described herein is the use of a leukemia inhibitory factor (LIF) binding antibody in combination with a platinum-based anti-tumor agent for treating cancer in an individual, wherein the LIF binding antibody comprises: (a) an immunoglobulin heavy chain complementary determining region 1 (VH-CDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 1-3; (b) an immunoglobulin heavy chain complementary determining region 2 (VH-CDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 4 or 5; (c) an immunoglobulin heavy chain complementary determining region 3 (VH-CDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 6-8; (d) an immunoglobulin light chain complementary determining region 1 (VL-CDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 9 or 10; (e) an immunoglobulin light chain complementary determining region 2 (VL-CDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: NO: the amino acid sequence set forth in any one of 11 or 12; and (f) an immunoglobulin light chain complementary determining region 3 (VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO:13.

[0054] In another aspect, described herein is a method of treating an individual having cancer comprising administering to the individual having cancer an effective amount of: (a) a leukemia inhibitory factor (LIF) binding polypeptide; and (b) a platinum-based anti-tumor agent.

[0055] In another aspect, described herein is a method of treating an individual having cancer, the method comprising administering to the individual having cancer an effective amount of a leukemia inhibitory factor (LIF) binding polypeptide, wherein a therapeutic amount of a platinum-based anti-tumor agent has been administered to the individual.

[0056] In another aspect, described herein is a method of treating an individual having cancer, the method comprising administering to the individual having cancer; an effective amount of a platinum-based anti-tumor agent, wherein a therapeutic amount of a leukemia inhibitory factor (LIF) binding polypeptide has been administered to the individual.

[0057] In another aspect, described herein is a method of treating an individual having cancer, comprising administering to the individual having cancer an effective amount of: (a) a leukemia inhibitory factor (LIF) binding antibody comprising: (i) an immunoglobulin heavy chain complementary determining region 1 (VH-CDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 1-3; (ii) an immunoglobulin heavy chain complementary determining region 2 (VH-CDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 4 or 5; (iii) an immunoglobulin heavy chain complementary determining region 3 (VH-CDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 6-8; (iv) an immunoglobulin light chain complementary determining region 1 (VL-CDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 9 or 10; (v) an immunoglobulin light chain complementary determining region 2 (VL-CDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: NO: 11 or 12; and (vi) an immunoglobulin light chain complementary determining region 3 (VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13; and (b) a platinum-based antitumor agent.

[0058] In another aspect, described herein is a kit comprising: (a) a leukemia inhibitory factor (LIF) binding polypeptide; and (b) a platinum-based anti-tumor agent.

[0059] In another aspect, described herein is a composition comprising: (a) a leukemia inhibitory factor (LIF) binding polypeptide; and (b) a platinum-based anti-tumor agent.

[0060] As used herein, the terms "individual," "subject," and "patient" are used interchangeably and include a human diagnosed with or suspected of having a tumor, cancer, or other neoplasm.

[0061] The terms "cancer" and "tumor" refer to the physiological condition in mammals characterized by unregulated cell growth. Cancer is a class of diseases in which a group of cells show uncontrolled growth or unwanted growth. Cancer cells can also spread to other locations, which can lead to the formation of metastases. The spread of cancer cells in the body can occur, for example, through the lymph or blood. Uncontrolled growth, invasion, and metastasis formation are also referred to as the malignant properties of cancer. These malignant properties distinguish cancer from benign tumors, which typically do not invade or metastasize.

[0062] As used herein, the term "effective amount" refers to the amount of a therapeutic agent that causes a biological effect when administered to a mammal. Biological effects include, but are not limited to, inhibiting or blocking receptor ligand interactions (e.g., LIF-LIFR), inhibiting signal transduction pathways (e.g., STAT3 phosphorylation), reducing tumor growth, reducing tumor metastasis, or prolonging the survival of animals carrying tumors. A "therapeutic amount" is a drug concentration that is calculated to exert a therapeutic effect. A therapeutic amount includes a dosage range that can induce a therapeutic response in a population of individuals. The mammal can be a human individual. The human individual may have or be suspected of having a tumor.

[0063] As used herein, unless otherwise indicated, the term "antibody" includes an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the antigen-specific binding ability to bind to a full-length antibody, e.g., a fragment that retains 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; heavy chain antibodies, single-chain antibody molecules, e.g., single-chain variable region fragments (scFv), nanobodies, and multispecific antibodies formed by antibody fragments with different specificities, such as bispecific antibodies. In certain embodiments, these antibodies are humanized in a manner that reduces an individual's immune response to the antibody. For example, the antibody can be chimeric, e.g., a non-human variable region with a human constant region, or CDR-grafted, e.g., a non-human CDR region with a human constant region and a variable region framework sequence. In certain embodiments, the antibody is deimmunized after humanization. Deimmunization involves removing or mutating one or more T cell epitopes in the constant region of the antibody. In certain embodiments, the antibodies described herein are monoclonal. As used herein, a "recombinant antibody" is an antibody comprising amino acid sequences derived from two different species or two different sources, and includes synthetic molecules, e.g., antibodies comprising non-human CDRs and human framework or constant regions. In certain embodiments, the recombinant antibodies of the present invention are produced or synthesized by recombinant DNA molecules.

[0064] The percentage (%) of sequence identity relative to a reference polypeptide or antibody sequence is the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide or antibody sequence, after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percentage of sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining amino acid sequence identity percentages can be achieved by known various means, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Suitable parameters for aligning sequences can be determined, including algorithms required for achieving maximum alignment over the full length of the compared sequences. However, for the purposes of this article, the sequence comparison computer program ALIGN-2 is used to generate % amino acid sequence identity values. The ALIGN-2 sequence comparison computer program is written by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington, D.C. 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from source code. The ALIGN-2 program should be compiled for use on a UNIX operating system (including digital UNIX V4.0D). All sequence comparison parameters are set by the ALIGN-2 program and are not varied.

[0065] In the case of amino acid sequence comparisons using ALIGN-2, the % amino acid sequence identity of a given amino acid sequence A to / with / relative to a given amino acid sequence B (alternatively this may be phrased as a given amino acid sequence A having or comprising a particular % amino acid sequence identity to / with / relative to a given amino acid sequence B) is calculated as follows: 100 multiplied by the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the program's alignment of A and B, and where Y is the total number of amino acid residues in B. It should be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0066] The term "epitope" includes any determinant that can be bound by an antigen binding protein such as an antibody. An epitope is an antigenic region bound by an antigen binding protein that targets the antigen, and when the antigen is a protein, the epitope includes specific amino acids that directly contact the antigen binding protein. Epitopes are most often located on proteins, but can be located on other types of molecules (such as sugars or lipids) in some cases. Epitope determinants can include chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural features and / or specific charge characteristics. Typically, antibodies that are specific for a particular target antigen will preferentially identify epitopes on the target antigen in a complex mixture of proteins and / or macromolecules.

[0067] The term "anti-tumor agent" refers to an agent that can prevent, inhibit or stop the development of cancer, tumors or neoplasms. Anti-tumor agents may also be referred to as chemotherapeutic agents. Anti-tumor agents can target and destroy cancer cells. Anti-tumor agents may include, but are not limited to, antimetabolites, biological response modifiers, bleomycins, DNA alkylating agents, DNA cross-linking agents, enzymes, hormones, monoclonal antibodies, platinum complexes, proteasome inhibitors, taxanes, vinblastines, topoisomerase inhibitors, tyrosine kinase inhibitors, nucleoside analogs, antifolates, anthracyclines, podophyllotoxins, alkylating agents, mTOR inhibitors, retinoids, histone deacetylase inhibitors and immunomodulators.

[0068] Structural properties of the antibodies described herein

[0069] The complementarity determining region ("CDR") is the portion of the immunoglobulin (antibody) variable region that is primarily responsible for the antigen-binding specificity of the antibody. Even though the antibodies specifically bind to the same target or epitope, the CDR regions are highly variable from one antibody to another. The heavy chain variable region includes three CDR regions, abbreviated as VH-CDR1, VH-CDR2, and VH-CDR3; the light chain variable region includes three CDR regions, abbreviated as VL-CDR1, VL-CDR2, and VL-CDR3. These CDR regions are arranged consecutively in the variable region, with CDR1 being the most N-terminal and CDR3 being the most C-terminal. Interspersed between the CDRs are framework regions, which contribute to the structure and show much less variability than the CDR regions. The heavy chain variable region includes four framework regions, abbreviated as VH-FR1, VH-FR2, VH-FR3, and VH-FR4; the light chain variable region includes four framework regions, abbreviated as VL-FR1, VL-FR2, VL-FR3, and VL-FR4. A complete, full-size bivalent antibody comprising two heavy chains and a light chain will include: 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 include at least three heavy chain CDRs. In certain embodiments, the antibodies described herein include at least three light chain CDRs. In certain embodiments, the antibodies described herein include at least three heavy chain CDRs and three light chain CDRs.The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Maryland, USA ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262: 732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography" ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Igsuperfamily V-like domains [IMGT unique immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains encoding]," Dev Comp Immunol, 2003 Jan;27(1):55-77 ("IMGT" numbering scheme); and Honegger A and Plückthun 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 are identified herein from variable sequences provided using different numbering systems, using the Kabat, IMGT, Josiah numbering systems, or any combination of the three. The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Josiah scheme is based on structural information.Both the Kabat and Josiah schemes are numbered based on the most common antibody region sequence lengths, with insertions made by inserting letters (e.g., "30a"), and deletions appearing in some antibodies. The two schemes place certain insertions and deletions ("indels") in different locations, resulting in different numbering. The contact scheme is based on analysis of complex crystal structures and is similar in many ways to the Josiah numbering scheme.

[0070] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of natural antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs (see, e.g., Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, VH or VL domains can be used to separate antibodies that bind to a specific antigen from antibodies that bind to the antigen to screen libraries of complementary VL or VH domains, respectively (see, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991)). 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 include variable regions of rat origin. In certain embodiments, the antibodies described herein include CDRs of rat origin. In certain embodiments, the antibodies described herein include variable regions of mouse origin. In certain embodiments, the antibodies described herein include CDRs of mouse origin.

[0071] Changes (e.g., substitutions) can be made in the CDRs, for example, to improve antibody affinity. Such changes can be made in the CDR encoding codons at a high mutation rate during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and the resulting variants can be tested for binding affinity. Affinity maturation (e.g., using error-prone PCR, chain shuffling, CDR randomization, or oligonucleotide-directed mutagenesis) can be used to improve antibody affinity (see, e.g., 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, e.g., Cunningham and Wells Science, 244: 1081-1085 (1989)). CDR-H3 and CDR-L3 are particularly commonly targeted. Alternatively or additionally, the crystal structure of the antigen-antibody complex is analyzed to identify the contact points between the antibody and the antigen. Such contact residues and neighboring residues can be targeted or eliminated as substitution candidates. Variants can be screened to determine whether they contain the desired properties.

[0072] In certain embodiments, the antibodies described herein include a constant region in addition to the variable region. H ) consists of four domains, abbreviated as C H 1. C H 2. C H 3 and C H 4, located at the C-terminus of the complete heavy chain polypeptide, i.e., the C-terminus of the variable region. L ) than the C H Much smaller and located at the C-terminus of the complete light chain polypeptide, i.e., the C-terminus of the variable region. The constant region is highly conserved and includes different isotypes associated with slightly different functions and properties. In certain embodiments, the constant region is dispensable for antibodies that bind to target antigens. In certain embodiments, the constant region, heavy chain, and light chain of the antibody are all dispensable. In certain embodiments, the antibodies described herein lack one or more of the light chain constant region, the heavy chain constant region, or both. Most monoclonal antibodies are IgG isotypes; further divided into four subclasses IgG1, IgG2, IgG3, and IgG4. In certain embodiments, the antibodies described herein include any IgG subclass. 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.

[0073] Antibodies include a fragment crystallizable region (Fc region) that is responsible for binding to complement and Fc receptors. The Fc region includes the C H 2. C H 3 and C H 4. The Fc region of an antibody is responsible for activating complement and antibody-dependent cellular cytotoxicity (ADCC). The Fc region also contributes to the serum half-life of the antibody. In certain embodiments, the Fc region of an antibody described herein includes one or more amino acid substitutions that promote complement-mediated cell lysis. In certain embodiments, the Fc region of an antibody described herein includes one or more amino acid substitutions that promote ADCC. In certain embodiments, the Fc region of an antibody described herein includes one or more amino acid substitutions that reduce complement-mediated cell lysis. In certain embodiments, the Fc region of an antibody described herein includes one or more amino acid substitutions that increase the binding of the antibody to an Fc receptor. In certain embodiments, the Fc receptor includes FcγRI (CD64), FcγRIIA (CD32), FcγRIIIA (CD16a), FcγRIIIB (CD16b) or any combination thereof. In certain embodiments, the Fc region of an antibody described herein includes one or more amino acid substitutions that increase the serum half-life of the antibody. In certain embodiments, the 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).

[0074] In some embodiments, the antibodies disclosed herein are variants with some but not all effector functions, making them ideal candidates for applications where the in vivo half-life of the antibody is important but certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. Non-limiting examples of in vitro assays for evaluating ADCC activity of target molecules are described in U.S. Patent Nos. 5,500,362 and 5,821,337. Alternatively, non-radioactive assays (e.g., ACTI TM and CytoTox Non-radioactive cytotoxicity assays). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs), monocytes, macrophages, and natural killer (NK) cells.

[0075] The antibody may have increased half-life and improved binding to the neonatal Fc receptor (FcRn) (see, e.g., US 2005 / 0014934). Such antibodies may include an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn, and include those having substitutions at one or more of the following Fc region residues: 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, e.g., U.S. Pat. No. 7,371,826). Other examples of Fc region variants are also contemplated (see, e.g., Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. Nos. 5,648,260 and 5,624,821; and WO 94 / 29351).

[0076] Clinically useful antibodies are usually "humanized" to reduce the immunogenicity of human individuals. Humanized antibodies improve the safety and efficacy of monoclonal antibody therapy. A common method of humanization is to produce monoclonal antibodies in any suitable animal (e.g., mouse, rat, hamster), and replace the constant region with a human constant region. The antibody transformed in this way is called "chimeric". Another common method is "CDR transplantation", which replaces non-human V-FR with human V-FR. In the CDR transplantation method, all residues except the CDR region are human. 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 CDR transplanted.

[0077] Humanization generally reduces the overall affinity of an antibody or has little effect on the overall affinity of an antibody. Antibodies that unexpectedly have greater affinity for their targets after humanization are described herein. 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%. Affinity is measured using surface plasmon resonance (SPR) when suitable. In certain embodiments, affinity is measured using glycosylated human LIF. In certain embodiments, the glycosylated human LIF is fixed to the surface of the SPR chip. In certain embodiments, the antibody has a K of less than about 300 nanomolar, 200 nanomolar, 150 nanomolar, 125 nanomolar, 100 nanomolar, 90 nanomolar, 80 nanomolar, 70 nanomolar, 60 nanomolar, 50 nanomolar, 40 nanomolar, or less. D Combine.

[0078] The compositions and methods described herein include a combination of a LIF-binding polypeptide and a platinum-based anti-tumor agent. In certain embodiments, the LIF-binding polypeptide includes a fragment of an immunoglobulin variable region or an immunoglobulin heavy chain constant region. In certain embodiments, the LIF-binding polypeptide is an antibody that specifically binds to LIF. In certain embodiments, the LIF-binding antibody includes at least one framework region derived from a human immunoglobulin framework region. In certain embodiments, the antibody that specifically binds to LIF is humanized. In certain embodiments, the antibody that specifically binds to LIF is deimmunized. In certain embodiments, the antibody that specifically binds to LIF includes two immunoglobulin heavy chains and two immunoglobulin light chains. In certain embodiments, the antibody that specifically binds to LIF is an IgG antibody. In certain embodiments, the antibody that specifically binds to LIF is a Fab, F(ab)2, a single domain antibody, a single chain variable fragment (scFv) or a nanobody. In certain embodiments, the antibody is the h5D8 antibody described herein.

[0079] In certain embodiments described herein, the antibody used or administered in combination with a platinum-based anti-tumor agent is the h5D8 antibody. The h5d8 antibody specifically binds to LIF, and the antibody includes a VH-CDR1 set forth in any one of SEQ ID NOs: 1-3, a VH-CDR2 set forth in any one of SEQ ID NOs: 4 or 5, and a VH-CDR3 set forth in any one of SEQ ID NOs: 6 to 8. In certain embodiments described herein, h5D8 specifically binds to LIF and includes a VL-CDR1 set forth in any one of SEQ ID NOs: 9 or 10, a VL-CDR2 set forth in SEQ ID NOs: 11 or 12, and a VL-CDR3 set forth in SEQ ID NO: 13. In certain embodiments described herein, h5D8 specifically binds LIF and includes a VH-CDR1 set forth in any one of SEQ ID NOs: 1-3, a VH-CDR2 set forth in any one of SEQ ID NOs: 4 or 5, and a VH-CDR3 set forth in any one of SEQ ID NOs: 6-8, a VL-CDR1 set forth in any one of SEQ ID NOs: 9 or 10, a VL-CDR2 set forth in any one of SEQ ID NOs: 11 or 12, and a VL-CDR3 set forth in SEQ ID NO: 13. The VH and VL regions of h5D8 are set forth in SEQ ID NOs: 42 and 46.

[0080] In certain embodiments, the 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 in any one of SEQ ID NOs: 14-17, a VH-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: 18 or 19, a VH-FR3 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: 20-22, or a VH-FR4 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: 23-25. In certain embodiments, the one or more human heavy chain framework regions include 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, a VH-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 SEQ ID NO:19, a VH-FR3 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:20, or a VH-FR4 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:24. In certain embodiments, the antibody that specifically binds LIF comprises one or more human light chain framework regions comprising: 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 one of SEQ ID NOs: 26-29, a 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 about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 34-37, or a VL-FR4 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: 38-40.In certain embodiments, the one or more human light chain framework regions include 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, a 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 SEQ ID NO:31, a VL-FR3 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:35, or a VL-FR4 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:38. In certain embodiments, the one or more human heavy chain framework regions and the one or more human light chain 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, a VH-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 SEQ ID NO:19, a VH-FR3 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:20, a VH-FR4 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:24, a VH-FR5 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: NO:27, 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, a 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 SEQ ID NO:31, a VL-FR3 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:35, and a VL-FR4 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:38.In certain embodiments, the antibody that specifically binds to LIF comprises one or more human heavy chain framework regions comprising: a VH-FR1 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs: 14-17, a VH-FR2 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs: 18 or 19, a VH-FR3 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs: 20-22, or a VH-FR4 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs: 23-25. In certain embodiments, the one or more human heavy chain framework regions comprise a VH-FR1 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 15, a VH-FR2 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 19, a VH-FR3 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 20, or a VH-FR4 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, the antibody that specifically binds to LIF comprises one or more human light chain framework regions comprising: a VL-FR1 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs: 26-29, a VL-FR2 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs: 30-33, a VL-FR3 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs: 34-37, or a VL-FR4 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs: 38-40. In certain embodiments, the one or more human light chain framework regions comprise a VL-FR1 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 27, a VL-FR2 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 31, a VL-FR3 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 35, or a VL-FR4 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 38.In certain embodiments, the one or more human heavy chain framework regions and the one or more human light chain regions include a VH-FR1 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 15, a VH-FR2 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 19, a VH-FR3 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 20, a VH-FR4 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 24, a VL-FR1 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 27, a VL-FR2 identical to the amino acid sequence set forth in SEQ ID NO: 31, a VL-FR3 identical to the amino acid sequence set forth in SEQ ID NO: 35, and a VL-FR4 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 38. In certain embodiments, the antibody specifically binds to human LIF.

