Methods of eliminating myeloid-derived suppressor cells using NEO-201 antibodies

Abstract

NEO-201, an antibody that specifically binds to glycosylated peptides carrying core-1 and / or extended core-1 O-glycans comprised in CEACAM5 and CEAMCAM6, but does not bind to non-glycosylated CEACAM5 or non-glycosylated CEAMCAM6, has surprisingly shown to bind to and kill granulocyte myeloid-derived suppressor cells (gMDSC). The gMDSC is known to inhibit innate immunity in different cancers and infectious diseases, as well as other conditions. Based thereon, there is provided the use of NEO-201, alone or in combination, for the treatment of cancer and infectious diseases, as well as other conditions in which gMDSC inhibits innate immunity against the disease. The methods optionally include detecting the gMDSC before, during, or after the NEO-201 treatment. Diagnostic methods, therapeutic methods, and combination therapies using NEO-201, optionally in combination with another agent, to eliminate gMDSC and disease cells are also described.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 343,559 filed on May 19, 2022 and U.S. Provisional Application No. 63 / 494,094 filed on April 4, 2023, the entire contents of both of which are incorporated by reference in their entirety.

[0003] Sequence Listing Information

[0004] This application includes a biological sequence listing as part of the present disclosure, the biological sequence listing file is named "1143282o005000.txt", created on May 18, 2022, and is 32,427 bytes in size, which is hereby incorporated by reference in its entirety. Background Art

[0005] The human carcinoembryonic antigen (CEA) family consists of 29 genes arranged in tandem on chromosome 19q13.2. Based on nucleotide homology, these genes are divided into two major subfamilies, namely CEACAM and pregnancy-specific glycoprotein subgroups. The proteins encoded by CEACAM include CEA (CEACAM5), CEA-related cell adhesion molecules (CEACAM1, CEACAM3, CEACAM4, CEACAM6, CEACAM7 and CEACAM8). The CEACAM family belongs to the Ig superfamily. Structurally, each of the human CEACAMs contains an N-terminal domain that includes 108-110 amino acids and is homologous to the Ig variable domain, followed by a different number (zero to six) of Ig C2-type constant-like domains. CEACAM proteins can interact with each other homophilically and heterophilically. CEACAM1 is a unique protein in this family because it contains ITIM (immunoreceptor tyrosine-based inhibition motif) in its cytoplasmic domain, like PD1. This inhibitory effect is triggered by phosphorylation of tyrosine residues with ITIM, which leads to the recruitment of tyrosine phosphatase 1 and tyrosine phosphatase 2 containing Src homology 2 domain. CEACAM1 protein is expressed on a variety of immune cells, including monocytes, granulocytes, activated T cells, B cells and NK cells. CEACAM1 appears in several isoforms, the two main isoforms are CEACAM1-L and CEACAM1-S, which have long (L) or short (S) cytoplasmic domains, respectively. CEACAM1-S expression is completely absent in human leukocytes. CEACAM1-L is expressed on a subset of activated human NK cells that are negative for CD16 but positive for CD56. The heterophilic interaction between CEA on tumor cells and CEACAM1 on NK cells inhibits NK cell cytotoxicity against tumor cells.

[0006] NEO-201 is a humanized IgG1 mAb that binds to cancer proteins carrying core-1 and / or extended core-1 O-glycans, including tumor-associated variants of CEACAM family members, particularly cancer-associated variants of CEACAM5 and CEACAM6 (Zeligs et al., Cancer Res. 2017 Jul 1(77)(13 Suppl)3025) and Tsang KY, Fantini M, Zaki A, Mavroukakis SA, Morelli MP, Annunziata CM, Arlen PM. "Identification of the O-Glycan Epitope Targeted by the Anti-Human Carcinoma Monoclonal Antibody (mAb) NEO-201", Cancers (Basel). 2022 Oct 12;14(20):4999. doi:10.3390 / cancers14204999. NEO-201 has been shown to be responsive to certain cancers but not to most normal tissues. Applicant's prior U.S. Patents Nos. 5,688,657, 7,314,622, 7,491,801, 7,763,720, 7,829,678, 8,470,326, 8,524,456, 8,535,667, 8,802,090, 9,034,588, 9,068,014, 9,371,375, 9,592,290, 9,718,866 and RE39,760 disclose the use of NEO-201 for the diagnosis and treatment of colon cancer and pancreatic cancer. Furthermore, recently in PCT Application No. XXX, Applicants disclosed the use of NEO-201 for the treatment of hematological malignancies expressing cancer proteins carrying core-1 and / or extended core-1 O-glycans, including CEACAM5 and / or CEACAM6.

[0007] However, to the knowledge of the applicant, the use of NEO-201 or any other antibody targeting CEACAM5 and / or CEACAM6 to detect and / or deplete myeloid-derived suppressor cells (MDSC) has not been reported before. As known in the art, MDSC is a myeloid cell population produced during a large number of pathological conditions ranging from cancer, infection to obesity. These cells represent a pathological state of activation of monocytes and relatively immature neutrophils. MDSC is characterized by a unique set of genomic and biochemical characteristics, and can be distinguished from granulocytes and other cells by the expression of its specific surface molecules. The notable feature of these cells is their ability to inhibit T cell function, and therefore contributes to the pathogenesis of various diseases. In particular, it is known that these cells contribute to the pathology of diseases (including cancer, infectious diseases, autoimmunity, obesity and pregnancy).

[0008] Therefore, methods for detecting and / or eliminating MDSCs have great therapeutic potential. Summary of the invention

[0009] NEO-201 has previously been shown to bind to cancer-associated variants of CEACAM5 and CEACAM6, specifically through cancer-associated glycosylation variants of these proteins carrying core-1 and / or extended core-1 O-glycans. NEO-201 is a humanized IgG1 monoclonal antibody derived from an immunogenic preparation of tumor-associated antigens from pooled allogeneic colon tumor tissue extracts. NEO-201 is reactive against most tumor tissues from many different carcinomas, but unresponsive to most normal tissues. Functional analyses have shown that NEO-201 is able to mediate antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against tumor cells. Previous studies have demonstrated that NEO-201 can attenuate the growth of human tumor xenografts in mice and is safe and tolerable in non-human primates, with a transient decrease in circulating neutrophils being the only adverse effect observed.

[0010] The applicant shows in this article that NEO-201 binds to granulocytes, and further binds to MDSC (gMDSC) derived from granulocytes, and can kill gMDSC by ADCC. As previously mentioned, MDSC is a myeloid cell population produced during a large number of pathological conditions from cancer to obesity, which especially inhibits T cell function and contributes to the pathogenesis of various diseases. In particular, these cells contribute to the pathology of diseases (including cancer, infectious diseases, autoimmunity, obesity and pregnancy).

[0011] MDSCs have become universal regulators of immune function in many pathological diseases. MDSCs are composed of two major types of cells: granulocytes or polymorphonuclear cells (PMN-MDSCs or gMDSCs) and monocytes (M-MDSCs). PMN-MDSCs or gMDSCs are phenotypically and morphologically similar to neutrophils, while M-MDSCs are more similar to monocytes (Gabrilovich DI et al., "Coordinated regulation of myeloid cells by tumors", Nat Rev Immunol. 2012; 12(4): 253–268). The existence of a third small MDSC population, represented by cells with colony-forming activity and other myeloid precursors, termed early MDSC (eMDSC) has also been reported (Dumitru CA et al., “Neutrophils and granulocyticmyeloid-derived suppressor cells: immunophenotyping, cell biology and clinical relevance in human oncology”, Cancer Immunol Immunother. 2012; 61(8): 1155–11673).

[0012] Therefore, based on its demonstrated ability to detect and / or eliminate gMDSC, NEO-201 is potentially useful for treating and / or monitoring the disease state of any condition (most particularly cancer) in which gMDSC are involved in the pathology of the disease, and is particularly useful for treating cancers that do not express CEACAM5 and / or CEACAM6, as well as for treating chronic infectious conditions in which gMDSC are known to suppress innate immunity. In addition, NEO-201 is particularly suitable for combination therapy, for example, in combination with other therapeutic agents (e.g., other drugs or treatments that eliminate and / or inhibit the activity of MDSC, as well as other therapeutic antibodies, checkpoint inhibitors, chemotherapeutic agents, etc.), because NEO-201, due to its ability to deplete gMDSC, should, for example, enhance the efficacy of other therapeutic agents by enhancing innate immunity (e.g., innate anti-tumor or anti-infective responses), even in subjects previously resistant to treatment.

[0013] Based on the foregoing, in one embodiment, the present invention provides a method for killing or eliminating granulocyte-derived myeloid-derived suppressor cells (gMDSCs) in a patient in need thereof, the method comprising administering to the patient an effective amount of an antibody or antibody fragment that binds to glycosylated CEACAM5 and CEACAM6 carrying core-1 and / or extended core-1 O-glycans, but does not bind to aglycosylated CEACAM5 or aglycosylated CEACAM6, optionally wherein the antibody or antibody fragment recognizes an O-glycosylation epitope that binds to a threonine in the region from amino acids 310 to 318 (RTTVTTITV) of CEACAM5 and to a threonine and a serine in the region from amino acids 312 to 320 (TVTMITVSG) of CEACAM6.

[0014] In another embodiment, the present invention provides a method of killing or eliminating granulocytic myeloid-derived suppressor cells (gMDSC) in a patient in need thereof, the method comprising administering to the patient an effective amount of NEO-201 or an antigen-binding fragment thereof.

[0015] In another embodiment, the present invention provides a method of reversing tolerance and / or restoring innate immunity (e.g., innate anti-tumor immunity or innate anti-infective immunity) in a patient in need thereof by killing or eliminating granulocytic myeloid-derived suppressor cells (gMDSCs) in the subject, the method comprising administering to the patient an effective amount of NEO-201 or an antigen-binding fragment thereof.

[0016] In another embodiment, the invention provides a method of reversing resistance or tolerance to an anti-cancer or anti-infective treatment (e.g., an immunomodulatory antibody, a checkpoint inhibitor antibody or fusion protein, or a chemotherapeutic agent) involving granulocytic myeloid-derived suppressor cells by administering NEO-201 alone or in combination with another treatment (e.g., an immunomodulatory antibody, a checkpoint inhibitor antibody or fusion protein, or a chemotherapeutic agent) to reverse such resistance or tolerance.

[0017] In another embodiment, the present invention provides a method of treating or preventing recurrence of cancer or infection by administering NEO-201 alone or in combination with another treatment (e.g., an immunomodulatory antibody, a checkpoint inhibitor antibody or fusion protein, or a chemotherapeutic agent) to inhibit the proliferation of MDSCs, thereby reconstituting innate immunity.

[0018] In an exemplary embodiment, in any of the foregoing methods, the gMDSC expresses O-glycans selected from Figure 2 and / or Figure 5One or more of the 01, 02, 06, 023, 026 and 039 O-glycans of the structures shown in the array in.

[0019] In an exemplary embodiment, in any of the foregoing methods, the gMDSCs are derived from neutrophils expressing 06, 01 or 02 O-glycans having Figure 2 and / or Figure 5 The structure shown in the array.

[0020] In an exemplary embodiment, in any of the foregoing methods, the gMDSCs are derived from a gene expressing Figure 2 and / or Figure 5 06O-glycans of neutrophils are shown in the array.

[0021] In an exemplary embodiment, in any of the foregoing methods, the gMDSCs may express Figure 1 Tn antigen or core 1, 2, 4 or 4 O-glycans of the structures shown in FIG.

[0022] In an exemplary embodiment, in any of the aforementioned methods, the patient suffers from cancer or an infectious disease, wherein gMDSCs are implicated in the disease pathology and / or immunosuppression of innate immunity against the disease.

[0023] In an exemplary embodiment, in any of the foregoing methods, the antibody or antigen-binding fragment is linked directly or indirectly to a cytotoxic agent, optionally a radionuclide or a chemotherapeutic agent.

[0024] In an exemplary embodiment, in any of the foregoing methods, the antibody or antigen-binding fragment is directly or indirectly linked to a label, optionally a fluorescent or radioactive label.

[0025] In an exemplary embodiment, in any of the aforementioned methods, the subject treated has a cancer in which MDSCs are involved in the disease pathology, optionally wherein the treated cancer cells do not express or overexpress an antigen bound by NEO-201.

[0026] In an exemplary embodiment, in any of the foregoing methods, the subject treated has a cancer in which MDSCs are involved in the pathology of the disease, optionally adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, adult brain / CNS tumors, childhood brain / CNS tumors, breast cancer, male breast cancer, adolescent cancer, childhood cancer, young adult cancer, cancer of unknown primary, Castleman's disease, Disease), Cervical Cancer, Colon / Rectal Cancer, Endometrial Cancer, Esophageal Cancer, Ewing Family of Tumors, Eye Cancer, Gallbladder Cancer, Gastrointestinal Carcinoid Tumors, Gastrointestinal Stromal Tumors (GIST), Gestational Trophoblastic Disease, Hodgkin's Disease, Kaposi's Sarcoma, Kidney Cancer, Laryngeal and Hypopharynx Cancer, Leukemia, Leukemia - Adult Acute Lymphocytic (ALL), Leukemia - Acute Myeloid (AML), Leukemia - Chronic Lymphocytic (CLL), Leukemia - Chronic Myeloid (CML), Leukemia - Chronic Myelomonocytic (CMML), Childhood Leukemia, Liver Cancer, Lung Cancer, Lung Cancer - Non-Small Cell, Lung Cancer - Small Cell, Lung Carcinoid Tumors, Lymphoma, Lymphoma of the Skin, Malignant Mesothelioma, Multi NEO-201 is a cancer cell therapy that is directed toward the treatment of a variety of diseases, including: primary myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, childhood non-Hodgkin's lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma - adult soft tissue cancer, skin cancer, skin cancer - basal and squamous cell, skin cancer - melanoma, skin cancer - Merkel cell, small intestine cancer, gastric cancer, testicular cancer, thymic cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor, optionally wherein the cancer cells being treated do not express or overexpress an antigen bound by NEO-201.

[0027] In an exemplary embodiment, in any of the aforementioned methods, the subject treated has a cancer in which MDSCs are involved in the pathology of the disease, the cancer optionally being a cancer and / or tumor selected from the group consisting of lung cancer, breast cancer, triple negative breast cancer (TNBC), colorectal cancer, liver cancer, gastric cancer, colon cancer, non-small cell lung cancer (NSCLC), bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, colorectal cancer, small intestine cancer, rectal cancer, anal cancer, fallopian tube cancer, endometrial cancer, cervical cancer. NEO-201 is a cancer cell that is present in the lungs, ovaries, ulcers, esophagus, small intestine, lymph nodes, bladder, gallbladder, endocrine, thyroid, parathyroid, adrenal, soft tissue, urethral, ​​penile, prostate, adenocarcinoma, chronic or acute leukemia, lymphocytic lymphoma, bladder, kidney or ureter, renal cell, renal pelvis, central nervous system, primary CNS, spinal cord, brain stem, and pituitary adenomas, wherein the cancer cells are not expressed or overexpress an antigen that is bound by NEO-201.

[0028] In exemplary embodiments, in any of the aforementioned methods, the cancer or infection being treated is not characterized by expression of glycosylated CEACAM5 and / or glycosylated CEACAM6; and / or is not characterized by increased expression of glycosylated CEACAM5 and / or glycosylated CEACAM6.

[0029] In an exemplary embodiment, in any of the foregoing methods, the subject being treated has a cancer in which MDSCs are involved in the disease pathology, and the treatment elicits one or more of: (i) increased T cell responses, (ii) increased antigen presentation, (iii) decreased MDSC proliferation and / or (iv) decreased Treg recruitment.

[0030] In exemplary embodiments, in any of the aforementioned methods, the subject treated has a stage I, stage II, stage III, or stage IV cancer involving MDSCs.

[0031] In an exemplary embodiment, in any of the foregoing methods, the antibody or fragment (optionally NEO-201) reduces, eliminates, slows or prevents the growth of a tumor, wherein in a patient whose anti-tumor immunity was previously suppressed by gMDSCs, the tumor burden in the individual is reduced, tumor growth is inhibited, and / or the survival of the individual is increased.

[0032] In an exemplary embodiment, in any of the aforementioned methods, the subject suffers from an infectious disorder in which MDSCs are implicated in the disease pathology.

[0033] In an exemplary embodiment, in any of the foregoing methods, the subject has a bacterial infection involving MDSCs, and the bacterial infection is optionally Bacillus anthracis, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis and Enterococcus faecium, Escherichia coli (-1, coli O157:H7, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia, Salmonella Typhi, Salmonella Typhimurium, Shigella sonnei, and / or Staphylococcus aureus.

[0034] In an exemplary embodiment, in any of the foregoing methods, the subject has a chronic or acute viral infection involving MDSCs, optionally associated with a respiratory virus such as adenovirus, avian influenza, influenza A virus, influenza B virus, measles, parainfluenza virus, respiratory syncytial virus (RSV), rhinovirus, SARS-CoV; a gastrointestinal virus such as coxsackie virus, enterovirus, polio virus, rotavirus; a hepatitis virus such as hepatitis B virus, hepatitis C virus; bovine viral diarrhea virus (surrogate); a herpes virus such as herpes simplex type 1, herpes simplex type 2, human cytomegalovirus, varicella zoster virus; a retrovirus such as human immunodeficiency virus type 1 (HIV-1), human immunodeficiency virus type 2 (HIV-2), simian immunodeficiency virus (SIV), simian human immunodeficiency virus (SHIV); a viral select factor (Viral Select Factor) Agent) / emerging viral pathogens such as avian influenza, dengue virus, hantavirus, hemorrhagic fever virus, lymphocytic choriomeningitis virus, smallpox virus surrogate, cowpox, monkeypox, rabbitpox, vaccinia virus, Venezuelan equine encephalitis virus (VEE), West Nile virus, and yellow fever virus.

[0035] In an exemplary embodiment, in any of the foregoing methods, the subject has a disorder involving MDSCs, wherein MDSCs are involved in suppressing innate immunity, the disorder optionally being acquired immunodeficiency syndrome (AIDS), acute disseminated encephalomyelitis (ADEM), Addison's disease, agammaglobulinemia, allergic diseases, alopecia areata, Alzheimer's disease, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, antisynthetase syndrome, arterial plaque disorders, asthma, atherosclerosis, atopic allergies. reaction, atopic dermatitis, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune hypothyroidism, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune urticaria, autoimmune uveitis, Balo disease disease) / Balo concentric sclerosis, Behcet's disease, Berger's disease, Bickerstaff's encephalitis, Blau syndrome, bullous pemphigoid, Castleman's disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy, chronic recurrent multifocal osteomyelitis, chronic obstructive pulmonary disease, chronic venous stasis ulcer, Churg-Strauss syndrome, cicatricial pemphigoid, Cogan syndrome, cold agglutinin disease, complement component 2 deficiency, contact dermatitis, cranial arteritis, CREST syndrome, Crohn's disease, Cushing's syndrome Syndrome), cutaneous leukocytoclastic vasculitis, Dego's disease, Dercum's disease, dermatitis herpetiformis, dermatomyositis, diabetes mellitus type 1, diabetes mellitus type 2, diffuse cutaneous systemic sclerosis, Dressler's syndrome, drug-induced lupus, discoid lupus erythematosus, eczema, emphysema, endometriosis, enthesitis-associated arthritis, eosinophilic fasciitis, eosinophilic gastroenteritis, eosinophilic pneumonia, epidermolysis bullosa acquisita, erythema nodosum, erythroblastosis fetalis, essential mixed cryoglobulinemia, Evan's syndromesyndrome), progressive fibrodysplasia ossificans progressiva, fibrosing alveolitis (or idiopathic pulmonary fibrosis), gastritis, gastrointestinal pemphigoid, Gaucher's disease, glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome (GBS), Hashimoto's encephalopathy, Hashimoto's thyroiditis, heart disease, Henlein-Schönlein purpura ( purpura), herpes gestationis (also known as pemphigoid gestationis), hidradenitis suppurativa, HIV infection, Hughes-Stovin syndrome, hypogammaglobulinemia, infectious diseases (including bacterial infections), idiopathic inflammatory demyelinating diseases, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, IgA nephropathy, inclusion body myositis, inflammatory arthritis, inflammatory bowel disease, inflammatory dementia, interstitial cystitis, interstitial pneumonia, juvenile idiopathic arthritis (also known as juvenile rheumatoid arthritis), Kawasaki's disease, Lambert-Eaton myasthenic syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, linear IgA disease (LAD), lupus hepatitis (also known as autoimmune hepatitis), lupus erythematosus, lymphomatoid granulomatosis, Majeed syndrome syndrome), malignancies including cancer (e.g.sarcoma, Kaposi's sarcoma, lymphoma, leukemia, carcinoma and melanoma), Meniere's disease, microscopic polyangiitis, Miller-Fisher syndrome, mixed connective tissue disease, morphea, Mucha-Habermann disease (also known as acute pityriasis lichen vulgaris), multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (also known as Devic's disease), neuromyotonia, ocular cicatricial pemphigoid, opsoclonus-myoclonic syndrome, Ord's thyroiditis, relapsing rheumatic disease, PANDAS (pediatric autoimmune neuropsychiatric disorder associated with Streptococcus), paraneoplastic cerebellar degeneration, Parkinson's disease, paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome (Parry-Romberg syndrome). syndrome, Parsonage-Turner syndrome, pars planitis, pemphigus vulgaris, peripheral arterial disease, pernicious anemia, perivenous encephalomyelitis, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, pyoderma gangrenosum, pure red cell aplasia, Rasmussen's encephalitis, Raynaud's phenomenon, relapsing polychondritis, Reiter's syndrome, restenosis, restless legs syndrome, retroperitoneal fibrosis, rheumatoid arthritis, rheumatic fever, sarcoidosis, schizophrenia, Schmidt's syndrome, Schnitzler's syndrome, scleritis, scleroderma, sepsis, serum sickness, Sjogren's syndrome, spondyloarthropathies, Still's disease disease (adult-onset), stiff-person syndrome, stroke, subacute bacterial endocarditis (SBE), Susac syndrome, Sweet's syndrome, Sydenham chorea, sympathetic ophthalmia, systemic lupus erythematosus, Takayasu'sarteritis, temporal arteritis (also known as "giant cell arteritis"), thrombocytopenia, Tolosa-Hunt syndrome), transplant (e.g., heart / lung) rejection, transverse myelitis, tuberculosis, ulcerative colitis, undifferentiated connective tissue disease, undifferentiated spondyloarthropathies, urticarial vasculitis, vasculitis, vitiligo, and Wegener's granulomatosis.

[0036] In an exemplary embodiment, in any of the foregoing methods, gMDSCs may be detected and monitored in a patient before, during, and after completion of treatment and / or after the patient enters remission.

[0037] In an exemplary embodiment, in any of the foregoing methods, gMDSCs can be detected in a patient prior to treatment to determine whether the patient will likely benefit from NEO-201 treatment.

[0038] In an exemplary embodiment, in any of the foregoing methods, gMDSCs in a biological sample can be detected using one or more ligands, such as antibodies that recognize specific biomarkers expressed on gMDSCs, optionally LOX-1, CD11b, CD15, CD66b, and glycosylated CEACAM5 and CEACAM6 antigens expressing core-1 and / or extended core-10 glycans recognized by NEO-201.

[0039] In an exemplary embodiment, in any of the foregoing methods, the number or concentration of gMDSCs in a sample from a subject having a cancer involving gMDSCs can be used to monitor the progression of the cancer (with or without treatment), wherein the patient is being treated for such cancer with NEO-201 alone or in combination with another therapeutic agent.

[0040] In an exemplary embodiment, in any of the foregoing methods, the number or concentration of gMDSCs in a sample from a subject having a cancer involving gMDSCs can be used to determine whether NEO-201 alone or in combination with another therapeutic agent may be beneficial in treating the cancer, wherein the patient is being treated for such cancer with NEO-201 alone or in combination with another therapeutic agent.

[0041] In an exemplary embodiment, in any of the foregoing methods, the number or concentration of gMDSCs in a sample from a subject having a cancer involving gMDSCs can be used to develop a dosing regimen for NEO-201 alone or in combination with another therapeutic agent.

[0042] In an exemplary embodiment, in any of the foregoing methods, the level of gMDSCs in a patient sample (such as a blood or biopsy sample) can be used to determine cancer prognosis before, during or after NEO-201 treatment, the method optionally comprising contacting the gMDSCs with a NEO-201 antibody.

[0043] In an exemplary embodiment, in any of the aforementioned methods, the detecting comprises cell sorting, optionally fluorescence activated cell sorting, thereby generating a sample enriched and / or depleted for cells positive for expression of the NEO-201 antigen (eg, gMDSCs).

[0044] In an exemplary embodiment, any of the foregoing methods may comprise detecting and / or staining gMDSCs by contacting cells with a NEO-201 antibody and detecting cells expressing NEO-201, wherein optionally NEO-201 is directly or indirectly labeled.

[0045] In an exemplary embodiment, in any of the foregoing methods, gMDSCs can be isolated by contacting a patient sample with a support comprising NEO-201 antibodies and / or using other antibodies or ligands that recognize other MDSC biomarkers, thereby retaining the MDSCs on the support.

[0046] In an exemplary embodiment, in any of the foregoing methods, the level of MDSCs in a patient sample (such as a blood or biopsy sample) can be used to determine whether the patient suffers from or is likely to develop MDSC-mediated immunosuppression.

[0047] In an exemplary embodiment, in any of the foregoing methods, the method can include administering another therapeutic agent.

[0048] In an exemplary embodiment, in any of the foregoing methods, the method may include administering at least one additional therapeutic agent, wherein administration of NEO-201 or other antibodies that bind to glycosylated CEACAM 5 and CEACAM6 carrying core-1 or extended core-1O glycans, but not to aglycosylated CEACAM5 or aglycosylated CEACAM6, potentiates the efficacy of the at least one additional therapeutic agent.

[0049] In an exemplary embodiment, in any of the foregoing methods, the other therapeutic agent comprises another therapeutic antibody, a checkpoint inhibitor, a chemotherapeutic agent, and / or comprises an immune cell, optionally a CAR-T or CAR-NK cell.

[0050] In an exemplary embodiment, in any of the foregoing methods, the additional therapeutic agent may comprise (i) another moiety that eliminates MDSCs, (ii) a moiety that promotes MDSC differentiation, (iii) a moiety that inhibits MDSC migration, (iv) an epigenetic therapy that targets MDSCs, a moiety, or (v) a chemotherapeutic agent that targets MDSCs, or a combination of one or more of the foregoing.

[0051] In an exemplary embodiment, in any of the foregoing methods, NEO-201, due to its ability to deplete gMDSCs, can enhance the efficacy of other therapeutic agents by enhancing innate immunity (e.g., innate anti-tumor or anti-infective agent responses), optionally in subjects previously resistant to treatment with the other therapeutic agent.

[0052] In an exemplary embodiment, in any of the aforementioned methods, the method may include one or more additional therapeutic agents, including but not limited to peptides, nucleic acid molecules, small molecule compounds, antibodies, and derivatives thereof.

[0053] In an exemplary embodiment, in any of the foregoing methods, the method may include one or more additional therapeutic agents, optionally an immune checkpoint inhibitor, optionally an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-CD28 antibody, an anti-TIGIT antibody, an anti-LAGS antibody, an anti-TIM3 antibody, an anti-GITR antibody, an anti-4-1BB antibody, or an anti-OX-40 antibody, and / or the one or more additional therapeutic agents include therapeutic agents targeting adenosine A2A receptor (AZAR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAGS), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V-domain Ig inhibitor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0054] In an exemplary embodiment, in any of the foregoing methods, the method may include one or more additional therapeutic agents, such as CSF-1 / 1R binders or inhibitors, or (a) microtubule inhibitors, topoisomerase inhibitors, platinum, alkylating agents, and antimetabolites; (b) MK-2206, ON 013105, RTA402, BI 2536, sorafenib, ISIS-STAT3Rx, microtubule inhibitors, topoisomerase inhibitors, platinum, alkylating agents, antimetabolites, paclitaxel, gemcitabine, doxorubicin, vinblastine, etoposide, 5-fluorouracil, carboplatin, hexamethylmelamine, aminoglutethimide, amsacrine, anastrozole, azacitidine, bleomycin, busulfan, carmustine, chlorambucil, 2-chlorodeoxyadenosine, cisplatin, colchicine, cyclophosphamide, cytarabine, cytoxan, dacarbazine, actinomycin D, daunorubicin, docetaxel, estramustine phosphate, floxuridine, fludarabine, gemtuzumab, hexamethylmelamine, hydroxyurea, ifosfamide, imatinib, interferon, irinotecan, lomustine, nitrogen mustard, melphalan, 6 - mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, pentostatin, procarbazine, rituximab, streptozotocin, tamoxifen, temozolomide, teniposide, 6-thioguanine, topotecan, trastuzumab, vincristine, vindesine and / or vinorelbine; (c) 1-D-ribofuranosyl-1,2,4-triazole-3-carboxamide, 9->2-hydroxy-ethoxymethylguanine, amantadine, 5-iodo-2'-deoxyuridine, trifluorothymidine, interferon, adenine arabinoside, protease inhibitors, thymidine kinase inhibitors, sugar or glycoprotein synthesis inhibitors, structural protein synthesis inhibitors, attachment and adsorption inhibitors and nucleoside analogs, such as acyclovir, penciclovir, valacyclovir and ganciclovir; (d) PD-1 inhibitors or anti-PD-1 antibodies, such as (pembrolizumab), (nivolumab) or LIBTAYO (cemiplimab); (e) a PD-L1 inhibitor or anti-PD-L1 antibody, such as TECENTRIQ (atezolizumab), IMFINZI (durvalumab), or BAVENCIO (avelumab); or (f) a CTLA-4 inhibitor or anti-CTLA-4 antibody, such as Ipilimumab.

[0055] In an exemplary embodiment, in any of the foregoing methods, the patient optionally has been determined to be resistant to treatment with one or more active substances due to gMDSC-mediated immunosuppression prior to NEO-201 treatment.

[0056] In an exemplary embodiment, in any of the foregoing methods, prior to treatment with NEO-201, the patient optionally has been determined to be resistant to treatment with a therapeutic antibody, optionally a therapeutic antibody that targets a checkpoint inhibitor.

[0057] In an exemplary embodiment, in any of the foregoing methods, prior to treatment with NEO-201, the patient optionally has been determined to be resistant to treatment with a PD-1 or CTLA-4 antagonist (optionally an antibody or fusion protein).

[0058] In exemplary embodiments, in any of the aforementioned methods, prior to treatment with NEO-201, the patient optionally has developed resistance to and / or is no longer responsive to treatment with the other therapeutic agent.

[0059] In an exemplary embodiment, in any of the aforementioned methods, the patient optionally responds more effectively clinically to other active substances, optionally another therapeutic antibody or fusion protein, further optionally a therapeutic antibody or fusion protein targeting a checkpoint inhibitor and / or immune cells (optionally CAR-T or CAR-NK cells) after treatment with NEO-201.

[0060] In an exemplary embodiment, in any of the foregoing methods, the patient optionally responds clinically more effectively to another active agent, optionally another therapeutic antibody or fusion protein, further optionally a PD-1 antagonist antibody (such as pembrolizumab, nivolumab, cemiplizumab, atezolizumab, dotalimumab, durvalumab, lambrolizumab, or avelumab) after treatment with NEO-201.

[0061] In an exemplary embodiment, in any of the foregoing methods, the patient optionally responds more effectively clinically to other active substances, optionally another therapeutic antibody or fusion protein targeting CTLA-4 (optionally Yervoy or Tremelimumab) and / or immune cells (optionally CAR-T or CAR-NK cells) after treatment with NEO-201.

[0062] In an exemplary embodiment, in any of the aforementioned methods, the NEO-201 antibody may comprise the VH and VL CDR sequences contained in SEQ ID NO:28 and SEQ ID NO:29.

[0063] In an exemplary embodiment, in any of the aforementioned methods, the method may include that the NEO-201 antibody may comprise a variable heavy chain sequence that is at least 90% identical to SEQ ID NO:38.

[0064] In an exemplary embodiment, in any of the aforementioned methods, the NEO-201 antibody can comprise a variable light chain sequence that is at least 90% identical to SEQ ID NO:39.

[0065] In an exemplary embodiment, in any of the aforementioned methods, the NEO-201 antibody can comprise a variable heavy chain sequence at least 90% identical to SEQ ID NO:38 and a variable light chain sequence at least 90% identical to SEQ ID NO:39.

[0066] In an exemplary embodiment, in any of the aforementioned methods, the NEO-201 antibody comprises a heavy chain sequence at least 90% identical to amino acids 20-470 of SEQ ID NO:28 and a light chain sequence at least 90% identical to amino acids 20-233 of SEQ ID NO:29.

