Method for eliminating myeloid-derived suppressor cells using NEO-201 antibody

Abstract

NEO-201, an antibody that specifically binds glycosylated peptides with core 1 and / or extended core 1 O-glycans contained in CEACAM5 and CEAMCAM6, but not non-glycosylated CEACAM5 or non-glycosylated CEAMCAM6, has surprisingly been shown to bind and kill granulocytic myeloid-derived suppressor cells (gMDSCs). gMDSCs are known to suppress natural immunity in other conditions, various cancers and infectious diseases. Based on this, the use of NEO-201 alone or in combination with the treatment of cancers and infectious diseases and other conditions in which gMDSCs suppress natural immunity to disease is provided. These methods optionally include detecting gMDSCs before, during or after treatment with NEO-201. Diagnostic methods, therapeutic methods and combination therapies using NEO-201, optionally in combination with another agent, to eliminate gMDSCs and disease cells are also described.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 343,559, filed May 19, 2022, and U.S. Provisional Application No. 63 / 494,094, filed April 4, 2023, the contents of each of which are incorporated by reference in their entirety.

[0002] Sequence Listing Information This application includes as part of its disclosure a biological sequence listing in a file entitled "1143282o005000.txt", created on May 18, 2022 and having a size of 32,427 bytes, which is incorporated herein by reference in its entirety. [Background technology]

[0003] The human carcinoembryonic antigen (CEA) family consists of 29 genes arranged in tandem on chromosome 19q13.2. Based on nucleotide homology, these genes are classified into two major subfamilies: the CEACAM subgroup and the pregnancy-specific glycoprotein subgroup. Proteins encoding CEACAMs 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 human CEACAM contains one N-terminal domain, containing 108–110 amino acids and homologous to Ig variable domains, followed by a different number (0–6) of Ig-specific glycoproteins. This is followed by a C2-type quorum-like domain. CEACAM proteins can interact homophilically and heterophilically with each other. CEACAM1 is a unique protein within this family because it contains an ITIM (immunoreceptor tyrosine-based inhibitory motif) like PD1 in its cytoplasmic domain. This inhibitory effect is caused by phosphorylation of tyrosine residues by the ITIM, which results in the recruitment of tyrosine phosphatases-1 and -2, which contain Src homology 2 domains. CEACAM1 protein is expressed in a variety of cells, including monocytes, granulocytes, activated T cells, B cells, and NK cells. It is expressed on a variety of immune cells. CEACAM1 occurs as several isoforms, the two major ones being CEACAM1-L and CEACAM1-S, with long (L) or short (S) cytoplasmic domains, respectively. Expression of CEACAM1-S on human leukocytes is completely absent. CEACAM1-L is expressed on a subpopulation of activated human NK cells that are negative for CD16 but positive for CD56. Heterophilic interaction between CEA on tumor cells and CEACAM1 on NK cells inhibits NK cell cytotoxicity against tumor cells.

[0004] NEO-201 is a humanized IgG1 mAb that binds to cancer proteins with core 1 and / or extended core 1 O-glycans (e.g., tumor-associated variants of CEACAM family members, in particular, cancer-associated variants of CEACAM5 and CEACAM6) (Zeligs et al., Cancer Res. July 1 2017(77)(13 Supplement)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 demonstrated to be reactive against certain cancers, but not against most normal tissues. Applicant's prior U.S. Patent 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 in the diagnosis and treatment of colon 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 having core 1 and / or extended core 1 O-glycans, including CEACAM5 and / or CEACAM6.

[0005] However, to the applicant's knowledge, the use of NEO-201, or any other antibody targeting CEACAM5 and / or CEACAM6 to detect and / or deplete myeloid-derived suppressor cells (MDSCs) has not been reported previously. As known in the art, MDSCs are a population of myeloid cells that are generated during a number of pathologies, from cancer, infections to obesity. These cells represent a pathological state of activation of monocytes and relatively immature neutrophils. MDSCs are characterized by a unique set of genomic and biochemical features and can be distinguished from granulocytes and other cells by the expression of certain surface molecules. A hallmark of these cells is their ability to inhibit the function of T cells, thereby contributing to the pathogenesis of various diseases. In particular, these cells are known to contribute to the pathology of diseases (e.g., cancer, infections, autoimmunity, obesity, and pregnancy).

[0006] Therefore, methods for detecting and / or eliminating MDSCs have great therapeutic potential.

[0007] BRIEF DESCRIPTION AND EXEMPLARY EMBODIMENTS We previously show that NEO-201 binds to cancer-associated variants of CEACAM5 and CEACAM6 (specifically via cancer-associated glycosylation variants of these proteins with 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 in pooled allogeneic colon tumor tissue extracts. NEO-201 reacts with most tumor tissues from many different carcinomas, but not with most normal tissues. Functional analysis revealed that NEO-201 can mediate both antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against tumor cells. Previous studies have demonstrated that NEO-201 attenuates the growth of human tumor xenografts in mice and is safe and tolerable in non-human primates, with a transient reduction in circulating neutrophils being the only side effect observed.

[0008] Applicant herein shows that NEO-201 can bind to granulocytes and further bind to granulocyte-derived MDSCs (gMDSCs) and kill gMDSCs via ADCC. As mentioned above, MDSCs are a population of myeloid cells that are generated during a number of pathologies, from cancer to obesity, which among other things inhibit T cell function and contribute to the pathogenesis of various diseases. In particular, these cells contribute to the pathology of diseases (e.g., cancer, infectious diseases, autoimmunity, obesity, and pregnancy).

[0009] MDSCs have emerged as universal regulators of immune function in many pathologies. MDSCs consist of two major cell populations: granulocytic or polymorphonuclear cells (PMN-MDSCs or gMDSCs) and monocytic cells (M-MDSCs). PMN-MDSCs or gMDSCs are phenotypically and morphologically similar to neutrophils, whereas 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 population of MDSCs, represented by cells with colony-forming activity and other myeloid progenitor cells, has also been reported, and these have been termed early MDSCs (eMDSCs) (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).

[0010] Therefore, based on its demonstrated ability to detect and / or eliminate gMDSCs, NEO-201 can potentially be used to treat and / or monitor disease states in any condition in which gMDSCs are involved in disease pathology (particularly cancer), and may be particularly useful in treating cancers that do not express CEACAM5 and / or CEACAM6, as well as chronic infection conditions in which gMDSCs are known to suppress natural immunity. Furthermore, NEO-201 should be particularly useful in combination therapy, e.g., in combination with other therapies (e.g., other drugs or treatments that eliminate and / or inhibit the activity of MDSCs, other therapeutic antibodies, checkpoint inhibitors, chemotherapeutic drugs, etc.). The reason is that NEO-201, with its ability to deplete gMDSCs, should enhance the efficacy of other therapies, e.g., by enhancing natural immunity (e.g., responses to innate antitumor or antiinfective drugs), even in subjects that were previously resistant to the treatment.

[0011] Based on the above, in one embodiment, the present invention provides a method for killing or depleting 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 having core 1 type and / or extended core 1 type O-glycans, but does not bind to aglycosylated CEACAM5 or aglycosylated CEACAM6, optionally wherein the antibody or antibody fragment recognizes an O-glycosylated epitope that binds to a threonine within the region of amino acids 310-318 (RTTVTTITV) of CEACAM5 and a threonine and serine within the region of amino acids 312-320 (TVTMITVSG) of CEACAM6.

[0012] In another embodiment, the present invention provides a method for killing or depleting granulocytic myeloid-derived suppressor cells (gMDSCs) 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.

[0013] In another embodiment, the present invention provides a method of reversing tolerance and / or restoring natural immunity (e.g., innate anti-tumor immunity or innate anti-infectious immunity) in a patient in need thereof by killing or ablating granulocytic myeloid-derived suppressor cells (gMDSCs) in the patient, the method comprising administering to the patient an effective amount of NEO-201 or an antigen-binding fragment thereof.

[0014] In another embodiment, the present 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), which resistance or tolerance involves 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.

[0015] In another embodiment, the invention provides a method of treating or preventing recurrence of cancer or infectious disease 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 suppress proliferation of MDSCs and restore innate immunity.

[0016] In an exemplary embodiment, in any one of the above methods, the gMDSC expresses an O-glycan selected from one or more of O-glycans 01, 02, 06, 023, 026, and 039 having the structures shown in the sequences of Figure 2 and / or Figure 5.

[0017] In an exemplary embodiment, in any one of the above-mentioned methods, the gMDSCs are derived from neutrophils expressing O6, O1, or O2 O-glycans having the structures shown in the sequences of Figure 2 and / or Figure 5.

[0018] In an exemplary embodiment, in any one of the methods described above, the gMDSCs are derived from neutrophils expressing O6 O-glycans as shown in the sequences of FIG. 2 and / or FIG.

[0019] In an exemplary embodiment, in any of the methods described above, the gMDSCs may express a Tn antigen having the structure shown in FIG. 1 or a core type 1, core type 2, core type 4, or type 4 O-glycan.

[0020] In an exemplary embodiment, in any one of the above methods, the patient is suffering from cancer or an infectious disease, and the disease pathology and / or immunosuppression of innate immunity to the disease involves gMDSC.

[0021] In an exemplary embodiment, in any of the above methods, the antibody or antigen-binding fragment is directly or indirectly conjugated to a cytotoxic agent (optionally a radionuclide or a chemotherapeutic agent).

[0022] In an exemplary embodiment, in any of the methods described above, the antibody or antigen-binding fragment is directly or indirectly conjugated to a label (optionally a fluorescent or radioactive label).

[0023] In an exemplary embodiment, in any of the above-described methods, the subject is afflicted with a cancer in which MDSCs are involved in the disease pathology, and optionally, the cancer cells being treated do not express or overexpress the antigen bound by NEO-201.

[0024] In an exemplary embodiment, in any one of the above-mentioned methods, the subject is treated with a cancer in which MDSCs are involved in the disease pathology (optionally adrenal gland cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, adult brain / CNS tumors, pediatric brain / CNS tumors, breast cancer, male breast cancer, adolescent cancer, pediatric cancer, young adult cancer, cancer of unknown primary, Castleman's disease, cervical cancer, colon / rectal 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 (acute lymphocytic) (ALL), adult leukemia (acute myeloid) (AML), chronic lymphocytic leukemia (CLL), leukemia (chronic myeloid) (CML), leukemia (chronic myelomonocytic) (CMML), pediatric leukemia, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, pulmonary carcinoid tumor, lymphoma, lymphoma of the skin, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, childhood non-Hodgkin's lymphoma, oral and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, 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), and optionally, the treated cancer cells do not express or overexpress the antigen to which NEO-201 binds.

[0025] In an exemplary embodiment, in any one of the above-mentioned methods, the subject is treated with a cancer in which MDSCs are involved in the disease pathology, optionally including 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, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, colon 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 or ureter cancer, renal cell carcinoma, renal pelvis cancer, central nervous system tumors, primary CNS tumors, spinal cord tumors, brain stem glioma, and pituitary adenoma, and optionally, the treated cancer cells do not express or overexpress the antigen bound by NEO-201.

[0026] In an exemplary embodiment, in any one of the above methods, the treated cancer or infectious disease 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.

[0027] In an exemplary embodiment, in any one of the above-mentioned methods, the subject is afflicted with a cancer in which MDSCs are involved in the disease pathology, and the treatment induces one or more of: (i) an increase in T cell response; (ii) an increase in antigen presentation; (iii) a decrease in MDSC proliferation; and / or (iv) a decrease in Treg recruitment.

[0028] In an exemplary embodiment, in any of the above methods, the subject is afflicted with stage I, stage II, stage III, or stage IV cancer in which MDSCs are involved.

[0029] In an exemplary embodiment, in any one of the above methods, the antibody or fragment (optionally NEO-201) reduces, eliminates, or slows or inhibits tumor growth in a patient whose anti-tumor immunity has previously been suppressed with gMDSC, reduces tumor burden in the individual, inhibits tumor growth, and / or increases survival of the individual.

[0030] In an exemplary embodiment, in any of the above methods, the subject is suffering from an infectious condition and the disease pathology involves MDSC.

[0031] In an exemplary embodiment, in any one of the above methods, the subject is diagnosed with a bacterial infection in which MDSCs are involved (optionally, Bacillus anthraces, 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, Corynebacterium diphtheriae, Enterococcus faecalis and Enterococcus faecium, Escherichia coli (generally), Enterotoxigenic Escherichia coli (ETEC), Enteropathogenic E. coli, E. 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 infection).

[0032] In an exemplary embodiment, in any one of the above-mentioned methods, the subject is optionally administered a HIV-infected antibody or mAb for a respiratory virus (e.g., adenovirus, avian influenza, influenza virus type A, influenza virus type B, measles, parainfluenza virus, respiratory syncytial virus (RSV), rhinovirus, SARS-CoV), gastrointestinal virus (e.g., coxsackievirus, enterovirus, poliovirus, rotavirus), hepatitis virus (e.g., hepatitis B virus, hepatitis C virus), bovine viral diarrhea virus (alternative), herpes virus (e.g., herpes simplex type 1, herpes simplex type 2), human cytomegalovirus, or combination therapy with a HIV-infected antibody or mAb. Patients suffering from chronic or acute viral infections involving MDSCs, including those related to viruses, varicella zoster virus, retroviruses (e.g., 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 (e.g., avian influenza, dengue virus, hantavirus, hemorrhagic fever viruses, lymphocytic choroiditis virus, smallpox virus surrogates, cowpox, monkeypox, rabbitpox, vaccinia virus, Venezuelan equine encephalomyelitis virus (VEE), West Nile virus, and yellow fever virus).

[0033] In an exemplary embodiment, in any one of the above-mentioned methods, the subject is suffering from a condition in which MDSCs are involved, and MDSCs are involved in suppressing innate immunity, optionally, in the treatment of diseases such as acquired immune deficiency 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 allergy, atopic dermatitis, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune pulmonary arterial disease, ... Infectious 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, Baro's disease / Baroconcentric sclerosis, Behçet's disease, Buerger's disease, Bickerstaff encephalitis, Blau syndrome, bullous pemphigoid, Castleman's disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy, chronic relapsing multiple myelitis , chronic obstructive pulmonary disease, chronic venous stasis ulcer, Churg-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, cutaneous leukocytoclastic vasculitis, Degos disease, Dercum's disease, dermatitis herpetiformis, dermatomyositis, diabetes mellitus type I, diabetes mellitus type II, diffuse cutaneous systemic sclerosis, Dressler's syndrome, drug-induced lupus, discoid lupus erythematosus, eczema, emphysema, endometriosis, enthesitis-related arthritis, eosinophilic fasciitis, eosinophilic gastroenteritis, eosinophilic pneumonia, epidermolysis bullosa acquisita , erythema nodosum, erythroblastosis fetalis, essential mixed cryoglobulinemia, Evans syndrome, fibrodysplasia ossificans progressiva, fibrosing alveolitis (or idiopathic pulmonary fibrosis), gastritis, gastrointestinal pemphigoid, Gaucher disease, glomerulonephritis, Goodpasture syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's encephalopathy, Hashimoto's thyroiditis, heart disease, Henoch-Schönlein purpura, herpes gestationis (also known as pemphigoid of gestation), hidradenitis suppurativa, HIV infection, Hughes-Stobin syndrome, hypogammaglobulinemia, infections (e.g., 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 disease, Lambert-Eaton myasthenic syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, linear IgA disease (LAD), lupoid hepatitis (also known as autoimmune hepatitis), lupus erythematosus, lymphomatoid granulomatosis, Majeed syndrome, malignant tumors, such as cancer (e.g., sarcoma, Kaposi's sarcoma, lymphoma, leukemia, carcinoma, and melanoma), Meniere's disease, microscopic Polyangiitis, Miller Fisher syndrome, localized scleroderma, Mucca-Habermann disease (aka pityriasis lichenoidis varioli), multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (aka Devic's disease), neuromyotonia, ophthalmic cicatricial pemphigoid, opsoclonus-myoclonus syndrome, Ord's thyroiditis, relapsing rheumatism, PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococci), paraneoplastic cerebellar degeneration, Parkinson's disease, paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, Parsonage-Turner syndrome -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 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 It has been implicated in idiopathies, serum sickness, Sjögren's syndrome, spondyloarthropathy, Still's disease (adult onset), stiff-person syndrome, stroke, subacute bacterial endocarditis (SBE), Susac syndrome, Sweet's syndrome, Sydenham chorea, sympathetic ophthalmia, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis (also known as "giant cell arteritis"), thrombocytopenia, Tolosa-Hunt syndrome, transplant (e.g., heart / lung transplant) rejection, transverse myelitis, tuberculosis, ulcerative colitis, undifferentiated connective tissue disease, undifferentiated spondyloarthropathy, urticarial vasculitis, vasculitis, vitiligo, and Wegener's granulomatosis.

[0034] In an exemplary embodiment, in any one of the above-described methods, a patient's gMDSCs may be detected and monitored before treatment, during treatment, after completion of treatment, and / or after the patient has entered remission.

[0035] In an exemplary embodiment, in any of the methods described above, gMDSCs in a patient may be detected pre-treatment in determining whether the patient would potentially benefit from NEO-201 treatment.

[0036] In an exemplary embodiment, in any one of the above-mentioned methods, gMDSCs may be detected in a biological sample using one or more ligands, for example, antibodies that recognize specific biomarkers expressed on gMDSCs (optionally, LOX-1, CD11b, CD15, CD66b, and glycosylated CEACAM5 and CEACAM6 antigens expressing core 1 type and / or extended core 1 type O-glycans recognized by NEO-201).

[0037] In an exemplary embodiment, in any of the methods described above, the number or concentration of gMDSC in a sample from a subject suffering from a cancer in which gMDSC is involved can be used to monitor the progression of the cancer (with or without treatment) when the patient is receiving treatment for such cancer with NEO-201 alone or in combination with another therapeutic agent.

[0038] In an exemplary embodiment, in any of the methods described above, the number or concentration of gMDSCs in a sample from a subject suffering from a cancer in which gMDSCs are involved, where the patient is receiving treatment for such cancer with NEO-201 alone or in combination with another therapeutic agent, may be used to determine whether NEO-201, alone or in combination with another therapeutic agent, may be beneficial in treating the cancer.

[0039] In an exemplary embodiment, in any of the above-described methods, the number or concentration of gMDSCs in a sample from a subject suffering from a cancer in which gMDSCs are involved can be used to develop a dosing regimen for NEO-201 alone or in combination with another therapeutic agent.

[0040] In an exemplary embodiment, in any of the above-described methods, the level of gMDSC in a patient sample (e.g., a blood or biopsy sample) may be used to determine the prognosis of cancer before, during, or after treatment with NEO-201, which method optionally includes contacting the gMDSC with a NEO-201 antibody.

[0041] In an exemplary embodiment, in any one of the above-mentioned methods, the detection includes cell sorting, optionally fluorescence-activated cell sorting, thereby generating a sample enriched and / or depleted of cells positive for NEO-201 antigen expression (e.g., gMDSCs).

[0042] In an exemplary embodiment, any one of the methods described above may include detecting and / or staining gMDSCs by contacting cells with a NEO-201 antibody and detecting cells that express NEO-201, optionally where the NEO-201 is directly or indirectly labeled.

[0043] In an exemplary embodiment, in any of the methods described above, gMDSCs may be isolated by contacting a patient sample with a support comprising NEO-201 antibody and / or using other antibodies or ligands that recognize other MDSC biomarkers, whereby the MDSCs are retained on the support.

[0044] In an exemplary embodiment, in any of the methods described above, the level of MDSC in a patient sample (e.g., a blood or biopsy sample) may be used to determine whether the patient has developed or is likely to develop MDSC-mediated immunosuppression.

[0045] In an exemplary embodiment, in any one of the methods described above, the method may include administration of another therapeutic agent.

[0046] In an exemplary embodiment, in any one of the above-described methods, the method may include administration of at least one other therapeutic agent, wherein administering NEO-201 or other antibodies that bind to glycosylated CEACAM5 and CEACAM6 having core 1 type or extended core 1 type O-glycans, but do not bind to non-glycosylated CEACAM5 or non-glycosylated CEACAM6, together with the at least one other therapeutic agent enhances the effectiveness of the at least one other therapeutic agent.

[0047] In an exemplary embodiment, in any one of the above methods, the other therapeutic agent comprises another therapeutic antibody, a checkpoint inhibitor, a chemotherapeutic agent, and / or comprises immune cells, optionally, CAR-T cells or CAR-NK cells.

[0048] In an exemplary embodiment, in any one of the above methods, the other therapeutic agent may include (i) another moiety that eliminates MDSCs, (ii) a moiety that promotes differentiation of MDSCs, (iii) a moiety that inhibits migration of MDSCs, (iv) an epigenetic therapy moiety that targets MDSCs, or (v) a chemotherapeutic agent that targets MDSCs, or a combination of one or more of the above.

[0049] In an exemplary embodiment, in any of the above-described methods, NEO-201, due to its ability to deplete gMDSCs, may optionally enhance the effectiveness of other therapeutic agents by enhancing natural immunity (e.g., innate response to anti-tumor or anti-infective agents) in subjects previously resistant to treatment with the other therapeutic agents.

[0050] In an exemplary embodiment, in any of the methods described above, the method may include another additional therapeutic agent(s), including, but not limited to, peptides, nucleic acid molecules, small molecule compounds, antibodies, and derivatives thereof.

[0051] In an exemplary embodiment, in any one of the methods described above, the method may include another therapeutic agent(s), 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 additional therapeutic agent(s) include a therapeutic agent targeting adenosine A2A receptor (AZAR), B7-H3 (also known as CD276); These target 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 genes 3 (LAGS), programmed cell death 1 (PD-1), T-cell immunoglobulin and mucin domain 3 (TIM-3), and V-domain Ig suppressor of T-cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0052] In an exemplary embodiment, in any one of the methods described above, the method may include another therapeutic agent(s) (e.g., a CSF-1 / 1R binding agent or inhibitor), or (a) microtubule inhibitors, topoisomerase inhibitors, platinums, alkylating agents, and antimetabolites; (b) MK-2206, ON 013105, RTA 402, BI 2536, sorafenib, ISIS-STAT3Rx, microtubule inhibitors, topoisomerase inhibitors, platins, alkylating agents, antimetabolites, paclitaxel, gemcitabine, doxorubicin, vinblastine, etoposide, 5-fluorouracil, carboplatin, altretamine, aminoglutethimide, amsacrine, anastrozole, azacitidine, bleomycin, busulfan, carmustine, chlorambucil, 2-chlorodeoxyadenosine, cisplatin. , colchicine, cyclophosphamide, cytarabine, cytoxan, dacarbazine, dactinomycin, daunorubicin, docetaxel, estramustine phosphate, floxuridine, fludarabine, gentuzumab, hexamethylmelamine, hydroxyurea, ifosfamide, imatinib, interferon, irinotecan, lomustine, mechlorethamine, melphalen, 6-mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, penicillin (c) 1-D-ribofuranosyl-1,2,4-triazole-3-carboxamide, 9->2-hydroxyethoxymethylguanine, adamantanamine, 5-iodo-2'-deoxyuridine, trifluorothymidine, interferon, adenine, riboflavin ... arabinosides, protease inhibitors, thymidine kinase inhibitors, sugar or glycoprotein synthesis inhibitors, structural protein synthesis inhibitors, attachment and adsorption inhibitors, and nucleoside analogs (e.g., acyclovir, penciclovir, valacyclovir, and ganciclovir); (d) PD-1 inhibitors or anti-PD-1 antibodies (e.g., KEYTRUDA® (pembrolizumab), OPDIVO® (nivolumab), or LIBTAYO (cemiplimab);(e) a PD-L1 inhibitor or anti-PD-L1 antibody (e.g., TECENTRIQ (atezolizumab), IMFINZI (durvalumab), or BAVENCIO (avelumab); or, (f) a CTLA-4 inhibitor or anti-CTLA-4 antibody (e.g., YERVOY® ipilimumab).

[0053] In an exemplary embodiment, in any of the above methods, the patient has optionally been determined to be resistant to treatment with one or more active agents due to gMDSC-mediated immunosuppression prior to NEO-201 treatment.