[0081] The r5D8 antibody described herein was generated by rats immunized with DNA encoding human LIF. r5D8 was cloned and sequenced and includes CDRs having the following amino acid sequences (using a combination of Kabat and IMGT CDR numbering methods): VH-CDR1 corresponding to SEQ ID NO: 1 (GFTFSHAWMH), VH-CDR2 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). The antibody has been humanized by CDR grafting, and the humanized version is referred to as h5D8.

[0082] In certain embodiments, antibodies that specifically bind to LIF are described herein, comprising a VH-CDR1 that is at least 80% or 90% identical to that set forth in SEQ ID NO: 1 (GFTFSHAWMH), a VH-CDR2 that is at least 80%, 90% or 95% identical to that set forth in SEQ ID NO: 4 (QIKAKSDDYATYYAESVKG), and a VH-CDR3 that is at least 80% or 90% identical to that set forth in SEQ ID NO: 6 (TCWEWDLDF). In certain embodiments, antibodies that specifically bind to LIF are described herein, comprising a VL-CDR1 that is at least 80% or 90% identical to that set forth in SEQ ID NO: 9 (RSSQSLLDSDGHTYLN), a VL-CDR2 that is at least 80% identical to that set forth in SEQ ID NO: 11 (SVSNLES), and a VL-CDR3 that is at least 80% or 90% identical to that set forth in SEQ ID NO: 13 (MQATHAPPYT). In certain embodiments, antibodies that specifically bind to LIF are described herein, comprising a VH-CDR1 set forth in SEQ ID NO: 1 (GFTFSHAWMH), a VH-CDR2 set forth in SEQ ID NO: 4 (QIKAKSDDYATYYAESVKG), a VH-CDR3 set forth in SEQ ID NO: 6 (TCWEWDLDF), a VL-CDR1 set forth in SEQ ID NO: 9 (RSSQSLLDSDGHTYLN), a VL-CDR2 set forth in SEQ ID NO: 11 (SVSNLES), and a VL-CDR3 set forth in SEQ ID NO: 13 (MQATHAPPYT). Certain conservative amino acid substitutions are contemplated in the amino acid sequences of the CDRs disclosed herein. In certain embodiments, the antibody comprises a CDR that differs from the amino acid sequence set forth in any one of SEQ ID NOs: 1, 4, 6, 9, 11, and 13 by 1, 2, 3, or 4 amino acids. In certain embodiments, the antibody comprises a CDR that differs from the amino acid sequence set forth in any one of SEQ ID NOs: 1, 4, 6, 9, 11 and 13 by 1, 2, 3 or 4 amino acids without affecting the binding affinity by more than 10%, 20% or 30%.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 in any one of SEQ ID NOs: 14-17, a VH-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: 18 or 19, a VH-FR3 amino acid sequence that is at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 20-22, or a VH-FR4 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: 23-25. In certain embodiments, the one or more human heavy chain framework regions include 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, a VH-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 SEQ ID NO:19, a VH-FR3 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:20, or a VH-FR4 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:24. In certain embodiments, the antibody that specifically binds LIF comprises one or more human light chain framework regions comprising: 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 one of SEQ ID NOs: 26-29, a 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 about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 34-37, or a VL-FR4 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: 38-40.In certain embodiments, the one or more human light chain framework regions include 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, a 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 SEQ ID NO:31, a VL-FR3 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:35, or a VL-FR4 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:38. In certain embodiments, the one or more human heavy chain framework regions and the one or more human light chain 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, a VH-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 SEQ ID NO:19, a VH-FR3 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:20, a VH-FR4 amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:24, 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, NO: 31, a 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 SEQ ID NO: 31, a VL-FR3 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: 35, and a VL-FR4 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: 38. In certain embodiments, the antibody specifically binds to human LIF.In certain embodiments, the antibody that specifically binds to LIF comprises one or more human heavy chain framework regions comprising: a VH-FR1 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs: 14-17, a VH-FR2 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs: 18 or 19, a VH-FR3 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs: 20-22, or a VH-FR4 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs: 23-25. In certain embodiments, the one or more human heavy chain framework regions comprise a VH-FR1 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 15, a VH-FR2 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 19, a VH-FR3 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 20, or a VH-FR4 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, the antibody that specifically binds to LIF comprises one or more human light chain framework regions comprising: a VL-FR1 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs: 26-29, a VL-FR2 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs: 30-33, a VL-FR3 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs: 34-37, or a VL-FR4 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs: 38-40. In certain embodiments, the one or more human light chain framework regions comprise a VL-FR1 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 27, a VL-FR2 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 31, a VL-FR3 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 35, or a VL-FR4 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 38.In certain embodiments, the one or more human heavy chain framework regions and the one or more human light chain regions include a VH-FR1 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 15, a VH-FR2 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 19, a VH-FR3 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 20, a VH-FR4 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 24, a VL-FR1 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 27, a VL-FR2 identical to the amino acid sequence set forth in SEQ ID NO: 31, a VL-FR3 identical to the amino acid sequence set forth in SEQ ID NO: 35, and a VL-FR4 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 38. In certain embodiments, the antibody specifically binds to human LIF.

[0083] In certain embodiments, described herein are antibodies that specifically bind to LIF, comprising a VH-CDR1 amino acid sequence that is at least 80% or 90% identical to that set forth in SEQ ID NO: 1 (GFTFSHAWMH), a VH-CDR2 amino acid sequence that is at least 80%, 90% or 95% identical to that set forth in SEQ ID NO: 4 (QIKAKSDDYATYYAESVKG), and a VH-CDR3 amino acid sequence that is at least 80% or 90% identical to that set forth in SEQ ID NO: 8 (TSWEWDLDF). In certain embodiments, described herein are antibodies that specifically bind to LIF, comprising a VL-CDR1 amino acid sequence at least 80% or 90% identical to that set forth in SEQ ID NO: 9 (RSSQSLLDSDGHTYLN), a VL-CDR2 amino acid sequence at least 80% identical to that set forth in SEQ ID NO: 11 (SVSNLES), and a VL-CDR3 amino acid sequence at least 80% or 90% identical to that set forth in SEQ ID NO: 13 (MQATHAPPYT). In certain embodiments, antibodies that specifically bind to LIF are described herein, comprising a VH-CDR1 amino acid sequence set forth in SEQ ID NO: 1 (GFTFSHAWMH), a VH-CDR2 amino acid sequence set forth in SEQ ID NO: 4 (QIKAKSDDYATYYAESVKG), a VH-CDR3 amino acid sequence set forth in SEQ ID NO: 8 (TSWEWDLDF), a VL-CDR1 amino acid sequence set forth in SEQ ID NO: 9 (RSSQSLLDSDGHTYLN), a VL-CDR2 amino acid sequence set forth in SEQ ID NO: 11 (SVSNLES), and a VL-CDR3 amino acid sequence set forth in SEQ ID NO: 13 (MQATHAPPYT). Certain conservative amino acid substitutions are contemplated in the amino acid sequences of the CDRs disclosed herein. In certain embodiments, the antibody comprises a CDR that differs from the amino acid sequence set forth in any one of SEQ ID NOs: 1, 4, 8, 9, 11, and 13 by 1, 2, 3, or 4 amino acids. In certain embodiments, the antibody comprises a CDR that differs from the amino acid sequence set forth in any one of SEQ ID NOs: 1, 4, 8, 9, 11 and 13 by 1, 2, 3 or 4 amino acids without affecting the binding affinity by more than 10%, 20% or 30%.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 in any one of SEQ ID NOs: 14-17, a VH-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: 18 or 19, a VH-FR3 amino acid sequence that is at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 20-22, or a VH-FR4 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: 23-25. In certain embodiments, the one or more human heavy chain framework regions include 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, a VH-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 SEQ ID NO:19, a VH-FR3 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:20, or a VH-FR4 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:24. In certain embodiments, the antibody that specifically binds LIF comprises one or more human light chain framework regions comprising: 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 one of SEQ ID NOs: 26-29, a 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 about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 34-37, or a VL-FR4 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: 38-40.In certain embodiments, the one or more human light chain framework regions include 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, a 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 SEQ ID NO:31, a VL-FR3 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:35, or a VL-FR4 amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:38. In certain embodiments, the one or more human heavy chain framework regions and the one or more human light chain 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, a VH-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 SEQ ID NO:19, a VH-FR3 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:20, a VH-FR4 amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:24, 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, a 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 SEQ ID NO: NO: 31, a 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 SEQ ID NO: 31, a VL-FR3 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: 35, and a VL-FR4 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: 38. In certain embodiments, the antibody specifically binds to human LIF.In certain embodiments, the antibody that specifically binds to LIF comprises one or more human heavy chain framework regions comprising: a VH-FR1 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs: 14-17, a VH-FR2 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs: 18 or 19, a VH-FR3 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs: 20-22, or a VH-FR4 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs: 23-25. In certain embodiments, the one or more human heavy chain framework regions comprise a VH-FR1 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 15, a VH-FR2 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 19, a VH-FR3 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 20, or a VH-FR4 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, the antibody that specifically binds to LIF comprises one or more human light chain framework regions comprising: a VL-FR1 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs: 26-29, a VL-FR2 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs: 30-33, a VL-FR3 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs: 34-37, or a VL-FR4 amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs: 38-40. In certain embodiments, the one or more human light chain framework regions comprise a VL-FR1 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 27, a VL-FR2 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 31, a VL-FR3 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 35, or a VL-FR4 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 38.In certain embodiments, the one or more human heavy chain framework regions and the one or more human light chain regions include a VH-FR1 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 15, a VH-FR2 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 19, a VH-FR3 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 20, a VH-FR4 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 24, a VL-FR1 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 27, a VL-FR2 identical to the amino acid sequence set forth in SEQ ID NO: 31, a VL-FR3 identical to the amino acid sequence set forth in SEQ ID NO: 35, and a VL-FR4 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 38. In certain embodiments, the antibody specifically binds to human LIF.

[0084] In certain embodiments, described herein is an antibody that specifically binds to 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 about 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 41, 42, and 44. In certain embodiments, described herein is an antibody that specifically binds to LIF, comprising a humanized heavy chain variable region comprising an amino acid sequence set forth in any one of SEQ ID NOs: 41, 42, and 44. In certain embodiments, described herein is an antibody that specifically binds to 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 set forth in any one of SEQ ID NOs: 45-48. In certain embodiments, described herein is an antibody that specifically binds to LIF, comprising a humanized light chain variable region comprising an amino acid sequence set forth in any one of SEQ ID NOs: 45 to 48. In certain embodiments, the antibody specifically binds to human LIF.

[0085] In certain embodiments, an antibody that specifically binds to LIF is described herein, 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 about 99% identical to the amino acid sequence set forth in 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, an antibody that specifically binds to LIF is described herein, comprising a humanized heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 42; and a humanized light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 46.

[0086] In certain embodiments, an antibody that specifically binds to LIF is described herein, 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 about 99% identical to the amino acid sequence set forth in 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, an antibody that specifically binds to LIF is described herein, comprising a humanized heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 66; and a humanized light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 46.

[0087] In certain embodiments, antibodies that specifically bind to LIF are 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 an amino acid sequence set forth in any one of SEQ ID NOs: 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 an amino acid sequence set forth in any one of SEQ ID NOs: 61-64. In certain embodiments, antibodies that specifically bind to LIF are described herein, comprising a humanized heavy chain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 57-60; and a humanized light chain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 61-64.

[0088] In certain embodiments, an antibody that specifically binds to 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: 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. In certain embodiments, an antibody that specifically binds to LIF is described herein, comprising a humanized heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 58; and a humanized light chain comprising the amino acid sequence set forth in SEQ ID NO: 62. In certain embodiments, described herein is an antibody that specifically binds to 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 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. In certain embodiments, described herein is an antibody that specifically binds to LIF, comprising a humanized heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 67; and a humanized light chain comprising the amino acid sequence set forth in SEQ ID NO: 62.

[0089] In certain embodiments, described herein are recombinant antibodies that specifically bind to leukemia inhibitory factor (LIF), comprising: a heavy chain complementary determining region 1 (VH-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 3; a heavy chain complementary determining region 2 (VH-CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 4; a heavy chain complementary determining region 3 (VH-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 7; a light chain complementary determining region 1 (VL-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 9; a light chain complementary determining region 2 (VL-CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 11; and a light chain complementary determining region 3 (VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13.

[0090] In certain embodiments, described herein are recombinant antibodies that specifically bind to leukemia inhibitory factor (LIF), comprising: a heavy chain complementary determining region 1 (VH-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 2; a heavy chain complementary determining region 2 (VH-CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 5; a heavy chain complementary determining region 3 (VH-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 6; a light chain complementary determining region 1 (VL-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 10; a light chain complementary determining region 2 (VL-CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 12; and a light chain complementary determining region 3 (VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13. Certain conservative amino acid substitutions are contemplated in the amino acid sequences of the CDRs disclosed herein. In certain embodiments, the antibody comprises a CDR that differs by 1, 2, 3, or 4 amino acids from the amino acid sequence set forth in any one of SEQ ID NOs: 2, 5, 6, 10, 12, and 13. In certain embodiments, the antibody comprises a CDR that differs by 1, 2, 3, or 4 amino acids from the amino acid sequence set forth in any one of SEQ ID NOs: 2, 5, 6, 10, 12, and 13, and does not affect the binding affinity by more than 10%, 20%, or 30%.

[0091] In certain embodiments, described herein are recombinant antibodies that specifically bind to leukemia inhibitory factor (LIF), comprising: a heavy chain complementary determining region 1 (VH-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 3; a heavy chain complementary determining region 2 (VH-CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 4; a heavy chain complementary determining region 3 (VH-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 7; a light chain complementary determining region 1 (VL-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 9; a light chain complementary determining region 2 (VL-CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 11; and a light chain complementary determining region 3 (VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13. Certain conservative amino acid substitutions are contemplated in the amino acid sequences of the CDRs disclosed herein. In certain embodiments, the antibody comprises a CDR that differs by 1, 2, 3, or 4 amino acids from the amino acid sequence set forth in any one of SEQ ID NOs: 3, 4, 7, 9, 11, and 13. In certain embodiments, the antibody comprises a CDR that differs by 1, 2, 3, or 4 amino acids from the amino acid sequence set forth in any one of SEQ ID NOs: 3, 4, 7, 9, 11, and 13, and does not affect the binding affinity by more than 10%, 20%, or 30%.

[0092] In certain embodiments, antibodies that specifically bind to LIF are 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 any one of SEQ ID NOs: 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 one of SEQ ID NOs: 53-56. In certain embodiments, antibodies that specifically bind to LIF are described herein, comprising a humanized heavy chain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 49-52; and a humanized light chain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 53-56.

[0093] In certain embodiments, antibodies that specifically bind to LIF are 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: 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. In certain embodiments, antibodies that specifically bind to LIF are described herein, 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 SEQ ID NO: 54.

[0094] Epitopes bound by therapeutically useful LIF antibodies

[0095] The unique epitope of human LIF is described herein, which inhibits the biological activity of LIF (e.g., STAT3 phosphorylation) when bound and inhibits tumor growth in vivo. The epitope described herein consists of two discontinuous amino acid fragments (from residue 13 to residue 32 and residue 120 to 138 of human LIF), which are present in two different topological domains (α helix A and C) of human LIF protein. This combination is a combination of weak (van der Waals attraction), medium (hydrogen binding) 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 an 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 forms a van der Waals force with a residue on an anti-LIF antibody and is within at least 5, 4 or 3 angstroms.

[0096] In certain embodiments, the methods and compositions described herein comprising a LIF binding antibody and cisplatin comprise an isolated antibody that binds 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 of SEQ ID NO:68. In certain embodiments, described herein are isolated antibodies that 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 of SEQ ID NO: 68. In certain embodiments, described herein are isolated antibodies that 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 of SEQ ID NO: 68. In certain embodiments, the antibody binds only to residues that participate in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues that participate in strong interactions with the antibody. In certain embodiments, the antibody interacts with helices A and C of LIF. In certain embodiments, the antibody blocks the interaction of LIF with gp130.

[0097] In certain embodiments, described herein is an antibody comprising a CDR having an amino acid sequence set forth in any one of SEQ ID NOs: 1, 4, 6, 9, 11, and 13, which binds 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 of SEQ ID NO:68. In certain embodiments, an antibody is described herein that includes a CDR having an amino acid sequence as set forth in any one of SEQ ID NOs: 1, 4, 6, 9, 11, and 13 that 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 of SEQ ID NO: 68. In certain embodiments, the antibody only binds to residues that participate in strong or moderate interactions with the antibody. In certain embodiments, the antibody only binds to residues that participate in strong interactions with the antibody.

[0098] In certain embodiments, described herein is an antibody comprising a CDR having an amino acid sequence set forth in any one of SEQ ID NOs: 1, 4, 8, 9, 11, and 13, which binds 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 of SEQ ID NO:68. In certain embodiments, an antibody is described herein that includes a CDR having an amino acid sequence as set forth in any one of SEQ ID NOs: 1, 4, 8, 9, 11, and 13 that 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 of SEQ ID NO: 68. In certain embodiments, the antibody only binds to residues that participate in strong or moderate interactions with the antibody. In certain embodiments, the antibody only binds to residues that participate in strong interactions with the antibody.

[0099] In certain embodiments, described herein is an antibody comprising a CDR that differs from the amino acid sequence set forth in any one of SEQ ID NOs: 1, 4, 6, 9, 11, and 13 by 1, 2, 3, 4, or 5 amino acids, and binds 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 of SEQ ID NO:68. In certain embodiments, an antibody is described herein that includes a CDR that differs from the amino acid sequence set forth in any one of SEQ ID NOs: 1, 4, 6, 9, 11, and 13 by 1, 2, 3, 4, or 5 amino acids, and 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 of SEQ ID NO: 68. In certain embodiments, the antibody only binds to residues that participate in strong or moderate interactions with the antibody. In certain embodiments, the antibody only binds to residues that participate in strong interactions with the antibody.

