WO2019197903A1 - Combination of lif inhibitors and pd-1 axis inhibitors for use in treating cancer - Google Patents

Combination of lif inhibitors and pd-1 axis inhibitors for use in treating cancer Download PDF

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Publication number
WO2019197903A1
WO2019197903A1 PCT/IB2019/000423 IB2019000423W WO2019197903A1 WO 2019197903 A1 WO2019197903 A1 WO 2019197903A1 IB 2019000423 W IB2019000423 W IB 2019000423W WO 2019197903 A1 WO2019197903 A1 WO 2019197903A1
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Prior art keywords
pdl
amino acid
acid sequence
cancer
seq
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PCT/IB2019/000423
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English (en)
French (fr)
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WO2019197903A8 (en
Inventor
Joan Seoane Suarez
Judit Anido Folgueira
Johan Fransson
Robin Matthew HALETT
Monica PASCUAL GARCIA
Ester BONFILL TEIXIDOR
Ester PLANAS RIGOL
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Institucio Catalana de Recerca i Estudis Avancats ICREA
Fundacio Privada Institut dInvestigacio Oncologica Vall dHebron
Mosaic Biomedicals SL
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Institucio Catalana de Recerca i Estudis Avancats ICREA
Fundacio Privada Institut dInvestigacio Oncologica Vall dHebron
Mosaic Biomedicals SL
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Priority to JP2021504589A priority Critical patent/JP7423598B2/ja
Priority to US17/046,599 priority patent/US20210130453A1/en
Priority to CN201980038348.0A priority patent/CN112585164A/zh
Priority to EP19733546.6A priority patent/EP3774895A1/en
Priority to CA3096779A priority patent/CA3096779A1/en
Priority to KR1020207032587A priority patent/KR20210021287A/ko
Priority to EA202092360A priority patent/EA202092360A1/ru
Priority to AU2019251289A priority patent/AU2019251289B2/en
Application filed by Institucio Catalana de Recerca i Estudis Avancats ICREA, Fundacio Privada Institut dInvestigacio Oncologica Vall dHebron, Mosaic Biomedicals SL filed Critical Institucio Catalana de Recerca i Estudis Avancats ICREA
Priority to SG11202009966SA priority patent/SG11202009966SA/en
Publication of WO2019197903A1 publication Critical patent/WO2019197903A1/en
Publication of WO2019197903A8 publication Critical patent/WO2019197903A8/en
Priority to IL277918A priority patent/IL277918A/en
Anticipated expiration legal-status Critical
Priority to JP2024005463A priority patent/JP2024054138A/ja
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4412—Non condensed pyridines; Hydrogenated derivatives thereof having oxo groups directly attached to the heterocyclic ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the LIF binding antibody comprises an immunoglobulin heavy chain variable region (VH) comprising an amino acid sequence at least about 90% identical to the amino acid sequence set forth in SEQ ID NO: 42; and an immunoglobulin light chain variable region (VL) at least about 90% identical to the amino acid sequence set forth in SEQ ID NO: 46.
  • VH immunoglobulin heavy chain variable region
  • VL immunoglobulin light chain variable region
  • the VH sequence is identical to the amino acid sequence set forth in SEQ ID NO: 42
  • the VL sequence is identical to the amino acid sequence set forth in SEQ ID NO: 46.
  • the antibody that specifically binds to LIF binds with a K D of less than about 100 picomolar.
  • the inhibitor of PD-l, PDL-l, or PDL-2 signaling comprises an antibody or PD-l, PDL-l, or PDL-2 binding fragment thereof.
  • the antibody specifically binds PD-l .
  • the antibody comprises Pembrolizumab, Nivolumab,
  • the antibody specifically binds PDL-l or PDL-2.
  • the antibody comprises Durvalumab, Atezolizumab, Avelumab, BMS-936559, or FAZ053, or a PDL-l or PD1-2 binding fragment thereof.
  • the inhibitor of PD-l, PDL-l, or PDL-2 signaling comprises an Fc-Fusion protein that binds PD-l, PDL-l, or PDL-2.
  • the Fc-Fusion protein comprises AMP-224 or a PD-l binding fragment thereof.
  • the antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy chain variable region (VH) sequence with the amino acid sequence 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 with the amino acid sequence 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
  • 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%,
  • 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.
  • the antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy chain sequence with the amino acid sequence 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 with the amino acid sequence 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.
  • the LIF-binding polypeptide and the inhibitor of PD-l, PDL-l, or PDL- 2 signaling are administered at the same time. In certain embodiments, the LIF-binding polypeptide and the inhibitor of PD-l, PDL-l, or PDL-2 signaling are administered in a single composition.
  • a method of treating an individual with a cancer comprising: administering to the individual with cancer an effective amount of a Leukemia Inhibitory Factor (LIF)-binding polypeptide, wherein the individual has been administered a therapeutic amount of an inhibitor of PD-1 (CD279), PDL-l (CD274), or PDL-2 (CD-273) signaling.
  • LIF Leukemia Inhibitory Factor
  • the antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy chain variable region (VH) sequence with the amino acid sequence 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 with the amino acid sequence 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.
  • VH immunoglobulin heavy chain variable region
  • VL immunoglobulin light chain variable region
  • a method of treating an individual with a cancer comprising: administering to the individual with cancer; an effective amount of an inhibitor of PD-l (CD279), PDL-l (CD274), or PDL-2 (CD273) signaling, wherein the individual has been administered a therapeutic amount of a Leukemia Inhibitory Factor (LIF) binding polypeptide.
  • the method inhibits growth or metastasis of the cancer.
  • the LIF-binding polypeptide comprises a fragment of an immunoglobulin variable region, or an immunoglobulin heavy chain constant region.
  • the LIF-binding polypeptide comprises an antibody that specifically binds to LIF.
  • the LIF-binding polypeptide comprises at least one framework region derived from a human immunoglobulin framework region.
  • the antibody that specifically binds to LIF is humanized.
  • the antibody that specifically binds to LIF is deimmunized.
  • the antibody that specifically binds to LIF comprises two immunoglobulin heavy chains and two immunoglobulin light chains.
  • the antibody that specifically binds to LIF is an IgG antibody.
  • the antibody that specifically binds to LIF is a Fab, F(ab h, single-domain antibody, a single chain variable fragment (scFv), or a nanobody.
  • VH-CDR1 immunoglobulin heavy chain complementarity determining region 1
  • VH-CDR2 immunoglobulin heavy chain complementarity determining region 2
  • the antibody that specifically binds to LIF comprises: (a) an immunoglobulin heavy chain sequence with the amino acid sequence 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 with the amino acid sequence 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.
  • the antibody that specifically binds to LIF binds with a K D of less than about 200 picomolar.
  • the antibody that specifically binds to LIF is an IgG antibody. In certain embodiments, the antibody that specifically binds to LIF is a Fab,
  • VH-CDR3 complementarity determining region 3
  • VL-CDR1 immunoglobulin light chain complementarity determining region 1
  • VL-CDR2 immunoglobulin light chain complementarity determining region 2
  • VL-CDR3 immunoglobulin light chain complementarity determining region 3
  • the checkpoint inhibitor is an inhibitor of PD-l, PDL-l, or PDL-2 signaling.
  • the inhibitor of PD-l, PDL-l, or PDL-2 signaling is an antibody or fragment thereof that binds to PD-L
  • the inhibitor of PD-l, PDL-l, or PDL-2 signaling is administered to the individual before the LIF-binding antibody is administered to the individual. In certain embodiments, the inhibitor of PD-l, PDL-l, or PDL-2 signaling is administered to the individual at the same time as the LIF-binding antibody is administered to the individual. In certain embodiments, the LIF-binding antibody is humanized.
  • the inhibitor ofPD-1, PDL-l, or PDL-2 signaling comprises an Fc-Fusion protein that binds PD-l, PDL-l, or PDL-2.
  • the Fc-Fusion protein comprises AMP -224 or a PD-l binding fragment thereof.
  • the inhibitor of PD-l, PDL-l, or PDL-2 signaling comprises a small molecule inhibitor of PD-l, PDL-l, or PDL-2.
  • FIG. 2A and 2B depicts a western blot showing inhibition of LIF-induced STAT3 phosphorylation by humanized and parental 5D8 antibodies.
  • Fig. 8A shows the effect of r5D8 on inhibition of growth of ovarian cancer cells in an syngeneic mouse model.
  • Fig. 8C illustrates that h5D8 shows a significant reduction in tumor growth when administered at 200 pg/mouse twice weekly (p ⁇ 0.05). Symbols are mean + SEM, statistical significance compared with vehicle (with unpaired non-parametric Mann-Whitney U-test).
  • Fig. 9B shows the individual measurements of tumors at day 17.
  • Fig. 13B illustrates detailed interactions between LIF and h5D8, showing residues forming salt bridges and h5D8 residues with buried surface areas greater than 100 A 2 .
