Targeted cytokine constructs for engineered cell therapy

Abstract

Provided herein are targeted cytokine constructs for use in treating diseases, e.g., proliferative diseases, e.g., cancer, and methods of genetically engineered cell therapy by administering the targeted cytokine constructs in combination with genetically engineered cell therapy.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 123,281, filed December 9, 2020, which is incorporated herein by reference in its entirety. [Summary of the invention]

[0002] One embodiment is a targeted cytokine construct for a genetically engineered cell, comprising: (i) a chimeric antigen receptor (CAR) or a T cell receptor (TCR) exogenously introduced into the genetically engineered cell; (ii) a tag molecule selectively expressed on the surface of genetically engineered cells, and (iii) a CAR exogenously introduced into genetically engineered cells. (iv) a polypeptide tag that is part of a TCR that is exogenously introduced into the genetically engineered cell; or (vi) any combination of (i) to (v). Targeted cytokine constructs are provided that include at least one targeting cell binding domain and a cytokine protein or functional fragment or variant thereof.

[0003] In some embodiments, the targeted cytokine construct selectively activates genetically engineered cells with greater than 10-fold potency compared to activation of non-genetically engineered cells. In some embodiments, efficacy is measured by a pSTAT5 activation assay or a pSTAT3 activation assay. In some embodiments, the domain of the CAR is an scFv. In some embodiments, a non-genetically engineered cell does not express a CAR, TCR, or tag molecule on its surface. In some embodiments, the cell binding domain comprises an antibody or antigen binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is bivalent or monovalent. In some embodiments, the cytokine is a group consisting of IL-2, IL-7, IL-10, IL-15, and IL-21, or a functional fragment or variant thereof, or any combination thereof. selected from. In some embodiments, the cytokine is an IL-2 polypeptide, or a functional fragment thereof, or a variant thereof. In some embodiments, the IL-2 polypeptide has a binding affinity of an IL-2Rα polypeptide having the amino acid sequence of SEQ ID NO:2 compared to the binding affinity of a wild-type IL-2 polypeptide having the amino acid sequence of SEQ ID NO:1. exhibits at least about 50% reduced binding affinity for the peptide. In some embodiments, the IL-2 polypeptide has a binding affinity of an IL-2Rβ polypeptide having the amino acid sequence of SEQ ID NO:3 compared to the binding affinity of a wild-type IL-2 polypeptide having the amino acid sequence of SEQ ID NO:1. exhibits at least about 50% reduced binding affinity for the peptide and / or at least about 50% reduced binding affinity for the IL-2Rγ polypeptide having the amino acid sequence of SEQ ID NO:4. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 1 with one or more amino acid substitutions relative to SEQ ID NO: 1, where the one or more substitution(s) are Q11, H16 , L18, L19, D20, Q22, R38, F42 , K43, Y45, E62, P65, E68, V69, L72, D84, S87, N88, V91, I92, T123, Q126, S127, I129, and S130. Including. In some embodiments, the one or more substitution(s) comprises a F42A amino acid substitution or a F42K amino acid substitution relative to SEQ ID NO:1. In some embodiments, the one or more substitution(s) further comprises an R38A, R38D, R38E, E62Q, E68A, E68Q, E68K, or E68R amino acid substitution to SEQ ID NO:1. In some embodiments, the one or more substitution(s) to SEQ ID NO:1 is H16E, H16D, D20N, M23A, M23R, M23K, S87K, S87A, D84L, D84N, D84V, D84H, D84Y, D84R, D84K, N88A, N88G, N88S, N88T, N8 8R, N88I, N88D, V91A, V91T, V91E, I92A, E95S, E95A, E95R, T123A, T123E, T123K, Further comprising an amino acid substitution of T123Q, Q126A, Q126S, Q126T, Q126E, S127A, S127E, S127K, or S127Q. In some embodiments, the one or more substitution(s) further comprises the amino acid mutation C125A compared to SEQ ID NO:1. In some embodiments, the IL-2 polypeptide has the following set of amino acid substitutions (relative to the sequence of SEQ ID NO: 1): R38E and F42A; R38D and F42A; F42A and E62Q; R38A and F42K; N88S;R38E, F42A, and N88A;R38E , F42A, and N88G; R38E, F42A, and N88D; R38E, F42A, and V91E; R38E, F42A, and D84H; R38 E, F42A, and D84K; R38E, F42A, and D84R; H16D, R38E, and F42A; H16E, R38E, and F42A; R38E, F42A and Q126S; R38D, F42A and N88S; R38D, F42A and N88A; R38D, F42A and N88G; R38D, F4 2A and N88D; R38D, F42A and V91E; R38D, F42A, and D84H; R38D, F42A, and D84K; R38D, F42A , and D84R; H16D, R38D and F42A; H16E, R38D and F42A; R38D, F42A and Q126S; R38A, F42K, and N88S; R38A, F42K, and N88A; R38A, F42K, and N88G; R38A, F42K, and N88D; R38A, F42K, and V91E; R38A, F42K, and D84H; R38A, F42K, and D84K; R38A, F42K, and D84R; H16D, R38A , and F42K; H16E, R38A, and F42K; R38A, F42K, and Q126S; F42A, E62Q, and N88S; F42A, E62 Q, and N88A; F42A, E62Q, and N88G; F42A, E62Q, and N88D; F42A, E62Q, and V91E; F42A, E6 2Q, and D84H; F42A, E62Q, and D84K; F42A, E62Q, and D84R; H16D, F42A, and E62Q; H16E, F4 2A, and E62Q; F42A, E62Q, and Q126S; R38E, F42A, and C125A; R38D, F42A, and C125A; F42 A, E62Q, and C125A; R38A, F42K, and C125A; R38E, F42A, N88S, and C125A; R38E, F42A, N8 8A, and C125A; R38E, F42A, N88G, and C125A; R38E, F42A, N88D, and C125A; R38E, F42A, V91E, and C125A; R38E, F42A, D84H, and C125A; R38E, F42A, D84K, and C125A; R38E, F42A , D84R, and C125A; H16D, R38E, F42A, and C125A; H16E, R38E, F42A, and C125A; R38E, F4 2A, C125A and Q126S; R38D, F42A, N88S, and C125A; R38D, F42A, N88A, and C125A; R38D, F 42A, N88G, and C125A; R38D, F42A, N88D, and C125A; R38D, F42A, V91E, and C125A; R38D , F42A, D84H, and C125A; R38D, F42A, D84K, and C125A; R38D, F42A, D84R, and C125A; H16 D, R38D, F42A, and C125A; H16E, R38D, F42A, and C125A; R38D, F42A, C125A, and Q126S; R38A, F42K, N88S, and C125A; R38A, F42K, N88G, and C125A; R38A, F42K, N88D, and C125A ;R38A, F42K, N88A, and C125A;R38A, F42K, V91E, and C125A;R38A, F42K, D84H, and C12 5A; R38A, F42K, D84K, and C125A; R38A, F42K, D84R, and C125A; H16D, R38A, F42K, and C1 25A; H16E, R38A, F42K, and C125A; R38A, F42K, C125A and Q126S; F42A, E62Q, N88S, and C125A;F42A, E62Q, N88A, and C125A;F42A, E62Q, N88G, and C125A;F42A, E62Q, N88D, and C125A; F42A, E62Q, V91E, and C125A; F42A, E62Q, and D84H, and C125A; F42A, E62Q, and D84K, and C125A; F42A, E62Q, and D84R, and C125A; H16D, F42A, and E62Q, and C125A; H16E, F42A, E62Q, and C125A; F42A, E62Q, C125A, and Q126S; F42A, N88S, and C125A; F42A, N 88A, and C125A; F42A, N88G, and C125A; F42A, N88D, and C125A; F42A, V91E, and C125A; F4 2A, D84H, and C125A; F42A, D84K, and C125A; F42A, D84R, and C125A; H16D, F42A, and C12 5A; H16E, F42A, and C125A; and one of F42A, C125A, and Q126S. In some embodiments, the IL-2 polypeptide comprises an amino acid sequence that is at least about 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 11-90. In some embodiments, the IL-2 polypeptide comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 43, 48, 52, 49, and 156.

[0004] In some embodiments, the cytokine is an IL-7 polypeptide, or a functional fragment thereof, or a variant thereof. In some embodiments, the IL-7 polypeptide has a binding affinity of SEQ ID NO: 94 compared to the binding affinity of a wild-type IL-7 polypeptide comprising the amino acid sequence of SEQ ID NO: 91 to the IL-7Ra polypeptide. exhibits at least about 50% reduced binding affinity for an IL-7Ra polypeptide comprising the amino acid sequence. In some embodiments, the IL-7 polypeptide has a binding affinity of SEQ ID NO: 4 compared to the binding affinity of a wild-type IL-7 polypeptide comprising the amino acid sequence of SEQ ID NO: 91 to an IL-2Ra polypeptide. exhibits at least about 50% reduced binding affinity for an IL-2Rg polypeptide comprising the amino acid sequence. In some embodiments, the IL-7 polypeptide comprises the sequence of SEQ ID NO: 91 with one or more substitutions to SEQ ID NO: 91. In some embodiments, one or more substitutions are at positions K10, Q11, S14, V15, V18, Q22, L35, N36, D74, L77, L80, K81, E84, I88, R133, Q136, E137, T140 , N143, and K144. In some embodiments, the substitution at position K81 is K81A and the substitution at position T140 is K140A. In some embodiments, the cytokine is an IL-10 polypeptide, or a functional fragment thereof, or a variant thereof. In some embodiments, the IL-10 polypeptide has a binding affinity of SEQ ID NO: 96 compared to the binding affinity of a wild-type IL-10 polypeptide comprising the amino acid sequence of SEQ ID NO: 95 to an IL-10RA polypeptide. exhibits at least about 50% reduced binding affinity for an IL-10RA polypeptide comprising the amino acid sequence. In some embodiments, the IL-10 polypeptide has a binding affinity of SEQ ID NO: 97 compared to the binding affinity of a wild-type IL-10 polypeptide comprising the amino acid sequence of SEQ ID NO: 95 to the IL-10RB polypeptide. exhibits at least about 50% increased binding affinity for an IL-10RB polypeptide comprising the amino acid sequence. In some embodiments, the IL-10 polypeptide comprises the sequence of SEQ ID NO: 95 with one or more substitutions to SEQ ID NO: 95. In some embodiments, the IL-10 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 99-112. In some embodiments, the cytokine is an IL-21 polypeptide, or a functional fragment thereof, or a variant thereof. In some embodiments, the IL-21 polypeptide has a binding affinity of SEQ ID NO: 93 compared to the binding affinity of a wild-type IL-21 polypeptide comprising the amino acid sequence of SEQ ID NO: 92 to an IL-21R polypeptide. exhibits at least about 50% reduced binding affinity for an IL-21R polypeptide comprising the amino acid sequence. In some embodiments, the IL-21 polypeptide has a binding affinity of SEQ ID NO:4 compared to the binding affinity of a wild-type IL-21 polypeptide comprising the amino acid sequence of SEQ ID NO:92 to an IL-2Rg polypeptide. exhibits at least about 50% reduced binding affinity for an IL-2Rg polypeptide comprising the amino acid sequence. In some embodiments, the IL-21 polypeptide comprises the sequence of SEQ ID NO: 115 with one or more substitutions to SEQ ID NO: 115. In some embodiments, substitutions at one or more positions include positions R5, I8, R9, R11, Q12, I14, D15, D18, Q19, Y23, R65, S70, K72, K73, K75, R76, K77, S80, Q116, and K117, and the position numbering is according to the amino acid sequence of SEQ ID NO: 115.

[0005] In some embodiments, the genetically engineered cells are T cells expressing T cell receptors (TCR-T cells), γδ T cells, T cells derived from pluripotent stem cells, or T cells derived from induced pluripotent stem cells. cells, natural killer cells (NK cells), NK cells derived from pluripotent stem cells or NK cells derived from induced pluripotent stem cells (iPSCs), genetically engineered to express chimeric antigen receptors. Child-engineered T cells (CAR-T cells), CD8-positive T cells, CD4-positive T cells, cytotoxic T cells, tumor-infiltrating lymphocytes, CAR-NK cells, γδ T cells, bone marrow cells, hematopoietic cells, hematopoietic cells It contains at least one of stem cells and progenitor cells (HSC), hematopoietic multipotent progenitor cells (MPP), pre-T cell progenitor cells, T cell progenitor cells, and NK cell progenitor cells. In some embodiments, targeted cytokine constructs are suitable for administration to a subject in combination with a therapy involving genetically engineered cells. In some embodiments, the genetically engineered cells are autologous to the subject. In some embodiments, the genetically engineered cells are allogeneic to the subject. In some embodiments, the subject is a human. In some embodiments, the subject has cancer. In some embodiments, the cancer is acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), acute myeloid leukemia, B-cell prolymphocytic leukemia, B-cell acute lymphoblastic leukemia (“BALL”), blastic plasmacytoid dendritic cell tumor, Burkitt lymphoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloid leukemia, chronic or acute leukemia, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), hairy cell leukemia, Hodgkin's disease, malignant lymphoproliferative disease, MALT lymphoma, mantle cell lymphoma, marginal Lymphoma, monoclonal gammaglobulinemia of undetermined significance (MGUS), multiple myeloma, myeloid dysmorphia myelodysplastic syndromes, non-Hodgkin lymphoma (NHL), plasma cell proliferative disorders (including asymptomatic myeloma (including smoldering multiple myeloma or indolent myeloma)), plasmablastic lymphoma , plasmacytoid dendritic cell tumor, plasmacytoma (plasma cell disease, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytoma) primary B-cell lymphoma (PMBC), small cell or large follicular lymphoma, POEMS syndrome (also known as Crow-Fukase syndrome, Takatsuki disease, and PEP syndrome), Splenic marginal zone lymphoma (SMZL), systemic amyloid light chain amyloidosis, T-cell acute lymphoblastic leukemia (TA LL), T-cell lymphoma, transformed follicular lymphoma or Waldenström's macroglobulinemia, mantle cell lymphoma (MCL), transformed follicular lymphoma (TFL), primary mediastinal large B-cell lymphoma (PMBCL) , multiple myeloma, hairy cell lymphoma / leukemia, lung cancer, small cell lung cancer, non-small cell lung (NS CL) cancer, bronchioloalveolar cell lung cancer, squamous cell carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, head and neck cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer cancer of the genital area, skin or intraocular melanoma, thyroid cancer, uterine cancer, gastrointestinal cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, stomach cancer, colon cancer, breast cancer, uterus endometrial cancer, uterine cancer, Carcinoma of the fallopian tubes, carcinoma of the cervix, carcinoma of the vagina, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, Adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureteral cancer, renal cell cancer, carcinoma of the renal pelvis, mesothelioma, bladder cancer , liver cancer, stem cell tumor, hepatocellular carcinoma, Cervical cancer, salivary gland cancer, biliary tract cancer, central nervous system (CNS) tumors, spinal axis tumors, brainstem gliomas, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, Medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, and Ewing's sarcoma, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers.

[0006] One embodiment is a pharmaceutical composition comprising a targeted cytokine construct according to the present disclosure and at least one of a pharmaceutically acceptable excipient, carrier, or diluent, or any combination thereof. Provide. In some embodiments, the pharmaceutical composition further comprises a population of genetically engineered cells. One embodiment provides a cell therapy kit that includes a pharmaceutical composition comprising a targeted cytokine construct according to the present disclosure and specified instructions for administering the targeted cytokine construct to a subject. In some embodiments, the cell therapy kit further comprises a pharmaceutical composition comprising a population of genetically engineered cells and instructions specifying for administering the population of genetically engineered cells to a subject. . In some embodiments, the pharmaceutical composition comprising the targeted cytokine construct and the population of genetically engineered cells are for sequential or simultaneous administration.

[0007] One embodiment provides a method of treating a disease in a subject, the method comprising: (a) a genetically engineered cell; and (b) (i) a receptor exogenously introduced into the genetically engineered cell. or (ii) a targeted cytokine construct comprising a cytokine protein or a functional fragment or variant thereof.