[0054] In an exemplary embodiment, in any of the above methods, the patient has been determined to be resistant to treatment with a therapeutic antibody, optionally a targeting checkpoint inhibitor, optionally prior to treatment with NEO-201.

[0055] In an exemplary embodiment, in any of the above methods, the patient has been determined to be resistant to treatment with a PD-1 or CTLA-4 antagonist, optionally an antibody or fusion protein, optionally prior to treatment with NEO-201.

[0056] In an exemplary embodiment, in any of the above methods, the patient optionally has developed resistance to and / or is no longer responsive to treatment with the other therapeutic agent prior to treatment with NEO-201.

[0057] In an exemplary embodiment, in any one of the above methods, the patient responds more effectively clinically to other active agents, optionally another therapeutic antibody or fusion protein, and optionally, a checkpoint inhibitor and / or targeting immune cells (optionally, CAR-T cells or CAR-NK cells), following treatment with NEO-201.

[0058] In an exemplary embodiment, in any one of the above-described methods, the patient responds more effectively clinically to other active agents, optionally another therapeutic antibody or fusion protein, and optionally a PD-1 antagonist antibody (e.g., pembrolizumab, nivolumab, cemiplimab, atezolizumab, atezolizumab, dostallimab, durvalumab, lambrolizumab, or avelumab), following treatment with NEO-201.

[0059] In an exemplary embodiment, in any one of the above methods, the patient responds more clinically effectively to other active agents targeting CTLA-4 (optionally Yervoy or tremelimumab and / or immune cells, optionally CAR-T or CAR-NK cells), optionally another therapeutic antibody or fusion protein, following treatment with NEO-201.

[0060] In an exemplary embodiment, in any one of the above methods, the NEO-201 antibody may comprise the VH and VL CDR sequences contained in SEQ ID NO:28 and SEQ ID NO:29.

[0061] In an exemplary embodiment, in any one of the above methods, the NEO-201 antibody may comprise a variable heavy chain sequence having at least 90% identity to SEQ ID NO:38.

[0062] In an exemplary embodiment, in any one of the above methods, the NEO-201 antibody may comprise a variable light chain sequence having at least 90% identity to SEQ ID NO:39.

[0063] In an exemplary embodiment, in any one of the above-mentioned methods, the NEO-201 antibody may comprise a variable heavy chain sequence having at least 90% identity to SEQ ID NO:38 and a variable light chain sequence having at least 90% identity to SEQ ID NO:39.

[0064] In an exemplary embodiment, in any one of the methods described above, the NEO-201 antibody may comprise a heavy chain sequence having at least 90% identity to amino acids 20-470 of SEQ ID NO:28 and a light chain sequence having at least 90% identity to amino acids 20-233 of SEQ ID NO:29.

[0065] In an exemplary embodiment, in any one of the above-described methods, the NEO-201 antibody may comprise or consist of a heavy chain sequence of amino acids 20 to 470 of SEQ ID NO:28 and a light chain sequence of amino acids 20 to 233 of SEQ ID NO:29.

[0066] In an exemplary embodiment, in any of the above methods, the NEO-201 antibody may comprise a human IgG1 constant domain.

[0067] In an exemplary embodiment, in any one of the above methods, the antibody preferably comprises the NEO-201 antibody or a variant thereof (eg, one that contains the same CDRs or variable regions as the NEO-201 antibody).

[0068] In an exemplary embodiment, in any one of the above methods, the NEO-201 antibody may be conjugated to another moiety.

[0069] In an exemplary embodiment, in any of the methods described above, the NEO-201 antibody may be conjugated to another cytotoxic moiety, a label, a radioactive moiety, or an affinity tag.

[0070] In an exemplary embodiment, in any one of the methods described above, the NEO-201 antibody (preferably, the NEO-201 antibody) is comprised in a chimeric antigen receptor (CAR) that is administered to the subject.

[0071] In an exemplary embodiment, in any one of the above methods, the NEO-201 antibody may 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.

[0072] In an exemplary embodiment, in any one of the aforementioned methods, the other antigen is a checkpoint inhibitor or a cytokine or a hormone or a growth factor.

[0073] In an exemplary embodiment, in any one of the above methods, the NEO-201 antibody is administered as an immune cell (optionally a human T cell or NK cell), which expresses a CAR comprising the antibody.

[0074] In another exemplary embodiment, the present invention provides a method of killing gMDSCs in vivo, the method comprising administering an effective amount of a NEO-201 antibody to a patient, optionally wherein the patient has been treated with CAR-T cells or CAR-NK cells.

[0075] In another exemplary embodiment, 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 a NEO-201 antibody.

[0076] In other exemplary embodiments, the present invention provides methods of enhancing the efficacy of CAR-T or CAR-NK therapy by administering NEO-201 in combination therewith, where the CAR may target any of the antigens disclosed herein.

[0077] In other exemplary embodiments, any one of the above methods further comprises administering to the patient another therapeutic agent, optionally the other therapeutic agent being selected from the group consisting of: (a) microtubule inhibitors, topoisomerase inhibitors, platinums, alkylating agents, and antimetabolites; (b) MK-2206, ON 013105, RTA 402, BI 2536, sorafenib, ISIS-STAT3Rx, microtubule inhibitors, topoisomerase inhibitors, platins, alkylating agents, antimetabolites, paclitaxel, gemcitabine, doxorubicin, vinblastine, etoposide, 5-fluorouracil, carboplatin, altretamine, aminoglutethimide, amsacrine, anastrozole, azacitidine, bleomycin, busulfan, carmustine, chlorambucil, 2-chlorodeoxyadenosine, cisplatin, Colchicine, cyclophosphamide, cytarabine, cytoxan, dacarbazine, dactinomycin, daunorubicin, docetaxel, estramustine phosphate, floxuridine, fludarabine, gentuzumab, hexamethylmelamine, hydroxyurea, ifosfamide, imatinib, interferon, irinotecan, lomustine, mechlorethamine, melphalen, 6-mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, pentostaphylococcus aureus, (c) 1-D-ribofuranosyl-1,2,4-triazole-3-carboxamide, 9->2-hydroxyethoxymethylguanine, adamantanamine, 5-iodo-2'-deoxyuridine, trifluorothymidine, interferon, adenine alavir, 1-methyl-2-propanediol ... vinosides, protease inhibitors, thymidine kinase inhibitors, sugar or glycoprotein synthesis inhibitors, structural protein synthesis inhibitors, adhesion and adsorption inhibitors, and nucleoside analogs (e.g., acyclovir, penciclovir, valacyclovir, and ganciclovir); (d) PD-1 inhibitors or anti-PD-1 antibodies (e.g., KEYTRUDA® (pembrolizumab), OPDIVO® (nivolumab), or LIBTAYO (cemiplimab));(e) a PD-L1 inhibitor or anti-PD-L1 antibody (e.g., TECENTRIQ (atezolizumab), IMFINZI (durvalumab), or BAVENCIO (avelumab)); or (f) a CTLA-4 inhibitor or anti-CTLA-4 antibody (e.g., YERVOY® ipilimumab, or optionally, the other agent comprises CAR-T cells or CAR-NK cells, and / or the method further comprises administering to the patient an anti-cancer vaccine or CAR-T cells or CAR-NK cells;

[0078] In another exemplary embodiment, the invention provides a method of killing gMDSC in vitro, the method comprising contacting a tissue, organ or cell sample suspected of containing gMDSC with a NEO-201 antibody, optionally wherein the tissue, organ or cell sample is obtained from a patient with cancer or an infectious disease condition or wherein the tissue, organ or cell sample is an autologous or allogeneic donor bone marrow sample, and optionally further comprising contacting the gMDSC with complement, and optionally wherein the gMDSC are killed by ADCC or CDC.

[0079] In another exemplary embodiment, the invention provides a method of killing gMDSC in vitro, the method comprising contacting a tissue, organ or cell sample suspected of containing gMDSC with a NEO-201 antibody, optionally wherein the tissue, organ or cell sample is obtained from a patient with cancer or an infectious disease condition, the method further comprising contacting the gMDSC with effector cells, optionally wherein the effector cells comprise natural killer cells, and further optionally wherein the gMDSC are killed by ADCC.

[0080] In another exemplary embodiment, in any one of the above methods, the NEO-201 antibody is conjugated to a cytotoxic moiety.

[0081] In another exemplary embodiment, the present invention provides a method of detecting gMDSC, the method comprising detecting expression of the NEO-201 antigen by the gMDSC and optionally one or more other gMDSC biomarkers, optionally wherein the level of gMDSC in a patient sample (e.g., a blood or biopsy sample) is used to determine a cancer prognosis or treatment regimen, the method optionally comprising contacting the gMDSC with a NEO-201 antibody, optionally wherein the NEO-201 antibody is directly or indirectly conjugated to a label, and / or optionally wherein the detection comprises cell sorting, optionally fluorescence activated cell sorting.

[0082] In another exemplary embodiment, the present invention provides a method for staining gMDSCs, the method comprising contacting the cells with a NEO-201 antibody, optionally wherein the NEO-201 antibody is directly or indirectly bound to a label.

[0083] In another exemplary embodiment, the present invention provides a method for isolating gMDSCs 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 the NEO-201 antibody being directly or indirectly labeled, and further optionally the sample being or comprising a blood or bone marrow or tumor biopsy sample, and further optionally the method comprising separating NEO-201 positive cells from NEO-201 negative cells, and optionally the gMDSCs being isolated by cell sorting, optionally by fluorescence activated cell sorting and / or optionally the gMDSCs being isolated by contacting the sample with a support comprising a NEO-201 antibody, whereby the gMDSCs are retained on the support.

[0084] In other exemplary embodiments, in any one 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 having at least 90% identity to SEQ ID NO:38, and / or the NEO-201 antibody comprises a variable light chain sequence having at least 90% identity to SEQ ID NO:39, and / or the NEO-201 antibody comprises a variable heavy chain sequence having at least 90% identity to SEQ ID NO:38 and a variable light chain sequence having at least 90% identity to SEQ ID NO:39, and / or the NEO-201 antibody comprises the CDR sequences contained in SEQ ID NO:28. and / or the NEO-201 antibody comprises a heavy chain sequence having at least 90% identity to amino acids 20-470 of SEQ ID NO:29 and a light chain sequence having at least 90% identity to amino acids 20-233 of SEQ ID NO:29, and / or the NEO-201 antibody comprises all six CDR sequences contained in SEQ ID NO:28 and SEQ ID 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, label, radioactive moiety, or affinity tag. [Brief description of the drawings]

[0085] [Figure 1] The table contains the O-glycan core structures found in mucins, including the O-glycan recognized by the NEO-201 antibody, where Gal is galactose; GalNAc is N-acetylgalactosamine; GlcNAc is N-acetylglucosamine; Sial is sialic acid; Ser is serine; and Thr is threonine. [Diagram 2] It contains an array of various O-glycan structures, including the O-glycan recognized by the NEO-201 antibody. [Diagram 3] Contains the amino acid sequence of CEACAM6. [Figure 4] Contains the amino acid sequence of CEACAM5. [Diagram 5] Contains the structures of O-glycans recognized by the NEO-201 antibody (eg, O-glycans 01, 02, 06, 023 026, and 039). [Figure 6-9] Contains the results of flow cytometry analysis of gMDSCs generated from GM-CSF and IL-6 treated neutrophils from four normal donors. [Figure 10] Contains a table showing that 47.59-52.58% 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%-88.09% of the HLA-DR negative and CD33 positive population were CD15 positive and CD14 negative. 66.44%-99.71% of the HLA-DR negative / CD33 positive / CD15 positive / CD14 negative population were CD66 positive and NEO-201 positive. [Figure 11] Contains the results of ADCC experiments demonstrating that when gMDSCs were incubated with PBMCs (E:T 100:1) and NEO-201, a reduction of 33.01% (18.29% vs. 27.23%) and 29.5% (25.95% vs. 36.83%) of CD33 positive / HLA-DR negative viable cells was observed compared to gMDSCs incubated with PBMCs alone (E:T 100:1) in healthy donors 1 and 2, respectively. A similar reduction of CD33 positive / HLA-DR negative viable cells was observed comparing gMDSCs incubated with PBMCs and NEO-201 (E:T 50:1) to gMDSCs incubated with PBMCs alone (E:T 50:1) in both healthy donors. [Figure 12]Comparison of the percentage of circulating gMDSC (HLA-DR- / CD33+ / CD15+ / CD14- / CD66b+ cells) in two patients with stable stage (SD) and two patients with progressive stage (PD) cancer at different time points by flow cytometry analysis. In these experiments, gMDSC were gated from live PBMCs. Data are presented as the median percentage of viable cells expressing gMDSC markers. Fluorescence minus 1 control was used to determine positivity. "HNSCC" in the figure refers to "head and neck squamous cell carcinoma." DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0086] The present disclosure provides a method of depleting or ablating gMDSCs in a patient in need thereof, comprising administering to the patient an effective amount of a NEO-201 antibody.

[0087] The name myeloid-derived suppressor cells (MDSC) was introduced into the scientific literature about 15 years ago (Gabrilovich D et al.,et al.,“The terminology issue for myeloid-derived suppressor cells”,Cancer Res.2007;67:42). It originally described a loosely defined group of myeloid cells with potent immunomodulatory activity. In recent years, the properties and biological roles of MDSC have become clear, and MDSC have emerged as universal regulators of immune function in many pathological conditions. MDSC consist of two major cell populations: granulocytic or polymorphonuclear cells (PMN-MDSC or g-MDSC) and monocytic cells (m-MDSC). PMN-MDSCs or gMDSCs are phenotypically and morphologically similar to neutrophils, whereas 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). Clinical studies in humans have demonstrated the existence of a third small population of MDSCs, represented by cells with colony-forming activity and other myeloid progenitor cells.

[0088] Furthermore, intensive clinical studies have identified MDSCs as valuable predictive markers in cancer prognosis, and therefore, as discussed below, a number of drugs and treatments targeting MDSCs have been developed to treat diseases in which such cells are involved in the disease pathology.

[0089] Morphologically and phenotypically, MDSCs resemble neutrophils and monocytes. The major population of bone marrow (BM)-derived myeloid cells includes granulocytes (neutrophils as their most abundant representative) and mononuclear cells (monocytes, terminally differentiated macrophages (MΦ) and dendritic cells (DC)). In contrast to in vitro experiments, where both MΦ and DC can easily differentiate from monocytes, in steady-state tissues, MΦ mainly proliferate in situ and most DC differentiate from specific BM progenitors (Geissmann F et al., “Development of monocytes, macrophages, and dendritic cells”, Science. 327(5966):656-661). However, in inflammation and cancer, BM-derived monocytes are the major progenitors of MΦs, particularly a population of tumor-associated macrophages (TAMs) and inflammatory DCs (Veglia F et al., “Dendritic cells in cancer: the role revisited”, Curr Opin Immunol. 2017;45:43-51).

[0090] Myeloid cells have emerged during evolution as one of the main defense mechanisms against pathogens and are an important component of tissue remodeling. Under physiological conditions, GM-CSF promotes myelopoiesis, while 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 promote 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). Thus, the accumulation of MDSCs follows the same differentiation pathway as neutrophils and monocytes.

[0091] Besides tumors and infection sites, MDSCs can be detected in the blood; for example, in some breast cancers, blood MDSC levels are 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).

[0092] The size of the myeloid suppressor cell compartment is thought to be a key factor in the clinical success or failure of cancer immunotherapy, highlighting the importance of this cell type to human pathophysiology. (Kodach LL,et al.,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.PMID 33798523).

[0093] As shown below, Applicants surprisingly show that NEO-201 binds to granulocytes and gMDSC derived therefrom and specifically induces the death or elimination of gMDSC, and thus NEO-201 can potentially be used in any condition in which gMDSC are involved in disease pathology, particularly cancer and chronic infectious conditions where MDSC are known to suppress innate immunity.

[0094] Conditions under which NEO-201 can be used to eliminate gMDSCs cancer Because NEO-201 specifically depletes gMDSCs, it can be used to treat any cancer in which gMDSCs influence innate anti-cancer immunity, including cancers that express the antigen that NEO-201 binds and cancers that do not express the antigen that NEO-201 binds.

[0095] Thus, cancer types in which MDSCs are involved in disease pathology and in which NEO-201 may be used to deplete or eliminate gMDSCs include both solid tumors and hematological cancers. Exemplary tumors in which MDSCs may be involved in disease pathology include adrenal gland cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, adult brain / CNS tumors, pediatric 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 / rectal 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 (acute lymphocytic) (ALL), adult leukemia (acute myeloid) (AML), chronic lymphocytic leukemia (CLL), leukemia (chronic myelogenous) (CML), leukemia (chronic myelomonocytic) (CML), and the like. and malignant leukemia (CMML), childhood leukemia, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, pulmonary carcinoid tumor, lymphoma, lymphoma of the skin, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, childhood non-Hodgkin's lymphoma, oral and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, 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, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0096] In some embodiments, such cancer patients are those with 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, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, colon 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, The patient may have a cancer and / or tumor selected from the group consisting of, but not limited to, 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 or ureter cancer, renal cell carcinoma, renal pelvis cancer, central nervous system tumors, primary CNS tumors, spinal cord tumors, brain stem glioma, and pituitary adenoma.

[0097] Some cancers are strongly influenced by MDSCs, and therefore patients suffering from these cancers will benefit more from the modulation of gMDSC suppressive function and differentiation by administration of NEO-201. Such benefits may be synergistic with the induction of T cell responses, as antigen presentation is improved and other immunosuppressive effects of MDSCs, including recruitment of Tregs, are alleviated.

[0098] In some aspects of the invention, the cancer patient may have stage I, stage II, stage III, or stage IV cancer involving MDSCs. In other aspects, NEO-201 reduces, eliminates, or slows or stops tumor growth, or alternatively, anti-tumor immunity is suppressed with gMDSCs, which may result in a reduction in tumor burden in the individual, inhibition of tumor growth, and / or increased survival of the individual.

[0099] In some aspects of the invention, a cancer patient may have developed resistance or tolerance to an anti-cancer 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-cancer treatment (e.g., an immunomodulatory antibody, a checkpoint inhibitor antibody or fusion protein, or a chemotherapeutic agent) to reverse such resistance or tolerance.

[0100] In some embodiments of the present invention, cancer patients may be in remission and NEO-201 administration may be used for 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 suppress the proliferation of MDSCs, thereby suppressing cancer recurrence by promoting innate anti-tumor immunity.

[0101] 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 suppress proliferation of MDSCs and thereby re-establish innate anti-tumor immunity.

[0102] Infection status NEO-201 could also potentially be used to treat bacterial conditions where gMDSCs are involved in disease pathology and may suppress innate immunity.

[0103] The types of bacterial infections for which NEO-201 can be used to deplete gMDSC include Bacillus anthraces, 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, Corynebacterium diphtheriae, Enterococcus faecalis and Enterococcus faecium, Escherichia coli (generally), Enterotoxigenic Escherichia coli (ETEC), Enteropathogenic E.coli, E.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 Staphylococcus aureus.

[0104] The types of viral infections in which MDSCs have been reported to play a role in disease pathology include both chronic and acute infections. Examples of infections for which NEO-201 may be used to deplete gMDSCs include respiratory viruses (e.g., adenovirus, avian influenza, influenza A virus, influenza B virus, measles, parainfluenza virus, respiratory syncytial virus (RSV), rhinovirus, SARS-CoV), gastrointestinal viruses (e.g., coxsackievirus, enterovirus, poliovirus, rotavirus), hepatitis viruses (e.g., hepatitis B virus, hepatitis C virus), bovine viral diarrhea virus (alternative), herpes viruses (e.g., herpes simplex 1, simplex herpes type 2), human cytomegalovirus, varicella zoster virus, retroviruses (e.g., 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 (e.g., avian influenza, dengue virus, hantavirus, hemorrhagic fever viruses, lymphocytic choroiditis virus, smallpox virus surrogates, cowpox, monkeypox, rabbitpox, vaccinia virus, Venezuelan equine encephalomyelitis virus (VEE), West Nile virus, yellow fever virus).

[0105] In some aspects of the invention, a patient may have developed resistance or tolerance to 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 promote innate anti-infective immunity.

[0106] In some aspects of the invention, an infectious disease patient (e.g., a herpes patient) may be in remission and administration of NEO-201 may be used for maintenance therapy, e.g., 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 suppress the proliferation of gMDSCs, thereby promoting innate anti-infective immunity, thereby suppressing cancer recurrence.

[0107] In some aspects of the invention, the infection may be recurrent and NEO-201 administration may be administered alone or in combination with another anti-infection treatment (e.g., an immunomodulatory antibody, a checkpoint inhibitor antibody or fusion protein, or a chemotherapeutic agent) to suppress proliferation of gMDSCs and thereby re-establish innate anti-infection immunity.

[0108] Other conditions associated with MDSCs Other diseases or conditions in which MDSCs may be involved in suppressing innate immunity include, but are not limited to, acquired immune deficiency 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 allergy, atopic dermatitis, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune enteropathy ... Immune 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, Baro's disease / Baroconcentric sclerosis, Behçet's disease, Buerger's disease, Bickerstaff encephalitis, Blau syndrome, bullous pemphigoid, Castleman's disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy, chronic relapsing multiple osteomyelitis, chronic obstructive pulmonary disease, chronic venous stasis ulcer, Char Strauss-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, cutaneous leukocytoclastic vasculitis, Degos disease, Dercum's disease, dermatitis herpetiformis, dermatomyositis, type 1 diabetes, type 2 diabetes, diffuse cutaneous systemic sclerosis, Dressler's syndrome, drug-induced lupus, discoid lupus erythematosus, eczema, emphysema, endometriosis, enthesitis-related arthritis, eosinophilic fasciitis, eosinophilic gastroenteritis, eosinophilic pneumonia, epidermolysis bullosa acquisita, erythema nodosum, erythroblastosis fetalis, essential mixed cryoglomerulonephritis pulmonary fibrosis, Evans syndrome, fibrodysplasia ossificans progressiva, fibrosing alveolitis (or idiopathic pulmonary fibrosis), gastritis, gastrointestinal pemphigoid, Gaucher disease, glomerulonephritis, Goodpasture syndrome, Graves disease, Guillain-Barré syndrome (GBS), Hashimoto's encephalopathy, Hashimoto's thyroiditis, heart disease, Henoch-Schönlein purpura, herpes gestationis (also known as pemphigoid of gestation), hidradenitis suppurativa, HIV infection, Hughes-Stobin syndrome, hypogammaglobulinemia, infections (e.g. 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 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, malignant tumors, such as cancer (e.g., sarcoma, Kaposi's sarcoma, lymphoma, leukemia, carcinoma, and melanoma), Meniere's disease, microscopic polyangiitis, Miller-Fisher syndrome, Mixed connective tissue disease, localized scleroderma, Mucca-Habermann disease (aka pityriasis lichenoidis variola), multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (aka Devic's disease), neuromyotonia, ocular cicatricial pemphigoid, opsoclonus-myoclonus syndrome, Ord's thyroiditis, relapsing rheumatism, PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococci), paraneoplastic cerebellar degeneration, Parkinson's disease, paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, Parsonage-Turner syndrome, tonsillitis, Plaque inflammation, 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 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, spondyloarthropathy, Still's disease (adult onset), stiff-person syndrome, stroke, subacute bacterial endocarditis (SBE), Susac syndrome, Sweet's syndrome, Sydenham chorea, sympathetic ophthalmia, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis (also known as "giant cell arteritis"), thrombocytopenia, Tolosa-Hunt syndrome, transplant (e.g., heart / lung transplant) rejection, transverse myelitis, tuberculosis, ulcerative colitis, undifferentiated connective tissue disease, undifferentiated spondyloarthropathy, urticarial vasculitis, vasculitis, vitiligo, and Wegener's granulomatosis.

[0109] Similarly, NEO-201 may be administered alone or in combination with another active agent to patients with such conditions to re-establish, maintain, or promote natural immunity.

[0110] Combination therapy Combination therapy with other drugs or biologics In particular, NEO-201 should be useful in combination therapy, e.g., in combination with other therapeutic agents (e.g., other biologics, e.g., therapeutic antibodies and fusion proteins, e.g., targeting cytokines or checkpoint inhibitors, chemotherapeutic agents, etc.) because, given its demonstrated ability to deplete gMDSCs, NEO-201 will enhance the efficacy of such other therapies (i.e., by restoring or enhancing natural immunity (e.g., innate responses to anti-tumor or anti-infective agents) that is otherwise suppressed by MDSCs) in subjects who have previously been resistant to treatment or have acquired resistance to treatment with a particular active agent.