[0067] In an exemplary embodiment, in any of the aforementioned methods, the NEO-201 antibody may comprise or consist of a heavy chain sequence of amino acids 20-470 of SEQ ID NO:28 and a light chain sequence of amino acids 20-233 of SEQ ID NO:29.

[0068] In an exemplary embodiment, in any of the aforementioned methods, the NEO-201 antibody may comprise a human IgG1 constant domain.

[0069] In an exemplary embodiment, in any of the aforementioned methods, the antibody preferably comprises the NEO-201 antibody or a variant thereof, eg, comprises the same CDRs or variable regions as the NEO-201 antibody.

[0070] In an exemplary embodiment, in any of the aforementioned methods, the NEO-201 antibody can be conjugated to another moiety.

[0071] In an exemplary embodiment, in any of the aforementioned methods, the NEO-201 antibody can be conjugated to another cytotoxic moiety, a label, a radioactive moiety, or an affinity tag.

[0072] In an exemplary embodiment, in any of the aforementioned methods, the NEO-201 antibody (preferably the NEO-201 antibody) is comprised in a chimeric antigen receptor (CAR) administered to the subject being treated.

[0073] In exemplary embodiments, in any of the aforementioned methods, the NEO-201 antibody can be a multispecific or bispecific antibody that targets at least one other antigen, optionally another tumor antigen or an antigen expressed on an immune cell.

[0074] In an exemplary embodiment, in any of the aforementioned methods, the other antigen is a checkpoint inhibitor or a cytokine or a hormone or a growth factor.

[0075] In an exemplary embodiment, in any of the aforementioned methods, the NEO-201 antibody may be administered as an immune cell, optionally a human T or NK cell, expressing a CAR comprising the antibody.

[0076] In other exemplary embodiments, the present invention provides a method of killing gMDSCs in vivo, the method comprising administering an effective amount of NEO-201 antibody to a patient, optionally wherein the patient is being treated with CAR-T or CAR-NK cells.

[0077] In other exemplary embodiments, the invention provides a method of treating or preventing or reversing gMDSC-mediated immunosuppression, the method comprising administering to a patient an effective amount of the NEO-201 antibody.

[0078] In other exemplary embodiments, the present invention provides a method of enhancing the efficacy of a CAR-T or CAR-NK therapy by administering NEO-201 in combination with the CAR-T or CAR-NK therapy, wherein the CAR can target any antigen disclosed herein.

[0079] In other exemplary embodiments, any of the foregoing methods may further comprise administering to the patient another therapeutic agent, optionally wherein the other therapeutic agent is selected from (a) microtubule inhibitors, topoisomerase inhibitors, platinum, alkylating agents, and antimetabolites; (b) MK-2206, ON 013105, RTA402, BI 2536, sorafenib, ISIS-STAT3Rx, microtubule inhibitors, topoisomerase inhibitors, platinum, alkylating agents, antimetabolites, paclitaxel, gemcitabine, doxorubicin, vinblastine, etoposide, 5-fluorouracil, carboplatin, hexamethylmelamine, aminoglutethimide, amsacrine, anastrozole, azacitidine, bleomycin, busulfan, carmustine, chlorambucil, 2-chlorodeoxyadenosine, cisplatin, colchicine, cyclophosphamide, cytarabine, cytoxan, dacarbazine, actinomycin D, daunorubicin, docetaxel, estramustine phosphate, floxuridine, fludarabine, gemtuzumab, hexamethylmelamine, hydroxyurea, ifosfamide, imatinib, interferon, irinotecan, lomustine, nitrogen mustard, melphalan, 6 - mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, pentostatin, procarbazine, rituximab, streptozotocin, tamoxifen, temozolomide, teniposide, 6-thioguanine, topotecan, trastuzumab, vincristine, vindesine and / or vinorelbine; (c) 1-D-ribofuranosyl-1,2,4-triazole-3-carboxamide, 9->2-hydroxy-ethoxymethylguanine, amantadine, 5-iodo-2'-deoxyuridine, trifluorothymidine, interferon, adenine arabinoside, protease inhibitors, thymidine kinase inhibitors, sugar or glycoprotein synthesis inhibitors, structural protein synthesis inhibitors, attachment and adsorption inhibitors and nucleoside analogs, such as acyclovir, penciclovir, valacyclovir and ganciclovir; (d) PD-1 inhibitors or anti-PD-1 antibodies, such as (pembrolizumab), (nivolumab) or LIBTAYO (cemiplimab); (e) a PD-L1 inhibitor or anti-PD-L1 antibody, such as TECENTRIQ (atezolizumab), IMFINZI (durvalumab), or BAVENCIO (avelumab); or (f) a CTLA-4 inhibitor or anti-CTLA-4 antibody, such as Ipilimumab, or optionally, the other agent comprises CAR-T or CAR-NK cells, and / or the method further comprises administering an anti-cancer vaccine or CAR-T or CAR-NK cells to the patient.

[0080] In other exemplary embodiments, the present invention provides a method of killing gMDSCs in vitro, the method comprising contacting a tissue, organ or cell sample suspected of containing gMDSCs with a NEO-201 antibody, optionally wherein the tissue, organ or cell sample is obtained from a patient suffering from cancer or an infectious disease, or wherein the tissue, organ or cell sample is a bone marrow sample from an autologous or allogeneic donor, further optionally further comprising contacting the gMDSCs with complement, further optionally wherein the gMDSCs are killed by ADCC or CDC.

[0081] In other exemplary embodiments, the present invention provides a method for killing gMDSCs in vitro, the method comprising contacting a tissue, organ or cell sample suspected of containing gMDSCs with a NEO-201 antibody, optionally wherein the tissue, organ or cell sample is obtained from a patient suffering from cancer or an infectious disease disorder, the method further comprising contacting the gMDSCs with effector cells, optionally wherein the effector cells comprise natural killer cells, further optionally wherein the gMDSCs are killed by ADCC.

[0082] In other exemplary embodiments, in any of the aforementioned methods, the NEO-201 antibody is conjugated to a cytotoxic moiety.

[0083] In other exemplary embodiments, the present invention provides a method of detecting gMDSCs, the method comprising detecting expression of the NEO-201 antigen and optionally one or more other gMDSC biomarkers by the gMDSCs, optionally wherein the level of gMDSCs in a patient sample (such as a blood or biopsy sample) is used to determine cancer prognosis or treatment regimen, the method optionally comprising contacting the gMDSCs with a NEO-201 antibody, wherein optionally the NEO-201 antibody is directly or indirectly coupled to a label, and / or optionally the detecting comprises cell sorting, optionally fluorescence activated cell sorting.

[0084] In other exemplary embodiments, the invention provides a method of staining gMDSCs, the method comprising contacting cells with a NEO-201 antibody, wherein optionally the NEO-201 antibody is directly or indirectly conjugated to a label.

[0085] In other exemplary embodiments, the invention provides a method of isolating gMDSCs, the method comprising isolating cells expressing the NEO-201 antigen and optionally at least one other gMDSC biomarker, the method optionally comprising contacting a sample containing gMDSCs with a NEO-201 antibody, optionally wherein the NEO-201 antibody is directly or indirectly labeled, further optionally wherein the sample is or comprises a blood or bone marrow or tumor biopsy sample, still further optionally the method comprises separating NEO-201 positive cells from NEO-201 negative cells, optionally wherein the gMDSCs are isolated by cell sorting, optionally fluorescence activated cell sorting, and / or optionally wherein the gMDSCs are isolated by contacting the sample with a support comprising the NEO-201 antibody, thereby retaining the gMDSCs on the support.

[0086] In other exemplary embodiments, in any of the preceding methods, the NEO-201 antibody comprises the CDR sequences contained in SEQ ID NO:28 and SEQ ID NO:29, and / or the NEO-201 antibody comprises a variable heavy chain sequence that is at least 90% identical to SEQ ID NO:38, and / or the NEO-201 antibody comprises a variable light chain sequence that is at least 90% identical to SEQ ID NO:39, and / or the NEO-201 antibody comprises a variable heavy chain sequence that is at least 90% identical to SEQ ID NO:38 and a variable light chain sequence that is at least 90% identical to SEQ ID NO:39, and / or the NEO-201 antibody comprises a heavy chain sequence that is at least 90% identical to amino acids 20-470 of SEQ ID NO:28 and a light chain sequence that is at least 90% identical to amino acids 20-233 of SEQ ID NO:29, and / or the NEO-201 antibody comprises SEQ ID NO:28 and SEQ ID NO:39. NO:29, and / or the NEO-201 antibody comprises a human IgG1 constant domain, and / or the NEO-201 antibody is humanized, and / or the NEO-201 antibody is conjugated to another moiety, and / or the NEO-201 antibody is conjugated to another cytotoxic moiety, a label, a radioactive moiety, or an affinity tag. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1Included are structures of the O-glycan cores found in mucins, including the O-glycans recognized by the NEO-201 antibody. In the table Gal is galactose; GalNAc is N-acetylgalactosamine; GlcNAc is N-acetylglucosamine; Sial is sialic acid; Ser is serine; and Thr is threonine.

[0088] Figure 2 Included are arrays of different O-glycan structures, including the O-glycan recognized by the NEO-201 antibody.

[0089] Figure 3 Includes the amino acid sequence of CEACAM6.

[0090] Figure 4 Includes the amino acid sequence of CEACAM5.

[0091] Figure 5 Included are structures of O-glycans recognized by the NEO-201 antibody, such as 01, 02, 06, 023, 026, and 039 O-glycans.

[0092] Figures 6 to 9 Included are the results of flow cytometric analysis of gMDSCs generated from GM-CSF- and IL-6-treated neutrophils from 4 normal donors.

[0093] Fig.10 A table is included showing that 47.59% to 52.58% of neutrophils treated with 10 ng / ml human GM-CSF and 10 ng / ml human IL-6 were HLA-DR negative and CD33 positive. 76.4% to 88.09% of the HLA-DR negative and CD33 positive population were CD15 positive and CD14 negative. 66.44% to 99.71% of the HLA-DR negative / CD33 positive / CD15 positive / CD14 negative population were CD66 positive and NEO-201 positive.

[0094] Fig.11 Results from ADCC experiments were included, demonstrating that when gMDSCs were incubated with PBMCs (E:T 100:1) plus NEO-201, CD33 expression was observed compared to gMDSCs incubated with PBMCs (E:T 100:1) alone from healthy donors 1 and 2. pos / HLA-DR neg The viable cells were reduced by 33.01% (18.29% vs. 27.23%) and 29.5% (25.95% vs. 36.83%), respectively. In two healthy donors, CD33 was observed in gMDSCs incubated with PBMCs (E:T 50:1) plus NEO-201 compared to gMDSCs incubated with PBMCs (E:T 50:1) alone.pos / HLA-DR neg A similar reduction in viable cells was observed.

[0095] Fig.12 Shown by flow cytometry analysis, at different time points, 2 cancer patients with stable disease (SD) and 2 cancer patients with progressive disease (PD) between the comparison of circulating gMDSC (HLA-DR- / CD33+ / CD15+ / CD14- / CD66b+ cells) percentage. In these experiments, gMDSC was gated from live PBMC. Data are presented as the median of the percentage of live cells expressing gMDSC markers. Positive is determined by using fluorescence minus one control. In the figure, "HNSCC" refers to "head and neck squamous cell carcinoma". DETAILED DESCRIPTION

[0096] The present disclosure provides a method of depleting or eliminating gMDSCs in a patient in need thereof, the method comprising administering to the patient an effective amount of a NEO-201 antibody.

[0097] The name myeloid-derived suppressor cell (MDSC) was introduced into the scientific literature about 15 years ago (Gabrilovich D et al., "The terminology issue for myeloid-derived suppressor cells", Cancer Res. 2007; 67: 42), which initially described a loosely defined group of myeloid cells with potent immunoregulatory activity. In recent years, the nature and biological role of MDSC have become clearer, and MDSC has become a universal regulator of immune function in many pathological conditions. MDSC consists of two major types of cells: granulocytes or polymorphonuclear cells (PMN-MDSC or g-MDSC) and monocytes (m-MDSC). PMN-MDSC or g-MDSC is phenotypically and morphologically similar to neutrophils, while M-MDSC is more similar to monocytes (Gabrilovich DI et al., "Coordinated regulation of myeloid cells by tumors", Nat Rev Immunol. 2012; 12 (4): 253–268). Clinical studies in humans have demonstrated the existence of a third, minor MDSC population represented by cells with colony-forming activity and other myeloid precursors.

[0098] In addition, a large number of clinical studies have identified MDSCs as valuable predictive markers in cancer prognosis. Therefore, as discussed below, a number of drugs and treatments are being developed to target MDSCs to treat diseases in which such cells are involved in the pathology of the disease.

[0099] MDSC is similar to neutrophils and monocytes in morphology and phenotype. The main populations of myeloid cells derived from bone marrow (BM) include granulocytes (the most abundant representative of which is neutrophils) and monocytes: monocytes and terminally differentiated macrophages (MΦ) and dendritic cells (DC). Compared with in vitro experiments (where both MΦ and DC can be easily differentiated from monocytes), in tissues under steady-state conditions, MΦ is amplified in situ, and most DCs are differentiated from their specific BM precursors (Geissmann F et al., " Development of monocytes, macrophages, and dendritic cells ", Science. 327 (5966): 656-661). However, during inflammation and cancer, BM-derived monocytes are the major precursors of MΦ, particularly tumor-associated macrophages (TAMs) and inflammatory DC populations (Veglia F et al., “Dendritic cells in cancer: the role revisited”, Curr Opin Immunol. 2017; 45: 43–51).

[0100] Myeloid cells have evolved to be one of the main protective mechanisms against pathogens and are important elements of tissue remodeling. Under physiological conditions, GM-CSF drives myelopoiesis, and G-CSF and M-CSF induce the differentiation of granulocytes and macrophages, respectively (Barreda DR et al., "Regulation of myeloid development and function by colony stimulating factors", Dev Comp Immunol. 2004; 28(5): 509–554). In cancer and other pathological conditions, these factors are overproduced and favor the generation of MDSCs (Gabrilovich DI et al., "Coordinated regulation of myeloid cells by tumors", Nat Rev Immunol. 2012; 12(4): 253–268; Marvel D, Gabrilovich DI. "Myeloid-derived suppressor cells in the tumor microenvironment: expect the unexpected", J Clin Invest. 2015; 125(9): 3356–3364. Therefore, the accumulation of MDSCs occurs along the same differentiation pathway as neutrophils and monocytes.

[0101] In addition to tumors and infection sites, MDSCs can also be detected in the blood, for example, in some breast cancers, the level of MDSCs in the blood is about 10 times higher than normal. (Safarzadeh E et al., April 2019, "Circulating myeloid-derived suppressor cells: An independent prognostic factor in patients with breast cancer", Journal of Cellular Physiology. 234(4): 3515–3525. doi: 10.1002 / jcp.26896. PMID 30362521.)

[0102] The size of the myeloid-derived suppressor compartment is thought to be an important factor in the clinical success or failure of cancer immunotherapy, highlighting the importance of this cell type to human pathophysiology. (Kodach LL, August 2021, "Targeting the Myeloid-Derived Suppressor Cell Compartment for Inducing Responsiveness to Immune Checkpoint Blockade Is Best Limited to Specific Subtypes of Gastric Cancers", Gastroenterology, 161(2): 727. doi: 10.1053 / j.gastro.2021.03.047. PMID33798523.)

[0103] As shown below, Applicants unexpectedly demonstrated that NEO-201 binds to granulocytes and gMDSCs derived therefrom and specifically triggers the killing or elimination of gMDSCs. Therefore, NEO-201 is potentially useful for treating any condition in which gMDSCs are involved in the pathology of the disease, most particularly cancers and chronic infectious conditions in which MDSCs are known to suppress innate immunity.

[0104] NEO-201 can be used to eliminate gMDSC diseases

[0105] cancer

[0106] Because NEO-201 specifically eliminates gMDSCs, NEO-201 can be used to treat any cancer in which gMDSCs affect innate anti-cancer immunity. This includes cancers that express the antigens bound by NEO-201 and cancers that do not express the antigens bound by NEO-201.

[0107] Thus, cancer types in which MDSCs are involved in disease pathology and in which NEO-201 can be used to deplete or eliminate gMDSCs include solid tumors and hematological cancers. Exemplary tumors in which MDSCs may be involved in disease pathology include adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, adult brain / CNS tumors, childhood brain / CNS tumors, breast cancer, male breast cancer, adolescent cancer, childhood cancer, young adult cancer, cancer of unknown primary, Castleman's disease, cervical cancer, colon / rectum cancer, endometrial cancer, esophageal cancer, Ewing's family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, leukemia-adult acute lymphocytic (ALL), leukemia-acute myeloid (AML), leukemia-chronic lymphocytic (CLL), leukemia-chronic myeloid (CML), leukemia-chronic Myelomonocytic (CMML), Childhood Leukemia, Liver Cancer, Lung Cancer, Lung Cancer - Non-Small Cell, Lung Cancer - Small Cell, Lung Carcinoid Tumors, Lymphoma, Lymphoma of the Skin, Malignant Mesothelioma, Multiple Myeloma, Myelodysplastic Syndrome, Nasal and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Childhood Non-Hodgkin Lymphoma, Oral and Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer, Pancreatic Cancer, Penile Cancer, Pituitary Tumors, Prostate Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma - Soft Tissue Cancer - Adults, Skin Cancer, Skin Cancer - Basal and Squamous Cell, Skin Cancer - Melanoma, Skin Cancer - Merkel Cell, Small Intestine Cancer, Gastric Cancer, Testicular Cancer, Thymic Cancer, Thyroid Cancer, Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer, Waldenstrom's Macroglobulinemia, and Wilms' Tumor.

[0108] In some embodiments, such cancer patients may have a cancer and / or tumor selected from the group consisting of lung cancer, breast cancer, triple negative breast cancer (TNBC), colorectal cancer, liver cancer, gastric cancer, colon cancer, non-small cell lung cancer (NSCLC), bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, colorectal cancer, small intestine cancer, rectal cancer, anal cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer. , vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, lymph node cancer, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, adenocarcinoma, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer or ureter cancer, renal cell carcinoma, renal pelvis cancer, central nervous system tumors, primary CNS tumors, spinal cord tumors, brain stem gliomas and pituitary adenomas, but are not limited to these.

[0109] Some cancers are strongly influenced by MDSCs, and therefore, patients affected by these cancers will experience greater benefit due to modulation of gMDSC suppressive function and differentiation through administration of NEO-201. This benefit will be synergistic with the induction of T cell responses, as antigen presentation is improved and other immunosuppressive effects of MDSCs, including Treg recruitment, are alleviated.

[0110] In some aspects of the invention, the cancer patient may have a stage I, II, III, or IV cancer involving MDSCs. In other aspects, NEO-201 reduces, eliminates, slows, or prevents the growth of a tumor in which anti-tumor immunity is otherwise suppressed by gMDSCs, which can result in a reduction in tumor burden, inhibition of tumor growth, and / or improved survival of the individual.

[0111] In some aspects of the invention, a cancer patient may have developed resistance or tolerance to an anti-cancer therapy (e.g., an immunomodulatory antibody, a checkpoint inhibitor antibody or fusion protein, or a chemotherapeutic agent), and NEO-201 may be administered alone or in combination with another anti-cancer therapy (e.g., an immunomodulatory antibody, a checkpoint inhibitor antibody or fusion protein, or a chemotherapeutic agent) to reverse such resistance or tolerance.

[0112] In some aspects of the invention, cancer patients may be in remission, and NEO-201 administration may be used in maintenance therapy, e.g., it may be administered alone or in combination with another anti-cancer treatment (e.g., an immunomodulatory antibody, a checkpoint inhibitor antibody or fusion protein, or a chemotherapeutic agent) to inhibit cancer recurrence by inhibiting the proliferation of MDSCs and thereby promoting innate anti-tumor immunity.

[0113] In some aspects of the invention, the cancer may have recurred, and NEO-201 administration may be administered alone or in combination with another anti-cancer treatment (e.g., an immunomodulatory antibody, a checkpoint inhibitor antibody or fusion protein, or a chemotherapeutic agent) to inhibit the proliferation of MDSCs and thereby reestablish innate anti-tumor immunity.

[0114] Infectious diseases

[0115] Furthermore, NEO-201 could potentially be used to treat bacterial conditions where gMDSCs are involved in disease pathology and may suppress innate immunity.

[0116] The types of bacterial infections for which NEO-201 can be used to deplete gMDSCs include Bacillus anthracis, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, and Clostridium tetani. tetani), Corynebacteriumdiphtheriae, Enterococcus faecalis and Enterococcus faecium, Escherichia coli (general), Enterotoxigenic Escherichia coli (ETEC), Enteropathogenic E.coli, Escherichia coli O157:H7, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia, Salmonella Typhi typhi), Salmonella typhimurium, Shigella sonnei and Staphylococcus aureus.

[0117] Types of viral infections in which MDSCs are reported to be involved in disease pathology include both chronic and acute infections. Exemplary infections in which NEO-201 can be used to deplete gMDSCs include respiratory viruses such as adenovirus, avian influenza, influenza A virus, influenza B virus, measles, parainfluenza virus, respiratory syncytial virus (RSV), rhinovirus, SARS-CoV; gastrointestinal viruses such as coxsackievirus, enterovirus, poliovirus, rotavirus; hepatitis viruses such as hepatitis B virus, hepatitis C virus; bovine viral diarrhea virus (surrogate); herpes viruses such as herpes simplex type 1, herpes simplex type 2, human cytomegalovirus, varicella zoster virus; retroviruses such as human immunodeficiency virus type 1 (HIV-1), human immunodeficiency virus type 2 (HIV-2), simian immunodeficiency virus (SIV), simian human immunodeficiency virus (SHIV); Viral Select Factors (Viral Select Factors) Agent) / emerging viral pathogens such as avian influenza, dengue virus, hantavirus, hemorrhagic fever virus, lymphocytic choriomeningitis virus, smallpox virus surrogate, cowpox, monkeypox, rabbitpox, vaccinia virus, Venezuelan equine encephalitis virus (VEE), West Nile virus, and yellow fever virus.

[0118] In some aspects of the invention, the patient may have developed resistance or tolerance to an anti-infective treatment (e.g., an immunomodulatory antibody, a checkpoint inhibitor antibody or fusion protein, or a chemotherapeutic agent), and NEO-201 may be administered alone or in combination with another anti-infective treatment (e.g., an immunomodulatory antibody, a checkpoint inhibitor antibody or fusion protein, or a chemotherapeutic agent) to boost innate anti-infective immunity.

[0119] In some aspects of the invention, patients with an infection may be in remission (e.g., herpes patients), and NEO-201 administration may be used in maintenance therapy, for example, it may be administered alone or in combination with another anti-infective treatment (e.g., an immunomodulatory antibody, a checkpoint inhibitor antibody or fusion protein, or a chemotherapeutic agent) to inhibit cancer recurrence by inhibiting the proliferation of gMDSCs and thereby promoting innate anti-infective immunity.

[0120] In some aspects of the invention, the infection may have recurred, and NEO-201 administration may be administered alone or in combination with another anti-infective treatment (e.g., an immunomodulatory antibody, a checkpoint inhibitor antibody or fusion protein, or a chemotherapeutic agent) to inhibit the proliferation of gMDSCs and thereby reestablish innate anti-infective immunity.

[0121] Other diseases involving MDSCs

[0122] Other diseases or conditions in which MDSC may be involved in suppressing innate immunity include, but are not limited to, acquired immunodeficiency syndrome (AIDS), acute disseminated encephalomyelitis (ADEM), Addison's disease, agammaglobulinemia, allergic diseases, alopecia areata, Alzheimer's disease, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, antisynthetase syndrome, arterial plaque disease, asthma, atherosclerosis, atopic allergy, atopic dermatitis, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune hypothyroidism, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral Neuropathy, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune urticaria, autoimmune uveitis, Barlow's disease / Barlow's concentric sclerosis, Behcet's disease, Buerger's disease, Bickerstaff encephalitis, Blau syndrome, bullous pemphigoid, Castleman's disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy, chronic relapsing multifocal osteomyelitis, chronic obstructive pulmonary disease, chronic venous stasis ulcer, Charg-Strauss syndrome, cicatricial pemphigoid, Cogan's syndrome, cold agglutinin disease, complement component 2 deficiency, contact dermatitis, cranial arteritis, CREST syndrome, Crohn's disease, Cushing's syndrome Syndrome, cutaneous leukocytoclastic vasculitis, Degos' disease, Derken's disease, dermatitis herpetiformis, dermatomyositis, diabetes mellitus type 1, diabetes mellitus type 2, diffuse cutaneous systemic sclerosis, Dressler syndrome, drug-induced lupus, discoid lupus erythematosus, eczema, emphysema, endometriosis, enthesitis-associated arthritis, eosinophilic fasciitis, eosinophilic gastroenteritis, eosinophilic pneumonia, epidermolysis bullosa acquisita, erythema nodosum, erythroblastosis fetalis, essential mixed cryoglobulinemia, Evan's syndrome, progressive fibrodysplasia ossificans, fibrosing alveolitis (or idiopathic pulmonary fibrosis), gastritis, gastrointestinal pemphigoid, Gaucher's disease, glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome GBS, Hashimoto's encephalopathy, Hashimoto's thyroiditis, heart disease, Henlein-Schönlein purpura, herpes gestationis (also known as gestational pemphigoid), hidradenitis suppurativa, HIV infection, Hof-Stauwen syndrome, hypogammaglobulinemia, infectious diseases (including bacterial infectious diseases), idiopathic inflammatory demyelinating diseases, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, IgA nephropathy, inclusion body myositis, inflammatory arthritis, inflammatory bowel disease, inflammatory dementia, interstitial cystitis, interstitial pneumonia, juvenile idiopathic arthritis (also known as juvenile rheumatoid arthritis), Kawasaki disease, Lebert-Eaton myasthenic syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, linear IgA disease (LAD),Lupus hepatitis (also known as autoimmune hepatitis), lupus erythematosus, lymphomatoid granulomatosis, Magid syndrome, malignancies including cancer (e.g., sarcomas, Kaposi's sarcoma, lymphomas, leukemias, carcinomas, and melanomas), Meniere's disease, microscopic polyangiitis, Miller-Fisher syndrome, mixed connective tissue disease, morphea, Muhlenberg-Harris disease (also known as acute pityriasis lichen vulgaris), multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (also known as Devic's disease), neuromyotonia, eye scars Scar pemphigoid, opsoclonus-myoclonus syndrome, Ord's thyroiditis, relapsing rheumatic disease, PANDAS (pediatric autoimmune neuropsychiatric disorder associated with Streptococcus), paraneoplastic cerebellar degeneration, Parkinson's disease, paroxysmal nocturnal hemoglobinuria, Parry-Roberg syndrome, Parsner-Turner syndrome, pars planitis, pemphigus vulgaris, peripheral arterial disease, pernicious anemia, perivenous encephalomyelitis, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, pyoderma gangrenosum, pure red cell aplasia, Rasmussen's encephalitis, Raynaud's phenomenon, relapsing polychondritis, Reiter's syndrome, restenosis, restless legs syndrome, retroperitoneal fibrosis, rheumatoid arthritis, rheumatic fever, sarcoidosis, schizophrenia, Schmidt's syndrome, Schnitzler's syndrome, scleritis, scleroderma, sepsis, serum sickness, Sjögren's syndrome, spondyloarthropathies, Still's disease (adult-onset), rigors syndrome, stroke, subacute bacterial endocarditis (SBE), Susac syndrome, Sweet's syndrome, Sydenham's chorea, sympathetic ophthalmia, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis (also known as "giant cell arteritis"), thrombocytopenia, Tolman syndrome), transplant (e.g., heart / lung transplant) rejection, transverse myelitis, tuberculosis, ulcerative colitis, undifferentiated connective tissue disease, undifferentiated spondyloarthropathies, urticarial vasculitis, vasculitis, vitiligo, and Wegener's granulomatosis.

[0123] Similarly, NEO-201 can be administered alone or in combination with another active agent to patients suffering from such disorders to reconstitute, maintain or boost innate immunity.

[0124] Combination therapy

[0125] Combination therapy with other drugs or biologics

[0126] NEO-201 should be particularly suitable for use in combination therapies, e.g., in combination with other therapies, e.g., other biologics (such as therapeutic antibodies and fusion proteins, e.g., those targeting cytokines or checkpoint inhibitors), chemotherapeutic agents, etc., because NEO-201, given its demonstrated ability to deplete gMDSCs, should potentiate the efficacy of such other therapeutic agents, i.e., by restoring or enhancing innate immunity (e.g., innate anti-tumor or anti-infective responses) that is otherwise suppressed by MDSCs, including in subjects that were previously resistant to treatment or have developed resistance to treatment with a particular active substance.

[0127] Specifically provided herein are combination therapies in which NEO-201 is administered with one or more additional therapeutic agents to kill or eliminate MDSCs that may inhibit the efficacy of such one or more additional therapeutic agents. Such one or more additional therapeutic agents include, but are not limited to, peptides, nucleic acid molecules, small molecule compounds, antibodies, and derivatives thereof.

[0128] Combination with other drugs targeting MDSCs

[0129] i. Combination of NEO-201 with chemotherapeutic agents targeting MDSCs

[0130] Reducing the tumor-promoting effects of MDSCs can be achieved by weakening the immunosuppressive function of MDSCs. STAT3 plays an indispensable role in MDSC-mediated tumorigenesis. Blocking STAT3 activation by applying specific small molecule inhibitors of p-STAT3 or STAT3-targeted siRNA can eliminate the suppressive activity of MDSCs by reducing the expression of ARG1 in MDSCs [Vasquez-Dunddel D. et al., “STAT3 regulates arginase-I in myeloid-derived suppressor cells from cancer patients”, J. Clin. Invest. 2013; 123: 1580–1589; Trovato R. et al., “Immunosuppression by monocytic myeloid-derived suppressor cells in patients with pancreatic ductal carcinoma is orchestrated by STAT3”, J. Immunother. Cancer. 2019; 7: 255]. Receptor tyrosine kinases, such as TYRO3 (a protein tyrosine kinase), AXL (a receptor tyrosine kinase), and C-Mer proto-oncogene tyrosine kinase (MERTK) and their ligands Gas 6 and protein S, can reverse the tumorigenic properties of MDSC, increase the number of tumor-infiltrating CD8+ T cells, and enhance anti-PD-1 immune checkpoint therapy. MERTK eliminates the suppressive capacity of MDSC by negatively regulating STAT3 [Holtzhausen A. et al., "TAM family receptor kinase inhibition reportedly reverses MDSC-mediated suppression and augmentations Anti-PD-1therapy in melanoma", Cancer Immunol. Res. 2019.In addition, it has been reported that STAT3 inhibitors (such as sunitinib, AZD9150, and BBI608) or conjugates of STAT3 antisense oligonucleotides (ASOs) tethered to the immunostimulatory toll-like receptor 9 (TLR9) agonist (CpG-STAT3ASOs) can significantly reduce the immunosuppressive function of MDSCs and rescue antitumor immunity [Guha P. et al., “STAT3 inhibition induces Bax-dependent apoptosis in liver tumor myeloid-derived suppressor cells”, Oncogene. 2019; 38: 533–548.; Moreira D et al. “STAT3 inhibition combined with CpG immunostimulation activates antitumor immunity to eradicate genetically distinct castration-resistant prostate cancers”, Clin. Cancer Res. 2018; 24: 5948–5962; Reilley MJ et al. “STAT3 antisense oligonucleotide AZD9150 in a subset of patients with heavily pretreated lymphoma: Results of a phase 1b trial", J. Immunother. Cancer. 2018; 6:119].

[0131] It is reported that PGE2 induces MDSC to upregulate the production of ARG1 and iNOS and exerts an inhibitory effect. Cyclooxygenase-2 (COX-2) is an upstream molecular signal of PGE2, which regulates the production of PGE2. Therefore, COX-2 can be targeted to negatively regulate the synthesis of PGE2. shRNA targeting of COX-2 significantly reduced MDSC in the spleen of tumor-bearing mice [Mao Y. et al., "Inhibition of tumor-derived prostaglandin-e2 blocks the induction of myeloid-derived suppressor cells and recovers natural killer cell activity", Clin. Cancer Res. 2014; 20: 4096–4106]. COX-2 expression can also be inhibited by acetylsalicylic acid, NS-398, and celecoxib, thereby hindering the activity of MDSCs and increasing the infiltration of CTLs in tumor sites [Wong JL et al., “Synergistic COX2Induction by IFNgamma and TNFalpha Self-Limits Type-1 Immunity in the Human Tumor Microenvironment”, Cancer Immunol. Res. 2016; 4: 303–311; Chen WC et al., “Inflammation-induced myeloid-derived suppressor cells associated with squamous cell carcinoma of the head and neck”, Head Neck. 2017; 39: 347–355; Fujita M. et al., “COX-2blockade suppresses gliomagenesis by inhibiting myeloid-derived suppressor cells”, Cancer Res. 2011; 71: 2664–2674.].