[0008] In some embodiments, the targeted cytokine construct selectively activates a population of genetically engineered cells with greater than 10-fold potency compared to activation of non-genetically engineered cells. In some embodiments, administering a targeted cytokine construct results in increased activation of genetically engineered cells compared to activation of a population of non-genetically engineered cells. In some embodiments, activation is measured by a pSTAT5 activation assay or a pSTAT3 activation assay. In some embodiments, administering the targeted cytokine construct increases the expansion and / or proliferation of a population of genetically engineered cells as compared to the expansion and / or proliferation of a population of non-genetically engineered cells. bring. In some embodiments, administration of the targeted cytokine construct results in an increase in the in vivo persistence of the population of genetically engineered cells compared to the in vivo persistence of the population of non-genetically engineered cells. In some embodiments, the non-genetically engineered cells do not express a CAR, TCR, or tag molecule. In some embodiments, upon administration of the targeted cytokine construct, the activation, expansion, and / or proliferation of the population of genetically engineered cells is greater than that of the genetically engineered cells when administered without the targeted cytokine construct. An increase is produced relative to activation, expansion, and / or proliferation of a population of cells. In some embodiments, expansion and / or proliferation occurs in vivo or in vitro. In some embodiments, the in vivo persistence of the genetically engineered population of cells when administered with the targeted cytokine construct is the same as the in vivo persistence of the genetically engineered population of cells when administered without the targeted cytokine construct. An increase is brought about compared to sex. In some embodiments, the in vivo persistence of the population of genetically engineered cells includes a period of about 15 days, about 30 days to about 1 year. In some embodiments, upon administration of the targeted cytokine construct, the rate and / or extent of depletion of the population of genetically engineered cells is greater than that of the genetically engineered cells when administered without the targeted cytokine construct. decrease relative to the rate and / or extent of population depletion. In some embodiments, administration of a targeted cytokine construct results in selective enhancement of genetically engineered cells, and when administered with a non-targeted cytokine or functional fragment or variant thereof, It becomes possible to enhance the specific enrichment of a genetically engineered population of cells compared to the specific enrichment of a population of genetically engineered cells. In some embodiments, administering a targeted cytokine construct increases the number of Treg cells in a biological sample isolated from a subject from a subject who has been administered a non-targeted cytokine or functional fragment or variant thereof. compared to the number of Treg cells in the biological sample. In some embodiments, the biological sample is at least one of a tumor biopsy or peripheral blood. In some embodiments, the subject has previously been administered a preconditioning regimen. In some embodiments, administering a targeted cytokine construct allows for a reduction in at least one of the severity or duration of a preconditioning regimen. In some embodiments, a preconditioning regimen is used to reduce the endogenous lymphocyte population so that the population of genetically engineered cells can be expanded. In some embodiments, the preconditioning regimen includes administering a lymphodepleting agent. In some embodiments, administration of a targeted cytokine construct reduces the degree of lymphocyte depletion required for engraftment of genetically engineered cells. In some embodiments, the preconditioning regimen includes administering a chemotherapeutic agent to the subject. In some embodiments, the chemotherapeutic agent is at least one of fludarabine and cyclophosphamide. In some embodiments, the preconditioning regimen includes radiation therapy. In some embodiments, the preconditioning regimen includes administering a depleting antibody. In some embodiments, the depleting antibody is alemtuzumab. In some embodiments, the subject is not administered a preconditioning regimen.

[0009] One embodiment provides a method that eliminates the need for, or minimizes the severity of, administering a preconditioning regimen prior to administering genetically engineered cell therapy, the method comprising: (a) genetically engineered cell therapy; and (b) (i) genetically engineered cells. (ii) a targeted cytokine construct comprising a cytokine protein or functional fragment or variant thereof; Including.

[0010] In some embodiments, the subject has not been administered a preconditioning regimen. In some embodiments, the targeted cytokine construct selectively activates genetically engineered cells with greater than 10-fold potency compared to activation of non-genetically engineered cells. In some embodiments, activation is measured by a pSTAT5 activation assay or a pSTAT3 activation assay. In some embodiments, a non-genetically engineered cell does not contain receptors or domains that have been exogenously introduced into the cell. In some embodiments, the targeting cytokine construct is administered for a period of about 2 days, 3 days, 6 days, 12 days, 15 days, 30 days, 60 days, or 90 days or more after administration of the genetically engineered cells. or within a period of about 2, 3, 6, 12, 15, 30, 60, or 90 days or more. In some embodiments, the targeted cytokine construct is administered at the same time as administering the genetically engineered cells. In some embodiments, the effective dose of the genetically engineered cells in the optimal treatment regimen is the same as that of the reference optimal treatment regimen that includes administering the genetically engineered cells but does not include administering the targeted cytokine construct. Lower than effective dose.

[0011] In some embodiments, the effective dose of genetically engineered cells in the optimal treatment regimen is at least about 1.5 times to about 1000 times lower than the effective dose of genetically engineered cells in the reference optimal treatment regimen.

[0012] One embodiment provides a method of increasing the effectiveness of genetically engineered cell therapy in a subject, the method comprising: (a) genetically engineered cells; administering to the subject an optimal treatment regimen comprising a cell binding domain that binds to the introduced receptor or domain; and (ii) a targeted cytokine construct comprising a cytokine protein or functional fragment or variant thereof; thereby increasing the efficacy of genetically engineered cell therapy in a subject.

[0013] In some embodiments, the targeted cytokine construct selectively activates genetically engineered cells with greater than 10-fold potency compared to activation of non-genetically engineered cells. In some embodiments, a non-genetically engineered cell does not contain receptors or domains that have been exogenously introduced into the cell. In some embodiments, efficacy is measured by a pSTAT5 activation assay.

[0014] One embodiment provides a method of treating a subject with loss of B cell aplasia, the method comprising: (i) a cell that binds to a receptor or domain exogenously introduced into the genetically engineered cell; and (ii) a cytokine protein or functional fragment or variant thereof.

[0015] One embodiment includes (a) a genetically engineered cell and (b) a cell binding domain that binds to (i) a receptor or domain exogenously introduced into the genetically engineered cell, and (ii) a cytokine protein. or a targeted cytokine construct comprising a functional fragment or variant thereof. provides a method of treating a disease, comprising administering genetically engineered cells by administering a targeted cytokine construct, but including genetically engineered cells in a reference optimal treatment regimen that does not include a targeted cytokine construct. It becomes possible to reduce the effective dose of genetically engineered cells in an optimal treatment regimen relative to the effective dose of .

[0016] In some embodiments, the effective dose of genetically engineered cells in the optimal treatment regimen is at least about 1.5 times to about 1000 times lower than the effective dose of genetically engineered cells in the reference optimal treatment regimen. In some embodiments, the genetically engineered cells are provided in a composition that is produced at the point of care and administered to the patient without culturing the population of cells. One embodiment provides a method of targeting genetically engineered cells in a subject, the method comprising administering to the subject a targeting cytokine construct comprising a cell binding domain and a modified cytokine or functional fragment or variant thereof. The genetically engineered cell expresses (i) a receptor for the modified cytokine or functional fragment or variant thereof, and (ii) a target antigen for the cell binding domain. One embodiment provides a method of enriching a population of genetically engineered cells in a subject, the method comprising administering to the subject a targeted cytokine construct comprising a cell binding domain and a modified cytokine or functional fragment or variant thereof. the genetically engineered cell expresses (i) a receptor for the modified cytokine or functional fragment or variant thereof, and (ii) a target antigen for the cell binding domain.

[0017] In some embodiments, genetically engineered cells are generated in vivo in a subject. In some embodiments, the subject has previously been administered a nucleic acid carrier comprising a nucleic acid expressing a chimeric antigen receptor (CAR) or T cell receptor protein (TCR). In some embodiments, the nucleic acid carrier is at least one of a linear polynucleotide, a polynucleotide associated with an ionic or amphipathic compound, a plasmid, and a virus. In some embodiments, the nucleic acid carrier is a nanocarrier. In some embodiments, the nucleic acid carrier is a viral vector, and the viral vector is at least one of a Sendai virus vector, an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, or a lentivirus vector. In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the RNA is messenger RNA (mRNA). In some embodiments, the nucleic acid carrier further comprises a targeting moiety for targeting immune cells. In some embodiments, the immune cells include myeloid cells, T cells, or NK cells. In some embodiments, T cells include T lymphocytes. In some embodiments, T cells or NK cells are induced with a vector or nucleic acid carrier to generate genetically engineered cells in vivo in a subject. In some embodiments, upon administration of the targeted cytokine construct, the activation, expansion, and / or proliferation of the population of genetically engineered cells generated in vivo is greater than when the subject is not administered the targeted cytokine construct. , resulting in an increase relative to the activation, expansion, and / or proliferation of a population of genetically engineered cells generated in vivo.

[0018] In some embodiments, upon administration of the targeted cytokine construct, the persistence of the population of genetically engineered cells generated in vivo is greater than that of the genetically engineered cells generated in vivo when the subject is not administered the targeted cytokine construct. An increase is produced relative to the persistence of the engineered cell population. In some embodiments, upon administration of the targeted cytokine construct, the rate and / or extent of depletion of the population of genetically engineered cells generated in vivo is no greater than that in vivo when administered without the targeted cytokine construct. The rate and / or extent of depletion of a population of genetically engineered cells generated in a. In some embodiments, administration of a targeted cytokine construct results in selective enhancement of genetically engineered cells generated in vivo, and when administered with a non-targeted cytokine or a functional fragment or variant thereof. It becomes possible to enhance the specific enrichment of a population of genetically engineered cells generated in vivo, compared to the specific enrichment of a population of genetically engineered cells generated in vivo. In some embodiments, administering a targeted cytokine construct increases the number of Treg cells in a biological sample isolated from a subject from a subject who has been administered a non-targeted cytokine or functional fragment or variant thereof. compared to the number of Treg cells in the biological sample. In some embodiments, the biological sample is at least one of a tumor biopsy or peripheral blood. In some embodiments, the persistence of the population of genetically engineered cells includes a period of at least about 30 days to about 1 year.

[0019] One embodiment provides a method of enriching a population of genetically engineered cells, the method comprising enriching a population of genetically engineered cells with a target comprising a cell binding domain and a modified cytokine or functional fragment or variant thereof. the genetically engineered cell expresses (i) a receptor for the modified cytokine or a functional fragment or variant thereof, and (ii) a target antigen for the cell binding domain. In some embodiments, the cytokine is a group consisting of IL-2, IL-7, IL-10, IL-15, and IL-21, or a functional fragment or variant thereof, or any combination thereof. selected from. In some embodiments, the cytokine is (i) an IL-2Rβγ agonist polypeptide that binds to and / or activates an IL-2Rβ polypeptide comprising the amino acid sequence of SEQ ID NO: 3; At least one IL-2Rβγ polypeptide agonist polypeptide that binds to and / or activates an IL-2Rγ polypeptide comprising the amino acid sequence number 4. In some embodiments, the cytokine is an IL-2 polypeptide, or a functional fragment thereof, or a variant thereof. In some embodiments, the method of claim 114, wherein the IL-2 polypeptide has a binding affinity of a wild-type IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 to an IL-2Rα polypeptide. exhibits at least about a 50% reduced binding affinity for an IL-2Rα polypeptide comprising the amino acid sequence of SEQ ID NO:2. In some embodiments, the IL-2 polypeptide has a binding affinity of SEQ ID NO: 3 compared to the binding affinity of a wild-type IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 to an IL-2Rβ polypeptide. At least about 50% reduced binding affinity for an IL-2Rβ polypeptide comprising the amino acid sequence and / or at least about 50% reduced binding affinity for an IL-2Rγ polypeptide comprising the amino acid sequence of SEQ ID NO: 4. shows. In some embodiments, the cytokine comprises the amino acid sequence of SEQ ID NO: 94 compared to the binding affinity of a wild-type IL-7 polypeptide comprising the amino acid sequence of SEQ ID NO: 91 to the IL-7Ra polypeptide. An IL-7 polypeptide that exhibits at least about 50% reduced binding affinity for an IL-7Ra polypeptide. In some embodiments, the IL-7 polypeptide has a binding affinity of SEQ ID NO:4 compared to the binding affinity of a wild-type IL-7 polypeptide comprising the amino acid sequence of SEQ ID NO:91 to an IL-2Rg polypeptide. exhibits a 50% or more reduced binding affinity for an IL-2Rg polypeptide comprising the amino acid sequence. In some embodiments, the cytokine comprises the amino acid sequence of SEQ ID NO: 96 compared to the binding affinity of a wild-type IL-10 polypeptide comprising the amino acid sequence of SEQ ID NO: 95 to the IL-10RA polypeptide. An IL-10 polypeptide that exhibits at least about 50% reduced binding affinity for an IL-10RA polypeptide. In some embodiments, the IL-10 polypeptide has a binding affinity of SEQ ID NO: 97 compared to the binding affinity of a wild-type IL-10 polypeptide comprising the amino acid sequence of SEQ ID NO: 95 to the IL-10RB polypeptide. exhibits at least about 50% increased binding affinity for an IL-10RB polypeptide comprising the amino acid sequence. In some embodiments, the cytokine comprises the amino acid sequence of SEQ ID NO: 93 compared to the binding affinity of a wild-type IL-21 polypeptide comprising the amino acid sequence of SEQ ID NO: 92 to the IL-21R polypeptide. An IL-21 polypeptide that exhibits a 50% or more reduced binding affinity for an IL-21R polypeptide. In some embodiments, the IL-21 polypeptide has a binding affinity of SEQ ID NO: 4 compared to the binding affinity of a wild-type IL-21 polypeptide comprising the amino acid sequence of SEQ ID NO: 92 to an IL-2Rg polypeptide. exhibits at least about 50% reduced binding affinity for an IL-2Rg polypeptide comprising the amino acid sequence. In some embodiments, the cytokine is an IL-2 polypeptide comprising the sequence of SEQ ID NO: 1 with one or more amino acid substitutions relative to SEQ ID NO: 1, and the one or more substitution(s) are: Q11, H16, L18, L19, D20, Q22, R38, F 42, K43, Y45, E62, P65, E68, V69, L72, D84, S87, N88, V91, I92, T123, Q126, S127, I129, and S130 at position SEQ ID NO: 1. Contains substitution(s). In some embodiments, the one or more substitution(s) comprises a F42A amino acid substitution or a F42K amino acid substitution relative to SEQ ID NO:1. In some embodiments, the one or more substitution(s) further comprises an R38A, R38D, R38E, E62Q, E68A, E68Q, E68K, or E68R amino acid substitution to SEQ ID NO:1. In some embodiments, the one or more substitution(s) to SEQ ID NO:1 is H16E, H16D, D20N, M23A, M23R, M23K, S87K, S87A, D84L, D84N, D84V, D84H, D84Y, D84R, D84K, N88A, N88G, N88S, N88T, N8 8R, N88I, N88D, V91A, V91T, V91E, I92A, E95S, E95A, E95R, T123A, T123E, T123K, Further comprising an amino acid substitution of T123Q, Q126A, Q126S, Q126T, Q126E, S127A, S127E, S127K, or S127Q. In some embodiments, the one or more substitution(s) further comprises the amino acid mutation C125A compared to SEQ ID NO:1. In some embodiments, the cytokine is an IL-2 polypeptide comprising an amino acid sequence that is at least about 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 11-90. In some embodiments, the cytokine is an IL-2 polypeptide comprising an amino acid sequence that is at least about 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 43, 48, 52, 49, and 156.In some embodiments, the cytokine has the following set of amino acid mutations (relative to the sequence of SEQ ID NO: 1): R38E and F42A; R38D and F42A; F42A and E62Q; R38A and F42K; F42A, and N88A; R38E, F42A , and N88G; R38E, F42A, and N88D; R38E, F42A, and V91E; R38E, F42A, and D84H; R38E, F42A , and D84K; R38E, F42A, and D84R; H16D, R38E, and F42A; H16E, R38E, and F42A; R38E, F42A, and Q126S; R38D, F42A and N88S; R38D, F42A and N88A; R38D, F42A and N88G; R38D, F42A and N8 8D; R38D, F42A and V91E; R38D, F42A, and D84H; R38D, F42A, and D84K; R38D, F42A, and D84 R; H16D, R38D and F42A; H16E, R38D and F42A; R38D, F42A and Q126S; R38A, F42K, and N88S; R38A, F42K, and N88A; R38A, F42K, and N88G; R38A, F42K, and N88D; R38A, F42K, and V91E; R38A, F42K, and D84H; R38A, F42K, and D84K; R38A, F42K, and D84R; H16D, R38A, and F42K ;H16E, R38A, and F42K;R38A, F42K, and Q126S;F42A, E62Q, and N88S;F42A, E62Q, and N88 A;F42A, E62Q, and N88G;F42A, E62Q, and N88D;F42A, E62Q, and V91E;F42A, E62Q, and D84 H;F42A, E62Q, and D84K;F42A, E62Q, and D84R;H16D, F42A, and E62Q;H16E, F42A, and E62 Q;F42A, E62Q, and Q126S;R38E, F42A, and C125A;R38D, F42A, and C125A;F42A, E62Q, and and C125A; R38A, F42K, and C125A; R38E, F42A, N88S, and C125A; R38E, F42A, N88A, and C12 5A; R38E, F42A, N88G, and C125A; R38E, F42A, N88D, and C125A; R38E, F42A, V91E, and C1 25A; R38E, F42A, D84H, and C125A; R38E, F42A, D84K, and C125A; R38E, F42A, D84R, and C 125A; H16D, R38E, F42A, and C125A; H16E, R38E, F42A, and C125A; R38E, F42A, C125A, and and Q126S; R38D, F42A, N88S, and C125A; R38D, F42A, N88A, and C125A; R38D, F42A, N88G, and C125A; R38D, F42A, N88D, and C125A; R38D, F42A, V91E, and C125A; R38D, F42A, D84H , and C125A; R38D, F42A, D84K, and C125A; R38D, F42A, D84R, and C125A; H16D, R38D, F42 A, and C125A; H16E, R38D, F42A, and C125A; R38D, F42A, C125A, and Q126S; R38A, F42K, N88S, and C125A; R38A, F42K, N88G, and C125A; R38A, F42K, N88D, and C125A; R38A, F42K , N88A, and C125A; R38A, F42K, V91E, and C125A; R38A, F42K, D84H, and C125A; R38A, F42 K, D84K, and C125A; R38A, F42K, D84R, and C125A; H16D, R38A, F42K, and C125A; H16E, R3 8A, F42K, and C125A; R38A, F42K, C125A and Q126S; F42A, E62Q, N88S, and C125A; F42A, E62Q, N88A, and C125A; F42A, E62Q, N88G, and C125A; F42A, E62Q, N88D, and C125A; F42A , E62Q, V91E, and C125A; F42A, E62Q, and D84H, and C125A; F42A, E62Q, and D84K, and C125A ;F42A, E62Q, and D84R, and C125A; H16D, F42A, and E62Q, and C125A; H16E, F42A, and E62Q, and C125A; F42A, E62Q, C125A and Q126S; F42A, N88S, and C125A; F42A, N88A, and C125A; F 42A, N88G, and C125A; F42A, N88D, and C125A; F42A, V91E, and C125A; F42A, D84H, and C12 5A; F42A, D84K, and C125A; F42A, D84R, and C125A; H16D, F42A, and C125A; H16E, F42A, and C125A; and the amino acid sequence of SEQ ID NO: 1, having one of the following: An IL-2 polypeptide comprising.