  • Fig. 14B illustrates an extensive network of Van der Waals interactions mediated by unpaired CyslOO. This residue is well-ordered, partakes in shaping the conformations of HCDR1 and HCDR3 and is not involved in undesired disulfide scrambling. Distances between residues are shown as dashed lines and labeled.
  • Fig. 15B illustrates binding of h5D8 Cl 00 mutants to mouse LIF by ELISA.
  • Fig. 19A-D show flow cytometry analysis detecting the abundance of functional CD8 T cells in h5D8/anti-PD-l treated tumors.
  • 19A shows CD8 T cell function defined based on their capacity to produce IFNy in response to tumor associated peptide (GP70).
  • 19B shows a representative FACs data plot. Significance determined by unpaired t-test.
  • Fig. 20A-P show LIF blockade decreases tumor growth and regulates immune cell infiltration in GBM and ovarian cancer models that express high levels of LIF.
  • 20A shows distribution of LIF mRNA expression (log2 RSEM) across 28 distinct solid tumors.
  • the black line represents the estimated cut-off between low expression/background noise.
  • the bottom panel shows correlation values (Pearson R 2 values) between LIF expression and the relative abundance of TAMs and Tregs based on ssGSEA of the gene signatures of the immune cell types. Correlation values are only shown if the correlation is significant (adjusted P-value ⁇ 0.1).
  • a heatmap representing the expression values of the indicated genes each column represents a sample and each row a gene. The last column represents the log2 fold change (log2 FC) of gene expression.
  • 21B shows mRNA expression for the indicated genes in isolated CD1 lb + cells from anti-LIF (r5D8) treated or untreated ID8 and GL261N tumors.
  • 21C shows percentage and mean fluorescent intensity (MFI) of CCL2 + and CXCL9 + in TAMs (CD1 lb + Ly6G Ly6C ) from anti-LIF (r5D8) treated or untreated GL261N tumors.
  • 21D shows percentage of double positive cells relative to the TAM marker positive cells.
  • 22H shows percentage of double positive cells relative to Ibal + cells and percentage of CXCL9 + cells in GBM organotypic slices (patients 1, 2, 3) incubated with 10 pg/ml anti-LIF (r5D8) for 3 days relative to the total number of cells. Data are mean of all patients ⁇ SEM. Statistical analyses by Mann-Whitney T test. *P ⁇ 0.05, **P ⁇ 0.0l; ***P ⁇ 0.001; ****P ⁇ 0.0001.
  • 23C shows FI of CD8 + T infiltrating cells detected by flow cytometry in organotypic tissues treated with anti-LIF (r5D8) for 72 h and then cultured with PBMCs for 48 h (patient 4, 5, 6).
  • 23D shows FI of CD8 + T infiltrating cells detected by flow cytometry in GBM organotypic tissues treated with anti-LIF (r5D8) and/or anti-CXCL9 for 72 h and then cultured with PBMCs for 48 h.
  • 23E shows CD8 + T infiltrating cells into subcutaneously engrafted GBM specimens in NSG mice. Bar graph represents the ratio of CD8 + T cells detected by flow cytometry in the tissue vs.
  • LIF Leukemia Inhibitory Factor
  • a method of treating an individual with a cancer comprising: administering to the individual with cancer an effective amount of a Leukemia Inhibitory Factor (LIF)-binding polypeptide, wherein the individual has been administered a therapeutic amount of an inhibitor of PD-l (CD279), PDL-l (CD274), or PDL-2 (CD-273) signaling.
  • LIF Leukemia Inhibitory Factor
  • VH-CDR2 an immunoglobulin heavy chain complementarity determining region 2
  • VH-CDR3 an immunoglobulin heavy chain complementarity determining region 3
  • VL-CDR1 an immunoglobulin light chain complementarity determining region 1
  • an“effective amount” refers to the amount of a therapeutic that causes a biological effect when administered to a mammal.
  • Biological effects include, but are not limited to, inhibition or blockade a receptor ligand interaction (e g., LIF-LIFR, PD-l- PDL1/PDL-2, inhibition of a signaling pathway (e.g., STAT3 phosphorylation), reduced tumor growth, reduced tumor metastasis, or prolonged survival of an animal bearing a tumor.
  • a “therapeutic amount” is the concertation of a drug calculated to exert a therapeutic effect.
  • a therapeutic amount encompasses the range of dosages capable of inducing a therapeutic response in a population of individuals.
  • the mammal can be a human individual.
  • the human individual can be afflicted with or suspected or being afflicted with a tumor.
  • 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 from antibody fragments with separate specificities, such as a bispecific antibody.
  • the antibodies are humanized in such a way as to reduce an individual’s immune response to the antibody.
  • the antibodies may be chimeric, e.g. non-human variable region with human constant region, or CDR grafted, e.g.
  • the ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code.
  • the ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX Y4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
  • the Fc region of antibodies described herein comprises one or more amino acid substitutions that increase binding of the antibody to an Fc receptor.
  • the Fc receptor comprises FcyRI (CD64), FcyRIIA (CD32), FcyRIIIA (CDl6a), FcyRIIIB (CD16b), or any combination thereof.
  • the Fc region of the antibodies described herein comprise one or more amino acid substitutions that increase the serum half-life of the antibody.
  • the one or more amino acid substitutions that increase the serum half-life of the antibody increase affinity of the antibody to the neonatal Fc receptor (FcRn).
  • the antibody that specifically binds LIF comprises one or more human light chain framework regions comprising: a VL-FR1 amino acid sequence 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 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 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 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.
  • 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 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 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 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 at least about 80%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 24, a VL-FR1 amino acid sequence 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-FR1 amino acid
  • the r5D8 antibody described herein was generated from rats immunized with DNA encoding human LIF.
  • r5D8 was cloned and sequenced and comprises CDRs (using the combination of the Rabat and IMGT CDR numbering methods) with the following amino acid sequences: a VH-CDR1 corresponding to SEQ ID NO: 1 (GFTF SHAWMH), a VH-CDR2 corresponding to SEQ ID NO: 4 (QDCAKSDDYATYYAESVKG), a VH-CDR3 corresponding to SEQ ID NO: 6 (T CWEWDLDF), a VL-CDR1 corresponding to SEQ ID NO: 9
  • an antibody that specifically binds LIF comprising a VH-CDRlat least 80% or 90% identical to that set forth in SEQ ID NO: 1
  • an antibody that specifically binds LIF comprising a VH-CDR1 set forth in SEQ ID NO: 1 (GFTF SHAWMH), 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).
  • the antibody specifically binds human LIF.
  • the antibody that specifically binds 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 region amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs: 23-25.
  • 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, and a VH-FR4 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 24.
  • the antibody that specifically binds 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 YL-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.
  • 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 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 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, and a VL-FR4 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 38.
  • the antibody specifically binds human LIF.
  • an antibody that specifically binds LIF comprising a VH-CDR1 amino acid sequence at least 80% or 90% identical to that set forth in SEQ ID NO: 1 (GFTFSHAWMH), a VH-CDR2 amino acid sequence at least 80%, 90%, or 95% identical to that set forth in SEQ ID NO: 4 (QIKAKSDD Y ATYY AES VKG), and a VH- CDR3 amino acid sequence at least 80% or 90% identical to that set forth in SEQ ID NO: 8 (TSWEWDLDF).
  • the one or more human light chain framework regions comprise a VL-FR1 amino acid sequence 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 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 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 at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 38.
  • 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 region amino acid sequence identical to the amino acid sequence set forth in any one of SEQ ID NOs: 23-25.
  • the antibody that specifically binds 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.
  • 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, and a VL-FR4 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 38.
  • 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 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 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, and a VL-FR4 amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 38.
  • the antibody specifically binds human LIF.
  • an antibody that specifically binds LIF comprising a humanized heavy chain variable region comprising an amino acid sequence 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 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.
  • an antibody that specifically binds LIF comprising a humanized heavy chain variable region comprising an amino acid sequence 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 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.
  • an antibody that specifically binds LIF 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.
  • an antibody that specifically binds LIF comprising a humanized heavy chain comprising an amino acid sequence 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: 57-60; and a humanized light chain comprising an amino acid sequence 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: 61-64.
  • an antibody that specifically binds LIF comprising a humanized heavy chain comprising an amino acid sequence 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 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.
  • an antibody that specifically binds LIF comprising a humanized heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 67; and a humanized light chain comprising an amino acid sequence set forth in SEQ ID NO: 62.
  • LIF Leukemia Inhibitory Factor
  • VH-CDR1 a heavy chain complementarity determining region 1
  • VH-CDR2 a heavy chain complementarity determining region 2
  • VH-CDR3 a heavy chain complementarity determining region 3
  • VL-CDR1 a light chain complementarity determining region 1
  • VL-CDR2 a light chain complementarity determining region 3
  • VL-CDR3 comprising an amino acid sequence set forth in SEQ ID NO: 13
  • VL-CDR1 a light chain complementarity determining region 1
  • VL-CDR2 a light chain complementarity determining region 2
  • VL-CDR3 a light chain complementarity determining region 3
  • the antibody comprises CDRs that differ from the amino acid sequence set forth in any one of SEQ ID NOs: 2, 5, 6, 10, 12, and 13 by 1, 2, 3, or 4 amino acids. In certain embodiments, the antibody comprises CDRs that differ from the amino acid sequence set forth in any one of SEQ ID NOs: 2, 5, 6, 10, 12, and 13 by 1, 2, 3, or 4 amino acids and does not affect the binding affinity by greater than 10%, 20%, or 30%.