[0132] RIPK3 induces cell necrosis by interacting with TLR3 / 4 [He S. et al., "Toll-like receptors activate programmed necrosis in macrophages through a receptor-interacting kinase-3-mediated pathway", Proc. Natl. Acad. Sci. USA. 2011; 108: 20054–20059.]. RIPK3 deficiency activates the NF-κB signaling pathway and upregulates the expression of downstream signaling molecules COX-2 and PGE2, which aggravates the immunosuppressive activity of MDSCs and accelerates tumor growth. It is reported that treatment with aspirin (ASA, COX inhibitor) significantly protected mice from tumorigenesis [Yan G. et al., "ARIPK3-PGE2 circuit mediates myeloid-derived suppressor cell-potentiated colorectal carcinogenesis", Cancer Res. 2018; 78: 5586–5599.]. In addition, overexpression of fatty acid transport protein 2 (FATP2) is also involved in the synthesis of PGE2 by activating the STAT5 signaling pathway. The selective FATP2 inhibitor lipofermata selectively inhibits the function of MDSCs and enhances immunotherapy [Veglia F. et al., "Fatty acid transport protein 2reprograms neutrophils in cancer", Nature. 2019; 569: 73–78. doi: 10.1038 / s41586-019-1118-2.].

[0133] Phosphodiesterase 5 (PDE5) is another target for MDSC therapy. It is a hydrolase that acts on the NO / cyclic guanosine monophosphate (cGMP) signaling pathway [Peak TC et al., “The Role of PDE5 inhibitors and the NO / cGMP pathway in cancer”, Sex. Med. Rev. 2016; 4:74–84.].The use of PDE5 inhibitors (including sildenafil, tadalafil, vardenafil, etc.) can reduce the production of ARG1 and iNOS in MDSCs, eliminate the inhibitory activity of MDSCs, reduce the number of Tregs, and thus greatly delay tumor progression [Tai LH et al., "Phosphodiesterase-5inhibition reduces postoperative metastatic disease by targeting surgery-induced myeloid derived suppressor cell-dependent inhibition of NaturalKiller cell cytotoxicity", OncoImmunology.2018;7:e1431082.doi:10.1080 / 2162402X.2018.1431082; Weed DT et al., "Tadalafil reduces myeloid-derivedsuppressor cells and regulatory T cells and promotes tumor immunity in patients with head and neck squamous cell carcinoma", Clin.Cancer Res.2015;21:39–48; Noonan ... KA et al., “Targeting immune suppression with PDE5 inhibition inend-stage multiple myeloma,” Cancer Immunol. Res., 2014; 2:725–731; Serafini P. et al., “Phosphodiesterase-5inhibition augments endogenous antitumor immunity by reducing myeloid-derived suppressor cell function", J. Exp. Med., 2006; 203:2691–270].It has been reported that combined treatment with tadalafil and cytokine-induced killer (CIK) cell-based immunotherapy enhanced the activity of CIK against human hepatocellular carcinoma (HCC) cell lines in vitro [Yu SJ et al., "Targeting the crosstalk between cytokine-induced killer cells and myeloid-derived suppressor cells in hepatocellular carcinoma", J. Hepatol., 2019; 70: 449–457]. Nitroaspirin is another inhibitor of ARG1 and iNOS that can reduce the production of ROS [De Santo C. et al., "Nitroaspirin corrects immune dysfunction in tumor-bearing hosts and promotes tumor eradication by cancer vaccination", Proc. Natl. Acad. Sci. USA. 2005; 102: 4185–4190.].

[0134] Nuclear factor E2-related factor 2 (Nrf2), a transcription factor, is reported to be a major regulator of resistance to oxidative stress. Nrf2 is associated with abnormal ROS accumulation in MDSCs, which has been confirmed by Nrf2-deficient mouse models. In Nrf2 knockout (KO) mice, the circulating levels of MDSCs were unchanged; however, as the amount of cellular ROS increased, the number of CD8+T cells decreased significantly, and the tumor growth rate increased [Satoh H. et al., "Nrf2-deficiency creates a responsive microenvironment for metastasis to the lung", Carcinogenesis. 2010; 31: 1833–1843.; Zhang D. et al., "Identification of an unfavorable immune signature in advanced lung tumors from Nrf2-deficient mice. Antioxid. Redox Signal. 2018; 29: 1535–1552.].Treatment with Nrf2-inducing triterpenoids, such as omaveloxolone (RTA-408), CDDO-Me (RTA-402), and CDDO-Im (RTA-403), reportedly increases the transcriptional activity of Nrf2, which attenuates ROS production, abolishes the immunosuppressive effects of MDSCs, and protects immune cells and tissues from oxidative stress [Hiramoto K. et al., “Myeloid lineage-specific deletion of antioxidant system enhances tumor metastasis”, Cancer Prev. Res. (Phila.) 2014; 7:835–844.; Creelan B. et al., “Safety, pharmacokinetics, and pharmacodynamics of oral omaveloxolone (RTA 408), a synthetic triterpenoid, in a first-in-human trial of patients with advanced solid tumors”, OncoTargets Ther. 2017; 10:4239–4250; Nagaraj ... S. et al., Youn J. "Anti-inflammatory triterpenoid blocks immune suppressive function of MDSCs and improves immune response in cancer", Clin. Cancer Res. 2010; 16: 1812–1823]. However, recent studies have demonstrated that Nrf2 is activated by PKR-like endoplasmic reticulum (ER) kinase (PERK) in tumor-infiltrating MDSCs, thereby endowing MDSCs with immunosuppressive potential [Mohamed E. et al., "The unfolded protein response mediator perk governs myeloid cell-driven immunosuppression in tumors through inhibition of STING signaling", Immunity, 2020; 52: 668–682.]. It has been reported that the loss of PERK or treatment with the selective inhibitor of PERK, AMG-44, reduces Nrf2 transcription, leading to ROS overexpression, causing mitochondrial damage, hindering MDSC immunosuppression, and increasing CD8. +Infiltration of T cells. This situation can be antagonized by adding the Nrf2 inducer sulforaphane [Mohamed E. et al., "The unfolded protein response mediator perk governsmyeloid cell-driven immunosuppression in tumors through inhibition of STING signaling", Immunity. 2020; 52: 668–682]. In summary, Nrf2 overexpression and loss affect the immunosuppressive activity of MDSC, and only when Nrf2 is maintained in a stable state can MDSC exert a normal tumor-promoting effect.

[0135] N-hydroxy-nor-L-arginine (nor-NOHA) is used as an ARG1 inhibitor. Blockade of ARG1 by nor-NOHA has been reported to reverse the immunosuppressive activity of MDSCs [Bak SP, “Murine ovarian cancer vascular leukocytes require arginase-1 activity for T cell suppression”, Mol. Immunol. 2008; 46: 258–268]. Inhibition of the VEGF / VEGFR-2 axis using the antibody DC101 suppressed primary tumor growth and metastasis in the 4T1 breast cancer model. In addition, arginase inhibition has been reported to suppress lung metastasis in the 4T1 breast cancer model, independent of the immunomodulatory and anti-metastatic effects of VEGFR-2 blockade. OncoImmunology. 2017; 6: e1316437.]. 1-methyl-DL-tryptophan (1-MT), a competitive inhibitor of IDO, has been reported to abrogate the immunosuppressive function of MDSCs on T cells. When 1-MT was combined with nor-NOHA, the T cell proliferation rate was almost completely restored [Du J. et al., “The study of CD14+HLA-DR- / low myeloid-derived suppressor cells (MDSC) in peripheral blood of peripheral T-cell lymphoma patients and its biological function” Cell. Mol. Biol. 2017; 63: 62–67.].

[0136] Bruton's tyrosine kinase (BTK) is reported to be a non-receptor intracellular kinase associated with the migration and proliferation of MDSCs. Treatment with the BTK inhibitory drug ibrutinib reduces cytokine production and motility of MDSCs [Molina-Cerrillo J. et al., "Bruton's tyrosine kinase (BTK) as a promising target in solid tumors", Cancer Treat. Rev. 2017; 58: 41–50.].

[0137] It is also reported that estrogen interacts with estrogen receptor α, drives the mobilization of MDSC by activating the STAT3 pathway, thereby promoting dysregulated myelopoiesis. The progression of tumors can be delayed by removing estrogen activity via anti-estrogen therapy [Svoronos N. et al., "Tumor cell-independent estrogen signaling drives disease progression through mobilization of myeloid-derived suppressor cells", Cancer Discov. 2017; 7: 72–85]. Castration-resistant prostate cancer shows resistance to androgen deprivation therapy, mainly because IL-23 secreted by MDSC activates androgen receptor (AR) and STAT3 / RORγ signaling axis in prostate tumor cells. Blocking IL-23 production can counteract MDSC-mediated CRPC by treatment with anti-IL-23 antibodies and the AR antagonist enzalutamide [Calcinotto A. et al., “IL-23secreted by myeloid cells drives castration-resistant prostate cancer”, Nature. 2018;559:363–369].

[0138] MDSCs have low glycolysis and mitochondrial respiration capacity, but contain high levels of methylglyoxal, which inhibits the anti-tumor activity of CD8+ effector T cells. Neutralization of methylglyoxal with guanidine-containing compounds such as metformin can effectively eliminate the immunosuppressive activity of MDSCs. The combination of metformin and anti-PD-1 can overcome the inhibition of MDSCs on immunotherapy [Baumann T. et al., "Regulatory myeloid cells paralyze T cells through cell-cell transfer of the metabolite methylglyoxal", Nat. Immunol. 2020; 21: 555–566.].

[0139] ii. Combination of NEO-201 with other drugs that deplete MDSCs

[0140] Treatment with low-dose chemotherapy drugs such as gemcitabine, 5-fluorouracil (5-FU), paclitaxel and cisplatin can effectively affect the activity of MDSCs [Wang Y. et al., “Metabolic regulation of myeloid-derived suppressor cell function in cancer”, Cells, 2020; 9: 1011.; Won WJ et al., “Metabolic and functional reprogramming of myeloid-derived suppressor cells and their therapeutic control in glioblastoma”, Cell Stress. 2019; 3: 47–65; Chaib M. et al., “Friend or foe? Recent strategies to target myeloid cells in cancer”, Front. Cell Dev. Biol. 2020; 8: 351.]. Gemcitabine is a selective inhibitor of MDSC, which reduces the number of circulating Tregs and the levels of TGFβ1 and PMN-MDSC in the peripheral blood of patients with pancreatic cancer, but does not reduce the level of M-MDSC, and restores the proliferation and anti-tumor ability of effector T cells [Eriksson E. et al., "Gemcitabine reduces MDSCs, Tregs and TGFbeta-1 while restoring the Teff / Treg ratio in patients with pancreatic cancer", J. Transl. Med. 2016; 14: 282]. 5-FU can equivalently induce the death of two subtypes of MDSC and has no obvious effect on other immune cells such as T cells, NK cells, DCs and B cells. It has been reported that treatment with 5-FU triggered apoptosis of MDSCs, promoted high levels of IFNγ production by tumor-infiltrating T cells, and enhanced T cell-dependent antitumor responses in the mouse EL4 model [Incent J. et al., “5-Fluorouracil selectively kills tumor-associated myeloid-derived suppressor cells resulting in enhanced T cell-dependent antitumor immunity”, Cancer Res. 2010; 70: 3052–3061].It has been reported that 5-FU significantly and specifically eliminates MDSCs by inducing apoptosis in the TME and spleen of tumor-bearing mice [Vincent J. et al., “5-Fluorouracil selectively kills tumor-associated myeloid-derived suppressor cells resulting in enhanced T cell-dependent antitumor immunity”, Cancer Res. 2010; 70: 3052–3061]. However, the assembly of NLRP3 in MDSCs is activated by 5-FU, which reportedly leads to the secretion of MDSC-derived IL-1β and CD4+ T cell-derived IL-17 and inhibits the antitumor effect of 5-FU. Based on this, combined administration of 5-FU and IL-1β inhibitors, such as the indirect inhibitors DHA and SP600125, may provide an effective means for inhibiting MDSC [Dumont A. et al., "Docosahexaenoic acid inhibits both NLRP3 inflammasome assembly and JNK-mediated mature IL-1beta secretion in 5-fluorouracil-treated MDSC: Implication in cancer treatment", Cell Death Dis. 2019; 10: 485.; Bruchard M. et al., "Chemotherapy-triggered cathepsin B release in myeloid-derived suppressor cells activates the Nlrp3 inflammasome and promotes tumor growth", Nat. Med. 2013; 19: 57–64]. Docetaxel, which has the same effect as paclitaxel, has been reported to significantly inhibit tumor growth. Docetaxel achieves its anti-tumor effect by polarizing MDSCs into M1 macrophages and reducing the proportion of MDSCs in the spleen [Kodumudi KN et al., “A novel chemoimmunomodulating property of docetaxel: Suppression of myeloid-derived suppressor cells in tumor bearers”, Clin. Cancer Res. 2010; 16: 4583–4594.].ApoE hinders tumor invasion and endothelial cell recruitment, but liver-X receptor (LXR) inhibits ApoE expression. It has been reported that LXR agonists GW3965 and RGX-104 impair MDSC survival and enhance the anti-tumor activity of CTLs by activating the LXR / ApoE axis [Tavazoie MF et al., "LXR / ApoE Activation Restricts InnateImmune Suppression in Cancer", Cell. 2018; 172: 825–840.; Liang H. et al., "LXR activation radiosensitizes non-small cell lung cancer by restricting myeloid-derived suppressor cells", Biochem. Biophys. Res. Commun. 2020; 528: 330–335]. CD33 is highly expressed on MDSCs in humans, particularly M-MDSCs, but CD33 is a therapeutic target on circulating and tumor-infiltrating MDSCs in multiple cancer types [Lamba JK et al., “CD33 splicing polymorphism determines gemtuzumabozogamicin response in de novo acute myeloid leukemia: Report from randomized phase III children's oncology group trial AAML0531”, J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. 2017; 35: 2674–2682].The immunotoxin gemtuzumab ozogamicin, a CD33 monoclonal antibody (mAb), effectively eliminates MDSCs and reactivates T cells to fight a variety of cancers [Lamba JK et al. “CD33splicing polymorphism determines gemtuzumab ozogamicin response in de novo acute myeloid leukemia: Report from randomized phase III children's oncology group trial AAML0531”, J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. 2017; 35: 2674–2682; Fultang L. et al., “MDSC targeting with Gemtuzumab ozogamicin restores T cell immunity and immunotherapy against cancers”, EBioMedicine. 2019; 47: 235–246.]. In addition, it was reported that targeting the bromodomain and extra-terminal domain (BET), a component of the endogenous transcriptional enhancer of MDSCs, by treating HCC patient-derived PBMCs with the small molecule inhibitor i-BET762 significantly reduced the number of CD14+HLA-DR- / low M-MDSCs and enhanced the effect of immunotherapy [Liu M. et al., “Targeting monocyte-intrinsic enhancer reprogramming improves immunotherapy efficacy in hepatocellular carcinoma”, Gut. 2020; 69: 365–379.].

[0141] iii. Combination of NEO-201 with drugs that block MDSC migration

[0142] It has been reported that blocking the migration of MDSCs can effectively reduce the proportion of MDSCs in the TME and periphery by hindering the response of MDSCs to chemokines [De Sanctis F. et al., “MDSCs in cancer: Conceiving new prognostic “The tumor microenvironment innately modulates cancer progression”, Cancer Res. 2019; 79: 4557–4566]. It has been reported that antagonists of chemokines can help prevent MDSCs, especially PMN-MDSCs, from reaching tumor sites and changing the immunosuppressive microenvironment [Zhou J et al., “Neutrophils and PMN-MDSC: Their biological role and interaction with stromal cells”, Semin. Immunol. 2018; 35: 19–28]. CXCR2 is an important chemokine receptor for MDSC trafficking [Park SM et al., “Role of myeloid-derived suppressor cells in immune checkpoint inhibitor therapy in cancer", Arch.Pharm.Res.2019;42:560–566.; Cheng Y et al., "Potential roles and targeted therapy of the CXCLs / CXCR2 axis in cancer and inflammatory diseases", Biochim.Biophys.Acta Rev.Cancer.2019;1871:289–312.].It has been reported that blocking the CXCR2 / CXCL pathway by CXCR2 inhibitors such as SX-682, reparixin, and SB225002 can effectively reduce the infiltration of MDSCs and improve the function of cytotoxic T cells [Yan G et al., “ARIPK3-PGE2 circuit mediates myeloid-derived suppressor cell-potentiated colorectal carcinogenesis”, Cancer Res. 2018; 78: 5586–5599.; Liao W et al., “KRAS-IRF2 axis drives immune suppression and immune therapy resistance in colorectal cancer”, Cancer Cell. 2019; 35: 559–572.; Ocana A. et al., “Neutrophils in cancer: Prognostic role and therapeutic strategies:, Mol. Cancer. 2017; 16: 137. doi: 10.1186 / s12943-017-0707-7.].It has been reported that the progression and invasiveness of various tumors can be inhibited by targeting the CCR5 / CCL axis [Tan MC et al., “Disruption of CCR5-dependent homing of regulatory T cells inhibits tumor growth in amurine model of pancreatic cancer”, J. Immunol. 2009; 182: 1746–1755.; Zhang X. et al., “Anibamine, a natural product CCR5 antagonist, as a novel lead for the development of anti-prostate cancer agents”, Bioorganic Med. Chem. Lett. 2010; 20: 4627–4630.; Velasco-Velázquez M. et al., “CCR5 antagonist blocks metastasis of basal breast cancer cells”, Cancer Res. 2012; 72: 3839–3850; Halama N. et al., “Tumoral immune cell exploitation in colorectal cancer metastases can be targeted effectively by Anti-CCR5 therapy in cancer patients”, Cancer Cell. 2016; 29: 587–60]. It has been reported that administration of mCCR5-Ig to neutralize CCR5 ligands reduces the migration of MDSCs and Tregs without affecting the recruitment of effector T cells to the TME [Blattner C. et al., “CCR5(+)myeloid-derived suppressor cells are enriched and activated in melanoma lesions”, Cancer Res. 2018; 78: 157–167]. The CXCR4 receptor of CXCL12 (also known as stromal cell-derived factor 1, SDF-1) also mediates the recruitment of MDSCs.Neutralization of CXCR4 by antagonists such as AMD3100 has been reported to reduce the number of MDSCs and Tregs and promote M2 to M1 macrophage polarization in the TME [Wang J. et al., “CXCR4 antagonist AMD3100 (plerixafor): “From an impurity to atherapeutic agent”, Pharmacol. Res. 2020: 105010.; Zhuang Y. et al., “CD8(+)T cells that produce interleukin-17regulate myeloid-derived suppressor cells and areassociated with survival time of patients with gastric cancer”, Gastroenterology, 2012; 143: 951–962.]. In addition, colony stimulating factor-1 receptor (CSF-1R) is a tyrosine kinase receptor that has been reported to induce the formation and trafficking of MDSCs to tumor sites when combined with the receptor.Recently, it has been reported that blocking the CSF-1R signaling pathway by CSF-1R inhibitors such as RG7155 and PLX647 reportedly leads to the elimination of MDSCs or the inhibition of their pro-tumor functions and the reprogramming of TAMs [Law AMK, “Myeloid-derived suppressor cells as a therapeutic target for cancer”, Cells 2020;9:561.; Holmgaard RB “Targeting myeloid-derived suppressor cells with colony stimulating factor-1 receptor blockade can reverse immune resistance to immunotherapy in indoleamine 2,3-dioxygenase-expressing tumors”, EBioMedicine, 2016;6:50–58; Mitchem JB et al., “Targeting tumor-infiltrating macrophages decreases tumor-initiating cells, relieves immunosuppression, and improves chemotherapeutic responses”, Cancer Res. 2013; 73:1128–1141; Lonardi S. et al., “Potential contribution of tumor-associated slan(+) cells as anti-CSF-1R targets in human carcinoma”, J. Leukoc. Biol. 2018; 103: 559–564.].

[0143] iv. Combination of NEO-201 with compounds that induce MDSC differentiation

[0144] Another means for targeting MDSC is by inducing MDSC differentiation into cells with a proinflammatory phenotype. All-trans retinoic acid (ATRA) is a metabolic intermediate of vitamin A and has been identified as an anticancer drug that induces MDSC differentiation into DCs and macrophages [Fleming V. et al., "Targeting myeloid-derived suppressor cells to bypass tumor-induced immunosuppression:, Front. Immunol. 2018; 9: 398.; Nefedova Y. et al., "Mechanism of all-trans retinoic acid effect on tumor-associated myeloid-derived suppressor cells", Cancer Res. 2007; 67: 11021–11028; Schneider AK et al., "The multifaceted immune regulation of bladder cancer”, Nat. Rev. Urol. 2019; 16: 613–630.]. ATRA reportedly induces differentiation of MDSCs in vivo and in vitro, which reportedly greatly reduces the number of MDSCs. The specific mechanism postulated is that the added ATRA activates ERK1 / 2 signaling, which further upregulates the expression of glutathione synthetase in MDSCs, leading to increased glutathione levels, neutralization of generated ROS, and inhibition of MDSC inhibitory activity [Ohl K., et al., “Reactive oxygen species as regulators of MDSC-mediated immunesuppression”, Front.Immunol.2018;9doi:10.3389 / fimmu.2018.02499]. In addition, myeloid cells were reported to differentiate in response to treatment with ATRA. In addition, vitamin D3 was also reported to promote the differentiation of MDSCs. MDSCs at tumor sites had higher levels of vitamin D receptors compared to MDSCs in the spleen and bone marrow. Treatment with the active form of vitamin D3 (1α,25-dihydroxyvitamin D3, 1,25(OH)D) was reported to significantly reduce the T cell suppressive capacity of MDSCs.In vitro derived MDSCs reduced NO production upon stimulation with 1,25(OH)D [Fleet JC et al., “1alpha,25Dihydroxyvitamin D (1,25(OH)2D) inhibits the T cell suppressive function of myeloid derived suppressor cells (MDSC)”, J. Steroid Biochem. Mol. Biol. 2020; 198: 105557]. Another study reported that the addition of 1,25(OH)D abolished IL-6-induced MDSC accumulation [Chen PT et al., “1alpha,25-Dihydroxyvitamin D3 Inhibits esophageal squamous cell carcinoma progression by reducing IL6 Signaling”, Mol. Cancer Ther. 2015; 14: 1365–1375.]. Based on the above, combining NEO-201 with MDSC-targeted therapies should further reduce the number and function of MDSCs at the tumor site and in the circulation.

[0145] Combination of NEO-201 and epigenetic therapy

[0146] Epigenetic therapy is another reported method for targeting MDSCs to treat cancer. Reported epigenetic treatment methods mainly include treatment with histone methyltransferase inhibitors (HMTis), histone deacetylase inhibitors (HDACis), and DNA methyltransferase inhibitors (DNMTis) [Gomez S. et al., "Combining epigenetic and immune therapy to overcome cancer resistance", Semin. Cancer Biol. 2019]. Zeste enhancer homolog 2 (EZH2), a gene encoding a histone methyltransferase, is often overexpressed in a variety of cancer types [Zhou J. et al. "Targeting EZH2 histone methyltransferase activity eases experiments intestinalitis", Nat. Commun. 2019; 10: 2427.]. It has been reported that treatment with the EZH2 inhibitor GSK343 ​​significantly increased the number of functional MDSCs in a mouse model of colorectal cancer or in vitro

[156] . Similarly, the use of another inhibitor, GSK126, also promoted the proliferation of MDSCs. The combination of anti-Gr1 antibody or gemcitabine / 5-FU with GSK126 can relieve the immunosuppression of MDSCs and increase the number of tumor-infiltrating T cells [Huang S., Wang Z., Zhou J., Huang J., Zhou L., Luo J., Wan YY, Long H., Zhu B. EZH2 inhibitor GSK126 suppresses antitumor immunity by driving production of myeloid-derived suppressor cells. Cancer Res. 2019; 79: 2009–2020].HDAC2 silences the transcription of the retinoblastoma (Rb) gene through epigenetic modification; therefore, M-MDSCs acquire partial phenotypes and functions of PMN-MDSCs in tumor-bearing mice [Youn JI, Kumar V., Collazo M., Nefedova Y., Condamine T., Cheng P., Villagra A., Antonia S., McCaffrey JC, Fishman M. et al. “Epigenetic silencing of retinoblastoma gene regulates pathologic differentiation of myeloid cells incancer”, Nat. Immunol. 2013; 14: 211–220]. It has been reported that the DNMTi 5-azacytidine (AZA) increases the proportion of CD8+ T cells and NK cells in the TME through type I IFN immune responses, reduces the accumulation of MDSCs and promotes antitumor effects. It has been reported that the addition of the HDACi entinostat (ENT) to AZA can further enhance the regulation of the immune microenvironment.Triple or quadruple therapy with AZA and ENT plus immunotherapy anti-PD-1 and anti-CTLA-4 showed highly effective tumor elimination [Stone ML et al., “Epigenetic therapy activates type I interferon signaling in murineovarian cancer to reduce immunosuppression and tumor burden”, Proc. Natl. Acad. Sci. USA. 2017;114:E10981–E10990; Kim K. et al., “Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells”, Proc. Natl. Acad. Sci. USA. 2014;111:11774–11779; Lu Z. et al. “Epigenetic therapy inhibits metastases by disrupting premetastatic niches”, Nature. 2020;579:284–290; Zhang Z. et al., “Glucocorticoidspromote the onset of acute experimental colitis and cancer by upregulatingmTOR signaling in intestinal epithelial cells,” Cancers. 2020; 12:945.].Adjuvant epigenetic therapy using AZA and ENT reportedly blocks the migration of MDSCs by downregulating CCR2 and CXCR2, which leads to the disturbance of MDSC differentiation into macrophages and pMNs [Lu Z. et al., “Epigenetic therapy inhibits metastases by disrupting premetastatic niches”, Nature. 2020; 579: 284–290.; Wang X., Bi Y. et al., “The calcineurin-NFAT axis controls allograft immunity in myeloid-derived suppressor cells through reprogramming Tcell differentiation”, Mol. Cell. Biol. 2015; 5: 598–609; Liu G. et al., “SIRT1 limits the function and fate of myeloid-derived suppressor cells in tumors by orchestrating HIF-1α-dependent glycolysis”, Cancer Res. 2014; 74: 727–737.].

[0147] Combination with immune checkpoint inhibitors

[0148] In some embodiments, the additional therapeutic agent administered with the NEO-201 antibody is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-CD28 antibody, an anti-TIGIT antibody, an anti-LAGS antibody, an anti-TIM3 antibody, an anti-GITR antibody, an anti-4-1BB antibody, or an anti-OX-40 antibody. In some embodiments, the additional therapeutic agent is an anti-TIGIT antibody. In some embodiments, the additional therapeutic agent is an anti-LAG-3 antibody selected from the group consisting of: BMS-986016 and LAG525. In some embodiments, the additional therapeutic agent is an anti-OX-40 antibody selected from the following: MEDI6469, MEDI0562, and MOXR0916. In some embodiments, the additional therapeutic agent is an anti-4-1BB antibody PF-05082566.

[0149] In some embodiments, the additional therapeutic agent is a therapeutic agent that targets immune checkpoints. Immune checkpoints are molecules in the immune system that turn up signals (e.g., co-stimulatory molecules) or turn down signals. Inhibitory checkpoint molecules that can be targeted by immune checkpoint blockade include adenosine A2A receptor (AZAR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAGS), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V domain Ig inhibitor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0150] Immune checkpoint inhibitors can be drugs, such as small molecules, recombinant forms of ligands or receptors, or in particular antibodies, such as human antibodies (e.g., International Patent Publication WO2015016718; Pardoll, Nat Rev Cancer, 12 (4): 252-64, 2012; both are incorporated herein by reference). Known inhibitors of immune checkpoint proteins or their analogs can be used, in particular chimeric, humanized or human antibodies can be used. As known to the skilled person, for some antibodies mentioned in the present disclosure, alternative and / or equivalent names can be used. In the context of the present invention, such alternative and / or equivalent names are interchangeable. For example, it is well known that lambrolizumab is also known as MK-3475 and pembrolizumab.

[0151] It is envisioned that any of the immune checkpoint inhibitors known in the art that stimulate immune responses can be used. This includes inhibitors that directly or indirectly stimulate or enhance antigen-specific T lymphocytes. These immune checkpoint inhibitors include, but are not limited to, agents that target immune checkpoint proteins and pathways involving PD-L2, LAG3, BTLA, B7H4, and TIM3. For example, LAG3 inhibitors known in the art include soluble LAG3 (IMP321, or LAG3-Ig disclosed in WO2009044273) and mouse or humanized antibodies that block human LAG3 (e.g., IMP701 disclosed in WO2008132601), or fully human antibodies that block human LAG3 (such as disclosed in EP 2320940). Another example is provided by using a blocker for BTLA, the blocker including, but not limited to, antibodies that block the interaction of human BTLA with its ligand (such as 4C7 disclosed in WO2011014438). Another example is provided by using an agent that neutralizes B7H4, including but not limited to antibodies against human B7H4 (disclosed in WO 2013025779 and WO2013067492) or a soluble recombinant form of B7H4 (as disclosed in US20120177645). Another example is provided by an agent that neutralizes B7-H3, including but not limited to antibodies that neutralize human B7-H3 (e.g., MGA271 disclosed as BRCA84D and derivatives in US 20120294796). Another example is provided by an agent that targets TIM3, including but not limited to antibodies that target human TIM3 (e.g., as disclosed in WO 2013006490 A2, or Jones et al., J Exp Med. 2008; 205 (12): anti-human TIM3 blocking antibody F38-2E2 disclosed in 2763-79).

[0152] In addition, more than one immune checkpoint inhibitor (e.g., anti-PD-1 antibody and anti-CTLA-4 antibody) can be used in combination with NEO-201. For example, p53 gene therapy and immune checkpoint inhibitors (e.g., anti-MR antibody and / or anti-PD-1 antibody) can be administered to enhance innate anti-tumor immunity, followed by IL24 gene therapy and immune checkpoint inhibitors (e.g., anti-PD-1 antibody) to induce adaptive anti-tumor immune responses.

[0153] The present invention specifically encompasses methods for treating cancer involving MDSC immunosuppression in an individual or delaying progression of cancer, the method comprising administering to the individual an effective amount of a PD-1 axis binding antagonist in combination with NEO-201. For example, PD-1 axis binding antagonists include PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists.

[0154] In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In a specific aspect, the PD-1 ligand binding partner is and / or PD-L2. In another embodiment, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In a specific aspect, the PD-L1 binding partner is PD-1 and / or B7-1. In another embodiment, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding partner. In a specific aspect, the PD-L2 binding partner is PD-1. The antagonist can be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Patents Nos. 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are known in the art, such as described in U.S. Patent Application Nos. US20140294898, US2014022021, and US20110008369, all of which are incorporated herein by reference.

[0155] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab (also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO) is an anti-PD-1 antibody described in WO2006 / 121168. Pembrolizumab (also known as MK-3475, Merck3475, lambrolizumab , KEYTRUDA and SCH-900475) are anti-PD-1 antibodies described in WO2009 / 114335. CT-011 (also known as hBAT or hBAT-1) is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224 (also known as B7-DCIg) is a PD-L2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342. Additional PD-1 binding antagonists include pidilizumab (also known as CT-011), MEDI0680 (also known as AMP-514) and REGN2810.

[0156] In some aspects, immune checkpoint inhibitors are PD-L1 antagonists, such as durvalumab (also known as MEDI4736), atezolizumab (also known as MPDL3280A) or avelumab (also known as MSB00010118C). In some aspects, immune checkpoint inhibitors are PD-L2 antagonists, such as rHIgM12B7. In some aspects, immune checkpoint inhibitors are LAG-3 antagonists, such as but not limited to IMP321 and BMS-986016. Immune checkpoint inhibitors can be adenosine A2a receptor (A2aR) antagonists, such as PBF-509.

[0157] Another immune checkpoint that may be strengthened by the elimination effect of NEO-201 on MDSC is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has GenBank accession number L15006. CTLA-4 is present on the surface of T cells and acts as a "close" switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA-4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA-4 is similar to T cell co-stimulatory protein CD28, and both molecules bind to CD80 and CD86 (also known as B7-1 and B7-2) on antigen-presenting cells. CTLA-4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA-4 is also present in regulatory T cells and may be important for its function. T cell activation by T cell receptors and CD28 leads to increased expression of CTLA-4 (inhibitory receptor for B7 molecules).