[0111] In particular, combination therapies are provided herein in which NEO-201 is administered with one or more additional therapeutic agent(s) to kill or eliminate MDSCs that may inhibit the effectiveness of such additional therapeutic agent(s). Such additional therapeutic agent(s) include, but are not limited to, peptides, nucleic acid molecules, small molecule compounds, antibodies, and derivatives thereof.

[0112] Combination with other agents targeting MDSCs i.NEO-201 in combination with chemotherapy drugs targeting MDSCs The tumor-promoting effect of MDSCs can be attenuated by weakening their immunosuppressive function. STAT3 plays an essential role in MDSC-mediated tumorigenesis. By applying a specific small molecule inhibitor of p-STAT3 or STAT3-targeting siRNA to block STAT3 activation, ARG1 expression in MDSCs can be reduced and the suppressive activity of MDSCs can be eliminated [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 (e.g., TYRO3 (a type of protein tyrosine kinase)), AXL (a type of receptor tyrosine kinase), and C-Mer proto-oncogene tyrosine kinase (MERTK) and its ligands, Gas6 and ProteinS, can reverse the tumorigenic properties of MDSCs, increase the number of tumor-infiltrating CD8+ T cells, and enhance anti-PD-1 immune checkpoint therapy. MERTK disrupts the suppressive ability of MDSCs by downregulating STAT3 [Holtzhausen A.et al.,“TAM family receptor kinase inhibition reportedly reverses MDSC-mediated suppression and augments Anti-PD-1 therapy in melanoma”,Cancer Immunol.Res.2019].In addition, it has been reported that STAT3 inhibitors (e.g., sunitinib, AZD9150, and BBI608) or conjugates of STAT3 antisense oligonucleotides (ASOs) linked to immunostimulatory Toll-like receptor 9 (TLR9) agonists (CpG-STAT3ASO) conjugates can significantly reduce the immunosuppressive function of MDSCs and restore 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].

[0113] It has been reported that PGE2 induces MDSCs to upregulate the production of ARG1 and iNOS, exerting an inhibitory effect. Cyclooxygenase-2 (COX-2) is an upstream molecular signal of PGE2 that regulates the production of PGE2. Therefore, COX-2 can be targeted to downregulate the synthesis of PGE2. shRNA targeting COX-2 significantly reduces MDSCs 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]. The expression of COX-2 can also be inhibited by acetylsalicylic acid, NS-398, and celecoxib, which can suppress the activity of MDSCs and increase the infiltration of CTLs at tumor sites [Wong JL et al., “Synergistic COX2 Induction 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-2 blockade suppresses gliomagenesis by inhibiting myeloid-derived suppressor cells”, Cancer Res. 2011; 71: 2664-2674.].

[0114] 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 exacerbates the immunosuppressive activity of MDSCs and promotes tumor growth. Treatment with aspirin (ASA, a COX inhibitor) has been reported to significantly prevent tumor formation in mice [Yan G.et al.,“A RIPK3-PGE2 circuit mediates myeloid-derived suppressor cell-potentiated colorectal carcinogenesis”,Cancer Res.2018;78:5586-5599.]. Moreover, overexpression of fatty acid transport protein 2 (FATP2) was also involved in the synthesis of PGE2 through activation of the STAT5 signaling pathway. Administration of the selective FATP2 inhibitor lipofermata selectively inhibits the function of MDSCs while enhancing immunotherapy [Veglia F.et al.,“Fatty acid transport protein 2 reprograms neutrophils in cancer”,Nature.2019;569:73-78.doi:10.1038 / s41586-019-1118-2.].

[0115] Phosphodiesterase 5 (PDE5) is another target for MDSC treatment, which 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 (e.g., sildenafil, tadalafil, and vardenafil) can reduce the production of ARG1 and iNOS in MDSCs, abolish the inhibitory activity of MDSCs, reduce the number of Tregs, and significantly delay tumor progression [Tai LHet al., “Phosphodiesterase-5 inhibition reduces postoperative metastatic disease by targeting surgery-induced myeloid derived suppressor cell-dependent inhibition of Natural Killer cell cytotoxicity”, OncoImmunology.2018;7:e1431082.doi:10.1080 / 2162402X.2018.1431082, Weed DTet al., “Tadalafil reduces myeloid-derived suppressor 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 KAet al.,“Targeting immune suppression with PDE5 inhibition in end-stage multiple myeloma”Cancer Immunol.Res.,2014;2:725-731,Serafini P.et al.,“Phosphodiesterase-5 inhibition augments endogenous antitumor immunity by reducing myeloid-derived suppressor cell function”, J. Exp. Med., 2006;203:2691-270].Treatment with tadalafil in combination with cytokine-induced killer (CIK) cell-based immunotherapy has been reported to enhance CIK activity 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 reduces ROS generation [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.].

[0116] The transcription factor nuclear factor E2-related factor 2 (Nrf2) has been reported to be a master regulator of antioxidant 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, but the number of CD8+ T cells was significantly reduced and tumor growth rates increased with increased amounts of intracellular ROS [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 (e.g., omaveloxolone (RTA-408), CDDO-Me (RTA-402), and CDDO-Im (RTA-403)) has been reported to enhance the transcriptional activity of Nrf2, which attenuates the generation of ROS, abolishes the immunosuppressive effect 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, resulting in immunosuppressive potential in MDSCs [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.].Deletion of PERK or treatment with a selective inhibitor of PERK (AMG-44) reduced the transcription of Nrf2, caused overexpression of ROS, triggered mitochondrial damage, and prevented the immunosuppression of MDSCs and CD8. + It has been reported that Nrf2 increases the infiltration of T cells. This situation can be antagonized by the addition of the Nrf2 inducer sulforaphane [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]. Based on the above, overexpression and deletion of Nrf2 affect the immune inhibitory activity of MDSCs, and only when Nrf2 maintains a steady state, MDSCs can exert normal pro-tumorigenic effects.

[0117] 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 et al”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 by the antibody DC101 suppressed primary tumor growth and metastasis in a 4T1 breast cancer model. Arginase inhibition has also been reported to suppress lung metastasis in a 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 eliminate the immunosuppressive function of MDSC on T cells. When 1-MT was combined with nor-NOHA, the proliferation rate of T cells was almost completely restored [Du J.et al.,,“The study of CD14+HLA-DR- / low myeloid-derived suppressor cell(MDSC) in peripheral blood of peripheral T-cell lymphoma patients and its biological function”Cell.Mol.Biol.2017;63:62-67.].

[0118] Bruton's tyrosine kinase (BTK) is a non-receptor intracellular kinase that has been reported to be involved in the migration and proliferation of MDSCs. Treatment with the BTK inhibitor ibrutinib reduces cytokine production and motility of MDSCs [Molina-Cerrillo J. et al., “Bruton's tyrosine kinase (BTK) as a thankful target in solid tumors”, CancerTreat.Rev.2017;58:41-50.].

[0119] It has also been reported that estrogen promotes MDSC recruitment by interacting with estrogen receptor alpha and activating the STAT3 pathway, which promotes dysregulation of myelopoiesis. Tumor progression can be delayed by removing estrogenic activity with anti-estrogen treatment [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 is resistant to androgen deprivation therapy mainly because IL-23 secreted by MDSC activates the androgen receptor (AR) and STAT3 / RORγ signaling axis in prostate tumor cells. Blocking IL-23 production can combat MDSC-mediated CRPC through treatment with anti-IL-23 antibodies and the AR antagonist enzalutamide [Calcinotto A.et al., “IL-23 secreted by myeloid cells drives castration-resistant prostate cancer”, Nature.2018;559:363-369].

[0120] MDSCs have low glycolytic and mitochondrial respiration capacity but contain high levels of methylglyoxal, which inhibits the antitumor activity of CD8+ effector T cells. Neutralizing methylglyoxal with compounds containing guanidine groups (e.g., metformin) can effectively abolish the immunosuppressive activity of MDSCs. The combination of metformin and anti-PD-1 overcomes the suppression of immunotherapy by MDSCs [Baumann T.et al.,“Regulatory myeloid cells paralyze T cells through cell-cell transfer of the metabolite methylglyoxal”, Nat.Immunol.2020;21:555-566.].

[0121] ii. Combination of NEO-201 with other agents that deplete MDSCs Treatment with low-dose chemotherapy drugs (e.g., gemcitabine, 5-fluorouracil (5-FU), paclitaxel, and cisplatin) effectively affects the survival rate of MDSCs [Wang Y.et al.,“Metabolic regulation of myeloid-derived suppressor cell function in cancer”,Cells,2020;9:1011.,Won WJet 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 MDSCs, which reduces the number of circulating Tregs and the levels of TGFβ1 and PMN-MDSCs in pancreatic cancer patients, but does not reduce the levels of M-MDSCs in peripheral blood, and restores the proliferation and antitumor 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 similarly induce the death of the two subtypes of MDSCs, but has no apparent effect on other immune cells (e.g., T cells, NK cells, DCs, and B cells).Treatment with 5-FU has been reported to induce apoptosis of MDSCs, promote tumor-infiltrating T cells to produce high levels of IFNγ, and enhance T cell-dependent antitumor responses in the murine EL4 model [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]. 5-FU has been reported to significantly and specifically eliminate 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, it has been reported that the assembly of NLRP3 in MDSCs is activated by 5-FU, which leads to the secretion of MDSC-derived IL-1β and CD4+ T cell-derived IL-17, inhibiting the antitumor effect of 5-FU.Based on these, the combined administration of 5-FU and IL-1β inhibitors (e.g., indirect inhibitors DHA and SP600125) could provide an effective means to inhibit MDSCs [Dumont A.et al., “Docosahexaenoic acid inhibits both NLRP3 inflammasome assembly and JNK-mediated mature IL-1beta secretion in 5-fluorouracil-treated MDSCs: 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 antitumor effects by polarizing MDSCs into M1-type macrophages and reducing the proportion of MDSCs in the spleen [Kodumudi KNet al., “A novel chemoimmunomodulating property of docetaxel: Suppression of myeloid-derived suppressor cells in tumor bearers”, Clin.Cancer Res.2010;16:4583-4594.]. ApoE inhibits tumor invasion and endothelial cell recruitment, while liver X receptor (LXR) inhibits ApoE expression.It has been reported that the LXR agonists GW3965 and RGX-104 worsen MDSC survival and enhance the antitumor activity of CTLs by activating the LXR / ApoE axis [Tavazoie MF et al., “LXR / ApoE Activation Restricts Innate Immune 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 human MDSCs (especially M-MDSCs) and is a therapeutic target for circulating and tumor-infiltrating MDSCs across multiple cancer types [Lamba JK et al., “CD33 splicing 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].The immunotoxin gemtuzumab ozogamicin, a CD33 monoclonal antibody (mAb), effectively eliminates MDSCs and reactivates T cells to fight multiple cancers [Lamba JK et al”CD33 splicing 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.]. Furthermore, it has been reported that targeting the bromodomain and extraterminal domain (BET), a component of the intrinsic transcriptional enhancer of MDSCs, by treating PBMCs derived from HCC patients with the small molecule inhibitor i-BET762 significantly reduced the number of CD14+HLA-DR- / low M-MDSCs and improved the efficacy of immunotherapy [Liu M.et al.,“Targeting monocyte-intrinsic enhancer reprogramming improves immunotherapy efficacy in hepatocellular carcinoma”,Gut.2020;69:365-379.].

[0122] iii. Combination of NEO-201 with agents that block MDSC migration It has been reported that blocking MDSC migration can effectively reduce the proportion of MDSC in the TME and periphery by interfering with MDSC responses 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 chemokine antagonists can help prevent MDSCs (especially PMN-MDSCs) from reaching tumor sites and altering 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 a critical 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 via CXCR2 inhibitors (e.g., SX-682, reparixin, and SB225002) effectively reduces MDSC infiltration and improves the function of cytotoxic T cells [Yan G et al., “A RIPK3-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 targeting the CCR5 / CCL axis can suppress the progression and invasion of several tumors [Tan MC et al., “Disruption of CCR5-dependent homing of regulatory T cells inhibits tumor growth in a murine 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-Velazquez 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]. Administration of the neutralizing CCR5 ligand mCCR5-Ig has been reported to reduce MDSC and Treg migration 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 for CXCL12 (also known as stromal cell-derived factor 1 (SDF-1)) also mediates the recruitment of MDSC.It has been reported that neutralization of CXCR4 by antagonists (e.g., AMD3100) reduces the number of MDSCs and Tregs in the TME and promotes polarization from M2 to M1 macrophages [Wang J.et al., “CXCR4 antagonist AMD3100 (plerixafor): “From an impurity to a therapeutic agent”, Pharmacol.Res.2020:105010., Zhuang Y.et al., “CD8(+)T cells that produce interleukin-17 regulate myeloid-derived suppressor cells and are associated with survival time of patients with gastric cancer”, Gastroenterology,2012;143:951-962.]. In addition, it has been reported that colony-stimulating factor 1 receptor (CSF-1R) is a tyrosine kinase receptor that, when combined with the receptor, can induce the formation and migration of MDSCs to tumor sites.Recently, it has been reported that CSF-1R inhibitors (e.g., RG7155 and PLX647) block the CSF-1R signaling pathway, leading to the elimination of MDSCs or inhibition of their tumor-promoting functions and the reprogramming of TAMs [Law AMK et al., “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.].

[0123] iv. Combination of NEO-201 with compounds that induce MDSC differentiation Another way to target MDSCs is by inducing them to differentiate into cells with a proinflammatory phenotype. All-trans retinoic acid (ATRA), a metabolic intermediate of vitamin A, has been identified as an anticancer drug that induces MDSCs to differentiate 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 AKet al.,“The multifaceted immune regulation of bladder cancer”,Nat.Rev.Urol.2019;16:613-630.]. ATRA has been shown to inhibit the differentiation of MDSCs into DCs and macrophages in vivo and in vivo. It has been reported that ATRA induces MDSC differentiation in both splenic and bone marrow tumors, thereby significantly reducing the number of MDSCs. The specific mechanism postulated is that the added ATRA activates ERK1 / 2 signaling, which in turn upregulates the expression of glutathione synthesis enzyme 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 immune suppression”,Front.Immunol.2018;9 doi:10.3389 / fimmu.2018.02499]. In addition, it has been reported that bone marrow cells differentiate in response to treatment with ATRA. Furthermore, it has been reported that vitamin D3 can also promote the differentiation of MDSCs. MDSCs at tumor sites have 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) has been reported to significantly reduce the T cell suppressive ability of MDSCs. In vitro derived MDSCs reduced NO production under stimulation with 1,25(OH)D [Fleet JC et al., “1alpha,25 Dihydroxyvitamin 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, the combination of NEO-201 and MDSC-targeted therapy should further reduce the number and function of MDSC in tumor sites and blood circulation.

[0124] NEO-201 in Combination with Epigenetic Therapy Epigenetic therapy has been reported as another way to target MDSCs to treat cancer. The reported epigenetic therapy approaches 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]. Enhancer of zeste homolog 2 (EZH2), a gene encoding a histone methyltransferase, is often overexpressed in many cancer types [Zhou J.,et al.“Targeting EZH2 histone methyltransferase activity alleviates experimental intestinal inflammation”,Nat.Commun.2019;10:2427.]. It has been reported that the number of functional MDSCs was significantly increased in colon cancer mouse models or in vitro after treatment with the EZH2 inhibitor GSK343 ​​

[0156] . Similarly, the use of another inhibitor, GSK126, also promoted the proliferation of MDSCs. Anti-Gr1 antibodies or gemcitabine / 5-FU in combination with GSK126 could reduce the immune suppression 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; thereby, M-MDSCs acquire partial phenotype and function 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 in DNMTi5-azacytidine (AZA) has been reported to increase the proportion of CD8+ T cells and NK cells in the TME via type I IFN immune responses, reduce MDSC accumulation, and promote antitumor effects. It has been reported that the addition of HDACi entinostat (ENT) to AZA further enhances the regulation of the immune microenvironment.Triple or quadruple treatment with AZA and ENT and immunotherapy (anti-PD-1 and anti-CTLA-4) showed highly effective tumor elimination [Stone M.L. et al., “Epigenetic therapy activates type I interferon signaling in murine ovarian 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. al., “Glucocorticoids promote the onset of acute experimental colitis and cancer by upregulating mTOR signaling in intestinal epithelial cells”, Cancers.2020;12:945.].Adjuvant epigenetic therapy with AZA and ENT has been reported to block MDSC migration by downregulating CCR2 and CXCR2, which leads to impaired differentiation of MDSC into macrophages and pMN [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 T cell 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.].

[0125] Combination with immune checkpoint inhibitors 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 MEDI6469, MEDI0562, and MOXR0916. In some embodiments, the additional therapeutic agent is the anti-4-1BB antibody PF-05082566.

[0126] In some embodiments, the additional therapeutic agent targets immune checkpoints. Immune checkpoints are molecules of the immune system that enhance signals (e.g., costimulatory molecules) or suppress signals. Inhibitory checkpoint molecules that can be targeted by immune checkpoint blockers include therapeutic agents that target 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 cell death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V domain Ig suppressor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0127] The immune checkpoint inhibitor may be a drug (e.g., a small molecule, a recombinant form of a ligand or receptor), or, in particular, an antibody (e.g., a human antibody) (e.g., International Patent Publication WO2015016718; Pardoll, Nat Rev Cancer, 12(4):252-64, 2012, both of which are incorporated herein by reference). Known inhibitors of immune checkpoint proteins or analogs thereof may be used, in particular chimeric, humanized, or human forms of antibodies. As the skilled artisan will know, alternative names and / or equivalent names may be used for the specific antibodies referred to in this disclosure. Such alternative names and / or equivalent names are interchangeable in the context of the present invention. For example, it is known that lambrolizumab is also known by the alternative and equivalent names MK-3475 and pembrolizumab.

[0128] It is contemplated that any immune checkpoint inhibitor known in the art that stimulates an immune response may 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, including PD-L2, LAG3, BTLA, B7H4, and TIM3. For example, LAG3 inhibitors known in the art include soluble LAG3 (IMP321, or LAG3-Ig, as disclosed in WO2009044273), murine or humanized antibodies that block human LAG3 (e.g., IMP701, as disclosed in WO2008132601), or fully human antibodies that block human LAG3 (as disclosed in EP2320940). Another example provides for the use of blocking agents against BTLA, including but not limited to antibodies that block the interaction of human BTLA with its ligands (e.g., 4C7, disclosed in WO2011014438). Yet another example provides for the use of agents that neutralize B7H4, including but not limited to antibodies against human B7H4 (disclosed in WO2013025779 and WO2013067492) or soluble recombinant forms of B7H4 (e.g., those disclosed in US20120177645). Yet another example provides for agents that neutralize B7-H3, including but not limited to antibodies that neutralize human B7-H3 (e.g., MGA271 and its derivatives, disclosed as BRCA84D in US20120294796). In yet another example, agents targeting TIM3 are provided, including, but not limited to, antibodies targeting human TIM3 (e.g., those disclosed in WO2013006490A2, or the anti-human TIM3, blocking antibody F38-2E2 disclosed in Jones et al., J Exp Med. 2008;205(12):2763-79).

[0129] In addition, multiple immune checkpoint inhibitors (e.g., anti-PD-1 and anti-CTLA-4 antibodies) can be used in combination with NEO-201. For example, p53 gene therapy and immune checkpoint inhibitors (e.g., anti-MR and / or anti-PD-1 antibodies) can be administered to boost innate anti-tumor immunity, followed by IL24 gene therapy and immune checkpoint inhibitors (e.g., anti-PD-1 antibodies) to induce adaptive anti-tumor immune responses.

[0130] In particular, methods of treating or delaying the progression of cancer in an individual with MDSC immunosuppression are encompassed by the present invention, 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.

[0131] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In certain aspects, the PD-1 ligand binding partner is and / or PD-L2. In another embodiment, the 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, the 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 may be an antibody, antigen-binding fragment thereof, immunoadhesin, fusion protein, or oligopeptide. Exemplary antibodies are described in U.S. Patent 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 as described in U.S. Patent Application Nos. US20140294898, US2014022021, and US20110008369, all of which are incorporated herein by reference.

[0132] 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 that includes an extracellular portion or a 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 is also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO, and is an anti-PD-1 antibody described in WO 2006 / 121168. Pembrolizumab, also known as MK-3475, Merck3475, Lambrolizumab, KEYTRUDA, and SCH-900475, is an anti-PD-1 antibody 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.

[0133] In some embodiments, the immune checkpoint inhibitor is a PD-L1 antagonist (e.g., durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, or avelumab, also known as MSB00010118C). In certain embodiments, the immune checkpoint inhibitor is a PD-L2 antagonist (e.g., rHIgM12B7). In some embodiments, the immune checkpoint inhibitor is a LAG-3 antagonist (e.g., but not limited to, IMP321 and BMS-986016). The immune checkpoint inhibitor may be an adenosine A2a receptor (A2aR) antagonist (e.g., PBF-509).

[0134] Another immune checkpoint that may be enhanced by the ablative effect of NEO-201 on MDSCs is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 is GenBank accession number L15006. CTLA-4 is found on the surface of T cells and functions as an "off" switch when it binds 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 the T cell costimulatory protein CD28, and both molecules bind to CD80 and CD86 (also called B7-1 and B7-2, respectively) on antigen-presenting cells. CTLA-4 transmits inhibitory signals to T cells, whereas CD28 transmits stimulatory signals. Intracellular CTLA-4 is also present in regulatory T cells and may be important for their function. Activation of T cells via the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules.

[0135] 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 present method can be generated 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 U.S. Patent No. 8,119,129, WO01 / 14424, WO98 / 42752, WO00 / 37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), U.S. Patent 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 can be used in the methods disclosed herein. The teachings of each of the above publications are incorporated herein by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 can 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 of which are incorporated herein by reference.

[0136] An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy) or antigen-binding fragments and variants thereof (see, e.g., WO01 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Accordingly, 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 to and / or binds to the same epitope on CTLA-4 as the above-mentioned antibody. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above-mentioned antibody (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).

[0137] Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors such as those described in U.S. Pat. Nos. 5,844,905, 5,885,796, International Patent Application Nos. WO1995001994 and WO1998042752, all of which are incorporated by reference herein, and immunoadhesins such as those described in U.S. Pat. No. 8,329,867, which is incorporated by reference herein.

[0138] Another immune checkpoint that may be enhanced by the ablation effect of NEO-201 on gMDSCs is a CSF-1 / 1R binding agent or inhibitor (e.g., anti-CSF1 or anti-CSF1R antibody), the combination of which is used to treat cancer, e.g., a cancer described herein, e.g., a solid tumor in which MDSCs are involved. In certain embodiments, the CSF-1 / 1R binding agent 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. WO2005 / 073224. In certain embodiments, the CSF-1 / 1R binding agent is an M-CSF inhibitor, compound A33, or a binding agent to CSF-1 disclosed in PCT Publication No. WO2004 / 045532 or PCT Publication No. WO2005 / 068503, including RX1 or 5H4 (e.g., an antibody molecule or Fab fragment to M-CSF). In certain embodiments, the CSF-1 / 1R binding agent 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. WO2007 / 121484. In certain embodiments, the CSF-1 / 1R binding agent is pexidartinib (CAS Registry Number 1029044-16-3). Pexidartinib 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. Pexidartinib is a small molecule receptor tyrosine kinase (RTK) inhibitor of KIT, CSF1R, and FLT3. In certain embodiments, the CSF-1 / 1R binding agent is emactuzumab. Emactuzumab is also known as RG7155 or R05509554. Emactuzumab is a humanized IgG1 mAb that targets CSF1R.In a particular embodiment, the CSF-1 / 1R binding agent is FPA008. FPA008 is a humanized mAb that inhibits CSF1R.