  • an antibody that specifically binds LIF comprising a humanized heavy chain comprising an amino acid sequence 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 at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in of SEQ ID NO: 54.
  • an antibody that specifically binds LIF comprising a humanized heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 50; and a humanized light chain comprising an amino acid sequence set forth in any one of SEQ ID NO: 54.
  • Described herein is a unique epitope of human LIF that when bound inhibits LIF biological activity (e.g., STAT3 phosphorylation) and inhibits tumor growth in vivo.
  • the epitope described herein consists of two discontinuous stretches of amino acids (from residue 13 to residue 32 and from residue 120 to 138 of human LIF), that are present in two distinct topological domains (alpha helixes A and C) of the human LIF protein. This binding is a combination of weak (Van der Waals attraction), medium (hydrogen binding), and strong (salt bridge) interactions.
  • a contact residue is a residue on LIF that forms a hydrogen bond with a residue on an anti-LIF antibody.
  • S127, N128, L130, C131, C134, S135, or H138 of SEQ ID NO: 68 is described herein.
  • an antibody comprising CDRs that differ 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, 114, R15, H16, P17, C18, H19, N20, Q25, Q29, Q32, D120, R123, S127, N128, L130, C131, C134, S135, or Hl38 of SEQ ID NO: 68.
  • CDRs that differ 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, 114, R15, H16, P17, C18, H19, N20, Q25, Q29, Q32, D120, R123, S127,
  • an antibody that specifically binds LIF comprising a humanized heavy chain variable region amino acid sequence 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 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 any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty of the following residues: A13, 114, R15, H16, P17,
  • the antibodies here are also highly specific for LIF compared to other IL-6 family member cytokines.
  • the PD-1 axis inhibitor utilized in the compositions and methods herein can inhibit signaling through PD-l (CD279), PDL-l (CD274), or PDL-2 (CD273).
  • the inhibitor can be an antibody or antibody fragment, a soluble ligand-Fc fusion construct, or a small molecule inhibitor.
  • the PD-l axis inhibitor comprises an antibody or PD-l binding fragment thereof.
  • the antibody or antigen binding fragment that specifically binds PD-l (CD279) comprises Pembrolizumab, Nivolumab, AMP-514,
  • the PD-l axis inhibitor comprises on or more a small molecule inhibitor such as N- ⁇ 2-[( ⁇ 2-methoxy-6-[(2-methyl[l, l'-biphenyl]-3-yl)methoxy]pyridin-3- yl ⁇ methyl)amino]ethyl ⁇ acetamide (BMS 202); (2-((3-cyanobenzyl)oxy)-4-((3-(2,3- dihydrobenzo[b][l,4]dioxin-6-yl)-2-methylbenzyl)oxy)-5-methylbenzyl)-D-serine hydrochloride; (2R,4R)- 1 -(5-chloro-2-((3 -cyanobenzyl)oxy)-4-((3 -(2,3-dihydrobenzo[b] [ 1 ,4]dioxin-6-yl)-2- methylbenzyl)oxy)benzyl)-4-hydroxypyr
  • a small molecule inhibitor
  • therapeutically acceptable amount is between about 5 mg/kg and about 15 mg/kg. In certain embodiments, the therapeutically acceptable amount is about 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, or 20 mg/kg.
  • Durvalumab can be administered at a dosage of about 10 mg/kg once every two weeks.
  • administration to an individual of the PD-l axis inhibitors can be at a flat dosage level of about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 milligrams.
  • administration to an individual of the PD-l axis inhibitors can be at level suitable for monotherapy.
  • Nivolumab can be administered at a dosage of about 240 milligrams every two weeks or about 480 milligrams every four weeks.
  • Pembrolizumab can be administered at about 200 milligrams once every three weeks.
  • a flat dose of h5D8 can be administered at about 75 milligrams.
  • a flat dose of h5D8 can be administered at about 225 milligrams.
  • a flat dose of h5D8 can be administered at about 750 milligrams.
  • a flat dose of h5D8 can be administered at about 1125 milligrams.
  • a flat dose of h5D8 can be administered at about 1500 milligrams.
  • a flat dose of h5D8 can be administered at about 2000 milligrams.
  • a flat dose of h5D8 can be administered at about 225 milligrams once every four weeks.
  • a flat dose of h5D8 can be administered at about 750 milligrams once every four weeks.
  • a flat dose of h5D8 can be administered at about 1125 milligrams once every four weeks.
  • a flat dose of h5D8 can be administered at about 1500 milligrams once every four weeks.
  • a flat dose of h5D8 can be administered at about 2000 milligrams once every four weeks.
  • a body weight adjusted dose of h5D8 can be administered at about 10 mg/kg.
  • a body weight adjusted dose of h5D8 can be administered at about 15 mg/kg.
  • a body weight adjusted dose of h5D8 can be administered at about 20 mg/kg.
  • a body weight adjusted dose of h5D8 can be administered at about 25 mg/kg.
  • a body weight adjusted dose of h5D8 can be administered at about 2 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 21 mg/kg, 22 mg/kg, 23 mg/kg, 24 mg/kg, 26 mg/kg, 27 mg/kg, 28 mg/kg, 29 mg/kg, or 30 mg/kg. Any of these doses can be administered once a week, once every two weeks, once every three weeks, or once every four weeks.
  • a PD-1 axis inhibitor can be administered to an individual one or more times before administration of a LIF-biding polypeptide.
  • a LIF-binding polypeptide can be administered within 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days of administration of a PD-1 axis inhibitor.
  • a LIF-binding polypeptide can be administered within 1 week, 2 weeks, 3 weeks, or 4 weeks of administration of a PD-1 axis inhibitor.
  • the h5D8 antibody can be administered within 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days of administration of a PD-1 axis inhibitor.
  • the h5D8 antibody can be administered within 1 week, 2 weeks, 3 weeks, or 4 weeks of administration of a PD-l axis inhibitor.
  • a LIF-biding polypeptide can be administered to an individual one or more times before administration of a PD-l axis inhibitor.
  • a PD-l axis inhibitor can be administered within 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days of administration of a LIF- binding polypeptide.
  • a PD-l axis inhibitor can be administered within 1 week, 2 weeks, 3 weeks, or 4 weeks of administration of a LIF -binding polypeptide.
  • a PD-l axis inhibitor can be administered within 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days of administration of the h5D8 antibody.
  • a PD-l axis inhibitor can be administered within 1 week, 2 weeks, 3 weeks, or 4 weeks of administration of the h5D8 antibody.
  • a PDl-axis inhibitor can be administered to an individual once every three weeks and a LIF-binding polypeptide can be administered to an individual every week, every two weeks, every three weeks or every four weeks.
  • a PDl-axis inhibitor can be administered to an individual once every four weeks and a LIF-binding polypeptide can be administered to an individual every week, every two weeks, every three weeks or every four weeks.
  • h5D8 can be administered to an individual one or more times before administration of a PD-l axis inhibitor.
  • a combination treatment according to the current disclosure may comprise
  • the cancer comprises breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head, neck, ovarian, prostate, brain, pancreatic, skin, bone, bone marrow, blood, thymus, uterine, testicular, and liver tumors.
  • the tumor/cancer is selected from the group of acral lentiginous melanoma, actinic keratosis, adenocarcinoma, adenoid cystic carcinoma, adenomas, adenosarcoma, adenosquamous carcinoma, astrocytic tumors, Bartholin gland carcinoma, basal cell carcinoma, bronchial gland carcinoma, capillary carcinoid, carcinoma, carcinosarcoma, cholangiocarcinoma, chondrosarcoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymal sarcoma, Swing's sarcoma, focal nodular hyperplasia
  • hemangioblastoma hemangioendothelioma
  • hemangioma hemangioma
  • hepatic adenoma hepatic
  • adenomatosis hepatocellular carcinoma, insulinite, intraepithelial neoplasia, intraepithelial squamous cell neoplasia, invasive squamous cell carcinoma, large cell carcinoma, liposarcoma, lung carcinoma, lymphoblastic leukemia, lymphocytic leukemia, leiomyosarcoma, melanoma, malignant melanoma, malignant mesothelial tumor, nerve sheath tumor, medulloblastoma, medulloepithelioma, mesothelioma, mucoepidermoid carcinoma, myeloid leukemia,
  • the tumor/cancer to be treated with one or more antibodies of the invention comprise brain cancer, head and neck cancer, colorectal carcinoma, acute myeloid leukemia, pre-B-cell acute lymphoblastic leukemia, bladder cancer, astrocytoma, preferably grade II, III or IV astrocytoma, glioblastoma, glioblastoma multiforme, small cell cancer, and non-small cell cancer, preferably non-small cell lung cancer, lung adenocarcinoma, metastatic melanoma, androgen-independent metastatic prostate cancer, androgen-dependent metastatic prostate cancer, prostate adenocarcinoma, and breast cancer, preferably breast ductal cancer, and/or breast carcinoma.