[0100] In certain embodiments, described herein is an antibody comprising a CDR that differs from the amino acid sequence set forth in any one of SEQ ID NOs: 1, 4, 8, 9, 11, and 13 by 1, 2, 3, 4, or 5 amino acids, and binds 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 of SEQ ID NO:68. In certain embodiments, an antibody is described herein that includes a CDR that differs from the amino acid sequence set forth in any one of SEQ ID NOs: 1, 4, 8, 9, 11, and 13 by 1, 2, 3, 4, or 5 amino acids, and 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 of SEQ ID NO: 68. In certain embodiments, the antibody only binds to residues that participate in strong or moderate interactions with the antibody. In certain embodiments, the antibody only binds to residues that participate in strong interactions with the antibody.

[0101] In certain embodiments, described herein are antibodies that specifically bind 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 in SEQ ID NO: 42; and a humanized light 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 in SEQ ID NO: 46, and binds 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: NO: 68 of A13, I14, R15, H16, P17, C18, H19, N20, Q25, Q29, Q32, D120, R123, S127, N128, L130, C131, C134, S135 or H138. In certain embodiments, described herein are antibodies that specifically bind to 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 in SEQ ID NO: 42; and a humanized light 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 in SEQ ID NO: 46, and 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 of SEQ ID NO: 68. In certain embodiments, the antibody binds only to residues that participate in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues that participate in strong interactions with the antibody.

[0102] In certain embodiments, described herein are antibodies that specifically bind 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 in SEQ ID NO: 66; and a humanized light 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 in SEQ ID NO: 46, and binds 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: NO: 68 of A13, I14, R15, H16, P17, C18, H19, N20, Q25, Q29, Q32, D120, R123, S127, N128, L130, C131, C134, S135 or H138. In certain embodiments, described herein are antibodies that specifically bind to 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 in SEQ ID NO: 66; and a humanized light 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 in SEQ ID NO: 46, and 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 of SEQ ID NO: 68. In certain embodiments, the antibody binds only to residues that participate in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues that participate in strong interactions with the antibody.

[0103] In certain embodiments, the antibodies disclosed herein inhibit LIF signaling in cells. In certain embodiments, the IC for bioinhibition of the antibodies in U-251 cells under serum starvation conditions is 50 less than or equal to about 100, 75, 50, 40, 30, 20, 10, 5 or 1 nanomolar. In certain embodiments, the IC for bioinhibition of the antibody in U-251 cells under serum starvation conditions is 50 Less than or equal to about 900, 800, 700, 600, 500, 400, 300, 200 or 100 nanomoles.

[0104] In certain embodiments, the antibodies disclosed herein can be used to treat tumors and cancers that express LIF. In certain embodiments, individuals treated with antibodies disclosed herein have been selected as having LIF-positive tumors / cancers for treatment. In certain embodiments, the tumor is LIF-positive or produces elevated levels of LIF. In certain embodiments, LIF positivity is determined compared to a reference value or a set of pathological criteria. In certain embodiments, LIF-positive tumors express 2 times, 3 times, 5 times, 10 times, 100 times or more LIF of non-transformed cells that derive the tumor. In certain embodiments, the tumor has obtained ectopic expression of LIF. LIF-positive tumors can be determined histologically using, for example, immunohistochemistry with anti-LIF antibodies; by commonly used molecular biology methods, such as mRNA quantification by real-time PCR or RNA-seq; or, for example, by Western blotting, flow cytometry, ELISA or homogenous protein quantification assay (e.g., ) for protein quantification. In certain embodiments, the antibodies can be used to treat patients diagnosed with cancer. In certain embodiments, the cancer comprises one or more cancer stem cells, or is one or more cancer stem cells.

[0105] In certain embodiments, the antibodies disclosed herein can be used to treat tumors in cancers expressing LIF receptors (CD118). LIF receptor-positive tumors can be determined by histopathology or flow cytometry, and in certain embodiments, they include cells that bind LIF receptor antibodies at 2 times, 3 times, 3 times, 4 times, 5 times, 10 times or more of isotype controls. In certain embodiments, the tumor has obtained ectopic expression of the LIF receptor. In a certain embodiment, the cancer is a cancer stem cell. In a certain embodiment, anti-LIF and anti-LIF antibodies can be used to determine LIF-positive tumors or cancers by immunohistochemistry. In a certain embodiment, LIF-positive tumors are determined by IHC analysis, wherein the LIF level is in the top 10%, 20%, 30%, 40% or the top 50% of the tumor.

[0106] The antibodies described herein affect many results. In a certain embodiment, compared with a control antibody (e.g., isotype control), the antibodies described herein can reduce the presence of M2 macrophages in tumor model tumors by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. M2 macrophages can be identified by staining CCL22 and CD206 in IHC sections or by flow cytometry of tumor-infiltrating immune cells or bone marrow cells. In a certain embodiment, compared with a control antibody (e.g., isotype control), the antibodies described herein can reduce the binding of LIF to gp130 tumors by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In certain embodiments, the antibodies described herein can reduce LIF signaling in LIF-responsive cell lines by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a control antibody (e.g., an isotype control). LIF signaling can be measured by, for example, Western blot of phosphorylated STAT3, a downstream target of LIF signaling. The antibodies herein are also highly specific for LIF compared to other IL-6 family member cytokines. In certain embodiments, the antibody binds to human LIF with an affinity of about 10x, about 50x, or about 100x that of any other IL-6 family member cytokine. In certain embodiments, the LIF antibody does not bind to other IL-6 family member cytokines produced in mammalian systems. In certain embodiments, the antibody does not bind to oncostatin M produced in mammalian systems.

[0107] In certain embodiments, the LIF binding polypeptide and antibody can be administered by any route suitable for administering a pharmaceutical composition containing an antibody, such as subcutaneously, intraperitoneally, intravenously, intramuscularly, intratumorally, or intracerebrally. In certain embodiments, the antibody is administered intravenously. In certain embodiments, the antibody is administered at a suitable dosage schedule, such as weekly, twice weekly, monthly, twice monthly, etc. In certain embodiments, the antibody is administered once every three weeks. The antibody can be administered in any therapeutically effective amount. In certain embodiments, the therapeutically acceptable amount is between about 0.1 mg / kg and about 50 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 1 mg / kg and about 40 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 5 mg / kg and about 30 mg / kg. The LIF binding polypeptide or antibody can be administered intravenously over a period of at least about 60 minutes; however, this period of time may vary somewhat depending on the conditions associated with each individual administration.

[0108] Platinum-based antineoplastic agents

[0109] Platinum-based anti-tumor agents, also known as platinums, are coordination complexes of platinum that are used to treat cancer. The LIF-binding polypeptides and antibodies described herein can be combined with platinum-based anti-tumor agents and used in methods for treating tumors, cancers, or other neoplasms. In certain embodiments, the LIF-binding polypeptides and antibodies described herein can be combined with platinum-based anti-tumor agents in pharmaceutical compositions useful for treating cancer, tumors, or other neoplasms. The h5D8 antibodies described herein can be combined with platinum-based anti-tumor agents and used in methods for treating tumors, cancers, or other neoplasms. In certain embodiments, the h5D8 antibodies described herein can be combined with platinum-based anti-tumor agents in pharmaceutical compositions useful for treating cancer, tumors, or other neoplasms.

[0110] The platinum-based anti-tumor agents used in the compositions and methods herein may cause cross-linking of DNA or binding to DNA. The cross-linking may inhibit DNA repair or DNA synthesis in cancer cells. The cross-linking may hinder cellular processes and lead to apoptosis. In some embodiments, the platinum-based anti-tumor agents include: cisplatin ( cis-diamminedichloroplatinum(II) (CDDP, cis-diamminedichloroplatinum(II), [SP-4-2]-diamminedichloroplatinum); carboplatin ( cis-diammine (1,1-cyclobutanedicarboxylate) platinum); oxaliplatin ( [SP-4-2]-(1R-trans)]-(1,2-cyclohexanediamine-N,N′)[oxalate (2-)-O,O′]platinum); Nedaplatin ( cis-Diamine(glycolate-O,O′)platinum, [SP-4-3]-Diamine(glycolate(2-)-O 1 , O 2 ) platinum); triplatinum tetranitrate (BBR3464, hexaammine dichloro-bis[μ-(1,6-hexanediamine-κN:κN′)] triplatinum tetranitrate (4 + )); Phenanthridine platinum ([SP-4-3]-diammine chloride (phenanthridine)-platinum nitrate (1 + ), picoplatin ([SP-4-3]-ammine dichloro (2-methylpyridine) platinum), satraplatin (JM216, [OC-6-43]-bis (acetyl-O) ammine dichloro (cyclohexylamine) platinum); lobaplatin ([SP-4-3-(S), (trans)]-(1,2-cyclobutanedimethylamine-N,N′) [2-hydroxypropionic acid (2-)-O 1 , O 2]-platinum); heptaplatin (Eptaplatin, [SP-4-2-[4R-(2α, 4α, 5β)]]-[2-(1-methylethyl)-1,3-dioxolane-4,5-dimethylamine-N,N′][malonic acid (2-)-O,O′]-platinum); a pharmaceutically acceptable salt thereof, or a combination thereof. In some embodiments, the platinum-based antitumor agent is cisplatin.

[0111] In certain embodiments, the platinum-based anti-tumor agent can be administered by any route suitable for administering a pharmaceutical composition containing a small molecule, such as subcutaneously, intraperitoneally, intravenously, intramuscularly, intratumorally, intracerebrally, or orally. In certain embodiments, the platinum-based anti-tumor agent is administered intravenously. In certain embodiments, the platinum-based anti-tumor agent is administered with a suitable dosage regimen, such as daily, once every two days, once every three days, once every four days, once every five days, once every five days, once every six days, once a week, twice a week, once a month, twice a month, once every two weeks, once every three weeks, or once every four weeks. The platinum-based anti-tumor agent can be administered in any therapeutically effective amount. In certain embodiments, the therapeutically acceptable amount is about 1 mg / m 2 About 1000 mg / m 2 In certain embodiments, the therapeutically acceptable amount is about 50 mg / m 2 Up to about 70 mg / m 2 In certain embodiments, the therapeutically acceptable amount is about 50 mg / m 2 About 100 mg / m 2 , about 50mg / m 2 About 150 mg / m 2 , or about 50 mg / m 2 About 200 mg / m 2 In certain embodiments, the therapeutically acceptable amount is about 10 mg / m 2 About 50 mg / m 2 , about 10mg / m 2 Up to about 70 mg / m 2 , or 10 mg / m 2 About 100 mg / m 2 In certain embodiments, the therapeutically acceptable amount is about 1 mg / m 2 About 50 mg / m 2 , about 1mg / m 2 Up to about 70 mg / m 2 , or about 1 mg / m 2 About 100 mg / m 2 In certain embodiments, the therapeutically acceptable amount is about 100 mg / m2 About 200 mg / m 2 , about 200mg / m 2 About 500 mg / m 2 , or about 300 mg / m 2 About 600 mg / m 2 In certain embodiments, the therapeutically acceptable amount is about 1 mg / m 2 , 2mg / m 2 , 3mg / m 2 , 4mg / m 2 , 5mg / m 2 , 6mg / m 2 , 7mg / m 2 , 8mg / m 2 , 9mg / m 2 , 10mg / m 2 , 20mg / m 2 , 30mg / m 2 , 40mg / m 2 , 50mg / m 2 , 60mg / m 2 , 70mg / m 2 , 80mg / m 2 , 90mg / m 2 , 100mg / m 2 , 110mg / m 2 , 120mg / m 2 , 130mg / m 2 , 140mg / m 2 , 150mg / m2, 160mg / m 2 , 170mg / m 2 , 180mg / m 2 , 190mg / m 2 , 200mg / m 2 , 300mg / m 2 , 400mg / m 2 , 500mg / m 2 , 600mg / m 2 , 700mg / m 2 , 800mg / m 2 , 900mg / m 2 or 1000 mg / m 2 Body surface area.

[0112] In certain embodiments, cisplatin may be administered in any therapeutically effective amount. In certain embodiments, the therapeutically acceptable amount of cisplatin is about 50 mg / m 2 About 70 mg / m 2In certain embodiments, the therapeutically acceptable amount of cisplatin is about 50 mg / m 2 About 100 mg / m 2 In certain embodiments, the therapeutically acceptable amount of cisplatin is about 10 mg / m 2 Up to about 70 mg / m 2 In certain embodiments, the therapeutically acceptable amount of cisplatin is about 10 mg / m 2 About 100 mg / m 2 In certain embodiments, the therapeutically acceptable amount of cisplatin is about 1 mg / m 2 About 50 mg / m 2 In certain embodiments, the therapeutically acceptable amount of cisplatin is about 1 mg / m 2 Up to about 70 mg / m 2 In certain embodiments, the therapeutically acceptable amount of cisplatin is about 1 mg / m 2 About 100 mg / m 2 In certain embodiments, the therapeutically acceptable amount of cisplatin is about 1 mg / m 2 , 2mg / m 2 , 3mg / m 2 , 4mg / m 2 , 5mg / m 2 , 6mg / m 2 , 7mg / m 2 , 8mg / m 2 , 9mg / m 2 , 10mg / m 2 , 20mg / m 2 , 30mg / m 2 , 40mg / m 2 , 50mg / m 2 , 60mg / m 2 , 70mg / m 2 , 80mg / m 2 , 90mg / m 2 or 100 mg / m 2 Body surface area.

[0113] In certain embodiments, the platinum-based anti-tumor agent can be administered to an individual at a flat dose level of between about 10 mg and about 1500 mg. In certain embodiments, the platinum-based anti-tumor agent can be administered to an individual at a flat dose level of between about 50 mg and about 1000 mg, between about 50 mg and about 800 mg, between about 50 mg and about 600 mg, between about 50 mg and about 500 mg, between about 100 mg and about 500 mg. In certain embodiments, the platinum-based anti-tumor agent may be administered to an individual at a flat dose level of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, or 1500 mg. In certain embodiments, the platinum-based anti-tumor agent may be administered to an individual at a level suitable for monotherapy.

[0114] In certain embodiments, cisplatin can be administered to an individual at a flat dose level between about 10 milligrams and about 500 milligrams. In certain embodiments, cisplatin can be administered to an individual at a flat dose level of between about 50 milligrams and about 500 milligrams, between about 50 milligrams and about 400 milligrams, between about 100 milligrams and about 400 milligrams, between about 100 milligrams and about 300 milligrams, or between about 100 milligrams and about 200 milligrams. In certain embodiments, cisplatin can be administered to an individual at a flat dose level of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 milligrams. In certain embodiments, cisplatin can be administered to an individual at a level suitable for monotherapy.

[0115] H5D8 Dosage

[0116] In certain embodiments, these antibodies can be administered by any route suitable for administering a pharmaceutical composition containing an antibody, such as subcutaneously, intraperitoneally, intravenously, intramuscularly, intratumorally or intracerebrally. In certain embodiments, these antibodies are administered intravenously. In certain embodiments, the antibody is administered with a suitable dosage schedule, such as weekly, twice weekly, monthly, twice monthly, etc. In certain embodiments, the antibody is administered once every three weeks. The antibody can be administered in any therapeutically effective amount. In certain embodiments, the therapeutically acceptable amount is between about 0.01 mg / kg and about 50 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 0.05 mg / kg and about 50 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 0.1 mg / kg and about 50 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 1 mg / kg and about 40 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 5 mg / kg and about 30 mg / kg. The h5D8 antibody can be administered in a flat dose, regardless of the weight or mass of the individual to which the h5D8 antibody is administered. Regardless of the weight or mass of the individual to whom the h5D8 antibody is administered, the h5D8 antibody can be administered in a flat dose, provided that the mass of the individual is at least about 37.5 kilograms. A flat dose of h5D8 can be administered from about 1 milligram to about 2000 milligrams. A flat dose of h5D8 can be administered from about 10 milligrams to about 2000 milligrams. A flat dose of h5D8 can be administered from about 25 milligrams to about 2000 milligrams. A flat dose of h5D8 can be administered from about 50 milligrams to about 2000 milligrams. A flat dose of h5D8 can be administered from about 75 milligrams to about 2000 milligrams. A flat dose of h5D8 can be administered from about 225 milligrams to about 2000 milligrams, about 750 milligrams to about 2000 milligrams, about 1125 milligrams to about 2000 milligrams, or about 1500 milligrams to about 2000 milligrams. A flat dose of h5D8 can be administered from about 1 milligram. A flat dose of h5D8 may be administered at about 10 mg. A flat dose of h5D8 may be administered at about 25 mg. A flat dose of h5D8 may be administered at about 50 mg. A flat dose of h5D8 may be administered at about 75 mg. A flat dose of h5D8 may be administered at about 225 mg. A flat dose of h5D8 may be administered at about 750 mg. A flat dose of h5D8 may be administered at about 1125 mg. A flat dose of h5D8 may be administered at about 1500 mg. A flat dose of h5D8 may be administered at about 2000 mg.

[0117] Other doses of h5D8 are contemplated. The dosage may be about 1, 10, 25, 50, 100, 150, 175, 200, 250, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 1100, 1150, 1175, 1200, 1225, 1250, 1275, 1300, 1325, 1350, 1375, 1400, 1425, 1450, 1475, 1525, 1550, 1575, 1600, 1625, 1650, 1675, 1700, 1725, 1750, 1775, 1800, 1825, 1850, 1875, 1900, 1925, 1950, 1975, 2025, 2050, 2075, or 2100 mg of h5D8. Any of these doses may be administered once a week, once every two weeks, once every three weeks, or once every four weeks.

[0118] A flat dose of h5D8 can be administered from about 1 mg to about 2000 mg once a week. A flat dose of h5D8 can be administered from about 10 mg to about 2000 mg once a week. A flat dose of h5D8 can be administered from about 25 mg to about 2000 mg once a week. A flat dose of h5D8 can be administered from about 50 mg to about 2000 mg once a week. A flat dose of h5D8 can be administered from about 75 mg to about 2000 mg once a week. A flat dose of h5D8 can be administered from about 75 mg to about 1500 mg once a week. A flat dose of h5D8 can be administered from about 225 mg to about 1500 mg, about 750 mg to about 1500 mg, about 1125 mg to about 1500 mg once a week. A flat dose of h5D8 can be administered from about 1 mg once a week. A flat dose of h5D8 can be administered from about 10 mg once a week. A flat dose of h5D8 can be administered from about 25 mg once a week. A flat dose of h5D8 may be administered at about 50 mg once a week. A flat dose of h5D8 may be administered at about 75 mg once a week. A flat dose of h5D8 may be administered at about 225 mg once a week. A flat dose of h5D8 may be administered at about 750 mg once a week. A flat dose of h5D8 may be administered at about 1125 mg once a week. A flat dose of h5D8 may be administered at about 1500 mg once a week. A flat dose of h5D8 may be administered at about 2000 mg once a week.