[0020] In some embodiments, the cytokine is an IL-7 polypeptide, or a functional fragment thereof, or a variant thereof. In some embodiments, the IL-7 polypeptide comprises the sequence of SEQ ID NO: 91 with one or more substitutions to SEQ ID NO: 91. In some embodiments, substitutions at one or more positions include positions K10, Q11, S14, V15, V18, Q22, L35, N36, D74, L77, L80, K81, E84, I88, R133, Q136, E137 , T140, N143, and K144. In some embodiments, the substitution at position K81 is K81A and the substitution at position T140 is K140A. In some embodiments, the cytokine is an IL-10 polypeptide, or a functional fragment thereof, or a variant thereof. In some embodiments, the IL-10 polypeptide comprises the sequence of SEQ ID NO: 95 with one or more substitutions to SEQ ID NO: 95. In some embodiments, the variant IL-10 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 99-112. In some embodiments, the cytokine is an IL-21 polypeptide, or a functional fragment thereof, or a variant thereof. In some embodiments, the IL-21 polypeptide comprises the sequence of SEQ ID NO: 115 with one or more substitutions to SEQ ID NO: 115. In some embodiments, substitutions at one or more positions include positions R5, I8, R9, R11, Q12, I14, D15, D18, Q19, Y23, R65, S70, K72, K73, K75, R76, K77, S80, Q116, and K117, and the position numbering is according to the amino acid sequence of SEQ ID NO: 115.

[0021] In some embodiments, the genetically engineered cells are T cells expressing T cell receptors (TCR-T cells), γδ T cells, T cells derived from pluripotent stem cells, or T cells derived from induced pluripotent stem cells. cells, natural killer cells (NK cells), NK cells derived from pluripotent stem cells or NK cells derived from induced pluripotent stem cells (iPSCs), genetically engineered to express chimeric antigen receptors. Child-engineered T cells (CAR-T cells), CD8+ T cells, CD4+ T cells, cytotoxic T cells, tumor-infiltrating lymphocytes, CAR-NK cells, γδT cells, myeloid cells, hematopoietic cells, hematopoietic cells It contains at least one of stem cells and progenitor cells (HSC), hematopoietic multipotent progenitor cells (MPP), pre-T cell progenitor cells, T cell progenitor cells, and NK cell progenitor cells.

[0022] In some embodiments, the genetically engineered cells are autologous to the subject. In some embodiments, the genetically engineered cells are allogeneic to the subject. In some embodiments, the subject is a human. In some embodiments, the subject has cancer. In some embodiments, the cancer is acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), acute myeloid leukemia, B-cell prolymphocytic leukemia, B-cell acute lymphoblastic leukemia (“BALL”), blastic plasmacytoid dendritic cell tumor, Burkitt lymphoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloid leukemia, chronic or acute leukemia, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), hairy cell leukemia, Hodgkin's disease, malignant lymphoproliferative disease, MALT lymphoma, mantle cell lymphoma, marginal Lymphoma, monoclonal gammaglobulinemia of undetermined significance (MGUS), multiple myeloma, myeloid dysmorphia myelodysplastic syndromes, non-Hodgkin lymphoma (NHL), plasma cell proliferative disorders (including asymptomatic myeloma (including smoldering multiple myeloma or indolent myeloma)), plasmablastic lymphoma , plasmacytoid dendritic cell tumor, plasmacytoma (plasma cell disease, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytoma) primary B-cell lymphoma (PMBC), small cell or large follicular lymphoma, POEMS syndrome (also known as Crow-Fukase syndrome, Takatsuki disease, and PEP syndrome), Splenic marginal zone lymphoma (SMZL), systemic amyloid light chain amyloidosis, T-cell acute lymphoblastic leukemia (TA LL), T-cell lymphoma, transformed follicular lymphoma or Waldenström's macroglobulinemia, mantle cell lymphoma (MCL), transformed follicular lymphoma (TFL), primary mediastinal large B-cell lymphoma (PMBCL) , multiple myeloma, hairy cell lymphoma / leukemia, lung cancer, small cell lung cancer, non-small cell lung (NS CL) cancer, bronchioloalveolar cell lung cancer, squamous cell carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, head and neck cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer cancer of the genital area, skin or intraocular melanoma, thyroid cancer, uterine cancer, gastrointestinal cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, stomach cancer, colon cancer, breast cancer, uterus endometrial cancer, uterine cancer, Carcinoma of the fallopian tubes, carcinoma of the cervix, carcinoma of the vagina, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, Adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureteral cancer, renal cell cancer, carcinoma of the renal pelvis, mesothelioma, bladder cancer , liver cancer, stem cell tumor, hepatocellular carcinoma, Cervical cancer, salivary gland cancer, biliary tract cancer, central nervous system (CNS) tumors, spinal axis tumors, brainstem gliomas, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, Medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, and Ewing's sarcoma, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers.

[0023] One embodiment provides a targeted cytokine construct for use in combination therapy with genetically engineered cells, wherein the fusion protein comprises (i) a cell binding domain, and (ii) a cytokine protein or functional fragment or fragment thereof. The cell-binding domain contains the variant (a) comprising an antibody or antigen-binding fragment thereof specific for the receptor or domain expressed exogenously on the surface of the genetically engineered cell; (b) comprising an antibody or antigen-binding fragment thereof specific for a domain of an antigen-binding protein expressed on the genetically engineered cell; (c) specific for the tag, the tag being co-expressed by or part of a receptor expressed by the genetically engineered cell; (d) is a domain derived from an antigen targeted by the genetically engineered cell; or (e) Contains any combination of (a) to (d).

[0024] In some embodiments, the receptor expressed by the genetically engineered cell is a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some embodiments, the cytokine is a group consisting of IL-2, IL-7, IL-10, IL-15, and IL-21, or a functional fragment or variant thereof, or any combination thereof. selected from. In some embodiments, the cytokine is an IL-2 polypeptide, or a functional fragment thereof, or a variant thereof. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 1 with one or more amino acid substitutions relative to SEQ ID NO: 1, where the one or more substitution(s) are Q11, H16 , L18, L19, D20, Q22, R38, F42, K4 3, a substitution at the position of SEQ ID NO: 1 selected from the group consisting of: (Multiple possible) In some embodiments, the one or more substitution(s) comprises a F42A amino acid substitution or a F42K amino acid substitution relative to SEQ ID NO:1. In some embodiments, the one or more substitution(s) further comprises an R38A, R38D, R38E, E62Q, E68A, E68Q, E68K, or E68R amino acid substitution to SEQ ID NO:1. In some embodiments, the one or more substitution(s) to SEQ ID NO:1 is H16E, H16D, D20N, M23A, M23R, M23K, S87K, S87A, D84L, D84N, D84V, D84H, D84Y, D84R, D84K, N88A, N88G, N88S, N88T, N8 8R, N88I, N88D, V91A, V91T, V91E, I92A, E95S, E95A, E95R, T123A, T123E, T123K, Further comprising an amino acid substitution of T123Q, Q126A, Q126S, Q126T, Q126E, S127A, S127E, S127K, or S127Q. In some embodiments, the one or more substitution(s) further comprises the amino acid mutation C125A compared to SEQ ID NO:1. In some embodiments, the IL-2 polypeptide comprises an amino acid sequence that is at least about 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 11-90. In some embodiments, the following set of amino acid mutations (relative to the sequence of SEQ ID NO: 1): R38E and F42A; R38D and F42A; F42A and E62Q; R38A and F42K; R38E, F42A, and N88S; N88A; R38E, F42A, and N88G ;R38E, F42A, and N88D;R38E, F42A, and V91E;R38E, F42A, and D84H;R38E, F42A, and D84 K; R38E, F42A, and D84R; H16D, R38E, and F42A; H16E, R38E, and F42A; R38E, F42A, and Q126S ;R38D, F42A and N88S;R38D, F42A and N88A;R38D, F42A and N88G;R38D, F42A and N88D;R3 8D, F42A and V91E; R38D, F42A, and D84H; R38D, F42A, and D84K; R38D, F42A, and D84R; H1 6D, R38D and F42A; H16E, R38D and F42A; R38D, F42A and Q126S; R38A, F42K, and N88S; R38 A, F42K, and N88A; R38A, F42K, and N88G; R38A, F42K, and N88D; R38A, F42K, and V91E; R3 8A, F42K, and D84H; R38A, F42K, and D84K; R38A, F42K, and D84R; H16D, R38A, and F42K; H 16E, R38A, and F42K; R38A, F42K, and Q126S; F42A, E62Q, and N88S; F42A, E62Q, and N88A ;F42A, E62Q, and N88G;F42A, E62Q, and N88D;F42A, E62Q, and V91E;F42A, E62Q, and D84 H;F42A, E62Q, and D84K;F42A, E62Q, and D84R;H16D, F42A, and E62Q;H16E, F42A, and E6 2Q; F42A, E62Q, and Q126S; R38E, F42A, and C125A; R38D, F42A, and C125A; F42A, E62Q, and C125A; R38A, F42K, and C125A; R38E, F42A, N88S, and C125A; R38E, F42A, N88A, and C 125A; R38E, F42A, N88G, and C125A; R38E, F42A, N88D, and C125A; R38E, F42A, V91E, and C125A; R38E, F42A, D84H, and C125A; R38E, F42A, D84K, and C125A; R38E, F42A, D84R , and C125A; H16D, R38E, F42A, and C125A; H16E, R38E, F42A, and C125A; R38E, F42A, C 125A and Q126S; R38D, F42A, N88S, and C125A; R38D, F42A, N88A, and C125A; R38D, F42A , N88G, and C125A; R38D, F42A, N88D, and C125A; R38D, F42A, V91E, and C125A; R38D, F4 2A, D84H, and C125A; R38D, F42A, D84K, and C125A; R38D, F42A, D84R, and C125A; H16D, R38D, F42A, and C125A; H16E, R38D, F42A, and C125A; R38D, F42A, C125A, and Q126S; R3 8A, F42K, N88S, and C125A; R38A, F42K, N88G, and C125A; R38A, F42K, N88D, and C125A; R38A, F42K, N88A, and C125A; R38A, F42K, V91E, and C125A; R38A, F42K, D84H, and C125 A; R38A, F42K, D84K, and C125A; R38A, F42K, D84R, and C125A; H16D, R38A, F42K, and C1 25A; H16E, R38A, F42K, and C125A; R38A, F42K, C125A and Q126S; F42A, E62Q, N88S, and C125A; F42A, E62Q, N88A, and C125A; F42A, E62Q, N88G, and C125A; F42A, E62Q, N88D, and C125A; F42A, E62Q, V91E, and C125A; F42A, E62Q, and D84H, and C125A; F42A, E62Q, and and D84K, and C125A; F42A, E62Q, and D84R, and C125A; H16D, F42A, and E62Q, and C125A; H16 E, F42A, and E62Q, and C125A; F42A, E62Q, C125A, and Q126S; F42A, N88S, and C125A; F42A , N88A, and C125A; F42A, N88G, and C125A; F42A, N88D, and C125A; F42A, V91E, and C125A ;F42A, D84H, and C125A;F42A, D84K, and C125A;F42A, D84R, and C125A;H16D, F42A, and The amino acid sequence of SEQ ID NO: 1 having one of C125A; H16E, F42A, and C125A; and F42A, C125A, and Q126S. In some embodiments, the cytokine is an IL-7 polypeptide comprising the sequence of SEQ ID NO: 91 with one or more substitutions to SEQ ID NO: 91. In some embodiments, substitutions at one or more positions include positions K10, Q11, S14, V15, V18, Q22, L35, N36, D74, L77, L80, K81, E84, I88, R133, Q136, E137 , T140, N143, and K144. In some embodiments, the substitutions at positions K81 and T140 are K81A and T140A. In some embodiments, the cytokine is an IL-10 polypeptide comprising the sequence of SEQ ID NO: 95, with one or more substitutions to SEQ ID NO: 95. In some embodiments, the IL-10 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 99-112. In some embodiments, the cytokine is an IL-21 polypeptide, or a functional fragment thereof, or a variant thereof. In some embodiments, the IL-21 polypeptide comprises the sequence of SEQ ID NO: 115 with one or more substitutions to SEQ ID NO: 115. In some embodiments, the IL-21 polypeptide has the sequence of SEQ ID NO: 115, or positions R5, I8, R9, R11, Q12, I14, D15, D18, Q19, Y23, R65, S70, K72, K73, K7 5, R76, K77, S80, Q116, and K117, the numbering of the positions being according to the amino acid sequence of SEQ ID NO: 115. be. In some embodiments, the tag co-expressed by the genetically engineered cell is an EGFRt tag.In some embodiments, the antigen targeted by the genetically engineered cells is a neoepitope from a tumor-associated antigen, TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvllll, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, Lew isY, CD24, PDGFR-β, SSEA-4, CD20, folate receptor α, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, prostase, PAP, ELF2M, ephrinB2, IGF- I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor β, TEM1 / CD248, TEM7R , CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51 E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 variant, prostein, survivin and telomerase, PCTA-1 / galectin 8, Melan A / MART1, Ras variant , hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1 B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut selected from the group consisting of hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, or IGLL1.