[0158] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the methods of the present invention can be produced using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies can be used. For example, anti-CTLA-4 antibodies disclosed in the following can be used in the methods disclosed herein: U.S. Pat. No. 8,119,129, WO 01 / 14424, WO 98 / 42752; WO 00 / 37504 (CP675,206, also known as tremelimumab; formerly known as tesimumab), U.S. Pat. No. 6,207,156; Hurwitz et al. (1998) Proc Natl Acad Sci USA 95(17):10067-10071; Camacho et al. (2004) J Clin Oncology 22(145):Abstract No. 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res 58:5301-5304. The teachings of each of the foregoing publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 may also be used. For example, humanized CTLA-4 antibodies are described in International Patent Application Nos. WO2001014424, WO2000037504, and U.S. Patent No. 8,017,114; all are incorporated herein by reference.

[0159] An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy) or an antigen-binding fragment and variant thereof (see, e.g., WO 01 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding and / or binds to the same epitope on CTLA-4 as the above antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above antibodies (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).

[0160] Other molecules used to modulate CTLA-4 include CTLA-4 ligands and receptors as described in U.S. Pat. Nos. 5,844,905, 5,885,796, and International Patent Application Nos. WO1995001994 and WO1998042752; all incorporated herein by reference, and immunoadhesins as described in U.S. Pat. No. 8,329,867 (incorporated herein by reference).

[0161] Another immune checkpoint that may enhance the elimination of gMDSCs by NEO-201 is a CSF-1 / 1R binder or inhibitor (e.g., an anti-CSF1 or anti-CSF1R antibody), wherein the combination is used to treat cancer, such as a cancer described herein, such as a solid tumor involving MDSCs. In certain embodiments, the CSF-1 / 1R binder is a CSF-1R tyrosine kinase inhibitor, 4-((2-(((1R,2R)-2-hydroxycyclohexyl)amino)benzo[d]thiazol-6-yl)oxy)-N-methylpicolinamide (Compound A15), or a compound disclosed in PCT Publication No. WO 2005 / 073224. In certain embodiments, the CSF-1 / 1R binder is an M-CSF inhibitor, compound A33, or a CSF-1 binder disclosed in PCT Publication No. WO 2004 / 045532 or PCT Publication No. WO 2005 / 068503, including RX1 or 5H4 (e.g., an antibody molecule or Fab fragment against M-CSF). In certain embodiments, the CSF-1 / 1R binder is 4-(2-((1R,2R)-2-hydroxycyclohexylamino)benzothiazol-6-yloxy)-N-methylpicolinamide or BLZ-945. 4-(2-((1R,2R)-2-hydroxycyclohexylamino)benzothiazol-6-yloxy)-N-methylpicolinamide is disclosed as Example 157 on page 117 of PCT Publication No. WO 2007 / 121484. In certain embodiments, the CSF-1 / 1R binder is pexidartinib (CAS Registry No. 1029044-16-3). Pyritinib is also known as PLX3397 or 5-((5-chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)methyl)-N-((6-(trifluoromethyl)pyridin-3-yl)methyl)pyridin-2-amine. Pyritinib is a small molecule receptor tyrosine kinase (RTK) inhibitor of KIT, CSF1R and FLT3. In certain embodiments, the CSF-1 / 1R binder is imiquizumab. imiquizumab is also known as RG7155 or R05509554. imiquizumab is a humanized IgG1 mAb targeting CSF1R. In certain embodiments, the CSF-1 / 1R binder is FPA008. FPA008 is a humanized mAb that inhibits CSF1R.

[0162] Other therapeutic agents whose combination with NEO-201 may induce synergistic effects in treating diseases in which MDSCs are involved in the pathology include (a) microtubule inhibitors, topoisomerase inhibitors, platinum, alkylating agents, and antimetabolites; (b) MK-2206, ON013105, RTA 402, BI 2536, sorafenib, ISIS-STAT3Rx, microtubule inhibitors, topoisomerase inhibitors, platinum, alkylating agents, antimetabolites, paclitaxel, gemcitabine, doxorubicin, vinblastine, etoposide, 5-fluorouracil, carboplatin, hexamethylmelamine, aminoglutethimide, amsacrine, anastrozole, azacitidine, bleomycin, busulfan, carmustine, chlorambucil, 2-chlorodeoxyadenosine, cisplatin, colchicine, cyclophosphamide, cytarabine, cytoxan, dacarbazine, actinomycin D, daunorubicin, docetaxel, estramustine phosphate, floxuridine, fludarabine, gemtuzumab, hexamethylmelamine, hydroxyurea, ifosfamide, imatinib, interferon, irinotecan, lomustine, nitrogen mustard, melphalan, 6 - mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, pentostatin, procarbazine, rituximab, streptozotocin, tamoxifen, temozolomide, teniposide, 6-thioguanine, topotecan, trastuzumab, vincristine, vindesine and / or vinorelbine; (c) 1-D-ribofuranosyl-1,2,4-triazole-3-carboxamide, 9->2-hydroxy-ethoxymethylguanine, amantadine, 5-iodo-2'-deoxyuridine, trifluorothymidine, interferon, adenine arabinoside, protease inhibitors, thymidine kinase inhibitors, sugar or glycoprotein synthesis inhibitors, structural protein synthesis inhibitors, attachment and adsorption inhibitors and nucleoside analogs, such as acyclovir, penciclovir, valacyclovir and ganciclovir; (d) PD-1 inhibitors or anti-PD-1 antibodies, such as (pembrolizumab), (nivolumab) or LIBTAYO (cemiplimab); (e) a PD-L1 inhibitor or anti-PD-L1 antibody, such as TECENTRIQ (atezolizumab), IMFINZI (durvalumab), or BAVENCIO (avelumab); or (f) a CTLA-4 inhibitor or anti-CTLA-4 antibody, such as Ipilimumab. It is predicted that the combination of immune checkpoint inhibition (PD-1 inhibition, PD-L1 inhibition and / or CTLA-4 inhibition) with NEO-201 may be particularly effective for treating hematological malignancies. See Vargas et al., Immunity. 2017 Apr 18; 46(4): 577–586 and Taylor et al., J Clin Invest. 2017; 127(9): 3472–3483, each of which is hereby incorporated by reference in its entirety.

[0163] Other treatment options include radiation therapy, the combination of which with NEO-201 may elicit a synergistic effect in treating conditions in which MDSCs are involved in the pathology. NEO-201 may increase the efficacy of such other therapeutic agents or treatment options, particularly in individuals who are resistant or develop resistance or intolerance to treatment with a particular therapeutic agent or treatment option due to MDSC-induced immunosuppression.

[0164] Combination of NEO-201 and cell therapy

[0165] NEO-201 should also improve the efficacy of immune cell therapies, such as CAR-T and CAR-NK cell therapies, due to its ability to eliminate gMDSCs, particularly during the use of CAR-T and CAR-NK cells to treat cancer, infection, autoimmune and inflammatory indications.

[0166] Use of NEO-201 and CAR-T cells

[0167] Chimeric antigen receptor T cells (also known as CAR T cells) are T cells that are genetically engineered to produce artificial T cell receptors for use in immunotherapy. Chimeric antigen receptors (CARs, also known as chimeric immune receptors, chimeric T cell receptors, or artificial T cell receptors) are receptor proteins that have been engineered to give T cells new abilities to target specific proteins. The receptors are chimeric because they combine antigen binding and T cell activation functions into a single receptor.

[0168] In some embodiments, CAR T cell therapy uses T cells engineered with CAR for cancer therapy. The premise of CAR-T immunotherapy is to modify T cells to recognize target cells, such as cancer cells, so as to more effectively target and destroy them. T cells are harvested, genetically altered, and then infused into the patient, where the resulting CAR T cells selectively attack target cells (e.g., tumor cells, infected cells, or autoimmune cells) or cause an effect on target cells. CAR T cells include CD4 + and CD8 + T cells and combinations thereof.

[0169] CAR T cells can be derived from T cells in the patient's own blood (autologous), or from T cells (allogeneic) from another healthy donor. Once separated from the human body, these T cells are genetically engineered to express specific CARs, which program them to target antigens present on the surface of tumors. After CAR-T cells are infused into the patient's body, they can act as "living drugs" for cancer cells. When they contact their target antigens on cells, CAR T cells bind to the target antigens and are activated, and then continue to proliferate and produce cytotoxicity. CAR T cells destroy cells through several mechanisms, including extensively stimulated cell proliferation, increasing their degree of toxicity to other living cells (cytotoxicity), and causing increased secretion of factors that can affect other cells (such as cytokines, interleukins, and growth factors).

[0170] CAR-T cells are used to treat various blood cancers as well as solid tumors. In addition, while most CAR-T cell research focuses on creating CAR-T cells that can eradicate specific cell populations (for example, CAR-T cells targeting lymphoma cells), the technology has other potential uses. T cells can also mediate autoimmune responses to self-antigens. Regulatory T cells equipped with CARs can be used to confer tolerance to specific antigens, such as in organ transplants or autoimmune or inflammatory diseases such as lupus and RA.

[0171] A "chimeric receptor" generally refers to a cell surface receptor comprising a combination of an extracellular ligand binding domain, a transmembrane domain, and a cytoplasmic co-stimulatory signaling domain that does not naturally occur together on a single protein. This particularly includes receptors in which the extracellular domain and the cytoplasmic domain do not naturally occur together on a single receptor protein. In addition, a chimeric receptor is different from the TCR expressed in natural T cell lymphocytes.

[0172] As described in U.S. Pat. Nos. 5,359,046, 5,686,281 and 6,103,521, the extracellular domain can be obtained from any of a variety of extracellular domains or secreted proteins associated with ligand binding and / or signal transduction. The extracellular domain can be part of a protein that is monomeric, homodimeric, heterodimeric, or associated with a large number of proteins in a non-covalent complex. In particular, the extracellular domain can be composed of an Ig heavy chain, which in turn can be covalently associated with an Ig light chain by the presence of a CH1 and hinge region, or can be covalently associated with other Ig heavy / light chain complexes by the presence of hinge, CH2 and CH3 domains. In the latter case, the heavy / light chain complex connected to the chimeric construct can constitute an antibody with a specificity different from the antibody specificity of the chimeric construct. Depending on the function, desired structure and signal transduction of the antibody, the entire chain may be used, or truncated chains may be used, in which all or part of the CH1, CH2 or CH3 domains may be removed, or all or part of the hinge region may be removed.

[0173] The extracellular domain of CAR is generally derived from an immunoglobulin and includes an antigen binding portion that retains the ability to bind to an antigen, i.e., an "antigen binding site" (e.g., fragments, subsequences, complementarity determining regions (CDRs)), including (i) a Fab fragment, which is a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of a VH domain (Ward et al., (1989) Nature 341:544-546); and (vi) an isolated complementarity determining region (CDR).

[0174] Chimeric receptors can be designed to treat any cancer for which there is or can be produced a specific monoclonal antibody. In particular, cancers such as neuroblastoma, small cell lung cancer, melanoma, ovarian cancer, renal cell carcinoma, colon cancer, Hodgkin's lymphoma, and acute lymphoblastic leukemia (e.g., childhood acute lymphoblastic leukemia) have antigens that can be targeted by such chimeric receptors.

[0175] The transmembrane domain can be provided by a protein that provides a multispecific extracellular inducer aggregation domain, a protein that provides an effector function signaling domain, a protein that provides a proliferation signaling portion, or a completely different protein. In most cases, it will be convenient to naturally associate the transmembrane domain with one of the domains. In some cases, it will be desirable to employ a transmembrane domain of a ζ, η, or FcεR1γ chain containing a cysteine ​​residue capable of disulfide bonding so that the resulting chimeric protein will be able to form a disulfide-linked dimer with itself or with an unmodified version of a ζ, η, or FcεR1γ chain or related protein. In some cases, the transmembrane domain will be selected or modified by amino acid substitution to avoid such domains from binding to the transmembrane domain of the same or different surface membrane proteins so as to minimize interactions with other members of the receptor complex. In other cases, it will be desirable to use ζ, η or FcεR1γ chain and -β, MB1 (Igα), B29 or CD3γ, ζ or ε transmembrane domains to maintain physical association with other members of the receptor complex. Examples of suitable transmembrane regions for the present invention include constant (Fc) regions of immunoglobulins, human CD8a and artificial joints, which are used to move the targeting portion away from the cell surface to improve access and binding to target cells, but any transmembrane region sufficient to anchor CAR in the membrane can be used. Those skilled in the art understand that transmembrane domains are produced in many membrane proteins and therefore can replace many transmembrane regions and structural elements (such as lipophilic amino acid regions) of any convenient sequence.

[0176] The cytoplasmic domain of the chimeric receptor of the present invention may include a signaling domain (e.g., a co-stimulatory signaling domain) by itself or in combination with any other desired cytoplasmic domain useful in the context of this chimeric receptor type (e.g., a 4-1BB signaling domain, a CD3 ζ signaling domain, and / or a CD28 signaling domain). 4-1BB, CD3 ζ, and CD28 signaling domains have been fully characterized, including, for example, their use in chimeric receptors. In one embodiment, the cytoplasmic domain of the chimeric receptor may include a 4-1BB signaling domain by itself or in combination with any other desired cytoplasmic domain useful in the context of this chimeric receptor type. In the most preferred embodiment of the present invention, the extracellular domain comprises a single chain variable domain of a monoclonal antibody, the transmembrane domain comprises a hinge and a transmembrane domain of CD8 α, and the cytoplasmic domain comprises a signaling domain of CD3 ζ and a signaling domain of 4-1BB. The CD8 alpha hinge and transmembrane domain consists of 69 amino acids translated from 207 nucleotides at positions 815-1021 of GenBank Accession No. NM_001768. The CD3 zeta signaling domain of the preferred embodiment contains 112 amino acids translated from 339 nucleotides at positions 1022-1360 of GenBank Accession No. NM_000734.

[0177] In adoptive immunotherapy, circulating lymphocytes or tumor-infiltrating lymphocytes of the patient are separated in vitro, activated by lymphokines such as IL-2 or transduced with genes for tumor necrosis, and re-administered. To achieve this, an immunologically effective amount of activated lymphocytes will be administered to an animal or human patient, the activated lymphocytes being genetically modified to express a tumor-specific chimeric receptor gene as described herein. The activated lymphocytes will most preferably be the patient's own cells, which have been previously isolated from blood or tumor samples and activated and expanded in vitro. Antigen-specific CAR-T cells can be expanded in vitro for adoptive cellular immunotherapy, where the infusion of such cells has been shown to have anti-tumor reactivity in tumor-bearing hosts.

[0178] Genetic modification for introducing CAR constructs into T cells can be achieved by transducing (or otherwise delivering) T cell compositions with recombinant DNA or RNA constructs (e.g., vectors). A carrier can be any agent capable of delivering or maintaining nucleic acids in a host cell, and includes viral vectors (e.g., retroviral vectors, lentiviral vectors, adenoviral vectors, or adeno-associated viral vectors), plasmids, naked nucleic acids, nucleic acids complexed with polypeptides or other molecules, and nucleic acids fixed to solid phase particles. Appropriate DNA sequences can be inserted into vectors by a variety of procedures. In general, DNA sequences are inserted into appropriate restriction endonuclease sites by procedures known in the art. Such programs and other programs are considered to be within the scope of those skilled in the art.

[0179] The selection of promoters and other regulatory sequences for protein expression is well known to those skilled in the art. Cell-specific promoters for expression in T cells include, but are not limited to, human CD2, distal Lck, and proximal Lck. In other embodiments, non-tissue-specific promoters may be used, such as non-tissue-specific promoters including viral promoters such as cytomegalovirus (CMV) promoters, beta-actin promoters, phosphoglycerate kinase (PGK) promoters, ubiquitin promoters, and EF-1α promoters. This list is not intended to be restrictive. The expression construct preferably also includes a sequence that allows the expression construct to replicate. The transcription of the DNA encoding the polypeptide of the present invention by higher eukaryotic cells can be increased by inserting an enhancer sequence into a vector. An enhancer is a cis-acting element of DNA, usually about 10 to 300 bp, which acts on a promoter to increase its transcription. Examples include the SV40 enhancer (bp 100 to 270) located at the rear side of the replication initiation point, the cytomegalovirus early promoter enhancer, the polyoma enhancer located at the rear side of the replication initiation point, and the adenovirus enhancer. Preferably, a retroviral vector (γ-retrovirus or slow virus) is used to introduce the CAR nucleic acid construct into the cell.For example, a polynucleotide encoding a co-stimulatory ligand protein (e.g., tumor necrosis factor (TNF) ligand, such as 4-1BBL, OX40L, CD70, LIGHT and CD30L, or an Ig superfamily ligand, such as CD80 and CD86) or a receptor or its variant or fragment of an antigen binding protein may be cloned into a retroviral vector, and expression may be driven from its endogenous promoter, a retroviral long terminal repeat sequence, or a promoter specific to the target cell type. Non-viral vectors may also be used.

[0180] CAR-T cells are usually expanded and activated in vitro to reach a sufficient number of treatments before being applied to a subject. Cells can be non-specifically expanded with mitogenic αCD3 and αCD28 antibodies, or by using genetically modified antigen presenting cell lines or particles to expand, the cell lines or particles display CAR binding domains (and in some cases, additional costimulatory molecules) targeted antigens. Other methods of selectively proliferating T cells to constitutively express CAR include co-expression with transgenes for selection and / or sorting at the cell killing concentration of the drug, such as using magnetic beads that recognize the introduction of proteins co-expressed with CAR. Antigen-specific amplification is preferred because CAR-mediated T cell activation is believed to depend on binding affinity to cognate antigens and increases with the increase in binding affinity to cognate antigens. If the CAR-T cells of the present invention are non-specifically expanded without activation before being treated with nucleic acid targeting agents, they can be activated in vitro before being applied to a subject, again using cell lines or particles that display antigens targeted by CAR binding domains.

[0181] Diseased cells can be from any type of cancer, any tissue or cell type source. Suitable target cells include, but are not limited to, cells from the following malignancies: leukemias, including chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), and acute lymphoblastic leukemia (ALL); multiple myeloma (MM); non-Hodgkin's lymphoma and Hodgkin's disease (lymphoma); solid tumors, including breast, lung, ovarian and testicular cancers, prostate cancer, colon cancer, melanoma, renal cell carcinoma, neuroblastoma, and head and neck tumors.

