Treatment of cancers with GM-CSF antagonists

Abstract

To provide treatment of cancers with GM-CSF antagonists.SOLUTION: The present invention provides, among other things, a method of treating cancer comprising administering a GM-CSF antagonist to a patient in need of treatment, where the administration of the GM-CSF antagonist results in inhibition of an immunosuppressive activity of myeloid-derived suppressor cells (MDSCs). The present invention also provides, among other things, a method of inhibiting immunosuppressive activity of myeloid-derived suppressor cells (MDSCs) in a patient suffering from cancer, comprising administering a GM-CSF antagonist to the patient.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 856,638, filed June 3, 2019, which is incorporated herein by reference in its entirety for all purposes.

[0002] Sequence Listing This specification references a Sequence Listing, which was submitted electronically as a .txt file entitled "KPL-035WO_SL.txt" on June 3, 2020. The .txt file was created on June 3, 2020 and is 4KB in size. The entire contents of the Sequence Listing are incorporated herein by reference. [Background technology]

[0003] Tumor cells express unique antigens that can be recognized by the host T cell repertoire and serve as potent targets for tumor immunotherapy. However, tumor cells express inhibitory cytokines that evade host immunity and suppress natural antigen presenting effector cell populations. One element in this immunosuppressive environment is the increased presence of regulatory T cells found in the tumor matrix, draining lymph nodes, and circulation of patients with malignant tumors. One area of ​​further research is the development of therapeutic agents to reverse tumor-associated anergy and stimulate effector cells to recognize and eliminate malignant cells.

[0004] The drawings are for purposes of illustration only, and not for purposes of limitation. [Brief description of the drawings]

[0005] [Figure 1]1 is an exemplary bar graph illustrating T cell proliferation assay using CD14+ cells (MDSC) from blood of a patient with pancreatic cancer.T cell proliferation is suppressed after co-culture of healthy donor allogeneic T cells with CD14+ cells selected from blood of a patient with pancreatic cancer, compared to those T cells cultured in complete medium alone.T cell proliferation is rescued after culture with anti-GM-CSFRα antibody and CD14+ cells selected from blood of a patient with pancreatic cancer. [Diagram 2] 1 is an exemplary graph illustrating GM-CSF expression levels in different cancer cell lines. [Diagram 3] 1 is a series of exemplary bar graphs illustrating that cancer cell conditioned medium can polarize monocytes to phenotype MDSC (CD14+ cells). To generate tumor conditioned medium (CM), four different cell lines were seeded and cultured according to methods known in the art. CD14+ monocyte cells were then cultured in the presence of CM for 6 days and analyzed for gene and protein expression. Low levels of HLA-DR biomarkers indicate MDSC phenotype. An increase in phenotype MDSC was observed when CD14+ monocytes were incubated with conditioned medium from GM-CSF expressing cancer cells, compared to CD14+ cells grown in normal culture medium (control). [Figure 4] 1 is a series of exemplary bar graphs illustrating PD-L1 expression on MDSCs cultured with various media. The data show that cancer cell conditioned medium (CM) and CM supplemented with recombinant GM-CSF can induce the expression of PD-L1 on MDSCs. Additionally, anti-GM-CSFRα antibodies (Abs) can reduce the expression level of PD-L1 on MDSCs. [Figure 5A]5A is a series of exemplary bar graphs illustrating PD-L1 expression on MDSCs cultured with various media. The data show that cancer cell conditioned medium (CM) and CM supplemented with recombinant GM-CSF on day 1 can induce the expression of PD-L1 on MDSCs compared to MDSCs grown in normal culture medium (medium). Additionally, anti-GM-CSFRα antibody (Ab) can reduce the PD-L1 expression level on MDSCs in CM. The data in FIG. 5A shows PD-L1 expression when CM and anti-GM-CSFRα antibody (Ab) are added simultaneously. PD-L1 expression was measured after 3 days of treatment. FIG. 5B shows PD-L1 expression when anti-GM-CSFRα antibody is added 72 hours after culture with CM. PD-L1 expression was measured 24 hours after treatment with anti-GM-CSFRα antibody. [Figure 5B] Same as above. [Figure 6]1 is a series of exemplary bar graphs illustrating T cell proliferation using monocytes treated with conditioned medium from a GM-CSF-expressing cancer cell line with or without supplementation of human recombinant GM-CSF and / or anti-GM-CSFRα antibody (Ab). Monocytes were cultured for 3 days in conditioned medium (CM) from a GM-CSF-expressing cancer cell line. T cells (1×105 cells) were prepared by labeling with 0.1 μM CFSE and stimulating with 10 ng / mL IL-2 and 10 uL soluble CD3 / CD28 T cell activator (ImmunoCult) in IMDM cell culture medium. Stimulated T cells were then co-cultured with CM-treated monocytes (at a monocyte:T cell ratio of 2:1) in a mixed lymphocyte reaction (MLR) with or without recombinant GM-CSF (10 ng / mL) and / or anti-GM-CSFRα antibody (100 μg / mL). T cells stimulated in IMDM culture medium together with healthy monocytes were used as a control. T cells were expanded for 5 days, harvested, and stained for CD4 and CD8, markers for helper and cytotoxic T cells. Cell proliferation was measured by flow cytometry and assessed by CFSE dilution. The left panel shows the results of the T cell proliferation assay in terms of % of proliferating cells, and the right panel shows the results in terms of % of maximum (MFI) by flow cytometry (signal detection of CFSE dilution in CD4+ or CD8+ cells). Summary of the Invention [Means for solving the problem]

[0006] The present invention provides, inter alia, an improved method for treating cancer based on the inhibition of the immunosuppressive activity of myeloid-derived suppressor cells using GM-CSF antagonists.The present invention is based, in part, on the surprising discovery that GM-CSF induces the expression of PD-L1 on MDSCs that have immunosuppressive activity, and that this expression can be inhibited by antagonizing GM-CSF.The present invention also provides a method for treating cancer using GM-CSF antagonists in combination with other cancer therapies, as further described herein.

[0007] In some aspects, the present invention provides a method of treating cancer comprising administering a GM-CSF antagonist to a patient in need of treatment, wherein administration of the GM-CSF antagonist results in inhibition of the immunosuppressive activity of myeloid-derived suppressor cells (MDSCs).

[0008] In some embodiments, the present invention provides a method of inhibiting the immunosuppressive activity of myeloid-derived suppressor cells (MDSCs) in a patient suffering from cancer, comprising administering to the patient a GM-CSF antagonist.

[0009] In some aspects, the present invention provides a method of enhancing an immune response for cancer treatment comprising administering a GM-CSF antagonist to a patient undergoing cancer treatment, wherein the immune response is increased compared to a control.

[0010] In some embodiments, the immune response is a percentage of T cell proliferation. In some embodiments, the T cells are CD8 positive (CD8+). In some embodiments, the T cells are CD4 positive (CD4+). In some embodiments, the T cells are double positive for CD8 and CD4 (CD8+ / CD4+) (CD8+ / CD4+).

[0011] In some embodiments, the control represents the level of immune response in the patient prior to administration of the GM-CSF antagonist, hi some embodiments, the control is a reference immune response level in a control patient undergoing cancer treatment without the GM-CSF antagonist, or a reference immune response level based on historical data.

[0012] In some embodiments, the cancer therapy is immunotherapy.

[0013] In some embodiments, administering a GM-CSF antagonist increases the effectiveness of immunotherapy.

[0014] In one aspect, the invention provides, inter alia, a method of inhibiting PD-L1 in a cancer patient comprising administering a GM-CSF antagonist to a patient in need of treatment compared to a control.

[0015] In some embodiments, administering a GM-CSF antagonist reduces the level of PD-L1 in a cancer patient.

[0016] In some embodiments, the control represents PD-L1 levels in the patient prior to administration of the GM-CSF antagonist.

[0017] In some embodiments, the control is a reference PD-L1 level in control patients undergoing cancer treatment without a GM-CSF antagonist, or a reference PD-L1 level based on historical data.

[0018] In some embodiments, the level of PD-L1 in the patient is decreased by at least 10% compared to a control. In some embodiments, the level of PD-L1 in the patient is decreased by at least 15% compared to a control. In some embodiments, the level of PD-L1 in the patient is decreased by at least 20% compared to a control. In some embodiments, the level of PD-L1 in the patient is decreased by at least 30% compared to a control. In some embodiments, the level of PD-L1 in the patient is decreased by at least 40% compared to a control. In some embodiments, the level of PD-L1 in the patient is decreased by at least 45% compared to a control. In some embodiments, the level of PD-L1 in the patient is decreased by at least 50% compared to a control. In some embodiments, the level of PD-L1 in the patient is decreased by at least 60% compared to a control. In some embodiments, the level of PD-L1 in the patient is decreased by at least 70% compared to a control. In some embodiments, the level of PD-L1 in the patient is decreased by at least 75% compared to a control. In some embodiments, the level of PD-L1 in the patient is decreased by at least 80% compared to a control. In some embodiments, the level of PD-L1 in the patient is decreased by at least 85% compared to a control. In some embodiments, the level of PD-L1 in the patient is reduced by at least 90% compared to the control.

[0019] In some embodiments, PD-L1 is expressed on MDSCs. In some embodiments, PD-L1 is expressed on circulating MDSCs. In some embodiments, PD-L1 is expressed on plasma-derived MDSCs. In some embodiments, PD-L1 is expressed on MDSCs. In some embodiments, PD-L1 is expressed on tumor-infiltrating immune cells.

[0020] In some embodiments, the patient has circulating myeloid-derived suppressor cells (MDSCs).

[0021] In some embodiments, the patient is afflicted with a cancer that has low levels of infiltrating T cells.

[0022] In some embodiments, the patient suffers from immune checkpoint inhibitor (ICI) refractory cancer.

[0023] In some embodiments, the patient is afflicted with late stage or metastatic cancer.

[0024] In some embodiments, the patient is afflicted with a cancer selected from breast cancer, colorectal cancer (CRC), prostate cancer, melanoma, bladder cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, hepatocellular carcinoma, gastric cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck squamous cell carcinoma, non-Hodgkin's lymphoma, cervical cancer, gastrointestinal cancer, genitourinary cancer, brain cancer, mesothelioma, renal cell carcinoma, gynecological cancer, ovarian cancer, endometrial cancer, lung cancer, gastrointestinal cancer, pancreatic cancer, esophageal cancer, hepatocellular carcinoma, cholangiocarcinoma, brain cancer, mesothelioma, malignant melanoma, Merkel cell carcinoma, multiple myeloma, acute myeloid leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, or acute lymphoblastic leukemia.

[0025] In some embodiments, the patient is afflicted with a cancer selected from stage IV breast cancer, stage IV colorectal cancer (CRC), prostate cancer, or melanoma.

[0026] 2. The method of any one of the preceding claims, wherein the method further comprises administering to the patient at least one other cancer therapy.

[0027] In some embodiments, the at least one other cancer therapy is chemotherapy, MDSC targeted therapy, immunotherapy, radiation therapy, and combinations thereof.