  • the cancer treated with the antibodies of this disclosure comprises glioblastoma. In certain embodiments, the cancer treated with one or more antibodies of this disclosure comprises pancreatic cancer. In certain embodiments, the cancer treated with one or more antibodies of this disclosure comprises ovarian cancer. In certain embodiments, the cancer treated with one or more antibodies of this disclosure comprises lung cancer. In certain embodiments, the cancer treated with one or more antibodies of this disclosure comprises prostate cancer. In certain embodiments, the cancer treated with one or more antibodies of this disclosure comprises colon cancer. In certain embodiments, the cancer treated comprises glioblastoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, or lung cancer. In a certain embodiment, the cancer is refractory to other treatment.
  • the cancer treated is relapsed.
  • the cancer is a relapsed/refractory glioblastoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, or lung cancer.
  • the cancer comprises an advanced solid tumor, glioblastoma, stomach cancer, skin cancer, prostate cancer, pancreatic cancer, breast cancer, testicular cancer, thyroid cancer, head and neck cancer, liver cancer, kidney cancer, esophageal cancer, ovarian cancer, colon cancer, lung cancer, lymphoma, or soft tissue cancer.
  • the PD-l axis inhibitors and the LIF-binding polypeptides of the current disclosure are a component of a pharmaceutical composition. In certain embodiments, the PD-l axis inhibitors and the LIF-binding polypeptides of the current disclosure are a component of the same pharmaceutical composition. In certain embodiments, the
  • polyol/disaccharide/polysaccharides for example, glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; amino acids, for example, histidine, glycine or arginine; antioxidants, for example, ascorbic acid, methionine; and chelating agents, for example, EDTA or EGTA.
  • the solution comprises a physiologically appropriate salt concentration (e.g., sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite., sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite
  • a highly concentrated stock solution of antibody may be diluted in about 0.9% NaCl.
  • the solution comprises about 0.9% NaCl.
  • the solution further comprises one or more of: buffers, for example, acetate, citrate, histidine, succinate, phosphate, bicarbonate and hydroxymethylaminomethane (Tris); surfactants, for example, polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), polysorbate and poloxamer 188; polyol/disaccharide/polysaccharides, for example, glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40 and combinations thereof; amino acids, for example, histidine, glycine or arginine; antioxidants, for example, ascorbic acid, methionine, and combinations thereof; and chelating agents, for example, EDTA or EGTA.
  • buffers for example, acetate, citrate, histidine
  • the h5D8 antibody can be included in a kit comprising a vial filled with a sterile solution comprising the h5D8 antibody at a concentration of about 20 mg/mL, about 25mM histidine, about 6% sucrose, and about 0.01% polysorbate 80.
  • the vial can be a single-use glass vial.
  • the single-use glass vial can be filled with about 10 milliliters of 5D8 antibody at a concentration of about 20 mg/mL h5D8 antibody, about 25mM histidine, about 6% sucrose, and about 0.01% polysorbate 80.
  • the pH of the solution is about 6.0.
  • H5D8 can be prepared at a concentration of about 8 mg/mL regardless of ultimate dose delivered to the patient. In certain embodiments, the h5D8 can be prepared at a level of no more than about 10, 9, 8, 7, 6, 5 or 4 mg/mL. In certain embodiments, the h5D8 can be prepared at a level of greater than about 1, 2,
  • a cDNA encoding amino acids 23-202 of human LIF was cloned into expression plasmids (Aldevron GmbH, Freiburg, Germany). Groups of laboratory rats (Wistar) were immunized by intradermal application of DNA-coated gold-particles using a hand-held device for particle-bombardment (“gene gun”). Cell surface expression on transiently transfected HEK cells was confirmed with anti-tag antibodies recognizing a tag added to the N-terminus of the LIF protein. Serum samples were collected after a series of immunizations and tested in flow cytometry on HEK cells transiently transfected with the aforementioned expression plasmids.
  • Antibody-producing cells were isolated and fused with mouse myeloma cells (Ag8) according to standard procedures. Hybridomas producing antibodies specific for LIF were identified by screening in a flow cytometry assay as described above. Cell pellets of positive hybridoma cells were prepared using an RNA protection agent (RNAlater, cat. #AM7020 by ThermoFisher Scientific) and further processed for sequencing of the variable domains of the antibodies.
  • RNA protection agent RNAlater, cat. #AM7020 by ThermoFisher Scientific
  • a cDNA encoding amino acids 23-202 of human LIF was cloned into expression plasmids (Aldevron GmbH, Freiburg, Germany). Groups of laboratory mice (NMRI) were immunized by intradermal application of DNA-coated gold-particles using a hand-held device for particle-bombardment (“gene gun”). Cell surface expression on transiently transfected HEK cells was confirmed with anti-tag antibodies recognizing a tag added to the N-terminus of the LIF protein. Serum samples were collected after a series of immunizations and tested in flow cytometry on HEK cells transiently transfected with the aforementioned expression plasmids.
  • Antibody-producing cells were isolated and fused with mouse myeloma cells (Ag8) according to standard procedures. Hybridomas producing antibodies specific for LIF were identified by screening in a flow cytometry assay as described above. Cell pellets of positive hybridoma cells were prepared using an RNA protection agent (RNAlater, cat. #AM7020 by ThermoFisher Scientific) and further processed for sequencing of the variable domains of the antibodies.
  • RNA protection agent RNAlater, cat. #AM7020 by ThermoFisher Scientific
  • the heavy chain and light chain CDR1, CDR2 and CDR3 of 5D8 were cloned into the 4 different heavy chain acceptor frameworks (Hl to H4), and 4 different light chain frameworks (Ll to L4). Then all 16 different antibodies were tested for: expression in CHO-S cells (Selexis); inhibition of LIF-induced STAT3 phosphorylation; and binding affinity by Surface Plasmon Resonance (SPR). These experiments are summarized in Table 1
  • the h5D8 antibody was immobilized to the sensor chip surface by non-covalent, Fc specific capturing.
  • Recombinant, Ig(Fc) specific S. aureus Protein A/G was used as capturing agent, allowing sterically uniform and flexible presentation of the anti-LIF antibody to the LIF analytes.
  • Sources of the LIF analytes are as follows: Human LIF (from E.coli ; Millipore reference LIF 1050); Human LIF (from HEK cells ACRO Biosystems LIF-H521); Mouse LIF ( E . coir. Millipore Cat.
  • proteins were obtained in radio-immunoprecipitation assay (RIP A) lysis buffer containing phosphatase and protease inhibitors, quantified (BCA-protein assay, Thermo Fisher Scientific) and used in western blot.
  • RIP A radio-immunoprecipitation assay
  • membranes were blocked for 1 hour in 5% non-fatty milk - TBST and incubated with the primary antibody overnight (p- STAT3, catalog #9145, Cell Signaling or STAT3, catalog #9132, Cell Signaling) or 30 minutes (b-actin-peroxidase, catalog #A3854, Sigma-Aldrich). Membranes were then washed with TBST, incubated with secondary antibody if necessary, and washed again. Proteins were detected by chemiluminescence (SuperSignal Substrate, catalog #34076, Thermo Fisher Scientific).
  • the U-251 cells were seeded at 600,000 cells per 6cm plate (per condition). Cells were treated with h5D8 in corresponding concentration (titration) overnight at 37°C, under serum starvation (0.1% FBS).
  • pSTAT3 recombinant LIF (R&D #7734-LF/CF) was used to stimulate the cells at 1.79 nM for lOmin at 37°C.
  • the JAK I inhibitor (Calbiochem #420099) was used at luM for 30min at 37°C.
  • Example 7 Additional antibodies that specifically bind to human LIF
  • Example 8-Additional anti LIF antibodies inhibit LIF-induced phosphorylation of ST A TSin vitro
  • RIP A radio-immunoprecipitation assay
  • BCA-protein assay Thermo Fisher Scientific
  • membranes were blocked for 1 hour in 5% non-fatty milk - TBST and incubated with the primary antibody overnight (p-STAT3, catalog #9145, Cell Signaling) or 30 minutes (b-actin-peroxidase, catalog #A3854, Sigma-Aldrich). Membranes were then washed with TBST, incubated with secondary antibody if necessary, and washed again. Proteins were detected by chemiluminescence (Super Signal Substrate, catalog #34076, Thermo Fisher Scientific).
  • Example 9- LIF is highly overexpressed across multiple tumor types
  • LIF glioblastoma multiforme
  • NBM non-small cell lung cancer
  • CRC colorectal cancer
  • pancreatic cancer pancreatic cancer
  • Example 10-Humanized clone h5D8 inhibits tumor growth in a mouse model of non-small cell lung carcinoma
  • Fig. 6A shows reduced tumor growth in mice treated with this antibody compared to a vehicle negative control.