[0182] CAR and CAR-T-derived effector cells targeting any desired antigen can be designed. Examples of target antigens include, for example: 0772P (CA125, MUC16; GenBank accession number AF36148); adipose differentiation-related protein (adipophilin) ​​(lipid droplet coating protein (perilipin)-2, adipose differentiation-related protein (Adipose differentiation-related protein), ADRP, ADFP, MGC10598; NCBI reference sequence: NP-001113.2); AIM-2 (melanoma deficiency factor, PYHIN4, interferon-induced protein AIM2; NCBI reference sequence: NP-004824.1); ALDH1 A1 (aldehyde dehydrogenase 1 family member A1, ALDH1, PUMB 1, retinal dehydrogenase 1, ALDC, ALDH-E1, ALHDII, RALDH 1, EC 1.2.1.36, ALDH11, HEL-9, HEL-S-53e, HEL12, RALDH1, aldehyde dehydrogenase 1, soluble aldehyde dehydrogenase 1, hepatic cytoplasmic aldehyde dehydrogenase, ALDH class 1, epididymal luminal protein 12, epididymal luminal protein 9, epididymal secretory sperm binding protein Li 53e, retinal dehydrogenase 1, RaIDH1, aldehyde dehydrogenase family 1 member A1, cytosolic aldehyde dehydrogenase, EC 1.2.1; NCBI Reference Sequence: NP-000680.2); alpha-actinin-4 (ACTN4, actinin alpha 4, FSGS1, focal segmental glomerulosclerosis 1, non-muscle alpha-actinin 4, F-actin Cross-Linking Protein, FSGS, ACTININ-4, actinin α4 isoform, α-actinin-4; NCBI reference sequence: NP-004915.2); alpha-fetoprotein (AFP, HPAFP, FETA, α-1-fetoprotein, α-fetoglobulin, α-1-fetoprotein, α-fetoglobulin, HP; GenBank: AAB58754.1); amphiregulin (AREG, SDGF, schwannoma-derived growth factor, colorectal cell-derived growth factor, AR, CRDGF; GenBank: AAA51781.1); ARTC1 (ART1, ADP-ribosyltransferase 1, mono(ADP-ribosyl)transferase 1, ADP-ribosyltransferase C2 and C3 toxin-like 1, ART2, CD296, RT6, ADP-ribosyltransferase 2, GPI-linked NAD(P)(+)-arginine ADP-ribosyltransferase 1, EC 2.4.2.31, CD296 antigen; NP); ASLG659; ASPHDI (aspartate β-hydroxylase domain-containing 1, aspartate β-hydroxylase domain-containing protein 1, EC 1.14.11., GenBank: AAI44153.1); B7-H4 (VTCN1, T cell activation inhibitor 1 containing V-set domain, B7H4, B7 superfamily member 1, immune co-stimulatory protein B7-H4, B7h.5, T cell co-stimulatory molecule B7x, B7S1, B7X, VCTN1, H4, B7 family member, PRO1291, B7 family member H4, T cell co-stimulatory molecule B7x, T cell activation inhibitor 1 containing V-Set domain, protein B7S1; GenBank: AAZ17406.1); BAFF-R (TNFRSF13C, tumor necrosis factor receptor superfamily member 13C, BAFFR, B cell activating factor receptor, BAFF receptor, BlyS receptor 3, CVID4, BROMIX, CD268, B cell activating factor receptor body, fluphenazine, tumor necrosis factor receptor superfamily member 13C, BR3, CD268 antigen; NCBI reference sequence: NP-443177.1); BAGE-1; BCLX (L); BCR-ABL fusion protein (b3a2); β-catenin (CTNNB1, catenin (cadherin-associated protein), β1, 88 kDa, CTNNB, MRD19, catenin (cadherin-associated protein), β1 (88 kD), armadillo, catenin β-1; GenBank: CAA61107.1); BING-4 (WDR46, WD repeat domain 46, C6orf11, BING4, WD repeat-containing protein BING4, chromosome 6 open reading frame 11, FP221, UTP7, WD repeat-containing protein 46; NP); BMPR1 B (bone morphogenetic protein receptor-IB type, GenBank accession number NM-00120; NP); B-RAF (Brevican (BCAN, BEHAB, GenBank accession number AF22905); Brevican (BCAN, chondroitin sulfate proteoglycan 7, brain-enriched hyaluronan binding protein, BEHAB, CSPG7, brevican proteoglycan, brevican core protein, chondroitin sulfate proteoglycan BEHAB; GenBank: AAH27971.1); CALCA (calcitonin-related polypeptide alpha, CALC1, calcitonin 1, calcitonin, alpha-type CGRP, calcitonin gene-related peptide I, CGRP-I, CGRP, CGRP1, CT, KC, calcitonin / calcitonin-related polypeptide alpha, calcitonin (katacalcin); NP); CASP-5 (CASP5, caspase 5, apoptosis-related cysteine ​​peptidase, caspase 5, apoptosis-related cysteine ​​protease, protease ICH-3, protease TY, ICE(rel)-111, ICE(rel)III, ICEREL-III, ICH-3, caspase-5, TY protease, EC 3.4.22.58, ICH3, EC 3.4.22; NP); CASP-8; CD19 (CD19 - B lymphocyte antigen CD19 isotype 2 receptor, B4, CVID3 [Homo sapiens], NCBI Reference Sequence: NP-001761.3); CD20 (CD20 - B lymphocyte antigen CD20, transmembrane 4-domain, subfamily A, member 1, B1, Bp35, CD20, CVID5, LEU-16, MS4A2, S7; NCBI Reference Sequence: NP-690605.1); CD21 (CD21 (CR2 (complement receptor or C3DR (C3d / Epstein-Barr virus receptor) or Hs.73792 GenBank accession number M2600); (CD22 (B cell receptor CD22-B isoform, BL-CAM, Lyb-8, LybB, SIGLEC-2, FLJ22814, GenBank accession number AK02646); CD2 2; CD33 (CD33 molecule, CD33 antigen (Gp67), sialic acid-binding Ig-like lectin 3, sialic acid-binding Ig-like lectin 3, SIGLEC3, gp67, SIGLEC-3, myeloid cell surface antigen CD33, p67, Siglec-3, CD33 antigen; GenBank: AAH28152.1); CD45; CD70 (CD70-tumor necrosis factor (ligand) superfamily member 7; Table CD70; Ki-24 antigen; CD27 ligand; CD27-L; tumor necrosis factor ligand superfamily member 7; NCBI reference sequence of Homo sapiens: NP-001243.1); CD72 (CD72 (B cell differentiation antigen CD72, Lyb-; 359aa, μl: 8.66, MW: 40225, TM: 1[P] gene chromosome: 9p13.3, GenBank accession number NP-001773.); CD79a (CD79a (CD79A, CD79a, immunoglobulin-associated alpha); CD79b (CD79b (CD79B, CD79b, IGb (immunoglobulin-associated beta), B29, GenBank accession number NM-000626 or 1103867); Cdc27 (cell division cycle 27, DOS1430E, D17S978E, complex subunit 3 that promotes anaphase of cell division, complex subunit 3 that promotes anaphase of cell division, ANAPC3, APC3, CDC27Hs, H-NUC, CDC27 homolog, cell division cycle 27 homolog (Saccharomyces cerevisiae), HNUC, NUC2, complex that promotes anaphase, protein 3, cell division cycle 27 homolog, cell division cycle protein 27 homolog, Nuc2 homolog; GenBank: AAH11656.1); CDK4 (cyclin-dependent kinase 4, cell division protein kinase 4, PSK-J3, EC 2.7.11.22, CMM3, EC 2.7.11; NCBI Reference Sequence: NP-000066.1); CDKN2A (cyclin-dependent kinase inhibitor 2A, MLM, CDKN2, MTS1, cyclin-dependent kinase inhibitor 2A (melanoma, P16, inhibiting CDK4), cyclin-dependent kinase 4 inhibitor A, multiple tumor suppressor 1, CDK4I, MTS-1, CMM2, P16, ARF, INK4, INK4A, P14, P 14ARF, p16-INK4A, p16INK4, p16INK4A, p19, p19ARF, TP16, CDK4 inhibitor p16-INK4, cell cycle negative regulator β, p14ARF, p16-INK4, p16-INK4a, p16INK4A, p19ARF; NP); CEA; CLL1 (CLL-1; CLPP (caseinolytic mitochondrial matrix peptidase proteolytic subunit, endopeptidase Clp, EC 3.4.21.92,PRLTS3,ATP-dependent protease ClpAP (Escherichia coli),ClpP (caseinolytic protease, ATP-dependent, proteolytic subunit, Escherichia coli) homolog,ClpP caseinolytic peptidase, ATP-dependent, proteolytic subunit homolog (Escherichia coli),ClpP caseinolytic protease, ATP-dependent, proteolytic subunit homolog (Escherichia coli),Human,proteolytic subunit,ATP-dependent protease ClpAP,proteolytic subunit,Human,ClpP caseinolytic peptidase ATP-dependent, proteolytic subunit,ClpP caseinolytic peptidase, ATP-dependent, proteolytic subunit homolog,ClpP caseinolytic peptidase, ATP-dependent, proteolytic subunit homolog,Putative ATP-dependent Clp protease proteolytic subunit,mitochondrial;NP);COA-1;CPSF;CRIPTO(CRIPTO(CR,CR1,CRGF,CRIPTO,TDGF 1, teratoma-derived growth factor, GenBank accession number NP-003203 or NM-00321); Cw6; CXCR5; CXORF61 CXORF61 - chromosome X open reading frame 61 [Homo sapiens], NCBI Reference Sequence: NP-001017978.1); cyclin Di (CCND1, BCL1, PRAD1, D11S287E, B-cell CLL / lymphoma 1, B-cell lymphoma 1 protein, BCL-1 oncogene, PRAD1 oncogene, cyclin Dl (PRAD1: parathyroid adenoma 1), G1 / S-specific cyclin Dl, parathyroid adenoma 1, U21B31, G1 / S-specific cyclin-D1, BCL-1; NCBI Reference Sequence: NP-444284.1); cyclin-A1 (CCNA1, CT146, cyclin A1; GenBank: AAH36346.1); dek-can fusion protein; DKK1 (Dickkopf WNT signaling pathway suppressor 1, SK, hDkk-1, Dickkopf (African clawed frog) homolog 1, Dickkopf 1 homolog (African clawed frog), DKK-1, Dickkopf 1 homolog, Dickkopf-related protein-1, Dickkopf-1-like, Dickkopf-like protein 1, Dickkopf-related protein 1, Dickkopf-1, Dkk-1; GenBank: AAQ89364.1); DR1 (transcriptional down-regulator 1, TBP-binding (negative cofactor 2), negative cofactor 2-β, TATA-binding protein-associated phosphoprotein, NC2, NC2-β, protein Dr1, NC2-β, transcriptional down-regulator 1; NCBI Reference Sequence: NP-001929.1); DR13 (major histocompatibility complex, class II, DRβ1, HLA-DR1B, DRw10, DW2.2 / DR2.2, SS1, DRB1, HLA-DRB, HLA class II histocompatibility antigen, DR-1β chain, human leukocyte antigen DRB1, lymphocyte antigen DRB1, MHC class II antigen, MHC class II HLA-DRβ1 chain, MHC class II HLA-DR-β cell surface glycoprotein, MHC class II HLA-DRw10-β, DR-1, DR-12, DR-13, DR-14, DR-16, DR-4, DR-5, DR-7, DR-8, DR-9, DR1, DR12, DR13, DR14, DR16, DR4, DR5, DR7, DRB, DR9, DRw11, DRw8, HLA-DRB2, clone P2-β-3, MHC Class II antigen DRB1*1, MHC class II antigen DRB1*10, MHC class II antigen DRB1*11, MHC class II antigen DRB1*12, MHC class II antigen DRB1*13, MHC class II antigen DRB1*14, MHC class II antigen DRB1*15, MHC class II antigen DRB1*16, MHC class II antigen DRB1*3, MHC class II antigen DRB1*4, MHC class II antigen DRB1*7, MHC Class II antigen DRB1*8, MHC class II antigen DRB1*9; NP); E16 (E16 (LAT1, SLC7A5, GenBank accession number NM-00348); EDAR (EDAR--tumor necrosis factor receptor superfamily member EDAR precursor, EDA-A1 receptor; anhedral homolog; exopolysaccharide protein-A receptor; ectodermal dysplasia receptor; anhidrotic exopolysaccharide protein receptor 1, DL; ECTD10A; ECTD10B; ED1R; ED3; ED5; EDA-AIR; EDA1R; EDA3; HRM1 [Homo sapiens]; NCBI reference sequence: NP-071731.1); EFTUD2 (elongation factor Tu GTP-binding domain-containing 2, elongation factor Tu GTP-binding domain-containing protein 2, hSNU114, SNU114 homolog, U5 SnRNP-specific protein, 116KDa, MFDGA, KIAA0031, 116KD, U5 SnRNP-specific protein, 116KDa U5 small nuclear ribonucleoprotein component, MFDM, SNRNP116, Snrp116, Snull 14, U5-116KD, SNRP116, U5-116 KDa; GenBank: AAH02360.1); EGFR (epidermal growth factor receptor, ERBB, proto-oncogene C-ErbB-1, receptor tyrosine-protein kinase ErbB-1, ERBB 1, HER1, EC 2.7.10.1, epidermal growth factor receptor (avian erythroblastic leukemia virus (V-Erb-B) oncogene homolog), erythroblastic leukemia virus (V-Erb-B) oncogene homolog (avian), P1G61, avian erythroblastic leukemia virus (V-Erb-B) oncogene homolog, cell growth inhibitory protein 40, cell proliferation inducing protein 61, mENA, EC 2.7.10; GenBank: AAH94761.1); EGFR-G719A; EGFR-G719C; EGFR-G719S; EGFR-L858R; EGFR-L861 Q; EGFR-57681; EGFR-T790M; Elongation factor 2 (EEF2, eukaryotic translation elongation factor 2, EF2, polypeptidyl-tRNA translocase, EF-2, SCA26, EEF-2; NCBI Reference Sequence: NP-001952.1); ENAH (hMena) (ENAH homolog (Drosophila), MENA, mammalian ENA, ENA, NDPP1, protein ENAH homolog; GenBank: AAH95481.1) - results for only "ENAH" and not "ENAH (hMena)"; EpCAM (epithelial cell adhesion molecule, M4S1, MIC18, tumor-associated calcium signal transducer 1, TACSTD1, TROP1, adenocarcinoma-associated antigen, cell surface glycoprotein Trop-1, epithelial glycoprotein 3) 14, Major gastrointestinal tumor-associated proteins GA733-2, EGP314, KSA, DIAR5, HNPCC8, antigen identified by monoclonal antibody AUA1, EGP-2, EGP40, ESA, KS1 / 4, MK-1, human epithelial glycoprotein-2, membrane component, chromosome 4, surface marker (35kD glycoprotein), EGP, Ep-CAM, GA733-2, M1S2, CD326 antigen, epithelial cell surface antigen, hEGP314, KS1 / 4 antigen, ACSTD1; GenBank: AAH14785.1); EphA3 (EPH receptor A3, ETK1, ETK, TYRO4, HEK, Eph-like tyrosine kinase 1, tyrosine-protein kinase receptor ETK1, EK4, EPH-like kinase 4, EC 2.7.10.1, EPHA3, HEK4, ephrin type A receptor 3, human embryonic kinase 1, TYRO4 protein tyrosine kinase, hEK4, human embryonic kinase, tyrosine-protein kinase TYRO4, EC 2.7.10; GenBank: AAH63282.1); EphB2R; epiregulin (EREG, ER, pro-epiregulin; GenBank: AAI36405.1); ETBR (EDNRB, endothelin receptor type B, HSCR2, HSCR, endothelin receptor non-selective type, ET-B, ET-BR, ETRB, ABCDS, WS4A, ETB, endothelin B receptor; NP); ETV6-AML1 fusion protein; EZH2 (Enhancer of Zeste homolog 2 (Drosophila), lysine N-methyltransferase 6, ENX-1, KMT6EC2.1.1.43, EZH1, WVS, Enhancer of Zeste (Drosophila) homolog 2, ENX1, EZH2b, KMT6A, WVS2, histone-lysine N-methyltransferase EZH2, Enhancer of Zeste homolog 2, EC 2.1.1; GenBank: AAH10858.1); FcRH1 (FCRL1, Fc receptor-like 1, FCRH1, Fc receptor homolog 1, FcR-like protein 1, immune receptor translocation-associated protein 5, IFGP1, IRTA5, hIFGP1, IFGP family protein 1, CD307a, Fc receptor-like protein 1, immunoglobulin superfamily Fc receptor, Gp42, FcRL1, CD307a antigen; GenBank: AAH33690.1); FcRH2 (FCRL2, Fc receptor-like 2, SPAP1, SH2 domain-containing phosphatase anchor protein 1, Fc receptor homolog 2, FcR-like protein 2, immunoglobulin receptor translocation-associated protein 4, FCRH2, IFGP4, IRTA4, IFGP family protein 4, SPAP1A, SPAP1 B, SPAP1C, CD307b, Fc receptor-like protein 2, immune receptor translocation-associated protein 4, immunoglobulin superfamily Fc receptor, Gp42, SH2 domain-containing phosphatase anchor protein 1, FcRL2, CD307b antigen; GenBank: AAQ88497.1); FcRH5 (FCRL5, Fc receptor-like 5, IRTA2, Fc receptor homolog 5, FcR-like protein 5, immune receptor translocation-associated protein 2, BXMAS1, FCRH5, CD307 , CD307e, PRO820, Fc receptor-like protein 5, immunoglobulin superfamily receptor translocation-associated 2 (IRTA2), FCRL5, CD307e antigen; GenBank: AAI01070.1); FLT3-ITD; FN1 (fibronectin 1, cold-insoluble globulin, FN, migration stimulating factor, CIG, FNZ, GFND2, LETS, ED-B, FINC, GFND, MSF, fibronectin; GenBank: AAI43764.1); G250 (MN, CAIX, carbonic anhydrase IX, carbonic anhydrase, RCC-associated protein G250, carbonic anhydrase IX, membrane antigen MN, renal cell carcinoma-associated antigen G250, CA-IX, p54 / 58N, pMW1, RCC-associated antigen G250, carbonic anhydrase 9; NP); -- also known as "G250" instead of "G250 / MN / CAIX"; GAGE-1,2,8; GAGE-3,4,5,6,7; GDNF-Ral (GDNF family receptor alpha 1; GFRA1; GDNFR; GDNFRA; RETL1; TRNR1; RET1 L; GDNFR-αl; GFR-ALPHA-; U95847; BC014962; NM-145793NM-005264); GEDA (GenBank accession number AY26076); GFRA1-GDNF family receptor alpha-1; GDNF receptor alpha-1; GDNFR-α-1; GFR-α-1; RET ligand 1; TGF-β-related neurotrophic factor receptor 1 [Homo sapiens]; ProtKB / Swiss-Prot: P56159.2; Glypican- 3 (GPC3, Glypican 3, SDYS, Glypican proteoglycan 3, intestinal protein OCI-5, GTR2-2, MXR7, SGBS1, DGSX, OCI-5.SGB, SGBS, heparan sulfate proteoglycan, secretory Glypican-3, OCI5; GenBank: AAH35972.1); GnTVf; gp100 (PMEL, premelanosomal protein, SILV, D12S53E, PMEL17, SIL, melanocyte protein Pmel 17, melanocyte lineage-specific antigen GP100, melanoma-associated ME20 antigen, silver locus protein homolog, ME20-M, ME20M, P1, P100, silver (mouse homolog)-like, silver homolog (mouse), ME20, SI, melanocyte protein Mel 17, melanocyte protein PMEL, melanosome matrix protein 17, silver, mouse, homolog; GenBank: AAC60634.1); GPC; GPNMB (glycoprotein (transmembrane) Nmb, glycoprotein NMB, glycoprotein Nmb-like protein, osteoactivin, transmembrane glycoprotein HGFIN, HGFIN, NMB, transmembrane glycoprotein, transmembrane glycoprotein NMB; GenBank: AAH32783.1); GPR172A (G protein-coupled receptor 172A; GPCR41; FLJ11856; D15Ertd747e); NP-078807.1; NM-024531.3); GPR19 (G protein-coupled receptor 19; Mm.478; NP-006134.1; NM-006143.2); GPR54 (KISS1 receptor; KISS1R; GPR54; HOT7T175; AXOR1; NP-115940.2; NM-032551.4); HAVCR1 (hepatitis A virus cellular receptor 1, T cell immunoglobulin mucin family member 1, kidney injury molecule 1, KIM-1, KIM1, TIM, TIM-1, TIM1, TIMD-1, TIMD1, T cell immunoglobulin mucin receptor 1, T cell membrane protein 1, HAVCR, HAVCR-1, T cell immunoglobulin domain and mucin domain protein 1, HAVcr-1, T cell immunoglobulin and mucin domain-containing protein 1; GenBank: AAH13325.1); HER2 (ERBB2, V-Erb-B2 avian erythroblastic leukemia virus oncogene homolog 2, NGL, NEU, neuro / glioblastoma-derived oncogene homolog, metastatic lymph node gene 19 protein, proto-oncogene C-ErbB-2, proto-oncogene Neu, tyrosine kinase-type cell surface receptor HER2, MLN 19, p185erbB2, EC 2.7.10.1, V-Erb-B2 avian erythroblastic leukemia virus oncogene homolog 2 (neuro / glioblastoma-derived oncogene homolog), CD340, HER-2, HER-2 / neu, TKR1, C-Erb B2 / Neu protein, herstatin, neuroblastoma / glioblastoma-derived oncogene homolog, receptor tyrosine-protein kinase ErbB-2, V-Erb-B2 erythroblastic leukemia virus oncogene homolog 2, neuro / glioblastoma-derived oncogene homolog, MLN19, CD340 antigen, EC 2.7.10; NP); HER-2 / neu-alias above; HERV-K-MEL; HLA-DOB (binds peptide and presents it to CD4. +β subunit of MHC class II molecule of T lymphocytes (la antigen)); 273aa, al: 6.56, MW: 30820.TM: 1 [P] gene chromosome: 6p21.3, GenBank accession number NP-002111); hsp70-2 (HSPA2, heat shock 70kDa protein 2, heat shock 70kD protein 2, HSP70-3, heat shock associated 70KDa protein 2, heat shock 70KDa protein 2; GenBank: AAD2 1815.1); IDO1 (indoleamine 2,3-dioxygenase 1, IDO, INDO, indoleamine-pyrrole 2,3-dioxygenase, IDO-1, indoleamine-pyrrole 2,3 dioxygenase, indoleamine 2,3 dioxygenase, indole 2,3 dioxygenase, EC 1.13.11.52; NCBI Reference Sequence: NP-002155.1); IGF2B3; IL13Rα2 (IL13RA2, interleukin 13 receptor, α2, cancer / testis Pellet antigen 19, interleukin-13-binding protein, IL-13R-α-2, IL-13RA2, IL-13 receptor subunit α-2, IL-13R subunit α-2, CD213A2, CT19, IL-13R, IL13BP, interleukin 13 binding protein, interleukin 13 receptor α2 chain, interleukin-13 receptor subunit α-2, IL13R, CD213a2 antigen; NP); IL20Rα; intestinal carboxylesterase; IRTA2( alias for FcRH5); Kallikrein 4 (KLK4, Kallikrein-related peptidase 4, PRSS17, EMSP1, enamel matrix serine protease 1, kallikrein-like protein 1, serine protease 17, KLK-L1, PSTS, AI2A1, Kallikrein 4 (protease, enamel matrix, prostate), ARM1, EMSP, androgen regulated information 1, enamel matrix serine protease 1, kallikrein, kallikrein-4, protease, EC 3.4.21.-, prostate, EC 3.4.21; GenBank: AAX30051.1); KIF20A (kinesin family member 20A, RAB6KIFL, RAB6 interacting, kinesin-like (Rab kinesin 6), mitosis a; LAGE-1; LDLR-fucosyltransferase AS fusion protein; Lengsin (LGSN, Lengsin, lens protein with glutamine synthetase domain, GLULD1, glutamate-ammonia ligase domain-containing protein 1, LGS, glutamate-ammonia ligase (glutamine synthetase) domain-containing 1, glutamate-ammonia ligase (glutamine synthetase) domain-containing 1, lens glutamine synthetase-like; GenBank: AAF61255.1); LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5; GPR49,GPR6; NP-003658.1; NM-003667.2; LY64 (lymphocyte antigen 64); Ly6E (lymphocyte antigen 6 complex, locus E; Ly67, RIG-E, SCA-2, TSA-; NP-002337.1; NM-002346.2); Ly6G6D (lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT; NP-067079.2; NM-021246.2); LY6K (lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348; FLJ3522; NP-059997.3; NM-017527.3); Ly PD1-LY6 / PLAUR domain 1, PHTS [Homo sapiens], GenBank: AAH17318.1); MAGE-A1 (melanoma antigen family A, 1 (directs expression of antigen MZ2-E, MAGE1, melanoma antigen family A1, MAGEA1, melanoma antigen MAGE-1, melanoma-associated antigen 1, melanoma-associated antigen MZ2-E, antigen MZ2-E, cancer / testis antigen 1.1, CT1.1, MAGE-1 antigen, cancer / testis antigen family 1 member 1, cancer / testis antigen family 1 member 1, MAGE1A; NCBI reference sequence: NP-004979.3); MAGE-A10 (MAGEA10, melanoma antigen family A, 1; NCBI reference sequence: NP-004979.3); MAGE-A12 (MAGEA12, melanoma antigen family A, 12, MAGE12, cancer / testis antigen 1.12, CT1.12, MAGE12F antigen, cancer / testis antigen family 1 member 12, cancer / testis antigen family 1 member 12, melanoma associated antigen 12, MAGE-12 antigen; NCBI reference sequence: NP-00115985.1). 9.1); MAGE-A2 (MAGEA2, melanoma antigen family A, 2, MAGE2, cancer / testis antigen 1.2, CT1.2, MAGEA2A, MAGE-2 antigen, cancer / testis antigen family 1 member 2, cancer / testis antigen family 1 member 2, melanoma antigen 2, melanoma associated antigen 2; NCBI Reference Sequence: NP-001269434.1); MAGE-A3 (MAGEA3, melanoma antigen family A, 3, MAGE3, MAGE-3 antigen, antigen MZ2-D, melanoma associated antigen 3, cancer / testis antigen 1.3, CT1.3, cancer / testis antigen family 1 member 3, HIPS, HYPD, MAGEA6,Cancer / testis antigen family 1 member 3; NCBI Reference Sequence: NP-005353.1); MAGE-A4 (MAGEA4, melanoma antigen family A, 4, MAGE4, ​​melanoma-associated antigen 4, cancer / testis antigen 1.4, CT1.4, MAGE-4 antigen, MAGE-41 antigen, MAGE-X2 antigen, MAGE4A, MAGE4B, cancer / testis antigen family 1 member 4, MAGE-41, MAGE-X2, cancer / testis antigen family 1 member 4; NCBI Reference Sequence: NP-001011550.1); MAGE- A6 (MAGEA6, melanoma antigen family A, 6, MAGE6, MAGE-6 antigen, melanoma-associated antigen 6, cancer / testis antigen 1.6, CT1.6, MAGE3B antigen, cancer / testis antigen family 1, melanoma antigen family A6, member 6, MAGE-3b, MAGE3B, cancer / testis antigen family 1, member 6; NCBI Reference Sequence: NP-787064.1); MAGE-A9 (MAGEA9, melanoma antigen family A, 9, MAGE9, MAGE-9 antigen, melanoma-associated antigen 9, cancer / testis antigen 1.9, CT 1.9, cancer / testis antigen family 1 member 9, cancer / testis antigen family 1 member 9, MAGEA9A; NCBI Reference Sequence: NP-005356.1); MAGE-C1 (MAGEC1, melanoma antigen family C, 1, cancer / testis antigen 7.1, CT7.1, MAGE-C1 antigen, cancer / testis antigen family 7 member 1, CT7, cancer / testis antigen family 7 member 1, melanoma-associated antigen C1; NCBI Reference Sequence: NP-005453.2); MAGE-C2 (MAGEC2, melanoma antigen family C, 2, MAGEE1 , cancer / testis antigen 10, CT10, HCA587, melanoma antigen family E, 1, cancer / testis specific, hepatocellular carcinoma-associated antigen 587, MAGE-C2 antigen, MAGE-E1 antigen, hepatocellular carcinoma antigen 587, melanoma-associated antigen C2; NCBI Reference Sequence: NP-057333.1); Mammaglobin-A (SCGB2A2, secretory globulin, family 2A member 2, MGB1, Mammaglobin 1, UGB2, Mammaglobin A, Mammaglobin-A, Mammaglobin-1, secretory globulin family 2A member 2; NP); MART2 (H HAT, hedgehog acyltransferase, SKI1, melanoma antigen recognized by T cells 2, thin hedgehog 1, Skn, melanoma antigen recognized by T cells 2, protein-cysteine ​​N-palmitoyltransferase HHAT, EC 2.3.1.-; GenBank: AAH39071.1); M-CSF (CSF1, colony stimulating factor 1 (macrophages), MCSF, CSF-1, lanimostim,Macrophage colony stimulating factor 1 (Lanimostim; GenBank: AAH21117.1); MCSP (SMCP, sperm mitochondria-associated cysteine-rich protein, MCS, mitochondrial envelope selenoprotein, HSMCSGEN1, sperm mitochondria-associated cysteine-rich protein; NCBI Reference Sequence: NP-109588.2); XAGE-lb / GAGED2a; WT1 (Wilms tumor 1, WAGR, GUD, WIT-2, WT33, amino-terminal domain of EWS, NPHS4, the last three zinc fingers of the DNA binding domain of WT 1, AWT 1, Wilms tumor protein, EWS-WT1; GenBank: AAB33443.1); VEGF; tyrosinase (TYR; OCAIA; OCA1A; tyrosinase; SHEP; NP-000363.1; NM-000372.4; GenBank: AAB60319.1); TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel subfamily M member 4, GenBank accession number NM-01763); TRP2-INT2; TRP-2; TRP-1 / gp75 (tyrosinase-associated protein 1, 5,6-dihydroxyindole-2-carboxylate oxidase, CAS2, CATB, TYRP, OCAS, catalase B, b-protein, glycoprotein 75, EC 1.14.18., melanoma antigen Gp75, TYRP1, TRP, TYRRP, TRP1, SHEP11, DHICA oxidase, EC 1.14.18, GP75, EC 1.14.18.1; triosephosphate isomerase (triosephosphate isomerase 1, TPID, triosephosphate isomerase, HEL-S-49, TIM, epididymal secretory protein Li 49, TPI, triosephosphate isomerase, EC5.3.1.1; TRAG-3 (CSAG family member 2, cancer / testis antigen family 24, CSAG3B, member 2, CSAG family member 3B, cancer / testis antigen family 24 member 2, cancer / testis antigen 24.2, chondrosarcoma-associated gene 2 / 3 protein, paclitaxel resistance-associated gene 3 protein, chondrosarcoma-associated gene 2 / 3 protein-like, CT24.2, paclitaxel resistance-associated gene 3, TRAG-3, CSAG3A, TRAG3;); TMEM46 (shisa homolog 2 (African clawed frog); SHISA; NP-001007539.1; NM-001007538.1; TMEM118 (RING finger protein,Transmembrane 2; RNFT2; FLJ1462; NP-001103373.1; NM-001109903.1; TMEFF1 (transmembrane protein with EGF-like and two follistatin-like domains 1; brain tumor suppressor protein (Tomoregulin)-; H7365; C9orf2; C9ORF2; U19878; X83961; NM-080655; NM-003692; TGF-βRII (TGFBR2, transforming growth factor, beta receptor II (70 / 80 kDa), TGFβ-RII, MFS2, tβR-II, TGFR-2, TGF-β receptor type IIB, TGF-β receptor type II, TGF-β receptor type 2, EC 2.7.11.30, transforming growth factor beta receptor type IIC, AAT3, TβR-II, transforming growth factor, beta receptor II (70-80 kD), TGF-β receptor type II, FAA3, transforming growth factor-β receptor type II, LDS1 B, HNPCC6, LDS2B, LDS2, RITC, EC 2.7.11, TAAD2; TENB2 (TMEFF2, brain tumor suppressor protein, TPEF, HPP1, TR, putative transmembrane proteoglycan, related to the EGF / helix family of growth factors and follistatin); 374aa, NCBI accession: AAD55776, AAF91397, AAG49451, NCBI RefSeq: NP-057276; NCBI gene: 23671; OMIM: 605734; SwissProt Q9UIK5; GenBank accession number AF179274; AY358907, CAF85723, CQ782436; TAG-2; TAG-1 (contactin 2 (axonal), TAG-1, AXT, axonin-1 cell adhesion molecule, TAX, contactin 2 (transiently expressed), TAXI, contactin-2, axonin TAG-1, transiently expressed axonin, transient axonin, axonin-1, TAX-1, TAG1, FAMES; PRF: 444868); SYT-SSX1 or SSX2 fusion protein; survivin; STEAP2 (HGNC 8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-associated gene 1, prostate cancer-associated protein 1, prostate six-transmembrane epithelial antigen 2, six-transmembrane prostate protein, GenBank accession number AF45513; STEAP 1 (prostate six-transmembrane epithelial antigen, GenBank accession number NM-01244; SSX-4; SSX-2 (SSX2, synovial sarcoma, X breakpoint 2, X breakpoint 2, SSX, X breakpoint 2B, cancer / testis antigen 5.2, X-chromosome-associated 2,Tumor antigen HOM-MEL-40, CT5.2, HD21, cancer / testis antigen family 5, HOM-MEL-40, isoform B, cancer / testis antigen family 5 member 2a, member 2a, protein SSX2, sarcoma, sarcoma, synovium, X-chromosome related 2, synovium, synovial sarcoma, X breakpoint 2B, synovial sarcoma, SSX2A; Sp17; SOX10 (SRY (sex determining region Y)-box 10, mouse, PCWH , DOM, WS4, WS2E, WS4C, dominant megacolon, mouse, human homolog, dominant megacolon, SRY-related HMG-box gene 10, human homolog, transcription factor SOX-10; GenBank: CAG30470.1); SNRPD1 (small nuclear ribonucleoprotein D1, small nuclear ribonucleoprotein D1, polypeptide 16 kDa, polypeptide (16 kD), SNRPD, HsT2456, Sm-D1, SMD 1, Sm-D autoantigen, small nuclear ribonucleoprotein D1 polypeptide 16 kDa pseudogene, SnRNP core protein D1, small nuclear ribonucleoprotein Sm D1; SLC35D3 (solute carrier protein family 35 member D3, FRCL1, fringe-like protein 1, bA55K22.3, Frc, fringe-like 1, solute carrier protein family 35 member D3; NCBI GenBank: NC-000006.11 NC-018917.2 NT-025741.16); SIRT2 (semperceptin 2, NAD-dependent deacetylase semperceptin-2, SIRL2, silent information regulator 2, regulatory protein SIR2 homolog 2, Sir2-related protein type 2, SIR2-like protein 2, semperceptin type 2, semperceptin (silent mating type information regulatory 2 homolog) 2 (Saccharomyces cerevisiae), semperceptin-2, semperceptin (silent mating type information regulatory 2, Saccharomyces cerevisiae, homolog) 2, EC 3.5.1., SIR2; GenBank: AAK51133.1); Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, guidance protein 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain.TM. and short cytoplasmic domain, (guidance protein) 5B, GenBank accession number AB04087; secernin 1 (SCRN1, SES1, KIAA0193, secernin-1; GenBank: EAL24458.1); SAGE (SAGE1, sarcoma antigen 1, cancer / testis antigen 14, CT14, putative tumor antigen; NCBI reference sequence: NP-061136.2); RU2AS (KAAG1, kidney-associated antigen 1, RU2AS, RU2 antisense gene protein,Kidney-associated antigen 1 (RAA1; GenBank: AAF23613.1); RNF43-E3 ubiquitin-protein ligase RNF43 precursor [Homo sapiens], RNF 124; URCC; NCBI Reference Sequence: NP-060233.3; RhoC (RGS5 (Regulator of G protein signaling 5, MSTP032, Regulator of G protein signaling 5, MSTP092, MST092, MSTP106, MST106, MSTP129, MST129; GenBank: AAB84001.1); RET (ret proto-oncogene; MEN2A; HSCR1; MEN2B; MTC1; PTC; CDHF12; Hs.168114; RET51; RET-ELE; NP-066124.1; NM-020975.4); RBAF600 (UBR4, ubiquitin protein ligase E3 component N-recognition protein 4, zinc finger, UBR1 Type 1, ZUBR1, E3 ubiquitin-protein ligase UBR4, RBAF600, 600KDa retinoblastoma protein-associated factor, zinc finger UBR1-type protein 1, EC 6.3.2., N-recognition protein-4, KIAA0462, p600, EC 6.3.2, KIAA1307; GenBank: AAL83880.1); RAGE-1 (MOK, MOK protein kinase, renal tumor antigen, RAGE, MAPK / MAK / MRK overlapping kinase, renal tumor antigen 1, renal cell carcinoma antigen, RAGE-1, EC 2.7.11.22, RAGE1; UniProtKB / Swiss-Prot: Q9UQ07.1); RAB38 / NY-MEL-1 (RAB38, NY-MEL-1, RAB38, member RAS oncogene family, melanoma antigen NY-MEL-1, Rab-related GTP-binding protein, Ras-related protein Rab-38, rrGTPbp; GenBank: AAH15808.1); PTPRK (DJ480J14.2.1 (protein tyrosine phosphatase, receptor type, K R-PTP-KAPPA, protein tyrosine phosphatase kappa, protein tyrosine phosphatase kappa), protein tyrosine phosphatase, receptor type, K, protein-tyrosine phosphatase kappa, protein-tyrosine phosphatase, receptor type, kappa, R-PTP-κ, receptor type tyrosine-protein phosphatase kappa, EC3.1.3.48, PTPK; GenBank: AAI44514.1); PSMA; PSCAhIg (2700050C12Rik, C530008016Rik, RIKEN cDNA2700050C12, RIKEN cDNA2700050C12 gene, GenBank accession number AY358628); PSCA (prostate stem cell antigen precursor,GenBank accession number AJ29743; PRDX5 (peroxiredoxin 5, EC 1.11.1.15, TPx type VI, B166, antioxidant enzyme B166, HEL-S-55, liver tissue 2D-Page spot 71B, PMP20, peroxisomal antioxidant enzyme, PRDX6, thioredoxin peroxidase PMP20, PRXV, AOEB 166, epididymal secretory protein Li 55, Alu co-repressor 1, peroxidoxin-5, mitochondrial, peroxidoxin V, prx-V, thioredoxin reductase, Prx-V, ACR1, Alu co-repressor, PLP; GenBank: CAG33484.1); PRAME (preferentially expressed antigen in melanoma, preferentially expressed antigen in melanoma, MAPE, 01P-4, OIPA, CT130, cancer / testis antigen 130, melanoma antigen preferentially expressed in tumor, Opa-interacting protein 4, Opa-interacting protein 01P4; GenBank: CAG30435.1); pml-RARα fusion protein; PMEL17 (silver homolog; SILV; D12S53E; PMEL17; SI; SIL); ME20; gp10 BC001414; BT007202; M32295; M77348; NM-006928; PBF (ZNF395, zinc finger protein 395, PRF-1, Huntington's disease regulatory, HD gene regulatory region-binding protein, region binding protein 2, protein 2, papillomavirus regulatory factor 1, HD-regulatory factor 2, papillomavirus regulatory factor, PRF1, HDBP-2, Si-1-8-14, HDBP2, Huntington's disease gene regulatory region-binding protein 2, HDRF-2, papillomavirus regulatory factor PRF-1, PBF; GenB ank: AAH01237.1); PAX5 (paired box 5, paired box homeotic gene 5, BSAP, paired box protein Pax-5, B cell lineage-specific activator, paired domain gene 5, paired box gene 5 (B cell lineage-specific activator protein), B cell-specific transcription factor, paired box gene 5 (B cell lineage-specific activator); PAP (REG3A, regenerating islet-derived 3 Α, INGAP, PAP-H, enterohepatic pancreatin, PBBCGF, human proislet peptide, REG-Ill, pancreatitis-associated protein 1, Regi, Reg III-Α, hepatocarcinoma-intestinal-pancreatic, regenerative islet-derived protein III-Α, pancreatic β-cell growth factor, HIP, PAP homologous protein, HIP / PAP, proliferation-inducing protein 34, PAP1, proliferation-inducing protein 42, REG-3-α, regenerative islet-derived protein 3-Α, pancreatitis-associated protein; GenBank: AAH36776.1); p53 (TP53, tumor protein P53, TPR53,P53, cellular tumor antigen P53, antigen NY-CO-13, mutant tumor protein 53, phosphoprotein P53, P53 tumor suppressor gene, BCC7, transformation-related protein 53, LFS1, tumor protein 53, Li-Fraumeni syndrome, tumor suppressor gene P53; P2X5 (purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, and its deficiency may lead to the pathophysiology of idiopathic detrusor instability); 422aa), al: 7.63, MW: 47206TM: 1 [P] gene chromosome: 17p13.3, GenBank accession number NP-002552.; OGT (O-linked N-acetylglucosamine (GlcNAc) transferase, O -GlcNAc transferase P110 subunit, 0-linked N-acetylglucosamine (GlcNAc) transferase (UDP-N-acetylglucosamine: polypeptide-N-acetylglucosamine transferase, UDP-N-acetylglucosamine-peptide N-acetylglucosamine transferase 110KDa subunit, UDP-N-acetylglucosamine: polypeptide-N-acetylglucosamine transferase, uridine diphosphate-N-acetylglucosamine: polypeptide β-N-acetylglucosamine transferase, O-GlcNAc transferase subunit P110, EC 2.4.1.255, O-linked N-acetylglucosamine transferase 110Kda subunit, EC 2.4.1, HRNT1, EC 2.4.1.186, O-GLCNAC; GenBank: AAH38180.1); OA1 (osteoarthritis QTL 1, OASD; GenBank: CAA88742.1); NY-ESO-1 / LAGE-2 (cancer / testis antigen 1B, CTAG1) B, NY-ESO-1, LAGE-2, ESO1, CTAG1, CTAG, LAGE2B, cancer / testis antigen 1, autoimmunogenic cancer / testis antigen NY-ESO-1, cancer antigen 3, cancer / testis antigen 6.1, New York esophageal squamous cell carcinoma 1, L antigen family member 2, LAGE2, CT6.1, LAGE2A; GenBank: AAI30365.1); NY-BR-1 (ANKRD30A, ankyrin repeat domain 30A, breast cancer antigen NY-BR-1, serologically defined breast cancer antigen NY-BR-1, ankyrin repeat domain-containing protein 30A; NCBI reference sequence: NP-443723.2); N-ras (NRAS, neuroblastoma RAS viral (V-Ras) oncogene homolog, NRAS1, transforming protein N-Ras, GTPase NRas, ALPS4, N-Ras protein part 4, NS6,Oncogene homolog HRAS1; GenBank: AAH05219.1); NFYC (nuclear transcription factor Y, gamma, HAPS, HSM, nuclear transcription factor Y subunit C, transactivator HSM-1 / 2, CCAAT binding factor subunit C, NF-YC, CCAAT transcription binding factor subunit gamma, CAAT box DNA-binding protein subunit C, histone H1 transcription factor large subunit 2A, CBFC, nuclear transcription factor Y subunit gamma, CBF-C, transactivator HSM-1, H1TF2A, transcription factor NF-Y, subunit C); neo-PAP (PAPOLG, Poly(A) polymerase gamma, Neo-Poly(A) polymerase, nuclear Poly(A) polymerase gamma, polynucleotide adenylyltransferase gamma, SRP RNA3 adenylase / Pap2, PAP-gamma, Neo-PAP, SRP RNA3'-adenylase, PAP2, EC 2.7.7.19, PAPG; NCBI Reference Sequence: NP-075045.2); NCA (CEACAM6, GenBank Accession No. M1872); Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute transporter family 34 (sodium phosphate) member 2, type II sodium-dependent phosphate transporter 3b, GenBank Accession No. NM-00642); myosin class I; MUM-3; MUM-2 (TRAPPC1, transporter particle complex 1, B ETS, BETS homolog, MUM2, melanoma ubiquitously mutated 2, multiple myeloma protein 2, transporter particle complex subunit 1; MUM-if; mucins (MUC1, mucin 1, cell surface associated, PEMT, PUM, CA15-3, MCKD1, ADMCKD, medullary cystic kidney disease 1 (autosomal dominant), ADMCKD1, mucin 1, transmembrane, CD227, breast cancer associated antigen DF3, MAM6, cancer antigen 15-3, MCD, cancer associated mucin, MCKD, Krebs Von Den Lungen-6, MUC-1 / SEC, peanut reactive urinary mucin, MUC1 / ZD, tumor associated epithelial membrane antigen, DF3 antigen, tumor associated mucin, episialin, EMA, H23 antigen, H23AG, mucin-1, KL-6, tumor associated epithelial mucin, MUC-1, Episialin, PEM, CD227 antigen; UniProtKB / Swiss-Prot: P15941.3); MUCSAC (mucin SAC, oligomeric mucus / gel forming, MUC5 of tracheobronchial, TBM, mucin 5, subtypes A and C, tracheobronchial / gastric, leB, gastric mucin, mucin SAC, oligomeric mucus / gel forming pseudogene, Lewis B blood group antigen, LeB, major airway glycoprotein, MUC-SAC,Mucin-5 subtype AC, tracheobronchial; MUC1 (mucin 1, cell surface associated, PEMT, PUM, CA15-3, MCKD1, ADMCKD, medullary cystic kidney disease 1 (autosomal dominant), ADMCKD1, mucin 1, transmembrane, CD227, breast cancer associated antigen DF3, MAM6, cancer antigen 15-3, MCD, cancer associated mucin, MCKD, Krebs Von Den Lungen-6, MUC-1 / SEC, peanut reactive urinary mucin, MUC-1 / X, polymorphic epithelial mucin, MUC 1 / ZD, tumor associated epithelial membrane antigen, DF3 antigen, tumor associated mucin, episialin, EMA, h23 antigen, H23AG, mucin-1, KL-6, tumor associated epithelial mucin, MUC-1, Episialin, PEM, CD227 antigen; MSG783 (RNF 124, hypothetical protein FLJ20315, GenBank accession number NM-01776; MRP4 - multidrug resistance-associated protein 4 isoform 3, MOAT-B; MOATB [Homo sapiens]; NCBI Reference Sequence: NP-001288758.1; MPF (MPF, MSLN, SMR, megakaryocyte potentiator factor, mesothelin, GenBank accession number NM-00582; MMP-7 (MMP7, matrix metalloproteinase, MPSL1, matrix metalloproteinase, matrix metalloproteinase-7, EC 3.4.23; MMP-1 (matrix metalloproteinase, uterine), matrix metalloproteinase, PUMP-1. 3.4.24, PUMP-1; GenBank: AAC37543.1); MMP-2 (MMP2, matrix metalloproteinase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase), MONA, CLG4A, matrix metalloproteinase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase), CLG4, 72 kDa gelatinase, 72 kDa type IV collagenase), matrix metalloproteinase-2, MMP-II, 72 KDa gelatinase, collagenase type IV-A, MMP-2, matrix metalloproteinase-II, TBE-1, neutrophil gelatinase, EC 3.4.24.24, EC 3.4.24; GenBank: AAH02576.1); Meloe; 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RUB, ADAM,ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, glial cell line-derived neurotrophic factor (Artemin), anti Id, ASPARTIC, atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B lymphocyte stimulator (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bel, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2BMP- 2a, BMP-3 osteogenic factor, BMP-4BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 ( C3), C3a, C4, C5, C5a, CIO, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCLll, CCL12, CCL13, CCL 14, CCL15, CCL16, CCL1 7. CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR , CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CDlla, CDllb, CDllc, CD13,CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56 , CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXC L4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CX CR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, decay-accelerating factor, des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, DNAse, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, EN A, endothelin receptor, enkephalin, eNOS, Eot, eotaxin l, EpCAM, ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, factor IIa, factor VII, factor VIIIc, factor IX, fibroblast activation protein (FAP), Fas, FcRI, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrinogen, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractal chemokine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1,GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-α1, GFR-α2, GFR-α3, GITR, Glucagon, Glut 4, glycoprotein Ilb / IIIa (GP Ilb / IIIa), GM-CSF, gp130, gp72, GRO, growth hormone releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV) gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma associated antigen (HMW-MAA), HIV gp120, HIVIIIB gp 120V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon ( INF)-α, INF-β, INF-γ, inhibin, iNOS, insulin A-chain, insulin B-chain, insulin-like growth factor 1, integrin α2, integrin α3, integrin α4, integrin α4 / β1, integrin, α4 / β7, integrin α5 (αV), integrin α5 / β1, integrin α5 / β3, integrin α6, integrin β1, integrin β2, interferon gamma, IP-10, 1-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein LI, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR,Keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1bpl, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP 2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloproteinases, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-α, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Mucl), MUC 18, Müllerian-inhibin substance, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4 or -6, neurotrophin, nerve growth factor (NGF), NGFR, NGF-β, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK- 1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), P1GF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, relaxin A-chain, relaxin B-chain, renin, respiratory syncytial virus (RSV) F, RSV Fgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat,STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor α / β), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testis PLAP-like alkaline phosphatase, TfR, TGF, TGF-α, TGF-β, TGF-β pan-specific, TGF-βRI (ALK-5), TGF-βR II, TGF-βRllb, TGF-βRIII, TGF-βl, TGF-β2, TGF-β3, TGF-β4, TGF-β5, thrombin, thymus Ck-1, thyroid stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-α, TNF-αβ, TNF-β2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSFIOB (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcRI, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSFllB (OPG OCIF, TR1), TNFRSF12 (TWEAKR FN14), TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF 18 (GITR AITR), TNFRSF 19(TROYTAJ,TRADE),TNFRSF 19L (RELT), TNFRSFIA (TNF RI CD120a, p55-60), TNFRSFIB (TNFRIICD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFC R), TNFRSF4 (OX40ACT35, TXGP1R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6),TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25 (DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 ligand, TL2), TNFSF11 (TRANCE / RANK ligand ODF, OPG ligand), TNFSF 12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF 18 (GITR ligand AITR ligand, TL6), TNFSFIA (TNF-a Conectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transfer receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA 125, Lewis expression Y-related carbohydrate tumor-associated antigen, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1(flt-1), VEGF, VEGFR, VEGFR-3(flt-4), VEGI, VIM, viral antigens, VLA, VLA-1, VLA-4, VNR integrin, von Würz Bland factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD,CTLA4 (cytotoxic T-lymphocyte antigen-4), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), LAG-3 (lymphocyte activation gene-3), TIM-3 (T cell immunoglobulin and mucin-3), receptors for hormones and growth factors. In certain embodiments, the CAR may be specific for BCMA, CTLA4 (cytotoxic T lymphocyte antigen-4), PD 1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), LAG-3 (lymphocyte activation gene-3), TIM-3, CD20, CD2, CD19, Her2, EGFR, EpCAM, FcyRIIIa (CD16), FcyRIIa (CD32a), FcyRIIb (CD32b), FcyRI (CD64), Toll-like receptor (TLR), TLR4, TLR9, cytokines, IL-2, IL-5, IL-13, IL-6, IL-17, IL-12, IL-23, TNFa, TGFb, cytokine receptor, IL-2R, chemokine, chemokine receptor, growth factor, VEGF, and HGF. ,

[0183] CAR-T cells can be used in combination with NEO-201 to treat any cancer, infectious, inflammatory or autoimmune disorder in which CAR-T cells can be used as described above.