[0028] In some embodiments, the GM-CSF antagonist and the other cancer therapy are administered simultaneously.

[0029] In some embodiments, the GM-CSF antagonist and the other cancer therapy are administered sequentially.

[0030] In some embodiments, the patient has been treated with another cancer therapy prior to administration of the GM-CSF antagonist.

[0031] In some embodiments, the patient has been treated with a GM-CSF antagonist prior to administration of the other cancer therapy.

[0032] In some embodiments, the other cancer therapy is an ICI.

[0033] In some embodiments, the ICI antagonizes the activity of PD-1, CTLA-4, B7, BTLA, HVEM, TIM-3, GAL-9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, or A2aR.

[0034] In some embodiments, the ICI is selected from an anti-PD-1 antibody (optionally pembrolizumab, nivolumab, cemiplimab), an anti-PD-L1 antibody (optionally atezolizumab, avelumab, durvalumab), an anti-CTLA-4 antibody (optionally ipilimumab), an anti-PD-L2 antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-BTLA antibody, an anti-HVEM antibody, an anti-TIM-3 antibody, an anti-GAL-9 antibody, an anti-LAG3 antibody, an anti-VISTA antibody, an anti-KIR antibody, an anti-2B4 antibody, an anti-CD160 antibody, an anti-CGEN-15049 antibody, an anti-CHK1 antibody, an anti-CHK2 antibody, an anti-A2aR antibody, an anti-B-7 antibody, and combinations thereof.

[0035] In some embodiments, the ICI is an anti-PD-L1 antibody.In some embodiments, the ICI is an anti-PD-L1 antibody.

[0036] In some embodiments, the method further comprises administering to the patient a chemotherapeutic agent.

[0037] In some embodiments, the MDSC targeted therapy is selected from an anti-CFS-1R antibody, an anti-IL-6 antibody, all-trans retinoic acid, axitinib, entinostat, gemcitabine, or phenformin, and combinations thereof.

[0038] In some embodiments, the immunotherapy is selected from a monoclonal antibody, a cytokine, a cancer vaccine, a T cell engaging therapy, and combinations thereof.

[0039] In some embodiments, the monoclonal antibody is selected from an anti-CD3 antibody, an anti-CD52 antibody, an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-CD20 antibody, an anti-BCMA antibody, a bispecific antibody, or a bispecific T cell engager (BiTE) antibody, and combinations thereof.

[0040] In some embodiments, the cytokine is selected from IFNa, IFNp, IFNy, IFNy, IFN, IL-2, IL-7, IL-15, IL-21, IL-11, IL-12, IL-18, hGM-CSF, TNFa, or any combination thereof.

[0041] In some embodiments, the GM-CSF antagonist is an anti-GM-CSF antibody or a fragment thereof.

[0042] In some embodiments, the GM-CSF antagonist is a soluble GM-CSF receptor.

[0043] In some embodiments, the GM-CSF antagonist is an anti-GM-CSF receptor antibody or a fragment thereof.

[0044] In some embodiments, the anti-GM-CSF receptor antibody or fragment thereof is an anti-GM-CSFRα antibody or fragment thereof.

[0045] In some embodiments, the anti-GM-CSFRα antibody or fragment thereof is a monoclonal antibody specific for human GM-CSFRα.

[0046] In some embodiments, the anti-GM-CSFRα antibody is a human or humanized IgG4 antibody.

[0047] In some embodiments, the anti-GM-CSFRα antibody is mavrilimumab.

[0048] In some embodiments, the anti-GM-CSFRα antibody fragment thereof comprises a light chain complementarity determining region 1 (LCDR1) defined by SEQ ID NO:6, a light chain complementarity determining region 2 (LCDR2) defined by SEQ ID NO:7, and a light chain complementarity determining region 3 (LCDR3) defined by SEQ ID NO:8, as well as a heavy chain complementarity determining region 1 (HCDR1) defined by SEQ ID NO:3, a heavy chain complementarity determining region 2 (HCDR2) defined by SEQ ID NO:4, and a heavy chain complementarity determining region 3 (HCDR3) defined by SEQ ID NO:5.

[0049] In some embodiments, administration of a GM-CSF antagonist and / or an ICI results in reduced levels of MDSCs in the patient compared to a control.

[0050] In some embodiments, administration of a GM-CSF antagonist and / or an ICI results in reduced levels of MDSC-mediated immunosuppressive activity in the patent compared to a control.

[0051] In some embodiments, administration of a GM-CSF antagonist and / or an ICI results in a reduced percentage of Lin-CD14+HLA-DR-M-MDSCs in the peripheral blood of a patient compared to a control.

[0052] In some embodiments, administration of a GM-CSF antagonist and / or an ICI results in an increased percentage of mature MDSC cells in the patient compared to a control.

[0053] In some embodiments, administration of a GM-CSF antagonist and / or an ICI results in reduced levels of Treg cells, macrophages, and / or neutrophils compared to a control.

[0054] In some embodiments, administration of a GM-CSF antagonist and / or an ICI results in reduced levels of inhibitory cytokines.

[0055] In some embodiments, the inhibitory cytokines are selected from IL-10 and TGFβ.

[0056] In some embodiments, administration of a GM-CSF antagonist and / or an ICI results in reduced levels of immunosuppressive factors.

[0057] In some embodiments, the immunosuppressant is selected from arginase 1, inducible nitric oxide synthase (iNOS), peroxynitrite, nitric oxide, reactive oxygen species, tumor-associated macrophages, and combinations thereof.

[0058] In some embodiments, administration of a GM-CSF antagonist and / or an ICI results in increased levels of CD4+ T effector cells compared to a control.

[0059] In some embodiments, the control is a pre-treatment level or percentage in the patient, or a reference level or percentage based on historical data.

[0060] In some embodiments, the present invention provides a pharmaceutical composition for treating cancer comprising a GM-CSF antagonist and an ICI.

[0061] In some embodiments, the GM-CSF antagonist is an anti-GM-CSF antibody or a fragment thereof.

[0062] In some embodiments, the GM-CSF antagonist is a soluble GM-CSF receptor.

[0063] In some embodiments, the GM-CSF antagonist is an anti-GM-CSF receptor antibody or a fragment thereof.

[0064] In some embodiments, the anti-GM-CSF receptor antibody or fragment thereof is an anti-GM-CSFRα antibody or fragment thereof.

[0065] In some embodiments, the anti-GM-CSFRα antibody or fragment thereof is a monoclonal antibody specific for human GM-CSFRα.

[0066] In some embodiments, the anti-GM-CSFRα antibody is a human or humanized IgG4 antibody.

[0067] In some embodiments, the anti-GM-CSFRα antibody is mavrilimumab.

[0068] In some embodiments, the anti-GM-CSFRα antibody fragment thereof comprises a light chain complementarity determining region 1 (LCDR1) defined by SEQ ID NO:6, a light chain complementarity determining region 2 (LCDR2) defined by SEQ ID NO:7, and a light chain complementarity determining region 3 (LCDR3) defined by SEQ ID NO:8, as well as a heavy chain complementarity determining region 1 (HCDR1) defined by SEQ ID NO:3, a heavy chain complementarity determining region 2 (HCDR2) defined by SEQ ID NO:4, and a heavy chain complementarity determining region 3 (HCDR3) defined by SEQ ID NO:5.

[0069] In some embodiments, the ICI antagonizes the activity of PD-1, CTLA-4, B7, BTLA, HVEM, TIM-3, GAL-9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, and combinations thereof.

[0070] In some embodiments, the ICI is selected from an anti-PD-1 antibody (optionally pembrolizumab, nivolumab, cemiplimab), an anti-PD-L1 antibody (optionally atezolizumab, avelumab, durvalumab), an anti-CTLA-4 antibody (optionally ipilimumab), an anti-PD-L2 antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-BTLA antibody, an anti-HVEM antibody, an anti-TIM-3 antibody, an anti-GAL-9 antibody, an anti-LAG3 antibody, an anti-VISTA antibody, an anti-KIR antibody, an anti-2B4 antibody, an anti-CD160 antibody, an anti-CGEN-15049 antibody, an anti-CHK1 antibody, an anti-CHK2 antibody, an anti-A2aR antibody, an anti-B-7 antibody, and combinations thereof.

[0071] In some aspects, the present invention provides kits for treating cancer comprising a pharmaceutical composition comprising a GM-CSF antagonist and a pharmaceutical composition comprising at least one other cancer therapy selected from chemotherapy, MDSC targeted therapy, immunotherapy, radiation therapy, and combinations thereof.

[0072] In some embodiments, the immunotherapy is an ICI selected from an anti-PD-1 antibody (optionally pembrolizumab, nivolumab, cemiplimab), an anti-PD-L1 antibody (optionally atezolizumab, avelumab, durvalumab), an anti-CTLA-4 antibody (optionally ipilimumab), an anti-PD-L2 antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-BTLA antibody, an anti-HVEM antibody, an anti-TIM-3 antibody, an anti-GAL-9 antibody, an anti-LAG3 antibody, an anti-VISTA antibody, an anti-KIR antibody, an anti-2B4 antibody, an anti-CD160 antibody, an anti-CGEN-15049 antibody, an anti-CHK1 antibody, an anti-CHK2 antibody, an anti-A2aR antibody, an anti-B-7 antibody, and combinations thereof.

[0073] definition In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions for the following terms, as well as other terms, are defined throughout the specification. Publications and other reference materials referred to herein to describe the background of the invention and to provide additional details regarding its practice are incorporated herein by reference.

[0074] Antibody: As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that binds (immunoreacts with) an antigen. By "binds" or "immunoreacts" it is meant that the antibody reacts with one or more antigenic determinants of interest. Antibodies include antibody fragments. Antibodies also include, but are not limited to, polyclonal, monoclonal, chimeric dAb (domain antibodies), single chain, Fab, Fab', F(ab')2 fragments, scFv, and Fab expression libraries. Antibodies can be whole antibodies, or immunoglobulins, or antibody fragments.

[0075] Amino acid: As used herein, the term "amino acid" in its broadest sense refers to any compound and / or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid, in some embodiments, an amino acid is a d-amino acid, and in some embodiments, an amino acid is an l-amino acid. "Standard amino acid" refers to any of the 20 standard l-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid" refers to any amino acid other than the standard amino acids, whether prepared synthetically or obtained from a natural source. As used herein, "synthetic amino acid" encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and / or substitutions. Amino acids, including the carboxyl terminal amino acid and / or amino terminal amino acid in a peptide, can be modified by methylation, amidation, acetylation, protecting groups, and / or substitutions with other chemical groups that can alter the circulating half-life of the peptide without adversely affecting their activity. The amino acids may participate in disulfide bonds. The amino acids may include one or post-translational modifications, such as association with one or more chemicals (e.g., methyl groups, acetate groups, acetyl groups, phosphate groups, formyl moieties, isoprenoid groups, sulfate groups, polyethylene glycol moieties, lipid moieties, carbohydrate moieties, biotin moieties, etc.). The term "amino acid" is used interchangeably with "amino acid residue" and may refer to free amino acids and / or amino acid residues of peptides. Whether the term refers to a free amino acid or a residue of a peptide will be clear from the context in which the term is used.