[0025] In some embodiments, the genetically engineered cells are T cells expressing the αβ T cell receptor, γδ T cells, NK T cells, regulatory T cells, T cells derived from pluripotent stem cells or T cells derived from induced pluripotent stem cells, natural killer cells (NK cells), NK cells derived from pluripotent stem cells or induced pluripotent stem cells ( iPSC-derived NK cells, T cells genetically engineered to express chimeric antigen receptors (CAR-T cells), T cells derived from T cells genetically engineered to express cell receptors (TCR-T cells), CD8+ T cells, CD4+ T cells, cytotoxic T cells, tumor-infiltrating lymphocytes, and chimeric antigen receptors. NK cells genetically engineered to express chimeric antigen receptors (CAR-NK cells), NK cells genetically engineered to express chimeric antigen receptors T cells (CAR-NK T cells), bone marrow cells, hematopoietic cells, hematopoietic stem and progenitor cells (HSC), hematopoietic multipotent progenitor cells (MPP), pre-T cell progenitors, T cell progenitors, NK cell progenitors containing at least one of the cells.

[0026] One embodiment provides a method of treating cancer, the method comprising administering a targeted cytokine construct according to any one of claims 147-168 in combination therapy with genetically engineered cells. Including. Some embodiments further include administering an additional therapeutic agent. In some embodiments, the cancer is acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), acute myeloid leukemia, B-cell prolymphocytic leukemia, B-cell acute lymphoblastic leukemia (“BALL”), blastic plasmacytoid dendritic cell tumor, Burkitt lymphoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloid leukemia, chronic or acute leukemia, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), hairy cell leukemia, Hodgkin's disease, malignant lymphoproliferative disease, MALT lymphoma, mantle cell lymphoma, marginal Lymphoma, monoclonal gammaglobulinemia of undetermined significance (MGUS), multiple myeloma, myeloid dysmorphia myelodysplastic syndromes, non-Hodgkin lymphoma (NHL), plasma cell proliferative disorders (including asymptomatic myeloma (including smoldering multiple myeloma or indolent myeloma)), plasmablastic lymphoma , plasmacytoid dendritic cell tumor, plasmacytoma (plasma cell disease, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytoma) primary B-cell lymphoma (PMBC), small cell or large follicular lymphoma, POEMS syndrome (also known as Crow-Fukase syndrome, Takatsuki disease, and PEP syndrome), Splenic marginal zone lymphoma (SMZL), systemic amyloid light chain amyloidosis, T-cell acute lymphoblastic leukemia (TA LL), T-cell lymphoma, transformed follicular lymphoma or Waldenström's macroglobulinemia, mantle cell lymphoma (MCL), transformed follicular lymphoma (TFL), primary mediastinal large B-cell lymphoma (PMBCL) , multiple myeloma, hairy cell lymphoma / leukemia, lung cancer, small cell lung cancer, non-small cell lung (NS CL) cancer, bronchioloalveolar cell lung cancer, squamous cell carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, head and neck cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer cancer of the genital area, skin or intraocular melanoma, thyroid cancer, uterine cancer, gastrointestinal cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, stomach cancer, colon cancer, breast cancer, uterus endometrial cancer, uterine cancer, Carcinoma of the fallopian tubes, carcinoma of the cervix, carcinoma of the vagina, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, Adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureteral cancer, renal cell cancer, carcinoma of the renal pelvis, mesothelioma, bladder cancer , liver cancer, stem cell tumor, hepatocellular carcinoma, Cervical cancer, salivary gland cancer, biliary tract cancer, central nervous system (CNS) tumors, spinal axis tumors, brainstem gliomas, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, Medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, and Ewing's sarcoma, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers.

[0027] One embodiment is a pharmaceutical composition comprising a targeted cytokine construct according to the present disclosure and at least one of a pharmaceutically acceptable excipient, carrier, or diluent, or any combination thereof. Provide. Some embodiments further include a population of genetically engineered cells. One embodiment provides a cell therapy kit having a pharmaceutical composition comprising a targeted cytokine construct according to the present disclosure and specified instructions for administering the targeted cytokine construct to a subject. In some embodiments, the cell therapy kit further comprises a pharmaceutical composition comprising a population of genetically engineered cells and instructions specifying for administering the population of genetically engineered cells to a subject. . In some embodiments, the pharmaceutical composition comprising the targeted cytokine construct and the population of genetically engineered cells are for sequential or simultaneous administration. Inclusion by reference

[0028] All publications, patents, and patent applications mentioned herein are mentioned as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Incorporated herein by reference to the extent.

[0029] The novel features of the disclosure are pointed out with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure may be gained by reference to the following detailed description and accompanying drawings that describe illustrative embodiments in which the principles of the present disclosure are utilized. [Brief explanation of the drawing]