  • Fig. 6B shows data generated using the r5D8 version.
  • the murine non-small cell lung cancer (NSCLC) cell line KLN205 with high LIF levels was stably infected with lentivirus expressing the firefly luciferase gene for in vivo
  • mice received an intraperitoneal injection of 0.2 mL of 15 mg/mL D-luciferin under 1-2% inhaled isoflurane anesthesia.
  • the bioluminescence signals were monitored using the IVIS system 2000 series (Xenogen Corp., Alameda, CA, USA) consisting of a highly sensitive cooled CCD camera. Living Image software (Xenogen Corp.) was used to grid the imaging data and integrate the total bioluminescence signals in each boxed region. Data were analyzed using the total photon flux emission (photons/second) in the regions of interest (ROI). The results demonstrate that treatment with the h5D8 antibody promote tumor regression. Data are presented as mean ⁇ SEM.
  • U251 cells stably expressing luciferase were harvested, washed in PBS, centrifuged at 400g for 5min, resuspended in PBS and counted with an automated cell counter (Countess, Invitrogen). Cells were kept on ice to maintain optimal viability. Mice were anaesthetized with intraperitoneal administration of Ketamine (Ketolar50®) / Xylacine (Rompiin®) (75 mg/kg and 10 mg/kg respectively). Each mouse was carefully placed in the stereotactic device and immobilized. Hair from the head was removed with depilatory cream, and the head skin was cut with a scalpel to expose the skull.
  • Ketamine Ketamine
  • Rompiin® Xylacine
  • mice were treated twice a week with h5D8 administered intraperitoneally. Treatment was initiated on day 0, immediately after tumor cell inoculation. Mice received a total of 2 doses of h5D8 or vehicle control.
  • Body weight and tumor volume Body weight was measured 2 times/week and tumor growth was quantified by bioluminescence on day 7 (Xenogen IVIS Spectrum). To quantify bioluminescence activity in vivo, mice were anaesthetized using isofluorane, and injected intraperitoneally with luciferin substrate (PerkinElmer) (167 pg/kg).
  • Example 12- h5D8 inhibits tumor growth in a mouse model of ovarian cancer [00200]
  • the efficacy of r5D8 was evaluated in two other syngeneic tumor models.
  • Results in Fig. 8C show that h5D8 also reduced tumor volume at a dose of 200 pg and above.
  • the ID8 cells were harvested, washed in PBS, centrifuged at 400 g for 5min and resuspended in PBS. Cells were kept on ice to maintain optimal viability and 200 pL of the cell suspension was injected intraperitoneally with a 27G needle. The final cell number implanted into mice was 5xl0 6 .
  • mice were treated twice weekly with h5D8 administered i.p. at different doses as indicated. Body weights were measured 2 times/week and tumor progression was monitored by measuring abdominal girth using a caliper (Fisher Scientific).
  • Example 13- r5D8 inhibits tumor growth in a mouse model of colorectal cancer
  • mice with subcutaneous colon CT26 tumors r5D8 (administered 300 pg IP twice weekly) significantly inhibited tumor growth (Fig. 9A and 9B).
  • 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 pg/mL streptomycin (PenStrep) and 0.25 pg/mL Plasmocin.
  • Example 14- r5D8 reduces inflammatory infiltration in tumor models
  • H5D8 treatment also programmed macrophages towards an immune-stimulatory phenotype in the syngeneic CT26 tumor model (Fig. 10E ).
  • h5D8 treatment increased macrophages with an Ml phenotype as indicated by an increased CD206
  • Example 15- r5D8 increases non-myeloid effector cells
  • r5D8 the effect of r5D8 on T cells and other non-myeloid immune effector cells within the tumor microenvironment were evaluated.
  • r5D8 treatment resulted in an increase in intratumoral NK cells and an increase in total and activated CD4 + and CD8 + T cells as shown in Fig. 11A.
  • r5D8 increased intratumoral NK cells, increased CD4+and CD8+T cells and trended to decrease CD4 + CD25 + FoxP3 + T-reg cells as shown in Fig. 11B.
  • CT26 cells were administered in both flanks to mice via subcutaneous injection using a 27G syringe. Mice were treated twice weekly with r5D8 administered intraperitoneally as indicated in the study design. Vehicle control (PBS), rat r5D8, and/or anti-CD4 and anti-CD8 was administered to the mice via intraperitoneal injection (IP) twice weekly as stated in the study design. All antibody treatments were administered concomitantly.
  • h5D8 The crystal structure of h5D8 was solved to a resolution of 3.1 angstroms in order to determine the epitope on LIF that h5D8 was bound to and to determine residues of h5D8 that participate in binding.
  • the co-crystal structure revealed that the N-terminal loop of LIF is centrally positioned between the light and heavy chain variable regions of h5D8 (Fig. 13A).
  • h5D8 interacts with residues on helix A and C of LIF, thereby forming a discontinuous and conformational epitope. 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 of interaction with gpl30. See Boulanger, M.J., Bankovich, A.J., Kortemme, T., Baker, D. & Garcia, K.C. Convergent mechanisms for recognition of divergent cytokines by the shared signaling receptor gpl30. Molecular cell 12, 577-589 (2003). The results are summarized below in Table 7 and depicted in Fig. 13.
  • Octet Binding Reagents were used and prepared as per manufacturer’s provided manual. A Basic Kinetics Experiment was performed using Octet Data Acquisition software ver. 9.0.0.26 as follows: Setup of sensors/program: i) Equilibration (60 seconds); ii) Loading (15 seconds); iii) Baseline (60 seconds); iv) Association (180 seconds); and v) Dissociation (600 seconds)
  • Octet Affinity of h5D8 for cytokines A Basic Kinetics Experiment was performed using Octet Data Acquisition software ver. 9.0.0.26 as follows: Amine Reactive 2ndGeneration Biosensors (AR2G) were hydrated for a minimum of 15 minutes in water. Amine conjugation of h5D8 to the biosensors was performed according to ForteBio Technical Note 26 (please see References) using the Amine Coupling Second Generation Kit.
  • Dip steps were as performed at 30°C, lOOOrpm as follows: i) 60 seconds Equilibration in water; ii) 300 seconds Activation in 20mM ECD, lOmM sulfo-NHS in water; iii) 600 second Immobilization of 10 pg/ml h5D8 in lOmM Sodium Acetate, pH 6.0; iv) 300 seconds Quench in 1M Ethanolamine, pH 8.5; v) 120 seconds Baseline in water.
  • Human recombinant LIF produced from mammalian cells was from ACROBiosystems (LIF-H52lb); 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).
  • 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 vapor diffusion methods and allowed to determine five crystal structures ranging between 1.65 A to 2.0 A in resolution. All structures were solved in the same crystallographic space group and with similar unit cell dimensions (P212121, a ⁇ 53.8 A, b ⁇ 66.5 A, c ⁇ 143.3 A), despite crystallization conditions ranging across five different pH levels: 5.6, 6.0, 6.5, 7.5 and 8.5. As such, these crystal structures allow for comparison of the three-dimensional disposition of h5D8 Fab unimpeded by crystal packing artefacts and across a wide spectrum of chemical conditions.
  • the Protein A flow-through, which contained the h5D8 Fab was recovered and buffer-exchanged into 20 mM sodium acetate, pH 5.6 using a 10K MWCO concentrator (Millipore).
  • the resulting sample was loaded onto a Mono S cation exchange column (GE Healthcare) using an AKTA Pure chromatography system (GE Healthcare). Elution with a gradient of 1 M potassium chloride resulted in a predominant h5D8 Fab peak that was recovered, concentrated and purified to size homogeneity using a Superdex 200 lncrease gel filtration column (GE Healthcare) in 20 mM Tris-HCl, 150 mM sodium chloride, at pH 8.0. The high purity of the h5D8 Fab was confirmed by SDS-PAGE under reducing and non-reducing conditions.
  • Crystals were obtained and harvested after four days in the following five crystallization conditions: 1) 0.085 M sodium citrate, 25.5% (w/v) PEG 4000, 0.17 M ammonium acetate, 15% (v/v) glycerol, pH 5.6; 2) 0.1 M MES, 20% (w/v) PEG 6000, 1 M lithium chloride, pH 6.0; 3) 0.1 M MES, 20% (w/v) PEG 4000, 0.6 M sodium chloride, pH 6.5; 4) 0.085 M sodium HEPES, 17% (w/v) PEG 4000, 8.5% (v/v) 2-propanol, 15% (v/v) glycerol, pH 7.5; and 5) 0.08 M Tris, 24% (w/v) PEG 4000, 0.16 M magnesium chloride, 20% (v/v) glycerol, pH 8.5.
  • Crystals Prior to flash-freezing in liquid nitrogen, mother liquors containing the crystals were supplemented with 5-15% (v/v) glycerol or 10% (v/v) ethylene glycol, as required. Crystals were subjected to X-ray synchrotron radiation 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 structures were determined by molecular replacement using Phaser. Refinement was carried out in PHENIX with iterative model building in Coot. Figures were generated in PyMOL. All software was accessed through SBGrid.