[0076] Enhancement: As used herein, the term "enhancement" refers to the prevention, reduction, or alleviation of a condition, or the improvement of a subject's condition. Enhancement includes, but does not require, a complete amelioration or complete prevention of a disease condition. In some embodiments, enhancement includes an increased level of an associated protein or its activity that is lacking in the associated disease tissue.

[0077] Approximately or about: As used herein, the term "approximately" or "about" as applied to one or more values ​​of interest refers to a value that is similar to a specified reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater or less) of the specified reference value, unless otherwise specified or otherwise clear from the context (except where such number exceeds 100% of possible values).

[0078] Delivery: As used herein, the term "delivery" encompasses both local and systemic delivery.

[0079] Improve, increase, inhibit or reduce: As used herein, the terms "improve," "increase," "inhibit," or "reduce," or grammatical equivalents thereof, refer to a value relative to a baseline measurement, such as a measurement in the same individual prior to the initiation of a treatment described herein, or a measurement in a control subject (or control subjects) not receiving a treatment described herein. A "control subject" is a subject suffering from the same form of disease as the subject being treated and who is approximately the same age as the subject being treated.

[0080] "Inhibit" or "Inhibiting": As used herein, "inhibit" or "inhibiting", or grammatical equivalents, means a reduction, decrease or inhibition of a biological activity. Neutralization: As used herein, neutralization refers to a reduction or decrease in the biological activity of the protein to which the neutralizing antibody binds, in this case GM-CSFRα. Inhibition means, for example, a reduction or inhibition of GM-CSFRα signaling, as measured by GM-CSFRα binding or, for example, GM-CSFRα-mediated responses. The reduction or inhibition of biological activity can be partial or total. The degree to which an antibody neutralizes GM-CSFRα is referred to as its neutralizing potency.

[0081] Patient: As used herein, the term "patient" refers to any organism to which a provided composition may be administered, for example, for experimental, diagnostic, preventative, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals, such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is a human. Human includes prenatal and postnatal forms.

[0082] Pharmaceutically acceptable: As used herein, the term "pharmacologically acceptable" refers to a material that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0083] Substantial identity: In this specification, the phrase "substantial identity" is used to refer to the comparison between amino acid sequences or nucleic acid sequences.As will be understood by those skilled in the art, two sequences are generally considered to be "substantially identical" if they contain the same residues at corresponding positions.As is well known in the art, amino acid sequences or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercially available computer programs, such as BLAS TN for nucleotide sequences, and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Examples of such programs are described in Altschul, et al., Basic local alignment search tool, J Mal. Biol., 215(3):403-410, 1990, Altschul, et al., Methods in Enzymology, Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997, Baxevanis et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998, and Misener, et al., (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying identical sequences, the above-mentioned programs typically provide a numerical value of the degree of identity. In some embodiments, sequences are considered to be substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the corresponding residues are identical over the relevant stretch of residues, in some embodiments the relevant stretch is the complete sequence.In some embodiments the relevant sequences are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues.

[0084] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Human includes prenatal and postnatal forms. In many embodiments, the subject is a human. A subject may be a patient, which refers to a human presenting to a health care provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient." A subject may be afflicted with or susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[0085] Substantially: As used herein, the term "substantially" refers to the qualitative state of exhibiting all or nearly all extent or degree of a desired characteristic or property. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, reach completion and / or progress to perfection, achieve perfect results, or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of perfection inherent in many biological and chemical phenomena.

[0086] Systemic distribution or delivery: As used herein, the terms "systemic distribution", "systemic delivery", or grammatical equivalents refer to a delivery or distribution mechanism or approach that affects the entire body or organism. Typically, systemic distribution or delivery is achieved via the body's circulatory system, e.g., the bloodstream. Compare with the definition of "local distribution or delivery".

[0087] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount that, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, is sufficient to treat, diagnose, prevent, and / or delay the onset of symptoms of the disease, disorder, and / or condition. It will be understood by those skilled in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[0088] Treatment: As used herein, the terms "treat", "treatment" or "treating" refer to any method used to partially or completely alleviate, improve, alleviate, inhibit, prevent, delay onset, reduce severity, and / or reduce incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. Treatment may be administered to subjects who do not exhibit signs of the disease and / or who exhibit only early signs of the disease for the purpose of reducing the risk of developing pathologies associated with the disease. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0089] The present invention provides, inter alia, a method for treating cancer by inhibiting the immunosuppressive activity of myeloid-derived suppressor cells (MDSCs) in patients in need of treatment using a GM-CSF antagonist. In some embodiments, the GM-CSF antagonist is used in combination with an immune checkpoint inhibitor. It is contemplated that the present invention is particularly effective in treating immune checkpoint inhibitor (ICI) refractory or resistant cancers, or late-stage or metastatic cancers.

[0090] Various aspects of the invention are described in detail in the following sections. The use of the sections is not meant to limit the invention. Each section may be applicable to any aspect of the invention. In this application, the use of "or" means "and / or" unless otherwise specified.

[0091] Myeloid-derived suppressor cells (MDSCs) MDSCs are a heterogeneous population of immune cells from the myeloid lineage. They proliferate aggressively in pathological conditions such as chronic infection and cancer, and are distinct from other myeloid cell types in that they possess potent immunosuppressive rather than immunostimulatory properties. + HLA-DR lo / neg Monocytes with reduced or no HLA-DR expression, called monocytes, can group into MDSCs and alter adaptive immunity to produce immunosuppression.

[0092] MDSCs accumulate in peripheral blood, lymphoid organs, spleen, and tumor sites in cancer, infection, chronic inflammation, transplantation, and autoimmunity. Although the specific pathways by which tumors recruit, expand, and activate MDSCs remain unknown, there is growing evidence for the involvement of interleukin (IL-1β), IL-6, cyclooxygenase 2 (COX2)-generated PGE2, high concentrations of GM-CSF, M-CSF, vascular endothelial growth factor (VEGF), IL-10, transforming growth beta (TGFβ), indoleamine 2,3-dioxygenase (IDO), FLT3 ligand, and stem cell factor.

[0093] Co-culture of immune competent cells with tumor cell lines has been shown to induce tolerogenic DCs or MDSCs. Previous studies also suggest that tumor cells release GM-CSF, which induces granulocyte ROS production to inhibit T cell function. Additionally, expression of programmed death ligand 1 (PD-L1) is increased on the surface of MDSCs in mouse tumor models, but the role of this in MDSC-mediated suppression remains unclear.

[0094] Programmed Death-Ligand 1 (PD-L1) Programmed death-ligand 1 (PD-L1, also known as CD274) is an immune checkpoint protein that binds to its receptor PD-1. PD-L1 is widely expressed on various cell types, mainly on tumor cells, MDSCs, monocytes, macrophages, natural killer (NK) cells, dendritic cells (DCs), and activated T cells, as well as in immune-privileged sites such as the brain, cornea, and retina. Under normal physiological conditions, activation of the PD-1 / PD-L1 signaling pathway is closely related to inducing and maintaining peripheral tolerance, maintaining T cell immune homeostasis, avoiding overactivation, and protecting against immune-mediated tissue damage. In pathological conditions, PD-L1 interacts with its receptor programmed death 1 (PD-1) and transmits negative signals to control a series of processes of T cell-mediated cellular immune responses, including priming, growth, proliferation, and apoptosis, as well as functional maturation, resulting in immune escape.

[0095] Immune checkpoint inhibitors (ICIs) have changed the therapeutic landscape for many tumors, inducing durable responses in some cases and improving tumor mutation load, CD8 + T cell density and PD-L1 expression have each been proposed as separate biomarkers of response to PD-1 / -L1 antagonists. One of the main challenges for immune checkpoint blocking antibodies is in malignant tumors with limited T cell response or immunologically "cold" tumors. These cold tumors contain few infiltrating T cells, are not recognized, and do not induce a strong response by the immune system, making them difficult to treat with current immunotherapy. The inventors surprisingly found that GM-CSF upregulates PD-L1 and contributes to immunosuppressive activity. Converting "cold" tumors to "hot" tumors is one of the milestones in cancer treatment.

[0096] GM-CSF antagonists GM-CSF signaling GM-CSF is a type I proinflammatory cytokine that enhances the survival and proliferation of a wide range of hematopoietic cell types. It is a growth factor that was first identified as an inducer of differentiation and proliferation of myeloid cells (e.g., neutrophils, basophils, eosinophils, monocytes, and macrophages) (Wicks IP and Roberts AW. Nat Rev Rheumatol. 2016, 12(1):37-48). Studies using different approaches have demonstrated that with GM-CSF overexpression, pathological changes almost always follow (Hamilton JA et al., Growth Factors. 2004, 22(4):225-31). GM-CSF enhances the trafficking of myeloid cells through the activated endothelium of blood vessels and may also contribute to the accumulation of monocytes and macrophages in blood vessels during inflammation. GM-CSF also promotes the activation, differentiation, survival, and proliferation of monocytes and macrophages as well as resident tissue macrophages in inflamed tissues. It regulates the phenotype of antigen-presenting cells in inflamed tissues by promoting the differentiation of infiltrating monocytes into MM1 macrophages and monocyte-derived dendritic cells (MoDCs). Furthermore, the production of IL-23 by macrophages and MoDCs, in combination with other cytokines such as IL-6 and IL-1, regulates T cell differentiation.

[0097] Together with M-CSF (macrophage-colony stimulating factor), GM-CSF regulates the number and function of macrophages. Macrophages activated by GM-CSF acquire a series of effector functions, all of which identify them as inflammatory macrophages. GM-CSF-activated macrophages produce proinflammatory cytokines, including TNF, IL-1β, IL-6, IL-23 and IL-12, as well as chemokines such as CCL5, CCL22, and CCL24, and recruit T cells and other inflammatory cells into the tissue microenvironment.

[0098] The GM-CSF receptor is a member of the haematopoietin receptor superfamily. It is a heterodimer consisting of an alpha and a beta subunit. The alpha subunit is highly specific for GM-CSF, whereas the beta subunit is shared with other cytokine receptors, including IL-3 and IL-5. This is reflected in the broader tissue distribution of the beta receptor subunit. The alpha subunit, GM-CSFRα, is expressed primarily on myeloid and non-hematopoietic cells, such as neutrophils, macrophages, eosinophils, dendritic cells, endothelial cells and respiratory epithelial cells. The full-length GM-CSFRα is a 400 amino acid type I membrane glycoprotein belonging to the type I cytokine receptor family, consisting of a 22 amino acid signal peptide (positions 1-22), a 298 amino acid extracellular domain (positions 23-320), a transmembrane domain from positions 321-345 and a short 55 amino acid intracellular domain. The signal peptide is cleaved to provide the mature form of GM-CSFRα as a 378 amino acid protein. Complementary DNA (cDNA) clones of human and mouse GM-CSFRα are available, and at the protein level, the receptor subunits share 36% identity. GM-CSF can bind with relatively low affinity (Kd 1-5 nM) to the α subunit alone, but cannot bind at all to the β subunit alone. However, the presence of both α and β subunits results in a high affinity ligand-receptor complex (Kd ≈100 pM). GM-CSF signaling occurs through its initial binding to the GM-CSFRα chain, then crosslinking with the larger subunit common β chain, generating a high affinity interaction that phosphorylates the JAK-STAT pathway. This interaction can also signal through tyrosine phosphorylation and activation of the MAP kinase pathway.