[0119] A flat dose of h5D8 can be administered from about 1 mg to about 2000 mg once every two weeks. A flat dose of h5D8 can be administered from about 10 mg to about 2000 mg once every two weeks. A flat dose of h5D8 can be administered from about 25 mg to about 2000 mg once every two weeks. A flat dose of h5D8 can be administered from about 50 mg to about 2000 mg once every two weeks. A flat dose of h5D8 can be administered from about 75 mg to about 2000 mg once every two weeks. A flat dose of h5D8 can be administered from about 75 mg to about 1500 mg once every two weeks. A flat dose of h5D8 can be administered from about 225 mg to about 1500 mg, about 750 mg to about 1500 mg, about 1125 mg to about 1500 mg once every two weeks. A flat dose of h5D8 can be administered from about 1 mg once every two weeks. A flat dose of h5D8 can be administered from about 10 mg once every two weeks. A flat dose of h5D8 can be administered from about 25 mg once every two weeks. A flat dose of h5D8 may be administered at about 50 mg once every two weeks. A flat dose of h5D8 may be administered at about 75 mg once every two weeks. A flat dose of h5D8 may be administered at about 225 mg once every two weeks. A flat dose of h5D8 may be administered at about 750 mg once every two weeks. A flat dose of h5D8 may be administered at about 1125 mg once every two weeks. A flat dose of h5D8 may be administered at about 1500 mg once every two weeks. A flat dose of h5D8 may be administered at about 2000 mg once every two weeks.

[0120] A flat dose of h5D8 can be administered from about 1 mg to about 2000 mg once every three weeks. A flat dose of h5D8 can be administered from about 10 mg to about 2000 mg once every three weeks. A flat dose of h5D8 can be administered from about 25 mg to about 2000 mg once every three weeks. A flat dose of h5D8 can be administered from about 50 mg to about 2000 mg once every three weeks. A flat dose of h5D8 can be administered from about 75 mg to about 2000 mg once every three weeks. A flat dose of h5D8 can be administered from about 75 mg to about 1500 mg once every three weeks. A flat dose of h5D8 can be administered from about 225 mg to about 1500 mg, about 750 mg to about 1500 mg, about 1125 mg to about 1500 mg once every three weeks. A flat dose of h5D8 can be administered from about 1 mg once every three weeks. A flat dose of h5D8 can be administered from about 10 mg once every three weeks. A flat dose of h5D8 may be administered at about 25 mg once every three weeks. A flat dose of h5D8 may be administered at about 50 mg once every three weeks. A flat dose of h5D8 may be administered at about 75 mg once every three weeks. A flat dose of h5D8 may be administered at about 225 mg once every three weeks. A flat dose of h5D8 may be administered at about 750 mg once every three weeks. A flat dose of h5D8 may be administered at about 1125 mg once every three weeks. A flat dose of h5D8 may be administered at about 1500 mg once every three weeks. A flat dose of h5D8 may be administered at about 2000 mg once every three weeks.

[0121] A flat dose of h5D8 can be administered from about 1 mg to about 2000 mg once every four weeks. A flat dose of h5D8 can be administered from about 10 mg to about 2000 mg once every four weeks. A flat dose of h5D8 can be administered from about 25 mg to about 2000 mg once every four weeks. A flat dose of h5D8 can be administered from about 50 mg to about 2000 mg once every four weeks. A flat dose of h5D8 can be administered from about 75 mg to about 2000 mg once every four weeks. A flat dose of h5D8 can be administered from about 75 mg to about 1500 mg once every four weeks. A flat dose of h5D8 can be administered from about 225 mg to about 1500 mg, about 750 mg to about 1500 mg, about 1125 mg to about 1500 mg once every four weeks. A flat dose of h5D8 can be administered from about 1 mg once every four weeks. A flat dose of h5D8 can be administered from about 10 mg once every four weeks. A flat dose of h5D8 can be administered from about 25 mg once every four weeks. A flat dose of h5D8 may be administered at about 50 mg once every four weeks. A flat dose of h5D8 may be administered at about 75 mg once every four weeks. A flat dose of h5D8 may be administered at about 225 mg once every four weeks. A flat dose of h5D8 may be administered at about 750 mg once every four weeks. A flat dose of h5D8 may be administered at about 1125 mg once every four weeks. A flat dose of h5D8 may be administered at about 1500 mg once every four weeks. A flat dose of h5D8 may be administered at about 2000 mg once every four weeks.

[0122] The h5D8 antibody can be administered based on the weight or mass of the individual to whom the h5D8 antibody is administered. A weight-adjusted dose of h5D8 can be administered from about 0.01 mg / kg to about 25 mg / kg. A weight-adjusted dose of h5D8 can be administered from about 0.05 mg / kg to about 25 mg / kg. A weight-adjusted dose of h5D8 can be administered from about 0.1 mg / kg to about 25 mg / kg. A weight-adjusted dose of h5D8 can be administered from about 0.5 mg / kg to about 25 mg / kg. A weight-adjusted dose of h5D8 can be administered from about 1 mg / kg to about 25 mg / kg. A weight-adjusted dose of h5D8 can be administered from about 3 mg / kg to about 25 mg / kg, about 10 mg / kg to about 25 mg / kg, about 15 mg / kg to about 25 mg / kg, or about 20 mg / kg to about 25 mg / kg. A weight-adjusted dose of h5D8 can be administered at about 1 mg / kg. A weight-adjusted dose of h5D8 may be administered at about 3 mg / kg. A weight-adjusted dose of h5D8 may be administered at about 10 mg / kg. A weight-adjusted dose of h5D8 may be administered at about 15 mg / kg. A weight-adjusted dose of h5D8 may be administered at about 20 mg / kg. A weight-adjusted dose of h5D8 may be administered at about 25 mg / kg.

[0123] A weight-adjusted dose of h5D8 may be administered from about 0.01 mg / kg to about 25 mg / kg once every three weeks. A weight-adjusted dose of h5D8 may be administered from about 0.05 mg / kg to about 25 mg / kg once every three weeks. A weight-adjusted dose of h5D8 may be administered from about 0.1 mg / kg to about 25 mg / kg once every three weeks. A weight-adjusted dose of h5D8 may be administered from about 0.5 mg / kg to about 25 mg / kg once every three weeks. A weight-adjusted dose of h5D8 may be administered from about 1 mg / kg to about 25 mg / kg once every three weeks. A weight-adjusted dose of h5D8 may be administered 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, from about 20 mg / kg to about 25 mg / kg once every, two, three or four weeks.

[0124] Other weight-adjusted doses of h5D8 are contemplated. A weight-adjusted dose of h5D8 may be administered at about 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.5 mg / kg, 1 mg / kg, 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 may be administered weekly, biweekly, every three weeks, or every four weeks.

[0125] A weight-adjusted dose of h5D8 may be administered at about 0.01 mg / kg once per week. A weight-adjusted dose of h5D8 may be administered at about 0.05 mg / kg once per week. A weight-adjusted dose of h5D8 may be administered at about 0.1 mg / kg once per week. A weight-adjusted dose of h5D8 may be administered at about 0.5 mg / kg once per week. A weight-adjusted dose of h5D8 may be administered at about 1 mg / kg once per week. A weight-adjusted dose of h5D8 may be administered at about 3 mg / kg once per week. A weight-adjusted dose of h5D8 may be administered at about 10 mg / kg once per week. A weight-adjusted dose of h5D8 may be administered at about 15 mg / kg once per week. A weight-adjusted dose of h5D8 may be administered at about 20 mg / kg once per week. A weight-adjusted dose of h5D8 may be administered at about 25 mg / kg once per week.

[0126] A weight-adjusted dose of h5D8 may be administered at about 0.01 mg / kg once every two weeks. A weight-adjusted dose of h5D8 may be administered at about 0.05 mg / kg once every two weeks. A weight-adjusted dose of h5D8 may be administered at about 0.1 mg / kg once every two weeks. A weight-adjusted dose of h5D8 may be administered at about 0.5 mg / kg once every two weeks. A weight-adjusted dose of h5D8 may be administered at about 1 mg / kg once every two weeks. A weight-adjusted dose of h5D8 may be administered at about 3 mg / kg once every two weeks. A weight-adjusted dose of h5D8 may be administered at about 10 mg / kg once every two weeks. A weight-adjusted dose of h5D8 may be administered at about 15 mg / kg once every two weeks. A weight-adjusted dose of h5D8 may be administered at about 20 mg / kg once every two weeks. A weight-adjusted dose of h5D8 may be administered at about 25 mg / kg once every two weeks.

[0127] A weight-adjusted dose of h5D8 may be administered at about 0.01 mg / kg once every three weeks. A weight-adjusted dose of h5D8 may be administered at about 0.05 mg / kg once every three weeks. A weight-adjusted dose of h5D8 may be administered at about 0.1 mg / kg once every three weeks. A weight-adjusted dose of h5D8 may be administered at about 0.5 mg / kg once every three weeks. A weight-adjusted dose of h5D8 may be administered at about 1 mg / kg once every three weeks. A weight-adjusted dose of h5D8 may be administered at about 3 mg / kg once every three weeks. A weight-adjusted dose of h5D8 may be administered at about 10 mg / kg once every three weeks. A weight-adjusted dose of h5D8 may be administered at about 15 mg / kg once every three weeks. A weight-adjusted dose of h5D8 may be administered at about 20 mg / kg once every three weeks. A weight-adjusted dose of h5D8 may be administered at about 25 mg / kg once every three weeks.

[0128] A weight-adjusted dose of h5D8 may be administered at about 0.01 mg / kg once every four weeks. A weight-adjusted dose of h5D8 may be administered at about 0.05 mg / kg once every four weeks. A weight-adjusted dose of h5D8 may be administered at about 0.1 mg / kg once every four weeks. A weight-adjusted dose of h5D8 may be administered at about 0.5 mg / kg once every four weeks. A weight-adjusted dose of h5D8 may be administered at about 1 mg / kg once every four weeks. A weight-adjusted dose of h5D8 may be administered at about 3 mg / kg once every four weeks. A weight-adjusted dose of h5D8 may be administered at about 10 mg / kg once every four weeks. A weight-adjusted dose of h5D8 may be administered at about 15 mg / kg once every four weeks. A weight-adjusted dose of h5D8 may be administered at about 20 mg / kg once every four weeks. A weight-adjusted dose of h5D8 may be administered at about 25 mg / kg once every four weeks.

[0129] Any dose detailed herein may be administered intravenously over a period of at least about 60 minutes; however, this period of time may vary somewhat depending on the conditions associated with each individual administration.

[0130] Dosing schedule for combination therapy

[0131] Combination therapy comprising a LIF-binding polypeptide and a platinum-based antitumor agent can be administered in a variety of ways. The LIF-binding polypeptide and the platinum-based antitumor agent can be administered simultaneously on the same schedule or at different times on different schedules. When administered simultaneously, administration can be by separate formulations or a single formulation comprising the LIF-binding polypeptide and the platinum-based antitumor agent. Administration methods can be mixed, for example, the LIF-binding polypeptide can be administered intravenously, while the platinum-based antitumor agent can be administered orally or by parenteral injection. In certain embodiments, the LIF-binding polypeptide is administered intravenously, parenterally, subcutaneously, intratumorally or orally. In certain embodiments, the platinum-based antitumor agent is administered intravenously, parenterally, subcutaneously, intratumorally or orally.

[0132] When the combination therapy is given to an individual on the same schedule, the LIF-binding polypeptide and the platinum-based anti-tumor agent may be administered once a week, once every two weeks, or once every four weeks. The LIF-binding polypeptide and the platinum-based anti-tumor agent may be administered separately or as a single formulation.

[0133] When the combination therapy is performed on an individual with different schedules, the LIF-binding polypeptide and the platinum-based anti-tumor agent can be used alternately. In certain embodiments, the individual can be administered one or more platinum-based anti-tumor agents prior to the administration of the LIF-binding polypeptide. The LIF-binding polypeptide can be administered within 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days of the administration of the platinum-based anti-tumor agent. The LIF-binding polypeptide can be administered within 1 week, 2 weeks, 3 weeks, or 4 weeks of the administration of the platinum-based anti-tumor agent. The h5D8 antibody can be administered within 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days of the administration of the platinum-based anti-tumor agent. The h5D8 antibody can be administered within 1 week, 2 weeks, 3 weeks, or 4 weeks of the administration of the platinum-based anti-tumor agent.

[0134] The LIF-binding polypeptide may be administered to an individual one or more times prior to administration of a platinum-based anti-tumor agent. In certain embodiments, a platinum-based anti-tumor agent may be administered within 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days of administering the LIF-binding polypeptide. In certain embodiments, a platinum-based anti-tumor agent may be administered within 1 week, 2 weeks, 3 weeks, or 4 weeks of administering the LIF-binding polypeptide. In certain embodiments, a platinum-based anti-tumor agent may be administered within 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days of administering the h5D8 antibody. In certain embodiments, a platinum-based anti-tumor agent may be administered within 1 week, 2 weeks, 3 weeks, or 4 weeks of administering the h5D8 antibody.

[0135] In certain embodiments, LIF-binding polypeptides may be administered to an individual once a week, and a platinum-based anti-tumor agent may be administered to an individual weekly, every two weeks, every three weeks, or every four weeks. In certain embodiments, LIF-binding polypeptides may be administered to an individual once every two weeks, and a platinum-based anti-tumor agent may be administered to an individual weekly, every two weeks, every three weeks, or every four weeks. In certain embodiments, LIF-binding polypeptides may be administered to an individual once every three weeks, and a platinum-based anti-tumor agent may be administered to an individual weekly, every two weeks, every three weeks, or every four weeks. In certain embodiments, LIF-binding polypeptides may be administered to an individual once every four weeks, and a platinum-based anti-tumor agent may be administered to an individual weekly, every two weeks, every three weeks, or every four weeks. In certain embodiments, LIF-binding polypeptides may be administered to an individual once every four weeks, and a platinum-based anti-tumor agent may be administered to an individual weekly, every two weeks, every three weeks, or every four weeks. In certain embodiments, LIF-binding polypeptides may be administered to an individual once a week, and a platinum-based anti-tumor agent may be administered to an individual weekly, every two weeks, every three weeks, or every four weeks. In certain embodiments, LIF-binding polypeptides may be administered to an individual once every two weeks, and a platinum-based anti-tumor agent may be administered to an individual weekly, every two weeks, every three weeks, or every four weeks. In certain embodiments, a platinum-based anti-tumor agent may be administered to an individual once every three weeks, and a LIF-binding polypeptide may be administered to an individual weekly, every two weeks, every three weeks, or every four weeks. In certain embodiments, a platinum-based anti-tumor agent may be administered to an individual once every four weeks, and a LIF-binding polypeptide may be administered to an individual weekly, every two weeks, every three weeks, or every four weeks. In certain embodiments, h5D8 may be administered to an individual once or multiple times before administering a platinum-based anti-tumor agent. In certain embodiments, h5D8 may be administered to an individual once a week, and a platinum-based anti-tumor agent may be administered to an individual weekly, every two weeks, every three weeks, or every four weeks. In certain embodiments, h5D8 may be administered to an individual once every two weeks, and a platinum-based anti-tumor agent may be administered to an individual weekly, every two weeks, every three weeks, or every four weeks. In certain embodiments, h5D8 may be administered to an individual once every three weeks, and a platinum-based anti-tumor agent may be administered to an individual weekly, every two weeks, every three weeks, or every four weeks. In certain embodiments, h5D8 can be administered to the subject once every four weeks, and the platinum-based anti-tumor agent can be administered to the subject every week, every two weeks, every three weeks, or every four weeks.

[0136] Combination therapies according to the present disclosure may include a combination of one or two active components (e.g., LIF-binding polypeptides and platinum-based anti-tumor agents) that are ineffective as monotherapy by themselves but effective when administered as part of a combination therapy. In certain embodiments, platinum-based anti-tumor agents are administered at levels that are ineffective for monotherapy but effective in combination with LIF-binding polypeptides. In certain embodiments, platinum-based anti-tumor agents are administered at levels that are ineffective for monotherapy but effective in combination with h5D8 antibodies. In certain embodiments, LIF-binding polypeptides are administered at levels that are ineffective for monotherapy but effective in combination with platinum-based anti-tumor agents. In certain embodiments, h5D8 is administered at levels that are ineffective for monotherapy but effective in combination with platinum-based anti-tumor agents. In certain embodiments, both LIF-binding polypeptides and platinum-based anti-tumor agents are administered at levels that are ineffective for monotherapy but effective in combination with platinum-based anti-tumor agents. In certain embodiments, both h5D8 and platinum-based anti-tumor agents are administered at levels that are ineffective for monotherapy but effective in combination. In certain embodiments, both h5D8 and platinum-based anti-tumor agents are administered at levels that are ineffective for monotherapy but effective in combination.