[0139] Other therapeutic agents that may elicit synergistic effects in combination with NEO-201 in the treatment of conditions in which MDSCs are involved in the pathology include (a) microtubule inhibitors, topoisomerase inhibitors, platinums, alkylating agents, and antimetabolites; (b) MK-2206, ON 013105, RTA 402, BI 2536, sorafenib, ISIS-STAT3Rx, microtubule inhibitors, topoisomerase inhibitors, platins, alkylating agents, antimetabolites, paclitaxel, gemcitabine, doxorubicin, vinblastine, etoposide, 5-fluorouracil, carboplatin, altretamine, aminoglutethimide, amsacrine, anastrozole, azacitidine, bleomycin, busulfan, carmustine, chlorambucil, 2-chlorodeoxyadenosine, cisplatin. , colchicine, cyclophosphamide, cytarabine, cytoxan, dacarbazine, dactinomycin, daunorubicin, docetaxel, estramustine phosphate, floxuridine, fludarabine, gentuzumab, hexamethylmelamine, hydroxyurea, ifosfamide, imatinib, interferon, irinotecan, lomustine, mechlorethamine, melphalen, 6-mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, penicillin (c) 1-D-ribofuranosyl-1,2,4-triazole-3-carboxamide, 9->2-hydroxyethoxymethylguanine, adamantanamine, 5-iodo-2'-deoxyuridine, trifluorothymidine, interferon, adenine, riboflavin ... arabinosides, protease inhibitors, thymidine kinase inhibitors, sugar or glycoprotein synthesis inhibitors, structural protein synthesis inhibitors, attachment and adsorption inhibitors, and nucleoside analogs (e.g., acyclovir, penciclovir, valacyclovir, and ganciclovir); (d) PD-1 inhibitors or anti-PD-1 antibodies (e.g., KEYTRUDA® (pembrolizumab), OPDIVO® (nivolumab), or LIBTAYO (cemiplimab);(e) a PD-L1 inhibitor or anti-PD-L1 antibody (e.g., TECENTRIQ (atezolizumab), IMFINZI (durvalumab), or BAVENCIO (avelumab); or (f) a CTLA-4 inhibitor or anti-CTLA-4 antibody (e.g., YERVOY® ipilimumab). It is expected that combinations of immune checkpoint inhibitors (PD-1 inhibitors, PD-L1 inhibitors, and / or CTLA-4 inhibitors) with NEO-201 may be particularly effective in 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 incorporated herein by reference in its entirety.

[0140] Other therapeutic regimens that may be combined with NEO-201 to elicit synergistic effects in the treatment of conditions in which MDSCs are involved in the pathology include radiation therapy. NEO-201 may increase the efficacy of such other therapeutics or regimens, particularly in individuals who are or become resistant or refractory to treatment with a particular therapeutic or regimen due to MDSC-induced immunosuppression.

[0141] NEO-201 in combination with cell therapy NEO-201, due to its ability to eliminate gMDSCs, should also improve the efficacy of immune cell therapies (e.g., CAR-T cell and CAR-NK cell therapies), particularly during the use of CAR-T cell and CAR-NK cells for cancer, infectious disease, autoimmune, and inflammatory indications.

[0142] Use of NEO-201 with CAR-T cells Chimeric antigen receptor T cells (also called CART cells) are T cells that have been genetically engineered to produce artificial T cell receptors used in immunotherapy. Chimeric antigen receptors (also called CARs, chimeric immune receptors, chimeric T cell receptors, or artificial T cell receptors) are receptor proteins that have been engineered to give T cells new capabilities to target specific proteins. The receptors are chimeric because they combine both antigen-binding and T cell activation functions in one receptor.

[0143] In some embodiments, CART cell therapy uses T cells engineered with CARs for cancer therapy. The premise of CART immunotherapy is to modify T cells to recognize target cells (e.g., cancer cells) so as to more effectively target and destroy them. T cells are collected, genetically modified, and then infused into the patient, and the resulting CART cells selectively attack or cause an effect on the target cells (e.g., tumor cells, infected cells, or autoimmune cells). CART cells are CD4 + T cells and CD8 + T cells, and combinations thereof.

[0144] CART cells can be obtained from T cells in the patient's own blood (autologous) or from the T cells of another healthy donor (allogeneic). After being isolated from the person, these T cells are genetically engineered to express a specific CAR, which programs them to target antigens present on the surface of the tumor. After being infused into the patient, the CART cells act as a "living drug" against the cancer cells. When CART cells come into contact with a target antigen on a cell, they bind to it and become activated, then continue to proliferate and become cytotoxic. CART cells destroy cells by causing increased secretion of factors that can affect other cells (e.g., cytokines, interleukins, and growth factors) through several mechanisms (e.g., extensively stimulated cell proliferation), which increases the degree of toxicity (cytotoxicity) to other living cells.

[0145] CAR-T cells are used to treat a variety of blood cancers as well as solid tumors. And while most CAR-T cell research has focused on creating CAR-T cells that can eradicate specific cell populations (e.g., CAR-T cells that target lymphoma cells), this technology has other potential applications. T cells can also mediate autoimmune responses against self-antigens. CAR-equipped regulatory T cells can be used to confer tolerance to specific antigens, for example, in organ transplants or autoimmune or inflammatory diseases (e.g., lupus and RA).

[0146] "Chimeric receptor" generally refers to a cell surface receptor that includes an extracellular ligand binding domain, a transmembrane domain, and a cytoplasmic costimulatory signaling domain in a combination that does not naturally occur together on a single protein. This specifically includes receptors in which the extracellular and cytoplasmic domains do not naturally occur together on a single receptor protein. Furthermore, chimeric receptors are distinct from TCRs expressed on natural T cell lymphocytes.

[0147] As described in U.S. Patent Nos. 5,359,046, 5,686,281, and 6,103,521, the extracellular domain may be obtained from any of a wide variety of extracellular domains or secreted proteins associated with ligand binding and / or signal transduction. The extracellular domain may be part of a protein that is associated with multiple proteins in a monomer, homodimer, heterodimer, or non-covalent complex. In particular, the extracellular domain may consist of an Ig heavy chain, which may in turn be covalently linked to an Ig light chain due to the presence of a CH1 and hinge region, or may be covalently linked to other Ig heavy / light chain complexes due to the presence of hinge, CH2, and CH3 domains. In the latter case, the heavy / light chain complex that becomes bound to the chimeric construct may constitute an antibody with a specificity different from the antibody specificity of the chimeric construct. Depending on the antibody function, desired structure, and signaling, entire chains can be used or truncated chains can be used, all or part of the CH1, CH2, or CH3 domains can be removed, or all or part of the hinge region can be removed.

[0148] The extracellular domain of a CAR is often derived from an immunoglobulin and comprises an antigen-binding portion (i.e., an "antigen-binding site") (e.g., fragments, subsequences, complementarity determining regions (CDRs)), which include: (i) a Fab fragment (a monovalent fragment consisting of the VL, VH, CL, and CH1 domains); (ii) a F(ab')2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at 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 (Ward et al., (1989) Nature 341:544-546); and (vi) an isolated complementarity determining region (CDR).

[0149] Chimeric receptors can be designed to treat any cancer for which a specific monoclonal antibody exists or can be made. In particular, cancers (e.g., 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.

[0150] The transmembrane domain may be contributed by a protein that contributes to the clustering domain of the multispecific extracellular inducer, a protein that contributes to the effector function signaling domain, a protein that contributes to the growth signaling portion, or a completely different protein. In most cases, it will be convenient to have a transmembrane domain that is naturally associated with one of the domains. In some cases, it will be desirable to use a transmembrane domain of the ζ, η, or FcεR1γ chain that contains a cysteine ​​residue capable of disulfide bonding, so that the resulting chimeric protein can form a disulfide-bonded dimer with itself or with an unmodified version of the 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 binding of such domains to the transmembrane domain of the same or a different surface membrane protein, in order to minimize interactions with other members of the receptor complex. In other cases, it may be desirable to use transmembrane domains of the ζ, η, or FcεR1γ chains and -β, MB1 (Igα), B29, or CD3γ, ζ, or ε to maintain physical association with other members of the receptor complex. Examples of transmembrane regions suitable for use in the present invention include the constant (Fc) region of immunoglobulins, human CD8a, and artificial linkers that serve to move targeting moieties away from the cell surface to improve access and binding to target cells, although any transmembrane region sufficient to anchor the CAR to the membrane can be used. Those skilled in the art are aware of the structural elements (e.g., lipophilic amino acid regions) that generate transmembrane regions and transmembrane domains in many membrane proteins, and therefore any convenient sequence can be substituted.

[0151] The cytoplasmic domain of the chimeric receptor of the present invention may comprise a signaling domain (e.g., a costimulatory signaling domain) alone or in combination with any other desired cytoplasmic domain(s) useful in the context of this chimeric receptor type (e.g., 4-1BB, CD3ζ signaling domain and / or CD28 signaling domain). For example, the 4-1BB, CD3ζ and CD28 signaling domains, including those used in chimeric receptors, have been well characterized. In one embodiment, the cytoplasmic domain of the chimeric receptor may comprise a 4-1BB signaling domain alone or in combination with any other desired cytoplasmic domain(s) 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 the hinge and transmembrane domains of CD8α, and the cytoplasmic domain comprises the signaling domain of CD3ζ and the signaling domain of 4-1BB. The CD8α hinge and transmembrane domain consists of 69 amino acids translated from 207 nucleotides at positions 815-1021 of GenBank Accession No. NM_001768. The CD3ζ signaling domain of a preferred embodiment contains 112 amino acids translated from 339 nucleotides at positions 1022-1360 of GenBank Accession No. NM_000734.

[0152] In adoptive immunotherapy, the patient's circulating or tumor-infiltrating lymphocytes are isolated in vitro, activated with lymphokines (e.g., IL-2), or transduced with tumor necrosis genes and re-administered. To accomplish this, an immunologically effective amount of activated lymphocytes genetically modified to express the tumor-specific chimeric receptor genes described herein will be administered to an animal or human patient. Most preferably, the activated lymphocytes are the patient's own cells, previously isolated from a blood or tumor sample and activated and expanded in vitro. When such cells are injected, antigen-specific CAR-T cells can be expanded in vitro for use in adoptive cellular immunotherapy, which has been shown to have anti-tumor reactivity in tumor-bearing hosts.

[0153] For example, genetic modification to introduce a CAR construct into a T cell can be achieved by transducing (or otherwise delivering) a T cell composition with a recombinant DNA or RNA construct, such as a vector. A vector can be any agent capable of delivering or maintaining a nucleic acid in a host cell, including viral vectors (e.g., retroviral, lentiviral, adenoviral, or adeno-associated viral vectors), plasmids, naked nucleic acids, nucleic acids complexed with polypeptides or other molecules, and nucleic acids immobilized on solid-phase particles. An appropriate DNA sequence can be inserted into a vector by a variety of procedures. In general, a DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.

[0154] 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 (e.g., viral promoters, such as the cytomegalovirus (CMV) promoter, β-actin promoter, phosphoglycerate kinase (PGK) promoter, ubiquitin promoter, and EF-1α promoter) can be used. This list is not limiting. The expression construct preferably also includes a sequence that allows replication of the expression construct. Transcription of the DNA encoding the polypeptide of the present invention by higher eukaryotes can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp, that act on a promoter to increase transcription. Examples include the SV40 enhancer at 100-270 behind the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer at the rear of the replication origin, and the adenovirus enhancer. Preferably, a retroviral vector (either gamma retrovirus or lentivirus) is used to introduce the CAR nucleic acid construct into the cell. For example, a polynucleotide encoding a costimulatory ligand protein (e.g., tumor necrosis factor (TNF) ligand, e.g., 4-1BBL, OX40L, CD70, LIGHT, and CD30L, or an Ig superfamily ligand, e.g., CD80 and CD86) or a receptor that binds to an antigen, or a variant or fragment thereof, can be cloned into a retroviral vector and expression can be driven from an endogenous promoter, from a retroviral long terminal repeat sequence, or from a promoter specific to the target cell type of interest. Non-viral vectors can also be used.

[0155] CAR-T cells are usually expanded and activated in vitro to reach therapeutically sufficient numbers before being administered to a subject. The cells can be expanded non-specifically using mitogenic αCD3 and αCD28 antibodies, or through the use of recombinant antigen-presenting cell lines or particles that display the antigen targeted by the CAR binding domain (and, in some cases, additional costimulatory molecules). Other methods for selectively expanding T cells to constitutively express the CAR include co-expression with a transgene for selection under cytocidal concentrations of drugs and / or sorting (e.g., using magnetic beads that recognize the introduced protein co-expressed with the CAR). Antigen-specific expansion is preferred, since CAR-mediated T cell activation is believed to depend on and increase with binding affinity to the cognate antigen. If the CAR-T cells of the present invention are expanded non-specifically without activation before treatment with a nucleic acid targeting drug, they can be activated in vitro before being administered to a subject, again using a cell line or particle that displays the antigen targeted by the CAR binding domain.

[0156] The diseased cells can be of any type of cancer, any tissue or cell type origin. Suitable target cells include, but are not limited to, the following malignant tumor cells: leukemia (e.g., chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), and acute lymphoblastic leukemia (ALL); multiple myeloma (MM); non-Hodgkin's lymphoma and Hodgkin's disease (lymphoma); solid tumors (e.g., breast cancer, lung cancer, ovarian and testicular cancer, prostate cancer, colon cancer, melanoma, renal cancer cells, neuroblastoma, and head and neck tumors).