[0099] Pathologically, GM-CSF has been shown to play a role in exacerbating inflammatory, respiratory, and autoimmune diseases. Thus, neutralization of GM-CSF binding to GM-CSFRα is a therapeutic approach to treat diseases and conditions mediated through GM-CSFR. Thus, the present invention relates to binding members that bind to human GM-CSF or GM-CSFRα, or inhibit the binding of human GM-CSF to GM-CSFRα, and / or inhibit signaling resulting from GM-CSF ligand binding to the receptor. Upon ligand binding, GM-CSFR induces stimulation of multiple downstream signaling pathways, including the JAK2 / STAT5, MAPK pathway, and PI3K pathway, all associated with activation and differentiation of myeloid cells. The binding members can be reversible inhibitors of GM-CSF signaling through GM-CSFR.

[0100] GM-CSF antagonists GM-CSF antagonists suitable for the present invention include those therapeutic agents capable of reducing, inhibiting or neutralizing one or more GM-CSF-mediated signaling, including those described herein. For example, suitable GM-CSF antagonists according to the present invention include, but are not limited to, anti-GM-CSF antibodies and fragments thereof, soluble GM-CSF receptor and variants thereof, including fusion proteins such as GM-CSF soluble receptor-Fc fusion proteins, anti-GM-CSF receptor antibodies or fragments thereof, to name a few.

[0101] In some embodiments, a suitable GM-CSF antagonist is an anti-GM-CSFRα antibody. Exemplary anti-GM-CSFRα monoclonal antibodies are described in International Application No. PCT / GB2007 / 001108, filed March 27, 2007, published as WO2007 / 110631, EP Application No. 120770487, filed October 10, 2010, U.S. Application No. 11 / 692,008, filed March 27, 2007, U.S. Application No. 12 / 294,616, filed September 25, 2008, U.S. Application No. 13 / 941,409, filed July 12, 2013, U.S. Application No. 13 / 291,363, filed November 30, 2010, and U.S. Application No. 13 / 10,409, filed November 30, 2010. No. 14 / 753,792, filed on 2007-02-29, International Application No. PCT / EP2012 / 070074, filed on 10 October 2012 and published as WO / 2013 / 053767, International Application No. PCT / EP2015 / 060902, filed on 18 May 2015 and published as WO2015 / 177097, and International Application No. PCT / EP2017 / 062479, filed on 23 May 2017, each of which is incorporated herein by reference in its entirety. In one embodiment, the anti-GM-CSFRα monoclonal antibody is mavrilimumab. WO2007 / 110631 reports the isolation and characterization of the anti-GM-CSFRα antibody mavrilimumab and variants thereof that share the ability to neutralize the biological activity of GM-CSFRα with high potency. The functional properties of these antibodies are believed to be at least in part due to binding the Tyr-Leu-Asp-Phe-Gln motif at positions 226-230 of human GM-CSFRα, thereby inhibiting the association between GM-CSFRα and its ligand GM-CSF. Mavrilimumab is a human IgG4 monoclonal antibody designed to regulate macrophage activation, differentiation, and survival by targeting GM-CSFRα. It has been shown to be a potent neutralizer of the biological activity of GM-CSFRα and exerts its therapeutic effect by binding to GM-CSFRα on leukocytes within the synovial joints of RA patients, resulting in reduced cell survival and activation.The safety profile of the GM-CSFRα antibody mavrilimumab for in vivo use to date has been established in a Phase II trial for rheumatoid arthritis (RA).

[0102] In a particular embodiment, the antibody is composed of two light chains and two heavy chains. The heavy chain variable domain (VH) comprises the amino acid sequence specified in SEQ ID NO: 1. The light chain variable domain (VL) comprises the amino acid sequence specified in SEQ ID NO: 2. The heavy and light chains each comprise the complementarity determining regions (CDRs) and framework regions in the following arrangement: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0103] The mavrilimumab antibody heavy chain comprises the CDRs: HCDR1, HCDR2, HCDR3 as specified by the amino acid sequences of SEQ ID NOs: 3, 4 and 5, respectively. The light chain comprises the CDRs: LCDR1, LCDR2, LCDR3 as specified by the amino acid sequences of SEQ ID NOs: 6, 7 and 8, respectively.

[0104] Anti-GM-CSFRα heavy chain variable domain amino acid sequence QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSIHWVRQAPGKGLEWMGGFDPEENEIVYAQRFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCAIVGSFSPLTLGLWGQGTMVTVSS (SEQ ID NO: 1)

[0105] Anti-GM-CSFRα light chain variable domain amino acid sequence QSVLTQPPSVSGAPGQRVTISCTGSGSNIGAPYDVSWYQQLPGTAPKLLIYHNNKRPSGVPDRFSGSSKSGTSASLAITGLQAEDEADYYCATVEAGLSGSVFGGGTKLTVL (SEQ ID NO: 2)

[0106] Anti-GM-CSFRa heavy chain variable domain CDR1 (HCDR1) amino acid sequence ELSIH (SEQ ID NO: 3)

[0107] Anti-GM-CSFRα heavy chain variable domain CDR2 (HCDR2) amino acid sequence GFDPEENEIVYAQRFQG (SEQ ID NO: 4)

[0108] Anti-GM-CSFRα heavy chain variable domain CDR3 (HCDR3) amino acid sequence VGSFSPLTLGL (SEQ ID NO:5)

[0109] Anti-GM-CSFRα light chain variable domain CDR1 (LCDR1) amino acid sequence TGSGSNIGAPYDVS (SEQ ID NO: 6)

[0110] Anti-GM-CSFRα light chain variable domain CDR2 (LCDR2) amino acid sequence HNNKRPS (SEQ ID NO: 7)

[0111] Anti-GM-CSFRa light chain variable domain CDR3 (LCDR3) amino acid sequence ATVEAGLSGSV (SEQ ID NO:8)

[0112] In some embodiments, the anti-GM-CSFRα antibody for cancer treatment is a variant of mavrilimumab selected from the GM-CSFα binding members disclosed in applications WO2007 / 11063 and WO2013 / 053767, the entireties of which are incorporated by reference in their entireties.

[0113] In some embodiments, an anti-GM-CSFRα antibody for cancer treatment comprises a CDR amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to one or more of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.

[0114] In some embodiments, an anti-GM-CSFRα antibody comprises a light chain variable domain having an amino acid sequence at least 90% identical to SEQ ID NO:2, and a heavy chain variable domain having an amino acid sequence at least 90% identical to SEQ ID NO:1. In some embodiments of the invention, an anti-GM-CSFRα antibody has a light chain variable domain amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:2, and a heavy chain variable domain amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:1. In some embodiments of the invention, the anti-GM-CSFRα antibody comprises a light chain variable domain having the amino acid sequence set forth in SEQ ID NO:2 and a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO:1. In some embodiments of the invention, the heavy chain constant region of the anti-GM-CSFRα antibody comprises a CH1, hinge and CH2 domain from an IgG4 antibody fused to a CH3 domain from an IgG1 antibody. In some embodiments of the invention, the heavy chain constant region of the anti-GM-CSFRα antibody is or is derived from an IgG1, IgG2 or IgG4 heavy chain constant region. In some embodiments of the invention, the light chain constant region of the anti-GM-CSFRα antibody is or is derived from a lambda or kappa light chain constant region.

[0115] In some embodiments, the anti-GM-CSFRα antibody is a fragment of the mavrilimumab antibody. In some embodiments, the inhibitor comprises a single chain variable fragment (ScFv) comprising at least one of the CDR sequences of SEQ ID NO: 3, 4, 5, 6, 7, or 8. In some embodiments, the inhibitor is a fusion molecule comprising at least one of the CDR sequences of SEQ ID NO: 3, 4, 5, 6, 7, or 8. In some embodiments, the anti-GM-CSFRα inhibitor sequence is a bispecific antibody comprising at least one of the CDR sequences of SEQ ID NO: 3, 4, 5, 6, 7, or 8.

[0116] In other embodiments, the suitable GM-CSF antagonist is an anti-GM-CSF antibody. Exemplary anti-GM-CSF monoclonal antibodies include those described in International Application No. PCT / EP2006 / 004696, filed May 17, 2006, published as WO2006 / 122797, International Application No. PCT / EP2016 / 076225, filed October 31, 2016, published as WO2017 / 076804, and International Application No. PCT / US2018 / 053933, filed October 2, 2018, published as WO / 2019 / 070680, each of which is incorporated herein by reference in its entirety. In one embodiment, the anti-GM-CSF monoclonal antibody is otilimab.

[0117] The anti-GM-CSFRα or anti-GM-CSF antibody of the present disclosure may be multispecific, e.g., bispecific. The antibody may be mammalian (e.g., human or murine), humanized, chimeric, recombinant, synthetically produced, or naturally isolated. Exemplary antibodies of the present disclosure include, without limitation, IgG (e.g., IgG1, IgG2, IgG3, and IgG4), IgM, IgA (e.g., IgA1, IgA2, and IgAsec), IgD, IgE, Fab, Fab'2, F(ab')2, Fd, Fv, Feb, scFv, scFv-Fc, and SMIP binding moieties. In certain embodiments, the antibody is an scFv. The scFv may include a flexible linker that allows the scFv to be oriented in different directions, for example, to allow antigen binding. In various embodiments, the antibody may be a cytosol-stable scFv or intrabody that retains its structure and function in the reducing environment inside the cell (see, e.g., Fisher and DeLisa, J. Mol. Biol. 385(1):299-311, 2009, incorporated herein by reference). In certain embodiments, the scFv is converted to an IgG or chimeric antigen receptor according to methods known in the art. In embodiments, the antibody binds to both denatured and native protein targets. In embodiments, the antibody binds to both denatured and native proteins.

[0118] In most mammals, including humans, whole antibodies have at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3), and a hinge region between CH1 and CH2. Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

[0119] Antibody includes all known forms of antibody and other protein scaffolds with antibody-like properties.For example, anti-GM-CSFRα antibody can be monoclonal antibody, polyclonal antibody, human antibody, humanized antibody, bispecific antibody, monovalent antibody, chimeric antibody, or protein scaffolds with antibody-like properties such as fibronectin or ankyrin repeat.Antibody can have any of the following isotypes: IgG (e.g., IgG1, IgG2, IgG3, and IgG4), IgM, IgA (e.g., IgA1, IgA2, and IgAsec), IgD, or IgE.