  • H5D8 revealed a free cysteine residue at position 100 (C l 00) in the variable region of the heavy chain.
  • H5D8 variants were generated by substituting C l 00 with each naturally occurring amino acid in order to characterize binding to and affinity for human and mouse LIF. Binding was characterized using ELISA and Octet assay. Results are summarized in Table 9. ELISA EC50 curves are shown in Fig. 15 (Fig.l5A human LIF and Fig. 15B Mouse LIF).
  • 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. Plates were blocked with lx blocking buffer for 2 hours at room temperature. Titrations of each h5D8 Cl 00 variants were added and allowed to bind for 1 hour at room temperature. Plates were washed three times with PBS+0.05% Tween-20. HRP-conjugated anti human IgG was added and allowed to bind for 30 min at room temperature. Plates were washed three times with PBS+0.05% Tween-20 and developed using lx TMB substrate. The reaction was stopped with 1M HC1 and absorbance at 450 nm was measured. Generation of figures and non-linear regression analysis was performed using Graphpad Prism.
  • Octet RED96 The affinity of h5D8 C100 variants to human and mouse LIF was determined by BLI using the Octet RED96 system. h5D8 Cl 00 variants were loaded onto Anti- Human Fc biosensors at 7.5 ug/mL following a 30 second baseline in lx kinetics buffer.
  • Example 20- h5D8 blocks binding of LIF to gpI30 in vitro
  • h5D8 prevented LIF from binding to LIFR
  • a molecular binding assay using the Octet RED 96 platform was performed. H5D8 was loaded onto AHC biosensors by anti-human Fc capture. Then, the biosensors were dipped in LIF and, as expected, association was observed (Fig. 16A, middle third). Subsequently, the biosensors were dipped in different concentrations of LIFR. A dose-dependent association was observed (Fig. 16A, right third). The control experiment demonstrated that this association was LIF-specific (not shown), and not due to a non-specific interaction of LIFR with h5D8 or with the biosensors.
  • LIFR mRNA expression was highest in human adipose tissue (mesenteric-ileum [1]), blood vessel tissue (pulmonary [9]), brain tissue [11-28] and thyroid [66] tissue; and was lowest in PBMCs [31] LIF and LIFR mRNA expression levels in cynomolgus tissues were similar to those observed in human tissues, wherein LIF expression was high in adipose tissue and LIFR expression was high in adipose tissue and low in PBMCs (data not shown).
  • tissue numbering for Fig. 17A and Fig. 17B is: 1 - adipose (mesenteric-ileum); 2 - adrenal gland; 3 - bladder; 4 - bladder (trigone); 5 - blood-vessel (cerebral: middle-cerebral- artery); 6 - blood vessel (choroid-plexus); 7 - blood vessel (coronary artery); 8 - blood vessel (mesenteric (colon)); 9 - blood vessel (pulmonary); 10 - blood vessel (renal); 11 - brain
  • hypothalamus anterior
  • 24 - brain hypothalamus: posterior
  • 25 - brain locus coeruleus
  • 26 - brain medulla oblongata
  • 27 - brain nucleus accumbens
  • 28 - brain substantially nigra
  • 29 - breast 30 - caecum; 31- peripheral blood mononuclear cell (PBMCs); 32 - colon; 33 - dorsal root ganlia (DRG); 34 - duodenum; 35 - fallopian tube; 36 - gallbladder; 37 - heart (left atrium); 38 - heart (left ventricle); 39 - ileum; 40 - jejunum; 41 - kidney (cortex); 42 - kidney (medulla); 43 - kidney (pelvis); 44 - liver (parenchyma); 45 - liver (bronchus: primary); 46 - liver (bronchus: tertiary); 47 - lung (parenchym
  • Example 22-H5D8 and anti-PD-1 antibody inhibit tumor growth in a mouse model of colorectal cancer
  • the efficacy of h5D8 was evaluated in combination with a PD-l inhibitor in the syngeneic CT26 and MC38 models. Mice treated with a combination of PD-l inhibitor and h5D8 exhibited decreased CT26 tumor growth when compared to mice treated with PD-l inhibitor or h5D8 alone, as shown in Figs. 18A and 18B. Whereas durable survival benefit with h5D8 monotherapy was not observed (Fig. 18C) and only rarely observed with anti -PD 1 therapy (Fig. 18C), h5D8 and anti -PD 1 combination resulted in long-term survival benefit in
  • CD8 TIL were functionally different on a per cell basis in tumors harvested from mice treated with either anti-PDl monotherapy or the h5D8 and anti-PDl combination, the capacity of the CD8 TIL to produce IFNy in response to tumor specific antigens was examined.
  • CDS TIL isolated from anti-PDl or h5D8 and anti-PDl combination treated CT26 tumors showed no difference in function when stimulated ex vivo with AH1 peptide (gp70; 423-43 la.a.), which comprises the immunodominant rejection antigen of CT26 (Fig. 19C).
  • h5D8 and the PD-l inhibitor, antibody clone RMP1-14 (BioXCell), were administered twice weekly at l5mg/kg and lOmg/kg doses respectively, and tumor volume was monitored through caliper-based measurements.
  • the effect of LIF on the cancer immune system was determined by measuring the relative abundance of tumor-associated macrophages (TAMs) and regulatory T cells (Tregs) across 28 types of solid tumors from the cancer genome atlas (TCGA).
  • TAMs tumor-associated macrophages
  • Tregs regulatory T cells
  • TCGA cancer genome atlas
  • a significant correlation between LIF and TAMs and Tregs was observed across several tumor types (Fig. 20A and 20B).
  • Glioblastoma (GBM), prostate adenocarcinoma, thyroid cancer and ovarian cancer were the 4 tumor types exhibiting the highest correlations between LIF, TAMs and Tregs, while showing a high LIF expression across samples (Fig. 20A and 20B).
  • a wide range of LIF expression was observed in GBM tumors being expressed by tumor cells and the immune cell infiltrates (Fig.
  • LIF assumes a crucial role in the exclusion of CD8 + T cells, while promoting the presence of pro-tumoral TAMs.
  • a blockade of LIF in tumors expressing high levels of LIF was observed to decrease CD206, CD 163 and CCL2 and induced CXCL9 expression in TAMs.
  • the blockade of LIF released the epigenetic silencing of CXCL9 triggering CD8 + T cell tumor infiltration.
  • the combination of LIF neutralizing antibodies with the inhibition of the PD1 immune checkpoint promoted tumor regression and an increase in overall survival.
  • RNA-seq data for 9,403 patients suffering from 28 distinct solid tumors was downloaded from The Cancer Genome Atlas (TCGA), Firebrowse server (firebrowse.org, version 20l6_0l_28).
  • the expression data (RSEM) was log2 transformed for all downstream analyses.
  • the gene signatures of the four immune populations of interest were obtained: TAMs, Tregs, CD4 + T cells and CD8 + T cells.
  • the correlation between LIF expression and the gene signatures of the four immune populations and the correlation between LIF and a set of genes of interest were then computed.
  • the GBM cell line, GL261N (a derivative of the GL261 cell line), the GFAP-tv-a RCAS-PDGFA, shp53, shNFl (RCAS) transgenic model, and the ovarian cancer cell line, ID8, that generated tumors in the brain (GL261N and RCAS) and peritoneum (ID8) of mice were identified as expressing high levels of LIF (Fig. 25).
  • intraparenchymal of 8-week-old C57BL/6 mice.
  • 5xl0 6 ID8 ovarian cancer cells were intraperitoneally injected into 8-week-old C57BL/6 mice.
  • a dose of 300 pg (ID8) or 600 pg (GL261N, GL261, and RCAS) of anti-LIF or a control IgG was administered intraperitoneally twice a week.
  • mice used were RAG , CCL2 / , and CXCL9 " from Jackson Laboratories and NOD SCID gamma (NSG) from Charles River.
  • TAMs derived from recruited monocytes (CD1 lb + Ly6G Ly6C CD49d + ) 12 were decreased in response to anti-LIF (Fig. 26D) and no major effect was observed on the dendritic cell population (CD1 lb + , CD1 lc + , MHCII + ) (Fig. 26E) nor on the levels of IL-10 or IL-12 in the tissue (Fig. 26F).
  • mice were euthanized and tumors were isolated.
  • GL261N and RCAS tumors were enzymatically digested with Brain Tumor Dissociation kit and myelin was removed with Myelin Removal Beads II (all from Miltenyi Biotec).
  • ID8 tumors were processed with Mouse Tumor Dissociation kit (Miltenyi Biotec) and ascitic liquids were collected.
  • Human GBM specimens of the organotypic model and the patient derived xenografts were enzymatically digested with Human Tumor Dissociation kit (Miltenyi Biotec).
  • Example 27-CD8+ T cell infiltration is not the result of the anti -tumor response to the blockade ofLIF
  • the genes related to an oncogenic phenotype that were downregulated were determined by isolating CD1 lb + cells from the ID8 mouse model, treating the cells with anti-LIF antibodies, and performing a transcriptomic analysis.