[0030] [Figure 1] A wild-type cytokine protein (e.g., a mutein cytokine) that has a cell-binding domain that recognizes a target antigen (domain or receptor) selectively expressed on genetically engineered cells (e.g., CAR-transduced T cells). , or a fragment or variant thereof, in some examples, mutant IL-2 (mutant IL2, or mutein A targeted cytokine construct comprising a cytokine polypeptide (also referred to as in-IL-2, or mutein-IL2), or a non-targeted cytokine polypeptide, in a genetically engineered cell that expresses the target antigen or in another cell that does not express the target antigen. FIG. 2 illustrates the general mechanism of how cells of the human body work to stimulate cells. [Figure 2-1] Assay results demonstrating preferential activity of an exemplary targeted cytokine construct containing a cell-binding domain that is an anti-CAR antibody, specifically genetically engineered cells generated from one donor (CAR-T cells) ) with the indicated constructs: non-CAR-binding antibody fused to the IL-2Rβγ-binding polypeptide IL2m1 (control IL2m1), CAR-binding antibody fused to the IL-2Rβγ-binding polypeptide IL2m1 (anti-CAR FIG. 3 shows the results of a phosphorylated STAT5 assay using IL2m1) or a non-targeting cytokine (IL-2) as a control. The percentage of pSTAT5 expressing cells is shown for either CAR+ or CAR- T cells at the indicated concentrations. [Figure 2-2]Assay results demonstrating preferential activity of an exemplary targeted cytokine construct containing a cell-binding domain that is an anti-CAR antibody, specifically genetically engineered cells generated from one donor (CAR-T cells) ) with the indicated constructs: non-CAR-binding antibody fused to the IL-2Rβγ-binding polypeptide IL2m1 (control IL2m1), CAR-binding antibody fused to the IL-2Rβγ-binding polypeptide IL2m1 (anti-CAR FIG. 3 shows the results of a phosphorylated STAT5 assay using IL2m1) or a non-targeting cytokine (IL-2) as a control. The percentage of pSTAT5 expressing cells is shown for either CAR+ or CAR- T cells at the indicated concentrations. [Figure 2-3] Assay results demonstrating preferential activity of an exemplary targeted cytokine construct containing a cell-binding domain that is an anti-CAR antibody, specifically genetically engineered cells generated from one donor (CAR-T cells) ) with the indicated constructs: non-CAR-binding antibody fused to the IL-2Rβγ-binding polypeptide IL2m1 (control IL2m1), CAR-binding antibody fused to the IL-2Rβγ-binding polypeptide IL2m1 (anti-CAR FIG. 3 shows the results of a phosphorylated STAT5 assay using IL2m1) or a non-targeting cytokine (IL-2) as a control. The percentage of pSTAT5 expressing cells is shown for either CAR+ or CAR- T cells at the indicated concentrations. [Figure 3-1] Results of assays demonstrating preferential activity of an exemplary targeted cytokine construct containing a cell-binding domain that is an anti-CAR antibody, specifically shown in Figure 2. Genetically engineered cells generated from a different donor (CAR - T cells) with the indicated constructs: a non-CAR-binding antibody fused to the IL-2Rβγ-binding polypeptide IL2m2 (control IL2m2), a CAR-binding antibody fused to the IL-2Rβγ-binding polypeptide IL2m2 (anti-CAR FIG. 3 shows the results of a phosphorylated STAT5 assay using IL2m2) or a non-targeting cytokine (IL-2) as a control. The percentage of pSTAT5 expressing cells is shown for either CAR+ or CAR- T cells at the indicated concentrations. [Figure 3-2] Results of assays demonstrating preferential activity of an exemplary targeted cytokine construct containing a cell-binding domain that is an anti-CAR antibody, specifically shown in Figure 2. Genetically engineered cells generated from a different donor (CAR - T cells) with the indicated constructs: a non-CAR-binding antibody fused to the IL-2Rβγ-binding polypeptide IL2m2 (control IL2m2), a CAR-binding antibody fused to the IL-2Rβγ-binding polypeptide IL2m2 (anti-CAR FIG. 3 shows the results of a phosphorylated STAT5 assay using IL2m2) or a non-targeting cytokine (IL-2) as a control. The percentage of pSTAT5 expressing cells is shown for either CAR+ or CAR- T cells at the indicated concentrations. [Figure 3-3] Results of assays demonstrating preferential activity of an exemplary targeted cytokine construct containing a cell-binding domain that is an anti-CAR antibody, specifically shown in Figure 2. Genetically engineered cells generated from a different donor (CAR - T cells) with the indicated constructs: a non-CAR-binding antibody fused to the IL-2Rβγ-binding polypeptide IL2m2 (control IL2m2), a CAR-binding antibody fused to the IL-2Rβγ-binding polypeptide IL2m2 (anti-CAR FIG. 3 shows the results of a phosphorylated STAT5 assay using IL2m2) or a non-targeting cytokine (IL-2) as a control. The percentage of pSTAT5 expressing cells is shown for either CAR+ or CAR- T cells at the indicated concentrations. [Figure 4-1] achieved in long-term culture with either a non-targeting cytokine (IL-2) or a targeted cytokine construct containing an anti-CAR antibody cell-binding domain and an IL-2Rβγ-binding polypeptide (IL2m1 or IL2m2). FIG. 3 shows the expression frequency (left panel) and number (right panel) of CAR+ T cells in culture. Anti-CAR IL2m1, anti-CAR IL2m2, control IL-2, or cytokine-free cell binding domain anti-CAR antibodies were cultured at a concentration of 0.1 nM for up to 10 days. The number and expression frequency of CAR+ T cells were determined by flow cytometry at the indicated time points. [Figure 4-2] achieved in long-term culture with either a non-targeting cytokine (IL-2) or a targeted cytokine construct containing an anti-CAR antibody cell-binding domain and an IL-2Rβγ-binding polypeptide (IL2m1 or IL2m2). FIG. 3 shows the expression frequency (left panel) and number (right panel) of CAR+ T cells in culture. Anti-CAR IL2m1, anti-CAR IL2m2, control IL-2, or cytokine-free cell binding domain anti-CAR antibodies were cultured at a concentration of 0.1 nM for up to 10 days. The number and expression frequency of CAR+ T cells were determined by flow cytometry at the indicated time points. [Figure 5A] FIG. 3 shows an exemplary design of a CAR-targeting cytokine construct to target CAR-transduced gene-engineered T cells in accordance with the present disclosure. [Figure 5B] FIG. 3 shows an exemplary design of a TCR-targeted cytokine construct to target TCR-transduced engineered T cells in accordance with the present disclosure. [Figure 6A] Mature IL-2 (Figure 6A, SEQ ID NO: 1), IL-2Rα (Figure 6B, SEQ ID NO: 2), IL-2Rβ (Figure 6C, SEQ ID NO: 3), and IL-2Rγ (Figure 6D, SEQ ID NO: 4) polypeptides It is a figure showing the amino acid sequence of. [Figure 6B] Mature IL-2 (Figure 6A, SEQ ID NO: 1), IL-2Rα (Figure 6B, SEQ ID NO: 2), IL-2Rβ (Figure 6C, SEQ ID NO: 3), and IL-2Rγ (Figure 6D, SEQ ID NO: 4) polypeptides It is a figure showing the amino acid sequence of. [Figure 6C] Mature IL-2 (Figure 6A, SEQ ID NO: 1), IL-2Rα (Figure 6B, SEQ ID NO: 2), IL-2Rβ (Figure 6C, SEQ ID NO: 3), and IL-2Rγ (Figure 6D, SEQ ID NO: 4) polypeptides It is a figure showing the amino acid sequence of. [Figure 6D] Mature IL-2 (Figure 6A, SEQ ID NO: 1), IL-2Rα (Figure 6B, SEQ ID NO: 2), IL-2Rβ (Figure 6C, SEQ ID NO: 3), and IL-2Rγ (Figure 6D, SEQ ID NO: 4) polypeptides It is a figure showing the amino acid sequence of. [Figure 7] FIG. 2 shows the amino acid sequence of wild-type mature IL-2 polypeptide (SEQ ID NO: 1). "X" indicates an amino acid that is substituted with another amino acid in the sequence of a wild-type IL-2 polypeptide to generate a variant IL-2 polypeptide of the present disclosure. [Figure 8A] 1A and 1B are schematic illustrations of three different exemplary CAR-targeting cytokine constructs according to the present disclosure, respectively. Figure 8A shows a bivalent antibody with a mutant IL-2 polypeptide fused to the C-terminus of one of the heavy chains. FIG. 8B shows a monovalent antibody with a mutant IL-2 polypeptide fused to the C-terminus of a heavy chain lacking the variable region. FIG. 8C shows a monovalent antibody with a mutant IL-2 polypeptide fused to the N-terminus of a heavy chain lacking the variable region. [Figure 8B] 1A and 1B are schematic illustrations of three different exemplary CAR-targeting cytokine constructs according to the present disclosure, respectively. Figure 8A shows a bivalent antibody with a mutant IL-2 polypeptide fused to the C-terminus of one of the heavy chains. FIG. 8B shows a monovalent antibody with a mutant IL-2 polypeptide fused to the C-terminus of a heavy chain lacking the variable region. FIG. 8C shows a monovalent antibody with a mutant IL-2 polypeptide fused to the N-terminus of a heavy chain lacking the variable region. [Figure 8C] 1A and 1B are schematic illustrations of three different exemplary CAR-targeting cytokine constructs according to the present disclosure, respectively. Figure 8A shows a bivalent antibody with a mutant IL-2 polypeptide fused to the C-terminus of one of the heavy chains. FIG. 8B shows a monovalent antibody with a mutant IL-2 polypeptide fused to the C-terminus of a heavy chain lacking the variable region. FIG. 8C shows a monovalent antibody with a mutant IL-2 polypeptide fused to the N-terminus of a heavy chain lacking the variable region. [Figure 9A] Mature IL-10 (Figure 9A, SEQ ID NO: 95), IL-10RA (Figure 9B, SEQ ID NO: 96), IL-10RB (Figure 9C, SEQ ID NO: 97), and mature monomeric IL-10 (Figure 9D, SEQ ID NO: 97). 98) is a diagram showing the amino acid sequence of the polypeptide No. 98). [Figure 9B] Mature IL-10 (Figure 9A, SEQ ID NO: 95), IL-10RA (Figure 9B, SEQ ID NO: 96), IL-10RB (Figure 9C, SEQ ID NO: 97), and mature monomeric IL-10 (Figure 9D, SEQ ID NO: 97). 98) is a diagram showing the amino acid sequence of the polypeptide No. 98). [Figure 9C] Mature IL-10 (Figure 9A, SEQ ID NO: 95), IL-10RA (Figure 9B, SEQ ID NO: 96), IL-10RB (Figure 9C, SEQ ID NO: 97), and mature monomeric IL-10 (Figure 9D, SEQ ID NO: 97). 98) is a diagram showing the amino acid sequence of the polypeptide No. 98). [Figure 9D] Mature IL-10 (Figure 9A, SEQ ID NO: 95), IL-10RA (Figure 9B, SEQ ID NO: 96), IL-10RB (Figure 9C, SEQ ID NO: 97), and mature monomeric IL-10 (Figure 9D, SEQ ID NO: 97). 98) is a diagram showing the amino acid sequence of the polypeptide No. 98). [Figure 10A] FIG. 10 shows the amino acid sequences of wild-type mature IL-10 polypeptide (FIG. 10A, SEQ ID NO: 95) and mature monomeric IL-10 (FIG. 10B, SEQ ID NO: 98). "X" indicates an amino acid that is substituted with another amino acid in the sequence of a wild-type IL-10 polypeptide to generate a variant IL-10 polypeptide of the present disclosure. [Figure 10B]FIG. 10 shows the amino acid sequences of wild-type mature IL-10 polypeptide (FIG. 10A, SEQ ID NO: 95) and mature monomeric IL-10 (FIG. 10B, SEQ ID NO: 98). "X" indicates an amino acid that is substituted with another amino acid in the sequence of a wild-type IL-10 polypeptide to generate a variant IL-10 polypeptide of the present disclosure. [Figure 11A-1] Figure 11 shows the amino acid sequences of wild type mature IL-10 polypeptide (Figure 1 IA, SEQ ID NO: 95) and mature monomeric IL-10 (Figure 1 IB, SEQ ID NO: 98). White boxes indicate residues substituted to reduce the affinity of IL-10 for IL-10RA, gray boxes indicate residues substituted to modify the affinity of IL-10 for IL-10RB. Substituted residues are indicated. The substituted amino acid in place of the wild type residue at each position is indicated. [Figure 11A-2] Figure 11 shows the amino acid sequences of wild type mature IL-10 polypeptide (Figure 1 IA, SEQ ID NO: 95) and mature monomeric IL-10 (Figure 1 IB, SEQ ID NO: 98). White boxes indicate residues substituted to reduce the affinity of IL-10 for IL-10RA, gray boxes indicate residues substituted to modify the affinity of IL-10 for IL-10RB. Substituted residues are indicated. The substituted amino acid in place of the wild type residue at each position is indicated. [Figure 11B-1] Figure 11 shows the amino acid sequences of wild-type mature IL-10 polypeptide (Figure 1 IA, SEQ ID NO: 95) and mature monomeric IL-10 (Figure 1 IB, SEQ ID NO: 98). White boxes indicate residues substituted to reduce the affinity of IL-10 for IL-10RA, gray boxes indicate residues substituted to modify the affinity of IL-10 for IL-10RB. Substituted residues are indicated. The substituted amino acid in place of the wild type residue at each position is indicated. [Figure 11B-2] Figure 11 shows the amino acid sequences of wild type mature IL-10 polypeptide (Figure 1 IA, SEQ ID NO: 95) and mature monomeric IL-10 (Figure 1 IB, SEQ ID NO: 98). White boxes indicate residues substituted to reduce the affinity of IL-10 for IL-10RA, gray boxes indicate residues substituted to modify the affinity of IL-10 for IL-10RB. Substituted residues are indicated. The substituted amino acid in place of the wild type residue at each position is indicated. [Figure 12A] (CAR cell-binding domain, an anti-scFv antibody (targeting scFv expressed by T cells), and IL-2 muteins (m1 (Figure 12B), m2 (Figure 12C), m3 (Figure 12D), m4 (Figure 12E) of some exemplary targeting cytokine constructs, including m5 (Figure 12F)). Assay results demonstrating preferential activity, specifically, genetically engineered cells generated from one donor (CAR-T cells (CAR+) in a mixture with CAR-nonexpressing T cells (CAR-)) FIG. 3 shows the results of a phosphorylated STAT5 assay when cells were incubated with the indicated constructs. The percentage of pSTAT5 expressing cells is shown for either CAR+ or CAR- T cells at the indicated concentrations. Figure 12A is a diagram of the constructs tested in this assay. [Figure 12B] (CAR cell-binding domain, an anti-scFv antibody (targeting scFv expressed by T cells), and IL-2 muteins (m1 (Figure 12B), m2 (Figure 12C), m3 (Figure 12D), m4 (Figure 12E) of some exemplary targeting cytokine constructs, including m5 (Figure 12F)). Assay results demonstrating preferential activity, specifically, genetically engineered cells generated from one donor (CAR-T cells (CAR+) in a mixture with CAR-nonexpressing T cells (CAR-)) FIG. 3 shows the results of a phosphorylated STAT5 assay when cells were incubated with the indicated constructs. The percentage of pSTAT5 expressing cells is shown for either CAR+ or CAR- T cells at the indicated concentrations. Figure 12A is a diagram of the constructs tested in this assay. [Figure 12C] (CAR cell-binding domain, an anti-scFv antibody (targeting scFv expressed by T cells), and IL-2 muteins (m1 (Figure 12B), m2 (Figure 12C), m3 (Figure 12D), m4 (Figure 12E) of some exemplary targeting cytokine constructs, including m5 (Figure 12F)). Assay results demonstrating preferential activity, specifically, genetically engineered cells generated from one donor (CAR-T cells (CAR+) in a mixture with CAR-nonexpressing T cells (CAR-)) FIG. 3 shows the results of a phosphorylated STAT5 assay when cells were incubated with the indicated constructs. The percentage of pSTAT5 expressing cells is shown for either CAR+ or CAR- T cells at the indicated concentrations. Figure 12A is a diagram of the constructs tested in this assay. [Figure 12D] (CAR cell-binding domain, an anti-scFv antibody (targeting scFv expressed by T cells), and IL-2 muteins (m1 (Figure 12B), m2 (Figure 12C), m3 (Figure 12D), m4 (Figure 12E) of some exemplary targeting cytokine constructs, including m5 (Figure 12F)). Assay results demonstrating preferential activity, specifically, genetically engineered cells generated from one donor (CAR-T cells (CAR+) in a mixture with CAR-nonexpressing T cells (CAR-)) FIG. 3 shows the results of a phosphorylated STAT5 assay when cells were incubated with the indicated constructs. The percentage of pSTAT5 expressing cells is shown for either CAR+ or CAR- T cells at the indicated concentrations. Figure 12A is a diagram of the constructs tested in this assay. [Figure 12E] (CAR cell-binding domain, an anti-scFv antibody (targeting scFv expressed by T cells), and IL-2 muteins (m1 (Figure 12B), m2 (Figure 12C), m3 (Figure 12D), m4 (Figure 12E) of some exemplary targeting cytokine constructs, including m5 (Figure 12F)). Assay results demonstrating preferential activity, specifically, genetically engineered cells generated from one donor (CAR-T cells (CAR+) in a mixture with CAR-nonexpressing T cells (CAR-)) FIG. 3 shows the results of a phosphorylated STAT5 assay when cells were incubated with the indicated constructs. The percentage of pSTAT5 expressing cells is shown for either CAR+ or CAR- T cells at the indicated concentrations. Figure 12A is a diagram of the constructs tested in this assay. [Figure 12F] (CAR cell-binding domain, an anti-scFv antibody (targeting scFv expressed by T cells), and IL-2 muteins (m1 (Figure 12B), m2 (Figure 12C), m3 (Figure 12D), m4 (Figure 12E) of some exemplary targeting cytokine constructs, including m5 (Figure 12F)). Assay results demonstrating preferential activity, specifically, genetically engineered cells generated from one donor (CAR-T cells (CAR+) in a mixture with CAR-nonexpressing T cells (CAR-)) FIG. 3 shows the results of a phosphorylated STAT5 assay when cells were incubated with the indicated constructs. The percentage of pSTAT5 expressing cells is shown for either CAR+ or CAR- T cells at the indicated concentrations. Figure 12A is a diagram of the constructs tested in this assay. [Figure 13A] The cell-binding domain, which is an anti-tag molecule (targets the tag molecule selectively expressed by CAR-T cells), and the IL-2 mutein (m1 (Figure 13B), m2 (Figure 13C), m3 (Figure 13D) ), m4 (Figure 13E), m5 (Figure 13F)). Assay results demonstrating the preferential activity of the construct specifically showed that genetically engineered cells generated from a single donor (CAR-T cells (CAR-T cells) in a mixture with non-CAR-expressing T cells (CAR-) CAR+)) were cultured with the indicated constructs and show the results of a phosphorylated STAT5 assay. The percentage of pSTAT5 expressing cells is shown for either CAR+ or CAR- T cells at the indicated concentrations. Figure 13A is a diagram of the constructs tested in this assay. [Figure 13B] The cell-binding domain, which is an anti-tag molecule (targets the tag molecule selectively expressed by CAR-T cells), and the IL-2 mutein (m1 (Figure 13B), m2 (Figure 13C), m3 (Figure 13D) ), m4 (Figure 13E), m5 (Figure 13F)). Assay results demonstrating preferential activity of the construct specifically showed that genetically engineered cells generated from a single donor (CAR-T cells (CAR-T cells) in a mixture with non-CAR-expressing T cells (CAR-) CAR+)) were cultured with the indicated constructs and show the results of a phosphorylated STAT5 assay. The percentage of pSTAT5 expressing cells is shown for either CAR+ or CAR- T cells at the indicated concentrations. Figure 13A is a diagram of the constructs tested in this assay. [Figure 13C] The cell-binding domain, which is an anti-tag molecule (targets the tag molecule selectively expressed by CAR-T cells), and the IL-2 mutein (m1 (Figure 13B), m2 (Figure 13C), m3 (Figure 13D) ), m4 (Figure 13E), m5 (Figure 13F)). Assay results demonstrating preferential activity of the construct specifically showed that genetically engineered cells generated from a single donor (CAR-T cells (CAR-T cells) in a mixture with non-CAR-expressing T cells (CAR-) CAR+)) were cultured with the indicated constructs and show the results of a phosphorylated STAT5 assay. The percentage of pSTAT5 expressing cells is shown for either CAR+ or CAR- T cells at the indicated concentrations. Figure 13A is a diagram of the constructs tested in this assay. [Figure 13D]The cell-binding domain, which is an anti-tag molecule (targets the tag molecule selectively expressed by CAR-T cells), and the IL-2 mutein (m1 (Figure 13B), m2 (Figure 13C), m3 (Figure 13D) ), m4 (Figure 13E), m5 (Figure 13F)). Assay results demonstrating the preferential activity of the construct specifically showed that genetically engineered cells generated from one donor (CAR-T cells (CAR-T cells) in a mixture with non-CAR-expressing T cells (CAR-) CAR+)) were cultured with the indicated constructs and show the results of a phosphorylated STAT5 assay. The percentage of pSTAT5 expressing cells is shown for either CAR+ or CAR- T cells at the indicated concentrations. Figure 13A is a diagram of the constructs tested in this assay. [Figure 13E] The cell-binding domain, which is an anti-tag molecule (targets the tag molecule selectively expressed by CAR-T cells), and the IL-2 mutein (m1 (Figure 13B), m2 (Figure 13C), m3 (Figure 13D) ), m4 (Figure 13E), m5 (Figure 13F)). Assay results demonstrating the preferential activity of the construct specifically showed that genetically engineered cells generated from one donor (CAR-T cells (CAR-T cells) in a mixture with non-CAR-expressing T cells (CAR-) CAR+)) were cultured with the indicated constructs and show the results of a phosphorylated STAT5 assay. The percentage of pSTAT5 expressing cells is shown for either CAR+ or CAR- T cells at the indicated concentrations. Figure 13A is a diagram of the constructs tested in this assay. [Figure 13F] The cell-binding domain, which is an anti-tag molecule (targets the tag molecule selectively expressed by CAR-T cells), and the IL-2 mutein (m1 (Figure 13B), m2 (Figure 13C), m3 (Figure 13D) ), m4 (Figure 13E), m5 (Figure 13F)). Assay results demonstrating the preferential activity of the construct specifically showed that genetically engineered cells generated from one donor (CAR-T cells (CAR-T cells) in a mixture with non-CAR-expressing T cells (CAR-) CAR+)) were cultured with the indicated constructs and show the results of a phosphorylated STAT5 assay. The percentage of pSTAT5 expressing cells is shown for either CAR+ or CAR- T cells at the indicated concentrations. Figure 13A is a diagram of the constructs tested in this assay. [Figure 14A] Cell-binding domain anti-tag antibodies (targeting tags expressed by CAR-T cells) and IL-2 muteins (m1 (Figure 14B), m2 (Figure 14C), m3 (Figure 14D), m4 ( Some exemplary examples include Figure 14E), m5 (Figure 14F)) Results of assays demonstrating preferential activity of targeted cytokine constructs, specifically, genetically engineered cells (CAR-T cells) generated from one donor were cultured with the indicated constructs. , is a diagram showing the results of phosphorylated STAT5 assay. The percentage of pSTAT5 expressing cells is shown for either CAR+ or CAR- T cells at the indicated concentrations. Figure 14A is a diagram of the constructs tested in this assay. [Figure 14B] Cell-binding domain anti-tag antibodies (targeting tags expressed by CAR-T cells) and IL-2 muteins (m1 (Figure 14B), m2 (Figure 14C), m3 (Figure 14D), m4 ( Some exemplary examples include Figure 14E), m5 (Figure 14F)) Results of assays demonstrating preferential activity of targeted cytokine constructs, specifically, genetically engineered cells (CAR-T cells) generated from one donor were cultured with the indicated constructs. , is a diagram showing the results of phosphorylated STAT5 assay. The percentage of pSTAT5 expressing cells is shown for either CAR+ or CAR- T cells at the indicated concentrations. Figure 14A is a diagram of the constructs tested in this assay. [Figure 14C] Cell-binding domain anti-tag antibodies (targeting tags expressed by CAR-T cells) and IL-2 muteins (m1 (Figure 14B), m2 (Figure 14C), m3 (Figure 14D), m4 ( Some exemplary examples include Figure 14E), m5 (Figure 14F)) Results of assays demonstrating preferential activity of targeted cytokine constructs, specifically, genetically engineered cells (CAR-T cells) generated from one donor were cultured with the indicated constructs. , is a diagram showing the results of phosphorylated STAT5 assay. The percentage of pSTAT5 expressing cells is shown for either CAR+ or CAR- T cells at the indicated concentrations. Figure 14A is a diagram of the constructs tested in this assay. [Figure 14D] Cell-binding domain anti-tag antibodies (targeting tags expressed by CAR-T cells) and IL-2 muteins (m1 (Figure 14B), m2 (Figure 14C), m3 (Figure 14D), m4 ( Some exemplary examples include Figure 14E), m5 (Figure 14F)) Results of assays demonstrating preferential activity of targeted cytokine constructs, specifically, genetically engineered cells (CAR-T cells) generated from one donor were cultured with the indicated constructs. , is a diagram showing the results of phosphorylated STAT5 assay. The percentage of pSTAT5 expressing cells is shown for either CAR+ or CAR- T cells at the indicated concentrations. Figure 14A is a diagram of the constructs tested in this assay. [Figure 14E] Cell-binding domain anti-tag antibodies (targeting tags expressed by CAR-T cells) and IL-2 muteins (m1 (Figure 14B), m2 (Figure 14C), m3 (Figure 14D), m4 ( Some exemplary examples include Figure 14E), m5 (Figure 14F)) Results of assays demonstrating preferential activity of targeted cytokine constructs, specifically, genetically engineered cells (CAR-T cells) generated from one donor were cultured with the indicated constructs. , is a diagram showing the results of phosphorylated STAT5 assay. The percentage of pSTAT5 expressing cells is shown for either CAR+ or CAR- T cells at the indicated concentrations. Figure 14A is a diagram of the constructs tested in this assay. [Figure 14F] Cell-binding domain anti-tag antibodies (targeting tags expressed by CAR-T cells) and IL-2 muteins (m1 (Figure 14B), m2 (Figure 14C), m3 (Figure 14D), m4 ( Some exemplary examples include Figure 14E), m5 (Figure 14F)) Results of assays demonstrating preferential activity of targeted cytokine constructs, specifically, genetically engineered cells (CAR-T cells) generated from one donor were cultured with the indicated constructs. , is a diagram showing the results of phosphorylated STAT5 assay. The percentage of pSTAT5 expressing cells is shown for either CAR+ or CAR- T cells at the indicated concentrations. Figure 14A is a diagram of the constructs tested in this assay. [Figure 15A] FIG. 3 shows the results of an assay demonstrating preferential binding of an exemplary targeted cytokine construct containing a cell-binding domain that is an anti-tag antibody (targeting a tag expressed by TCR-transduced T cells). FIG. 15A is a diagram of a targeting cytokine construct containing the anti-tag antibody used in FIG. 15B with an IL-2 mutein. [Figure 15B] FIG. 3 shows the results of an assay demonstrating preferential binding of an exemplary targeted cytokine construct containing a cell-binding domain that is an anti-tag antibody (targeting a tag expressed by TCR-transduced T cells). FIG. 15A is a diagram of a targeting cytokine construct containing the anti-tag antibody used in FIG. 15B with an IL-2 mutein. [Details for carrying out the invention]

[0031] specific definition As used in this specification and the claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "chimeric transmembrane receptor polypeptide" includes multiple chimeric transmembrane receptor polypeptides.