[0120] An antibody fragment may comprise one or more segments derived from an antibody. Segments derived from an antibody may retain the ability to specifically bind to a particular antigen. An antibody fragment may be, for example, a Fab, Fab', Fab'2, F(ab')2, Fd, Fv, Feb, scFv, or SMIP. An antibody fragment may be, for example, a diabody, a triabody, an affibody, a nanobody, an aptamer, a domain antibody, a linear antibody, a single chain antibody, or any of the various multispecific antibodies that can be formed from antibody fragments.

[0121] Examples of antibody fragments include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment, a fragment consisting of the VH and CH1 domains; (iv) an Fv fragment, a fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment, a fragment comprising the VH and VL domains; (vi) a dAb fragment, a fragment which is a VH domain; (vii) a dAb fragment, a fragment which is a VL domain; (viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more isolated CDRs, which may optionally be linked by one or more synthetic linkers. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked using recombinant methods, for example, by a synthetic linker that allows the VL and VH regions to be expressed as a single protein that pairs to form a monovalent binding moiety (known as single-chain Fv (scFv)). Antibody fragments can be obtained using conventional techniques known to those skilled in the art, and in some instances can be used in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. Antibody fragments can further include any of the antibody fragments described above, with the addition of additional C-terminal amino acids, N-terminal amino acids, or amino acids that separate the individual fragments.

[0122] When an antibody contains one or more antigenic determining regions or constant regions from a first species and one or more antigenic determining regions or constant regions from a second species, it can be called chimeric. Chimeric antibodies can be constructed, for example, by genetic engineering. Chimeric antibodies can contain immunoglobulin gene segments from different species (e.g., from mouse and human).

[0123] The antibody can be a human antibody. Human antibody refers to a binding moiety having variable regions in which both framework and CDR regions are derived from human immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also derived from human immunoglobulin sequences. Human antibodies can, for example, include one or more sequence variations, such as mutations, that are not specified amino acid residues in human immunoglobulin sequences. The variations or additional amino acids can, for example, be introduced by human engineering. The human antibodies of the present disclosure are not chimeric.

[0124] An antibody may be humanized, meaning that an antibody that contains one or more antigen-determining regions (e.g., at least one CDR) substantially derived from a non-human immunoglobulin or antibody is engineered to contain at least one immunoglobulin domain substantially derived from a human immunoglobulin or antibody. An antibody may be humanized using the conversion methods described herein, for example, by inserting an antigen recognition sequence from a non-human antibody encoded by a first vector into a human framework encoded by a second vector. For example, a first vector may contain a polynucleotide encoding a non-human antibody (or a fragment thereof) and a site-specific recombination motif, while a second vector may contain a polynucleotide encoding a human framework and a site-specific recombination motif complementary to the site-specific recombination motif on the first vector. The site-specific recombination motif may be positioned on each vector such that a recombination event results in the insertion of one or more antigen-determining regions from the non-human antibody into the human framework, thereby forming a polynucleotide encoding a humanized antibody.

[0125] In certain embodiments, the antibody is converted from scFv to IgG (e.g., IgG1, IgG2, IgG3, and IgG4). There are various methods in the art for converting scFv fragments to IgG. One such method for converting scFv fragments to IgG is disclosed in US Patent Application Publication No. 2016 / 0362476, the contents of which are incorporated herein by reference.

[0126] Combination therapy Immune checkpoint inhibitors (ICIs) In some embodiments, the methods of treating cancer according to the present invention comprise administering to a subject in need thereof a GM-CSF antagonist in combination with an ICI.

[0127] In some embodiments, the ICI is a biotherapeutic agent or a small molecule. In some embodiments, the ICI is a monoclonal antibody, a humanized antibody, a fully human antibody, a fusion protein, or a combination thereof.

[0128] In some embodiments, the ICI inhibits a checkpoint protein, which may be CTLA-4, PD-L1, PD-L2, PD-1, B7-H3, B7-H4, BTLA, HVEM, TIM-3, GAL-9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, a B-7 family ligand, or a combination thereof. In some embodiments, the ICI interacts with a ligand of a checkpoint protein, which may be CTLA-4, PD-L1, PD-L2, PD-1, B7-H3, B7-H4, BTLA, HVEM, TIM-3, GAL-9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, a B-7 family ligand, or a combination thereof.

[0129] In some embodiments, the ICI is an anti-CTLA-4 antibody. In some embodiments, the ICI is an anti-PD-L1 antibody. In some embodiments, the ICI is an anti-PD-L2 antibody. In some embodiments, the ICI is an anti-PD-1 antibody. In some embodiments, the ICI is an anti-B7-H3 antibody. In some embodiments, the ICI is an anti-B7-H4 antibody. In some embodiments, the ICI is an anti-BTLA antibody. In some embodiments, the ICI is an anti-HVEM antibody. In some embodiments, the ICI is an anti-TIM-3 antibody. In some embodiments, the ICI is an anti-GAL-9 antibody. In some embodiments, the ICI is an anti-LAG-3 antibody. In some embodiments, the ICI is an anti-VISTA antibody. In some embodiments, the ICI is an anti-KIR antibody. In some embodiments, the ICI is an anti-2B4 antibody. In some embodiments, the ICI is an anti-CD160 antibody. In some embodiments, the ICI is an anti-CGEN-15049 antibody. In some embodiments, the ICI is an anti-CHK1 antibody. In some embodiments, the ICI is an anti-CHK2 antibody. In some embodiments, the ICI is an anti-A2aR antibody. In some embodiments, the checkpoint inhibitor is an anti-B-7 antibody.

[0130] In some embodiments, the PD-1 antibody is pembrolizumab. In some embodiments, the PD-1 antibody is nivolumab. In some embodiments, the PD-1 antibody is cemiplimab. In some embodiments, the PD-L1 antibody is atezolizumab. In some embodiments, the PD-L1 antibody is avelumab. In some embodiments, the PD-L1 antibody is durvalumab. In some embodiments, the CTLA-4 antibody is ipilimumab.

[0131] Additional Therapeutic Agents In some embodiments, the method of treating cancer according to the present invention comprises administering to a subject in need thereof a GM-CSF antagonist in combination with an additional therapeutic agent. In certain embodiments, the additional agent is a cancer therapy comprising chemotherapy and / or radiation therapy. In certain embodiments, the additional therapeutic agent comprises a recombinant protein or a monoclonal antibody. In certain embodiments, the recombinant protein or monoclonal antibody comprises Etaracizumab (Abegrin), Tacatuzumab tetraxetan, Bevacizumab (Avastin), Labetuzumab, Cetuximab (Erbitux), Obinutuzumab (Gazyva), Trastuzumab (Herceptin), Clivatuzumab, Trastuzumab Emtansine (Kadcyla), Ramucirumab, Rituximab (MabThera, Rituxan), Gemtuzumab Ozogamicin (Mylotarg), Pertuzumab (Omnitarg), Girentuximab (Rencarex), or Nimotuzumab (Theracim, Theraloc).

[0132] In certain embodiments, the GM-CSF antagonist comprises an immunomodulatory agent that targets a checkpoint inhibitor as described in the checkpoint inhibitor section above. In certain embodiments, the immunomodulatory agent comprises nivolumab, ipilimumab, atezolizumab, or pembrolizumab. In certain embodiments, the additional therapeutic agent is a chemotherapeutic agent. In certain embodiments, the chemotherapeutic agent is an alkylating agent (e.g., cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan, mechlorethamine, uramustine, thiotepa, nitrosourea, or temozolomide), an anthracycline (e.g., doxorubicin, adriamycin, daunorubicin, epirubicin, or mitoxantrone), a cytoskeletal disruptor (e.g., paclitaxel or docetaxel), a histone deacetylase inhibitor (e.g., vorinostat or romidepsin), an inhibitor of topoisomerase (e.g., irinotecan, topotecan, amsacrine, etoposide, or teniposide), a kinase inhibitor (e.g., bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, or vismodegib), a nucleoside analog or precursor analog (e.g., azacitidine, azathioprine, capecitabine, cytarabine, fluorouracil, gemcitabine, hydroxyurea, mercaptopurine, methotrexate, or thioguanine), a peptide antibiotic (e.g., actinomycin or bleomycin), a platinum-based drug (e.g., cisplatin, oxaloplatin, or carboplatin), or a plant alkaloid (e.g., vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin, paclitaxel, or docetaxel). In some embodiments, the chemotherapeutic agent is a nucleoside analog. In some embodiments, the chemotherapeutic agent is gemcitabine. In certain embodiments, the additional therapeutic agent is radiation therapy.

[0133] Cancer Treatment The present invention may be used to treat a variety of cancers, particularly those that are refractory or resistant to immune checkpoint inhibition (ICI), or late stage or metastatic cancers.

[0134] In some embodiments, the cancer is any solid or liquid tumor, including genitourinary cancer (such as prostate cancer, renal cell carcinoma, bladder cancer), gynecological cancer (such as ovarian cancer, cervical cancer, endometrial cancer), lung cancer, gastrointestinal cancer (such as non-metastatic or metastatic colorectal cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma, cholangiocarcinoma, etc.), head and neck cancer (e.g., head and neck squamous cell carcinoma), brain cancer, including malignant glioma and brain metastases, malignant mesothelioma, non-metastatic or metastatic breast cancer (e.g., hormone-refractory metastatic breast cancer), malignant melanoma, Merkel cell carcinoma or bone and soft tissue sarcoma, and hematological neoplasms, such as multiple myeloma, acute myeloid leukemia, chronic myelogenous leukemia, myelodysplastic syndromes, and acute lymphoblastic leukemia. In some embodiments, the disease is non-small cell lung cancer (NSCLC), breast cancer (e.g., stage IV breast cancer, hormone-refractory metastatic breast cancer), head and neck cancer (e.g., head and neck squamous cell carcinoma), metastatic colorectal cancer, hormone-sensitive or hormone-refractory prostate cancer, colon cancer (e.g., stage IV colorectal cancer), ovarian cancer, hepatocellular carcinoma, renal cell carcinoma, soft tissue sarcoma, or small cell lung cancer.

[0135] As used herein, the term "cancer" refers to a broad class of disorders characterized by hyperproliferative cell growth, either in vitro (e.g., transformed cells) or in vivo. Conditions that may be treated or prevented by the compositions and methods of the invention include, for example, various neoplasms, including benign or malignant tumors, various hyperplasias, and the like. The compounds and methods of the invention can achieve inhibition and / or reversal of the unwanted hyperproliferative cell growth involved in such conditions.