[0157] CAR and CAR-T derived effector cells can be designed to target any desired antigen. Examples of target antigens include, for example: 0772P (CA125, MUC16, GenBank Accession Number AF36148); Adipophilin (Perilipin-2, Adipose Differentiation Related Protein, ADRP, ADFP, MGC10598; NCBI Reference Sequence: NP-001113.2); AIM-2 (Absent in Melanoma 2, PYHIN4, Interferon Inducible Protein AIM2, NCBI Reference Sequence: NP-004824.1); ALDH1 A1 (aldehyde dehydrogenase 1 family, member A1, ALDH1, PUMB1, retinaldehyde dehydrogenase 1, ALDC, ALDH-E1, ALHDII, RALDH1, EC1.2.1.36, ALDH11, HEL-9, HEL-S-53e, HEL12, RALDH1, acetaldehyde dehydrogenase 1, aldehyde dehydrogenase 1, soluble, aldehyde dehydrogenase, hepatic cytoplasmic, ALDH class 1, epididymal intraluminal protein 12, epididymal intraluminal protein 9, epididymal secretory sperm-binding protein Li 53e, retinal dehydrogenase 1, RaIDH1, aldehyde dehydrogenase family 1 member A1, aldehyde dehydrogenase, cytoplasmic, EC1.2.1; NCBI Reference Sequence: NP-000680.2; alpha actinin-4 (ACTN4, actinin, alpha 4, FSGS1, focal segmental glomerulosclerosis 1, nonmuscle alpha actinin 4, F-actin cross-linking protein, FSGS, ACTININ-4, actinin alpha 4 isoform, alpha actinin-4; NCBI Reference Reference sequence: NP-004915.2); alpha-fetoprotein (AFP, HPAFP, FETA, alpha-1-fetoprotein, alpha-fetoglobulin, alpha-1-fetoprotein, alpha-fetoglobulin, HP; GenBank: AAB58754.1); amphiregulin (AREG, SDGF, Schwann cell-derived growth factor, colorectal cell-derived growth factor, AR, CRDGF; GenBank: AAA51781.1); ARTC1 (ART1, ADP-ribo silyltransferase 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 beta-hydroxylase domain containing 1, aspartate beta-hydroxylase domain-containing protein 1, EC1.14.11., GenBank: AAI44153.1); B7-H4 (VTCN1, V-set domain-containing T-cell activation inhibitor 1, B7H4, B7 superfamily member 1, immune costimulatory protein B7-H4, B7h.5, T-cell costimulatory molecule B7x, B7S1, B7X, VCTN1, H4, B7 family member, PRO1291, B7 family member, H4, T-cell costimulatory molecule B7x, V-set domain-containing T-cell activation inhibitor 1, 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, prolixin, 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); beta-catenin (CTNNB1, Catenin (cadherin-associated protein), beta 1, 88 kDa, CTNNB, MRD19, Catenin (cadherin-associated protein), beta 1 (88 kD), Armadillo, Catenin beta-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); BMPR1B (bone morphogenetic protein receptor type IB, 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, 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 isoform 2 precursor, B4, CVID3 [Homo sapiens], NCBI reference sequence: NP-0 01761.3); CD20 (CD20-B lymphocyte antigen CD20, transmembrane 4 domains, 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, SIG. LEC-2, FLJ22814, GenBank Accession No. AK02646; CD22; 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 family, member 7; surface antigen CD70; Ki-24 antigen; CD27 ligand; CD27-L; tumor necrosis factor ligand superfamily member 7; NCBI reference sequence for Homo sapiens species: 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 (C D79a (CD79A, CD79a, immunoglobulin-related alpha; CD79b (CD79b (CD79B, CD79b, IGb (immunoglobulin-related beta), B29, GenBank accession numbers NM-000626 or 1103867); Cdc27 (cell division cycle 27, DOS1430E, D17S978E, anaphase-promoting complex subunit 3, anaphase-promoting complex subunit 3, ANAPC3, APC3, CDC27Hs, H-NUC, CD C27 homolog, cell division cycle 27 homolog (S. Cerevisiae), HNUC, NUC2, anaphase-promoting complex, 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, EC2.7.11.22, CMM3, EC2.7.11; NCBI reference sequence: NP-000066.1);CDKN2A (cyclin-dependent kinase inhibitor 2A, MLM, CDKN2, MTS1, cyclin-dependent kinase inhibitor 2A (inhibits melanoma, P16, CDK4), cyclin-dependent kinase 4 inhibitor A, multiple tumor suppressor 1, CDK4I, MTS-1, CMM2, P16, ARF, INK4, INK4A, P14, P14ARF, P16-INK4A, P16INK4, P16INK4A, P19, P19ARF, TP16, CDK4 inhibitor P16-INK4, cell cycle negative regulator beta, 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 (E. coli), ClpP (caseinolytic protease, ATP-dependent, proteolytic subunit, E. coli) homolog, ClpP caseinolytic peptidase, ATP-dependent, proteolytic subunit homolog (E. coli), ClpP caseinolytic protease, ATP-dependent, proteolytic subunit homolog (E. 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 protease, ATP-dependent, proteolytic subunit homolog, putative ATP-dependent Clp protease proteolytic subunit, mitochondrial; NP; COA-1; CPSF; CRIPTO (CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-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); Psychi. linDi (CCND1, BCL1, PRAD1, D11S287E, B-cell CLL / lymphoma 1, B-cell lymphoma 1 protein, BCL-1 oncogene, PRAD1 oncogene, cyclin Dl (PRAD1:parathyroid adenomatosis 1), G1 / S-specific cyclin Dl, parathyroid adenomatosis 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 inhibitor 1, SK, hDkk-1, Dickkopf (Xenopus laevis) homolog 1, Dickkopf-1 homolog (Xenopus laevis), DKK-1, Dickkopf1 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 downregulator 1, TBP-binding (negative cofactor 2), negative cofactor 2-beta, TATA-binding protein-associated phosphoprotein, NC2, NC2-beta, protein Drl, NC2-beta, transcriptional downregulator 1, NCBI reference sequence: NP-001929.1); DR13 (major histocompatibility complex, class II, DR beta 1, HLA-DR1B, DRw10, DW2.2 / DR2.2, SS1, DRB1, HLA-DRB, HLA class II histocompatibility antigen, DR-1 beta chain, human leukocyte antigen DRB1, lymphocyte antigen DRB1, MHC class II antigen, MHC class II HLA-DR beta 1 chain, MHC class II HLA-DR beta cell surface glycoprotein, MHC class II HLA-DRw10-beta, 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 beta 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 D RB1*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, downless homolog, ectodysplasin A receptor, ectodermal dysplasia receptor; anhidrotic ectodysplasin 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, Snull14, U5-116KD, SNRP116, U5-116KDa; GenBank:AAH02360.1);EGFR (epidermal growth factor receptor, ERBB, proto-oncogene C-ErbB-1, receptor tyrosine protein kinase ErbB-1, ERBB1, HER1, EC2.7.10.1, 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 inducer protein 61, mENA, EC2.7.10, GenBank:AAH94761.1; EGFR-G719A; EGFR-G719C; EGFR-G719S; EGFR-L858R; EGFR-L861Q; EGFR-57681; EGFR-T790M; elongation factor 2 (EEF2, eukaryotic translation elongation factor 2, EF2, polypeptide-tRNA translocase, EF-2, SCA26, EEF-2; NCBI reference sequence:NP-001952.1); ENAH(hMena)(Enabled Homolog(Drosophila), MENA, Mammalian Enabled, ENA, NDPP1, Protein Enabled Homolog;GenBank:AAH95481.1) - Results for "ENAH" only, not "ENAH (hMena)";EpCAM (epithelial cell adhesion molecule, M4S1, MIC18, tumor-associated calcium signaling factor 1, TACSTD1, TROP1, adenocarcinoma-associated antigen, cell surface glycoprotein Trop-1, epithelial glycoprotein 314, major gastrointestinal tumor-associated protein 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 (glycoprotein of 35 kD), 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, proepiregulin; GenBank: AAI36405.1);ETBR (EDNRB, endothelin receptor type B; HSCR2, HSCR, endothelin receptor nonselective; 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, KMT6 EC2.1.1.43, EZH1, WVS, Enhancer of Zeste (Drosophila) homolog 2, ENX1, EZH2b, KMT6A, WVS2, Histone-lysine N-methyltransferase EZH2, Enhancer of Zeste homolog 2, EC2.1.1; 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, SPAP1B, SPAP1C, CD307b, Fc receptor-like protein 2, immunoreceptor translocation-associated protein 4, immunoglobulin superfamily Fc receptor, Gp42, SH2 domain-containing phosphatase anchor protein 1, FcRL2, CD307b antigen; GenBank:AAQ8849 7.1);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 stimulator 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); -- Alias ​​result for "G250", not "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; RET1L; GDNFR-alpha 1; GFR-alpha; U95847; BC014962; NM-145793NM-005264); GEDA (GenBank accession number AY26076); GFRA1-GDNF family receptor alpha-1; GDNF receptor alpha-1; GDNF R-alpha-1; GFR-alpha-1; RET ligand 1; TGF-beta-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, secreted glypican-3, OCI5; GenBank:AAH35972.1);GnTVf;gp100 (PMEL, pre-melanosome protein; SILV, D12S53E, PMEL17; SIL, melanocyte protein Pmel17; 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 Mel17; melanocyte protein PMEL; melanocyte nosomal 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; FLJ1185 6;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 cell receptor 1, T cell immunoglobulin mucin family member 1, kidney injury 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 viral oncogene homolog 2, NGL, NEU, neuro / neuron. glioblastoma-derived oncogene homolog, metastatic lymph node gene 19 protein, proto-oncogene C-ErbB-2, proto-oncogene Neu, tyrosine kinase cell surface receptor HER2, MLN19, p185erbB2, EC2.7.10.1, V-Erb-B2 avian erythroblastic leukemia virus oncogene homolog 2 (neuronal / glioma-derived oncogene homolog), CD340, HER-2, HER-2 / neu, TKR1, C- ErbB2 / Neu protein, herstatin, neuroblastoma / glioblastoma-derived oncogene homolog, receptor tyrosine protein kinase ErbB-2, V-Erb-B2 erythroblastic leukemia viral oncogene homolog 2, neuro / glioblastoma-derived oncogene homolog, MLN19, CD340 antigen, EC2.7.10, NP); HER-2 / neu above; HERV-K-MEL; HLA-DOB (peptide binding to CD4 +beta subunit of MHC class II molecule (Ia antigen) presented to T lymphocytes); 273aa, al:6.56, MW:30820.TM:1[P] gene chromosome:6p21.3, GenBank accession number NP-002111); hsp70-2 (HSPA2, heat shock 70 kDa protein 2, heat shock 70 kDa protein 2, HSP70-3, heat shock associated 70 KDa protein 2, heat shock 70 KDa protein 2, GenBank:AAD21815.1); IDO1 (indole amine 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; IL13R alpha 2 (IL13RA2, interleukin-13 receptor, alpha 2, cancer / testis antigen 19, interleukin-13 binding protein, I L-13R-alpha-2, IL-13RA2, IL-13 receptor subunit alpha-2, IL-13R subunit alpha-2, CD213A2, CT19, IL-13R, IL13BP, interleukin-13 binding protein, interleukin-13 receptor alpha 2 chain, interleukin-13 receptor subunit alpha 2, IL13R, CD213a2 antigen, NP; IL20Rα; intestinal carboxylesterase; IRTA2 (another name for FcRH5); kallikrein 4 (KLK4, kallikrein-associated prostase 4, PRSS17, EMSP1, enamel matrix serine protease 1, kallikrein-like protein 1, serine protease 17, KLK-L1, PSTS, AI2A1, kallikrein 4 (prostase, enamel matrix, prostate), ARM1, EMSP, androgen-regulated message 1, enamel matrix serine protease 1, kallikrein, kallikrein-4, prostase, EC3.4.21.-, prostase, EC3.4.21; 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; Lensin (LGSN, Lensin, 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; glutamine synthase) domain-containing 1, lens glutamine synthase-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); LyPD1-LY6 / PLAUR domain-containing 1, PHTS [Homo sapiens], GenBank: AAH17318.1); MAGE-A1 (Melanoma antigen family A, 1 (induces expression of antigen MZ2-E, MAGE1, melanoma antigen family A1, MAGEA1, melanoma antigen MAGE-1, melanoma-associated antigen Gen1, 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, 10, MAGE10, MAGE-10 antigen, melanoma associated antigen 10, cancer / testis antigen 1.10, CT1.10, cancer / testis antigen family 1, member 10, cancer / testis antigen family 1, member 10; NC NCBI Reference Sequence: NP-001238757.1; 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-001159859.1); MAGE-A2 (MAGEA2, melanoma antigen family A, 2, MAGE2, cancer / testis antigen 1.2, CT1.2, MAGEA2A, MAG E-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 (MA GEA6, 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; CT1.9, 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, secretoglobin, family 2A, member 2; MGB1, mammaglobin 1; UGB2, mammaglobin A, mammaglobin-A, mammaglobin-1, secretoglobin family 2A member 2, NP); MART2 (HHAT, hedgehog acyltransferase; SKI1, melanoma antigen 2 recognized by T cells; skinny hedgehog protein 1; Skn, melanoma antigen 2 recognized by T cells; protein cysteine ​​N-palmitoyltransferase HHAT, EC 2.3.1.-; GenBank :AAH39071.1); M-CSF (CSF1, colony-stimulating factor 1 (macrophage); MCSF, CSF-1; lanimostim, macrophage colony-stimulating factor 1, lanimostim; GenBank:AAH21117.1); MCSP (SMCP, sperm mitochondrial-associated cysteine-rich protein; MCS, mitochondrial capsule selenoprotein; HSMCSGEN1, sperm mitochondrial-associated cysteine-rich protein; NCBI reference sequence:NP-109588.2); XAGE-lb / GAGED2a; WT1 (Wilms tumor tumor 1, WAGR, GUD, WIT-2, WT33, amino-terminal domain of EWS, NPHS4, last three zinc fingers of DNA-binding domain of WT1, AWT1, 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 catalysis on channel, subfamily M, member 4, GenBank accession number NM-01763);TRP2-INT2;TRP-2;TRP-1 / gp75 (tyrosinase-related protein 1, 5,6-dihydroxyindole-2-carboxylic acid oxidase, CAS2, CATB, TYRP, OCAS, catalase B, b-protein, glycoprotein 75, EC1.14.18, melanoma antigen Gp75, TYRP1, TRP, TYRRP, TRP1, SHEP11, DHICA oxidase, EC1.14.18, GP75, EC1.14.18.1, triosephosphate isomerase (triosephosphate isomerase 1, TPID, triosephosphate isomerase, HEL-S-49, TIM, epididymal secretory protein Li49, TPI, triosephosphate isomerase, EC 5.3.1.1; 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, taxol resistance-associated gene 3 protein, chondrosarcoma-associated gene 2 / 3 protein-like, CT24.2, taxol resistance-associated gene 3, TRAG-3, CSAG3A, TRAG3;); TMEM46 (shisa homolog 2 (Xenopus); SHISA; NP-001007539.1; NM-001007538.1; TMEM118 (ribosomal protein 1, ribosomal protein 2, ribosomal protein 3 ... Binding finger protein, transmembrane 2;RNFT2;FLJ1462;NP-001103373.1;NM-001109903.1;TMEFF1 (transmembrane protein with EGF-like and two follistatin-like domains 1)Tomoregulin-;H7365;C9orf2;C90RF2;U19878;X83961;NM-080655;NM-003692;TGF-betaRII (TGFBR2, transforming growth factor tbetaR-II, TGFR-2, TGF-beta receptor type IIB, TGF-beta type II receptor, TGF-beta receptor type 2, EC2.7.11.30, transforming growth factor beta receptor type IIC, AAT3, TbetaR-II, transforming growth factor, beta receptor II (70-80kD), TGF-beta receptor ta. type II, FAA3, transforming growth factor beta receptor type II, LDS1B, HNPCC6, LDS2B, LDS2, RITC, EC2.7.11, TAAD2; TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative transmembrane proteoglycan, related to the EGF / heregulin 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 numbers AF179274; AY358907; CAF85723, CQ782436; TAG-2; TAG-1 (contactin 2 (axonal), TAG-1, AXT, axonal 1 cell adhesion molecule, TAX, contactin 2 (transiently expressed), TAXI, contactin 2, axonal glycoprotein TAG-1, transiently expressed axonal glycoprotein, transient axonal glycoprotein, axonin-1, TAX-1, TAG1, FAMES; PRF:444868); SYT-SSX1 or SSX2 fusion protein; survivin; STEAP2 (HGNC863 9, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, prostate six-transmembrane epithelial antigen 2, prostate six-transmembrane protein, GenBank accession number AF45513; STEAP1 (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-linked 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 of dominant megacolon, SRY-related HMG box gene 10, human homolog of transcription factor SOX-10, GenBank: CAG3 0470.1); SNRPD1 (small nuclear ribonucleoprotein Dl, small nuclear ribonucleoprotein Dl, polypeptide 16 kDa, polypeptide (16 kD), SNRPD, HsT2456, Sm-D1, SMD1, Sm-D autoantigen, small nuclear ribonucleoprotein D1 polypeptide 16 kDa pseudogene, SnRNP core protein Dl, small nuclear ribonucleoprotein SmDl); SLC35D3 (solute carrier family 35, member D3, FRCL1, Fringe connection-like protein 1, bA55K22.3, Frc, Fringe-like 1, solute carrier family 35 member D3; NCBI GenBank:NC-000006.11 NC-018917.2 NT-025741.16);SIRT2 (Sirtuin 2, NAD-dependent deacetylase sirtuin 2, SIRL2, silent information regulator 2, regulatory protein SIR2 homolog 2, Sir2-related protein type 2, SIR2-like protein 2, sirtuin type 2, sirtuin (silent mating type regulation 2 homolog) 2 (S. cerevisiae), sirtuin 2, sirtuin (silent mating type regulation 2, S. cerevisiae, homolog) 2, EC 3.5.1., SIR2, GenBank:AAK51133.1);Sema5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain.TM. and a short cytoplasmic domain, (semaphorin) 5B, GenBank accession number AB04087; seserin 1 (SCRN1, SES1, KIAA0193, seserin-1; GenBank: EAL2445.8.1);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; GenBank: AAF23613.1);RNF43-E3 ubiquitin protein ligase RNF43 precursor [Homo sapiens], RNF124;URCC; NCBI Reference Sequence: NP-060233.3;RhoC (RGS5 (G protein 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 4, zinc finger, UBR1 type 1, ZUBR1, E3 ubiquitin protein ligase UBR4, RBAF600, retinoblastoma protein-associated factor of 600 kDa, zinc finger UBR1 type protein 1, EC 6.3.2., N-recognin-4, KIAA0462, p600, EC 6.3.2, KIAA1307, GenBank: AAL83880.1); RAGE-1 (MOK, MOK protein kinase, renal tumor antigen, RAGE, MAPK / MAK / MR K overlapping kinase, renal tumor antigen 1, renal cell carcinoma antigen, RAGE-1, EC2.7.11.22, RAGE1; UniProtKB / Swiss-Prot:Q9UQ07.1; RAB38 / NY-MEL-1 (RAB38, NY-MEL-1, RAB38, member of the RAS oncogene family, melanoma antigen NY-MEL-1, Rab-associated GTP-binding protein, Ras-related protein Rab-38, rrGTPbp; GenBank:AAH15808.1); PTPRK (DJ480J14.2.1 (protein tyrosine phosphatase, receptor type, KR-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-kappa, receptor type tyrosine protein phosphatase kappa, EC3.1.3.48, PTPK; GenBank: AAI44514.1); PSMA; PSCA hIg (2700050C12Rik, C530008016Rik, RIKEN cDNA2700050C12, RIKEN cDNA2700050C12 gene, GenBank accession number AY358628; PSCA (prostate stem cell antigen precursor, GenBank accession number AJ29743); PRDX5 (peroxiredoxin 5, EC1.11.1.15, 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; AOEB166, epididymal secretory protein Li55; Alu corepressor 1; peroxiredoxin-5; mitochondrial; peroxiredoxin prxin V, prx-V, thioredoxin reductase, Prx-V, ACR1, Alu corepressor, 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 tumors, Opa interacting protein 4, Opa interacting protein 01P4; GenBank: CAG30435.1); pml-RAR alpha fusion protein; PMEL17 (Silver homolog; SILV; D12S53E; PMEL17; SI; SIL); ME20; gp10BC0. 01414;BT007202;M32295;M77348;NM-006928;PBF (ZNF395, zinc finger protein 395, PRF-1, Huntington's disease regulated, 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; GenBank: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 alpha, INGAP, PAP-H, hepatic enteropancreatic protein; PBBCGF, human proislet peptide, REG -III, pancreatitis-associated protein 1; Regi, RegIII-alpha, hepatocellular-intestinal-pancreatic; regenerating islet-derived protein III-alpha, pancreatic beta cell growth factor; HIP, PAP homologous protein; HIP / PAP, proliferation-inducing protein 34; PAP1, proliferation-inducing protein 42; REG-3-alpha, regenerating islet-derived protein 3-alpha, pancreatitis-associated protein; GenBank: AAH36776.1; p53 (TP53, tumor protein P53; TPR53; P53, tumor cell antigen P53; NY-CO-13 antigen) , mutated tumor protein 53, phosphoprotein P53, P53 tumor suppressor, BCC7, transformation-associated protein 53, LFS1, tumor protein 53, Li-Fraumeni syndrome, tumor suppressor P53; P2X5 (purinergic receptor P2X ligand-gated ion channel 5, an ion channel regulated by extracellular ATP that may be involved in synaptic transmission and neurogenesis, defects may contribute 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, O-linked N-acetylglucosamine (GlcNAc) transferase (UDP-N-acetylglucosamine:polypeptide-N-acetylglucosaminyltransferase, UDP-N-acetylglucosamine-peptide N-acetylglucosaminyltransferase 110KDa subunit, UDP-N-acetylglucosamine:polypeptide-N-acetylglucosaminyltransferase, uridine diphospho-N-acetylglucosamine:polypeptide beta-N-acetylglucosaminyltransferase, O-GlcNAc transferase subunit P110, EC 2.4.1.255, O-linked N-acetylglucosamine transferase 110KDa subunit, EC 2 .4.1, HRNT1, EC2.4.1.186, O-GLCNAC; GenBank:AAH38180.1);OA1 (Osteoarthritis QTL1, OASD; GenBank:CAA88742.1);NY-ESO-1 / LAGE-2 (Cancer / Testis antigen 1B, CTAG1B, 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 virus. (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, C subunit; neo-PAP (PAPOLG, poly(A) polymerase gamma, neopoly(A) polymerase, nuclear poly(A) polymerase gamma, polynucleotide adenyltransferase gamma, SRP RNA3 adenylase / Pap2, PAP-gamma, Neo-PAP, SRP RNA3'-adenylation enzyme, PAP2, EC2.7.7.19, PAPG, NCBI reference sequence: NP-075045.2); NCA (CEACAM6, GenBank accession number M1872); Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b, GenBank accession number NM-00642); myosin class I; MUM-3; MUM-2 (TRAPPC1, transport protein particle complex 1, BETS, BE TS homolog, MUM2, melanoma ubiquitous mutated 2, multiple myeloma protein 2, transport protein particle complex subunit 1, MUM-if, mucin (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, tracheobronchial mucin MUC5, 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, 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; MSG783 (RNF124, 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 potentiating factor, mesothelin, GenBank accession number NM-00582;MMP-7 (MMP7, matrilysin, MPSL1, matrine, matrix metalloproteinase 7 (matrilysin, uterine), matrilla. isin, matrix metalloproteinase-7, EC 3.4.24.23, PUMP-1 protease; matrine, uterine metalloproteinase, PUMP1, MMP-7, EC 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; collagenase, matrix metalloproteinase-2, MMP-II, 72KDa 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 RIBALK-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, artemin, anti-Id, ASPART IC, atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B lymphocyte stimulating factor (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 osteogenin, 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), BMP, 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, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL 8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD4, CD5, CD6, CD 7, 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, CD5 5. CD56, CD61, CD64, CD66e, CD74, CD80(B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Toxin, Toxin, Toxin, Toxin, 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, CXCL 4. CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR 2. CXCR3, CXCR4, CXCR5, CXCR6, DAN, DCC, DcR3, DC-SIGN, collapse-promoting factor, des(1-3)-IGF-I (IGF-1), Dhh, Dゴキシン, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, 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, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, G CSF, 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-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, glutamate 4, glycoprotein IIb / IIIa (GPIIb / 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, HIV IIIB gp120 V3 loop, HLA, HLA-DR, HM1.24, HMFGPEM, 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)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, integrin alpha 2, interferon integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta 1, integrin, alpha 4 / beta 7, integrin alpha 5 (alpha V), integrin alpha 5 / beta 1, integrin alpha 5 / beta 3, integrin alpha 6, integrin beta 1, integrin beta 2, 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, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloprotease tease, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, 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), MUC18, Müllerian inhibitory substance, Mug, MuSK, NAIP, NAP, NCAD, N -cadherin, NCA90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, 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, P DK-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, RSVFgp, 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 alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, T fR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan specificity, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta Rllb, TGF-beta RIII, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, Thrombin, Thymic Ck-1, Thyroid stimulating hormone, Tie, TIMP, TIQ, Tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF IOB(TRAIL R2 DR5、KILLER、TRICK-2A、TRICK-B)、TNFRSF10C(TRAIL R3 DcRI、LIT、TRID)、TNFRSF10D(TRAIL R4 DcR2、TRUNDD)、TNFRSF11A(RANK ODFR、TRANCE R)、TNFRSFllB(OPG OCIF、TR1)、TNFRSF12(TWEAK R FN14)、TNFRSF13B(TACI)、TNFRSF13C(BAFF R)、TNFRSF14(WHEM ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR)、TNFRSF17(BCMA)、TNFRSF 18(GITR AITR)、TNFRSF 19(TROY CROWN)、TNFRSF 19L(RELT)、TNFRSFIA(TNF RI CD120a、p55-60) TNFRSFIB(TNF RII). CD120b, p75-80, TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1 R), TNFRSF5(CD40 p50)), TNFRSF6(Fas). Apo-1, APT1, CD95, TNFRSF6B(DcR3 M68, 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 polymer TL2) TNFSF11(TRANCE / RANK dimer ODF) TNFSF12(TWEAK). Apo-3リンド, DR3リンド, TNFSF13(APRIL TALL2), TNFSF13B(BAFF LIGHT, TALL1, THANK, TNFSF20), TNFSF14(LIGHTHVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSFIA (TNF-a connectin, 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, metastasis receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expressing Lewis Y-related carbohydrate, 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 antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand 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 G1), globulin and mucin protein 3), hormone receptors, and growth factors. In certain embodiments, the CAR may have specificity for BCMA, 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, CD20, CD2, CD19, Her2, EGFR, EpCAM, FcyRIIIa (CD16), FcyRIIa (CD32a), FcyRIIb (CD32b), FcyRI (CD64), Toll-like receptors (TLR), TLR4, TLR9, cytokines, IL-2, IL-5, IL-13, IL-6, IL-17, IL-12, IL-23, TNFa, TGFb, cytokine receptors, IL-2R, chemokines, chemokine receptors, growth factors, VEGF, and HGF.

[0158] CAR-T cells may be used in combination with NEO-201 to treat any cancer, infectious disease, inflammation, or autoimmune condition for which the CAR-T cells find application.

[0159] NEO-201 in combination with CAR-NK cell therapy NEO-201 should also improve the efficacy of CAR-NK cell therapy because of its ability to eliminate gMDSC. 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 as they are in CAR-T cells. CAR-NK cells often have CD3 as the first signaling domain and CD28 or CD137 (4-1BB) as the costimulatory domain to form an intracellular signaling motif. NK cells further increase their cytotoxicity and cytokine production through two costimulatory molecules, namely NKG2D and CD244 (2B4). CAR-modified NK cells are often used to target cancer cells because they have more enhanced tumor-specific targeting and cytotoxicity than CAR-T cells.

[0160] CAR-NK cell therapy has advantageous characteristics (e.g., fewer safety concerns, lower cost, and higher tumorigenicity than CAR-T cells). Allogeneic haploidentical NK cells are safe for adoptive cell therapy (ACT) since they do not usually mediate and can reduce GVHD. CAR-NK cells also have significantly fewer safety concerns (e.g., on-target / off-tumor effects, CRS, and tumor lysis syndrome) than CAR-T cells. In addition, NK cells secrete only small amounts of IFN-γ and GM-CSF, and do not produce IL-1 and IL-6, which induce CRS. Secondly, tumor cells may not be detected by CAR-T cells due to tumor evasion, since either MHC class I expression or tumor-specific antigens are lost. CAR-NK cells lack self-antigens and retain their innate cytotoxicity against germline-encoded tumor / stress ligands, so they may detect MHC class I-negative tumor cells. Furthermore, both HLA-A and HLA-B bind to the KIR3D receptor, whereas HLA-C only binds to the KIR2D receptor. CD94-NKG2A, which detects HLA-E, LILRB1, and all MHC class I molecules, is another inhibitory receptor that distinguishes MHC class I molecules expressed by NK cells. Normal MHC class I-sufficient cells are ignored by NK cells because their inhibitory receptors can detect MHC class I molecules, but are not inhibited after interacting with abnormal MHC class I-low cells. Third, the low expression level of MHC class I in cancer stem cells (CSCs) and the presence of NKp30, NKp44, and NKG2D (activating receptors) are thought to cause cytokine-activated NK cell-mediated killing of CSCs. Fourth, CAR-NK cells can modulate activating receptors (e.g., NKp30, NKp44, NKp46, NKG2D, KIR-2DS, KIR-3DS, 2B4, CD226, CD94 / NKG2C, and DNAM-1), reducing the chance of relapse due to loss of CAR target antigens. Furthermore, while T lymphocytes only kill targets through CAR-specific mechanisms, NK cells exhibit spontaneous cytotoxic activity and can kill target cells regardless of the presence of tumor-specific antigens.Although tumor cells downregulate antigens to avoid immune detection, NK cells are still effective against tumor cells. Furthermore, the cytokines (e.g., IFN-γ, IL-3, and GM-CSF) produced by primary human NK cells are different from the proinflammatory cytokines released by T cells that induce CRS. Individual NK cells may persist after interacting with and destroying some target cells, potentially reducing the number of adoptively transferred cells. Fifth, the availability of off-the-shelf CAR-NK therapy would significantly enhance the pace of administration and minimize the lag time from the decision to treat, making the first dose a day. Sixth, CAR-NK therapy should reduce enormous overhead costs since CAR-NK infusions can be administered with follow-up monitoring in an outpatient setting, and would not require long-term hospitalization after treatment, as it is safer and free of potential toxicity. Furthermore, NK cells can be harvested from multiple sources (e.g., iPSC, PB, UCB, human embryonic stem cells, and NK cell lines). Similar to CAR-T cells, CAR-NK cell therapy has been used to treat hematological and solid tumors. CD19 (NCT02742727), CD7 (NCT02742727), and CD33 (NCT02944162) are the targets of CAR-NK cell therapy used in reported clinical studies on lymphoma and leukemia. Also, HER2 targeting GBM (NCT03383978) and costimulatory transducing receptor have been used to treat non-small cell lung cancer (NSCLC) (NCT03656705). CAR-NK cell therapy targeting mucin 1 (MUC1) for multiple refractory solid tumors (e.g., pancreatic tumors, HCC, NSCLC, and triple-negative invasive breast cancer) is also under investigation (NCT02839954).

[0161] CAR-NK cells can be used in combination with NEO-201 to treat any cancer, infectious disease, inflammation, or autoimmune condition for which CAR-T cells are used as described above. The CAR expressed by such NK cells can be specific for any of the antigens targeted by CAR-T cells. The CAR can also include any of the signaling domains, hinge domains, and other domains typically used in CARs expressed in CAR-T cells. Such domains and sequences used in CARs are generally known in the art and described above.

[0162] Monitoring / detection of MDSCs in patients In some embodiments, gMDSCs in a patient will be detected and monitored before, during, and after treatment, or after the patient is in remission. Such methods can help determine whether a patient would potentially benefit from NEO-201 treatment.

[0163] Methods for detecting and monitoring gMDSCs in patient samples are known in the art and are described in: U.S. Published Application No. 20210318310 by Gabrilovich; Dmitry I., published on October 14, 2021; U.S. Published Application No. 20170261507 by BANIYASH; Michal, published on September 14, 2017 (these applications are incorporated by reference in their entireties).

[0164] Methods for identifying and isolating gMDSC from a sample can include contacting the biological sample with a ligand (e.g., an antibody that recognizes a particular biomarker expressed on gMDSC). Such biomarkers include LOX-1, CD11b, CD15, and CD66b.

[0165] These methods may provide accurate enumeration or enrichment of the gMDSC cell population in a suitable biological sample of a subject.

[0166] In some embodiments, these methods of determining the exact cell number / concentration of gMDSC in a subject suffering from cancer or receiving treatment for cancer with NEO-201 alone or in combination with another therapeutic agent can be used to monitor the progression of cancer (with or without treatment).

[0167] In some embodiments, these methods of determining the number or concentration of gMDSCs in a subject with cancer may be used to determine whether NEO-201, alone or in combination with another therapeutic agent, may be beneficial in treating cancer.

[0168] In some embodiments, these methods of determining the number or concentration of gMDSCs within a tumor can be used to develop dosing regimens for NEO-201 alone or in combination with another therapeutic agent.

[0169] In some embodiments, the present disclosure provides a method for detecting gMDSC, and the level of gMDSC in a patient sample (e.g., blood or biopsy sample) is used to determine the prognosis of cancer before, during, or after treatment with NEO-201.For example, if gMDSC that reacts with NEO-201 cells is detected in the patient sample, the patient can be designated or administered an effective amount of NEO-201 to kill gMDSC.The method can include contacting the gMDSC with NEO-201 antibody.

[0170] The detection may involve cell sorting, optionally fluorescence-activated cell sorting, to generate a sample that is enriched and / or depleted of cells positive for NEO-201 antigen expression (e.g., gMDSCs).

[0171] In another aspect, the present disclosure provides a method for detecting gMDSCs, comprising contacting a cell with a NEO-201 antibody and detecting a cell expressing a NEO-201 target antigen. The NEO-201 antibody can be directly or indirectly labeled.

[0172] In another aspect, the present disclosure provides a method of staining gMDSCs, comprising contacting the cells with a NEO-201 antibody. The NEO-201 antibody can be directly or indirectly labeled.

[0173] In another aspect, the present disclosure provides a method for isolating or enriching MDSC, comprising isolating cells expressing NEO-201 target antigen. The method may comprise contacting a sample (e.g., a tumor biopsy sample containing gMDSC) with NEO-201 antibody, and optionally, the NEO-201 antibody is directly or indirectly labeled. The sample may also comprise blood or bone marrow. The method may comprise separating NEO-201 positive gMDSC from NEO-201 negative cells. The method may further comprise carrying out additional diagnostic assays on the cell sample to detect the expression of other MDSC biomarkers.