[0032] The term "about" or "approximately" means within a tolerance range of a particular value as determined by one of ordinary skill in the art; Partly depends. For example, "about" can mean within 1 standard deviation or more than 1 standard deviation, according to practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with regard to biological systems or biological processes, the term may mean within an order of magnitude, preferably within 5 times, more preferably within 2 times the value. When a particular value is recited in this application and claims, the term "about" should be assumed to mean within a tolerance range for the particular value, unless stated otherwise.

[0033] As used herein, "cell" generally refers to a biological cell. A cell may be the basic structural, functional, and / or biological unit of an organism. A cell can be derived from any organism that has one or more cells. Some non-limiting examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, unicellular eukaryotic cells, protozoan cells, cells of plant origin (e.g., plant crops, fruits, vegetables, Grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hay, potatoes, cotton, taima, tobacco, flowering plants, conifers, gymnosperms, ferns, lizards, hornworts, liverworts, mosses), algal cells (e.g. Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, etc.), seaweeds (e.g., kelp), fungal cells (e.g., yeast cells, mushroom-derived cells), animal cells, invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.) cells of origin, spine Cells derived from vertebrates (e.g. fish, amphibians, reptiles, birds, mammals), mammals (e.g. pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.) Examples include cells such as. In some cases, cells are not derived from a natural organism (eg, cells can be made synthetically and are sometimes referred to as artificial cells).

[0034] As used herein, the term "antigen" refers to a molecule or fragment thereof that can be bound by a selective binding agent. As an example, an antigen can be a ligand that can be bound by a selective binding agent, such as a receptor. As another example, an antigen can be an antigenic molecule that can be bound by a selective binding agent such as an immune protein (eg, an antibody). An antigen can also refer to a molecule or a fragment thereof that can be used in an animal to produce antibodies capable of binding to that antigen.

[0035] A "cytokine" is a type of immunomodulatory polypeptide that can mediate crosstalk between progenitor / primary cells and target / effector cells. Cytokines can function in soluble form or on the cell surface to bind to "cytokine receptors" on target immune cells and activate signal transduction. As used herein, a "cytokine receptor" is a polypeptide on the cell surface that activates intracellular signaling upon binding to a cytokine on the extracellular cell surface. Cytokines can include, but are not limited to, chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. Cytokines are produced by a wide range of cells including immune cells, endothelial cells, fibroblasts, and stromal cells. A given cytokine may be produced by multiple cell types. Cytokines are pleiotropic, and because receptors are expressed on multiple immune cell subsets, one cytokine can activate signaling pathways in multiple cells. However, depending on the cell type, cytokine signaling events can trigger different downstream cellular events, such as activation, proliferation, survival, apoptosis, effector functions, and secretion of other immunomodulatory proteins. In some embodiments, a given cytokine is a wild-type cytokine polypeptide, such as a mutant cytokine polypeptide (also referred to herein as a mutein cytokine, e.g., mutein IL-2, mutein IL-7); fragment or variant thereof.

[0036] As used herein, "antigen binding domain" refers to a molecule that specifically binds to an antigenic determinant. A targeting moiety or antigen binding domain can be a protein, carbohydrate, lipid, or other chemical compound. These include antibodies, antibody fragments (Chames et al, 2009; Chan & Carter, 2010; Leavy, 2010; Holliger & Hudson, 2005), scaffold antigen binding proteins (Gebauer and Skerra, 2009; Stumpp et al, 2008), Single domain antibodies (sdAb), minibodies (Tramontano et al. al, 1994), heavy chain antibody variable domains (nanobodies, VHHs), novel antigen receptor variable domains (VNARs), carbohydrate binding domains (CBDs) (Blake et al, 2006), collagen binding domains (Knight et al. al, 2000), lectin-binding proteins (tetranectin), collagen-binding proteins, adnectin / fibronectin (Lipovsek, 2011), serum transferrin (transbodies), shrimp bodies, protein A-derived molecules such as protein A Z domain (Affibodies), (Nygren et al, 2008), A domain (Avimer / Maxibody), alpha body (WO 2010 / 066740), Avimer / Maxibody, engineered ankyrin-repeat domains (DARPins) (Stumpp et al, 2008), anticalin (Skerra et al, 2008), human gamma-crystallin or ubiquitin (Affilin molecules), the Kunitz-type domain of human protease inhibitors, Knottin (Kolmar et al, 2008), linear or Restricted peptides, e.g. (Fc fusion-peptibodies) (Rentero Rebollo & Heinis,2013;European Patent No. 1144454(B2);Shimamoto et al, 2012; US Patent No. 7205275 (B2)), constrained bicyclic peptides (US Patent Application Publication No. 2018 / 0200378 (A1)), aptamers, genetically engineered CH2 domains (nano antibodies, Dimitrov, 2009) ), and a genetically engineered CH3 domain “Fcab” domain (Wozniak-Knopp et al. al, 2010), but are not limited to these.

[0037] As used herein, the term "antibody" refers to a proteinaceous binding molecule with immunoglobulin-like functionality. The term antibody includes antibodies (eg, monoclonal and polyclonal antibodies), as well as derivatives, variants, and fragments thereof. Antibodies include, but are not limited to, different classes of immunoglobulins (Ig) (ie, IgA, IgG, IgM, IgD, and IgE) and subclasses (IgG1, IgG2, etc.). These derivatives, variants, or fragments refer to functional derivatives or fragments that retain the binding specificity (eg, complete and / or partial) of the corresponding antibody. Antigen-binding fragments include Fab, Fab’, F(ab’) 2 , variable fragments (Fv), single chain variable fragments (scFv), minibodies, diabodies, and single domain antibodies (“sdAbs” or “nanobodies” or “camelids”). The term antibody includes optimized, genetically engineered, or chemically conjugated antibodies and antigen-binding fragments of antibodies. Examples of optimized antibodies include affinity matured antibodies. Examples of genetically engineered antibodies include Fc-optimized antibodies (eg, antibodies optimized in the fragment crystallizable region) and multispecific antibodies (eg, bispecific antibodies).

[0038] As used herein, the term "Fc receptor" or "FcR" generally refers to a receptor, or any derivative, variant or fragment thereof, that is capable of binding to the Fc region of an antibody. In certain embodiments, the FcR is one that binds IgG antibodies (γ receptors, FcγR), including allelic variants and alternatively spliced ​​forms of these receptors, such as FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16) subclass receptors. FcγRII receptors include FcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibitory receptor”), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The term "FcR" also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus.

[0039] As used herein, "effector function" refers to the biochemical events that result from the interaction of an antibody Fc region with an Fc receptor or ligand, which varies depending on the antibody isotype. Effector functions include antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), cytokine secretion, immune complex antigen uptake by antigen-presenting cells, These include, but are not limited to, downregulation of cell surface receptors (eg, B cell receptors), and B cell activation. “Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to nonspecific cytotoxic cells that express FcR (such as natural killer (NK) cells, neutrophils, and macrophages) that bind antibodies on target cells. refers to a cellular response that recognizes a target cell and subsequently causes lysis of the target cell. ADCC is correlated with binding to FcγRIIIa, and increased binding to FcγRIIIa leads to increased ADCC activity. To assess the ADCC activity of a molecule of interest, in vitro ADCC assays such as those described in US Pat. No. 5,500,362 or US Pat. No. 5,821,337 may be performed. As used herein, "ADCP" or antibody-dependent cellular phagocytosis refers to the recognition of bound antibodies on target cells by non-specific cytotoxic cells expressing FcγR, and subsequent phagocytosis of the target cells. Refers to a cellular response that causes an effect.

[0040] "Fc null" and "Fc null variant" are used interchangeably herein to describe a modified Fc with reduced or eliminated effector function. Such Fc nulls or Fc null variants have reduced or eliminated FcγRs and / or complement receptors. Preferably, such an Fc null or Fc null variant has lost effector function. Exemplary methods of modification include, but are not limited to, chemical alterations, amino acid residue substitutions, insertions, deletions, and the like. To reduce the effector function of the resulting variant, position i) IgG1:C220, C226, C229, E233, L234, L235, G237, P238, S239, D265, S267, N297, L328, P331, K322, A327 , and P329, ii)I gG2:V234, G237, D265, H268, N297, V309, A330, A331, K322; Amino acid position (numbering based on the EU numbering system). Exemplary Fc molecules with reduced effector function include the following substitutions: i) IgG1:N297A, N297Q, D265A / N297A, D265A / N297Q, C220S / C2 26S / C229S / P238S, S267E / L328F, C226S / C229S / E233P / L234V / L235A, L234F / L235E / P331S, L234A / L2 35A, L234A / L235A / G237A, L234A / L235A / G237A / K322A, L234A / L235A / G237A / A330S / A331S, L234A / L2 35A / P329G, E233P / L234V / L235A / G236del / S239K, E233P / L234V / L235A / G236del / S267K, E233P / L234 V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G236del / S267K / A327G and E233P / L234V / L235 A / G236del, L234A / L235A / G237 deleted, ii) IgG2:A330S / A331S, V234A / G237A, V234A / G237A / D265A, D265A / A330S / A331S, V234A / G237A / D265A / A330S / A331S, and H268Q / V309L / A330S / A331S, iii) IgG4:L235A / Included are Fc molecules having one or more of G237A / E318A, D265A, L235A / G237A / D265A, and L235A / G237A / D265A / E318A.

[0041] As used herein, "epitope" refers to a determinant capable of specifically binding to a variable region of an antibody molecule, known as a paratope. An epitope is a group of molecules, such as amino acids or sugar side chains, usually having specific structural characteristics as well as specific charge characteristics. A single antigen may have more than one epitope. Epitopes are amino acid residues that are directly involved in binding and other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively inhibited by antigen-binding peptides (in other words, amino acid residues that are within the footprint of ). Epitopes can be either conformational or linear. Epitopes usually include at least 3, more usually at least 5 or 8-10 amino acids. Antibodies that recognize the same epitope can be identified by simple immunoassays that demonstrate the ability of one antibody to inhibit the binding of another antibody to a target antigen, such as "binning."

[0042] As used herein, "linker" refers to a molecule that connects two polypeptide chains. The linker may be a polypeptide linker or a synthetic chemical linker (see, for example, those disclosed in Protein Engineering, 9(3), 299-305, 1996). The length and sequence of the polypeptide linker are not particularly limited and can be selected depending on the purpose by those skilled in the art. A polypeptide linker includes one or more amino acids. Preferably, the polypeptide linker is a peptide having a length of at least 5 amino acids, preferably 5 to 100 amino acids, more preferably 10 to 50 amino acids. In one embodiment, the peptide linkers are G, S, GS, SG, SGG, GGS, and GSG (G=glycine and S=serine). In another embodiment, the peptide linker is (GGGS)xGn (SEQ ID NO: 5) or (GGGGS)xGn (SEQ ID NO: 6) or (GGGGGS)xGn (SEQ ID NO: 7), where x=1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, or 12, and n=0, 1, 2, or 3. Preferably, the linker is (GGGGS)xGn, where x=2, 3, or 4 and n=0 (SEQ ID NO: 8); more preferably, the linker is (GGGGS)xGn, x=3, and n=0 (SEQ ID NO: 9). Synthetic chemical linkers include cross-linking agents routinely used to cross-link peptides, such as N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), and bis(succinimidyl suberate). dithiobis(succinimidyl propionate) (BS3), dithiobis(succinimidyl propionate) (DSP), dithiobis(succinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) ) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2 -(succinimidoxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(succinimidoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES).

[0043] As used herein, the term "nucleotide" generally refers to a base-sugar-phosphate combination. Nucleotides can include synthetic nucleotides. Nucleotides can include synthetic nucleotide analogs. Nucleotides can be monomeric units of nucleic acid sequences, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The term nucleotide includes the ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP), and dATP, dCTP, dITP, Deoxyribonucleoside triphosphates such as dUTP, dGTP, dTTP, or derivatives thereof may be included. Such derivatives can include, for example, [αS]dATP, 7-deaza-dGTP, and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. As used herein, the term nucleotide refers in some examples to dideoxyribonucleoside triphosphates (ddNTPs) and derivatives thereof. Examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides can be unlabeled or detectably labeled by well-known techniques. Labeling with quantum dots can also be performed. Detectable labels can include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. Fluorescent labels for nucleotides include fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N, N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, Cyanine, and 5-(2'-aminoethyl)aminonaphthalene Mention may be made, without limitation, of -1-sulfonic acid (EDANS). Specific examples of fluorescently labeled nucleotides include Perkin Elmer (Foster [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, available from City, Calif.) [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP, Amersham (Arlington FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Boehringer Fluorescein-15-dATP, Fluorescein-12-dUTP, Tetramethyl-rhodamine-6-dUTP, IR770-9-dATP, Fluorescein-12-ddUTP, Fluorescein-12-UTP, available from Mannheim (Indianapolis, Ind.) and fluorescein-15-2'-dATP, and Molecular Chromosome-labeled nucleotides available from Probes (Eugene, Oreg.), BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR -14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, Full Luorescein-12-UTP, Fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, Tetramethylrhodamine-6-UTP, Tetramethylrhodamine-6-dUTP, Mention may be made of Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP. Nucleotides can also be labeled or marked by chemical modification. The chemically modified single nucleotide can be biotin-dNTP. Some non-limiting examples of biotinylated dNTPs include biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-1 1-dCTP, biotin-14-dCTP), and biotin-dUTP (eg, biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).

[0044] The terms "polynucleotide," "oligonucleotide," and "nucleic acid" refer to any single-stranded, double-stranded, or multi-stranded form of deoxyribonucleotides or ribonucleotides, or analogs thereof. used interchangeably to refer to polymeric forms of nucleotides of any length. The polynucleotide may be exogenous to the cell or endogenous. Polynucleotides can also exist in a cell-free environment. A polynucleotide can be a gene or a fragment thereof. A polynucleotide can be DNA. Polynucleotides can be RNA. Polynucleotides can have any three-dimensional structure and perform any function, known or unknown. A polynucleotide can include one or more analogs (eg, modified backbones, sugars, or nucleobases). If modifications are present in the nucleotide structure, they can be made before or after construction of the polymer. Some non-limiting examples of analogs include 5-bromouracil, peptide nucleic acids, xenonucleic acids, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores ( For example, sugar-linked rhodamine or flu (olecein), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiuridine, pseudourdine, dihydrouridine, cuosine, and wyosine. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, multiple genetic loci (loci) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA. (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), small hairpin RNA (sh RNA), microRNA (miRNA), ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, cell-free DNA (cfDNA) and Included are cell-free polynucleotides, including cellular RNA (cfRNA), nucleic acid probes, and primers. A sequence of nucleotides may be interrupted by non-nucleotide components.