[0184] NEO-201 in combination with CAR-NK cell therapy

[0185] NEO-201 should also improve the efficacy of CAR-NK cell therapy due to its ability to eliminate gMDSCs. In particular, the great success of CAR-T therapy in clinical trials has led to the development of CAR-NK cells. Extracellular, transmembrane, and intracellular signaling domains are present in CAR-NK cells, just as they are in CAR-T cells. CAR-NK cells typically have CD3 as their initial signaling domain and CD28 or CD137 (4-1BB) as co-stimulatory domains to form intracellular signaling domains. NK cells increase their cytotoxic capacity and cytokine production through two additional co-stimulatory molecules, NKG2D and CD244 (2B4). CAR-modified NK cells are often used to target cancer cells due to their enhanced tumor-specific targeting and cytotoxicity than CAR-T cells.

[0186] Compared with CAT-T cells, CAR-NK cell therapy has advantages such as low safety issues, low cost and higher tumor potential. Allogeneic haploidentical NK cells are safe for adoptive cell therapy (ACT) because they usually do not mediate GVHD and may alleviate GVHD. In addition, the safety issues of CAR-NK cells (such as on-target / off-tumor effects, CRS and tumor lysis syndrome) are much less than CAR-T cells. In addition, NK cells only secrete a small amount of IFN-γ and GM-CSF, and do not produce IL-1 and IL-6 that initiate CRS. Secondly, tumor cells may not be detected by CAR-T cells due to tumor escape caused by loss of MHC class I expression or tumor-specific antigens. CAR-NK cells lack self-antigens and can detect MHC class I negative tumor cells because they retain the innate cytotoxic potential for tumor / stress ligands encoded by the germline. In addition, both HLA-A and HLA-B bind to KIR3D receptors, while HLA-C only binds to KIR2D receptors. CD94-NKG2A, which detects HLA-E, LILRB1, and all MHC class I molecules, is another inhibitory receptor that identifies MHC class I molecules expressed by NK cells. Normal MHC class I sufficient cells are ignored by NK cells because their inhibitory receptors detect MHC class I molecules; however, they are not inhibited after interacting with abnormal MHC class I low cells. Third, it is believed that low levels of MHC class I expression in cancer stem cells (CSCs) and the presence of NKp30, NKp44, and NKG2D (activating receptors) lead to cytokine-activated NK cell-mediated CSC death. Fourth, CAR-NK cells can regulate their activating receptors, including NKp30, NKp44, NKp46, NKG2D, KIR-2DS, KIR-3DS, 2B4, CD226, CD94 / NKG2C, and DNAM-1; therefore, the chance of relapse due to the loss of CAR-targeted antigens is reduced. In addition, T lymphocytes kill their targets only through CAR-specific mechanisms, while NK cells exhibit spontaneous cytotoxic activity and can kill target cells regardless of the presence of tumor-specific antigens. Tumor cells downregulate antigens to escape immune detection; however, NK cells are still effective against them. In addition, cytokines such as IFN-γ, IL-3, and GM-CSF produced by primary human NK cells are different from proinflammatory cytokines released by T cells that induce CRS. A single NK cell can survive after interacting with and destroying several target cells, potentially reducing the number of cells transferred by adoptive transfer. Fifth, the availability of ready-made CAR-NK therapies significantly increases the speed of administration and the first dose on day 1 by minimizing the lag time in determining treatment.Sixth, CAR-NK therapy should reduce huge indirect costs because CAR-NK infusions can be administered under outpatient follow-up monitoring and do not require long-term hospitalization after treatment because they are safer and have no potential toxicity. In addition, NK cells can be harvested from a variety of sources (including iPSC, PB, UCB, human embryonic stem cells, and NK cell lines). Like CAR-T cells, CAR-NK cell therapy is used to treat hematological tumors and solid tumors. CD19 (NCT02742727), CD7 (NCT02742727), and CD33 (NCT02944162) are targets of CAR-NK cell therapy used in reported clinical studies of lymphoma and leukemia. In addition, HER2-targeted GBM (NCT03383978) and co-stimulatory conversion receptors are being used to treat non-small cell lung cancer (NSCLC) (NCT03656705). CAR-NK cell therapy targeting mucin 1 (MUC1) is also under investigation for a variety of refractory solid tumors, including pancreatic tumors, HCC, NSCLC, and triple-negative invasive breast tumors (NCT02839954).

[0187] CAR-NK cells can be used in combination with NEO-201 to treat any cancer, infectious, inflammatory or autoimmune disease, wherein CAR-T cells are used as described above. The CAR expressed by such NK cells can be specific to any antigen targeted by the CAR-T cells. In addition, the CAR may include any of the signaling, hinges, and other domains commonly used in CARs expressed in CAR-T cells. Such domains and sequences used in CARs are generally known in the art and as described above.

[0188] Monitoring / detection of MDSCs in patients

[0189] In some embodiments, gMDSCs will be detected and monitored in patients before, during, and after treatment is completed or after the patient enters remission. Such methods may help determine whether a patient will likely benefit from NEO-201 treatment.

[0190] Methods for detecting and monitoring gMDSCs in patient samples are known in the art and disclosed in U.S. Published Application 20210318310, published on October 14, 2021, by Gabrilovich; Dmitry I.; U.S. Published Application 20170261507, published on September 14, 2017, by BANIYASH; Michal, which are incorporated by reference in their entirety.

[0191] Methods for identifying and isolating gMDSCs from a sample may include contacting the biological sample with a ligand, such as an antibody that recognizes a specific biomarker expressed on gMDSCs. Such biomarkers include LOX-1, CD11b, CD15, and CD66b.

[0192] These methods can provide an accurate count or concentration of a gMDSC cell population from a suitable biological sample from a subject.

[0193] In some embodiments, these methods of determining accurate cell counts / concentrations of gMDSCs in a subject having cancer or being treated for cancer with NEO-201 alone or in combination with another therapeutic agent can be used to monitor the progression of the cancer (with or without treatment).

[0194] In some embodiments, these methods of determining the number or concentration of gMDSCs in a subject having cancer can be used to determine whether NEO-201 alone or in combination with another therapeutic agent may be beneficial in treating the cancer.

[0195] In some embodiments, these methods of determining the number or concentration of gMDSCs in a tumor can be used to develop dosing regimens for NEO-201, alone or in combination with another therapeutic agent.

[0196] In some embodiments, the present disclosure provides a method for detecting gMDSCs, wherein the level of gMDSCs in a patient sample (such as a blood or biopsy sample) is used to determine cancer prognosis before, during, or after NEO-201 treatment. For example, if gMDSCs reactive to NEO-201 cells are detected in the patient sample, NEO-201 in an amount effective to kill gMDSCs may be assigned to the patient or may be administered to the patient. The method may include contacting the gMDSCs with a NEO-201 antibody.

[0197] The detection may include cell sorting, optionally fluorescence activated cell sorting, thereby generating a sample enriched and / or depleted for cells positive for expression of the NEO-201 antigen (eg, gMDSCs).

[0198] In another aspect, the present disclosure provides a method for detecting gMDSCs, the method comprising contacting cells with a NEO-201 antibody and detecting cells expressing a NEO-201 target antigen. The NEO-201 antibody may be labeled directly or indirectly.

[0199] In another aspect, the present disclosure provides a method of staining gMDSCs, the method comprising contacting cells with a NEO-201 antibody. The NEO-201 antibody may be labeled directly or indirectly.

[0200] On the other hand, the present disclosure provides a method for separating or enriching MDSC, the method comprising separating cells expressing NEO-201 target antigens. The method may include contacting a sample (e.g., a tumor biopsy sample containing gMDSC) with a NEO-201 antibody, optionally wherein the NEO-201 antibody is directly or indirectly labeled. The sample may also include blood or bone marrow. The method may include separating NEO-201 positive gMDSC from NEO-201 negative cells. The method may further include performing further diagnostic assays on the cell sample to detect the expression of other MDSC biomarkers.

[0201] The gMDSCs can also be isolated by cell sorting (optionally fluorescence activated cell sorting) based on the expression of the NEO-201 target antigen and the expression of other MDSC biomarkers.

[0202] The gMDSCs can be isolated by contacting the sample with a support comprising NEO-201 antibodies and / or using other antibodies or ligands that recognize other MDSC biomarkers, thereby retaining the MDSCs on the support.

[0203] On the other hand, the present disclosure provides a method for detecting gMDSC, the method comprising detecting the expression of the MDSC to the NEO-201 target antigen, optionally wherein the level of gMDSC in a patient sample (such as blood or a biopsy sample) is used to determine whether the patient has or may develop MDSC-mediated immunosuppression. Optionally, the method may also include allocating or administering NEO-201 treatment to the patient based on the detection of the gMDSC. For example, if gMDSC and / or other biomarkers reactive to NEO-201 are detected in the patient sample, NEO-201 in an amount effective to kill gMDSC may be allocated to the patient or may be administered to the patient. The method may include contacting the gMDSC with a NEO-201 antibody.

[0204] The detection may include cell sorting, optionally fluorescence activated cell sorting, thereby generating a sample enriched and / or depleted for cells positive for expression of the NEO-201 target antigen (eg, gMDSCs).

[0205] In another aspect, the present disclosure provides a method for detecting gMDSCs, the method comprising contacting cells with a NEO-201 antibody and detecting cells expressing a NEO-201 target antigen. The NEO-201 antibody may be labeled directly or indirectly.

[0206] In another aspect, the present disclosure provides a method of staining gMDSCs, the method comprising contacting cells with a NEO-201 antibody. The NEO-201 antibody may be labeled directly or indirectly.

[0207] On the other hand, the present disclosure provides a method for separating gMDSC, the method comprising separating cells expressing NEO-201 target antigens and optionally other MDSC biomarkers. The method may include contacting a sample containing a cell sample (e.g., a tumor biopsy sample) with a NEO-201 antibody, optionally wherein the NEO-201 antibody is directly or indirectly labeled. The sample may alternatively include a blood or bone marrow sample. The method may include separating NEO-201 positive gMDSC from NEO-201 negative cells. The method may further include, for example, using a ligand bound to other MDSC biomarkers to further diagnose the putative gMDSC.

[0208] The gMDSCs can be isolated by cell sorting (optionally fluorescence activated cell sorting) based on NEO-201 expression.

[0209] The gMDSCs can be isolated by contacting the sample with a support comprising NEO-201 antibodies, thereby retaining the gMDSCs on the support.

[0210] Cancer vaccines

[0211] The subject treatment method may also include administering a cancer vaccine to the patient. Exemplary cancer vaccines that may be administered are disclosed, for example, in Fisher et al., Immun Inflamm Dis. 2017 Mar; 5(1): 16–28; Klages et al., Cancer Res 2010 Oct 15 (70)(20) 7788-7799; Reginato et al., Br J Cancer. 2013 Oct 15; 109(8): 2167–2174; Litzinger MT et al., Blood 2007, 110: 3192, each of which is hereby incorporated by reference in its entirety.

[0212] In vitro elimination of gMDSCs using NEO-201

[0213] On the other hand, the present disclosure provides a method of killing gMDSC cells in vitro, the method comprising contacting the gMDSC cells with NEO-201 antibodies. The method may also include contacting the gMDSC cells with complement. The gMDSC cells may be killed by CDC. The method may also include contacting the gMDSC with effector cells (such as natural killer cells). The gMDSC may be killed by ADCC.

[0214] In another aspect, the present disclosure provides a method of killing MDSCs in vitro, the method comprising contacting a sample containing gMDSCs with an effective amount of NEO-201 antibody. The sample may be obtained from a patient. In addition, in some cases, the NEO-201 antibody may be coupled to a cytotoxic moiety.

[0215] NEO-201 antibody sequence

[0216] In any of the foregoing or following methods, the NEO-201 antibody may comprise at least one, two, three, four, five, or preferably all six CDR sequences contained in SEQ ID NO:28 and SEQ ID NO:29.

[0217] In any of the foregoing or following methods, the NEO-201 antibody may comprise a variable heavy chain sequence that is at least 90% identical to SEQ ID NO:38.

[0218] In any of the foregoing or following methods, the NEO-201 antibody may comprise a variable light chain sequence that is at least 90% identical to SEQ ID NO:39.

[0219] In any of the foregoing or following methods, the NEO-201 antibody may comprise a variable heavy chain sequence that is at least 90% identical to SEQ ID NO:38 and a variable light chain sequence that is at least 90% identical to SEQ ID NO:39.

[0220] In any of the foregoing or following methods, the NEO-201 antibody may comprise a heavy chain sequence at least 90% identical to amino acids 20-470 of SEQ ID NO:28 and a light chain sequence at least 90% identical to amino acids 20-233 of SEQ ID NO:29.

[0221] In any of the foregoing or following methods, the NEO-201 antibody may comprise all six CDR sequences contained in SEQ ID NO:28 and SEQ ID NO:29.

[0222] In any of the foregoing or following methods, the NEO-201 antibody may comprise a human IgG1 constant domain. Alternatively, the NEO-201 antibody may comprise a human IgG2, human IgG3, or human IgG4 constant domain, or a hybrid or chimeric domain of two or more of human IgG1, human IgG2, human IgG3, or human IgG4.

[0223] In any of the foregoing or following methods, the antibody comprises a NEO-201 antibody or a variant thereof, eg, a NEO-201 antibody or a variant thereof comprising the same CDRs or variable regions as NEO-201.

[0224] In any of the foregoing or following methods, the NEO-201 antibody may be conjugated to another moiety.

[0225] In any of the foregoing or following methods, the NEO-201 antibody may be conjugated to another cytotoxic moiety, a label, a radioactive moiety, or an affinity tag.

[0226] In any of the foregoing or following methods, the NEO-201 antibody can compete with the antibodies contained in SEQ ID NO:28 and SEQ ID NO:29 for binding to the NEO-201 antigen.

[0227] definition

[0228] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those terms commonly understood by one of ordinary skill in the art to which the invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the present invention or testing of the present invention, suitable methods and materials are described herein. The materials, methods, and examples are exemplary only and are not intended to be limiting.

[0229] As used in the description herein and throughout the claims that follow, the meaning of "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0230] As used herein, "amino acid" refers generally to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those amino acids encoded by the genetic code, as well as those amino acids that are later modified, for example, hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, that is, the basic chemical structure is carbon, carboxyl, amino, and R groups bound to hydrogen, for example, homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to compounds that have a general chemical structure different from that of amino acids, but function in a manner similar to naturally occurring amino acids.

[0231] As used herein, the term "NK depletion" or "natural killer depletion" refers to a patient with a low level of natural killer (NK) cells relative to the normal range. NK cells are cytotoxic innate immune lymphocytes. Typically, NK cells account for 5%-20% of peripheral blood mononuclear cells (PBMCs) in healthy individuals. Patients with NK cells that account for less than 5% of PMBCs are called NK depleted. In addition, if NK cells account for less than 3% of PMBCs, the patient is called severely NK depleted. In addition, in normal individuals, up to 90% of PBMC NK cells are CD56 dim CD16 + NK cells, and these cells are considered the most cytotoxic subset. If less than 70% of PBMC NK cells are CD56 dim CD16 + If less than 50% of PBMC NK cells are CD56 dim CD16 + NK cells, the patient is referred to as severe NK depletion. Based on meeting one or two of these individual criteria, a given patient may be referred to as NK depletion or severe NK depletion. In general, by testing a sample taken from the patient (e.g., a blood sample, such as a sample obtained and tested within the previous week or two weeks), it is determined that the patient's status is NK depletion or severe NK depletion. It is also possible to infer that the patient's status is NK depletion or severe NK depletion from disease diagnosis and / or the course of treatment associated with such NK cell depletion.

[0232] As used herein, "antibody" refers to any molecular structure containing polypeptide chains with a specific shape suitable for and recognizing epitopes, wherein one or more non-covalent binding interactions make the compound between the molecular structure and the epitope more stable. The prototype antibody molecule is an immunoglobulin, and all types of immunoglobulins (IgG, IgM, IgA, IgE, IgD) from all sources (e.g., humans, rodents, rabbits, cattle, sheep, pigs, dogs, chickens) are considered "antibodies". Antibodies include, but are not limited to, chimeric antibodies, human antibodies and other non-human mammalian antibodies, humanized antibodies, single-chain antibodies (scFv), camel antibodies, nanobodies, IgNARs (single-chain antibodies derived from sharks), small molecule immunopharmaceuticals (SMIPs) and antibody fragments (e.g., Fab, Fab', F(ab')2). Numerous antibody coding sequences have been described; and others can be produced by methods well known in the art. See Streltsov et al. (2005) Protein Sci. 14(11):2901–9; Greenberg et al. (1995) Nature 374(6518):168–173; Nuttall et al. (2001) Mol Immunol. 38(4):313–26; Hamers-Casterman et al. (1993) Nature 363(6428):446–8; Gill et al. (2006) Curr Opin Biotechnol. 17(6):653–8.

[0233] "NEO-201 antibody" refers to an antibody containing a heavy chain of SEQ ID NO: 28 and a light chain or variable region of SEQ ID NO: 29, optionally together with a constant region contained therein, and fragments and variants thereof. Such variants include sequences containing one, two, three, four, five or preferably all six CDR sequences contained in SEQ ID NO: 28 and SEQ ID NO: 29, i.e., a heavy chain CDR1 of SEQ ID NO: 32, a heavy chain CDR2 of SEQ ID NO: 33, a heavy chain CDR3 of SEQ ID NO: 34, a light chain CDR1 of SEQ ID NO: 35, a light chain CDR2 of SEQ ID NO: 36, and a light chain CDR3 of SEQ ID NO: 37. Such variants also include antibodies that compete with NEO-201 for binding to the NEO-201 antigen. The antibodies may be humanized. The antibodies may be expressed to contain one or more leader sequences, which may be removed during expression and / or processing and secretion of the antibodies. The antibodies may be present in monovalent, bivalent or higher multivalent forms, including but not limited to bispecific or multispecific antibodies containing the NEO-201 antibody sequence and a binding fragment of a different antibody. Typically, the antibodies specifically bind to cancerous cells and compete with antibodies comprising a variable heavy chain of SEQ ID NO:38 and a variable light chain of SEQ ID NO:39 or a heavy chain of SEQ ID NO:28 and a light chain of SEQ ID NO:29 for binding to cancerous cells. One or more of those CDR sequences contained in SEQ ID NO:28 and / or SEQ ID NO:29 may be replaced by a variant sequence, such as a light chain CDR1 of SEQ ID NO:1 or 4; a light chain CDR2 of SEQ ID NO:2 or 5; a light chain CDR3 of SEQ ID NO:3 or 6; a heavy chain CDR1 of SEQ ID NO:7; a heavy chain CDR2 of SEQ ID NO:8, 10, 30 or 31; a heavy chain CDR3 of SEQ ID NO:9 or 11; or SEQ ID NO:30-31. The light chain may comprise the CDRs contained in the light chain sequence of SEQ ID NO: 14, 16, 17, 18, 19, 20, 21 or 29. The heavy chain may comprise the CDRs contained in the heavy chain sequence of SEQ ID NO: 15, 22, 23, 24, 25, 26, 27 or 29.The antibody may comprise a variable heavy chain sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:38, and / or a variable light chain sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:39, optionally wherein the heavy and / or light chain sequences contain one, two, three, four, five or preferably all six CDR sequences contained in SEQ ID NO:28 and SEQ ID NO:29, i.e., the heavy chain CDR1 of SEQ ID NO:32, the heavy chain CDR2 of SEQ ID NO:33, the heavy chain CDR3 of SEQ ID NO:34, the light chain CDR1 of SEQ ID NO:35, the light chain CDR2 of SEQ ID NO:36 and the light chain CDR3 of SEQ ID NO:37. The antibody may be conjugated to another moiety, such as a cytotoxic moiety, a radioactive moiety, a label, or a purification tag.

[0234] As used herein, "antigen" refers generally to a molecule or a portion of a molecule that can be bound by an antibody, and furthermore, the antigen can induce an animal to produce an antibody that can bind to an epitope of the antigen. An antigen may have one epitope, or more than one epitope. The specific reactions mentioned herein indicate that an antigen will react with its corresponding antibody in a highly selective manner, and will not react with a plurality of other antibodies that may be stimulated by other antigens. Antigens may be tumor specific (e.g., expressed by neoplastic cells of pancreatic cancer and colon cancer).

[0235] As used herein, "cancer" refers broadly to any neoplastic disease (whether invasive or metastatic) characterized by abnormal and uncontrolled cell division resulting in malignant growth or tumors.

[0236] As used herein, "cancer vaccine" refers to an immunogenic composition that elicits or is intended to elicit an immune response against cancer cells.

[0237] As used herein, "chimeric antibody" refers broadly to antibody molecules in which the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and / or species, or an entirely different molecule, e.g., an enzyme, toxin, hormone, growth factor, drug, that confers new properties to the chimeric antibody; or the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigenic specificity.

[0238] As used herein, "conservatively modified variants" are applicable to both amino acid sequences and nucleic acid sequences, and for specific nucleic acid sequences, generally refer to conservatively modified variants, which refer to those nucleic acids encoding the same or substantially identical amino acid sequences, or in the case where the nucleic acid does not encode an amino acid sequence, refer to substantially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. Such nucleic acid variations are "silent variations", which are one of conservatively modified variations. Each nucleic acid sequence encoding a polypeptide herein also describes each possible silent variation of the nucleic acid. The technician will recognize that each codon in the nucleic acid (except AUG (which is typically the only codon for methionine) and TGG (which is typically the only codon for tryptophan)) can be modified to produce functionally identical molecules.

[0239] As used herein, "complementarity determining region", "hypervariable region" or "CDR" refers generally to one or more of the hypervariable regions or complementary determining regions (CDRs) found in the variable region of the light chain or heavy chain of an antibody. See Kabat et al. (1987) "Sequences of Proteins of Immunological Interest" National Institutes of Health, Bethesda, MD. These expressions include hypervariable regions or hypervariable loops in the three-dimensional structure of an antibody as defined by Kabat et al., (1983) "Sequences of Proteins of Immunological Interest" U.S. Pat. of Health and Human Services. Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917. The CDRs in each chain are closely connected by the framework region and, together with the CDRs in the other chain, contribute to the formation of the antigen binding site. In the CDRs, there are selected amino acids described as selectivity determining regions (SDRs), which represent the key contact residues used by the CDRs in antibody-antigen interactions. Kashmiri (2005) Methods 36:25-34.

[0240] As used herein, "control amount" generally refers to any amount or range of amounts of a marker that is compared to a test amount of the marker. For example, a control amount of a marker can be the amount of the marker in a patient with a particular disease or condition or in a person without such a disease or condition. A control amount can be an absolute amount (e.g., micrograms / ml) or a relative amount (e.g., relative intensity of a signal).

[0241] As used herein, "differentially present" refers generally to the difference in the amount or quality of a marker present in a sample taken from a patient with a disease or condition, compared to a comparable sample taken from a patient without one of the disease and condition. For example, if the amount of nucleic acid fragments in one sample is significantly different from the amount of nucleic acid fragments in another sample, such as measured by hybridization and / or based on a NAT assay, the nucleic acid fragments may optionally be differentially present between the two samples. If the amount of a polypeptide in one sample is significantly different from the amount of a polypeptide in another sample, the polypeptide is differentially present between the two samples. It should be noted that if a marker is detectable in one sample and undetectable in another sample, it can be considered that such a marker is differentially present. Optionally, a relatively small amount of upregulation can be used as a marker.

[0242] As used herein, "diagnostic" refers generally to identifying the presence or nature of a pathological condition. Diagnostic methods differ in sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals that test positive (the percentage of "true positives"). Diseased individuals that are not detected by the assay are "false negatives." Subjects who are not diseased and test negative in the assay are called "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of patients who test positive for those who do not have the disease. Although a particular diagnostic method may not provide a definitive diagnosis of a condition, it is sufficient as long as the method provides a positive indication that helps in diagnosis.

[0243] As used herein, "diagnosis" refers broadly to classifying a disease or symptom, determining the severity of a disease, monitoring the progression of a disease, predicting the outcome of a disease and / or the prospects for recovery. The term "detection" may also optionally include any of the foregoing. In some embodiments, according to the present invention, the diagnosis of a disease may be affected by determining the level of a polynucleotide or polypeptide of the present invention in a biological sample obtained from a subject, wherein the determined level may be related to susceptibility to the disease or the presence or absence of the disease. It should be noted that a "biological sample obtained from a subject" may also optionally include a sample that has not been physically removed from the subject.

[0244] As used herein, "effective amount" refers generally to the amount of a compound, antibody, antigen or cell that achieves the desired result. When administered to a patient for the treatment of a disease, an "effective amount" is sufficient to achieve such treatment of the disease. An effective amount can be an amount effective for prevention, and / or an amount effective for prevention. An effective amount can be an amount that is effectively reduced, effectively preventing the occurrence of signs / symptoms, reducing the severity of the occurrence of signs / symptoms, eliminating the occurrence of signs / symptoms, slowing down the development of the occurrence of signs / symptoms, preventing the development of the occurrence of signs / symptoms and / or achieving the amount of prevention of the occurrence of signs / symptoms. An "effective amount" can vary according to the disease and its severity, as well as the age, weight, medical history, susceptibility and pre-existing conditions of the patient being treated. For the purposes of this disclosure, the term "effective amount" is synonymous with "therapeutically effective amount".

[0245] As used herein, "expression vector" refers to any recombinant expression system for constitutively or inducibly expressing the nucleic acid sequence of the present disclosure in any cell (including prokaryotic, yeast, fungal, plant, insect or mammalian cells) in vitro or in vivo. The term includes linear or circular expression systems. The term includes expression systems that remain free or integrated into the host cell genome. The expression system may have the ability to self-replicate or not, i.e., only drive transient expression in the cell. The term includes a recombinant expression cassette that contains only the minimum elements required for transcription of the recombinant nucleic acid.

[0246] As used herein, "framework region" or "FR" refers generally to one or more of the framework regions within the variable regions of the light and heavy chains of an antibody. See Kabat et al. (1987) "Sequences of Proteins of Immunological Interest" National Institutes of Health, Bethesda, MD. These expressions include those amino acid sequence regions interposed between the CDRs within the variable regions of the light and heavy chains of an antibody.

[0247] "Hematologic malignancies" refers to a form of cancer that begins in hematopoietic tissues such as the bone marrow or immune system cells. Examples of hematologic malignancies include leukemia, lymphoma, multiple myeloma, and myelodysplastic syndrome (MDS). More specific examples of hematologic malignancies include, but are not limited to, marginal zone lymphoma (MZL) (including splenic marginal zone lymphoma (SMZL)), Burkitt's lymphoma (BL), multiple myeloma (MM) (including plasma cell leukemia (PCL) and myeloma extramedullary disease (EMD)), myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) (including B-cell AML), acute lymphocytic leukemia (ALL), T-cell lymphoma (TCL) (including anaplastic large cell lymphoma (ALCL) and Sezary syndrome) and Hodgkin's lymphoma (HL).

[0248] As used herein, "heterologous" refers broadly to portions of a nucleic acid that indicate that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids are typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0249] As used herein, "high affinity" refers to a binding affinity of at least 10 -8 M, more preferably at least 10 -9 M, even more preferably at least 10 -10 "High affinity" binding may be different for other antibody isotypes. For example, "high affinity" binding for an IgM isotype refers to an antibody with a KD of at least 10 -7 M, more preferably at least 10 -8 Antibodies for KD of M.

[0250] As used herein, "homology" refers generally to the degree of similarity between a nucleic acid sequence and a reference nucleic acid sequence or between a polypeptide sequence and a reference polypeptide sequence. Homology can be partial or complete. Complete homology indicates that the nucleic acid sequences or amino acid sequences are identical. A partially homologous nucleic acid sequence or amino acid sequence is a sequence that is different from a reference nucleic acid sequence or amino acid sequence. The degree of homology can be determined by sequence alignment. The terms "sequence identity" and "homology" are used interchangeably.