[0136] Examples of cancer are: acute lymphoblastic leukemia, adult;acute lymphoblastic leukemia, pediatric;acute myeloid leukemia, adult;adrenal cortical carcinoma;adrenal cortical carcinoma, pediatric;AIDS-related lymphoma;AIDS-related malignancies;anal cancer;astrocytoma, pediatric cerebellar;astrocytoma, pediatric cerebral;bile duct cancer, extrahepatic;bladder cancer;bladder cancer, pediatric;bone cancer, osteosarcoma / malignant fibrous histiocytoma;brain stem glioma, pediatric;brain tumor, adult;brain tumor, brain stem glioma, small children;brain tumor, cerebellar astrocytoma, children;brain tumor, cerebral astrocytoma / malignant glioma, children;brain tumor, ependymoma, children;brain tumor, medulloblastoma, children;brain tumor, supratentorial primitive neuroectodermal tumor, children;brain tumor, visual pathway and hypothalamic glioma, children;brain tumor, children (other);breast cancer;breast cancer and pregnancy;breast cancer, children;breast cancer, male;bronchial adenoma / carcinoid, children:carcinoid tumor, children;carcinoid tumor, digestive gastrointestinal;carcinoma, adrenal cortex;carcinoma, islet cell;carcinoma of unknown primary;central nervous system lymphoma, primary;cerebellar astrocytoma, pediatric;cerebral astrocytoma / malignant glioma, pediatric;cervical cancer;childhood cancer;chronic lymphocytic leukemia;chronic myelogenous leukemia;chronic myeloproliferative disorder;clear cell sarcoma of tendon sheath;colon cancer;colorectal cancer, pediatric;cutaneous T-cell lymphoma;endometrial cancer;ependymoma, pediatric;epithelial carcinoma, ovarian;esophageal cancer;esophageal cancer, pediatric;yu Includes: -ing's family of tumors;Extracranial Germ Cell Tumors, Childhood;Extragonadal Germ Cell Tumors;Extrahepatic Bile Duct Cancer;Eye Cancer, Intraocular Melanoma;Eye Cancer, Retinoblastoma;Gallbladder Cancer;Gastric (Stomach) Cancer;Gastric (Stomach) Cancer, Childhood;Gastrointestinal Carcinoid Tumors;Germ Cell Tumors, Extracranial, Childhood;Germ Cell Tumors, Extragonadal;Germ Cell Tumors, Ovarian;Gestational Trophoblastic Tumors;Glioma.Childhood Brain Stem;Glioma. Visual pathway and hypothalamus in children;Hairy cell leukemia;Head and neck cancer;Hepatocellular (liver) cancer, adult (primary);Hepatocellular (liver) cancer, childhood (primary);Hodgkin lymphoma, adult;Hodgkin lymphoma, childhood;Hodgkin lymphoma in pregnancy;Hypopharyngeal cancer;Hypothalamus and visual pathway glioma, children;Intraocular melanoma;Islet cell carcinoma (endocrine pancreas);Kaposi's sarcoma;Kidney cancer;Laryngeal cancer;Laryngeal cancer, children;Leukemia, acute lymphoblastic, adult;Leukemia, acute lymphoblastic, childhood;Leukemia, acute myeloid, adult;Leukemia, acute myeloid, childhood;Leukemia, chronic lymphocytic;Leukemia, chronic myeloid;Leukemia, hairy cell;Cancer of the lip and oral cavity;Liver cancer, adult (primary);Liver cancer, childhood (primary);Lung cancer, non-small cell;Lung cancer, small cell;Lymphoblastic leukemia, adult acute;Lymphoblastic leukemia, childhood acute;Lymphocytic leukemia, chronic;Lymphoma, AIDS-related;Lymphoma, central nervous system (primary);Lymphoma, cutaneous T-cell;Lymphoma, Hodgkin, adult;Lymphoma, Hodgkin;Childhood;Lymphoma, Hodgkin during pregnancy;Lymphoma, non-Hodgkin, adult;Lymphoma, non-Hodgkin, childhood;Lymphoma, non-Hodgkin during pregnancy;Lymphoma, primary central nervous system;Macroglobulinemia, Waldenstrom;Male breast cancer;Malignant mesothelioma, Adult;Malignant mesothelioma, childhood;Malignant thymoma;Medulloblastoma, childhood;Melanoma;Melanoma, intraocular;Merkel cell carcinoma;Mesothelioma, malignant;Occult primary metastatic squamous cell neck cancer;Multiple endocrine neoplasia syndrome, childhood;Multiple myeloma / plasma cell neoplasm;Mycosis fungoides;Myelodysplastic syndrome;Myeloid leukemia, chronic;Myeloid leukemia, childhood acute;Myeloma, multiple;Myeloproliferative disorder, chronic;Nasal cavity and paranasal sinus cancer;Nasopharyngeal carcinoma;Nasopharyngeal carcinoma, childhood;Neuroblastoma;Neurofibroma;Non-Hodgkin's lymphoma, adult;Non-Hodgkin's lymphoma, childhood;Non-Hodgkin's lymphoma in pregnancy;Non-small cell lung cancer;Oral cancer, childhood;Oral Cavity) and lip cancer;oropharyngeal cancer;osteosarcoma / malignant fibrous histiocytoma of bone;ovarian cancer, children;ovarian epithelial cancer;ovarian germ cell tumors;ovarian low malignant potential tumors;pancreatic cancer;pancreatic cancer, children, pancreatic cancer, islet cell;sinoasal and nasal cancer;parathyroid cancer;penile cancer;pheochromocytoma, pineal and supratentorial primitive neuroectodermal tumors, children;pituitary tumors;plasma cell neoplasms / multiple myeloma;pleuropulmonary blastoma;pregnancy and breast cancer;pregnancy and Hodgkin's lymphoma;pregnancy and non-Hodgkin's lymphoma;primary central nervous system lymphoma;primary liver cancer, adults;primary liver Cancer, children;Prostate cancer;Rectal cancer;Renal cell (kidney) cancer;Renal cell carcinoma, children;Renal pelvis and ureter, transitional cell carcinoma;Retinoblastoma;Rhabdomyosarcoma, children;Salivary gland cancer;Salivary gland cancer, children;Sarcoma, Ewing's tumor family;Sarcoma, Kaposi's sarcoma (osteosarcoma) / malignant fibrous histiocytoma of bone;Sarcoma, rhabdomyosarcoma, children;Sarcoma, soft tissue, adults;Sarcoma, soft tissue, children;Sezary syndrome;Skin cancer;Skin cancer, children;Skin cancer (melanoma);Skin cancer, Merkel cell;Small cell lung cancer;Small intestine cancer;Soft tissue sarcoma, adults;Soft tissue sarcoma, children;Occult primary squamous cell neck cancer, metastatic;Stomach (Gastric) cancer;Stomach (Gastric) cancer, childhood;Supratentorial primitive neuroectodermal tumor, childhood;T-cell lymphoma, skin;Testicular cancer;Thymoma, childhood;Thymoma, malignant;Thyroid cancer;Thyroid cancer, childhood;Transitional cell carcinoma of the renal pelvis and ureter;Chloroblastic tumor, gestational;Carcinoma of unknown primary site, childhood;Unusual cancer of childhood;Ureter and renal pelvis, transitional cell carcinoma;Urethral cancer;Uterine sarcoma;Vaginal cancer;Visual pathway and hypothalamic glioma, childhood;Vulvar cancer;Waldenström's macroglobulinemia and Wilms' tumor.;

[0137] Pharmaceutical Compositions and Administration The antibody or agent of the present invention (also referred to herein as "active compound") and their derivatives, fragments, analogs and homologs can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include the antibody or agent and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as liposomes and fixed oils can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the composition is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0138] The pharmaceutical composition of the present invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. The solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates, and agents for adjusting osmotic pressure such as sodium chloride or dextrose. The pH may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0139] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents in the composition, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0140] Sterile injectable solution can be prepared by incorporating active compound in the required amount in suitable solvent with one or combination of the above-listed components as required, followed by filtration sterilization.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains basic dispersion medium and other components required from above-listed.For the preparation of sterile injectable solution, the preparation method is vacuum drying and freeze-drying, which produces powder of active ingredient plus any additional desired components from its previously sterile-filtered solution.

[0141] Oral compositions generally include an inert diluent or an edible carrier. These can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with an excipient and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, where the compound in the fluid carrier is applied orally, swished, expectorated, or swallowed. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The tablets, capsules, troches and the like may contain any of the following ingredients: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring, or compounds of a similar nature.

[0142] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0143] Systemic administration can also be by transmucosal or transdermal means.For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated is used in the formulation.Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.Transmucosal administration can be achieved through the use of nasal sprays or suppositories.For transdermal administration, the active compound is formulated into ointments, salves, gels, or creams generally known in the art.

[0144] The compounds can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0145] In one embodiment, the active compound is prepared with a carrier that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Methods for the preparation of such formulations will be apparent to those skilled in the art. Materials can also be commercially obtained from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions, including liposomes targeted to infected cells using monoclonal antibodies against viral antigens, can also be used as pharma-ceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0146] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate oral or parenteral compositions in dosage unit form.As used herein, dosage unit form refers to a physically separate unit suitable as a unitary dosage for the subject to be treated, and each unit contains a predetermined amount of active compound calculated to produce desired therapeutic effect in association with required pharmaceutical carrier.The specification for dosage unit form of the present invention is dictated by and directly depends on the inherent characteristics of active compound and the specific therapeutic effect to be achieved, and the inherent limitations of the technology of compounding such active compound for individual treatment.

[0147] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. EXAMPLES

[0148] The present invention is further illustrated, but not limited, by the slides accompanying this specification. While certain compounds, compositions and methods of the present invention are specifically described according to certain embodiments, the following examples serve only to illustrate the present invention and are not intended to limit the present invention.

[0149] Example 1. Anti-GM-CSFRα antibodies rescue T cell proliferation The studies in this example demonstrate that the suppressive capacity of myeloid populations on T cell proliferation can be rescued by a GM-CSF antagonist.

[0150] T cell proliferation assay was performed using CD14+ cells with or without anti-GM-CSFRα antibody treatment. Briefly, CD14+ (MDSC) cells were isolated from blood samples (PBMCs) obtained from pancreatic cancer patients according to methods known in the art. The isolated CD14+ cells were treated with anti-GM-CSFRα antibody for 48 hours. Then, CD3+ T cells labeled with carboxy-fluorescein diacetate succinimidyl ester (CFSE) were co-cultured with anti-GM-CSFRα antibody-treated or untreated CD14+ cells for 96 hours, and proliferation was determined by CFSE dilution (split cells). T cells without co-culture were used as negative control.

[0151] As shown in Figure 1, T cell proliferation was significantly suppressed after culture with untreated CD14+ cells, whereas anti-GM-CSFRα antibody-treated CD14+ cells showed increased T cell proliferation, suggesting that the addition of anti-GM-CSFRα antibody rescued T cell proliferation and prevented the suppressive ability of MDSCs.

[0152] Example 2. Cancer cell conditioned medium polarizes monocytes towards the phenotype MDSC In this study, various cancer cell lines were compared to determine whether CD4+14 monocytes phenotyped MDSCs (HLA-DR1, HLA-DR2, HLA-DR3) when incubated with conditioned medium from GM-CSF-expressing cancer cells. 低 ) was used to illustrate the increase in

[0153] Cancer cell lines were analyzed for expression of GM-CSF. In this particular study, two colon cancer (HCT116 and SW-480), two pancreatic cancer (Panc-1 and Capan-1), cervical adenocarcinoma (HeLa), and malignant melanoma (A375) cell lines were measured for GM-CSF expression. As shown in Figure 2, cancer cell lines express GM-CSF at different levels. In particular, SW480 and Capan-1 have relatively high expression of GM-CSF, while HeLa cells have relatively low expression of GM-CSF.