[0045] As used herein, the term "gene" refers to its corresponding nucleotide sequence that is responsible for encoding nucleic acids (eg, DNA such as genomic DNA and cDNA) and RNA transcripts. The term used herein with respect to genomic DNA includes intervening non-coding and control regions and can include 5' and 3' ends. In some uses, the term includes transcribed sequences, including 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons, and introns. For some genes, the transcribed region includes an "open reading frame" that encodes a polypeptide. In some uses of the term, a "gene" includes only the coding sequences (eg, an "open reading frame" or "coding region") necessary to encode a polypeptide. In some cases, the gene does not encode a polypeptide, such as ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some cases, the term "gene" includes not only transcribed sequences, but also non-transcribed regions, including upstream and downstream control regions, enhancers, and promoters. Gene, as the case may be, refers to an "endogenous gene" or a naturally occurring gene in its natural location within the genome of an organism. Gene refers to a "foreign gene" or a non-natural gene, as the case may be. A non-natural gene, in some cases, refers to a gene that is not normally found in the host organism, but that is introduced into the host organism by gene transfer. A non-natural gene, as the case may be, refers to a gene that is not in its natural location within the genome of an organism. Non-natural genes also refer to naturally occurring nucleic acid or polypeptide sequences (eg, non-natural sequences) that, in some cases, include mutations, insertions, and / or deletions.

[0046] The term "regulate" with respect to expression or activity, as used herein, refers to altering the level of expression or activity. Regulation may occur at the transcriptional and / or translational level.

[0047] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to a polymer of at least two amino acid residues joined by peptide bond(s). This term does not imply a specific length of the polymer and does not imply or distinguish whether the peptide is produced using recombinant technology, chemical synthesis, or enzymatic synthesis, or is naturally occurring. It is not intended to be. This term applies to naturally occurring amino acid polymers as well as amino acid polymers containing at least one modified amino acid. In some cases, the polymer may be interrupted by non-amino acids. The term includes amino acid chains of any length, including full-length proteins, and proteins with or without secondary and / or tertiary structure (eg, domains). The term also includes amino acid polymers modified by, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulations such as conjugation with labeling moieties. As used herein, the terms "amino acid" and "amino acids" generally refer to natural and unnatural amino acids, including, but not limited to, modified amino acids and amino acid analogs. Modified amino acids can include natural amino acids and non-natural amino acids that are chemically modified to include groups or chemical moieties that do not occur naturally on the amino acid. Amino acid analogs can refer to amino acid derivatives. The term "amino acid" includes both D-amino acids and L-amino acids.

[0048] The terms "derivative," "variant," and "fragment," as used herein with respect to polypeptides, refer to, for example, amino acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic refers to a polypeptide that is related to the wild-type polypeptide, either by activity) and / or function. Derivatives, variants, and fragments of polypeptides may contain one or more amino acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof as compared to the wild-type polypeptide. can.

[0049] As used herein, the term "residue" refers to a position within a protein and its associated amino acid identity. For example, Leu 234 (also referred to as Leu234 or L234) is the residue at position 234 of the human antibody IgG1.

[0050] As used herein, the term "wild type" refers to an amino acid or nucleotide sequence found in nature, including allelic variations. A wild-type protein has an amino acid or nucleotide sequence that is not intentionally modified.

[0051] The term "substitution" or "mutation" refers to a change to a polypeptide backbone in which an amino acid that is naturally occurring in the wild-type sequence of the polypeptide is replaced by another amino acid that is not naturally occurring at the same position in the polypeptide. refers to In some cases, a mutation or mutations are introduced to modify the affinity of a polypeptide for a receptor, thereby altering its activity to be different from the affinity and activity of the wild-type cognate polypeptide. Mutations can also improve the biophysical properties of a polypeptide. Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, and the like. It is contemplated that methods of altering the side chain groups of amino acids by methods other than genetic engineering, such as chemical modification, may also be useful.

[0052] The term "affinity" or "binding affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Point. Unless otherwise indicated, "binding affinity" as used herein refers to an inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). Refers to gender. Affinity is generally determined by the dissociation constant (K D ), which is the ratio of the dissociation rate constant to the association rate constant (koff and kon, respectively). Thus, equivalent affinities may include different rate constants as long as the ratio of rate constants remains the same. Affinity can be determined using enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) technology (e.g., BIAcore), BioLayer Interferometry (BLI) technology (e.g., Octet), and other conventional binding assays (Heeley, Endocr. Res 28, 217-229 (2002)).

[0053] As used herein, the terms "binding" or "specific binding" refer to the ability of a polypeptide or antigen-binding domain to selectively interact with a receptor of a polypeptide or target antigen, respectively. This specific interaction can be distinguished from non-target interactions, or unwanted interactions, or non-specific interactions. Examples of specific binding include IL-2 cytokines that bind to specific receptors for IL-2 cytokines (e.g., IL-2Rα, IL-2Rβ, and IL-2Rγ), and specific antigens (e.g., CD8 or Examples include, but are not limited to, antigen-binding domains that bind to PD-1).

[0054] The terms "subject," "individual," and "patient" are used interchangeably herein and refer to a vertebrate, preferably a mammal, such as a human. Mammals include, but are not limited to, rodents, monkeys, humans, livestock, sport animals, and pets. Also included are tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro.

[0055] As used herein, the terms "treatment" and "treating" refer to obtaining a beneficial or desired result, including but not limited to therapeutic and / or prophylactic efficacy. Refers to the method for For example, treatment can include administering a system or cell population disclosed herein. Therapeutic efficacy refers to any therapeutically relevant improvement or effect in one or more diseases, diseases, or conditions being treated. For prophylactic efficacy, the composition may be administered to a subject at risk of developing a particular disease, disease, or condition, or to the physiology of the disease, even if the disease, disorder, or condition has not yet appeared. Can be administered to subjects reporting one or more of the following symptoms:

[0056] The term "effective amount" or "therapeutically effective amount" or "effective dose" or "effective dosage" refers to the composition, e.g., immune cells such as lymphocytes (e.g., T lymphocytes and / or NK cells). Refers to the amount of a composition that can be combined with a targeted cytokine construct of the present disclosure sufficient to produce the desired activity upon administration to a subject in need thereof. Within the context of this disclosure, the term "therapeutically effective" refers to a composition that is sufficient to delay the onset, arrest the progression, reduce or alleviate at least one symptom of the disorder treated by the methods of this disclosure. Refers to the amount of something. overview

[0057] In summary, the present disclosure provides methods for treating diseases or diseases, e.g. The present invention relates to methods, compositions, kits, systems, and regimens for the treatment of diseases, such as cancer. In some embodiments, described herein are combination methods, e.g., genetically engineered cells, that can improve the therapeutic efficacy of genetically engineered cells for diseases, e.g., proliferative diseases, e.g., cancer. and a targeted cytokine construct. In some embodiments, in vitro methods are provided for improving therapeutic efficacy of genetically engineered cells by contacting a population of genetically engineered cells described herein with a targeted cytokine construct. .

[0058] In some embodiments, the targeted cytokine constructs have the following effects on genetically engineered cells: enhancing proliferation of such genetically engineered cells, altering cytokine secretion by genetically engineered cells. That is, genetically engineered reducing the dependence on exogenous cytokines for cell survival and proliferation; enhancing the cytotoxicity of such genetically engineered cells; increasing the survival rate of such genetically engineered cells; apoptosis in genetically engineered cells such as blocking, delaying the aging of such genetically engineered cells, preventing or delaying the depletion of such genetically engineered cells, enhancing the in vivo persistence of the cells, enhancing the in vivo efficacy of genetically engineered cells, enhancing the penetration of genetically engineered cells into diseased organs or tissues (e.g., tumors), or the like when administered to a subject; any combination of triggering one or more of the following.

[0059] In some embodiments, the present disclosure provides methods and compositions for improving in vivo persistence and therapeutic efficacy of genetically engineered cells. In some embodiments, administering an optimal treatment regimen that includes a population of genetically engineered cells and a targeted cytokine construct results in a greater amount of genetically engineered cells than when the population of genetically engineered cells is administered alone. The in vivo persistence of the population of cells that has been improved is increased by at least about 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%. In some embodiments, a population of genetically engineered cells is combined with a non-targeted cytokine or a functional fragment or variant thereof by administering an optimal treatment regimen that includes the population of genetically engineered cells and a targeted cytokine construct. The in vivo persistence of the population of genetically engineered cells is increased by at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% as compared to administration. In some embodiments, a population of genetically engineered cells is combined with a cell binding domain that is not fused to a cytokine protein by administering an optimal treatment regimen that includes the population of genetically engineered cells and a targeted cytokine construct. The in vivo persistence of the genetically engineered cells is increased by at least about 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% compared to administration. In some embodiments, the targeted cytokine constructs of the present disclosure provide a population of genetically engineered cells with an in vitro persistence of at least about 10 %, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% improvement.

[0060] In some embodiments, the present disclosure provides methods and compositions for improving in vivo persistence and therapeutic efficacy of genetically engineered cells. In some embodiments, administering an optimal treatment regimen that includes a population of genetically engineered cells and a targeted cytokine construct results in a greater amount of genetically engineered cells than when the population of genetically engineered cells is administered alone. The in vivo persistence of the cell population is improved by at least about 2-fold. In some embodiments, a population of genetically engineered cells is combined with a non-targeted cytokine or a functional fragment or variant thereof by administering an optimal treatment regimen that includes the population of genetically engineered cells and a targeted cytokine construct. The in vivo persistence of the genetically engineered cell population is increased by at least 2-fold compared to administration of the genetically engineered cell population. In some embodiments, a population of genetically engineered cells is combined with a cell binding domain that is not fused to a cytokine protein by administering an optimal treatment regimen that includes the population of genetically engineered cells and a targeted cytokine construct. The persistence of genetically engineered cells in vivo is increased by at least 2-fold compared to when administered directly.

[0061] In some embodiments, the present disclosure provides effective dosages of genetically engineered cells in genetically engineered cell therapy comprising administering populations of genetically engineered cells and targeted cytokine constructs described herein. Regarding how to reduce In some embodiments, a composition that includes a population of genetically engineered cells and a targeted cytokine construct has an amount equal to that in a reference composition that includes a population of genetically engineered cells but does not include a targeted cytokine construct. In comparison, both compositions, containing lower amounts of genetically engineered cells, show similar efficacy.

[0062] In some embodiments, a composition comprising a population of genetically engineered cells and a targeted cytokine construct is in a reference composition comprising a population of genetically engineered cells and a non-targeted cytokine or functional fragment or variant thereof. contains a lower amount of genetically engineered cells compared to the amount of. In some embodiments, a composition comprising a population of genetically engineered cells and a targeted cytokine construct comprises a population of genetically engineered cells and a cell binding domain, but no cytokine protein in a reference composition. Contains a lower amount of genetically engineered cells compared to the amount. In some embodiments, the effective dose or amount described above is about 2 times, about 4 times, about 8 times, about 16 times, about 32x, approx. 50x, approx. 64x, approx. 70x, Approximately 75 times, approximately 80 times, approximately 90 times, approximately 91 times, approximately 92 times, approximately 93 times, approximately 94 times, approximately 95 times, approximately 96 times, approximately 97 times, approximately 98 times, approximately 99 times, or approximately About 1.5 times to about 1000 times lower, such as 1000 times.

[0063] Studies have shown that CAR T cell loss is related to either T cell-specific problems that prevent expansion, or external immune rejection of T cells. Patients who experience relapse after CAR T cell-induced remission have very early loss of CAR T cells or loss of B cell aplasia (absolute B cell counts typically >50-100 / μL). (as measured by flow) and their disappearance is generally associated with relapse (eg, relapse of CD19+ B cells). For example, see Nie et al. "Mechanisms underlying CD19-positive ALL relapse after anti-CD19 CAR T cell therapy and associated strategies", Biomarker Research volume 8, Article number: 18 (2020). In patients who relapse after treatment with genetically engineered cells (e.g., CAR-T cell therapy, such as CD19 CAR-T cell therapy), administration of targeted cytokine constructs of the present disclosure may serve as a salvage therapy. . Accordingly, some embodiments of the present disclosure provide methods of treatment by administering targeted cytokine constructs to patients who experience loss of B cell aplasia following genetically engineered cell therapy.

[0064] In some embodiments, an optimal treatment regimen is provided that includes administering to a subject a population of genetically engineered cells and a targeted cytokine construct described herein, the method comprising: (a) administering to the subject a population of genetically engineered cells and a targeted cytokine construct as described herein; (b) reducing the degree of prior lymphodepletion required for expansion, engraftment of the genetically engineered cells; Make it. Non-limiting examples of lymphocyte depleting agents include chemotherapeutic agents such as fludarabine, cyclophosphamide, and depleting antibodies such as alemtuzumab.

[0065] In some embodiments, the targeted cytokine construct is administered to genetically engineered targeted cells generated in vivo in the subject. Various improvements to such in vivo generated, genetically engineered cells, such as increased persistence, decreased rate and / or extent of depletion, increased expansion and / or proliferation, and no increase in Treg cell numbers. , selective enhancement and specific enrichment of genetically engineered cells can be achieved by administering targeted cytokine constructs. Exemplary techniques for generating genetically engineered cells (e.g., CAR-T cells or TCR-T cells) in vivo include administering to a subject a nucleic acid carrier that includes a CAR gene or a TCR gene. . In some embodiments, the nucleic acid carrier is a non-viral vector, a plasmid, a linear polynucleotide, a polynucleotide associated with an ionic or amphipathic compound, a plasmid, or a virus (such as a viral vector). In some embodiments, the viral vector is at least one of a Sendai virus vector, an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, or a lentivirus vector. The nucleic acid carrier optionally includes a targeting moiety specific for immune cells (eg, T cells, NK cells, T lymphocytes, myeloid cells). Immune cells targeted by the nucleic acid carrier are, in some embodiments, induced to generate genetically engineered cells in vivo that are targeted by the targeted cytokine constructs of the present disclosure. combination therapy

[0066] In one aspect, the present disclosure provides methods for targeting optimal treatment regimens that include (1) genetically engineered cells, e.g., CAR-expressing cells, e.g., T cells, populations thereof, and (2) targeted cytokine constructs. Combination therapies, such as treatment methods and treatment regimens, for treating diseases or disorders, such as cancer or proliferative diseases, are provided. In another aspect, the present disclosure improves in vitro activation and / or expansion of a population of genetically engineered cells by contacting the population of genetically engineered cells described herein with a targeted cytokine construct. Provided are methods for genetically engineered cell therapy, in some embodiments, further comprising administering a population of genetically engineered cells to treat a disease or disorder, such as cancer or a proliferative disease. provide. Administration as described above can be combined with the targeted cytokine constructs of the present disclosure.

[0067] In some embodiments, the genetically engineered cells specifically recognize and / or target antigens associated with a disease or disorder, such as cancer or a proliferative disease. In some embodiments, the genetically engineered cell expresses a domain or receptor recognized by the cytokine of the targeted cytokine construct. It also includes combinations and articles of manufacture, such as kits, comprising compositions comprising populations of genetically engineered cells and / or compositions comprising targeted cytokine constructs, and such combinations and products for treating diseases, diseases, and disorders, including cancer. Also provided are compositions and uses of combinations. Such methods include prior to, concurrent with, during, or during the course of administration (e.g., during the course of administration) of a therapy containing genetically engineered cells (e.g., CAR-expressing T cells). (including once and / or periodically) and / or subsequent administrations of the targeted cytokine construct. In some embodiments, the administration can include continuous or intermittent administration of the targeted cytokine construct and / or population of genetically engineered cells (administering the population of genetically engineered cells includes Also referred to herein as genetically engineered cell therapy). Dosage and treatment regimen

[0068] In some embodiments, the present disclosure provides an optimal treatment regimen that includes a first pharmaceutical composition comprising a population of genetically engineered cells and a second pharmaceutical composition comprising a targeted cytokine construct. In some embodiments, the second pharmaceutical composition comprises a targeted cytokine construct in an amount sufficient to enhance the therapeutic efficacy of the population of genetically engineered cells.