[0251] As used herein, "host cell" refers generally to a cell that contains an expression vector and supports replication or expression of the expression vector. The host cell can be a prokaryotic cell such as E. coli, or a eukaryotic cell such as yeast, insect (e.g., SF9), amphibian or mammalian cell (such as CHO, HeLa, HEK-293), e.g., cultured cells, explants, and in vivo cells.

[0252] As used herein, "hybridization" broadly refers to the physical interaction of complementary (including partially complementary) polynucleotide chains through the formation of hydrogen bonds between complementary nucleotides when the complementary nucleotide chains are arranged antiparallel to each other.

[0253] As used herein, "K-assoc" or "Ka" refers generally to the association rate of a particular antibody-antigen interaction, while as used herein, the term "Kdiss" or "Kd" refers to the dissociation rate of a particular antibody-antigen interaction. As used herein, the term "KD" is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed as a molar concentration (M). The KD value of an antibody can be determined using methods well established in the art.

[0254] As used herein, "immunoassay" refers broadly to an assay that uses an antibody to specifically bind an antigen. An immunoassay can be characterized by using the specific binding properties of a particular antibody to isolate, target, and / or quantify an antigen.

[0255] As used herein, "isolated" refers generally to a substance that is removed from its naturally occurring original environment and thus artificially altered from its natural environment. An isolated substance can be, for example, an exogenous nucleic acid contained in a vector system, an exogenous nucleic acid contained in a host cell, or any substance that has been removed from its original environment and thus artificially altered (e.g., an "isolated antibody").

[0256] As used herein, "label" or "detectable moiety" refers broadly to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.

[0257] As used herein, "low stringency", "medium stringency", "high stringency" or "very high stringency conditions" generally refer to conditions for nucleic acid hybridization and washing. Guidance for performing hybridization reactions can be found in Ausubel et al. (2002) "Short Protocols in Molecular Biology", (5th Edition) John Wiley & Sons, NY. Exemplary specific hybridization conditions include, but are not limited to: (1) low stringency hybridization conditions, performed in 6X sodium chloride / sodium citrate (SSC) at about 45°C, followed by two washes in 0.2X SSC, 0.1% SDS at at least 50°C (the wash temperature can be increased to 55°C for low stringency conditions); (2) moderate stringency hybridization conditions, performed in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60°C; (3) high stringency hybridization conditions, performed in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C; and (4) very high stringency hybridization conditions, performed in 0.5 M sodium phosphate, 7% SDS at 65°C, followed by one or more washes in 0.2X SSC, 1% SDS at 65°C.

[0258] The terms "low level" or "low" used with respect to markers such as CD127 are well known in the art and refer to the expression level of a cell marker of interest (e.g., CD 127) where the expression level of the cell marker is low by comparison to the expression level of the cell marker in other cells of the cell population being analyzed as a whole. More specifically, the term "low" refers to a distinct cell population that expresses the cell marker at a level lower than one or more other distinct cell populations. Thus, CD127 低 Refers to cells of a type that stain lightly or dimly when contacted with a labeled CD127 antibody, for example, at a level higher than the CD127- subpopulation but lower than the CD127+ subpopulation.

[0259] As used herein, "mammal" refers to any and all warm-blooded vertebrates of the class Mammalia, including humans, characterized by hair covering the skin, and in females, characterized by milk-producing mammary glands for feeding young. Examples of mammals include, but are not limited to, alpacas, armadillos, capybaras, cats, camels, chimpanzees, chinchillas, cattle, dogs, goats, gorillas, hamsters, horses, humans, lemurs, llamas, mice, non-human primates, pigs, rats, sheep, shrews, squirrels, and tapirs. Mammals include, but are not limited to, bovine, canine, equine, feline, murine, ovine, swine, primates, and rodent species. Mammals also include any and all species listed in the Mammal Species of the World maintained by the National Museum of Natural History, Smithsonian Institution, Washington DC.

[0260] "Myeloid-derived suppressor cells" or "MDSCs" are a heterogeneous group of myeloid immune cells (a family of cells derived from bone marrow stem cells). MDSCs strongly proliferate in pathological conditions such as chronic infections and cancers due to altered hematopoiesis. MDSCs differ from other myeloid cell types in that they have strong immunosuppressive activity rather than immunostimulatory properties. Similar to other myeloid cells, MDSCs interact with other immune cell types (including T cells, dendritic cells, macrophages, and natural killer cells) to regulate their function.Clinical evidence shows that cancer tissues with high MDSC infiltration are associated with poor patient prognosis and resistance to therapy (Mantovani A., December 2010, "The growing diversity and spectrum of action of myeloid-derived suppressor cells", European Journal of Immunology. 40(12): 3317–20. doi: 10.1002 / eji.201041170. PMID 21110315; Allavena P, Mantovani A., February 2012, "Immunology in the clinic review series; focus on cancer: tumour-associated macrophages: undisputed stars of the inflammatory tumour microenvironment", Clinical and Experimental Immunology. 167(2): 195–205. doi: 10.1111 / j.1365-2249.2011.04515.x, PMID 21110315). 3278685.PMID 22235995; Galdiero MR et al., (November 2013). "Tumor associated macrophages and neutrophils in cancer", Immunobiology.218(11):1402–10.doi:10.1016 / j.imbio.2013.06.003.PMID 23891329; Gabrilovich DI et al., "Coordinated regulation of myeloid cells by tumors", NatureReviews.Immunology.12(4):253–68.doi:10.1038 / nri3175.PMC 3587148.PMID22437938.

[0261] MDSC is composed of two major types of cells: granulocytes or polymorphonuclear cells (PMN-MDSC or gMDSC) and monocytes (M-MDSC). PMN-MDSC or gMDSC are similar to neutrophils in phenotype and morphology, while M-MDSC is more similar to monocytes (Gabrilovich DI et al., "Coordinated regulation of myeloid cells by tumors", Nat Rev Immunol. 2012; 12 (4): 253–268). It has also been reported that there is a third small MDSC population, which is represented by cells with colony-forming activity and other myeloid precursors, which are referred to as early MDSC (eMDSC) (Dumitru CA et al., "Neutrophils and granulocytic myeloid-derived suppressor cells: immunophenotyping, cell biology and clinical relevance in human oncology", Cancer Immunol Immunother. 2012; 61 (8): 1155–11673).

[0262] As used herein, "nucleic acid" or "nucleic acid sequence" refers generally to deoxyribonucleotides or ribonucleotide oligonucleotides in single-stranded or double-stranded form. The term includes nucleic acids containing known analogs of natural nucleotides, i.e., oligonucleotides. The term also includes nucleic acid-like structures with synthetic backbones. Unless otherwise indicated, a specific nucleic acid sequence also implicitly includes variants and complementary sequences of its conservative modifications (e.g., degenerate codon substitutions), as well as the sequence explicitly indicated. The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide and polynucleotide.

[0263] As used herein, "operably linked" generally refers to a situation where two DNA fragments are linked so that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0264] As used herein, "paratope" refers generally to the portion of an antibody that recognizes an antigen (e.g., the antigen binding site of an antibody). A paratope can be a small region (e.g., 15-22 amino acids) of the Fv region of an antibody and can contain portions of both the heavy and light chains of an antibody. See Goldsby et al. Antigens (Chapter 3) Immunology (5th ed.) New York: WH Freeman and Company, pp. 57–75.

[0265] As used herein, "patient" refers generally to any animal in need of treatment to alleviate a disease state or prevent the occurrence or recurrence of a disease state. As used herein, "patient" also refers generally to any animal that has risk factors, a history of disease, susceptibility, symptoms, signs, has been previously diagnosed, is at risk for a disease, or is a member of a patient population for a disease. A patient can be a clinical patient (such as a human), or a veterinary patient (such as a companion animal, domestic animal, livestock, exotic animal, or zoo animal). The term "subject" is used interchangeably with the term "patient". In a preferred embodiment of the invention disclosed herein, the patient is a human.

[0266] The terms "polypeptide," "peptide," and "protein" are used interchangeably and refer generally to polymers of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an analog or mimetic of a corresponding naturally occurring amino acid, and to naturally occurring amino acid polymers. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, and to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Polypeptides may be modified, for example, by the addition of carbohydrate residues to form glycoproteins. The terms "polypeptide," "peptide," and "protein" include glycoproteins as well as non-glycoproteins.

[0267] As used herein, "promoter" refers generally to a group of nucleic acid sequences that direct the transcription of a nucleic acid. As used herein, a promoter comprises essential nucleic acid sequences near the transcription start site, such as, in the case of a type II polymerase promoter, a TATA element. A promoter also optionally includes a distal enhancer or repressor element, which may be located as far as several thousand base pairs from the transcription start site. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulatory conditions.

[0268] As used herein, a "prophylactically effective amount" generally refers to an amount of a compound that, when administered to a patient for the prevention of a disease or prevention of the recurrence of a disease, is sufficient to achieve such prevention of the disease or recurrence. A prophylactically effective amount may be an amount effective to prevent the occurrence of signs and / or symptoms. A "prophylactically effective amount" may vary depending on the disease and its severity, as well as the age, weight, medical history, susceptibility to the condition, pre-existing conditions of the patient being treated.

[0269] As used herein, "prevention" refers broadly to a course of treatment in which signs and / or symptoms are absent in a patient, are in remission, or were previously present in a patient. Prevention includes preventing the development of a disease following treatment of a disease in a patient. In addition, prevention includes treating a patient who may develop a disease, particularly a patient who is susceptible to the disease (e.g., members of a patient population, those with risk factors or at risk of developing a disease).

[0270] As used herein, the term "recombinant" refers broadly to, for example, a cell, or a nucleic acid, protein, or vector, with respect to a product, indicating that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or by the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes not found in the native (non-recombinant) form of the cell or expresses native genes that are otherwise abnormally expressed, underexpressed, or not expressed at all.

[0271] As used herein, "specific (or selective) binding" or "specific (or selective) immunoreaction" or "specific interaction or binding" with an antibody refers broadly to a protein or peptide (or other epitope), and in some embodiments, refers to a binding reaction that determines the presence of a protein in a heterogeneous population of proteins and other biologicals. For example, under specified immunoassay conditions, a particular antibody binds to a particular protein at least two times greater than background (non-specific signal) and does not substantially bind to other proteins present in the sample in significant amounts. Typically, a specific or selective reaction will be at least two times the background signal or noise, and more typically about 10 to 100 times the background.

[0272] As used herein, "specifically hybridizable" and "complementary" generally refer to a nucleic acid that can form hydrogen bonds with another nucleic acid sequence by traditional Watson-Crick or other non-traditional types. The binding free energy of a nucleic acid molecule to its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, such as RNAi activity. Determination of the binding free energy of nucleic acid molecules is well known in the art. See, for example, Turner, et al. (1987) CSH Symp. Quant. Biol. LII: 123-33; Frier, et al. (1986) PNAS 83: 9373-77; Turner, et al. (1987) J. Am. Chem. Soc. 109: 3783-85. The complementarity percentage indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., at least about 5, 6, 7, 8, 9, 10 out of 10, which is at least about 50%, 60%, 70%, 80%, 90%, and 100% complementary, including the end values). "Fully complementary" or 100% complementarity generally refers to all consecutive residues of a nucleic acid sequence hydrogen bonding with the same number of consecutive residues in a second nucleic acid sequence. "Substantial complementarity" refers to polynucleotide chains that exhibit at least about 90% complementarity, excluding regions of the polynucleotide chain that are selected to be non-complementary, such as overhangs. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under conditions where specific binding is required (i.e., in the case of an in vivo assay or therapeutic treatment or under physiological conditions in the case of an in vitro assay, under the conditions under which the assay is performed). Non-target sequences can typically differ by at least 5 nucleotides.

[0273] As used herein, a "sign" of disease refers broadly to any abnormality indicative of disease that can be found upon examination of a patient; it is an objective indicator of disease, as distinguished from symptoms, which are subjective indicators of disease.

[0274] As used herein, "solid support," "support," and "substrate" broadly refer to any material that provides a solid or semisolid structure to which another material can be attached, including but not limited to smooth supports (e.g., metal, glass, plastic, silicon, and ceramic surfaces) and textured and porous materials. Exemplary solid supports include beads, such as activated beads, magnetically responsive beads, or fluorescently labeled beads.

[0275] As used herein, "subject" refers to any individual suitable for treatment according to the invention disclosed herein, including but not limited to avian and mammalian subjects, and preferably mammalian subjects. Mammals in the context of the invention disclosed herein include but are not limited to canines, felines, bovines, caprines, equines, ovines, swine, rodents (e.g., rats and mice), lagomorphs, primates, and humans. Any mammalian subject that needs to be treated according to the invention disclosed herein is suitable. Human subjects (i.e., neonates, infants, teenagers, adolescents, adults, elderly) of any gender and at any stage of development can be treated according to the present invention. The present invention can also be carried out for veterinary purposes and for drug screening and drug development purposes to animal subjects, particularly mammalian subjects (such as mice, rats, dogs, cats, cattle, goats, sheep, and horses). "Subject" and "patient" are used interchangeably. In a preferred embodiment of the disclosed invention, the subject is a human.

[0276] As used herein, "symptoms" of a disease broadly refer to any morbid phenomena or deviations from normal in structure, function, or sensation experienced by a patient and indicative of the disease.

[0277] As used herein, "therapy", "therapeutic", "treating" or "treatment" refers generally to treating a disease, preventing or reducing the development of a disease or its clinical symptoms, and / or alleviating a disease, causing regression of a disease or its clinical symptoms. Treatment includes prevention, treatment, remediation, reduction, alleviation and / or alleviation of a disease, signs and / or symptoms of a disease. Treatment includes alleviating the signs and / or symptoms of a patient with ongoing signs and / or symptoms of a disease (e.g., tumor growth, metastasis). Treatment also includes "prevention". For therapeutic purposes, the term "reduction" refers generally to a clinically significant reduction in signs and / or symptoms. Treatment includes treating worsening or recurring signs and / or symptoms (e.g., tumor growth, metastasis). Treatment includes, but is not limited to, excluding the appearance of signs and / or symptoms at any time, as well as reducing existing signs and / or symptoms, and eliminating existing signs and / or symptoms. Treatment includes treating chronic diseases ("maintenance") and acute diseases. For example, treatment includes treating or preventing worsening or recurrence of signs and / or symptoms (e.g., tumor growth, metastasis).

[0278] As used herein, "variable region" or "VR" refers generally to the domain within each pair of light and heavy chains in an antibody that is directly involved in binding the antibody to an antigen. Each heavy chain has a variable domain (V H ), followed by multiple constant domains. Each light chain has a variable domain at one end (V L) and at its other end has a constant domain; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.

[0279] As used herein, "vector" refers generally to plasmids, cosmids, phagemids, phage DNA, or other DNA molecules capable of autonomous replication in a host cell and characterized by one or a small number of restriction endonuclease recognition sites, where such DNA sequences can be cut in a determinable manner without losing the basic biological function of the vector, and where DNA can be inserted so that it can be replicated and cloned. The vector may also contain a marker suitable for identifying cells transformed with the vector.

[0280] Techniques and processes are generally performed according to conventional methods well known in the art, and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al. (2001) Molec. Cloning: Lab. Manual [3rd Edition] Cold Spring Harbor Laboratory Press. Standard techniques can be used for recombinant DNA, oligonucleotide synthesis and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions or as generally implemented in the art or as described herein. The nomenclature used for analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry as described herein and the laboratory procedures and techniques for analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry as described herein are well known in the art and commonly used. Standard techniques can be used for chemical synthesis, chemical analysis, drug preparation, preparation and delivery and treatment of patients.

[0281] Example

[0282] Having now generally described the invention, the invention will be more readily understood by reference to the following examples, which are included merely for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to limit the invention.

[0283] Example 1: NEO-201 mAb targets and can be used to deplete human granulocytic myeloid-derived suppressor cells

[0284] background

[0285] Myeloid-derived suppressor cells

[0286] Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that are increased in cancer, inflammation, and infection. Myeloid-derived suppressor cells are associated with cancer evasion as well as tumor progression and metastasis by suppressing antitumor immune responses. The heterogeneous population of immature myeloid cells includes monocytic-MDSCs (mMDSCs) and granulocytic-MDSCs (gMDSCs) (Zilio S. and Serafini P., “Neutrophils and granulocytic MDSCs: The Janus God of cancer immunotherapy”, Vaccine 2016; 4(3):31; Aarts CEM and Kuijpers TW., “Neutrophils as myeloid-derived suppressor cells”, Eur J Clin Invest 2018; Nov, 48 Suppl 2: e12989.) Human MDSCs express myeloid cell markers such as CD11b positive and CD33 positive, but are usually negative for HLA-DR, CD3, CD19, and CD57. Monocyte MDSCs usually have a HLA-DR negative, CD11b positive, CD33 positive, and CD14 positive phenotype. Granulocyte MDSCs are usually characterized by a HLA-DR negative, CD11b positive, CD33 positive, and CD15 positive phenotype (Zilio S. and Serafini P., "Neutrophils and granulocytic MDSC: The Janus God of cancer immunotherapy", Vaccine 2016; 4(3): 31).

[0287] As discussed above, clinical studies have demonstrated the prognostic role of tumor-infiltrating neutrophils, elevated blood neutrophils, and elevated blood neutrophil / lymphocyte ratios, and in particular, such cells are associated with poor clinical outcomes in different human cancers. These results emphasize the importance and relevance of neutrophils in cancer biology.

[0288] Neutrophils are the primary inflammatory cells and are essential for protecting the host from invading pathogens such as bacteria and fungi. In recent years, neutrophils have been shown to be highly functionally plastic and can have both pro- and anti-tumor activities. Pro-tumor neutrophils function as suppressors of adaptive immune responses in cancer. Expansion of both immature and mature neutrophils has been observed to suppress T cell proliferation. Pro-tumor neutrophils are functionally related to gMDSCs. Myeloid-derived suppressor cells play an important role in suppressing host immune responses through multiple mechanisms, such as producing (a) arginase 1, (b) releasing reactive oxygen species (ROS), (c) releasing nitric oxide, and (d) secreting suppressive cytokines (Donskov F. et al., “Immunomonitoring and prognostic relevance of neutrophils in clinical trials”, Seminars in Cancer Biology 2013; 23: 200-2071; Sagiv J et al., “Phenotypicdiversity and plasticity in circulating neutrophil subpopulations in cancer”, Cell Reports 2015; 10: 562-573.)

[0289] Granulocytes are derived from hematopoietic stem cells in the bone marrow, which are controlled by granulocyte colony stimulating factor (G-CSF). Under pathological conditions, MDSCs can be produced in the bone marrow in response to factors such as G-CSF, GM-CSF, IL-6, IL-1-β, prostaglandin E2 (PGE2), TNF-α and VEGF caused by cancer and infection (Lechner MG et al., "Characterization of cytokine-induced myeloid-derived suppressor cells from normal peripheral blood mononuclear cells", J Immunol 2010; 185: 2273-2284).

[0290] NEO-201 monoclonal antibody

[0291] NEO-201 is a therapeutic IgG1 humanized mAb that is reactive against many different cancers but not against most normal epithelial tissues. No reactivity with NEO-201 was observed in hematopoietic cell subsets, except for CD15+ granulocytes and circulating Treg cells. Functional analysis revealed that NEO-201 can engage in ADCC and CDC to kill tumor cells. Previous studies have shown that NEO-201 can attenuate the growth of human tumor xenografts in mice and is safe / tolerable in non-human primates, with a transient decrease in neutrophils as the only adverse effect observed. The NIH Clinical Center is conducting the first human clinical trial evaluating NEO-201 in adults with chemotherapy-resistant solid tumors (Fantini M et al., “Preclinical characterization of a novel monoclonal antibody NEO-201 for the treatment of human carcinoma”, Front Immunol 2018; 8: 1899; Zeligs KP et al., “Evaluation of the anti-tumor activity of the humanized monoclonal antibody NEO-201 in preclinical models of ovarian cancer”, Front Oncol. 2020; 10: 805).

[0292] NEO-201 recognizes tumor-associated variants of CEACAM5 and 6 that carry core-1 and / or extended core-1 O-glycans. CEACAM1 is a potent inhibitor of natural killer (NK) cell function; binding between CEACAM1 on NK cells and CEACAM1 or CEACAM5 on tumor cells inhibits activation signaling of NKG2D, which prevents NK cell cytolysis and allows tumor cells to evade NK killing (Fantini M et al., "The monoclonal antibody NEO-201 enhances natural killer cell cytotoxicity against tumor cells through blockade of the inhibitory CEACAM5 / CEACAM1 immune checkpoint pathway", Cancer Biotherapy and Radiopharm 2020; 35(3): 190-198).

[0293] Materials and methods

[0294] In vitro generation of human gMDSCs

[0295] EasySep was used according to the manufacturer's protocol. Tm The Direct Human Neutrophil Isolation Kit (STEMCELL, catalog number 19257) immunomagnetically isolates neutrophils directly from whole blood from healthy donors.

[0296] The isolated neutrophils were cultured at 5 x 10 5 The cells were cultured in complete RPMI1640 medium at a concentration of 10 cells / ml. The medium was supplemented with human IL-6 (10 ng / mL, PeproTech, Inc.) and GM-CSF (10 ng / mL, PeproTech, Inc.) for 7 days at 37°C. The medium and cytokines were renewed every 2-3 days. Cells cultured for 7 days were collected from flow cytometric analysis of cell phenotype.

[0297] Phenotypic analysis by flow cytometry

[0298] Basically according to the method of Lechner et al. (Lechner MG et al., "Characterization of cytokine-induced myeloid-derived suppressor cells from normal peripheral blood mononuclear cells", J Immunol 2010; 185: 2273-2284), the expression of HLA-DR, CD33, CD66b, CD14, CD15 and NEO-201 target antigens in the phenotype of gMDSC generated in vitro was evaluated by flow cytometry. First, gMDSC was incubated with 1 μL of LIVE / DEAD Fixable Aqua (Thermo Fisher Scientific, Waltham, MA, USA) per test in 1 mL of 1X phosphate buffered saline (PBS) (VWR International, Radnor, PA, USA) for 30 minutes at 4°C to complete the identification of live cells and dead cells. Then, the cells were washed with 1X PBS and incubated at room temperature with 2-5 μL of human TruStain FcX in 100 μL 1X PBS. TM(BioLegend, San Diego, CA, USA) and incubate together for 5-10 minutes. In order to detect surface markers, the cells were then stained with 2-4 μL / sample of the following anti-human mAbs for 30 minutes in 100 μL of 1X PBS+1% BSA (Teknova, Hollister, CA, USA) at 4 ° C: HLA-DR-PE, CD33-APC, CD14-PerCP-Cy5.5, CD15-FITC, CD66b-PE-Cy7, NEO-201-Pacific Blue (BioLegend, San Diego, CA, USA). After staining, the cells were washed twice with cold 1X PBS and examined using a FACSVerse flow cytometer (BD Biosciences, San Jose, CA, USA). Cell fluorescence analysis was performed using BD FACSuite software (BD Biosciences, San Jose, CA, USA). Positive was determined by using a fluorescence minus one control.

[0299] ADCC assay

[0300] Flow cytometry was used to analyze NEO-201-mediated ADCC activity against gMDSCs. ADCC assays were performed essentially according to Lechner et al. (Lechner MG et al., "Characterization of cytokine-induced myeloid-derived suppressor cells from normal peripheral blood mononuclear cells", J Immunol 2010; 185: 2273-2284).

[0301] For ADCC determination, gMDSCs generated from neutrophils from 2 healthy donors were used as target cells. After culturing for 7 days in complete RPMI1640 medium supplemented with human IL-6 and GM-CSF, the gMDSCs produced were harvested and centrifuged at 1500rpm for 5 minutes. The supernatant was then discarded and the precipitate was washed with 1X PBS. The cells were then incubated with 1 μL of LIVE / DEAD Fixable Aqua per test in 1mL of 1X PBS for 30 minutes at 4°C to complete the identification of live cells from dead cells. The cells were then washed with 1XPBS and stained with 2-4 μL / sample of the following anti-human mAbs in 100 μL of 1X PBS+1% BSA at 4°C for 30 minutes: HLA-DR-PE, CD33-APC. After staining, the cells were washed twice with cold 1X PBS.

[0302] On the day of ADCC determination, PMBC from different healthy donors were thawed and cultured in RPMI complete medium. PBMC was used as effector cells, and with 100:1 and 50:1 effectors: target (E:T) ratios were added to the tube containing gMDSC stained with HLA-DR-PE and CD33-APC antibodies with or without NEO-201 (10 μg / mL). In these experiments, gMDSC treated only with culture medium were used as controls.

[0303] The cells were then incubated at 37°C for 4 hours. After incubation, the cells were washed twice with cold 1X PBS and examined using a FACSVerse flow cytometer (BD Biosciences, San Jose, CA, USA). Cell fluorescence analysis was performed using BD FACSuite software (BD Biosciences, San Jose, CA, USA). Positive was determined by using a fluorescence minus one control. To evaluate NEO-201-mediated ADCC activity, CD33 in gMDSCs incubated with medium alone was expressed. pos / HLA-DR neg The percentage of live cells was compared with CD33 incubated with PBMC alone and PBMC plus NEO-201 pos / HLA-DR neg The percentages of viable cells were compared.

[0304] result

[0305] Phenotypic analysis of gMDSCs generated from human neutrophils

[0306] Whole blood from four normal donors was used in this study. Fig.10 As shown in the table, 47.59% to 52.58% of neutrophils treated with 10 ng / ml human GM-CSF and 10 ng / ml human IL-6 were HLA-DR negative and CD33 positive. 76.4% to 88.09% of the HLA-DR negative and CD33 positive population were CD15 positive and CD14 negative. 66.44% to 99.71% of the HLA-DR negative / CD33 positive / CD15 positive / CD14 negative population were CD66 positive and NEO-201 positive.

[0307] Figure 6-9 Included are the results of flow cytometric analysis of gMDSCs generated from GM-CSF- and IL-6-treated neutrophils from four normal donors.

[0308] ADCC assay results

[0309] To evaluate whether NEO-201 can eliminate human gMDSCs by ADCC, gMDSCs generated from neutrophils from 2 healthy donors were used as target cells in an ADCC assay performed by flow cytometry. pos / HLA-DR neg The percentage of live cells was compared with CD33 incubated with PBMC alone and PBMC plus NEO-201 pos / HLA-DR neg The percentage of viable cells was compared to evaluate the ADCC activity of NEO-201.

[0310] like Fig.11 As shown in Figure 2, when gMDSCs were incubated with PBMCs (E:T 100:1) plus NEO-201, we observed an increase in CD33 compared to gMDSCs incubated with PBMCs (E:T 100:1) alone from healthy donors 1 and 2. pos / HLA-DR neg The viable cells were reduced by 33.01% (18.29% vs. 27.23%) and 29.5% (25.95% vs. 36.83%), respectively. In two healthy donors, CD33 was observed in gMDSCs incubated with PBMCs (E:T 50:1) plus NEO-201 compared to gMDSCs incubated with PBMCs (E:T 50:1) alone. pos / HLA-DR neg A similar reduction in viable cells was observed.

[0311] These data provide convincing evidence that NEO-201 can deplete or eliminate gMDSC by ADCC-mediated in vitro lysis. Based on these results, we expect that NEO-201 alone and in combination with other active substances (e.g., antibodies targeting checkpoint inhibitors and other biological agents or chemotherapeutic agents) should be used to alleviate immunosuppression and resistance to treatment caused by MDSC. In particular, NEO-201 can be used to alleviate immunosuppression and resistance to treatment in individuals with cancers and chronic diseases involving MDSC-mediated immunosuppression and resistance to treatment.

[0312] Example 2: The percentage of granulocytic myeloid-derived suppressor cells (gMDSCs) in peripheral blood mononuclear cells (PBMCs) is reduced after treatment with NEO-201 and pembrolizumab

[0313] Materials and methods

[0314] Phenotypic analysis of gMDSCs in PBMCs from cancer patients by flow cytometry

[0315] To investigate whether NEO-201 can bind to and consume gMDSCs in cancer patients, PBMCs from four patients were profiled by flow cytometry for the expression of specific gMDSC markers (including HLA-DR, CD33, CD66b, CD14, CD15, and NEO-201). PBMCs were thawed and first incubated at 4°C with 1 μL of LIVE / DEAD Fixable Aqua (Thermo Fisher Scientific, Waltham, MA, USA) per test in 1 mL of 1X phosphate buffered saline (PBS) (VWR International, Radnor, PA, USA) for 30 minutes to complete the identification of live and dead cells. Then, the cells were washed with 1X PBS and incubated at room temperature with 2-5 μL of human TruStain FcX in 100uL 1X PBS. TM (BioLegend, San Diego, CA, USA) and incubated together for 5-10 minutes. For gMDSC markers, the cells were then stained with 2-4 μL / sample of the following anti-human mAbs in 100 uL of 1XPBS+1%BSA (Teknova, Hollister, CA, USA) at 4 °C for 30 minutes: HLA-DR-PE, CD33-APC, CD14-PerCP-Cy5.5, CD15-FITC, CD66b-PE-Cy7, NEO-201-PacificBlue (BioLegend, San Diego, CA, USA). After staining, the cells were washed twice with cold 1X PBS and examined using a FACSVerse flow cytometer (BD Biosciences, San Jose, CA, USA). Cell fluorescence analysis was performed using BD FACSuite software (BD Biosciences, San Jose, CA, USA). Positive was determined by using a fluorescence minus one control.

[0316] result

[0317] Phenotypic analysis of gMDSCs from PBMCs of cancer patients

[0318] To evaluate whether treatment with NEO-201 affects the percentage of circulating gMDSCs in cancer patients, PBMCs from four cancer patients enrolled in a Phase IIa clinical trial combining NEO-201 with pembrolizumab in adults with chemotherapy-resistant solid tumors who had failed prior checkpoint inhibitor therapy (Clinical Trial NCT03476681) were used in this study.

[0319] Each treatment cycle was 42 days in length and consisted of 3 doses of 1.5 mg / kg NEO-201 IV every 2 weeks and 1 dose of 400 mg pembrolizumab IV every 6 weeks.

[0320] Radiologic assessments, including CT, MRI, or PET-CT (as appropriate), were performed prior to the initial infusion and repeated every 2 cycles (every 84 days) thereafter to correlate clinical response with modulation of the percentage and function of immune cells, including gMDSCs.

[0321] In four cancer patients, the percentage of gMDSCs in PBMCs was analyzed before starting treatment with NEO-201 (C1D1 PRE), 14 days after the first infusion with NEO-201 (C1D15), before cycle 2 (C2D1 PRE; 42 days after the first infusion), and before cycle 3 (C3D1 PRE; 84 days after the first infusion).

[0322] The gMDSC population in live PBMCs was defined as HLA-DR neg / CD33 + / CD15+ / CD14neg / CD66b + cell.

[0323] The results include Fig.12 Figure 2 and depicts the comparison of the percentage of circulating gMDSC (HLA-DR- / CD33+ / CD15+ / CD14- / CD66b+ cells) between 2 cancer patients with stable disease (SD) and 2 cancer patients with progressive disease (PD) at different time points by flow cytometry analysis. gMDSCs are gated from live PBMCs. Data are presented as the median of the percentage of live cells expressing gMDSC markers. Positive is determined by using fluorescence minus one control. In the figure, "HNSCC" refers to "head and neck squamous cell carcinoma".

[0324] like Fig.12 As shown, a patient with head and neck squamous cell carcinoma (HNSCC) showed a significant reduction in gMDSCs, i.e., a 93.64% reduction in gMDSCs after 84 days of treatment (C3D1 PRE) compared to baseline levels (C2D1 PRE: 0.22% vs. 0.22%; C3D1 PRE: 0.014% vs. 0.22%). This patient showed stable disease (SD) after more than 5 months of treatment after the first infusion of NEO-201 and pembrolizumab and is currently still receiving treatment.

[0325] like Fig.12As further shown in , another patient with SD (a patient with cervical cancer) showed an increase in circulating gMDSCs at C1D15 and C2D1PRE compared to baseline levels (C1D15: 0.15% vs. 0.11%; C2D1 PRE: 0.25% vs. 0.11%), but the percentage of gMDSCs began to decrease toward baseline levels at C3D1 (C3D1 PRE: 0.16% vs. 0.11%). This patient showed SD after more than 8 months of treatment following the first infusion of NEO-201 and pembrolizumab, suggesting that the reduction in circulating gMDSCs that began at C3D1PRE may persist beyond this time point and may contribute to disease stabilization.

[0326] On the contrary, Fig.12 As further shown in Figure 2, in one patient with uterine cancer (patient 4), the percentage of circulating gMDSCs increased after treatment. This phenomenon correlated with the progression of the disease (PD) reported at the time of the first restaging (previously C3D1).