[0174] The gMDSCs can also be separated by cell sorting, optionally by fluorescence-activated cell sorting, based on expression of the NEO-201 target antigen and expression of other MDSC biomarkers.

[0175] The gMDSCs may be isolated by contacting the sample with a support comprising the NEO-201 antibody and / or by using other antibodies or ligands that recognize other MDSC biomarkers, thereby retaining the MDSCs on the support.

[0176] In another aspect, the present disclosure provides a method for detecting gMDSC, the method includes detecting the expression of NEO-201 target antigen by the MDSC, and optionally the level of gMDSC in a patient sample (e.g., blood or biopsy sample) is used to determine whether the patient is suffering from or likely to develop MDSC-mediated immunosuppression. Optionally, the method may further include allocating or administering NEO-201 treatment to the patient based on the detection of the gMDSC. For example, if gMDSC that reacts with NEO-201 and / or other biomarkers is detected in the patient sample, the patient may be designated to receive or may be administered an amount of NEO-201 that is effective for killing gMDSC. The method may include contacting the gMDSC with a NEO-201 antibody.

[0177] The detection may involve cell sorting, optionally fluorescence-activated cell sorting, to generate a sample that is enriched and / or depleted of cells positive for NEO-201 antigen expression (e.g., gMDSCs).

[0178] In another aspect, the present disclosure provides a method for detecting gMDSCs, comprising contacting a cell with a NEO-201 antibody and detecting a cell expressing a NEO-201 target antigen. The NEO-201 antibody can be directly or indirectly labeled.

[0179] In another aspect, the present disclosure provides a method of staining gMDSCs, comprising contacting the cells with a NEO-201 antibody. The NEO-201 antibody can be directly or indirectly labeled.

[0180] In another aspect, the present disclosure provides a method for isolating gMDSC, comprising isolating cells expressing NEO-201 target antigen and optionally other MDSC biomarkers. The method may comprise contacting a sample containing a cell sample (e.g., a tumor biopsy sample) with a NEO-201 antibody, and optionally, the NEO-201 antibody is directly or indirectly labeled. The sample may alternatively comprise a blood or bone marrow sample. The method may comprise separating NEO-201 positive gMDSC from NEO-201 negative cells. The method may further comprise performing further diagnostic assays on the putative gMDSC, for example, using a ligand that binds to other MDSC biomarkers.

[0181] The gMDSCs can be isolated by cell sorting (optionally, fluorescence-activated cell sorting) based on NEO-201 expression.

[0182] The gMDSCs may be isolated by contacting the sample with a support comprising a NEO-201 antibody, whereby the gMDSCs are retained on the support.

[0183] Cancer Vaccines The treatment method may further comprise administering a cancer vaccine to the patient.Exemplary cancer vaccines that can be administered are disclosed, for example, in:Fisher et al.,Immun Inflamm Dis.2017 Mar;5(1):16-28;Klages et al.,Cancer Res October 15 2010(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 incorporated herein by reference in its entirety).

[0184] In vitro depletion of gMDSCs using NEO-201 In another aspect, the present disclosure provides a method for killing gMDSC cells in vitro, comprising contacting the gMDSC cells with NEO-201 antibody.The method may further comprise contacting the gMDSC cells with complement.The gMDSC cells may be killed by CDC.The method may further comprise contacting the gMDSC with effector cells (e.g., natural killer cells).The gMDSC cells may be killed by ADCC.

[0185] In another aspect, the present disclosure provides a method for killing MDSCs ex vivo, comprising contacting a sample containing gMDSCs with an effective amount of NEO-201 antibody. The sample can be obtained from a patient. In some cases, the NEO-201 antibody can be conjugated to a cytotoxic moiety.

[0186] NEO-201 antibody sequence In any of the above or below methods, the NEO-201 antibody may comprise at least one, two, three, four, five, or preferably all six of the CDR sequences contained in SEQ ID NO:28 and SEQ ID NO:29.

[0187] In any of the above or below methods, the NEO-201 antibody may comprise a variable heavy chain sequence having at least 90% identity to SEQ ID NO:38.

[0188] In any of the above or below methods, the NEO-201 antibody may comprise a variable light chain sequence having at least 90% identity to SEQ ID NO:39.

[0189] In any of the above or below methods, the NEO-201 antibody may comprise a variable heavy chain sequence having at least 90% identity to SEQ ID NO:38 and a variable light chain sequence having at least 90% identity to SEQ ID NO:39.

[0190] In any of the above or below methods, the NEO-201 antibody can comprise a heavy chain sequence having at least 90% identity to amino acids 20 to 470 of SEQ ID NO:28 and a light chain sequence having at least 90% identity to amino acids 20 to 233 of SEQ ID NO:29.

[0191] In any of the above or below described methods, the NEO-201 antibody may comprise all six of the CDR sequences contained in SEQ ID NO:28 and SEQ ID NO:29.

[0192] In any of the above or below methods, the NEO-201 antibody can comprise a human IgG1 constant domain. Alternatively, the NEO-201 antibody can comprise a human IgG2, human IgG3, or human IgG4 constant domain, or a hybrid or chimeric domain comprising two or more of human IgG1, IgG2, IgG3, or IgG4.

[0193] In any of the above or below methods, the antibody comprises a NEO-201 antibody or a variant thereof (eg, one that contains the same CDRs and / or variable regions as NEO-201).

[0194] In any of the above or following methods, the NEO-201 antibody may be conjugated to another moiety.

[0195] In any of the methods described above or below, the NEO-201 antibody may be conjugated to another cytotoxic moiety, a label, a radioactive moiety, or an affinity tag.

[0196] In any of the above or following methods, the NEO-201 antibody may compete with the antibodies contained in SEQ ID NO:28 and SEQ ID NO:29 for binding to the NEO-201 antigen.

[0197] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used in the present invention or testing of the present invention, but suitable methods and materials are described herein. The materials, methods, and examples are for illustrative purposes only and are not intended to be limiting.

[0198] As used throughout this specification and the claims that follow, the meanings of "a," "an," and "the" include plural referents unless the context clearly indicates otherwise.

[0199] The term "amino acid" as used herein refers to natural amino acids, synthetic amino acids, and amino acid analogs and amino acid mimetics that function in a manner similar to natural amino acids. Natural amino acids are those encoded by the genetic code and those amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as natural amino acids, i.e., an α carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as natural amino acids. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid, but function similarly to a naturally occurring amino acid.

[0200] The term "NK depletion" or "natural killer depletion" as used herein refers to patients with low natural killer (NK) cell levels compared to the normal range. NK cells are cytotoxic innate immune lymphocytes. Typically, NK cells comprise 5-20% of peripheral blood mononuclear cells (PBMCs) in healthy individuals. A patient with NK cells comprising less than 5% of PMBCs is referred to as NK depleted. Additionally, if NK cells comprise less than 3% of PMBCs, the patient is referred to as severely NK cell depleted. Additionally, in normal individuals, up to 90% of PBMC NK cells are CD56 dim CD16 + NK cells, which are considered the most cytotoxic subset. Less than 70% of PBMC NK cells express CD56 dim CD16 + If it is NK cells, the patient is said to be NK depleted. In addition, less than 50% of PBMC NK cells express CD56 dim CD16 + If it is NK cells, the patient is referred to as severely NK depleted. A given patient may be referred to as NK depleted or severely NK depleted based on meeting either or both of these individual criteria. Generally speaking, the status of a patient as NK depleted or severely NK depleted is determined by examining a sample taken from the patient, such as a blood sample, for example, a sample obtained and examined 1 or 2 weeks ago. The status of a patient as NK depleted or severely NK depleted may also be inferred from the diagnosis of disease and / or course of treatment associated with such depletion of NK cells.

[0201] "Antibody" as used herein refers broadly to a polypeptide chain-containing molecular structure with a specific shape that fits and recognizes an epitope, and one or more non-covalent interactions stabilize the complex between the molecular structure and the epitope. A typical antibody molecule is an immunoglobulin, and all types of immunoglobulins (IgG, IgM, IgA, IgE, IgD) from all sources (e.g., human, rodent, rabbit, bovine, ovine, porcine, canine, chicken) are considered "antibodies." Antibodies include chimeric antibodies, human antibodies and other non-human mammalian antibodies, humanized antibodies, single chain antibodies (scFv), camelidbodies, nanobodies, IgNAR (single chain antibodies from sharks), small modular immunopharmaceuticals (SMIPs), and antibody fragments (e.g., Fab, Fab', F(ab')). 2 ), including, but not limited to, the antibody coding sequences described; others can be generated by methods well known in the art. See, for example, 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.

[0202] "NEO-201 antibody" refers to an antibody comprising the heavy and light chains or variable regions of SEQ ID NOs:28 and 29, and optionally the constant regions contained therein, as well as fragments and variants thereof. Such variants include sequences that comprise one, two, three, four, five, or preferably all six of the CDR sequences contained in SEQ ID NOs:28 and 29, i.e., heavy chain CDR1 of SEQ ID NO:32, heavy chain CDR2 of SEQ ID NO:33, heavy chain CDR3 of SEQ ID NO:34, light chain CDR1 of SEQ ID NO:35, light chain CDR2 of SEQ ID NO:36, and 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 antibody may be humanized. The antibody may be expressed with one or more leader sequences, which may be removed during expression and / or during antibody processing and secretion. The antibodies may be presented in a monovalent, bivalent, or higher polyvalent format, including, but not limited to, bispecific or multispecific antibodies comprising the NEO-201 antibody sequence and a binding fragment of a different antibody. Typically, the antibodies specifically bind to cancer cells and compete for binding to cancer cells with an antibody comprising a variable heavy chain of SEQ ID NO: 38 and a variable light chain of SEQ ID NO: 39, or comprising a heavy chain of SEQ ID NO: 28 and a light chain of SEQ ID NO: 29. One or more of the CDR sequences contained in SEQ ID NO: 28 and / or SEQ ID NO: 29 may be replaced with a variant sequence, e.g., 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 having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:38 and / or a variable light chain sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:39, and optionally the heavy and / or light chain sequences include one, two, three, four, five or preferably all six of the CDR sequences contained in SEQ ID NO:28 and SEQ ID NO:29, i.e., heavy chain CDR1 of SEQ ID NO:32, heavy chain CDR2 of SEQ ID NO:33, heavy chain CDR3 of SEQ ID NO:34, light chain CDR1 of SEQ ID NO:35, light chain CDR2 of SEQ ID NO:36 and 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.

[0203] "Antigen" as used herein broadly refers to a molecule or a portion of a molecule to which an antibody can bind, which can further induce an animal to produce an antibody capable of binding to the epitope of that antigen. An antigen may have one epitope or more than one epitope. The specific reaction referred to herein indicates that the antigen reacts in a highly selective manner with its corresponding antibody and not with the multitude of other antibodies that may be elicited by other antigens. An antigen may be tumor-specific (e.g., expressed by neoplastic cells of pancreatic and colon cancer).

[0204] "Cancer," as used herein, refers broadly to any neoplastic disease, whether invasive or metastatic, characterized by abnormal and uncontrolled cell division that gives rise to malignant growth or tumors.

[0205] As used herein, "cancer vaccine" refers to an immunogenic composition that induces or is intended to induce an immune response against cancer cells.

[0206] As used herein, a "chimeric antibody" refers broadly to an antibody molecule in which the constant region or a portion thereof has been altered, replaced or exchanged so that the antigen binding site (variable region) is conjugated to a constant region of a different or altered class, effector function and / or species, or to an entirely different molecule that confers new properties to the chimeric antibody, such as an enzyme, toxin, hormone, growth factor, drug, etc., or the variable region or a portion thereof has been altered, replaced or exchanged with a variable region having a different or altered antigen specificity.

[0207] "Conservatively modified variants" as used herein applies to both amino acid and nucleic acid sequences, and refers broadly to conservatively modified variants with respect to a particular nucleic acid sequence, and refers to nucleic acids that code for identical or essentially identical amino acid sequences, or, if the nucleic acid does not code for an amino acid sequence, refers to essentially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids code for any given protein. Such nucleic acid variations are "silent variations" and are one species of conservatively modified variations. All nucleic acid sequences herein that code for a polypeptide also represent all possible silent variations of the nucleic acid. Those skilled in the art will understand that each codon in a nucleic acid can be modified to produce a functionally identical molecule (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan).

[0208] As used herein, "complementarity determining region", "hypervariable region", or "CDR" refers broadly to one or more hypervariable or complementarity determining regions (CDRs) found in the variable region of an antibody light or heavy chain. See Kabat, et al. (1987) "Sequences of Proteins of Immunological Interest" National Institutes of Health, Bethesda, MD. These terms include the hypervariable regions defined by Kabat, et al. (1983) "Sequences of Proteins of Immunological Interest" USDept. of Health and Human Services or the hypervariable loops in 3-dimensional structures of antibodies. Included are the hypervariable regions defined by Chothia and Lesk (1987) J Mol. Biol. 196:901-917. The CDRs within each chain are held in close proximity by framework regions and, together with the CDRs of the other chain, contribute to the formation of the antigen binding site. Within the CDRs there are select amino acids described as selectivity determining regions (SDRs) which represent essential contact residues used by the CDRs in antibody-antigen interactions. Kashmiri (2005) Methods 36:25-34.

[0209] As used herein, "control amount" refers broadly to a marker that can be any amount or range of amounts compared to the test amount of the marker.For example, the 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 disease or condition.The control amount can be either an absolute amount (e.g., micrograms / ml) or a relative amount (e.g., the relative intensity of signals).

[0210] As used herein, "differentially present" broadly refers to the difference in the quantity 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 diseases.A nucleic acid fragment can be differentially present between two samples, for example, when the amount of the nucleic acid fragment in one sample is significantly different from the amount of the nucleic acid fragment in another sample, as measured, for example, by hybridization and / or NAT-based assay.A polypeptide is differentially present between two samples, when the amount of the polypeptide in one sample is significantly different from the amount of the polypeptide in another sample.It should be noted that if a marker is detectable in one sample but not detectable in the other sample, such a marker can be considered to be differentially present.Optionally, a relatively small amount of upregulation can serve as a marker.

[0211] "Diagnostic" as used herein refers broadly to identifying the presence or nature of a disease state. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the proportion of diseased individuals who test positive (proportion of "true positives"). Diseased individuals not detected by the assay are "false negatives." Non-diseased subjects who test negative are referred to as "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 those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.

[0212] As used herein, "diagnosing" broadly refers to classifying a disease or condition, determining the severity of a disease, monitoring the progression of a disease, and predicting the outcome and / or prognosis of recovery from a disease. The term "detection" may optionally encompass any of the above. Diagnosis of a disease according to the present invention may, in some embodiments, be effected by determining the level of a polynucleotide or polypeptide of the present invention in a biological sample obtained from a subject, and the determined level may correlate with a predisposition to a disease, or the presence or absence of a 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.

[0213] An "effective amount" as used herein broadly refers to an amount of a compound, antibody, antigen, or cell that achieves a desired result. An "effective amount" is sufficient when administered to a patient to treat a disease to effect such treatment for the disease. An effective amount can be a prophylactically effective amount and / or a preventively effective amount. An effective amount can be an amount effective to reduce, prevent the onset of signs / symptoms, reduce the severity of the onset of signs / symptoms, eliminate the onset of signs / symptoms, slow the progression of the onset of signs / symptoms, prevent the progression of the onset of signs / symptoms, and / or an effective amount effective in preventing the onset of signs / symptoms. An "effective amount" can vary depending on the disease and its severity, as well as the age, weight, medical history, susceptibility, and pre-existing conditions of the patient being treated. The term "effective amount" is synonymous with "therapeutically effective amount" for purposes of this disclosure.

[0214] "Expression vector" as used herein broadly refers to any recombinant expression system intended to constitutively or inducibly express the nucleic acid sequences 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 episomal or are integrated into the genome of a host cell. The expression system may or may not have the ability for autonomous replication, i.e., it may drive only transient expression in the cell. The term includes recombinant expression cassettes that contain only the minimum elements required for transcription of the recombinant nucleic acid.

[0215] As used herein, "framework region" or "FR" refers broadly to one or more framework regions in the light and heavy chain variable regions of an antibody. See Kabat, et al. (1987) "Sequences of Proteins of Immunological Interest" National Institutes of Health, Bethesda, MD. These terms include the amino acid sequence regions interposed between the CDRs in the light and heavy chain variable regions of an antibody.

[0216] "Hematologic malignancies" refers to forms of cancer that arise in hematopoietic tissues (e.g., bone marrow) or cells of the immune system. Examples of hematologic malignancies include leukemia, lymphoma, multiple myeloma, and myelodysplastic syndromes (MDS). More specific examples of hematologic malignancies include, but are not limited to, marginal zone lymphoma (MZL) (e.g., splenic marginal zone lymphoma (SMZL)), Burkitt's lymphoma (BL), multiple myeloma (MM) (e.g., plasma cell leukemia (PCL) and extramedullary myeloma disease (EMD)), myelodysplastic syndromes (MDS), acute myeloid leukemia (AML) (e.g., B-cell AML), acute lymphoblastic leukemia (ALL), T-cell lymphoma (TCL) (e.g., anaplastic large cell lymphoma (ALCL) and Sézary syndrome), and Hodgkin's lymphoma (HL).

[0217] "Heterologous" as used herein refers broadly to a portion of a nucleic acid and indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, a nucleic acid is typically produced recombinantly and has two or more sequences from unrelated genes arranged to create a new functional nucleic acid, such as 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).

[0218] As used herein, "high affinity" refers to an affinity of at least 10 for a target antigen. -8 M, more preferably at least 10 -9 M, and even more preferably at least 10 -10 The term "high affinity" broadly refers to an antibody having a KD of at least 10 M. However, "high affinity" binding may vary for other antibody isotypes. For example, "high affinity" binding for an IgM isotype is at least 10 -7 M, more preferably at least 10 -8 It refers to an antibody having a KD of M.

[0219] "Homology" as used herein broadly refers 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 or amino acid sequence is identical. A partially homologous nucleic acid or amino acid sequence is one that is not identical to the reference nucleic acid or amino acid sequence. The degree of homology can be determined by sequence comparison. The term "sequence identity" can be used interchangeably with "homology".

[0220] As used herein, a "host cell" refers broadly to a cell that contains an expression vector and supports the replication or expression of the expression vector. Host cells can be prokaryotic (e.g., E. coli) or eukaryotic (e.g., yeast, insect (e.g., SF9), amphibian, or mammalian cells (e.g., CHO, HeLa, HEK-293)), including cultured cells, tissue explants, and in vivo cells.

[0221] As used herein, "hybridization" refers broadly to the physical interaction of complementary (including partially complementary) polynucleotide strands through the formation of hydrogen bonds between complementary nucleotides when the strands are positioned antiparallel to one another.

[0222] As used herein, "K-assoc" or "Ka" broadly refers to the association rate of a particular antibody-antigen interaction, while the term "Kdiss" or "Kd" as used herein refers to the dissociation rate of a particular antibody-antigen interaction. As used herein, the term "KD" refers to the dissociation rate obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration (M). KD values ​​for antibodies can be determined using methods well established in the art.

[0223] As used herein, "immunoassay" refers broadly to an assay that uses an antibody to specifically bind an antigen. Immunoassays can be characterized by the use of the specific binding properties of a particular antibody to isolate, target, and / or quantitate the antigen.

[0224] As used herein, "isolated" refers broadly to a material that is removed from its original environment in which it naturally occurs and thus modified by the hand of man from its natural environment. An isolated material can be, for example, an exogenous nucleic acid contained in a vector system, an exogenous nucleic acid contained in a host cell, or any material that is removed from its original environment and thus modified by the hand of man (e.g., an "isolated antibody").

[0225] As used herein, a "label" or a "detectable moiety" refers broadly to a composition detectable by microscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.

[0226] As used herein, "low stringency conditions", "moderate stringency conditions", "high stringency conditions", or "very high stringency conditions" refer broadly to nucleic acid hybridization and washing conditions. Guidance for performing hybridization reactions can be found in: Ausubel, et al. (2002) "Short Protocols in Molecular Biology", (5 th Ed.) John Wiley & Sons, NY. Exemplary specific hybridization conditions include, but are not limited to, the following: (1) low stringency hybridization conditions 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. (for low stringency conditions, the wash temperature can be increased to 55° C.); (2) medium stringency hybridization conditions in 6X SSC at about 45° C. (3) high stringency hybridization conditions at about 45° C. in 6× SSC, followed by one or more washes in 0.2× SSC, 0.1% SDS at 65° C.; and (4) very high stringency hybridization conditions are 0.5 M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2× SSC, 1% SDS at 65° C.

[0227] The term "low level" or "low" as used with respect to a marker such as CD127 is well known in the art and refers to the expression level of a cell marker of interest (e.g., CD127) that is low compared to the expression level of that cell marker in other cells in the cell population being analyzed as a whole. More specifically, the term "low" refers to a distinct cell population that expresses a cell marker at a lower level than one or more other distinct cell populations. Thus, CD127 低 refers to a cell type that stains faintly or dimly when contacted with a labeled CD127 antibody, for example, at a level greater than that of the CD127- subpopulation but less than that of the CD127+ subpopulation.

[0228] "Mammal" as used herein broadly refers to any and all warm-blooded vertebrates of the class Mammalia, including humans, characterized by a hairy covering of the skin and, in females, milk-producing mammary glands for feeding their young. Examples of mammals include, but are not limited to, alpacas, armadillos, capybaras, cats, camels, chimpanzees, chinchillas, cows, 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, bovines, canines, equines, felines, murines, ovines, porcines, primates, and rodents. Mammals also include any and all of those listed in the "Mammalian Species of the World" maintained by the Smithsonian National Museum of Natural History in Washington, DC.

[0229] "Myeloid-derived suppressor cells" or "MDSCs" are a heterogeneous group of immune cells of the myeloid lineage (a family of cells derived from bone marrow stem cells). MDSCs undergo extensive expansion in pathological conditions (e.g., chronic infection and cancer) as a result of altered hematopoiesis. MDSCs are distinct from other myeloid cell types in that they possess potent immunosuppressive activity rather than immunostimulatory properties. Like other myeloid cells, MDSCs interact with other immune cell types (e.g., T cells, dendritic cells, macrophages, and natural killer cells) to regulate their function.Clinical evidence indicates that cancer tissues with high MDSC infiltration are associated with poor patient prognosis and resistance to treatment (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,PMC 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”,Nature Reviews.Immunology.12(4):253-68.doi:10.1038 / nri3175.PMC 3587148.PMID 22437938).

[0230] MDSCs consist of two major cell populations: granulocytic or polymorphonuclear cells (PMN-MDSC or gMDSC) and monocytic cells (M-MDSC). PMN-MDSC or gMDSC are phenotypically and morphologically similar to neutrophils, whereas M-MDSC 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 population of MDSCs, represented by cells with colony-forming activity and other myeloid progenitor cells, has also been reported; these are called early MDSCs (eMDSCs) (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).

[0231] As used herein, "nucleic acid" or "nucleic acid sequence" broadly refers to deoxyribonucleotide or ribonucleotide oligonucleotides in single-stranded or double-stranded form. The term encompasses nucleic acids (i.e., oligonucleotides) that contain known analogs of natural nucleotides. The term also encompasses nucleic acid-like structures with synthetic backbones. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

[0232] As used herein, "operably linked" refers broadly to when two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.

[0233] As used herein, "paratope" refers broadly to the portion of an antibody that recognizes an antigen (e.g., the antigen-binding site of an antibody). The paratope may be a small region (e.g., 15-22 amino acids) of the Fv region of an antibody, or may contain parts of the heavy and light chains of an antibody. See Goldsby, et al. Antigens (Chapter 3) Immunology (5 th Ed.) New York: WH Freeman and Company, pages 57-75.

[0234] As used herein, a "patient" refers broadly to any animal in need of treatment to alleviate a medical condition or to prevent the occurrence or recurrence of a medical condition. Also, as used herein, a "patient" refers broadly to any animal that has risk factors, medical history, 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 may be a clinical patient (e.g., a human) or a veterinary patient (e.g., a pet, a livestock animal, a domestic animal, an exotic animal, or a zoo animal. The term "subject" may be used interchangeably with the term "patient". In a preferred embodiment of the invention disclosed herein, the patient is a human.