[0154] To generate tumor conditioned medium (CM), cell lines were seeded and cultured according to methods known in the art. CD14+ cells were then cultured in the presence of CM for 6 days and analyzed for gene and protein expression. Low levels of HLA-DR biomarkers indicate MDSC phenotype. Figure 3 shows an increase in phenotype MDSC when CD14+ monocytes are incubated with conditioned medium from GM-CSF-expressing cancer cells, compared to CD+14 cells grown in normal culture medium. The results show that CM from cancer cell lines with high GM-CSF expression has high induction of MDSC, suggesting that GM-CSF contributes to the polarization of monocytes into phenotype MDSC.

[0155] Example 3. Anti-GM-CSFRα antibodies block PD-L1 upregulation in MDSCs The studies of this example illustrate the surprising discovery by the inventors that GM-CSF induces expression of PD-L1 on phenotypic MDSCs. In particular, treatment with a GM-CSF antagonist is sufficient to suppress expression of PD-L1 on monocytes treated with conditioned medium (CM) from a GM-CSF-expressing cancer cell line.

[0156] One of the main challenges for immune checkpoint blocking antibodies is in malignant tumors with limited T cell response or immunologically "cold" tumors. These cold tumors contain few infiltrating T cells, are not recognized, and do not induce a strong response by the immune system, making them difficult to treat with current immunotherapies. The inventors surprisingly found that GM-CSF upregulates PD-L1, a checkpoint protein on the surface of MDSCs that contributes to immune suppressive activity. The study of this example shows that anti-GM-CSFRα antibodies can be used to convert "cold" tumors into "hot" tumors, possibly increasing the efficacy and sensitivity of immunotherapies.

[0157] In this study, various cancer cell lines were used to evaluate the change in PD-L1 expression levels on MDSCs when CD14+ monocytes were incubated with conditioned medium from GM-CSF-expressing cancer cells. As shown in Figure 4, adding conditioned medium from GM-CSF-expressing cancer cell lines to CD14+ monocytes increased the levels of PD-L1 expression compared to baseline (culture medium alone). HeLa cell lines that exhibited low levels of GM-CSF expression (see Figure 2) did not upregulate PD-L1 expression. Adding recombinant GM-CSF in combination with CM (CM + GM-CSF) increased the expression of PD-L1, indicating that GM-CSF induces the expression of PD-L1 on phenotypic MDSCs. The spike in PD-L1 was more pronounced in cell lines with low baseline PD-L1 expression with CM alone (e.g., Panc-1 and HeLa cells). The effect of anti-GM-CSFRα antibodies on PD-L1 expression was also examined. Treatment of MDSCs with anti-GM-CSFRα antibodies in CM in the absence or presence of recombinant GM-CSF (CM+Ab and CM+GM-CSF+Ab, respectively) resulted in significantly reduced levels of PD-L1 compared with CM alone or CM+GM-CSF, respectively. These data show that anti-GM-CSFRα antibodies suppress PD-L1 upregulation in CD14+ monocytes (MDSCs) treated with conditioned medium from GM-CSF-expressing cancer cell lines.

[0158] Example 4. Anti-GM-CSFRα antibody suppresses PD-L1 expression in MDSCs The studies in this example show that a GM-CSF antagonist can suppress PD-L1 expression on MDSCs treated with conditioned medium from a GM-CSF-expressing cancer cell line (CM), regardless of whether the GM-CSF antagonist is added simultaneously with or after CM treatment, when PD-L1 levels on MDSCs are already increased.

[0159] As shown in Figure 5A, conditioned medium (shown in Table 1) from GM-CSF cancer cell lines (CM) with or without recombinant GM-CSF (at 10 ng / mL) and anti-GM-CSFRα antibody (at 100 μg / mL) was added to CD14+ monocytes (MDSC) on day 1. After 3 days of incubation, MDSC cells were analyzed for PD-L1 expression. [Table 1]

[0160] Consistent with Example 3, adding conditioned medium from a cancer cell line expressing GM-CSF to MDSCs (B; CM) or CM with recombinant GM-CSF (D; CM+GM-CSF) increased the levels of PD-L1 expression compared to baseline (A; culture medium only). Furthermore, as shown in samples C and E of Figure 5A, respectively, a decrease in PD-L1 was observed when anti-GM-CSFRα antibody was added simultaneously with CM or CM+GM-CSF, suggesting that anti-GM-CSFRα antibody blocks the upregulation of PD-L1 on MDSCs.

[0161] Next, the effect of adding anti-GM-CSFRα antibody after incubation of MDSCs with conditioned medium to investigate the effect of GM-CSF blockade on PD-L1 expression levels when elevated levels of PD-L1 on MDSCs have already been established. In this particular setup, MDSCs are cultured for 48 hours in conditioned medium with or without recombinant GM-CSF (samples B-E in Figure 5B). Anti-GM-CSFRα antibody was then added to samples C and E in Figure 5B on day 3 (after 48 hours). MDSCs in sample A were incubated for 3 days in culture medium as a control. Phenotypic analysis of MDSCs was performed on day 4. As shown in Figure 5B, treatment of MDSCs with anti-GM-CSFRα antibody after MDSCs were cultured with conditioned medium from GM-CSF-expressing cancer cell lines (samples C and E) suppressed the expression levels of PD-L1 on MDSCs. Notably, after just 24 hours of treatment with anti-GM-CSFRα antibody, PD-L1 expression was decreased in samples C and E compared to samples B and D, respectively.

[0162] Example 5. Anti-GM-CSFRα antibodies reduce MDSC-mediated T cell suppression The studies in this example further illustrate that the suppressive capacity of myeloid populations on T cell proliferation can be inhibited by a GM-CSF antagonist.

[0163] In this particular experiment, monocytes treated with conditioned medium from a GM-CSF-expressing cancer cell line were used in a T cell proliferation assay. Briefly, monocytes were cultured in conditioned medium from a GM-CSF-expressing cancer cell line (CM) for 3 days (CM-treated monocytes). T cells (1×10 5T cells) were prepared by labeling with 1 μM CFSE and stimulating with 10 ng / mL IL-2 and 10 uL soluble CD3 / CD28 T cell activator in IMDM cell culture medium. Stimulated T cells were then co-cultured with CM-treated monocytes (at a monocyte:T cell ratio of 2:1) in a mixed lymphocyte reaction (MLR) with or without supplemental human recombinant GM-CSF (10 ng / mL) and / or anti-GM-CSFRα antibody (100 μg / mL) as shown in FIG. 6. T cells stimulated in IMDM culture medium together with healthy monocytes were used as a control. T cells were expanded for 5 days, harvested, and stained for CD4 and CD8, markers for helper and cytotoxic T cells. Cell proliferation was measured by flow cytometry and assessed by CFSE dilution.

[0164] Figure 6 shows the results of a T cell proliferation assay for % of proliferating cells (left panel) and % of maximum (MFI) by flow cytometry (signal detection of CFSE dilution in CD4+ or CD8+ cells) (right panel). As shown in Figure 6, CM-treated monocytes suppressed T cell proliferation compared to controls, and the addition of recombinant GM-CSF further suppressed T cell proliferation. Treatment with anti-GM-CSFRα antibody (Ab) reduced MDSC-mediated T cell suppression.