[0137] Treatment Indications

[0138] In certain embodiments, methods and compositions useful for treating cancer or tumors are disclosed herein. In certain embodiments, the cancer includes breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head, neck, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testis, and liver tumors. In certain embodiments, tumors that can be treated with the antibodies of the present invention include adenomas, adenocarcinomas, angiosarcomas, astrocytomas, epithelial cancers, germ cell tumors, glioblastomas, gliomas, hemangioendotheliomas, angiosarcomas, hematomas, hepatoblastomas, leukemias, lymphomas, medulloblastomas, melanomas, neuroblastomas, osteosarcomas, retinoblastomas, rhabdomyosarcomas, sarcomas, and / or teratomas. In certain embodiments, the tumor / cancer is selected from the group consisting of acral lentigo melanoma, actinic keratosis, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, astrocytic tumor, Bartholin's gland carcinoma, basal cell carcinoma, bronchial adenocarcinoma, capillary carcinoid, carcinoma, carcinosarcoma, bile duct carcinoma, chondrosarcoma, cystadenoma, intradermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ventricular septal sarcoma, Ewing's sarcoma, focal nodular hyperplasia, gastric adenoma, germ cell tumor, glioblastoma, glucagonoma, hemangioblastoma, hemangioendothelioma, hemangioma, hemangioma, hepatic adenoma, liver adenocarcinoma, hepatocellular carcinoma, insulinoma, intraepithelial neoplasia, intraepithelial squamous cell neoplasia, invasive squamous cell carcinoma, large cell carcinoma, adipose Sarcoma, lung cancer, lymphoblastic leukemia, lymphocytic leukemia, leiomyosarcoma, melanoma, malignant melanoma, malignant mesothelioma, schwannoma, medulloblastoma, medullary epithelioma, mesothelioma, mucoepidermoid carcinoma, myeloid leukemia, neuroblastoma, neuroepithelial adenocarcinoma, nodular melanoma, osteosarcoma, ovarian cancer, papillary serous adenoma, pituitary tumor, plasmacytoma, pseudosarcoma, prostate cancer, pulmonary blastoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, squamous cell carcinoma, small cell carcinoma, soft tissue cancer, somatostatin-secreting tumor, squamous carcinoma, squamous cell carcinoma, undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, vaginal / vulvar cancer, VIPpoma and Wilm's tumor. In certain embodiments, tumors / cancers to be treated with one or more antibodies of the invention include brain cancer, head and neck cancer, colorectal cancer, 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 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 carcinoma and / or breast cancer). In certain embodiments, cancers treated with antibodies of the present disclosure include glioblastoma.In certain embodiments, cancers treated with one or more antibodies of the present disclosure include pancreatic cancer. In certain embodiments, cancers treated with one or more antibodies of the present disclosure include ovarian cancer. In certain embodiments, cancers treated with one or more antibodies of the present disclosure include lung cancer. In certain embodiments, cancers treated with one or more antibodies of the present disclosure include prostate cancer. In certain embodiments, cancers treated with one or more antibodies of the present disclosure include colon cancer. In certain embodiments, the cancers treated include glioblastoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, or lung cancer. In certain embodiments, the cancer is refractory to other treatments. In certain embodiments, the treated cancer is recurrent. In certain embodiments, the cancer is recurrent / refractory glioblastoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, or lung cancer. In certain embodiments, the cancer includes advanced solid tumors, glioblastoma, gastric 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 comprises non-small cell lung cancer, epithelial ovarian cancer, or pancreatic adenocarcinoma. In certain embodiments, the cancer comprises an advanced solid tumor. In certain embodiments, the individual is refractory to treatment with a previous LIF-binding antibody as a monotherapy. In certain embodiments, the individual is refractory to treatment with a previous platinum-based anti-tumor agent as a monotherapy.

[0139] Pharmaceutically acceptable excipients, carriers and diluents

[0140] In certain embodiments, the platinum-based antitumor agent and LIF-binding polypeptide of the present disclosure are components of a pharmaceutical composition. In certain embodiments, the platinum-based antitumor agent and LIF-binding polypeptide of the present disclosure are components of the same pharmaceutical composition. In certain embodiments, the pharmaceutical composition includes a physiologically suitable salt concentration (e.g., NaCl). In certain embodiments, the pharmaceutical composition includes about 0.6% to 1.2% NaCl. In certain embodiments, the pharmaceutical composition includes about 0.7% to 1.1% NaCl. In certain embodiments, the pharmaceutical composition includes about 0.8% to 1.0% NaCl. In certain embodiments, the pharmaceutical composition further comprises one or more of the following: a buffer, e.g., acetate, citrate, histidine, succinate, phosphate, bicarbonate, and hydroxymethylaminomethane (Tris); a surfactant, e.g., polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), polysorbate, and poloxamer 188; a polyol / disaccharide / polysaccharide, e.g., glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; an amino acid, e.g., histidine, glycine, or arginine; an antioxidant, e.g., ascorbic acid, methionine; and a chelating agent, e.g., EGTA or EGTA.

[0141] In certain embodiments, the platinum-based antitumor agent, LIF-binding polypeptide, or both the platinum-based antitumor agent and the LIF-binding polypeptide of the present disclosure are suspended in a sterile solution for administration. In certain embodiments, the platinum-based antitumor agent and the LIF-binding polypeptide are administered from the same solution. In certain embodiments, the solution includes a physiologically suitable salt concentration (e.g., NaCl). In certain embodiments, the solution includes about 0.6% to 1.2% NaCl. In certain embodiments, the solution includes about 0.7% to 1.1% NaCl. In certain embodiments, the solution includes about 0.8% to 1.0% NaCl. In certain embodiments, a highly concentrated mother solution of an antibody can be diluted in about 0.9% NaCl. In certain embodiments, the solution includes about 0.9% NaCl. In certain embodiments, the solution further comprises one or more of the following: a buffer, e.g., acetate, citrate, histidine, succinate, phosphate, bicarbonate, and hydroxymethylaminomethane (Tris); a surfactant, e.g., polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), polysorbate, and poloxamer 188; a polyol / disaccharide / polysaccharide, e.g., glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; an amino acid, e.g., histidine, glycine, or arginine; an antioxidant, e.g., ascorbic acid, methionine; and a chelating agent, e.g., EGTA or EGTA. In certain embodiments, the platinum-based anti-tumor agent and LIF-binding polypeptide of the present disclosure are lyophilized for transport / storage and reconstitution prior to administration. In certain embodiments, the lyophilized antibody formulation comprises a bulking agent, such as mannitol, sorbitol, sucrose, trehalose, and dextran 40. In certain embodiments, the anti-LIF antibody of the present disclosure can be transported and stored as a concentrated mother solution, which is diluted when used at the treatment site. In certain embodiments, the mother solution includes about 25mM histidine, about 6% sucrose, about 0.01% polysorbate, and about 20mg / mL of anti-LIF antibody. In certain embodiments, the pH of the solution is about 6.0. In certain embodiments, the form administered to an individual is an aqueous solution including about 25mM histidine, about 6% sucrose, about 0.01% polysorbate 80, and about 20mg / mL h5D8 antibody. In certain embodiments, the pH of the solution is about 6.0.

[0142] In certain embodiments, the platinum-based antitumor agent and LIF-binding polypeptide of the present disclosure are suspended in a sterile solution for administration. In certain embodiments, the platinum-based antitumor agent and the LIF-binding polypeptide are administered from the same solution. In certain embodiments, the platinum-based antitumor agent and the LIF-binding polypeptide are administered from separate solutions. In certain embodiments, the solution includes a physiologically suitable salt concentration (e.g., NaCl). In certain embodiments, the solution includes about 0.6% and 1.2% NaCl. In certain embodiments, the solution includes about 0.7% and 1.1% NaCl. In certain embodiments, the solution includes about 0.8% and 1.0% NaCl. In certain embodiments, a highly concentrated mother solution of an antibody can be diluted in about 0.9% NaCl. In certain embodiments, the solution includes about 0.9% NaCl. In certain embodiments, the solution further comprises one or more of the following: a buffer, e.g., acetate, citrate, histidine, succinate, phosphate, bicarbonate, and hydroxymethylaminomethane (Tris); a surfactant, e.g., polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), polysorbate, and poloxamer 188; a polyol / disaccharide / polysaccharide, e.g., glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; an amino acid, e.g., histidine, glycine, or arginine; an antioxidant, e.g., ascorbic acid, methionine; and a chelating agent, e.g., EGTA or EGTA. In certain embodiments, the platinum-based anti-tumor agent and LIF-binding polypeptide of the present disclosure are lyophilized for transport / storage and reconstitution prior to administration. In certain embodiments, the lyophilized antibody formulation comprises a bulking agent, such as mannitol, sorbitol, sucrose, trehalose, and dextran 40. In certain embodiments, the anti-LIF antibody of the present disclosure can be transported and stored as a concentrated mother solution, which is diluted when used at the treatment site. In certain embodiments, the mother solution includes about 25mM histidine, about 6% sucrose, about 0.01% polysorbate, and about 20mg / mL of anti-LIF antibody. In certain embodiments, the pH of the solution is about 6.0. In certain embodiments, the form administered to an individual is an aqueous solution including about 25mM histidine, about 6% sucrose, about 0.01% polysorbate 80, and about 20mg / mL h5D8 antibody. In certain embodiments, the pH of the solution is about 6.0.

[0143] Also described herein are kits for performing the combination therapies described herein. In certain embodiments, the kit includes a LIF binding polypeptide and a platinum-based antitumor agent. In certain embodiments, the kit includes h5D8 and a platinum-based antitumor agent. One or both of the two components can be contained in a vial of glass or other suitable material in a lyophilized or liquid form.

[0144] In certain embodiments, the present invention describes the use of a LIF-binding polypeptide in combination with a platinum-based anti-tumor agent to treat non-small cell lung cancer, epithelial ovarian cancer, or pancreatic adenocarcinoma in an individual. In certain embodiments, the LIF-binding polypeptide and the platinum-based anti-tumor agent are administered to the individual in separate formulations. In certain embodiments, the LIF-binding polypeptide and the platinum-based anti-tumor agent are administered to the individual in the same formulation. In certain embodiments, the LIF-binding polypeptide is administered to the individual before the platinum-based anti-tumor agent is administered to the individual. In certain embodiments, the platinum-based anti-tumor agent is administered to the individual before the LIF-binding polypeptide is administered to the individual. In certain embodiments, the platinum-based anti-tumor agent is administered to the individual while the LIF-binding polypeptide is administered to the individual. In certain embodiments, the LIF-binding polypeptide comprises a fragment of an immunoglobulin variable region or an immunoglobulin heavy chain constant region. In certain embodiments, the LIF-binding polypeptide comprises an antibody that specifically binds to LIF. In certain embodiments, the antibody that specifically binds to LIF comprises at least one framework region derived from a human antibody framework region. In certain embodiments, the antibody that specifically binds to LIF is humanized. In certain embodiments, the antibody that specifically binds to LIF is deimmunized. In certain embodiments, the antibody that specifically binds to LIF comprises two heavy immunoglobulin chains and two light immunoglobulin chains. In certain embodiments, the antibody that specifically binds to LIF is an IgG antibody. In certain embodiments, the antibody that specifically binds to LIF is a Fab, F(ab)2, a single domain antibody, a single chain variable fragment (scFv) or a nanobody. In certain embodiments, the antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy chain complementary determining region 1 (VH-CDR1) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 1-3; (b) an immunoglobulin heavy chain complementary determining region 2 (VH-CDR2) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 4 or 5; (c) an immunoglobulin heavy chain complementary determining region 3 (VH-CDR3) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 6-8; (d) an immunoglobulin light chain complementary determining region 1 (VL-CDR1) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 9 or 10; (e) an immunoglobulin light chain complementary determining region 2 (VL-CDR2) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 11 or 12; and (f) an immunoglobulin light chain complementary determining region 3 (VL-CDR3) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 12 or 13. The amino acid sequence described in NO:13.In certain embodiments, the antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy chain variable region (VH) sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 41, 42, 44 or 66; and (b) an immunoglobulin light chain variable region (VL) sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 45-48. 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 antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy chain sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one 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%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 61-64. In certain embodiments, the antibody that specifically binds to LIF has a K of less than about 200 picomolar. D In certain embodiments, the antibody that specifically binds to LIF has a K of less than about 100 picomolar. D In certain embodiments, the platinum-based anti-tumor agent comprises cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenaplatin, picoplatin, setraplatin, or a combination thereof. In certain embodiments, the cancer is refractory to treatment with a therapeutic amount of a LIF-binding polypeptide or a platinum-based anti-tumor agent administered as a monotherapy.

[0145] In certain embodiments, the use of a LIF-binding polypeptide in combination with a platinum-based anti-tumor agent for treating colorectal cancer in an individual is described herein. In certain embodiments, the LIF-binding polypeptide and the platinum-based anti-tumor agent are administered to the individual in separate formulations. In certain embodiments, the LIF-binding polypeptide and the platinum-based anti-tumor agent are administered to the individual in the same formulation. In certain embodiments, the LIF-binding polypeptide is administered to the individual prior to administering the platinum-based anti-tumor agent to the individual. In certain embodiments, the platinum-based anti-tumor agent is administered to the individual prior to administering the LIF-binding polypeptide to the individual. In certain embodiments, the platinum-based anti-tumor agent is administered to the individual while the LIF-binding polypeptide is administered to the individual. In certain embodiments, the platinum-based anti-tumor agent is administered to the individual at the same time as the LIF-binding polypeptide is administered to the individual. In certain embodiments, the LIF-binding polypeptide comprises a fragment of an immunoglobulin variable region or an immunoglobulin heavy chain constant region. In certain embodiments, the LIF-binding polypeptide comprises an antibody that specifically binds to LIF. In certain embodiments, the antibody that specifically binds to LIF comprises at least one framework region derived from a human antibody framework region. In certain embodiments, the antibody that specifically binds to LIF is humanized. In certain embodiments, the antibody that specifically binds to LIF is deimmunized. In certain embodiments, the antibody that specifically binds to LIF comprises two heavy immunoglobulin chains and two light immunoglobulin chains. In certain embodiments, the antibody that specifically binds to LIF is an IgG antibody. In certain embodiments, the antibody that specifically binds to LIF is a Fab, F(ab)2, a single domain antibody, a single chain variable fragment (scFv) or a nanobody. In certain embodiments, the antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy chain complementary determining region 1 (VH-CDR1) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 1-3; (b) an immunoglobulin heavy chain complementary determining region 2 (VH-CDR2) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 4 or 5; (c) an immunoglobulin heavy chain complementary determining region 3 (VH-CDR3) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 6-8; (d) an immunoglobulin light chain complementary determining region 1 (VL-CDR1) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 9 or 10; (e) an immunoglobulin light chain complementary determining region 2 (VL-CDR2) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 11 or 12; and (f) an immunoglobulin light chain complementary determining region 3 (VL-CDR3) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 12 or 13. The amino acid sequence described in NO:13.In certain embodiments, the antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy chain variable region (VH) sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 41, 42, 44 or 66; and (b) an immunoglobulin light chain variable region (VL) sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 45-48. 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 antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy chain sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one 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%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 61-64. In certain embodiments, the antibody that specifically binds to LIF has a K of less than about 200 picomolar. D In certain embodiments, the antibody that specifically binds to LIF has a K of less than about 100 picomolar. D In certain embodiments, the platinum-based anti-tumor agent comprises cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenaplatin, picoplatin, setraplatin, or a combination thereof. In certain embodiments, the cancer is refractory to treatment with a therapeutic amount of a LIF-binding polypeptide or a platinum-based anti-tumor agent administered as a monotherapy.

[0146] In certain embodiments, the present invention describes the use of a LIF-binding polypeptide in combination with a platinum-based anti-tumor agent to treat glioblastoma multiforme in an individual. In certain embodiments, the LIF-binding polypeptide and the platinum-based anti-tumor agent are administered to the individual in separate formulations. In certain embodiments, the LIF-binding polypeptide and the platinum-based anti-tumor agent are administered to the individual in the same formulation. In certain embodiments, the LIF-binding polypeptide is administered to the individual before the platinum-based anti-tumor agent is administered to the individual. In certain embodiments, the platinum-based anti-tumor agent is administered to the individual before the LIF-binding polypeptide is administered to the individual. In certain embodiments, the platinum-based anti-tumor agent is administered to the individual while the LIF-binding polypeptide is administered to the individual. In certain embodiments, the LIF-binding polypeptide comprises a fragment of an immunoglobulin variable region or an immunoglobulin heavy chain constant region. In certain embodiments, the LIF-binding polypeptide comprises an antibody that specifically binds to LIF. In certain embodiments, the antibody that specifically binds to LIF comprises at least one framework region derived from a human antibody framework region. In certain embodiments, the antibody that specifically binds to LIF is humanized. In certain embodiments, the antibody that specifically binds to LIF is deimmunized. In certain embodiments, the antibody that specifically binds to LIF comprises two heavy immunoglobulin chains and two light immunoglobulin chains. In certain embodiments, the antibody that specifically binds to LIF is an IgG antibody. In certain embodiments, the antibody that specifically binds to LIF is a Fab, F(ab)2, a single domain antibody, a single chain variable fragment (scFv) or a nanobody. In certain embodiments, the antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy chain complementary determining region 1 (VH-CDR1) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 1-3; (b) an immunoglobulin heavy chain complementary determining region 2 (VH-CDR2) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 4 or 5; (c) an immunoglobulin heavy chain complementary determining region 3 (VH-CDR3) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 6-8; (d) an immunoglobulin light chain complementary determining region 1 (VL-CDR1) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 9 or 10; (e) an immunoglobulin light chain complementary determining region 2 (VL-CDR2) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 11 or 12; and (f) an immunoglobulin light chain complementary determining region 3 (VL-CDR3) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 12 or 13. The amino acid sequence described in NO:13.In certain embodiments, the antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy chain variable region (VH) sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 41, 42, 44 or 66; and (b) an immunoglobulin light chain variable region (VL) sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 45-48. 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 antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy chain sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one 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%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 61-64. In certain embodiments, the antibody that specifically binds to LIF has a K of less than about 200 picomolar. D In certain embodiments, the antibody that specifically binds to LIF has a K of less than about 100 picomolar. D In certain embodiments, the platinum-based anti-tumor agent comprises cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenaplatin, picoplatin, setraplatin, or a combination thereof. In certain embodiments, the cancer is refractory to treatment with a therapeutic amount of a LIF-binding polypeptide or a platinum-based anti-tumor agent administered as a monotherapy.

[0147] As used herein, "treatment" refers to an intervention intended to produce one or more beneficial effects in a disease state. For cancer / tumor purposes, treatment includes methods intended to cause or actually cause disease stabilization, partial response, complete response, progression-free survival extension, overall survival extension, tumor shrinkage, tumor growth delay, tumor growth stagnation, or prevention or reduction of metastasis. In some cases, the methods of treatment described herein can be used as maintenance after successful treatment or to prevent the recurrence or metastasis of a specific tumor or cancer. It is understood that not all individuals will respond to the same degree or no response at all to a given therapeutic antibody administration, but even if no response is detected, these individuals are still considered to have been treated.