[0235] The terms "polypeptide," "peptide," and "protein" are used interchangeably and refer broadly to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are analogs or mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to non-naturally occurring amino acid polymers. Polypeptides can be modified, for example, by the addition of carbohydrate residues to form glycoproteins. The terms "polypeptide," "peptide," and "protein" include glycoproteins and non-glycoproteins.

[0236] A "promoter" as used herein broadly refers to a set of nucleic acid sequences that direct the transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter can also optionally include distal enhancer or repressor elements that can be located as far away as several thousand bases from the start site of transcription. 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 and developmental control.

[0237] As used herein, a "prophylactically effective amount" refers broadly to an amount of a compound that, when administered to a patient for the prevention of a disease or prevention of recurrence of a disease, is sufficient to effect 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, predisposition to the condition, and pre-existing conditions of the patient being treated.

[0238] "Prevention," as used herein, broadly refers to a course of treatment during which signs and / or symptoms are absent, in remission, or previously present in a patient. Prevention includes preventing a disease from occurring in a patient following treatment of the disease. Additionally, prevention includes treating patients who may potentially develop a disease, particularly those susceptible to a disease (e.g., members of a patient population, those with risk factors, or at risk of developing a disease).

[0239] "Recombinant" as used herein refers broadly to a product, e.g., a cell, or a nucleic acid, protein, or vector, and indicates that the cell, nucleic acid, protein, or vector is derived from the introduction of a heterologous or naturally occurring nucleic acid or protein, or that the cell has been so modified. Thus, for example, a recombinant cell expresses genes that are not found within the native (non-recombinant) form of the cell, or expresses otherwise abnormally expressed, poorly expressed, or not at all native genes.

[0240] As used herein, "specifically (or selectively) binds" or "specifically (or selectively) immunoreacts" or "specifically interacts with or binds to" an antibody refers broadly to a protein or peptide (or other epitope) and, in some embodiments, to a binding reaction that determines the presence of a protein in a heterogeneous population of proteins and other biologics. For example, under specified immunoassay conditions, a specified antibody binds to a particular protein at least twice as much as background (non-specific signal) and does not bind substantially in significant amounts to other proteins present in the sample. Typically, a specific or selective reaction is at least twice the background signal or noise, and more typically about 10-100 times more background.

[0241] As used herein, "specifically hybridizable" and "complementary" broadly refer to a nucleic acid being able to form hydrogen bond(s) with another nucleic acid sequence, either by conventional Watson-Crick or other unconventional types. The binding free energy of a nucleic acid molecule and its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, such as RNAi activity. Measurement of the binding free energy of a nucleic acid molecule is well known in the art. 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. Percent complementarity refers to 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 have at least about 50%, 60%, 70%, 80%, 90%, and 100% complementarity). "Fully complementary" or 100% complementarity refers broadly to all of the contiguous residues of a nucleic acid sequence that hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. "Substantial complementarity" refers to polynucleotide strands that exhibit at least about 90% complementarity, except for regions of the polynucleotide strands that are selected to be non-complementary (e.g., 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 desired, i.e., under physiological conditions for in vivo assays or therapeutic treatments or in vitro assays, under conditions where the assay is performed. The non-target sequences will usually differ by at least 5 nucleotides.

[0242] As used herein, a "sign" of disease refers broadly to any abnormality detectable on examination of a patient that is indicative of disease, as opposed to a symptom, which is a subjective indication of disease.

[0243] As used herein, "solid support," "support," and "substrate" refer broadly to any material that provides a solid or semi-solid structure to which another material can be attached, including, but not limited to, smooth supports (e.g., metal, glass, plastic, silicon, and ceramic surfaces), as well as rough and porous materials. Exemplary solid supports include beads, such as activated beads, magnetically responsive beads, or fluorescently labeled beads.

[0244] "Subject" as used herein refers broadly to any person suitable for treatment according to the invention disclosed herein, including but not limited to avian and mammalian subjects, and preferably mammalian. Mammals in the context of the invention disclosed herein include, but are not limited to, canines, felines, bovines, caprines, equines, ovines, porcines, rodents (e.g., rats and mice), lagomorphs, primates, and humans. Any mammalian subject in need of treatment according to the invention disclosed herein is suitable. Human subjects of both genders and at any stage of development (i.e., neonates, infants, juveniles, adolescents, adults) can be treated according to the invention. The invention can also be practiced on animal subjects (particularly mammalian subjects, e.g., mice, rats, dogs, cats, cows, goats, sheep, and horses) for veterinary purposes, and for drug screening and drug development purposes. "Subject" is used interchangeably with "patient". In a preferred embodiment of the disclosed invention, the subject is a human.

[0245] A "symptom" of disease, as used herein, refers broadly to any morbid phenomenon or departure from the normal in structure, function, or sensation experienced by a patient and that is indicative of disease.

[0246] As used herein, "therapeutic", "therapeutic", "treat" or "treatment" broadly refers to treating a disease, arresting 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. Therapy includes prevention, treatment, repair, reduction, relief, and / or providing relief of a disease, signs and / or symptoms of a disease. Therapy includes the reduction of signs and / or symptoms in patients with ongoing signs and / or symptoms of a disease (e.g., tumor growth, metastasis). Treatment also includes "prevention". The term "reduced" broadly refers to a clinically significant reduction of signs and / or symptoms for purposes of treatment. Therapy includes treating signs of relapsing or recurrent signs and / or symptoms (e.g., tumor growth, metastasis). Therapy includes, but is not limited to, eliminating signs and / or symptoms at any time, as well as reducing existing signs and / or symptoms, and eliminating existing signs and / or symptoms. Therapy includes treatment of chronic diseases ("maintenance") and acute diseases. For example, treatment includes treating or preventing the recurrence or recurrence of signs and / or symptoms (eg, tumor growth, metastasis).

[0247] As used herein, "variable region" or "VR" refers broadly to the domains within each pair of light and heavy chains of an antibody that are directly involved in binding the antibody to an antigen. Each heavy chain contains a variable domain (VR) at one end. H ), followed by several constant domains. Each light chain has at one end a variable domain (V L ) at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain and the variable domain of the light chain is aligned with the variable domain of the heavy chain.

[0248] As used herein, a "vector" refers broadly to a plasmid, cosmid, phagemid, phage DNA, or other DNA molecule capable of autonomously replicating in a host cell and characterized by one or a small number of restriction endonuclease recognition sites, such DNA sequences may be cleaved in a determinable manner without loss of the essential biological function of the vector, and into which DNA may be inserted to effect its replication and cloning. A vector may further contain a marker suitable for use in identifying cells transformed with the vector.

[0249] Techniques and procedures 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 [3 rd [Ed] Cold Spring Harbor Laboratory Press. Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The nomenclature utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients. EXAMPLES

[0250] Having now generally described the invention, the invention will be more readily understood by reference to the following examples, which are included solely for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to be limiting of the invention.

[0251] Example 1: NEO-201mAb targets and can be used for the depletion of human granulocytic myeloid-derived suppressor cells.

[0252] Background Myeloid-derived suppressor cells Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that increase in cancer, inflammation, and infection. Myeloid-derived suppressor cells are associated with cancer evasion and 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 Suppl2:e12989.). Human MDSCs express myeloid cell markers (e.g., CD11b positive and CD33 positive) but are usually negative for HLA-DR, CD3, CD19, and CD57. Monocytic MDSCs usually have an HLA-DR negative, CD11b positive, CD33 positive, and CD14 positive phenotype. Granulocytic MDSCs are usually characterized by an 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).

[0253] As described herein above, clinical studies have demonstrated the prognostic role of tumor-infiltrating neutrophils, elevated blood neutrophils, and elevated blood neutrophil / lymphocyte ratios, in particular, that such cells are associated with poor clinical outcomes in a variety of human cancers. These results highlight the importance and relevance of neutrophils in cancer biology.

[0254] Neutrophils are the primary inflammatory cells and are essential for protecting the host against invading pathogens (e.g., bacteria and fungi). Recently, neutrophils have shown high functional plasticity and can assume pro- and anti-tumor functions. Pro-tumorigenic neutrophils function as suppressors of adaptive immune responses in cancer. Proliferation of immature and mature neutrophils has been observed to suppress T-cell proliferation. Pro-tumorigenic neutrophils are functionally associated with gMDSCs. Myeloid-derived suppressor cells play an important role in suppressing the host immune response, for example, through several mechanisms: (a) production of arginase 1, (b) release of reactive oxygen species (ROS), (c) release of nitric oxide, and (d) secretion of inhibitory cytokines (Donskov F.et al., “Immunomonitoring and prognostic relevance of neutrophils in clinical trials”, Seminars in Cancer Biology 2013;23:200-207l; Sagiv J et al., “Phenotypic diversity and plasticity in circulating neutrophil subpopulations in cancer”, Cell Reports 2015;10:562-573).

[0255] Granulocytes are derived from hematopoietic stem cells in the bone marrow that are regulated by granulocyte colony-stimulating factor (G-CSF). Under pathological conditions, MDSCs can be generated in the bone marrow in response to cancer and infection-inducing factors (e.g., G-CSF, GM-CSF, IL-6, IL-1-beta, prostaglandin E2 (PGE2), TNF-alpha, and VEGF) (Lechner MG, et al., “Characterization of cytokine-induced myeloid-derived suppressor cells from normal peripheral blood mononuclear cells”, J Immunol 2010;185:2273-2284).

[0256] NEO-201 monoclonal antibody 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 may participate in ADCC and CDC to kill tumor cells. Previous studies have demonstrated that NEO-201 attenuates the growth of human tumor xenografts in mice and is safe / tolerable in non-human primates, with a transient reduction in neutrophils being the only side effect observed. First-in-human clinical trials evaluating NEO-201 in adults with chemotherapy-resistant solid tumors are ongoing at the NIH Clinical Center (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).

[0257] NEO-201 recognizes tumor-associated variants of CEACAM5 and 6 that have core 1 and / or extended core 1 O-glycans. CEACAM1 is a potent inhibitor of natural killer (NK) cell function; binding of CEACAM1 on NK cells to CEACAM1 or CEACAM5 on tumor cells inhibits activation signaling by NKG2D, which prevents NK cell cytolysis and allows tumor cells to avoid killing by NK (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).

[0258] Materials and Methods In vitro generation of human gMDSCs The EasySep™ Direct Human Neutrophil Isolation Kit (STEMCELL, Cat. No. 19257) was used for immunomagnetic isolation of neutrophils directly from whole blood of healthy donors according to the manufacturer's protocol.

[0259] The isolated neutrophils were diluted to 5 × 10 5 The cells were cultured in complete RPMI 1640 medium at a concentration of 1000 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. The cells cultured for 7 days were collected for flow cytometry analysis of cell phenotype.

[0260] Phenotypic analysis by flow cytometry The phenotype of in vitro generated gMDSCs was evaluated for expression of HLA-DR, CD33, CD66b, CD14, CD15 and NEO-201 target antigens by flow cytometry as follows (Lechner MG et al., “Characterization of cytokine-induced myeloid-derived suppressor cells from normal peripheral blood mononuclear cells”, J Immunol 2010;185:2273-2284). First, to discriminate between live and dead cells, gMDSCs were incubated with 1 μL of LIVE / DEAD Fixable Aqua (Thermo Fisher Scientific, Waltham, MA, USA) per test in 1× phosphate-buffered saline (PBS) (1 mL) (VWR International, Radnor, PA, USA) for 30 min at 4°C. Cells were then washed with 1X PBS and incubated with 2-5 μL of HumanTruStain FcX™ (BioLegend, San Diego, CA, USA) in 100 μL of 1X PBS for 5-10 min at room temperature. To detect surface markers, cells were then stained with 2-4 μL / sample of the following anti-human mAbs in 100 μL of 1X PBS + 1% BSA (Teknova, Hollister, CA, USA) for 30 min 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, cells were washed twice with cold 1X PBS and examined using a FACSVerse flow cytometer (BD Biosciences, San Jose, CA, USA). Analysis of cell fluorescence was performed using BD FACSuite software (BD Biosciences, San Jose, CA, USA). Fluorescence minus 1 controls were used to determine positivity.

[0261] ADCC assay For analysis of NEO-201-mediated ADCC activity against gMDSCs, flow cytometry was used. ADCC assays were performed essentially as described by 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).

[0262] In the ADCC assay, gMDSCs generated from neutrophils of two healthy donors were used as target cells. After 7 days of culture in complete RPMI1640 medium supplemented with human IL-6 and GM-CSF, the generated gMDSCs were harvested and centrifuged at 1500 rpm for 5 min. The supernatant was then discarded and the pellet was washed with 1X PBS. Next, to discriminate between live and dead cells, the cells were incubated with 1 μL of LIVE / DEAD Fixable Aqua per test in 1X PBS (1 mL) for 30 min at 4 °C. Next, the cells were washed with 1X PBS and stained with 2-4 μL / sample of anti-human mAb (HLA-DR-PE, CD33-APC) in 100 μL of 1X PBS + 1% BSA for 30 min at 4 °C. After staining, the cells were washed twice with cold 1X PBS.

[0263] On the day of the ADCC assay, PBMCs from another healthy donor were thawed and cultured in RPMI complete medium. PBMCs were used as effector cells and added to tubes containing gMDSCs stained with both HLA-DR-PE and CD33-APC antibodies at effector:target (E:T) ratios of 100:1 and 50:1 with or without NEO-201 (10 μg / mL). In these experiments, gMDSCs treated with medium alone were used as controls.

[0264] The cells were then incubated for 4 hours at 37°C. After incubation, the cells were washed twice with cold 1X PBS and examined using a FACSVerse flow cytometer (BD Biosciences, San Jose, CA, USA). Analysis of cell fluorescence was performed using BD FACSuite software (BD Biosciences, San Jose, CA, USA). Fluorescence minus 1 controls were used to determine positivity. To evaluate NEO-201-mediated ADCC activity, CD33 in gMDSCs incubated with medium alone was measured. 陽性 / HLA-DR 陰性 The percentage of live cells was determined by comparing CD33 cells incubated with PBMC alone and with PBMC and NEO-201. 陽性 / HLA-DR 陰性 The percentage of viable cells was compared.

[0265] result Phenotypic analysis of gMDSCs generated from human neutrophils In this study, whole blood from four normal donors was used. As shown in the table in Figure 10, 47.59-52.58% 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%-88.09% of the HLA-DR negative and CD33 positive population were CD15 positive and CD14 negative. 66.44%-99.71% of the HLA-DR negative / CD33 positive / CD15 positive / CD14 negative population were CD66 positive and NEO-201 positive.

[0266] Figures 6-9 contain the flow cytometry analysis results of gMDSCs generated from GM-CSF and IL-6 treated neutrophils taken from four normal donors.

[0267] ADCC assay results To evaluate whether NEO-201 can eliminate human gMDSCs via ADCC, gMDSCs generated from neutrophils of two healthy donors have been used as target cells in an ADCC assay performed by flow cytometry. The ADCC activity of NEO-201 was evaluated by detecting CD33 expression in gMDSCs. 陽性 / HLA-DR 陰性 The percentage of live cells was determined by comparing CD33 cells incubated with PBMC alone and with PBMC and NEO-201. 陽性 / HLA-DR 陰性 The percentage of viable cells was compared.

[0268] As shown in FIG. 11, when gMDSCs were incubated with PBMCs (E:T 100:1) and NEO-201, CD33 expression was significantly increased compared to gMDSCs incubated with PBMCs (E:T 100:1) alone in healthy donors 1 and 2, respectively. 陽性 / HLA-DR 陰性 We observed a reduction in viable cells of 33.01% (18.29% vs. 27.23%) and 29.5% (25.95% vs. 36.83%). In both healthy donors, CD33 陽性 / HLA-DR 陰性 A similar reduction in viable cells was observed.

[0269] These data provide compelling evidence that NEO-201 can deplete or eliminate gMDSCs via ADCC-mediated lysis in vitro. Based on these results, we expect that NEO-201 should be useful alone or in combination with other active agents (e.g., antibodies targeting checkpoint inhibitors and other biologics or chemotherapeutic drugs to reduce the immunosuppression and treatment resistance caused by MDSCs). In particular, NEO-201 can be used to reduce the immunosuppression and treatment resistance of individuals suffering from cancer and chronic conditions that involve MDSC-mediated immunosuppression and treatment resistance.

[0270] Example 2: Reduction in the percentage of granulocytic myeloid-derived suppressor cells (gMDSC) in peripheral blood mononuclear cells (PBMCs) following treatment with NEO-201 and pembrolizumab

[0271] Materials and Methods Phenotypic analysis of gMDSCs in PBMCs from cancer patients by flow cytometry To determine whether NEO-201 can bind and deplete gMDSCs in cancer patients, PBMCs from four patients were profiled by flow cytometry for expression of specific gMDSC markers (e.g., HLA-DR, CD33, CD66b, CD14, CD15, and NEO-201). To discriminate between live and dead cells, PBMCs were frozen and first incubated with 1 μL of LIVE / DEAD Fixable Aqua (Thermo Fisher Scientific, Waltham, MA, USA) per test in 1× phosphate-buffered saline (PBS) (1 mL) (VWR International, Radnor, PA, USA) for 30 min at 4°C. Cells were then washed with 1× PBS and incubated with 2–5 μL of HumanTruStain FcX™ (BioLegend, San Diego, CA, USA) in 100 μL of 1× PBS for 5–10 min at room temperature. Next, for gMDSC markers, cells were stained with 2–4 μL / sample of the following anti-human mAbs in 100 μL of 1X PBS + 1% BSA (Teknova, Hollister, CA, USA) for 30 min 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, cells were washed twice with cold 1X PBS and examined using a FACSVerse flow cytometer (BD Biosciences, San Jose, CA, USA). Analysis of cell fluorescence was performed using BD FACSuite software (BD Biosciences, San Jose, CA, USA). Fluorescence minus 1 controls were used to determine positivity.

[0272] result Phenotypic analysis of gMDSCs derived from PBMCs of cancer patients To evaluate whether treatment with NEO-201 affected the proportion of circulating gMDSCs in cancer patients, the study used 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 previous checkpoint inhibitor therapy (clinical trial NCT03476681).

[0273] Each treatment cycle was 42 days long and consisted of three doses of 1.5 mg / kg NEO-201 IV every 2 weeks and one dose of 400 mg IV pembrolizumab every 6 weeks.

[0274] Radiological assessment (e.g., CT, MRI, or PET-CT) was performed before the first infusion and then repeated every 2 cycles (every 84 days) to correlate clinical response with modulation of immune cell proportions and function, including gMDSC.

[0275] In four cancer patients, the percentage of gMDSCs in PBMCs was analyzed before the start of 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).

[0276] The gMDSC population within viable PBMCs expresses HLA-DR 陰性 / CD33 + / CD15+ / CD14 negative / CD66b + was defined as a cell.

[0277] The results are contained in FIG. 12, which shows a comparison of the percentage of circulating gMDSC (HLA-DR- / CD33+ / CD15+ / CD14- / CD66b+ cells) in two patients with stable stage (SD) and two patients with progressive stage (PD) cancer at different time points by flow cytometry analysis. gMDSC were gated from live PBMCs. Data are presented as the median percentage of viable cells expressing gMDSC markers. Fluorescence minus 1 control was used to determine positivity. "HNSCC" in the figure refers to "head and neck squamous cell carcinoma."

[0278] As shown in FIG. 12, one 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 compared to baseline levels (C3D1 PRE) (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 with NEO-201 and pembrolizumab. The patient is undergoing further treatment.

[0279] Further, as shown in Figure 12, another SD patient (cervical cancer patient) showed an increase in circulating gMDSCs at C1D15 and C2D1 PRE 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 relative to baseline levels at C3D1 (C3D1 PRE: 0.16% vs. 0.11%). This patient showed SD after more than 8 months of treatment after the first infusion with NEO-201 and pembrolizumab, suggesting that the decrease in circulating gMDSCs initiated at C3D1 PRE may continue beyond this time point and contribute to disease stabilization.

[0280] Conversely, as further shown in Figure 12, one patient with uterine cancer (patient 4) showed an increased proportion of circulating gMDSCs after treatment, a phenomenon that correlates with disease progression (PD) reported at first restaging (before C3D1).

[0281] As also shown in Figure 12, another uterine cancer patient (Patient 5) who showed PD on first restaging was found to have an initial increase at C1D15 and was subsequently observed to have a 60% reduction in circulating gMDSCs at C2D1 versus baseline (0.052% vs. 0.13%), suggesting that the observed reduction in circulating gMDSCs, which is highly clinically significant, may not be the only factor responsible for the clinical response.

[0282] These clinical results provide compelling evidence that the NEO-201 antibody, when administered alone or in conjunction with other treatments, can be used to deplete gMDSCs in patients in need thereof (e.g., adult cancer patients with chemotherapy-resistant solid tumors and / or who have failed previous checkpoint inhibitor therapy), and that such treatment can reverse or significantly reduce gMDSC-associated resistance or recalcitrance to chemotherapy and / or checkpoint inhibitor therapy.

[0283] Humanized NEO-201 monoclonal antibody sequence The sequence of the NEO-201 antibody used in these examples is as follows: [ka]

[0284] The boundaries between the expression leader sequence, variable region, and constant region are separated by a forward slash (" / ") within each sequence, and the CDR sequences are indicated in bold, underlined text. The antibody sequences used contained the variable and constant regions shown. These include 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.

[0285] Each document cited herein is incorporated herein by reference in its entirety.

Claims

1. An antibody or antibody fragment that binds to glycosylated CEACAM5 and CEACAM6 having core type 1 and / or extended core type 1 O-glycan, but does not bind to aglycosylated CEACAM5 or aglycosylated CEACAM6, and is optionally NEO-201 or its binding fragment, for the following uses: (i) Killing or removing granulocyte myeloid suppressor cells (gMDSCs) in patients for whom killing or removal of such granulocyte myeloid suppressor cells (gMDSCs) is necessary; (ii) Reducing resistance and / or restoring innate immunity (e.g., innate antitumor immunity or innate anti-infective immunity) in a patient who requires the reduction of resistance and / or the restoration of innate immunity (e.g., innate antitumor immunity or innate anti-infective immunity) by killing or removing the patient's granulocyte myeloid suppressor cells (gMDSCs); (iii) Reducing resistance or tolerance to treatment with anticancer drugs or anti-infective agents (e.g., immunomodulatory antibodies, checkpoint inhibitor antibodies, or fusion proteins, or chemotherapeutic agents), provided that such resistance or tolerance is associated with granulocyte myeloid suppressor cells; or To treat or prevent the recurrence of cancer or infection by suppressing the proliferation of (iv) gMDSCs, thereby re-establishing innate immunity.