[0327] like Fig.12 It was also shown in that another patient with uterine cancer (patient 5) who showed PD at the time of first restaging was found to have an initial increase at C1D15, followed by a 60% decrease in circulating gMDSCs compared to baseline at C2D1 (0.052% vs. 0.13%), suggesting that the observed clinically important decrease in circulating gMDSCs may not be the only factor involved in the clinical response.

[0328] These clinical results provide convincing evidence that administration of the NEO-201 antibody, alone or in combination with other therapies, can be used to deplete gMDSCs in patients in need (e.g., adult cancer patients with chemotherapy-resistant solid tumors and / or who have failed prior checkpoint inhibitor therapy), and that such treatment can reverse or significantly alleviate gMDSC-associated resistance or tolerance to chemotherapy and / or checkpoint inhibitor therapy.

[0329] Humanized NEO-201 monoclonal antibody sequence

[0330] The sequence of the NEO-201 antibody used in these examples is shown below:

[0331] H16C3-Abb* Heavy Chain:

[0332]

[0333] H16C3-Abb* Light chain:

[0334]

[0335] The boundaries between the expression leader sequence, variable region and constant region are delineated by forward slashes (" / ") in each sequence, and the CDR sequences are shown in bold, underlined text. The antibody sequences used include the variable and constant regions shown. These include the heavy chain CDR1 of SEQ ID NO:32, the heavy chain CDR2 of SEQ ID NO:33, the heavy chain CDR3 of SEQ ID NO:34, the light chain CDR1 of SEQ ID NO:35, the light chain CDR2 of SEQ ID NO:36, and the light chain CDR3 of SEQ ID NO:37.

[0336] Each document cited herein is hereby incorporated by reference in its entirety.

Claims

1. A method for killing or eliminating granulocyte-derived myeloid-derived suppressor cells (gMDSCs) in a patient in need thereof, the method comprising administering to the patient an effective amount of an antibody or antibody fragment that binds to glycosylated CEACAM5 and CEACAM6 carrying core-1 and / or extended core-1 O-glycans, but does not bind to aglycosylated CEACAM5 or aglycosylated CEACAM6.

2. The method of claim 1, wherein the antibody or antibody fragment recognizes an O-glycosylated epitope that binds to a threonine in the region of amino acids 310 to 318 (RTTVTTITV) of CEACAM5 and to a threonine and a serine in the region of amino acids 312 to 320 (TVTMITVSG) of CEACAM6.

3. A method of killing or eliminating granulocytic myeloid-derived suppressor cells (gMDSC) in a patient in need thereof, the method comprising administering to the patient an effective amount of NEO-201 or an antigen-binding fragment thereof.

4. A method of reversing tolerance and / or restoring innate immunity, such as innate anti-tumor immunity or innate anti-infective immunity, in a patient in need thereof by killing or eliminating granulocytic myeloid-derived suppressor cells (gMDSCs) in the subject, the method comprising administering to the patient an effective amount of NEO-201 or an antigen-binding fragment thereof.

5. A method of reversing resistance or tolerance to an anti-cancer or anti-infective treatment, such as an immunomodulatory antibody, a checkpoint inhibitor antibody or fusion protein, or a chemotherapeutic agent, wherein the resistance or tolerance involves granulocytic myeloid-derived suppressor cells, by administering NEO-201 alone or in combination with another treatment, such as an immunomodulatory antibody, a checkpoint inhibitor antibody or fusion protein, or a chemotherapeutic agent, so as to reverse such resistance or tolerance.

6. A method of treating or preventing recurrence of cancer or infection by administering NEO-201 alone or in combination with another treatment, such as an immunomodulatory antibody, a checkpoint inhibitor antibody or fusion protein, or a chemotherapeutic agent, to inhibit the proliferation of gMDSCs, thereby reconstituting innate immunity.

7. The method of any of the preceding claims, wherein the gMDSCs express O-glycans selected from one or more of 01, 02, 06, 023, 026 and 039 O-glycans having the structures shown in the arrays in Figures 2 and 5.

8. The method of any of the preceding claims, wherein the gMDSCs express 06, 01 or 02 O-glycans having the structure shown in the arrays in Figures 2 and / or 5.

9. The method of any of the preceding claims, wherein the gMDSCs express 06O-glycans as shown in the arrays in Figures 2 and / or 5.

10. The method of any one of the preceding claims, wherein the gMDSCs express Tn antigen or core 1, 2, 4 or 4 O-glycans having the structure shown in Figure 1 .

11. The method of any one of the preceding claims, wherein the patient suffers from cancer or an infectious disease, wherein gMDSCs are implicated in the disease pathology and / or immunosuppression of innate immunity against the disease.

12. The method of any one of the preceding claims, wherein the antibody or antigen-binding fragment is linked directly or indirectly to a cytotoxic agent, optionally a radionuclide or a chemotherapeutic agent.

13. The method of any one of the preceding claims, wherein the antibody or antigen-binding fragment is directly or indirectly linked to a label, optionally a fluorescent or radioactive label.

14. The method of any of the preceding claims, wherein the subject treated has a cancer in which gMDSCs are involved in the disease pathology, optionally wherein the treated cancer cells do not express or overexpress an antigen bound by NEO-201.

15. The method of any of the preceding claims, wherein the treated subject has a cancer in which gMDSCs are involved in the pathology of the disease, the cancer optionally being adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, adult brain / CNS tumors, childhood brain / CNS tumors, breast cancer, male breast cancer, adolescent cancer, childhood cancer, young adult cancer, cancer of unknown primary, Castleman's disease, cervical cancer, colon / rectum cancer, endometrial cancer, esophageal cancer, Ewing's family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, leukemia-adult acute lymphocytic (ALL), leukemia-acute myeloid (AML), leukemia-chronic lymphocytic (CLL), leukemia-chronic myeloid (CML), leukemia-chronic NEO-201 is a cancer cell-derived, non-small cell, myelomonocytic (CMML), childhood leukemia, liver cancer, lung cancer, lung cancer - non-small cell, lung cancer - small cell, carcinoid tumors of the lung, lymphoma, cutaneous lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, childhood non-Hodgkin lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma - adult soft tissue cancer, skin cancer, skin cancer - basal and squamous cell, skin cancer - melanoma, skin cancer - Merkel cell, small intestine cancer, gastric cancer, testicular cancer, thymic cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor, optionally wherein the treated cancer cells do not express or overexpress an antigen bound by NEO-201.

16. The method of any of the preceding claims, wherein the treated subject has a cancer in which gMDSCs are involved in the pathology of the disease, the cancer optionally being a cancer and / or tumor selected from the group consisting of lung cancer, breast cancer, triple negative breast cancer (TNBC), colorectal cancer, liver cancer, gastric cancer, colon cancer, non-small cell lung cancer (NSCLC), bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, colorectal cancer, small intestine cancer, rectal cancer, anal cancer, fallopian tube cancer, endometrial cancer, uterine cancer. Neck cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, lymph node cancer, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, adenocarcinoma, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer or ureteral cancer, renal cell carcinoma, renal pelvis cancer, central nervous system tumors, primary CNS tumors, spinal cord tumors, brain stem gliomas and pituitary adenomas, optionally wherein the treated cancer cells do not express or overexpress an antigen bound by NEO-201.

17. The method of any of the preceding claims, wherein the cancer or infection being treated is not characterized by expression of glycosylated CEACAM5 and / or glycosylated CEACAM6 carrying core-1 and / or extended core-1 O-glycans; and / or is not characterized by increased expression of glycosylated CEACAM5 and / or glycosylated CEACAM6.

18. The method of any of the preceding claims, wherein the treated subject has a cancer in which gMDSCs are involved in disease pathology and the treatment elicits one or more of: (i) increased T cell responses, (ii) increased antigen presentation, (iii) decreased MDSC proliferation and / or (iv) decreased Treg recruitment.

19. The method of any of the preceding claims, wherein the treated subject has a stage I, II, III, or IV cancer involving gMDSCs.

20. The method of any of the preceding claims, wherein the antibody or fragment, optionally NEO-201, reduces, eliminates, slows or prevents the growth of a tumor, wherein in a patient whose anti-tumor immunity was previously suppressed by gMDSCs, the tumor burden in the individual is reduced, tumor growth is inhibited, and / or the survival of the individual is increased.

21. The method of any one of the preceding claims, wherein the subject suffers from an infectious disorder wherein the disease pathology involves gMDSCs.

22. The method of any one of the preceding claims, wherein the subject has a bacterial infection involving gMDSCs, optionally Bacillus anthracis, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis and Enterococcus faecium, Escherichia coli ( coli O157:H7, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori), Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia, Salmonella Typhi, Salmonella Typhimurium, Shigella sonnei, and / or Staphylococcus aureus infections.

23. A method as described in any of the preceding claims, wherein the subject has a chronic or acute viral infection involving gMDSCs, optionally associated with the following viruses: respiratory viruses such as adenovirus, avian influenza, influenza A virus, influenza B virus, measles, parainfluenza virus, respiratory syncytial virus (RSV), rhinovirus, SARS-CoV; gastrointestinal viruses such as coxsackievirus, enterovirus, poliovirus, rotavirus; hepatitis viruses such as hepatitis B virus, hepatitis C virus; bovine viral diarrhea virus (surrogate); herpes viruses such as herpes simplex type 1 , herpes simplex type 2, human cytomegalovirus, varicella zoster virus; retroviruses, such as human immunodeficiency virus type 1 (HIV-1), human immunodeficiency virus type 2 (HIV-2), simian immunodeficiency virus (SIV), simian human immunodeficiency virus (SHIV); viral selectors / emerging viral pathogens, such as avian influenza, dengue virus, hantavirus, hemorrhagic fever virus, lymphocytic choriomeningitis virus, smallpox virus surrogate, cowpox, monkeypox, rabbitpox, vaccinia virus, Venezuelan equine encephalitis virus (VEE), West Nile virus, yellow fever virus.

24. The method of any of the preceding claims, wherein the subject has a condition involving gMDSCs, wherein gMDSCs are involved in suppressing innate immunity, the condition optionally being acquired immunodeficiency syndrome (AIDS), acute disseminated encephalomyelitis (ADEM), Addison's disease, agammaglobulinemia, allergic disease, alopecia areata, Alzheimer's disease, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, antisynthetase syndrome, arterial plaque disorder, asthma, atherosclerosis, atopic allergy, atopic dermatitis, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune thyroid function hypofunction, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune urticaria, autoimmune uveitis, Barlow's disease / Barlow's concentric sclerosis, Behcet's disease, Buerger's disease, Bickerstaff encephalitis, Blau syndrome, bullous pemphigoid, Castleman's disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy, chronic recurrent multifocal osteomyelitis, chronic obstructive pulmonary disease, chronic venous stasis ulcer, Charg-Strauss syndrome, cicatricial pemphigoid, Cogan's syndrome, cold agglutinin disease, complement component 2 deficiency Asthenia, contact dermatitis, cranial arteritis, CREST syndrome, Crohn's disease, Cushing's syndrome, cutaneous leukocytoclastic vasculitis, Degos' disease, Derken's disease, dermatitis herpetiformis, dermatomyositis, type 1 diabetes mellitus, type 2 diabetes mellitus, diffuse cutaneous systemic sclerosis, Dressler syndrome, drug-induced lupus, discoid lupus erythematosus, eczema, emphysema, endometriosis, enthesitis-associated arthritis, eosinophilic fasciitis, eosinophilic gastroenteritis, eosinophilic pneumonia, epidermolysis bullosa acquisita, erythema nodosum, erythroblastosis fetalis, essential mixed cryoglobulinemia, Evan's syndrome, progressive fibrodysplasia ossificans, fibrosing alveolitis (or idiopathic pulmonary fibrosis), gastritis, gastrointestinal pemphigoid sores, Gaucher's disease, glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's encephalopathy, Hashimoto's thyroiditis, heart disease, Henlein-Schönlein purpura, herpes gestationis (also known as pemphigoid gestationis), suppurative hidradenitis, HIV infection, Hof-Stauwen syndrome, hypogammaglobulinemia, infectious diseases (including bacterial infectious diseases), idiopathic inflammatory demyelinating diseases, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, IgA nephropathy, inclusion body myositis, inflammatory arthritis, inflammatory bowel disease, inflammatory dementia, interstitial cystitis, interstitial pneumonia, juvenile idiopathic arthritis (also known as juvenile rheumatoid arthritis), Kawasaki disease, Lebert-Eaton myasthenic syndrome,Leukocytoclastic vasculitis, lichen planus, lichen sclerosus, linear IgA disease (LAD), lupus hepatitis (also known as autoimmune hepatitis), lupus erythematosus, lymphomatous granulomatosis, Magid syndrome, malignancies including cancer (e.g., sarcomas, Kaposi's sarcoma, lymphomas, leukemias, carcinomas, and melanomas), Meniere's disease, microscopic polyangiitis, Miller-Fisher syndrome, mixed connective tissue disease, morphea, Muhlenberg-Harris disease (also known as acute pityriasis lichenoides), multiple sclerosis, myasthenia gravis, myositis, narcolepsy, optic nerve Myelitis (also known as Devic's disease), neuromyotonia, ocular cicatricial pemphigoid, opsoclonus-myoclonus syndrome, Ord's thyroiditis, relapsing rheumatic disease, PANDAS (pediatric autoimmune neuropsychiatric disorder associated with Streptococcus), paraneoplastic cerebellar degeneration, Parkinson's disease, paroxysmal nocturnal hemoglobinuria (PNH), Parry-Roberg syndrome, Parsena-Turner syndrome, pars planitis, pemphigus vulgaris, peripheral arterial disease, pernicious anemia, perivenous encephalomyelitis, POEMS syndrome, hyperactivity nodularis Arthritis, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, pyoderma gangrenosum, pure red cell aplasia, Rasmussen's encephalitis, Raynaud's phenomenon, relapsing polychondritis, Reiter's syndrome, restenosis, restless legs syndrome, retroperitoneal fibrosis, rheumatoid arthritis, rheumatic fever, sarcoidosis, schizophrenia, Schmidt's syndrome, Schnitzler's syndrome, scleritis, scleroderma, sepsis, serum sickness, Sjögren's syndrome, spondyloarthropathies, Still's disease (adult-onset), stiff-person syndrome, stroke, subacute bacterial endocarditis (SBE), Susac syndrome, Sweet's syndrome, Sydenham's chorea, sympathetic ophthalmia, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis (also known as "giant cell arteritis"), thrombocytopenia, Tolman-Schönlein syndrome), transplant (e.g., heart / lung) rejection, transverse myelitis, tuberculosis, ulcerative colitis, undifferentiated connective tissue disease, undifferentiated spondyloarthropathies, urticarial vasculitis, vasculitis, vitiligo, and Wegener's granulomatosis.

25. The method of any of the preceding claims, wherein gMDSCs are detected and monitored in the patient before, during and after completion of treatment and / or after the patient enters remission.

26. The method of any of the preceding claims, wherein gMDSCs are detected in the patient prior to treatment to determine whether the patient will likely benefit from NEO-201 treatment.

27. The method of claim 25 or 26, wherein gMDSCs in a biological sample are detected using one or more ligands, such as antibodies that recognize specific biomarkers expressed on gMDSCs, optionally LOX-1, CD11b, CD15, CD66b, and glycosylated CEACAM5 and CEACAM6 antigens recognized by NEO-201.

28. The method of claim 25, 26 or 27, wherein the number or concentration of gMDSCs in a sample from a subject having a cancer involving gMDSCs is used to monitor the progression of the cancer (with or without treatment), wherein the patient is being treated for such cancer with NEO-201 alone or in combination with another therapeutic agent.

29. The method of any one of claims 25-28, wherein the number or concentration of gMDSCs in a sample from a subject having a cancer involving gMDSCs is used to determine whether NEO-201 alone or in combination with another therapeutic agent may be beneficial in treating the cancer, wherein the patient is being treated for such cancer with NEO-201 alone or in combination with another therapeutic agent.

30. The method of any one of claims 25-29, wherein the number or concentration of gMDSCs in a sample from a subject having a cancer involving gMDSCs is used to develop a dosing regimen for NEO-201 alone or in combination with another therapeutic agent.

31. The method of any one of claims 25 to 30, wherein: Wherein the level of gMDSCs in a patient sample, such as blood or a biopsy sample, is used to determine cancer prognosis before, during or after NEO-201 treatment, the method optionally comprises contacting the gMDSCs with a NEO-201 antibody.

32. The method of any one of claims 25-31, wherein the detecting comprises cell sorting, optionally fluorescence activated cell sorting, thereby generating a sample enriched and / or depleted for cells positive for expression of the NEO-201 antigen, such as gMDSCs.

33. The method of any one of claims 25-32, comprising detecting and / or staining gMDSCs by contacting cells with a NEO-201 antibody and detecting cells expressing NEO-201, wherein optionally NEO-201 is directly or indirectly labeled.

34. The method of any one of claims 25-33, wherein gMDSCs are isolated by contacting a patient sample with a support comprising NEO-201 antibodies and / or using other antibodies or ligands that recognize other gMDSC biomarkers, thereby retaining the gMDSCs on the support.

35. The method of any one of claims 25-34, wherein the level of gMDSCs in a patient sample, such as blood or a biopsy sample, is used to determine whether the patient suffers from or is likely to develop gMDSC-mediated immunosuppression.

36. The method of any of the preceding claims, comprising administering an additional therapeutic agent.

37. The method of any of the preceding claims, comprising administering at least one additional therapeutic agent, wherein administration of NEO-201 or other antibodies that bind to glycosylated CEACAM 5 and CEACAM6 carrying core-1 and / or extended core-1O glycans, but not to aglycosylated CEACAM5 or aglycosylated CEACAM6, potentiates the efficacy of the at least one additional therapeutic agent.

38. The method of claim 36 or 37, wherein the other therapeutic agent comprises another therapeutic antibody, a checkpoint inhibitor, a chemotherapeutic agent, and / or comprises immune cells, optionally CAR-T or CAR-NK cells.

39. The method of claim 36 or 37, wherein the additional therapeutic agent comprises (i) another moiety that eliminates gMDSCs, (ii) a moiety that promotes gMDSC differentiation, (iii) a moiety that inhibits gMDSC migration, (iv) an epigenetic therapy that targets gMDSCs, a moiety, or (v) a chemotherapeutic agent that targets gMDSCs, or a combination of one or more of the foregoing.

40. The method of any of the preceding claims, wherein NEO-201, due to its ability to deplete gMDSCs, enhances the efficacy of the other therapeutic agent by boosting innate immunity, e.g., an innate anti-tumor or anti-infective response, optionally in a subject previously resistant to treatment with the other therapeutic agent.

41. The method of any one of claims 35-40, wherein such one or more additional therapeutic agents include but are not limited to peptides, nucleic acid molecules, small molecule compounds, antibodies, and derivatives thereof.

42. A method as described in any of claims 35-41, wherein such one or more additional therapeutic agents include an immune checkpoint inhibitor, optionally an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-CD28 antibody, an anti-TIGIT antibody, an anti-LAGS antibody, an anti-TIM3 antibody, an anti-GITR antibody, an anti-4-1BB antibody, or an anti-OX-40 antibody, and / or the one or more additional therapeutic agents include therapeutic agents targeting adenosine A2A receptor (AZAR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAGS), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V domain Ig inhibitor of T cell activation (VISTA). In particular, the immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

43. The method of any one of claims 35-42, wherein the one or more additional therapeutic agents comprises a CSF-1 / 1R binding agent or inhibitor.

44. The method of any one of claims 35-43, wherein the one or more additional therapeutic agents include (a) microtubule inhibitors, topoisomerase inhibitors, platinum, alkylating agents, and antimetabolites; (b) MK-2206, ON 013105, RTA 402, BI 2536, sorafenib, ISIS-STAT3Rx, microtubule inhibitors, topoisomerase inhibitors, platinum, alkylating agents, antimetabolites, paclitaxel, gemcitabine, doxorubicin, vinblastine, etoposide, 5-fluorouracil, carboplatin, hexamethylmelamine, aminoglutethimide, amsacrine, anastrozole, azacitidine, bleomycin, busulfan, carmustine, chlorambucil, 2-chlorodeoxyadenosine, cisplatin, colchicine, cyclophosphamide, cytarabine, cyclophosphamide, dacarbazine, actinomycin D, daunorubicin, docetaxel, estramustine phosphate, floxuridine, fludarabine, gemtuzumab, hexamethylmelamine, hydroxyurea, ifosfamide, imatinib, interferon, irinotecan, lomustine, nitrogen mustard, melphalan, 6-mercaptopurine, methotrexate, mitomycin (c) 1-D-ribofuranosyl-1,2,4-triazole-3-carboxamide, 9->2-hydroxy-ethoxymethylguanine, amantadine, 5-iodo-2'-deoxyuridine, trifluorothymidine, interferon, adenine arabinoside, protease inhibitors, thymidine kinase inhibitors, sugar or glycoprotein synthesis inhibitors, structural protein synthesis inhibitors, attachment and adsorption inhibitors and nucleoside analogs, such as acyclovir, penciclovir, valacyclovir and ganciclovir; (d) PD-1 inhibitors or anti-PD-1 antibodies, such as (pembrolizumab), (nivolumab) or LIBTAYO (cemiplimab); (e) a PD-L1 inhibitor or anti-PD-L1 antibody, such as TECENTRIQ (atezolizumab), IMFINZI (durvalumab), or BAVENCIO (avelumab); or (f) a CTLA-4 inhibitor or anti-CTLA-4 antibody, such as Ipilimumab.

45. The method of any of the preceding claims, wherein the patient has been determined to be resistant to treatment with one or more active substances due to gMDSC-mediated immunosuppression prior to NEO-201 treatment.

46. ​​The method of any of the preceding claims, wherein prior to treatment with NEO-201, the patient has been determined to be resistant to treatment with a therapeutic antibody, optionally a therapeutic antibody targeting a checkpoint inhibitor.

47. The method of any of the preceding claims, wherein prior to treatment with NEO-201, the patient has been determined to be resistant to treatment with a PD-1 or CTLA-4 antagonist, optionally an antibody or fusion protein.

48. The method of any of the preceding claims, wherein prior to treatment with NEO-201, the patient has developed resistance to and / or is no longer responsive to treatment with the other therapeutic agent, such as a chemotherapeutic agent and / or an inhibitor of interest.

49. The method of any of the preceding claims, wherein after treatment with NEO-201, the patient clinically responds to other active substances, optionally another therapeutic antibody or fusion protein, further optionally a therapeutic antibody or fusion protein targeting a checkpoint inhibitor and / or immune cells, optionally CAR-T or CAR-NK cells.

50. The method of any of the preceding claims, wherein after treatment with NEO-201, the patient clinically responds to the other active agent, optionally chemotherapy and / or another therapeutic antibody or fusion protein, further optionally a PD-1 antagonist antibody such as pembrolizumab, nivolumab, cemiplizumab, atezolizumab, dotalimumab, durvalumab, lambrolizumab or avelumab.

51. The method of any of the preceding claims, wherein after treatment with NEO-201, the patient clinically responds to the other active agent, optionally another therapeutic antibody or fusion protein targeting CTLA-4, optionally Yervoy or Tremelimumab and / or immune cells, optionally CAR-T or CAR-NK cells.

52. The method of any of the preceding claims, wherein the NEO-201 antibody comprises the VH and VL CDR sequences contained in SEQ ID NO:28 and SEQ ID NO:

29.

53. The method of any of the preceding claims, wherein the NEO-201 antibody comprises a variable heavy chain sequence that is at least 90% identical to SEQ ID NO:

38.

54. The method of any of the preceding claims, wherein the NEO-201 antibody comprises a variable light chain sequence that is at least 90% identical to SEQ ID NO:

39.

55. The method of any of the preceding claims, wherein the NEO-201 antibody comprises a variable heavy chain sequence that is at least 90% identical to SEQ ID NO:38 and a variable light chain sequence that is at least 90% identical to SEQ ID NO:

39.

56. The method of any of the preceding claims, wherein the NEO-201 antibody comprises a heavy chain sequence at least 90% identical to amino acids 20-470 of SEQ ID NO:28 and a light chain sequence at least 90% identical to amino acids 20-233 of SEQ ID NO:

29.

57. The method of any of the preceding claims, wherein the NEO-201 antibody comprises or consists of the heavy chain sequence of amino acids 20-470 of SEQ ID NO:28 and the light chain sequence of amino acids 20-233 of SEQ ID NO:

29.

58. The method of any of the preceding claims, wherein the NEO-201 antibody comprises a human IgG1 constant domain.

59. The method of any of the preceding claims, wherein the NEO-201 antibody is humanized.

60. The method of any of the preceding claims, wherein the NEO-201 antibody is conjugated to another moiety.

61. The method of claim 60, wherein the NEO-201 antibody is conjugated to another cytotoxic moiety, a label, a radioactive moiety, or an affinity tag.

62. The method of any of the preceding claims, wherein the antibody, preferably the NEO-201 antibody, is contained in a chimeric antigen receptor (CAR) administered to the treated subject.

63. The method of any of the preceding claims, wherein the antibody is comprised in a multispecific or bispecific antibody that targets at least one other antigen, optionally another tumor antigen or an antigen expressed on an immune cell.

64. The method of claim 63, wherein the other antigen is a checkpoint inhibitor or a cytokine or a hormone or a growth factor.

65. The method of any one of the preceding claims, wherein the antibody is administered as an immune cell, optionally a human T or NK cell, that expresses a CAR comprising the antibody or the antibody.

66. A method of killing gMDSCs in vivo, the method comprising administering an effective amount of NEO-201 antibody to a patient, optionally wherein the patient is being treated with CAR-T or CAR-NK cells.

67. A method of treating or preventing or reversing gMDSC-mediated immunosuppression, the method comprising administering to a patient an effective amount of NEO-201 antibody.

68. A method of enhancing the efficacy of a CAR-T or CAR-NK therapy by administering NEO-201 in combination with the CAR-T or CAR-NK therapy, wherein the CAR can target any of the antigens disclosed herein.

69. The method of any of the preceding claims, further comprising administering to the patient another therapeutic agent.

70. The method of claim 69, wherein the additional agent is selected from the group consisting of (a) microtubule inhibitors, topoisomerase inhibitors, platinum, alkylating agents, and antimetabolites; (b) MK-2206, ON013105, RTA 402, BI 2536, sorafenib, ISIS-STAT3Rx, microtubule inhibitors, topoisomerase inhibitors, platinum, alkylating agents, antimetabolites, paclitaxel, gemcitabine, doxorubicin, vinblastine, etoposide, 5-fluorouracil, carboplatin, hexamethylmelamine, aminoglutethimide, amsacrine, anastrozole, azacitidine, bleomycin, busulfan, carmustine, chlorambucil, 2-chlorodeoxyadenosine, cisplatin, colchicine, cyclophosphamide, cytarabine, cyclophosphamide, dacarbazine, actinomycin D, daunorubicin, docetaxel, estramustine phosphate, floxuridine, fludarabine, gemtuzumab, hexamethylmelamine, hydroxyurea, ifosfamide, imatinib, interferon, irinotecan, lomustine, nitrogen mustard, melphalan, 6-mercaptopurine, methotrexate, mitomycin (c) 1-D-ribofuranosyl-1,2,4-triazole-3-carboxamide, 9->2-hydroxy-ethoxymethylguanine, amantadine, 5-iodo-2'-deoxyuridine, trifluorothymidine, interferon, adenine arabinoside, protease inhibitors, thymidine kinase inhibitors, sugar or glycoprotein synthesis inhibitors, structural protein synthesis inhibitors, attachment and adsorption inhibitors and nucleoside analogs, such as acyclovir, penciclovir, valacyclovir and ganciclovir; (d) PD-1 inhibitors or anti-PD-1 antibodies, such as (pembrolizumab), (nivolumab) or LIBTAYO (cemiplimab); (e) a PD-L1 inhibitor or anti-PD-L1 antibody, such as TECENTRIQ (atezolizumab), IMFINZI (durvalumab), or BAVENCIO (avelumab); or (f) a CTLA-4 inhibitor or anti-CTLA-4 antibody, such as Ipilimumab.

71. The method of claim 69 or 70, wherein the other agent comprises CAR-T or CAR-NK cells.

72. The method of any of the preceding claims, further comprising administering an anti-cancer vaccine or CAR-T or CAR-NK cells to the patient.

73. A method for killing gMDSCs in vitro, the method comprising contacting a tissue, organ or cell sample suspected of containing gMDSCs with the NEO-201 antibody.

74. The method of claim 73, wherein the tissue, organ or cell sample is obtained from a patient suffering from cancer or an infectious disease condition.

75. The method of claim 73 or 74, wherein the tissue, organ or cell sample is a bone marrow sample from an autologous or allogeneic donor.

76. The method of any one of claims 73-75, further comprising contacting the gMDSCs with complement.

77. The method of any of the preceding claims, wherein the gMDSCs are killed by ADCC or CDC.

78. The method of any one of claims 73-77, further comprising contacting the gMDSC with an effector cell.

79. The method of claim 78, wherein the effector cells comprise natural killer cells.

80. The method of any one of the preceding claims, wherein the gMDSCs are killed by ADCC.

81. The method of any of the preceding claims, wherein the NEO-201 antibody is conjugated to a cytotoxic moiety.

82. A method of detecting gMDSCs, the method comprising detecting expression of NEO-201 antigen and optionally one or more other gMDSC biomarkers by said gMDSCs, optionally wherein the level of gMDSCs in a patient sample, such as blood or a biopsy sample, is used to determine cancer prognosis or treatment regimen.

83. The method of claim 82, comprising contacting the gMDSCs with a NEO-201 antibody, wherein optionally the NEO-201 antibody is directly or indirectly conjugated to a label.

84. The method of claim 82 or 83, wherein the detecting comprises cell sorting, optionally fluorescence activated cell sorting.

85. A method of staining gMDSCs, the method comprising contacting the cells with NEO-201 antibody.

86. The method of claim 85, wherein the NEO-201 antibody is directly or indirectly conjugated to a label.

87. A method of isolating gMDSCs, the method comprising isolating cells expressing the NEO-201 antigen and optionally at least one other gMDSC biomarker.

88. The method of claim 87, comprising contacting a sample containing gMDSCs with a NEO-201 antibody, optionally wherein the NEO-201 antibody is directly or indirectly labeled.

89. The method of claim 88, wherein the sample is or comprises a blood or bone marrow or tumor biopsy sample.

90. The method of any one of claims 82-89, comprising separating NEO-201 positive cells from NEO-201 negative cells.

91. The method of any one of claims 82-90, wherein the gMDSCs are isolated by cell sorting, optionally fluorescence activated cell sorting.

92. The method of any one of claims 82-91, wherein the gMDSCs are isolated by contacting the sample with a support comprising NEO-201 antibodies, thereby retaining the gMDSCs on the support.

93. The method of any of the preceding claims, wherein the NEO-201 antibody comprises the CDR sequences contained in SEQ ID NO:28 and SEQ ID NO:

29.

94. The method of any of the preceding claims, wherein the NEO-201 antibody comprises a variable heavy chain sequence that is at least 90% identical to SEQ ID NO:

38.

95. The method of any of the preceding claims, wherein the NEO-201 antibody comprises a variable light chain sequence that is at least 90% identical to SEQ ID NO:

39.

96. The method of any of the preceding claims, wherein the NEO-201 antibody comprises a variable heavy chain sequence at least 90% identical to SEQ ID NO:38 and a variable light chain sequence at least 90% identical to SEQ ID NO:

39.

97. The method of any of the preceding claims, wherein the NEO-201 antibody comprises a heavy chain sequence at least 90% identical to amino acids 20-470 of SEQ ID NO:28 and a light chain sequence at least 90% identical to amino acids 20-233 of SEQ ID NO:

29.

98. The method of any of the preceding claims, wherein the NEO-201 antibody comprises all six CDR sequences contained in SEQ ID NO:28 and SEQ ID NO:

29.

99. The method of any of the preceding claims, wherein the NEO-201 antibody comprises a human IgG1 constant domain, optionally comprising at least one mutated human IgG1 constant domain, wherein the at least one mutation enhances or inhibits one or more effector functions, wherein the one or more effector functions are optionally FcR binding, FcRN binding, glycosylation, complement (C1 q ) binding, phagocytosis, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-mediated neutralization, opsonization, or any combination of the foregoing.

100. The method of any of the preceding claims, wherein the NEO-201 antibody is humanized.

101. The method of any of the preceding claims, wherein the NEO-201 antibody is conjugated to another moiety.

102. The method of any of the preceding claims, wherein the NEO-201 antibody is conjugated to another cytotoxic moiety, a label, a radioactive moiety, or an affinity tag.

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