[0148] Exemplary Embodiments

[0149] In exemplary embodiments are: 1. Use of a leukemia inhibitory factor (LIF) binding polypeptide in combination with a platinum-based anti-tumor agent for treating cancer in an individual. 2. The use as described in Example 1, wherein the LIF binding polypeptide and the platinum-based anti-tumor agent are administered to the individual in separate formulations. 3. The use as described in Example 1, wherein the LIF binding polypeptide and the platinum-based anti-tumor agent are administered to the individual in the same formulation. 4. The use as described in Example 1 or 2, wherein the LIF binding polypeptide is administered to the individual before the platinum-based anti-tumor agent is administered to the individual. 5. The use as described in Example 1 or 2, wherein the platinum-based anti-tumor agent is administered to the individual before the LIF binding polypeptide is administered to the individual. 6. The use as described in any one of Examples 1 to 3, wherein the platinum-based anti-tumor agent is administered to the individual at the same time as the LIF binding polypeptide is administered to the individual. 7. The use as described in any one of Examples 1 to 6, wherein the LIF binding polypeptide comprises a fragment of an immunoglobulin variable region or an immunoglobulin heavy chain constant region. 8. The use as described in Example 7, wherein the LIF-binding polypeptide comprises an antibody that specifically binds to LIF. 9. The use as described in Example 8, wherein the antibody that specifically binds to LIF comprises at least one framework region derived from a human antibody framework region. 10. The use as described in Example 8, wherein the antibody that specifically binds to LIF is humanized. 11. The use as described in Example 8, wherein the antibody that specifically binds to LIF is deimmunized. 12. The use as described in Example 8, wherein the antibody that specifically binds to LIF comprises two immunoglobulin heavy chains and two immunoglobulin light chains. 13. The use as described in Example 8, wherein the antibody that specifically binds to LIF is an IgG antibody. 14. The use as described in Example 8, wherein the antibody that specifically binds to LIF is a Fab, F(ab)2, a single domain antibody, a single chain variable fragment (scFv) or a nanobody.15. The use of any one of embodiments 8 to 14, wherein the antibody that specifically binds to LIF comprises: a) an immunoglobulin heavy chain complementary determining region 1 (VH-CDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 1-3; b) an immunoglobulin heavy chain complementary determining region 2 (VH-CDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 4 or 5; c) an immunoglobulin heavy chain complementary determining region 3 (VH-CDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 6-8; d) an immunoglobulin light chain complementary determining region 1 (VL-CDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 9 or 10; e) an immunoglobulin light chain complementary determining region 2 (VL-CDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 11 or 12; and f) an immunoglobulin light chain complementary determining region 3 (VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NOs: NO: 13. 16. The use of any one of embodiments 8 to 15, wherein the antibody that specifically binds to LIF comprises: a) an immunoglobulin heavy chain variable region (VH) sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 41, 42, 44 or 66; and b) an immunoglobulin light chain variable region (VL) sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 45-48. 17. The use of embodiment 16, wherein 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. 18. The use of embodiment 17, wherein 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: 46.19. The use of any one of embodiments 8 to 18, wherein the antibody that specifically binds to LIF comprises: a) an immunoglobulin heavy chain sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one 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%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 61-64. 20. The use of any one of embodiments 8 to 19, wherein the antibody that specifically binds to LIF binds with a KD of less than about 200 picomolar. 21. The use of any one of embodiments 8 to 19, wherein the antibody that specifically binds to LIF binds with a KD of less than about 100 picomolar. 22. The use of any one of embodiments 10 to 21, wherein the platinum-based anti-tumor agent comprises cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatinium tetranitrate, phenaplatin, picoplatin, setraplatin or a combination thereof. 23. The use of embodiment 22, wherein the platinum-based anti-tumor agent is cisplatin. 24. The use of any one of embodiments 1 to 23, wherein the cancer comprises an advanced solid tumor, glioblastoma, gastric 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. 25. The use of embodiment 24, wherein the cancer comprises non-small cell lung cancer, epithelial ovarian cancer or pancreatic adenocarcinoma. 26. The use of any one of embodiments 1 to 25, wherein the cancer is refractory to treatment with a therapeutic amount of a LIF-binding polypeptide or a platinum-based anti-tumor agent administered as a monotherapy. 27. A method of treating an individual with cancer, comprising administering to the individual with cancer an effective amount of a combination of: a) a leukemia inhibitory factor (LIF) binding polypeptide; and b) a platinum-based anti-tumor agent. 28. The method of embodiment 27, comprising administering to the individual with cancer an effective amount of the LIF binding polypeptide. 29. The method of embodiment 27, comprising administering to the individual with cancer an effective amount of the platinum-based anti-tumor agent. 30. The method of any one of embodiments 27 to 29, wherein the LIF binding polypeptide comprises a fragment of an immunoglobulin variable region or an immunoglobulin heavy chain constant region. 31. The method of any one of embodiments 27 to 29, wherein the LIF binding polypeptide comprises an antibody that specifically binds to LIF. 32. The method of embodiment 31, wherein the antibody that specifically binds to LIF comprises at least one framework region derived from a human antibody framework region. 33. The method of embodiment 31, wherein the antibody that specifically binds to LIF is humanized.34. The method of embodiment 31, wherein the antibody that specifically binds to LIF is deimmunized. 35. The method of embodiment 31, wherein the antibody that specifically binds to LIF comprises two heavy immunoglobulin chains and two light immunoglobulin chains. 36. The method of embodiment 31, wherein the antibody that specifically binds to LIF is an IgG antibody. 37. The method of embodiment 31, wherein the antibody that specifically binds to LIF is a Fab, F(ab)2, a single domain antibody, a single chain variable fragment (scFv), or a nanobody. 38. The method of any one of embodiments 31 to 37, wherein the antibody that specifically binds to LIF comprises: a) an immunoglobulin heavy chain complementary determining region 1 (VH-CDR1) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 1-3; b) an immunoglobulin heavy chain complementary determining region 2 (VH-CDR2) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 4 or 5; c) an immunoglobulin heavy chain complementary determining region 3 (VH-CDR3) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 6-8; d) an immunoglobulin light chain complementary determining region 1 (VL-CDR1) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 9 or 10; e) an immunoglobulin light chain complementary determining region 2 (VL-CDR2) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 11 or 12; and f) an immunoglobulin light chain complementary determining region 3 (VL-CDR3) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 12 or 13. NO: 13. 39. The method of any one of embodiments 31 to 37, wherein the antibody that specifically binds to LIF comprises: a) an immunoglobulin heavy chain variable region (VH) sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 41, 42, 44 or 66; and b) an immunoglobulin light chain variable region (VL) sequence having an amino acid sequence that is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 45-48. 40. The method of embodiment 39, wherein the VH sequence is at least about 80%, 90%, 95%, 97%, 98%, 99% or 100% 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%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:46.41. The method of embodiment 40, wherein 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. 42. The method of any one of embodiments 31 to 37, wherein the antibody that specifically binds to LIF comprises: a) an immunoglobulin heavy 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 one 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 one of SEQ ID NOs:61-64. 43. The method of any one of embodiments 31 to 42, wherein the antibody that specifically binds to LIF binds with a KD of less than about 200 picomolar. 44. The method of any one of embodiments 31 to 42, wherein the antibody that specifically binds to LIF binds with a KD of less than about 100 picomolar. 45. The method of any one of embodiments 27 to 44, wherein the platinum-based anti-tumor agent comprises cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenaplatin, picoplatin, setraplatin, or a combination thereof. 46. The method of embodiment 45, wherein the platinum-based anti-tumor agent is cisplatin. 47. The method of any one of embodiments 27 to 46, wherein the cancer comprises an advanced solid tumor, glioblastoma, gastric 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. 48. The method of embodiment 47, wherein the cancer comprises non-small cell lung cancer, epithelial ovarian cancer, or pancreatic adenocarcinoma. 49. The method of any one of embodiments 27 to 48, wherein the cancer is refractory to treatment with a therapeutic amount of a LIF-binding polypeptide inhibitor. 50. The method of any one of embodiments 27 to 48, wherein the cancer is refractory to treatment with a therapeutic amount of a platinum-based anti-tumor agent. 51. The method of any one of embodiments 27 to 50, wherein the leukemia inhibitory factor (LIF)-binding polypeptide and the platinum-based anti-tumor agent are administered separately. 52. The method of any one of embodiments 27 to 50, wherein the LIF-binding polypeptide and the platinum-based anti-tumor agent are administered simultaneously. 53. The method of any one of embodiments 27 to 50, wherein the LIF-binding polypeptide and the platinum-based anti-tumor agent are administered as a single composition.54. Use of an antibody that specifically binds to leukemia inhibitory factor (LIF) in combination with a platinum-based anti-tumor agent to treat cancer in an individual, wherein the LIF-binding antibody comprises: a) an immunoglobulin heavy chain complementary determining region 1 (VH-CDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 1-3; b) an immunoglobulin heavy chain complementary determining region 2 (VH-CDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 4 or 5; c) an immunoglobulin heavy chain complementary determining region 3 (VH-CDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 6-8; d) an immunoglobulin light chain complementary determining region 1 (VL-CDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 9 or 10; e) an immunoglobulin light chain complementary determining region 2 (VL-CDR2) comprising the amino acid sequence set forth in SEQ ID NOs: NO: 11 or 12; and f) an immunoglobulin light chain complementary determining region 3 (VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13. 55. A method for treating an individual suffering from cancer, comprising administering to the individual suffering from cancer an effective amount of a combination of the following items: a) an antibody that specifically binds to leukemia inhibitory factor (LIF), comprising: i. an immunoglobulin heavy chain complementary determining region 1 (VH-CDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 1-3; ii. an immunoglobulin heavy chain complementary determining region 2 (VH-CDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 4 or 5; iii. an immunoglobulin heavy chain complementary determining region 3 (VH-CDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 6-8; iv. an immunoglobulin light chain complementary determining region 1 (VL-CDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 9 or 10; v. an immunoglobulin light chain complementary determining region 2 (VL-CDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 10-11; NO: 11 or 12; and vi. an immunoglobulin light chain complementary determining region 3 (VL-CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 13; and b) a platinum-based anti-tumor agent. 56. The method of embodiment 55, comprising administering an effective amount of the LIF-binding polypeptide to the individual suffering from cancer. 57. The method of embodiment 55, comprising administering an effective amount of the platinum-based anti-tumor agent to the individual suffering from cancer.

[0150] Examples

[0151] The following illustrative examples represent embodiments of the compositions and methods described herein and are not meant to be limiting in any way.

[0152] Example 1 - Generation of rat antibodies specific for LIF

[0153] The cDNA encoding the amino acid 23-202 of human LIF is cloned into an expression plasmid (Aldevron GmbH, Freiburg, Germany). Using a handheld particle bombardment device ("gene gun"), each group of experimental rats (Wistar) is immunized by intradermal administration of gold particles coated with DNA. The cell surface expression on transiently transfected HEK cells was confirmed with an anti-tag antibody that recognizes the tag added to the N-terminus of the LIF protein. Serum samples were collected after a series of immunizations, and HEK cells transiently transfected with the above-mentioned expression plasmid were tested in flow cytometry. The cells producing antibodies were separated and fused with mouse myeloma cells (Ag8) according to standard procedures. As described above, hybridomas producing antibodies specific to LIF were identified by screening in flow cytometry. Cell precipitation of positive hybridoma cells was prepared using RNA protectants (RNAlater, catalog number AM7020, Thermo Fisher Scientific), and further processed for sequencing of antibody variable domains.

[0154] Example 2 - Generation of mouse antibodies specific for LIF

[0155] The cDNA encoding the amino acid 23-202 of human LIF is cloned into an expression plasmid (Aldevron GmbH, Freiburg, Germany). Using a handheld particle bombardment device ("gene gun"), each group of experimental mice (NMRI) is immunized by intradermal administration of gold particles coated with DNA. The cell surface expression on transiently transfected HEK cells was confirmed with an anti-tag antibody that recognizes the tag added to the N-terminus of the LIF protein. Serum samples were collected after a series of immunizations, and HEK cells transiently transfected with the above-mentioned expression plasmid were tested in flow cytometry. The cells producing antibodies were separated and fused with mouse myeloma cells (Ag8) according to standard procedures. As described above, hybridomas producing antibodies specific to LIF were identified by screening in flow cytometry. Cell precipitation of positive hybridoma cells was prepared using RNA protectants (RNAlater, catalog number AM7020, Thermo Fisher Scientific), and further processed for sequencing of antibody variable domains.

[0156] Example 3 - Humanization of a rat antibody specific for LIF

[0157] Select a clone (5D8) from rat immunization for subsequent humanization. Humanization was performed using a standard CDR transplantation method. Heavy and light chain regions were cloned from 5D8 hybridomas using standard molecular cloning techniques and sequenced by the Sanger method. BLAST searches were then performed for human heavy and light chain variable sequences, and 4 sequences were selected from each variable sequence as receptor frameworks for humanization. These receptor frameworks were deimmunized to remove T cell response epitopes. The heavy and light chain CDR1, CDR2, and CDR3 of 5D8 were cloned into 4 different heavy chain receptor frameworks (H1 to H4) and 4 different light chain frameworks (L1 to L4). All 16 different antibodies were then tested: expression in CHO-S cells (Selexis); inhibition of STAT3 phosphorylation induced by LIF; and binding affinity by surface plasmon resonance (SPR). These experiments are summarized in Table 1.

[0158]

[0159]

[0160] After 10 days of cell culture, cells were plated in fed-batch conical flasks (seeded with 3x10 5 The expression performance of transfected cells was compared with that of control cells (cells / mL, 200 mL culture volume). At this point, the cells were harvested and the secreted antibodies were purified using a protein A column and then quantified. All humanized antibodies were expressed, except for the antibody using the H3 heavy chain (SEQ ID NO: 43).

[0161] Inhibition of LIF-induced STAT3 phosphorylation on tyrosine 705 was determined by Western blotting. U251 glioma cells were plated in 6-well plates at a density of 100.000 cells / well. Before any treatment, cells were cultured in complete medium for 24 hours, followed by serum starvation for 8 hours. Afterwards, cells containing the indicated antibodies were incubated overnight at a concentration of 10 μg / ml. After treatment, proteins were obtained in phosphatase and protease inhibitors containing radioimmunoprecipitation assay (RIPA) lysis buffer, quantified (BCA protein assay, Thermo Fisher Scientific) and used for Western blotting. For Western blotting, the membranes were incubated in 5% skim milk powder-TBST for 1 hour and incubated with primary antibodies overnight (p-STAT3, catalog number Block 9145, Cell Signaling or STAT3, catalog number 9132, Cell Signaling) or 30 minutes (β-actin peroxidase, catalog number A3854, Sigma-Aldrich). The membranes were then washed with TBST, incubated with secondary antibodies and washed again. Proteins were detected by chemiluminescence (SuperSignal substrate, catalog number 34076, Thermo Fisher Scientific). These results are shown in Figure 1 The darker the pSTAT3 band, the less inhibitory effect. In the lanes labeled 5D8 (non-humanized rat), A (H0L0), C (H1L2), D (H1L3), and G (H2L2), the inhibitory effect is high; in H (H2L3), O (H4L2), and P (H4L3), the inhibitory effect is moderate; in B (H1L1), E (H1L4), F (H2L1), I (H2L4), N (H4L1), and Q (H4L4), there is no inhibitory effect.

[0162] The antibodies that exhibited inhibition of LIF-induced STAT3 phosphorylation were then analyzed by SPR to determine binding affinity. TM The 2002 instrument observed the binding of A (H0L0), C (H1L2), D (H1L3) and G (H2L2), H (H2L3) and O (H4L2) humanized antibodies to amine-coupled hLIF. At six ligand concentrations, the kinetic constants and affinities were determined by mathematical sensorgram fitting (Langemuir interaction model [A+B=AB]) of all sensorgrams generated on all sensor chip surfaces. The best fit curve (minimum Chi2) for each concentration was used to calculate the kinetic constants and affinities. See Table 1.

[0163] Since the experimental setup used a bivalent antibody as the analyte, the best-fit sensorgrams were also analyzed according to the bivalent analyte fitting model [A+B=AB; AB+B=AB2] in order to gain more detailed insights into the target binding mechanism of the humanized antibody. Kinetic sensorgram analysis using the bivalent fitting model [A+B=AB; AB+B=AB2] confirmed the relative affinity ranking of the mAb samples.

[0164] Humanized 5D8 including H2 and L2 was selected for more in-depth analysis due to its high binding affinity and high batch culture productivity.

[0165] Example 4 - Humanization of clone 5D8 improves binding to LIF

[0166] The H2L2 clone (h5D8) was selected for further analysis and compared with the parent rat 5D8 (r5D8) and mouse clone 1B2 by SPR. The 1B2 antibody is a previously disclosed mouse anti-LIF antibody, previously deposited at Demsche Sammlungvon Mikroorganismen and Zellkulturen GmbH (DSM ACC3054), and used for comparison purposes. Recombinant human LIF purified from Escherichia coli and HEK-293 cells was used as a ligand, respectively. LIF from human or Escherichia coli sources was covalently coupled to the surface of the Biacore optical sensor chip using amine coupling chemistry, and binding affinity was calculated based on kinetic constants.

[0167] Materials and methods

[0168] Human LIF from E. coli was obtained from Millipore, reference LIF1010; human LIF from HEK-293 cells was obtained from ACRO Biosystems, reference LIF-H521b. LIF was coupled to the sensor chip using the Biacore amine coupling kit (BR-1000-50; GE Healthcare, Uppsala). CM5 optical sensor chips (BR-1000-12; GE Healthcare, Uppsala) were used in Biacore TMSamples were run on a 2002 instrument. Biacore HBS-EP buffer was used during machine runs (BR-1001-88; GE Healthcare, Uppsala). Kinetic analysis of binding sensorgrams was performed using BIAevaluation 4.1 software. Kinetic constants and affinities were determined by mathematical sensorgram fit (Langmuir interaction model [A+B=AB]) of all sensorgrams generated on all sensor chip surfaces at increasing analyte concentrations. Sensorgrams were also analyzed according to the bivalent analyte sensorgram fit model [A+B=AB; AB+B=AB2], including component analysis, in order to generate an estimate of the bivalent contribution to the determined Langmuir antibody-target affinity (e.g., affinity contribution). The best fit curve (minimum Chi) for each concentration was used as the 2 ) were used to calculate kinetic constants and affinities. An overview of these affinity experiments is shown in Table 2 (human LIF produced in E. coli) and Table 3 (human LIF produced in HEK 293 cells).

[0169]

[0170]

[0171] The Langmuir 1:1 sensorgram fit model from this set of experiments indicated that the affinity of the humanized 5D8 (h5D8) antibody to human LIF was approximately 10-25 fold higher than that of mouse 1B2 and r5D8.

[0172] Next, the h5D8 antibody was tested against LIF from multiple species by SPR. h5D8 SPR binding kinetics were performed on recombinant LIF analytes derived from different species and expression systems: human LIF (E. coli, HEK293 cells); mouse LIF (E. coli, CHO cells); rat LIF (E. coli); cynomolgus monkey LIF (yeast, HEK293 cells).

[0173] Materials and methods

[0174] The h5D8 antibody was immobilized on the sensor chip surface by non-covalent Fc-specific capture. Recombinant Ig (Fc)-specific Staphylococcus aureus protein A / G was used as a capture agent, so that the anti-LIF antibody could be presented to the LIF analyte in a spatially uniform and flexible manner. The sources of the LIF analyte were as follows: human LIF (from E. coli; Millipore reference number LIF1050); human LIF (from HEK cells, ACRO Biosystems LIF-H521); mouse LIF (E. coli; Millipore catalog number NF-LIF2010); mouse LIF (from CHO cells; Reprokine catalog number RCP09056); monkey LIF (yeast, Kingfisher Biotech catalog number RP1074Y); monkey LIF produced in HEK-293 cells. Overall, h5D8 exhibited binding to LIF from several species. An overview of the affinity experiments is shown in Table 4.