2. The antibody or antibody fragment according to claim 1, wherein the following: (i) The antibody or antibody fragment recognizes an O-glycosylated epitope that binds to threonine in the amino acid region 310-318 of CEACAM5 (RTTVTTTITV) and to threonine and serine in the amino acid region 312-320 of CEACAM6 (TVTMITVSG); (ii) Use (iii) described in claim 1 further comprises the use of an immunomodulatory antibody, a checkpoint inhibitor antibody or fusion protein, or a chemotherapeutic agent to reduce the resistance or tolerance; (iii) Use (iv) described in claim 1 further comprises the use of an immunomodulatory antibody, a checkpoint inhibitor antibody or fusion protein, or a chemotherapeutic agent; (iv) The gMDSC expresses an O-glycan selected from one or more O-glycans 01, 02, 06, 023, 026, and 039 having the structure shown in the sequence in Figures 2 and 5; (v) The gMDSC expresses an O-glycan of 06, 01, or 02 having the structure shown in the sequence in Figure 2 and / or Figure 5; (vi) The gMDSC expresses the O-glycan of 06 shown in the sequence in Figure 2 and / or Figure 5; (vii) The gMDSC expresses a Tn antigen or a core type 1, core type 2, core type 4, or core type 4 O-glycan having the structure shown in Figure 1; (viiii) The patient has cancer or an infectious disease, and the immunosuppression of the disease pathology and / or the innate immunity to the disease is involved in gMDSC; (ix) The antibody or antigen-binding fragment is directly or indirectly bound to a cytotoxic agent, optionally a radionuclide or chemotherapeutic agent; (x) The antibody or antigen-binding fragment is directly or indirectly conjugated to a label, optionally a fluorescent label or a radioactive label; (xi) The treated subject has a cancer in which gMDSCs are involved in the disease pathology, and optionally the treated cancer cells do not express or overexpress the antigen bound with NEO-201; (xi) The treated subjects have cancers in which gMDSCs are involved in disease pathology (optionally: adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, adult brain / CNS tumors, pediatric brain / CNS tumors, breast cancer, male breast cancer, adolescent cancer, pediatric cancer, young adult cancer, cancer of unknown primary origin, Castleman disease, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, Ewing tumor family, eye cancer, bile duct cancer) Cystic carcinoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, renal cancer, laryngeal and hypopharyngeal cancer, leukemia, adult leukemia (acute lymphoblastic) (ALL), leukemia (acute myeloid) (AML), leukemia (chronic lymphoblastic) (CLL), leukemia (chronic myeloid) (CML), leukemia (chronic myelomonocytic) (CMML), childhood leukemia, liver cancer , lung cancer, non-small cell lung cancer, small cell lung cancer, pulmonary carcinoid tumor, lymphoma, cutaneous lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, pediatric non-Hodgkin lymphoma, oral and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma (adult soft tissue cancer), skin cancer, skin cancer (basal and squamous cells), skin cancer (melanoma), skin cancer (Merkel cells), small intestine cancer, gastric cancer, testicular cancer, thymic cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström type macroglobulinemia, and Wilms tumor) and optionally, the cancer cells to be treated do not express or overexpress the antigen to which NEO-201 binds; (xiiii) The treated subjects have cancers in which gMDSCs are involved in disease pathology, optionally including 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, Having cancers and / or tumors selected from the group consisting of endocrine cancers, thyroid cancers, parathyroid cancers, adrenal cancers, soft tissue sarcomas, urethral cancers, penile cancers, prostate cancers, adenocarcinomas, chronic or acute leukemias, lymphoid lymphomas, bladder cancers, kidney cancers or ureteral cancers, renal cell carcinomas, renal pelvis cancers, central nervous system tumors, primary CNS tumors, spinal cord tumors, brainstem gliomas, and pituitary adenomas, and optionally, the cancer cells to be treated do not express or overexpress antigens to which NEO-201 binds; (xiv) The cancer or infection being treated is not characterized by the expression of glycosylated CEACAM5 and / or glycosylated CEACAM6 having core type 1 and / or extended core type 1 O-glycans, nor is it characterized by an increase in the expression of glycosylated CEACAM5 and / or glycosylated CEACAM6; (xv) The treated subject has a cancer in which gMDSCs are involved in the disease pathology, and the treatment induces one or more of the following: (i) increased T cell response, (ii) increased antigen presentation, (iii) reduced MDSC proliferation, and / or (iv) reduced Treg recruitment; (xvi) The treated subject has stage I, stage II, stage III, or stage IV cancer involving gMDSCs; (xvii) The antibody or fragment (optionally, NEO-201) reduces, eliminates, slows or stops the growth of the tumor, reduces the tumor burden in the individual, inhibits tumor growth, and / or increases the survival rate of the individual in a patient whose antitumor immunity had previously been suppressed by gMDSCs; (xviiii) The subject is suffering from an infectious state, and the disease pathology is related to gMDSC; (xix)The subject is suffering from a bacterial infection involving gMDSC (optionally, 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, Corynebacterium diphtheriae, Enterococcus faecalis and Enterococcus faecium, Escherichia coli (generally), Enterotoxigenic Escherichia coli (ETEC), Enteropathogenic E. coli, E. 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 infection); (xx) The above subject may optionally include: respiratory viruses (e.g., adenovirus, avian influenza, influenza virus type A, influenza virus type B, measles, parainfluenza virus, respiratory syncytial virus (RSV), rhinovirus, SARS-CoV), gastrointestinal viruses (e.g., coxsackievirus, enterovirus, poliovirus, rotavirus), hepatitis viruses (e.g., hepatitis B virus, hepatitis C virus), bovine viral diarrhea virus (substitute), herpesviruses (e.g., herpes simplex virus type 1, herpes simplex virus type 2), human cytomegalovirus, varicella-zoster virus Suffering from chronic or acute viral infections involving gMDSCs, associated with viruses, retroviruses (e.g., 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 (e.g., avian influenza, dengue virus, hantavirus, hemorrhagic fever virus, lymphocytic choroiditis virus, smallpox virus substitute, cowpox, monkeypox, rabbitpox, vaccinia virus, Venezuelan equine encephalomyelitis virus (VEE), West Nile virus, yellow fever virus); (xxi) The subject is suffering from a condition involving gMDSC, and gMDSC suppresses innate immunity, optionally 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, antisynthetic enzyme syndrome, arterial plaque disorder, 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, Baro disease / Baro concentric sclerosis, Behçet's disease, Buerger's disease, Vickerstaff encephalitis, Blau syndrome, bullous pemphigoid, Castleman disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy, chronic relapsing polymyelitis, chronic obstructive pulmonary disease, chronic venous stasis ulcer, Chardonnay Gu-Strauss syndrome, bullous pemphigoid, Cogan syndrome, cold agglutinin disease, complement component 2 deficiency, contact dermatitis, cranial arteritis, CREST syndrome, Crohn's disease, Cushing's syndrome, cutaneous leukocytosis, Degos disease, Darkham's disease, herpes zoster, dermatomyositis, type 1 diabetes, type 2 diabetes, diffuse cutaneous systemic sclerosis, Dressler syndrome, drug-induced lupus, discoid lupus erythematosus, eczema, emphysema, endometriosis, enthesitis-associated arthritis, eosinophilic fasciitis, eosinophilic gastroenteritis, eosinophilic pneumonia, acquired epidermolysis bullosa, erythema nodosum, erythroblastosis fetus, essential mixed cryoglomerulosis Brynemia, Evans syndrome, fibrodysplasia ossificans progressive, alveolitis fibrosis (or idiopathic pulmonary fibrosis), gastritis, pemphigoid gastroenteritis, Gaucher disease, glomerulonephritis, Goodpasture syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's encephalopathy, Hashimoto's thyroiditis, heart disease, Henoch-Schönlein purpura, herpes zoster of pregnancy (also known as bullous pemphigoid of pregnancy), hidradenitis suppurativa, HIV infection, Hughes-Stobin syndrome, hypogammaglobulinemia, infections (e.g., bacterial infections), idiopathic inflammatory demyelinating disease, 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, Lambert-Eaton myasthenic syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosing, linear IgA disease (LAD), lupus hepatitis (also known as autoimmune hepatitis), lupus erythematosus, lymphomatous granulomatosis, Magid syndrome, malignant tumors, e.g., cancer (e.g., sarcoma, Kaposi's sarcoma, lymphoma, leukemia, carcinoma, and melanoma), Meniere's disease, microscopic polyangiitis, Miller-Fischer syndrome, mixed Connective tissue disease, focal scleroderma, Mukka-Habermann disease (also known as acute pityriasis lichenoides), multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (also known as Devic's disease), neurogenic myotonica, ocular pemphigoid, opsoclonus-myoclonus syndrome, Odo thyroiditis, relapsing rheumatoid arthritis, PANDAS (Streptococcus-associated childhood autoimmune neuropsychiatric disorder), paraneoplastic cerebellar degeneration, Parkinson's disease, paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, Personage-Turner syndrome, squamous cellulitis, Pemphigus vulgaris, peripheral artery 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 syndrome, Schnitzler syndrome, scleritis, scleroderma, sepsis, serum sickness, It is involved in Sjögren's syndrome, spondyloarthritis, Still's disease (adult-onset), Stiffperson syndrome, stroke, subacute bacterial endocarditis (SBE), Suzac syndrome, Sweet's syndrome, Sydenham chorea, sympathetic ophthalmitis, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis (also known as "giant cell arteritis"), thrombocytopenia, Tolosa-Hunt syndrome, transplant (e.g., heart / lung transplant) rejection, transverse myelitis, tuberculosis, ulcerative colitis, undifferentiated connective tissue disease, undifferentiated spondyloarthritis, urticarial vasculitis, vasculitis, vitiligo, and Wegener's granulomatosis; (xxii) The use described above includes the detection and monitoring of the patient's gMDSCs before, during, and after the treatment is completed and / or after the patient has achieved remission; (xxiii) In order to assess whether the patient may potentially benefit from the NEO-201 treatment, the patient's gMDSC is detected prior to the use; In (xxiv), (xxii), or (xxiii), the gMDSC is detected in a biological sample using antibodies that recognize one or more ligands, for example, specific biomarkers expressed on the gMDSC (optionally, LOX-1, CD11b, CD15, CD66b, and glycosylated CEACAM5 and CEACAM6 antigens recognized by NEO-201); In (xxv), (xxii), (xxiiii), or (xxiv), the number or concentration of gMDSCs in a sample of a subject suffering from a cancer involving gMDSCs is used to monitor the progression of the cancer (with or without treatment), and the patient is being treated for such cancer with NEO-201 alone or in combination with another therapeutic agent; In (xxvi), (xxii), (xxiii), (xxiv), or (xxv), the number or concentration of gMDSCs in a sample of a subject suffering from a cancer involving gMDSCs is used to determine whether NEO-201, alone or in combination with another therapeutic agent, may be beneficial in treating said cancer, and said patient is being treated for such cancer with NEO-201 alone or in combination with other therapeutic agents; (xxvii) Using the aforementioned number or concentration of gMDSCs in a sample of a subject with a cancer involving gMDSCs, develop a regimen of NEO-201 alone or in combination with another therapeutic agent in (xxii), (xxiii), (xxiv), (xxv), or (xxvi); In (xxvii), (xxiii), (xxiv), (xxv), (xxvi), or (xxvii), the level of gMDSC in a patient sample (e.g., blood or biopsy sample) is used to determine the prognosis of cancer before, during, or after NEO-201 treatment, and this method optionally includes contacting the gMDSC with the NEO-201 antibody; In (xxix), (xxii), (xxiii), (xxiv), (xxv), (xxvi), (xxvii), or (xxviiii), the detection comprises cell sorting, optionally, fluorescence-activated cell sorting, thereby generating a sample from which cells positive for NEO-201 antigen expression (e.g., gMDSCs) are enriched and / or removed; In (xxx), (xxii), (xxiii), (xxiv), (xxv), (xxvi), (xxvii), (xxviiii), or (xxix), the use comprises detecting and / or staining gMDSCs by contacting cells with NEO-201 antibody and detecting cells expressing NEO-201, wherein NEO-201 is optionally directly or indirectly labeled; In any of (xxxi) (xxii) to (xxx), gMDSCs are isolated by contacting a patient sample with a support containing NEO-201 antibody and / or by using other antibodies or ligands that recognize other gMDSC biomarkers, thereby retaining the gMDSCs on the support; (xxxi) In any of (xxxi) to (xxxi), the level of gMDSC in a patient sample (e.g., blood or biopsy sample) is used to determine whether the patient has or is likely to develop gMDSC-mediated immunosuppression; (xxxii-2) The use described above includes the administration of another therapeutic agent; (xxxiii ) The use thereof comprises the use of at least one other therapeutic agent, wherein the use of NEO-201 or other antibodies, which bind to glycosylated CEACAM5 and CEACAM6 having core type 1 and / or extended core type 1 O-glycans but do not bind to non-glycosylated CEACAM5 or non-glycosylated CEACAM6, enhances the efficacy of the at least one other therapeutic agent, Optionally, the other therapeutic agent may include another therapeutic antibody, checkpoint inhibitor, chemotherapeutic agent, and / or immune cells (optionally, CAR-T cells or CAR-NK cells), or The other therapeutic agent comprises (i) another part that removes gMDSCs, (ii) a part that promotes the differentiation of gMDSCs, (iii) a part that inhibits the migration of gMDSCs, (iv) a part of an epigenetic therapy that targets gMDSCs, or (v) a chemotherapeutic agent that targets gMDSCs, or one or more of the above; (xxxiv) The use may optionally enhance the effectiveness of the other therapeutic agents in subjects previously resistant to treatment with the other therapeutic agents, because NEO-201 has the ability to deplete gMDSCs by enhancing innate immunity (e.g., the natural response to antitumor or anti-infective agents); (xxxv) The use described above includes, but is not limited to, additional therapeutic agents including peptides, nucleic acid molecules, small molecule compounds, antibodies, and derivatives thereof; (xxxvi) Such additional therapeutic agents include, optionally, immune checkpoint inhibitors, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-CD28 antibody, anti-TIGIT antibody, anti-LAGS antibody, anti-TIM3 antibody, anti-GITR antibody, anti-4-1BB antibody, or anti-OX-40 antibody, and / or the additional therapeutic agents include adenosine A2A receptor (AZAR), B7-H3 (also known as CD276); B and T lymphocyte attenuators (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, CD152 and This includes, but is not known, indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene 3 (LAGS), programmed cell death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and the T cell activation V domain Ig suppressor (VISTA), and in particular, the immune checkpoint inhibitor targets the PD-1 axis and / or CTLA-4, and optionally, the immune checkpoint inhibitor targets the PD-1 axis and / or CTLA-4; (xxxvii) Such additional therapeutic agents include CSF-1 / 1R conjugates or inhibitors; (xxxviiii) Such additional therapeutic agents may 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, altretamine, aminoglutethimide, amsacrine, anastrozole, azacitidine, bleomycin, busulfan, carmustine, chlorambucil, 2-chlorodeoxyadenosine, cisplatin Colchicine, cyclophosphamide, cytarabine, cytoxane, dacarbazine, dactinomycin, daunorubicin, docetaxel, estramustine phosphate, floxuridine, fludarabine, gentuzumab, hexamethylmelamine, hydroxyurea, ifosfamide, imatinib, interferon, irinotecan, lomustine, mechloretamine, melpharen, 6-mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, pen Tostatin, procarbazine, rituximab, streptozocin, tamoxifen, temozolomide, teniposide, 6-thioguanine, topotecan, trastuzumab, vincristine, vindesine, and / or vinorelbine; (c) 1-D-ribofuranosyl-1,2,4-triazole-3carboxamide, 9->2-hydroxyethoxymethylguanine, adamantanamine, 5-iodo-2'-deoxyuridine, trifluorothymidine, interferon, adenine (d) Arabinosides, protease inhibitors, thymidine kinase inhibitors, glycoprotein synthesis inhibitors, structural protein synthesis inhibitors, adhesion and adsorption inhibitors, and nucleoside analogs (e.g., acyclovir, penciclovir, valacyclovir, and ganciclovir); (d) PD-1 inhibitors or anti-PD-1 antibodies (e.g., KEYTRUDA® (pembrolizumab), OPDIVO® (nivolumab), or LIBTAYO (semiprimab);(e) PD-L1 inhibitors or anti-PD-L1 antibodies (e.g., TECONTRIQ (atezolizumab), IMFINZI (durvalumab), or BAVENCIO (avelumab); or (f) CTLA-4 inhibitors or anti-CTLA-4 antibodies (e.g., YERVOY® ipilimumab); (xxxix) The patient has been determined to be resistant to treatment with one or more active substances for gMDSC-mediated immunosuppression prior to treatment with NEO-201; (xl) The patient has been determined to be resistant to treatment with a therapeutic antibody (optionally, one that targets a checkpoint inhibitor) prior to treatment with NEO-201; (xli) The patient has been determined to be resistant to treatment with PD-1 or CTLA-4 antagonists, optionally, antibodies or fusion proteins, prior to treatment with NEO-201; (xli) The patient has developed resistance to the other therapeutic agents (e.g., chemotherapeutic agents and / or checkpoint inhibitors) prior to treatment with NEO-201, and / or no longer responds to treatment with the other therapeutic agents; (xliiii) The patient, after treatment with NEO-201, clinically responds to the other active substance, optionally another therapeutic antibody or fusion protein, and optionally a checkpoint inhibitor and / or immune cells, optionally targeting CAR-T cells or CAR-NK cells; The patient, after treatment with (xliv) NEO-201, clinically responds to the other active substance, optionally a chemotherapeutic agent and / or another therapeutic antibody or fusion protein, and optionally a PD-1 antagonist antibody (e.g., pembrolizumab, nivolumab, semiprimab, atezolizumab, dostallimab, durvalumab, lambrolizumab, or avelumab); The patient, after treatment with (xlv)NEO-201, clinically responds to CTLA-4, optionally Yervoy or tremelimumab and / or immune cells, optionally CAR-T or CAR-NK cells, optionally another active substance, optionally another therapeutic antibody or fusion protein; (xlvi) The NEO-201 antibody comprises the VH and VL CDR sequences contained in SEQ ID NO: 28 and SEQ ID NO: 29; (xlvii) The NEO-201 antibody comprises a variable heavy chain sequence having at least 90% identity with SEQ ID NO: 38; (xlviiii) The NEO-201 antibody comprises a variable light chain sequence having at least 90% identity with SEQ ID NO: 39; (xlix) The NEO-201 antibody comprises a variable heavy chain sequence having at least 90% identity with SEQ ID NO: 38 and a variable light chain sequence having at least 90% identity with SEQ ID NO: 39; (l) The NEO-201 antibody comprises a heavy chain sequence having at least 90% identity with amino acids 20-470 of SEQ ID NO: 28 and a light chain sequence having at least 90% identity with amino acids 20-233 of SEQ ID NO: 29; (i) The NEO-201 antibody or antibody fragment 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; (lii) The NEO-201 antibody or antibody fragment comprises a human IgG1 constant domain; (liii) The NEO-201 antibody or antibody fragment is humanized; (lv) The NEO-201 antibody or antibody fragment is conjugated to another portion; (lv) The NEO-201 antibody or antibody fragment is conjugated to another cytotoxic moiety, label, radioactive moiety, or affinity tag; (lvi) The antibody or antibody fragment (preferably NEO-201 antibody) is included in the chimeric antigen receptor (CAR) administered to the subject of treatment; (lvi) The antibody or antibody fragment is included 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, optionally the other antigen being a checkpoint inhibitor, cytokine, hormone or growth factor; (lviii) The antibody or antibody fragment is administered as an immune cell (optionally, a human T cell or an NK cell), and the immune cell expresses a CAR containing the antibody or antibody fragment; or (lix) Any combination of (i) to (lviiii).

3. NEO-201 antibody for the following uses: (i) Kill gMDSCs in a patient, optionally, the patient has been treated with CAR-T cells or CAR-NK cells. (ii) To treat, prevent or reduce gMDSC-mediated immunosuppression in a patient, (iii) To enhance the efficacy of CAR-T or CAR-NK therapy by administration in combination with NEO-201, the CAR may target any of the antigens disclosed herein.

4. The use described in Claim 3, which is as follows: (i) further comprising administering another therapeutic agent to the patient, Optionally, the other 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, altoretamine, aminoglutethimide, amsacrine, anastrozole, azacitidine, bleomycin, busulfan, carmustine, chlorambucil, 2-chlorodeoxyadenosine, and cisplatin. Colchicine, cyclophosphamide, cytarabine, cytoxane, dacarbazine, dactinomycin, daunorubicin, docetaxel, estramustine phosphate, floxuridine, fludarabine, gentuzumab, hexamethylmelamine, hydroxyurea, ifosfamide, imatinib, interferon, irinotecan, lomustine, mechloretamine, melpharen, 6-mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, pen Tostatin, procarbazine, rituximab, streptozocin, tamoxifen, temozolomide, teniposide, 6-thioguanine, topotecan, trastuzumab, vincristine, vindesine, and / or vinorelbine; (c) 1-D-ribofuranosyl-1,2,4-triazole-3carboxamide, 9->2-hydroxyethoxymethylguanine, adamantanamine, 5-iodo-2'-deoxyuridine, trifluorothymidine, interferon, adenine (d) Arabinosides, protease inhibitors, thymidine kinase inhibitors, glycoprotein synthesis inhibitors, structural protein synthesis inhibitors, adhesion and adsorption inhibitors, and nucleoside analogs (e.g., acyclovir, penciclovir, valacyclovir, and ganciclovir); (d) PD-1 inhibitors or anti-PD-1 antibodies (e.g., KEYTRUDA® (pembrolizumab), OPDIVO® (nivolumab), or LIBTAYO (semiprimab);(e) a PD-L1 inhibitor or anti-PD-L1 antibody (e.g., TECENTRIQ (atezolizumab), IMFINZI (durvalumab), or BAVENCIO (avelumab); or (f) a CTLA-4 inhibitor or anti-CTLA-4 antibody (e.g., YERVOY® ipilimumab), or the other agent comprises CAR-T cells or CAR-NK cells; or; (ii) The use described above further comprises administering an anti-cancer vaccine or CAR-T cells or CAR-NK cells to the patient.

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

6. The method according to claim 5, which is as follows: (i) The tissue, organ, or cell sample is obtained from a patient with cancer or an infectious condition; (ii) The tissue, organ, or cell sample is a bone marrow sample derived from the autologous or allogeneic donor; (iii) Furthermore, the gMDSC is brought into contact with the complement; (iv) Kill the gMDSC with ADCC or CDC; (v) further comprising contacting the gMDSC with effector cells, optionally the effector cells including natural killer cells; (vi) Kill the gMDSC with ADCC; (vii) The NEO-201 antibody is bound to the cytotoxic portion; or (viiii) Any combination of (i) to (vii).

7. A method for detecting gMDSCs, comprising detecting the expression of the NEO-201 antigen by the gMDSCs and optionally one or more other gMDSC biomarkers, wherein the level of gMDSCs in a patient sample (e.g., blood or biopsy sample) is used to determine the prognosis or treatment regimen for cancer.

8. The method according to claim 7, comprising contacting the gMDSC with an NEO-201 antibody, wherein the NEO-201 antibody is optionally conjugated directly or indirectly to a label; the detection comprises cell sorting, optionally fluorescence-activated cell sorting.

9. A method for staining gMDSCs, comprising contacting cells with an antibody NEO-201, wherein the antibody NEO-201 is conjugated directly or indirectly to a label.

10. A method for isolating gMDSCs, comprising isolating cells expressing the NEO-201 antigen and optionally at least one other gMDSC biomarker, optionally comprising contacting a sample containing gMDSCs with an NEO-201 antibody, optionally the NEO-201 antibody being directly or indirectly labeled, and further optionally the sample being or comprising a blood, bone marrow, or tumor biopsy sample.

11. The method according to any one of claims 7 to 10, further comprising: (i) Isolating cells positive for NEO-201 from cells negative for NEO-201, optionally the gMDSCs being isolated by cell sorting, optionally by fluorescence-activated cell sorting, or the gMDSCs being isolated by contacting the sample with a support containing NEO-201 antibody, thereby retaining the gMDSCs on the support; (ii) The NEO-201 antibody contains the CDR sequence contained in SEQ ID NO: 28 and SEQ ID NO: 29; (iii) The NEO-201 antibody comprises a variable heavy chain sequence having at least 90% identity with SEQ ID NO: 38; (iv) The NEO-201 antibody comprises a variable light chain sequence having at least 90% identity with SEQ ID NO: 39; (v) The NEO-201 antibody comprises a variable heavy chain sequence having at least 90% identity with SEQ ID NO: 38 and a variable light chain sequence having at least 90% identity with SEQ ID NO: 39; (vi) The NEO-201 antibody comprises a heavy chain sequence having at least 90% identity with amino acids 20-470 of SEQ ID NO: 28 and a light chain sequence having at least 90% identity with amino acids 20-233 of SEQ ID NO: 29; (vii) The NEO-201 antibody contains all six of the CDR sequences contained in SEQ ID NO: 28 and SEQ ID NO: 29; (viiii) The NEO-201 antibody has a human IgG1 constant domain, optionally one or more effector functions, optionally FcR binding, FcRN binding, glycation, complement (C1 ｑ A human IgG1 constant domain comprising at least one mutation that enhances or inhibits ) binding, phagocytosis, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cell-mediated cytotoxicity (CDC), antibody-mediated neutralization, opsonization, or any combination thereof; (ix) The NEO-201 antibody is humanized; (x) The NEO-201 antibody is conjugated to another portion; (xi) The NEO-201 antibody is conjugated to another cytotoxic moiety, label, radioactive moiety, or affinity tag; or (xi) Any combination of (i) to (xi).

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