  • the genes identified were CCL2, CCL3, CCL7, PF4, CTSK, CD206, and CD 163. And, interestingly, CXCL9 was upregulated (Fig. 21 A).
  • the aforementioned gene responses were validated by qRT-PCR in the ID8 and GL261N models (Fig. 21B)
  • CXCL9 and CCL2 stood out as chemokines critical for CD8 + T cell tumor infiltration, and the recruitment of TAMs and Tregs, respectively.
  • CXCL9 and CCL2 regulation by the neutralization of LIF in TAMs (CD1 lb + Ly6G Ly6C ) was confirmed (Fig. 21C).
  • RMA Robust-Microarray Average
  • CXCL9 and CCL2 knockout (CXCL9 / , CCL2 / ) mouse models were used to test for the relevance of the regulation of CXCL9 and CCL2 in LIF oncogenic function. Tumors in these mouse models were treated with blocking antibodies against CXCL9 and CCL2. Interestingly, the anti-tumor response to the inhibition of LIF was blunted in the CXCL9 / mice but not in the CCL2 mice (Fig. 21F). Similarly, the CXCL9 neutralizing antibody but not the CCL2 antibody impaired the anti -cancer response to anti-LIF (Fig. 21F). These results indicate that the main mediator of the anti-LIF response was CXCL9. As expected, the blockade of CXCL9 decreased CD8 + T cell tumor infiltration in response to anti-LIF (Fig. 21G).
  • Immunohistochemical antibodies human LIF (Atlas; 1 :200), murine LIF (AbCam; 1 :200), murine p-STAT3 (Cell Signaling; 1 :50), murine Ki67 (AbCam; 1 :200), murine Cleaved- Caspase3 (CC3) (Cell Signaling; 1 :500), murine CD8 (Bioss; 1 :200), human/murine CCL2 (Novus Biologicals, 1 :200), human CXCL9 (Thermo Fischer Scientific; 1 : 100), and human CD163 (Leica Novacastra; 1 :200).
  • Immunofluorescence antibodies human/murine CCL2 (Novus Biologicals, 1 :200), human/murine CDl lb (AbCam; 1 :2000), human/murine Ibal (Wako; 1 : 1000), murine CD68 (AbCam; 1 :200), human/murine CD206 (Abeam; 1 :500), murine CD163 (Abeam; 1 :200), CXCL9 (murine Novus Biologicals 1 :200; human Thermo Fischer Scientific; 1 :200), and human CD 8 (DAKO; 1 :200).
  • LIF regulated the expression of several Ml -like and M2-like markers induced by IFNy or IL4 in BMDMs (Fig. 22A). CXCL9 expression was not detected except when BMDMs were treated with IFNy. Recombinant LIF repressed the induction of CXCL9 by IFNy both at the mRNA and protein levels (Fig. 22B and 22C).
  • CXCL9 was also regulated by IFNy and LIF in patient- derived TAMs (CD1 lb + CDl4 + ) obtained from fresh human GBM tumors (Fig. 22D and 29A). These results were further validated upon observing that recombinant LIF repressed the induction of CXCL9 by LPS both at the mRNA and protein levels. (Fig. 29B). Thus, LIF acted as a repressor of CXCL9 induction. CXCL9 promoter binding of p-STAT3 upon treatment with LIF was not observed (data not shown).
  • H3K27me3 H3 lysine 27 trimethylated
  • H4ac acetylated H4
  • EZH2 binding was found to increase the levels of H3 lysine 27 trimethylated (H3K27me3)
  • H4ac acetylated H4
  • EZH2 binding was found to increase the levels of H3 lysine 27 trimethylated (H3K27me3)
  • H4ac acetylated H4
  • EZH2 binding EZH2 binding to the CXCL9 promoter region
  • Dissociation kit and CD1 lb + cells were isolated using CD1 lb magnetic beads and the
  • MultiMACS Cell24 separator Plus (all from Miltenyi Biotec). CD1 lb + cells obtained were cultured in RPMI medium supplemented with 10% heat-inactivated FBS (Life Technologies). Recombinant LIF, IFNy, LPS, and IL4 were purchased from Millipore, R&D Systems, Sigma and Creative BioMart, respectively.
  • Immunocomplexes were recovered using 20 j.tl of protein G magnetic beads, washed, and eluted. Cross-linking was reversed at 65 °C 4 h and immunoprecipitated DNA was recovered using the PCR purification kit from Qiagen. Genomic regions of interest were identified by real-time quantitative PCR (qPCR) using SYBR Green Master Mix
  • Example 3- LIF regulation of immune cell tumor infiltration in patients [00261] To confirm that LIF regulates immune cell tumor infiltration through the repression of CXCL9 in tumors from actual cancer patients, organotypic tissue cultures were generated from GBM specimens freshly obtained from patients. These organotypic models allow for the short term culture of slices of tumors that maintain the tissue architecture and stroma (including immune cells) of the tumor of the patient. Organotypic tissue cultures from 3 patients whose tumor cells expressed high levels of LIF (Fig. 22H). In all 3 cultures a large infiltration of TAMs was present as detected by the Ibal marker and most of the TAMs expressed CCL2, CD163 and CD206. Interestingly, a 3-day treatment of the organotypic culture with a neutralizing antibody against LIF promoted a decrease in CCL2, CD 163 and CD206 and an increase in CXCL9 expression (Fig. 22H).
  • GBM neurospheres were generated as described as follows. Briefly, tumor samples were processed within 30 min after surgical resection. Minced pieces of human GBM samples were digested with 200 U/ml collagenase I (Sigma) and 500 U/ml DNase I (Sigma) in PBS for 1 h at 37°C with constant vigorous agitation. The single-cell suspension was filtered through a 70 pm cell strainer (BD Falcon) and washed with PBS.
  • GBM medium consisted of Neurobasal medium supplemented with B27, penicillin/streptomycin (all from Life Technologies) and growth factors (20 ng/ml EGF and 20 ng/ml FGF-2 (PeproTech).
  • GBM organotypic slice cultures were generated as follows. After resection, surgical specimens were cut with a scalpel into rectangular blocks of 5-10 mm length and 1-2 mm width and individually transferred into 0.4 pm membrane culture inserts (Millipore) within 6-well plates. Before placing the inserts into 6-well plates, 1.2 ml of Neurobasal medium (Life Technologies) supplemented with B27 (Life Technologies), penicillin/ streptomycin (Life Technologies) and growth factors (20 ng/ml EGF and 20 ng/ml FGF-2) (PeproTech) were placed into each well. The cultures were kept at 37°C with constant humidity, 95% air and 5% CO 2 .
  • PBMCs peripheral blood mononuclear cells
  • control or anti-LIF slices were embedded into Matrigel (Coming) with subsequent addition of lxlO 6 PBMCs into 24-well plate in complete RPMI medium.
  • supernatants were collected and organotypic slices were recovered from Matrigel and further processed for IF and flow cytometry.
  • PBMCs were resuspended with PBS at a concentration of 10 6 cells/ml and incubated for 20 min with 5 mM Cell Trace CFSE (Invitrogen). After the incubation, cells were washed with RPMI and added to the sections embedded into Matrigel. After 24 h, fluorescent PBMCs invasion into Matrigel was evaluated under microscope by counting migrating cells in five different areas per each condition.
  • Example 32-Treatment with anti-LIF increased CXCL9 and decreased CCL2 expression in tumors expressing high levels ofLIF
  • LIF LIF’s impact on the immune cell tumor infiltration was assessed.
  • organotypic slices from 3 patients with tumors expressing high levels of LIF were incubated with peripheral blood mononuclear cells (PBMCs) from the same patient (Fig. 23A).
  • PBMCs peripheral blood mononuclear cells
  • Treatment with anti-LIF increased CXCL9 and decreased CCL2 expression (Fig. 23B), and induced immune cell infiltration into the Matrigel surrounding the tumor specimen (Fig. 23B).
  • CD8 + T cells were recruited to the tumor tissue upon LIF blockade (Fig. 23B, 23C) and this effect was dependent on CXCL9 since the neutralization of CXCL9 prevented CD8 + T cell infiltration (Fig. 23D).
  • mice Similar results were confirmed in the context of an in vivo model. Tumor fragments from 4 patients, whose tumors expressed high LIF levels, were inoculated in NSG mice and these mice were treated with the LIF neutralizing antibody for 5 days. Next, each patient’s PBMCs were inoculated in the mice. Interestingly, mice treated with anti-LIF showed an increase in CD8 + T cell tumor infiltration and most of the infiltrating CD8 + T cells expressed the CXCL9 receptor, CXCR3 (Fig. 23E).
  • mice exhibiting complete tumor regression were collected and reinoculated 3xl0 5 tumor cells. No tumor appeared in these mice while tumors rapidly grew in naive mice inoculated in parallel with the same number of cells (Fig. 23H). The result of this rechallenge experiment indicated that the combined treatment with anti-LIF and anti -PD 1 generated immunological memory.