[0175]

[0176] Example 5 - Humanized clone 5D8 inhibits LIF-induced STAT3 phosphorylation in vitro

[0177] To determine the biological activity of h5D8, humanized and parental forms were tested in a cell culture model of LIF activation. Figure 2A It is shown that humanized clones exhibited increased inhibition of STAT3 phosphorylation (Tyr 705) when glioma cell lines were incubated with human LIF. Figure 2B Shown are repeated experiments with different dilutions of h5D8 antibody. Figure 2A Experiments with the same setup.

[0178] method

[0179] U251 glioma cells were plated in 6-well plates at a density of 150,000 cells / well. Cells were cultured in complete medium for 24 hours before any treatment. Afterwards, they were treated with r5D8 anti-LIF antibody or h5D8 anti-LIF antibody at a concentration of 10 μg / ml overnight or left untreated (control cells).

[0180] After treatment, proteins were obtained in phosphatase and protease inhibitors containing radioimmunoprecipitation assay (RIPA) lysis buffer, quantified (BCA protein assay, Thermo Fisher Scientific) and used for western blotting. For western blotting, the membrane was in 5% non-fat milk-TBST for 1 hour and incubated with the primary antibody overnight (p-STAT3, catalog number blocking 9145, cell signaling company or STAT3, catalog number 9132, cell signaling company) or 30 minutes (β-actin peroxidase, catalog number A3854, Sigma Aldrich). The membrane was then washed with TBST, incubated with the secondary antibody if necessary, and washed again. Proteins were detected by chemiluminescence (SuperSignal substrate, catalog number 34076, Thermo Fisher Scientific).

[0181] Example 6 - IC of h5D8 Antibody Treatment on Endogenous Levels of LIF in U-251 Cells 50 value.

[0182] The IC of the bioinhibitory effect of h5D8 in U-251 cells under serum starvation conditions was also determined. 50 As low as 490 pmol ( Figure 3A ). See Figure 3A and 3B and Table 5 for representative results.

[0183]

[0184] method

[0185] The U-251 cells were seeded at 600,000 cells per 6 cm plate (each condition). Under serum starvation (0.1% FBS), cells were treated with h5D8 at the corresponding concentration (titration) at 37°C overnight. As a positive control for pSTAT3, recombinant LIF (R&D#7734-LF / CF) was used to stimulate cells at 1.79nM for 10 minutes at 37°C. As a negative control for pSTAT3, JAK I inhibitor (Calbiochem#420099) was used for 30 minutes at 1uM at 37°C. Cells were then harvested on ice as lysates according to the protocols of the Meso ScaleDiscovery multipoint detection system total STAT3 (Catalog No. K150SND-2) and Phospho-STAT3 (Tyr705) (Catalog No. K150SVD-2) kits to measure protein levels detectable by MSD Meso Sector S600.

[0186] Example 7 - Other Antibodies That Bind Specifically to Human LIF

[0187] Additional rat antibody clones (10G7 and 6B5) were identified that specifically bound human LIF, an overview of their binding characteristics is shown below in Table 6, with clone 1B2 used for comparison.

[0188] method

[0189] Kinetic real-time binding assays were performed on anti-LIF mAbs 1B2, 10G7 and 6B5 immobilized on the surface of a CM5 optical sensor chip using recombinant LIF target proteins [human LIF (E. coli); Millipore Catalog No. LIF 1010 and human LIF (HEK293 cells); ACRO Biosystems Catalog No. LIF-H521b] as analytes.

[0190] Kinetic constants and affinities were obtained by mathematical sensorgram fitting using the Langmuir 1:1 binding model applying global (simultaneous fitting of sensorgram sets) as well as single curve fitting algorithms. obs Analyze and evaluate the rationality of the global fit.

[0191]

[0192] Example 8 - Other anti-LIF antibodies inhibit LIF-induced STAT3 phosphorylation in vitro

[0193] Other clones were tested for their ability to inhibit LIF-induced STAT3 phosphorylation in cell culture. Figure 4 As shown, clone 10G7 and previously described r5D8 exhibited high inhibition of LIF-induced STAT3 phosphorylation compared to clone 1B2. Anti-LIF polyclonal antiserum (positive) was included as a positive control. Although 6B5 did not exhibit an inhibitory effect, this may be due to the possible lack of binding of 6B5 to the unglycosylated LIF used in this experiment.

[0194] method

[0195] Patient-derived glioma cells were seeded in 6-well plates at a density of 150,000 cells / well. Before any treatment, cells were cultured in a GBM medium consisting of a neurobasal medium (Life Technologies) supplemented with B27 (Life Technologies), penicillin / streptomycin and growth factors (20ng / ml EGF and 20ng / ml FGF-2 [PeproTech]) for 24 hours. The next day, cells were treated with a mixture of recombinant LIF produced in Escherichia coli or recombinant LIF and the indicated antibody for 15 minutes (the final concentration of the antibody was 10 μg / ml and the final concentration of the recombinant LIF was 20ng / ml). After treatment, proteins were obtained in a phosphatase and protease inhibitor containing a radioimmunoprecipitation assay (RIPA) lysis buffer, quantified (BCA protein assay, Thermo Fisher Scientific) and used for Western blotting. For Western blotting, the membrane was incubated in 5% non-fat milk-TBST for 1 hour and incubated with the primary antibody overnight (p-STAT3, catalog number Block 9145, Cell Signaling) or 30 minutes (β-actin peroxidase, catalog number A3854, Sigma-Aldrich). The membrane was then washed with TBST, incubated with the secondary antibody if necessary, and washed again. Proteins were detected by chemiluminescence (SuperSignal substrate, catalog number 34076, Thermo Fisher Scientific).

[0196] Example 9 - LIF is highly overexpressed in multiple tumor types

[0197] Immunohistochemistry experiments were performed on a variety of human tumor types to determine the extent of LIF expression. Figure 5 As shown, LIF is highly expressed in glioblastoma multiforme (GBM), non-small cell lung cancer (NSCLC), ovarian cancer, colorectal cancer (CRC), and pancreatic cancer.

[0198] Example 10 - Humanized clone h5D8 inhibits tumor growth in a mouse model of non-small cell lung cancer

[0199] To determine the ability of the humanized 5D8 clone to inhibit LIF-positive cancers in vivo, the antibody was tested in a non-small cell lung cancer (NSCLC) mouse model. Fig. 6A It is shown that tumor growth was reduced in mice treated with the antibody compared to vehicle negative control. Figure 6B Data generated using version r5D8 are shown.

[0200] method

[0201] The murine non-small cell lung cancer (NSCLC) cell line KLN205 with high LIF levels was stably infected with a lentivirus expressing the firefly luciferase gene for in vivo bioluminescence monitoring. To establish the mouse model, 5x10 5 KLN205 non-small cell lung cancer (NSCLC) cells were orthotopically implanted in the left lung of 8-week-old immune-competent homologous DBA / 2 mice. Mice were treated intraperitoneally with control vehicles or with 15mg / kg or 30mg / kg of h5D8 antibodies twice a week, and the growth of tumors was monitored by bioluminescence. For bioluminescent imaging, mice received an intraperitoneal injection of 0.2mL 15mg / mL D-luciferin under 1%-2% inhalation isoflurane anesthesia. The IVIS system 2000 series (Xenogen Corp., Alameda, California, the United States) was used to monitor bioluminescent signals, which consisted of a highly sensitive cooling CCD camera. The imaging data was gridded using Living image software (Xenogen Corp.), and the total bioluminescent signal was integrated in each framed area. The total photon flux emission (photons / second) in the region of interest (ROI) was used to analyze the data. The results show that treatment with h5D8 antibodies promotes tumor regression. Data are presented as mean ± SEM.

[0202] Example 11 - h5D8 inhibits tumor growth in a mouse model of glioblastoma multiforme

[0203] In an orthotopic GBM tumor model using the human cell line U251 expressing luciferase, r5D8 significantly reduced tumor volume in mice administered 300 μg of r5D8 and h5D8 by intraperitoneal (IP) injection twice weekly. The results of this study are shown in Fig. 7A (quantification at day 26 after treatment). This experiment was also performed using 200 μg or 300 μg of humanized h5D8 to treat mice, showing statistically significant tumor reduction after 7 days of treatment. Figure 7B Data from mice are shown, and the mice were inoculated with human U251 GBM cells expressing luciferase, and then treated with 100, 200 or 300 μg of h5D8 or vehicle twice a week. Tumor size was determined by bioluminescence (Xenogen IVIS spectrum) on the 7th day. The figure shows individual tumor measurements, and horizontal bars represent mean ± SEM. Statistical significance was calculated using unpaired nonparametric Mann-Whitney U test.

[0204] method

[0205] U251 cells stably expressing luciferase were harvested, washed in PBS, centrifuged at 400 g for 5 min, resuspended in PBS, and counted using an automated cell counter (Countess, Invitrogen). Cells were kept on ice to maintain optimal viability. Ketamine was injected intraperitoneally / Xylosamine (75 mg / kg and 10 mg / kg, respectively). Each mouse was carefully placed in a stereotaxic apparatus and fixed. The hair on the head was removed with a depilatory cream, and the head skin was cut with a scalpel to expose the skull. A small incision was carefully made with a drill bit 1.8 mm lateral to the λ and 1 mm in front. 5 μL of cells were inoculated into the right striatum at a depth of 2.5 mm using a Hamilton 30G syringe. The head incision was closed with Hystoacryl tissue adhesive (Braun), and the mice were injected subcutaneously with the analgesic meloxicam. (1mg / kg). The final number of cells implanted into each mouse was 3x10 5 .

[0206] Mice were treated with h5D8 intraperitoneally twice a week. Treatment began immediately after tumor cell inoculation on day 0. Mice received a total of 2 doses of h5D8 or vehicle control.

[0207] Body weight and tumor volume: Body weight was measured 2 times / week, and tumor growth was quantified by bioluminescence (Xenogen IVIS Spectrum) on day 7. To quantify bioluminescent activity in vivo, mice were anesthetized with isoflurane and injected intraperitoneally with luciferin substrate (PerkinElmer) (167 μg / kg).

[0208] Tumor size, determined by bioluminescence (Xenogen IVIS Spectrum), was assessed on day 7. Individual tumor measurements and mean ± SEM were calculated for each treatment group. Statistical significance was determined by the unpaired, nonparametric Mann-Whitney U test.

[0209] Example 12 - h5D8 inhibits tumor growth in an ovarian cancer mouse model

[0210] The efficacy of r5D8 was evaluated in two other syngeneic tumor models. In the ovarian orthotopic tumor model ID8, twice-weekly intraperitoneal administration of 300 μg of r5D8 significantly inhibited tumor growth as measured by abdominal volume ( Fig. 8A and 8B ). Figure 8C The results showed that h5D8 also reduced tumor volume at doses of 200 μg and above.

[0211] method

[0212] ID8 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) 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).

[0213] ID8 cells were harvested, washed in PBS, centrifuged at 400 g for 5 minutes, and resuspended in PBS. Cells were placed on ice to maintain optimal viability, and 200 μL of the cell suspension was injected intraperitoneally using a 27G needle. The final number of cells implanted into mice was 5x10 6 .

[0214] Mice were treated with h5D8 intraperitoneally twice a week at various doses as indicated. Body weight was measured 2 times / week, and tumor progression was monitored by measuring abdominal circumference using a caliper (Fisher Scientific).

[0215] Example 13 - r5D8 inhibits tumor growth in a mouse model of colorectal cancer

[0216] In mice bearing subcutaneous colon CT26 tumors, r5D8 (300 μg administered intraperitoneally twice a week) significantly inhibited tumor growth ( Fig.9A and 9B ).

[0217] method

[0218] 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.

[0219] CT26 cells (8x10 5 ), rinsed with PBS, centrifuged at 400 g for 5 minutes, and resuspended in 100 μL PBS. The cells were placed on ice to avoid cell death. The CT26 cells were administered to mice by subcutaneous injection using a 27G needle.

[0220] Starting from day 3 after CT26 cell implantation, mice were administered 300 μg r5D8 or vehicle control by intraperitoneal injection (IP) twice a week.

[0221] Body weight and tumor volume were measured three times a week. Tumor volume was measured using a caliper (Fisher Scientific).

[0222] Example 14 - r5D8 reduces inflammatory infiltration in a tumor model

[0223] like Fig. 10A As shown, in the U251 GBM orthotopic model, the expression of CCL22, a marker for M2 polarized macrophages, was significantly reduced in tumors treated with r5D8. Fig. 10B This finding was also confirmed in a physiologically relevant organotypic tissue slice culture model using h5D8, where three patient samples showed a significant decrease in CCL22 and CD206 (MRC1) expression (also a marker for M2 macrophages) after treatment (compare upper left (control) and lower right (treated) for MRC1 and CCL22). In addition, r5D8 also reduced syngeneic ID8 ( Fig. 10C ) and CT26( Fig. 10D ) CCL22 in tumors + H5D8 treatment also programmed macrophages toward an immunostimulatory phenotype in the syngeneic CT26 tumor model ( Fig.10E h5D8 treatment increased macrophages with an M1 phenotype, as shown by an increased CD206-negative / MHCII-positive fraction, while macrophages with an M2 phenotype decreased, as shown by a decreased CD206-positive / MHCII-negative fraction. Fig.10F Gene expression data from monocytes cultured in conditioned media from LIF knockdown U251 cells are shown. MRC1, CCL2, CCL1, and CTSK (indicated by triangles) all showed significant reductions in expression.

[0224] Example 15-r5D8 increases non-myeloid effector cells

[0225] To investigate other immune mechanisms, the effects of r5D8 on T cells and other non-myeloid immune effector cells within the tumor microenvironment were evaluated. Fig.11A As shown, in an orthotopic ID8 syngeneic model, r5D8 treatment resulted in an increase in intratumoral NK cells and total and activated CD4 + and CD8 + T cells increase. Fig. 11B Similarly, in a syngeneic colon CT26 tumor model, r5D8 increased intratumoral NK cells, increased CD4+ and CD8+ T cells, and tended to decrease CD4 + CD25 + FoxP3 + T-reg cells. Fig. 11CAs shown, CD4 + CD25 + FoxP3 + There is a trend of a decrease in T-reg cells. Fig.12 As shown, consistent with the requirement for T cell-mediated efficacy, CD4 + and CD8 + Depletion of T cells suppressed the antitumor efficacy of r5D8.

[0226] T cell depletion method

[0227] CT26 cells were cultured in RPMI 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 plasminogen activator (Invitrogen). CT26 cells (5×10 5 ), rinsed with PBS, centrifuged at 400g for 5 minutes, and resuspended in 100 μL PBS. Cells were placed on ice to avoid cell death. CT26 cells were administered to mice in both ribs by subcutaneous injection using a 27G syringe. As shown in the study design, r5D8 was administered intraperitoneally twice a week to treat mice. As described in the study design, vehicle control (PBS), rat r5D8 and / or anti-CD4 and anti-CD8 were administered to mice by intraperitoneal injection (IP) twice a week. All antibody treatments were administered concomitantly.

[0228] Example 16 - Crystal structure of h5D8 in complex with human LIF

[0229] The crystal structure of h5D8 was solved to a resolution of 3.1 angstroms in order to identify the epitope on LIF to which h5D8 binds and to determine the h5D8 residues involved in binding. The co-crystal structure revealed that the N-terminal loop of LIF is located centrally between the variable regions of the h5D8 light and heavy chains ( Fig.13A In addition, h5D8 interacts with residues on helices A and C of LIF, forming discontinuous and conformational epitopes. Binding is driven by several salt bridges, H-bonds, and van der Waals interactions (Table 7, Fig. 13B). The h5D8 epitope of LIF spans the region that interacts with gp130. See Boulanger, MJ, Bankovich, AJ, Kortemme, T., Baker, D. & Garcia, KC Convergent mechanisms for recognition of divergent cytokines by the shared signaling receptor gp130. Molecular cell 12, 577-589 (2003). The results are summarized in Table 7 below and depicted in Figure 13.

[0230]

[0231]

[0232] method

[0233] LIF was added to HEK 293S (Gnt I - / - ) cells and purified using Ni-NTA affinity chromatography followed by gel filtration chromatography in 20mM Tris pH 8.0 and 150mM NaCl. Recombinant h5D8 Fab was transiently expressed in HEK 293F cells and purified using KappaSelect affinity chromatography followed by cation exchange chromatography. The purified h5D8 Fab and LIF were mixed at a molar ratio of 1:2.5 and incubated at room temperature for 30 minutes, followed by deglycosylation using EndoH. The complex was then purified using gel filtration chromatography. The complex was concentrated to 20 mg / mL and crystallization trials were performed using sparse matrix screening. Crystals formed at 4°C under conditions containing 19% (v / v) isopropanol, 19% (w / v) PEG 4000, 5% (v / v) glycerol, and 0.095M sodium citrate pH 5.6. The crystals diffracted to The data were collected, processed and scaled using XDS according to Kabsch et al. Xds. Acta crystallographica. Section D, Biological crystallography 66, 125-132 (2010). The structure was determined by molecular replacement using Phaser according to McCoy et al. Phaser crystallographic software. J Appl Crystallogr 40, 658-674 (2007). Several iterations of model building and refinement were performed using Coot and phenix.refine until the structure converged to an acceptable R 工作 and R 自由 . See, respectively, 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). These figures were developed in PyMOL (PyMOL Molecular Graphics System, Version 2.0, Schrodinger GmbH & Co. KG). LLC)).

[0234] Example 17 - h5D8 has high specificity for LIF

[0235] The binding of h5D8 to other LIF family members was tested to determine binding specificity. When both proteins were produced in E. coli, h5D8 bound to human LIF approximately 100 times better than the binding to Oncostatin M (OSM), the IL-6 family member with the highest homology to LIF, using Octet96 analysis. When both proteins were produced in mammalian systems, h5D8 did not exhibit binding to OSM. The data are summarized in Table 8.

[0236]

[0237]

[0238] method

[0239] Octet binding experiments: Reagents were used and prepared according to the manufacturer's manual. Basic kinetic experiments were performed using Octet data acquisition software version 9.0.0.26 as follows: Sensor / Program settings: i) equilibration (60 seconds); ii) loading (15 seconds); iii) baseline (60 seconds); iv) association (180 seconds); and v) dissociation (600 seconds)

[0240] Octet affinity of h5D8 for cytokines: Basic kinetic experiments were performed using Octet data acquisition software version 9.0.0.26 as follows: Amine-reactive second generation biosensors (AR2G) were hydrated in water for a minimum of 15 minutes. Conjugation of h5D8 to the biosensor was performed using the amine coupling second generation kit according to ForteBio technical note 26 (see references). The immersion steps were performed at 30°C 1000rpm as follows: i) equilibration in water for 60 seconds; ii) activation in 20mM ECD, 10mM sulfo-NHS in water for 300 seconds; iii) fixation of 10μg / ml h5D8 in 10mM sodium acetate, pH 6.0 for 600 seconds; iv) quenching in 1M ethanolamine, pH 8.5 for 300 seconds; v) baseline in water for 120 seconds. Kinetic experiments were then performed at 30°C, 1000 rpm with the following immersion and reading steps: vi) baseline in 1X kinetic buffer for 60 seconds; vii) association of appropriate cytokine serial dilutions in 1X kinetic buffer for 180 seconds; viii) dissociation in 1X kinetic buffer for 300 seconds; ix) three regeneration / neutralization cycles alternating between 10 mM glycine pH 2.0 and 1X kinetic buffer (5 seconds each, 3 cycles). After regeneration, the biosensor was reused for subsequent binding analysis.