[0165] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above description, but rather is as set forth in the following claims. In certain embodiments, for example, the following items are provided: (Item 1) A method for treating cancer, comprising administering a GM-CSF antagonist to a patient in need of treatment, wherein administration of the GM-CSF antagonist results in inhibition of the immunosuppressive activity of myeloid-derived suppressor cells (MDSCs). (Item 2) A method for inhibiting the immunosuppressive activity of myeloid-derived suppressor cells (MDSCs) in a patient suffering from cancer, comprising administering to the patient a GM-CSF antagonist. (Item 3) A method for enhancing an immune response for cancer treatment, comprising administering a GM-CSF antagonist to a patient undergoing cancer treatment, wherein the immune response is increased compared to a control. (Item 4) 4. The method of claim 3, wherein the immune response is a percentage of T cell proliferation. (Item 5) 5. The method of claim 3 or 4, wherein the control represents the level of immune response in the patient prior to administration of the GM-CSF antagonist. (Item 6) 5. The method of claim 3 or 4, wherein the control is a reference immune response level in a control patient undergoing the cancer treatment without a GM-CSF antagonist, or a reference immune response level based on historical data. (Item 7) 7. The method according to any one of items 3 to 6, wherein the cancer treatment is immunotherapy. (Item 8) 8. The method of claim 7, wherein said administering said GM-CSF antagonist increases the efficacy of said immunotherapy. (Item 9) A method of inhibiting PD-L1 in a cancer patient, comprising administering a GM-CSF antagonist to a patient in need of treatment compared to a control. (Item 10) 10. The method of claim 9, wherein said administering said GM-CSF antagonist reduces the level of PD-L1 in the cancer patient. (Item 11) 11. The method of item 9 or 10, wherein the control represents the PD-L1 level in the patient prior to administration of the GM-CSF antagonist. (Item 12) 11. The method of item 9 or 10, wherein the control is a reference PD-L1 level in a control patient undergoing the cancer treatment without a GM-CSF antagonist, or a reference PD-L1 level based on historical data. (Item 13) 13. The method of any one of items 10 to 12, wherein the level of PD-L1 in the patient is reduced by at least 10%, 20%, 30%, 50%, 60%, 70%, 80% or 90% compared to the control. (Item 14) 14. The method of any one of items 9 to 13, wherein the PD-L1 is expressed on MDSCs. (Item 15) The method of claim 14, wherein the PD-L1 is expressed on circulating MDSCs. (Item 16) 15. The method of claim 14, wherein the PD-L1 is expressed on plasma-derived MDSCs. (Item 17) The method of any one of the preceding items, wherein the patient has circulating myeloid-derived suppressor cells (MDSCs). (Item 18) The method of any one of the preceding items, wherein the patient is afflicted with a cancer that has low levels of infiltrating T cells. (Item 19) The patient suffers from immune checkpoint inhibitor (ICI) refractory cancer. 2. The method according to claim 1 . (Item 20) The method of any one of the preceding items, wherein the patient is suffering from late stage or metastatic cancer. (Item 21) 20. The method of any one of the preceding items, wherein the patient is afflicted with a cancer selected from breast cancer, colorectal cancer (CRC), prostate cancer, melanoma, bladder cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, hepatocellular carcinoma, gastric cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck squamous cell carcinoma, non-Hodgkin's lymphoma, cervical cancer, gastrointestinal cancer, genitourinary cancer, brain cancer, mesothelioma, renal cell carcinoma, gynecological cancer, ovarian cancer, endometrial cancer, lung cancer, gastrointestinal cancer, pancreatic cancer, esophageal cancer, hepatocellular carcinoma, cholangiocarcinoma, brain cancer, mesothelioma, malignant melanoma, Merkel cell carcinoma, multiple myeloma, acute myeloid leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, or acute lymphoblastic leukemia. (Item 22) The method of any one of the preceding claims, wherein the patient is afflicted with a cancer selected from stage IV breast cancer, stage IV colorectal cancer (CRC), prostate cancer, or melanoma. The method of any one of the preceding items, wherein the method further comprises administering to the patient at least one other cancer therapy. (Item 24) 24. The method of claim 23, wherein the at least one other cancer therapy is chemotherapy, MDSC targeted therapy, immunotherapy, radiation therapy, and combinations thereof. (Item 25) The method of claim 23 or claim 24, wherein the GM-CSF antagonist and the other cancer therapy are administered simultaneously. (Item 26) The method of claim 23 or claim 24, wherein the GM-CSF antagonist and the other cancer therapy are administered sequentially. (Item 27) The method of claim 23 or 24, wherein the patient has been treated with the other cancer therapy prior to the administration of the GM-CSF antagonist. (Item 28) The method of claim 23 or 24, wherein the patient has been treated with the GM-CSF antagonist prior to administration of the other cancer therapy. (Item 29) 29. The method according to any one of items 23 to 28, wherein the other cancer therapy is an ICI. (Item 30) 30. The method of claim 29, wherein the ICI antagonizes the activity of PD-1, CTLA-4, B7, BTLA, HVEM, TIM-3, GAL-9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, or A2aR. (Item 31) 30. The method of item 29, wherein the ICI is selected from an anti-PD-1 antibody (optionally pembrolizumab, nivolumab, cemiplimab), an anti-PD-L1 antibody (optionally atezolizumab, avelumab, durvalumab), an anti-CTLA-4 antibody (optionally ipilimumab), an anti-PD-L2 antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-BTLA antibody, an anti-HVEM antibody, an anti-TIM-3 antibody, an anti-GAL-9 antibody, an anti-LAG3 antibody, an anti-VISTA antibody, an anti-KIR antibody, an anti-2B4 antibody, an anti-CD160 antibody, an anti-CGEN-15049 antibody, an anti-CHK1 antibody, an anti-CHK2 antibody, an anti-A2aR antibody, an anti-B-7 antibody, and combinations thereof. (Item 32) 30. The method of item 29, wherein the ICI is an anti-PD-L1 antibody. (Item 33) 33. The method of any one of items 24 to 32, wherein the method further comprises administering a chemotherapeutic agent to the patient. (Item 34) The method according to any one of items 24 to 28, wherein the MDSC targeted therapy is selected from an anti-CFS-1R antibody, an anti-IL-6 antibody, all-trans retinoic acid, axitinib, entinostat, gemcitabine, or phenformin, and combinations thereof. (Item 35) 29. The method according to any one of items 24 to 28, wherein the immunotherapy is selected from a monoclonal antibody, a cytokine, a cancer vaccine, a T cell engaging therapy, and a combination thereof. (Item 36) 36. The method of claim 35, wherein the monoclonal antibody is selected from an anti-CD3 antibody, an anti-CD52 antibody, an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-CD20 antibody, an anti-BCMA antibody, a bispecific antibody, or a bispecific T cell engager (BiTE) antibody, and combinations thereof. (Item 37) 36. The method of claim 35, wherein the cytokine is selected from IFNa, IFNp, IFNy, IFNy, IFN, IL-2, IL-7, IL-15, IL-21, IL-11, IL-12, IL-18, hGM-CSF, TNFa, or any combination thereof. (Item 38) The method of any one of the preceding items, wherein the GM-CSF antagonist is an anti-GM-CSF antibody or a fragment thereof. (Item 39) 38. The method of any one of items 1 to 37, wherein the GM-CSF antagonist is a soluble GM-CSF receptor. (Item 40) 38. The method of any one of items 1 to 37, wherein the GM-CSF antagonist is an anti-GM-CSF receptor antibody or a fragment thereof. (Item 41) 41. The method of claim 40, wherein the anti-GM-CSF receptor antibody or fragment thereof is an anti-GM-CSFRα antibody or fragment thereof. (Item 42) 42. The method of claim 41, wherein the anti-GM-CSFRα antibody or fragment thereof is a monoclonal antibody specific for human GM-CSFRα. (Item 43) 43. The method of claim 42, wherein the anti-GM-CSFRα antibody is a human or humanized IgG4 antibody. (Item 44) 44. The method of claim 42 or 43, wherein the anti-GM-CSFRα antibody is mavrilimumab. (Item 45) 45. The anti-GM-CSFRα antibody or fragment thereof according to any one of items 41 to 44, comprising a light chain complementarity determining region 1 (LCDR1) defined by SEQ ID NO: 6, a light chain complementarity determining region 2 (LCDR2) defined by SEQ ID NO: 7, and a light chain complementarity determining region 3 (LCDR3) defined by SEQ ID NO: 8, and a heavy chain complementarity determining region 1 (HCDR1) defined by SEQ ID NO: 3, a heavy chain complementarity determining region 2 (HCDR2) defined by SEQ ID NO: 4, and a heavy chain complementarity determining region 3 (HCDR3) defined by SEQ ID NO: 5. The method described. (Item 46) The method of any one of the preceding items, wherein the administration of the GM-CSF antagonist and / or the ICI results in a reduced level of MDSC in the patient compared to a control. (Item 47) The method of any one of the preceding items, wherein the administration of the GM-CSF antagonist and / or the ICI results in a reduced level of MDSC-mediated immunosuppressive activity in the subject compared to a control. (Item 48) The administration of the GM-CSF antagonist and / or the ICI increases Lin in the peripheral blood of the patient compared to a control. - CD14 + HLA-DR - The method of any one of the preceding items, resulting in a reduced percentage of M-MDSC. (Item 49) The method of any one of the preceding items, wherein the administration of the GM-CSF antagonist and / or the ICI results in an increased percentage of mature MDSC cells in the patient compared to a control. (Item 50) The method of any one of the preceding items, wherein the administration of the GM-CSF antagonist and / or the ICI results in reduced levels of Treg cells, macrophages, and / or neutrophils compared to a control. (Item 51) The method of any one of the preceding items, wherein the administration of the GM-CSF antagonist and / or the ICI results in reduced levels of inhibitory cytokines. (Item 52) 52. The method of claim 51, wherein the inhibitory cytokine is selected from IL-10 and TGFβ. (Item 53) The method of any one of the preceding items, wherein said administration of said GM-CSF antagonist and / or said ICI results in reduced levels of immunosuppressive factors. (Item 54) 54. The method of claim 53, wherein the immunosuppressant is selected from arginase 1, inducible nitric oxide synthase (iNOS), peroxynitrite, nitric oxide, reactive oxygen species, tumor-associated macrophages, and combinations thereof. (Item 55) The administration of the GM-CSF antagonist and / or the ICI increases the CD4 + The method of any one of the preceding items, which results in increased levels of T effector cells. (Item 56) 56. The method of any one of items 46 to 55, wherein the control is a pre-treatment level or percentage in the patient, or a reference level or percentage based on historical data. (Item 57) A pharmaceutical composition for treating cancer comprising a GM-CSF antagonist and an ICI. (Item 58) 58. The pharmaceutical composition of item 57, wherein the GM-CSF antagonist is an anti-GM-CSF antibody or a fragment thereof. (Item 59) 58. The pharmaceutical composition of claim 57, wherein the GM-CSF antagonist is a soluble GM-CSF receptor. (Item 60) 58. The pharmaceutical composition of item 57, wherein the GM-CSF antagonist is an anti-GM-CSF receptor antibody or a fragment thereof. (Item 61) 61. The pharmaceutical composition of claim 60, wherein the anti-GM-CSF receptor antibody or fragment thereof is an anti-GM-CSFRα antibody or fragment thereof. (Item 62) 62. The pharmaceutical composition of claim 61, wherein the anti-GM-CSFRα antibody or fragment thereof is a monoclonal antibody specific for human GM-CSFRα. (Item 63) 63. The pharmaceutical composition of claim 62, wherein the anti-GM-CSFRα antibody is a human or humanized IgG4 antibody. (Item 64) 64. The pharmaceutical composition according to any one of items 60 to 63, wherein the anti-GM-CSFRα antibody is mavrilimumab. (Item 65) 64. The pharmaceutical composition according to any one of items 60 to 63, wherein the anti-GM-CSFRα antibody or fragment thereof comprises light chain complementarity determining region 1 (LCDR1) defined by SEQ ID NO: 6, light chain complementarity determining region 2 (LCDR2) defined by SEQ ID NO: 7, and light chain complementarity determining region 3 (LCDR3) defined by SEQ ID NO: 8, as well as heavy chain complementarity determining region 1 (HCDR1) defined by SEQ ID NO: 3, heavy chain complementarity determining region 2 (HCDR2) defined by SEQ ID NO: 4, and heavy chain complementarity determining region 3 (HCDR3) defined by SEQ ID NO: 5. (Item 66) 66. The pharmaceutical composition according to any one of items 57 to 65, wherein said ICI antagonizes the activity of PD-1, CTLA-4, B7, BTLA, HVEM, TIM-3, GAL-9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, and combinations thereof. (Item 67) 64. The pharmaceutical composition according to any one of items 57 to 63, wherein the ICI is selected from an anti-PD-1 antibody (optionally pembrolizumab, nivolumab, cemiplimab), an anti-PD-L1 antibody (optionally atezolizumab, avelumab, durvalumab), an anti-CTLA-4 antibody (optionally ipilimumab), an anti-PD-L2 antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-BTLA antibody, an anti-HVEM antibody, an anti-TIM-3 antibody, an anti-GAL-9 antibody, an anti-LAG3 antibody, an anti-VISTA antibody, an anti-KIR antibody, an anti-2B4 antibody, an anti-CD160 antibody, an anti-CGEN-15049 antibody, an anti-CHK1 antibody, an anti-CHK2 antibody, an anti-A2aR antibody, an anti-B-7 antibody, and combinations thereof. (Item 68) A kit for treating cancer comprising a pharmaceutical composition comprising a GM-CSF antagonist and a pharmaceutical composition comprising at least one other cancer therapy selected from chemotherapy, MDSC targeted therapy, immunotherapy, radiation therapy, and combinations thereof. (Item 69) 69. The kit of item 68, wherein the immunotherapy is an ICI selected from an anti-PD-1 antibody (optionally pembrolizumab, nivolumab, cemiplimab), an anti-PD-L1 antibody (optionally atezolizumab, avelumab, durvalumab), an anti-CTLA-4 antibody (optionally ipilimumab), an anti-PD-L2 antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-BTLA antibody, an anti-HVEM antibody, an anti-TIM-3 antibody, an anti-GAL-9 antibody, an anti-LAG3 antibody, an anti-VISTA antibody, an anti-KIR antibody, an anti-2B4 antibody, an anti-CD160 antibody, an anti-CGEN-15049 antibody, an anti-CHK1 antibody, an anti-CHK2 antibody, an anti-A2aR antibody, an anti-B-7 antibody, and combinations thereof.

Claims

[Claim 1] The invention described in the specification.

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