  • embodiment 8 wherein the antibody that specifically binds to LIF is an IgG antibody.
  • the antibody that specifically binds to LIF is a Fab, F(ab) 2 , single-domain antibody, a single chain variable fragment (scFv), or a nanobody.
  • VH-CDR1 an immunoglobulin heavy chain complementarity 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 complementarity 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 complementarity 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 complementarity 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 complementarity determining region 2 (VL-CDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 11 or 12; and
  • VL-CDR3 immunoglobulin light chain complementarity determining region 3
  • an immunoglobulin heavy chain variable region (VH) sequence with the amino acid sequence 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 with the amino acid sequence 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.
  • VH immunoglobulin heavy chain variable region
  • VL immunoglobulin light chain variable region
  • 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.
  • any one of embodiments 8 to 15, wherein the antibody that specifically binds to LIF comprises: a) an immunoglobulin heavy chain sequence with the amino acid sequence 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
  • an immunoglobulin light chain sequence with the amino acid sequence 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.
  • Atezolizumab Avelumab, BMS-936559, or FAZ053, or a PDL-l or PDL-2 binding fragment thereof.
  • any one of embodiments 1 to 30, wherein the cancer comprises an advanced solid tumor, glioblastoma, stomach cancer, skin cancer, prostate cancer, pancreatic cancer, breast cancer, testicular cancer, thyroid cancer, head and neck cancer, liver cancer, kidney cancer, esophageal cancer, ovarian cancer, colon cancer, lung cancer, lymphoma, a soft tissue cancer, or any combination thereof.
  • embodiment 31 wherein the cancer comprises non-small cell lung cancer, epithelial ovarian carcinoma, or pancreatic ductal adenocarcinoma.
  • a method of treating an individual with a cancer comprising administering to the individual with the cancer an effective amount of a combination of:
  • LIF Leukemia Inhibitory Factor
  • LIF-binding polypeptide comprises a fragment of an immunoglobulin variable region, or an immunoglobulin heavy chain constant region.
  • VH-CDRT immunoglobulin heavy chain complementarity determining region 1
  • VH-CDR2 immunoglobulin heavy chain complementarity determining region 2
  • VH-CDR3 immunoglobulin heavy chain complementarity determining region 3
  • VL-CDR1 immunoglobulin light chain complementarity determining region 1
  • VL-CDR2 immunoglobulin light chain complementarity determining region 2
  • VL-CDR3 immunoglobulin light chain complementarity determining region 3
  • an immunoglobulin heavy chain variable region (VH) sequence with the amino acid sequence 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 with the amino acid sequence 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.
  • VH immunoglobulin heavy chain variable region
  • VL immunoglobulin light chain variable region
  • 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:
  • Atezolizumab Avelumab, BMS-936559, or FAZ053, or a PDL-l or PDL-2 binding fragment thereof.
  • inhibitor of PD-l, PDL-l, or PDL-2 signaling comprises an Fc-Fusion protein that binds PD-l, PDL-l, or PDL-2.
  • inhibitor of PD-l, PDL-l, or PDL-2 signaling comprises a small molecule inhibitor of PD-l, PDL-l, or PDL-2.
  • the small molecule inhibitor of signaling through PD-l, PDL-l, or PDL-2 comprises one or more of: N- ⁇ 2-[( ⁇ 2-methoxy-6-[(2- methyl[l,l’-biphenyl]-3-yl)methoxy]pyridin-3-yl ⁇ methyl)amino]ethyl ⁇ acetamide (BMS 202);
  • the cancer comprises an advanced solid tumor, glioblastoma, stomach cancer, skin cancer, prostate cancer, pancreatic cancer, breast cancer, testicular cancer, thyroid cancer, head and neck cancer, liver cancer, kidney cancer, esophageal cancer, ovarian cancer, colon cancer, lung cancer, lymphoma, a soft tissue cancer, or combinations thereof.
  • cancer comprises non-small cell lung cancer, epithelial ovarian carcinoma, or pancreatic adenocarcinoma.
  • LIF binding antibody comprises: a) an immunoglobulin heavy chain complementarity 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 complementarity 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 complementarity 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 complementarity 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 complementarity determining region
  • VL-CDR3 immunoglobulin light chain complementarity determining region 3
  • LIF Leukemia Inhibitory Factor
  • VH-CDR1 immunoglobulin heavy chain complementarity determining region 1
  • VH-CDR2 immunoglobulin heavy chain complementarity determining region 2
  • VL-CDR1 immunoglobulin light chain complementarity determining region 1
  • VL-CDR2 immunoglobulin light chain complementarity determining region 2
  • VL-CDR3 immunoglobulin light chain complementarity determining region 3
  • 74 The method of any one of embodiments 70 to 73, wherein the cancer is glioblastoma multiforme (GBM), NSCLC (non-small cell lung carcinoma), ovarian cancer, colorectal cancer, thyroid cancer, pancreatic cancer, or combinations thereof.
  • GBM glioblastoma multiforme
  • NSCLC non-small cell lung carcinoma
  • ovarian cancer colorectal cancer
  • thyroid cancer pancreatic cancer, or combinations thereof.
  • a method of decreasing pro-tumoral tumor-associated macrophages (TAMs) in a tumor of an individual with cancer comprising administering to the individual with cancer an effective amount of a combination of:
  • LIF Leukemia Inhibitory Factor
  • VH-CDR1 immunoglobulin heavy chain complementarity determining region 1
  • VH-CDR3 immunoglobulin heavy chain complementarity determining region 3
  • VL-CDR1 immunoglobulin light chain complementarity determining region 1
  • VL-CDR2 immunoglobulin light chain complementarity determining region 2
  • VL-CDR3 immunoglobulin light chain complementarity determining region 3
  • VH-CDR2 immunoglobulin heavy chain complementarity determining region 2
  • VH-CDR3 immunoglobulin heavy chain complementarity determining region 3
  • LIF Leukemia Inhibitory Factor
  • VH-CDR1 immunoglobulin heavy chain complementarity determining region 1
  • VH-CDR2 immunoglobulin heavy chain complementarity determining region 2
  • VL-CDR1 immunoglobulin light chain complementarity determining region 1
  • VL-CDR2 immunoglobulin light chain complementarity determining region 2
  • VL-CDR3 immunoglobulin light chain complementarity determining region 3
  • T lymphocytes comprise CD8+ T cells.
  • T lymphocytes comprise CD4+ T cells.
  • the tumor comprises a lung tumor, a brain tumor, a pancreatic tumor, a breast tumor, a kidney tumor, a colorectal tumor, an ovarian tumor, or a combination thereof.

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EA202092360A EA202092360A1 (ru) 2019-02-22 2019-04-11 Комбинация ингибиторов lif и ингибиторов оси pd-1 для применения в лечении рака
CN201980038348.0A CN112585164A (zh) 2018-04-12 2019-04-11 用于治疗癌症的、lif抑制剂和pd-1轴抑制剂的组合
EP19733546.6A EP3774895A1 (en) 2018-04-12 2019-04-11 Combination of lif inhibitors and pd-1 axis inhibitors for use in treating cancer
CA3096779A CA3096779A1 (en) 2018-04-12 2019-04-11 Combination of lif inhibitors and pd-1 axis inhibitors for use in treating cancer
KR1020207032587A KR20210021287A (ko) 2018-04-12 2019-04-11 암을 치료하는 데 사용하기 위한 lif 억제제와 pd-1 축 억제제의 조합
AU2019251289A AU2019251289B2 (en) 2018-04-12 2019-04-11 Combination of LIF inhibitors and PD-1 axis inhibitors for use in treating cancer
SG11202009966SA SG11202009966SA (en) 2018-04-12 2019-04-11 Combination of lif inhibitors and pd-1 axis inhibitors for use in treating cancer
JP2021504589A JP7423598B2 (ja) 2018-04-12 2019-04-11 がんの治療で使用するためのｌｉｆ阻害剤とｐｄ－１軸阻害剤との組み合わせ
US17/046,599 US20210130453A1 (en) 2018-04-12 2019-04-11 Combination of lif inhibitors and pd-1 axis inhibitors for use in treating cancer
IL277918A IL277918A (en) 2018-04-12 2020-10-11 A combination of LIF inhibitors and PD-1 AXIS inhibitors for use in cancer therapy
JP2024005463A JP2024054138A (ja) 2018-04-12 2024-01-17 がんの治療で使用するためのｌｉｆ阻害剤とｐｄ－１軸阻害剤との組み合わせ

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WO2021110873A1 (en) * 2019-12-04 2021-06-10 Medimmune Limited Antibodies against lif and uses thereof
CN114929741A (zh) * 2019-12-04 2022-08-19 免疫医疗有限公司 针对lif的抗体及其用途
JP2023516494A (ja) * 2020-03-11 2023-04-19 パーデュー・リサーチ・ファウンデーション 免疫調節活性を持つ化合物およびその治療的使用
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IL277918A (en) 2020-11-30
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WO2019197903A8 (en) 2020-02-20
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