[0241] Human recombinant LIF produced in mammalian cells was from ACRO Biosystems (LIF-H521b); human recombinant OSM produced in mammalian cells was from R&D (8475-OM / CF); and human recombinant OSM produced in E. coli cells was from R&D (295-OM-050 / CF).

[0242] Example 18 - Crystal structure of h5D8fab

[0243] Five crystal structures of h5D8 Fab under a wide spectrum of chemical conditions were determined. The high resolution of these structures indicated that the conformations of the CDR residues were associated with little flexibility and were highly similar in different chemical environments. A unique feature of this antibody is the presence of an atypical cysteine ​​at position 100 of the variable heavy region. Structural analysis showed that the cysteine ​​is unpaired and largely solvent inaccessible.

[0244] H5D8 Fab was obtained by papain digestion of its IgG followed by purification using standard affinity, ion exchange, and size chromatography techniques. Crystals were obtained using the vapor diffusion method and the resolution was determined to be to Despite the crystallization conditions varying across five different pH levels: 5.6, 6.0, 6.5, 7.5, and 8.5, all structures were solved in the same crystallographic space group and had similar unit cell dimensions (P212121, ). Thus, these crystal structures allow comparison of the three-dimensional arrangement of h5D8 Fab unhindered by crystal packing artifacts and across a wide spectrum of chemical conditions.

[0245] The electron density of all complementarity determining region (CDR) residues was observed and subsequently modeled. Notably, LCDR1 and HCDR2 adopted an elongated conformation that, together with the shallow LCDR3 and HCDR3 regions, formed a binding groove in the center of the paratope ( Fig.14A ). The five structures are very similar at all residues, with a root mean square deviation of and Changes between Fig.14A ). These results indicate that the conformation of the CDR residues is maintained in a variety of chemical environments, including pH levels varying between 5.6 and 8.5 and ionic strengths varying between 150 mM and 1 M. Analysis of the electrostatic surface of the h5D8 paratope revealed that positively and negatively charged regions contribute equally to hydrophilicity, with no general hydrophobic patches. h5D8 has the rare feature of an atypical cysteine ​​at the base of HCDR3 (Cys100). In all five structures, this free cysteine ​​is ordered and does not form any disulfide mismatches. Additionally, it is not modified by the addition of Cys (cystylated) or glutathione (glutathionylated) and undergoes van der Waals interactions ( distance)( Fig. 14BFinally, Cys100 is a largely buried structural residue that appears to be involved in mediating the conformation of CDR1 and HCDR3. Therefore, it is unlikely to be reactive with other cysteines, as observed by the uniform distribution of this region in our five crystal structures.

[0246] method

[0247] H5D8-1 IgG was obtained from Catalent Biologics and prepared in 25 mM histidine, 6% sucrose, 0.01% polysorbate 80 (at pH 6.0). The prepared IgG was extensively buffer exchanged to PBS using a 10K MWCO concentrator (Millipore), followed by digestion with 1:100 micrograms of papain (Sigma) in PBS, 1.25 mM EDTA, 10 mM cysteine ​​at 37°C for 1 hour. The papain-digested IgG was passed through a protein A column (GE Healthcare) using an AKTA Start chromatography system (GE Healthcare). The protein A flow-through containing h5D8 Fab was recovered and buffer exchanged to 20 mM sodium acetate at pH 5.6 using a 10K MWCO concentrator (Millipore). The resulting sample was loaded onto a Mono S cation exchange column (GE Healthcare) using an AKTA Pure chromatography system (GE Healthcare). Gradient elution with 1 M KCl produced a major h5D8 Fab peak that was recovered, concentrated, and purified to size homogeneity using a Superdex 200 Increase gel filtration column (GE Healthcare) in 20 mM Tris-HCl, 150 mM NaCl, pH 8.0. The high purity of h5D8 Fab was confirmed by SDS-PAGE under reducing and non-reducing conditions.

[0248] Purified h5D8 Fab was concentrated to 25 mg / mL using a 10K MWCO concentrator (Millipore). Vapor diffusion crystallization experiments were set up using an Oryx4 dispenser (Douglas Instruments) at 20°C using a sparse matrix 96 condition commercial screen, JCSG TOP96 (Rigaku Reagents) and MCSG-1 (Anatrace). Crystals were obtained and harvested after four days under the following five crystallization conditions: 1) 0.085 M sodium citrate, 25.5% (w / v) PEG4000, 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 sodium HEPES, 17% (w / v) PEG 4000, 8.5% (v / v) 2-propanol, 15% (v / v) glycerol, pH 7.5; 5) 0.08 M Tris, 24% (w / v) PEG4000, 0.16 M magnesium chloride, 20% (v / v) glycerol, pH 8.5. Before snap freezing in liquid nitrogen, the mother liquor containing the crystals was supplemented with 5%-15% (v / v) glycerol or 10% (v / v) ethylene glycol as needed. Crystals were subjected to X-ray synchrotron irradiation at the Advanced Photon Source beamline 23-ID-D (Chicago, IL), and diffraction patterns were recorded on a Pilatus3 6M detector. Data were processed using XDS, and the structure was determined by molecular replacement using Phaser. Refinements were performed in PHENIX, and iterative models were built in Coot. Figures were generated in PyMOL. All software was accessed through SBGrid.

[0249] Example 19 - Mutation of h5D8 at Cysteine ​​100 Maintains Binding

[0250] Analysis of h5D8 revealed a free cysteine ​​residue at position 100 (C100) of the heavy chain variable region. H5D8 variants were generated by replacing C100 with each naturally occurring amino acid to characterize binding to human and mouse LIF and affinity for human and mouse LIF. Binding was characterized using ELISA and Octet assays. The results are summarized in Table 9. The ELISA EC50 curves are shown in Figure 15 ( Fig.15A Human LIF and Fig. 15B Mouse LIF).

[0251]

[0252] method

[0253] ELISA: Binding of h5D8 C100 variants to human and mouse LIF was determined by ELISA. Recombinant human or mouse LIF protein was coated on Maxisorp 384-well plates at 1 ug / mL overnight at 4°C. The plates were blocked with 1x blocking buffer for 2 hours at room temperature. A titration of each h5D8 C100 variant was added and allowed to bind for 1 hour at room temperature. The plates were washed three times with PBS + 0.05% Tween-20. Anti-human IgG conjugated with HR was added and allowed to bind for 30 minutes at room temperature. The plates were washed three times with PBS + 0.05% Tween-20 and developed using 1x TMB substrate. The reaction was stopped with 1M HCl and the absorbance at 450nm was measured. Graph generation and nonlinear regression analysis were performed using Graphpad Prism.

[0254] Octet RED96: The affinity of h5D8 C100 variants for human and mouse LIF was determined by BLI using the Octet RED96 system. After a 30-second baseline in 1x kinetic buffer, h5D8 C100 variants were loaded onto anti-human Fc biosensors at 7.5 μg / mL. Titrations of human or mouse LIF protein were associated with the loaded biosensors for 90 seconds and dissociated for 300 seconds in 1x kinetic buffer. KD was calculated using a 1:1 global fit model by the data analysis software.

[0255] Example 20 - h5D8 blocks the binding of LIF to gp130 in vitro

[0256] 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 the AHC biosensor via anti-human Fc capture. The biosensor was then immersed in LIF and association was observed as expected ( Fig.16A , middle third). Subsequently, the biosensor was immersed in different concentrations of LIFR. A dose-dependent association was observed ( Fig.16A , right third). Control experiments demonstrated that this association was specific for LIF (not shown) and not due to nonspecific interactions of LIFR with h5D8 or with the biosensor.

[0257] To further characterize the binding of h5D8 and LIF, a series of ELISA binding experiments were performed. H5D8 and LIF were pre-incubated and then introduced to plates coated with recombinant human LIFR (hLIFR) or gp130. The lack of binding between the h5D8 / LIF complex and the coated substrate indicated that h5D8 somehow disrupted the binding of LIF to the receptor. Additionally, control antibodies that did not bind LIF (isotype control, indicated by (-)) or that bound LIF at a known binding site were also used (B09 did not compete with gp130 or LIFR for LIF binding; r5D8 is the rat parental 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 were unable to prevent LIF / LIFR association ( Fig. 16B In contrast, the h5D8 / LIF complex (and r5D8 / LIF complex) were unable to bind recombinant human gp130 ( Fig. 16C ). This indicates that when LIF binds to h5D8, the gp130 binding site of LIF is affected.

[0258] Example 21 - LIF and LIFR expression in human tissues

[0259] To determine the expression levels of LIF and LIFR, real-time quantitative PCR was performed on many different types of human tissues. Fig.17A and 17B The average expression levels shown in are given as copies per 100 ng of total RNA. Most tissues express at least 100 copies per 100 ng of total RNA. LIF mRNA expression is highest in human adipose tissue (mesenteric ileum [1]), vascular tissue (choroid plexus [6] and mesentery [8]), and umbilical cord

[68] tissues, and lowest in brain tissue (cortex

[20] and substantia nigra

[28] ). LIFR mRNA expression is highest in human adipose tissue (mesenteric ileum [1]), vascular tissue (lung [9]), brain tissue [11-28], and thyroid tissue

[66] ; and lowest in PBMC

[31] . LIF and LIFR mRNA expression levels in cynomolgus monkey tissues are similar to those observed in human tissues, with high LIF expression in adipose tissue and high LIFR expression in adipose tissue and low PBMC (data not shown).

[0260] Fig.17A and Fig. 17BThe tissue numbers are: 1-fat (mesentery and ileum); 2-adrenal glands; 3-bladder; 4-bladder (trigendum); 5-blood vessels (brain: middle cerebral artery); 6-blood vessels (choroid plexus); 7-blood vessels (coronary artery); 8-blood vessels (mesentery (colon)); 9-blood vessels (lung); 10-blood vessels (kidney); 11-brain (amygdala); 12-brain (caudate nucleus); 13-brain (cerebellum); 14-brain (cortex: anterior cingulate area); 15-brain (cortex: posterior cingulate area); 16-brain (cortex: lateral frontal lobe) ; 17-brain (cortex: medial frontal lobe); 18-brain (cortex: occipital); 19-brain (cortex: parietal lobe); 20-brain (cortex: temporal lobe); 21-brain (dorsal-raphe-nucleus); 22-brain (hippocampus); 23-brain (hypothalamus: anterior region); 24-brain (hypothalamus: posterior region); 25-brain (locus coeruleus); 26-brain (medulla oblongata); 27-brain (nucleus accumbens); 28-brain (substantia nigra); 29-breast; 30-cecum; 31-peripheral blood mononuclear cells (P BMC); 32-colon; 33-dorsal root ganglion (DRG); 34-duodenum; 35-fallopian tube; 36-gall bladder; 37-heart (left atrium); 38-heart (left ventricle); 39-ileum; 40-jejunum; 41-kidney (cortex); 42-kidney (medulla oblongata); 43-kidney (pelvis); 44-liver (parenchyma); 45-liver (bronchus: first level); 46-liver (bronchus: third level); 47-lung (parenchyma); 48-lymph gland (tonsils); 49-muscle (bone); 5 0 - esophagus; 51 - ovary; 52 - pancreas; 53 - pineal gland; 54 - pituitary gland; 55 - placenta; 56 - prostate; 57 - rectum; 58 - skin (foreskin); 69 - spinal cord; 60 - spleen (parenchyma); 61 - stomach (antrum); 62 - stomach (body); 63 - stomach (fundus); 64 - stomach (pyloric duct); 65 - testicles; 66 - thyroid gland; 67 - trachea; 68 - umbilical cord; 69 - ureters; 70 - uterus (cervix); 71 - uterus (myometrium); and 72 - vas deferens.

[0261] Example 22-h5D8 combined with cisplatin, doxorubicin or paclitaxel in a subcutaneous tumor implantation model The efficacy of h5D6 was evaluated in combination with cisplatin, doxorubicin or paclitaxel in a subcutaneous implantation CT26 model. CT26 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and penicillin / streptomycin (both 100 μg / ml). 10,000 cells / mouse were implanted subcutaneously into BALB / c mice, and mice were treated as follows: h5D8 (15 mg / kg) twice a week and / or one of the following: cisplatin (10 mg / kg) once a week, paclitaxel (15 mg / kg) twice a week, doxorubicin (4 mg / kg) once a week. Each treatment cohort had 12 mice. Tumor volume and mouse weight were measured twice a week using calipers.

[0262] Mice treated with a combination of cisplatin and h5D8 exhibited reduced CT26 tumor growth compared to mice treated with cisplatin or h5D8 alone. Fig.18 Mice treated with the combination of doxorubicin and h5D8 did not show reduced CT26 tumor growth compared with mice treated with doxorubicin or h5D8 alone, as shown in Figure 2. Fig.19A Mice treated with the combination of paclitaxel and h5D8 did not show reduced CT26 tumor growth compared with mice treated with paclitaxel or h5D8 alone, as shown in Figure 2 Fig.19B Table 10 shows the tumor volumes of all animals in all treatment groups measured at the end point on day 21.

[0263]

[0264]

[0265] Example 23 - h5D8 combined with cisplatin in an intradermal tumor implantation model

[0266] In the intradermal implantation CT26 model, the efficacy of h5D6 was evaluated in combination with cisplatin, doxorubicin or paclitaxel. CT26 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and penicillin / streptomycin (both 100 μg / ml). 50,000 cells / mouse were implanted intradermally into BALB / c mice, and the mice were treated as follows: h5D8 (15 mg / kg) twice a week and / or cisplatin (10 mg / kg) once a week. There were 12 mice in each treatment cohort. Tumor volume and mouse weight were measured twice a week using calipers.

[0267] Mice treated with a combination of cisplatin and h5D8 exhibited reduced CT26 tumor growth compared to mice treated with cisplatin or h5D8 alone. Fig. 20 Table 11 shows the tumor volumes of all animals in all treatment groups measured at the end point on day 21.

[0268]

[0269]

[0270] Although preferred embodiments of the present invention are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided in an exemplary manner only. Without departing from the present invention, those skilled in the art will now be able to imagine numerous variations, changes and substitutions. It should be understood that in implementing the present invention, various alternatives to the embodiments of the present invention described herein may be employed.

[0271] All publications, patent applications, issued patents, and other documents mentioned in this specification are incorporated herein by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference. To the extent that a definition in this disclosure is inconsistent with a definition in the disclosure, the definition contained in the text incorporated by reference is excluded.

[0272] sequence

[0273]

[0274]

[0275]

[0276]

Claims

1. Use of an antibody that specifically binds to leukemia inhibitory factor (LIF) and a platinum-based antitumor agent in the preparation of a medicament for treating cancer, wherein the antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy chain complementarity determining region 1 (VH-CDR1), which consists of the amino acid sequence shown in SEQ ID NO: 1; (b) an immunoglobulin heavy chain complementarity determining region 2 (VH-CDR2), which consists of the amino acid sequence shown in SEQ ID NO: 4; (c) an immunoglobulin heavy chain complementarity determining region 3 (VH-CDR3), which consists of the amino acid sequence shown in SEQ ID NO: 6; (d) an immunoglobulin light chain complementary determining region 1 (VL-CDR1) consisting of the amino acid sequence shown in SEQ ID NO: 9; (e) an immunoglobulin light chain complementary determining region 2 (VL-CDR2) consisting of the amino acid sequence shown in SEQ ID NO: 11; and (f) an immunoglobulin light chain complementary determining region 3 (VL-CDR3) consisting of the amino acid sequence shown in SEQ ID NO: 13; And wherein the cancer is selected from small cell lung cancer, glioblastoma multiforme, ovarian cancer and colorectal cancer.

2. The use according to claim 1, wherein the antibody that specifically binds to LIF is humanized.

3. The use according to claim 1, wherein the antibody that specifically binds to LIF is an IgG antibody.

4. The use according to claim 1, wherein the antibody that specifically binds to LIF is Fab, F(ab)2, or a single-chain variable fragment (scFv).

5. The use according to claim 1, wherein the antibody that specifically binds to LIF comprises: a) an immunoglobulin heavy chain variable region (VH) sequence having an amino acid sequence as shown in any one of SEQ ID NOs: 41, 42, and 44; as well as b) an immunoglobulin light chain variable region (VL) sequence having an amino acid sequence shown in any one of SEQ ID NOs: 45-48.

6. The use according to claim 5, wherein the VH sequence is identical to the amino acid sequence shown in SEQ ID NO:42; and the VL sequence is identical to the amino acid sequence shown in SEQ ID NO:

46.

7. The use according to claim 1, wherein the antibody that specifically binds to LIF comprises: a) an immunoglobulin heavy chain sequence having an amino acid sequence shown in any one of SEQ ID NOs: 57-60; as well as b) an immunoglobulin light chain sequence having an amino acid sequence shown in any one of SEQ ID NOs: 61-64.

8. The use of claim 1, wherein the antibody that specifically binds to LIF has a K of less than 200 picomolar. D Combine.

9. The use of claim 1, wherein the antibody that specifically binds to LIF has a K of less than 100 picomolar. D Combine.

10. The use as claimed in claim 1, wherein the platinum-based antitumor agent comprises cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenaplatin, picoplatin, setraplatin or a combination thereof.

11. The use according to claim 10, wherein the platinum-based anti-tumor agent is cisplatin.

12. The use of claim 1, wherein the cancer is refractory to a therapeutic amount of an antibody that specifically binds to LIF.

13. The use of claim 1, wherein the cancer is refractory to treatment with therapeutic amounts of a platinum-based anti-tumor agent.

14. The use according to claim 1, wherein the antibody that specifically binds to LIF and the platinum-based anti-tumor agent are administered separately.

15. The use of claim 1, wherein the antibody that specifically binds to LIF and the platinum-based anti-tumor agent are administered on different schedules.

16. The use of claim 1, wherein the antibody that specifically binds to LIF and the platinum-based anti-tumor agent are administered as a single composition.

17. The use according to claim 7, wherein the immunoglobulin heavy chain sequence consists of the amino acid sequence shown in SEQ ID NO: 58; and the immunoglobulin light chain sequence consists of the amino acid sequence shown in SEQ ID NO: 62.

Images