[0069] In some embodiments, a first composition comprising a population of genetically engineered cells and a second pharmaceutical composition comprising a targeted cytokine construct are administered to the subject simultaneously or sequentially. In some embodiments, the subject first receives administration of a therapy comprising a population of genetically engineered cells, e.g., first finishes therapy comprising a population of genetically engineered cells, and then receive an conjugated cytokine construct.

[0070] In some embodiments, a therapy comprising a population of genetically engineered cells and a targeted cytokine construct is administered to a subject according to a predetermined treatment regimen, eg, simultaneously, intermittently, or sequentially. In some embodiments, the subject is administered according to a predetermined dosing regimen, e.g., 1, 2, 3, 4, 5, 6, or 7 times per week for several consecutive weeks, or for a given period of time. For example, one receives a therapy comprising a population of genetically engineered cells once every 1, 2, 3, 4, 5, 6, or 7 days for a week, a month, or a year. In some embodiments, the subject also follows the same dosing regimen as the therapy comprising the population of genetically engineered cells or, in some cases, a dosing regimen that overlaps with the regimen of the therapy comprising the population of genetically engineered cells. subject to administration of a targeted cytokine construct. For example, a subject can receive both a therapy comprising a population of genetically engineered cells, eg, a T cell infusion and a targeted cytokine construct, simultaneously, eg, both via intravenous infusion. In other cases, a subject can receive both a therapy involving a population of genetically engineered cells, eg, a T cell infusion and a targeted cytokine construct, on the same day. In some cases, when the two dosing regimens overlap, the subject receives a therapy containing a population of genetically engineered cells on one day and then receives a therapy containing a population of genetically engineered cells on the next day, or 2, 3, 4, 5, 6, The targeted cytokine construct can be received after 7 days or more and before receiving the next dose of therapy comprising the population of genetically engineered cells. Alternatively, in some cases, a subject can be administered a therapy comprising a population of genetically engineered cells more times than a targeted cytokine construct, or vice versa.

[0071] In some embodiments, a therapy comprising a population of genetically engineered cells is administered concurrently with, or after or after administering the targeted cytokine construct. In some embodiments, a therapy comprising a population of genetically engineered cells is administered for 0-30 days, 0-15 days, 0-6 days, 0-6 days, 0-30 days, 0-15 days, 0-6 days, ~96 hours, 0-24 hours, 0-12 hours, 0-6 hours, or 0-2 hours , 2 hours to 30 days, 2 hours to 15 days, 2 hours to 6 days, 2 hours to 96 hours, 2 hours to 24 hours, 2 hours to 12 hours, 2 hours to 6 hours, 6 hours to 90 days, 6 Time ~ 30 days, 6 hours ~ 15 days, 6 hours ~ 6 days, 6 hours ~ 96 hours, 6 hours ~ 24 hours, 6 hours to 12 hours, 12 hours to 90 days, 12 hours to 30 days, 12 hours to 15 days, 12 hours to 6 days, 12 hours to 96 hours, 12 hours to 24 hours, 24 hours to 90 days, 24 hours ~30 days, 24 hours ~ 15 days, 24 hours ~ 6 days, 24 hours ~ 96 hours time, 96 hours to 90 days, 96 hours to 30 days, 96 hours to 15 days, 96 hours to 6 days, 6 days to 90 days, 6 days to 30 days, 6 days to 15 days, 15 days to 90 days, Administered for 0 to 90 days, such as 15 days to 30 days, or 30 days to 90 days. In some embodiments, the therapy comprising the population of genetically engineered cells is administered at least or about at least or about 1 hour, 2 hours, 6 hours, 12 hours after beginning or beginning administration of the targeted cytokine construct. Administered for hours, 24 hours, 2 days, 3 days, 6 days, 12 days, 15 days, 30 days, 60 days, or 90 days.

[0072] In some embodiments, a therapy comprising a population of genetically engineered cells is administered concurrently with, or prior to, beginning administration of a targeted cytokine construct. In some embodiments, a therapy comprising a population of genetically engineered cells is administered for 0-30 days, 0-15 days, 0-6 days, 0-6 days, 0-30 days, 0-15 days, 0-6 days, or ~96 hours, 0-24 hours, 0-12 hours, 0-6 hours, or 0-2 hours , 2 hours to 30 days, 2 hours to 15 days, 2 hours to 6 days, 2 hours to 96 hours, 2 hours to 24 hours, 2 hours to 12 hours, 2 hours to 6 hours, 6 hours to 90 days, 6 Time ~ 30 days, 6 hours ~ 15 days, 6 hours ~ 6 days, 6 hours ~ 96 hours, 6 hours ~ 24 hours, 6 hours to 12 hours, 12 hours to 90 days, 12 hours to 30 days, 12 hours to 15 days, 12 hours to 6 days, 12 hours to 96 hours, 12 hours to 24 hours, 24 hours to 90 days, 24 hours ~30 days, 24 hours ~ 15 days, 24 hours ~ 6 days, 24 hours ~ 96 hours time, 96 hours to 90 days, 96 hours to 30 days, 96 hours to 15 days, 96 hours to 6 days, 6 days to 90 days, 6 days to 30 days, 6 days to 15 days, 15 days to 90 days, Administered for 0 to 90 days, such as 15 days to 30 days, or 30 days to 90 days. In some embodiments, a therapy comprising a population of genetically engineered cells is administered for at least or about at least or about 1 hour, 2 hours, 6 hours, 12 hours before beginning or beginning administration of the targeted cytokine construct. Administered for hours, 24 hours, 2 days, 3 days, 6 days, 12 days, 15 days, 30 days, 60 days, or 90 days.

[0073] In some embodiments, following an initial administration of the targeted cytokine construct and the population of genetically engineered cells, subsequent doses of the targeted cytokine construct are targeted to maintain the genetically engineered cells in vivo. administered to In some embodiments, subsequent administration is once every week, once every two weeks, once every three weeks, or monthly after the initial administration.

[0074] In some embodiments, the dosage and timing of the targeted cytokine construct is such that the dosage and timing of the targeted cytokine construct is such that the dosage and timing of the targeted cytokine construct is such that the dosage and timing of the targeted cytokine construct is such that the dosage and timing of the targeted cytokine construct are such that the dosage and timing of the targeted cytokine construct are such that the dosage and timing of the targeted cytokine construct are such that the dosage and timing of the targeted cytokine construct are such that the dosage and timing of the targeted cytokine construct are such that the dosage and timing of the targeted cytokine construct are such that the dosage and timing of the targeted cytokine construct are such that the dosage and timing of the targeted cytokine construct are such that the dosage and timing of the targeted cytokine construct are such that the dosage and timing of the targeted cytokine construct are such that the dosage and timing of the targeted cytokine construct are such that the dosage and timing of the targeted cytokine construct are such that the dosage and timing of the targeted cytokine construct are such that the dosage and timing of the targeted cytokine construct are such that the dosage and timing of the targeted cytokine construct are such that the dosage and timing of the targeted cytokine construct are such that the amount and timing of the targeted cytokine construct is such that the dosage and timing of the targeted cytokine construct are determined in a sample from the subject after administering the therapy that includes the population of genetically engineered cells. Adjustments are made based on one or more performance-related treatment effect measurements. In some cases, the dosing regimen for compositions comprising populations of genetically engineered cells and compositions comprising targeted cytokine constructs will be determined by the subject's attending physician. The physician's decision can be based on a number of factors, including, but not limited to, the subject's medical history and the subject's other medical test results, such as tumor pathological test results. The specific dosage of the targeted cytokine construct will vary depending on the particular combination of cytokine and cell binding domain selected, the dosing regimen followed, the health status of the subject, the tissue to which it is administered, and the physical delivery system. . In some embodiments, the targeted cytokine construct is administered on average about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 per week over the treatment cycle. , 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40 , 41, 42 , 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 , 68, 69 , 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 mg. For example, targeting cytokine constructs may be administered at approximately 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 , 54, or 55 mg. In some embodiments, the targeted cytokine construct is about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mg per week. be administered to the subject within the range of

[0075] In some embodiments, the targeted cytokine construct is administered on average per day over the treatment cycle at 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8 , 8.5, 9, 9.5, or 10 mg. For example, the targeted cytokine construct is administered to a subject in an amount of about 6-10 mg, about 6.5-9.5 mg, about 6.5-8.5 mg, about 6.5-8 mg, or about 7-9 mg per day on average over a treatment cycle. Ru.

[0076] In some embodiments, the targeted cytokine construct is about 0.01 mg / kg, about 0.02 mg / kg, about 0.03 mg / kg, about 0.04 mg / kg, about 0.05 mg / kg, about 0.06 mg / kg per day. , about 0.07mg / kg, approximately 0.08mg / kg, approximately 0.09mg / kg, approximately 0.1mg / kg, approximately 0.2mg / kg, approximately 0.3mg / kg, approximately 0.4mg / kg, approximately 5mg / kg, approximately 6mg / kg, approximately 7mg / kg, approx. 8mg / kg, approx. 9mg / kg, approx. 10mg / kg, about 11mg / kg, about 12mg / kg, about 13mg / kg, about 14mg / kg, about 15mg / kg, about 16mg / kg, about 17mg / kg, about 18mg / kg, about 19mg / kg, about 20mg / kg, about 25mg / kg , about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, or about 50 mg / kg, less than or more than these, in the range of about 0.01 mg / kg to 50 mg / kg per day. administered to the subject within. In some embodiments, the targeted cytokine construct is administered at about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 m per week. g / kg, approx. 0.7mg / kg, approx. 0.8mg / kg, approx. 0.9mg / kg, approx. 1mg / kg, approx. 5mg / kg, approx. 10mg / kg, approx. 15mg / kg, approx. 20mg / kg, approx. 25mg / kg, approx. 30mg / kg, about 35mg / kg, about 40mg / kg, about 45mg / kg, about 50mg / kg, about 100mg / kg, about 150mg / kg, about 200mg / kg, about 250mg / kg, about 30 The subject is administered in a range of about 0.1 mg / kg to 400 mg / kg per week, such as less than or greater than 0 mg / kg, about 350 mg / kg, or about 400 mg / kg. In some embodiments, the targeted cytokine construct is administered to the subject weekly or biweekly at a dose of about 1 mg / kg.

[0077] In some embodiments, the targeted cytokine construct is administered at about 0.4 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20mg / kg, approx. 25mg / kg, approximately 30mg / kg, approximately 35mg / kg, approximately 40mg / kg, approximately 45mg / kg, approximately 50mg / kg, approximately 100mg / kg, approximately 150mg / kg, approximately 200mg / kg, approximately 250mg / kg, approximately 300mg / kg, approx. 3 50mg / kg, about 400mg / kg, about 450mg / kg, about 500mg / kg, about 550mg / kg, about 600mg / kg, about 650mg / kg, about 700mg / kg, about 750mg / kg, about 800mg / kg, about 8 50mg / kg, about 900mg / kg, about 950mg / kg, or about 1000mg / kg, less than these, or more than these, administered to the subject within the range of about 0.4mg / kg to 1500mg / kg per month. be done. In some embodiments, the targeted cytokine construct is administered at a dosage of about 1 mg / m per week. 2 , about 5mg / m 2 , about 10mg / m 2 , about 15mg / m 2 , about 20mg / m 2 , about 25mg / m 2 , about 30mg / m 2 , about 35mg / m 2 , about 40mg / m 2 , about 45mg / m 2 , about 50mg / m 2 , about 55mg / m 2 , about 60mg / m 2 , about 65mg / m 2 , about 70mg / m 2 , about 75mg / m 2 , about 100mg / m 2 , about 125mg / m 2 , about 150mg / m 2 , about 175mg / m 2 , or about 200mg / m 2 , less than these, or more than these, approximately 0.1mg / m per week 2 ~200mg / m 2 be administered to the subject within the range of A target dose may be administered in a single dose. Alternatively, the target doses are approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 These or more doses may be administered. For example, a dose of about 1 mg / kg per week may be delivered weekly at a dose of about 1 mg / kg each week over the week, about 2 mg / kg may be administered every two weeks, or Approximately 4 mg / kg may be administered every 4 weeks. The dosing schedule may be repeated according to any regimen described herein, including any dosing schedule described herein. In some embodiments, the targeted cytokine construct is about 1 mg / m 2 , about 5mg / m 2 , about 10mg / m 2 , about 15mg / m 2 , about 20mg / m 2 , about 25mg / m 2 , about 30mg / m 2 , about 35mg / m 2 , about 40mg / m 2 , about 45mg / m 2 , about 50mg / m 2 , about 55mg / m 2 , about 60mg / m 2 , about 65mg / m 2 , about 70mg / m 2 , about 75mg / m 2 , about 100mg / m 2 , about 130mg / m 2 , about 135mg / m 2 , about 155mg / m 2 , about 175mg / m 2 , about 200mg / m 2 , about 225mg / m 2 , about 250mg / m 2 , about 300mg / m 2 , about 350mg / m 2 , about 400mg / m 2 , about 420mg / m 2 , about 450mg / m 2 , or about 500mg / m 2 , less than these, or more than these, approximately 0.1mg / m 2 ~500mg / m 2 administered to the subject within the range of

[0078] In some embodiments, the dose of targeted cytokine construct is about, at least about, or up to about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 2 75, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 7 50, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000 mg or mg / kg, or any range derivable therein. It is contemplated that a dose of mg / kg refers to the amount of targeted cytokine construct in mg per kg of total body weight of the subject. It is contemplated that when multiple doses are given to a patient, the doses may vary in amount or may be the same. therapeutic effect

[0079] In some embodiments, the methods described herein involve in vitro expansion and / or propagation of a population of genetically engineered cells (genetically engineered cell therapy), or genetically engineered cells that are administered to a subject. allows for regulation of the rate of in vitro growth of populations. In some embodiments, the methods of treatment described herein are performed in vivo on genetically engineered cells that are administered to a subject in the absence of a targeting cytokine construct. Increase cell expansion and / or proliferation. In some embodiments, the methods of treatment described herein are performed in vivo on genetically engineered cells that are administered to a subject in the absence of a targeting cytokine construct. Increases cell expansion and / or proliferation by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times. In some embodiments, the targeted cytokine constructs of the present disclosure are used to expand genetically engineered cells in vitro to genetically engineered cells that have not been contacted with the targeted cytokine constructs described herein. and / or increases proliferation by at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times. In some cases, increased in vivo expansion and / or proliferation of a population of genetically engineered cells is achieved when the population of genetically engineered cells is administered in combination with a targeting cytokine construct. , (a) cell-binding domain without cytokines, (b) cell-binding at least about 2, 3, 4, 5, 6, 7, 8, 9, 10 for administration in combination with at least one non-targeting cytokine or functional fragment or variant thereof that does not have a domain. , 20, 30, 40, 50, 60, 70, 80, 90, or 100 times. Similarly, in some cases, increased in vitro expansion and / or proliferation upon contacting a population of genetically engineered cells with a targeted cytokine construct may cause the population of genetically engineered cells to (a) (b) cell-binding domain without cytokine; (b) cell-binding domain with no cytokine; at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 , 30, 40, 50, 60, 70, 80, 90, or 100 times.

[0080] In some embodiments, the methods described herein involve administering a population of genetically engineered cells alone or in combination with a non-targeting cytokine or functional fragment or variant thereof that does not have a cell-binding domain. In contrast, it makes it possible to enhance the specific enrichment of a population of genetically engineered cells administered to a subject. In some embodiments, the genetically engineered cells express a receptor for a cytokine protein, and the methods described herein can be used to grow genetically engineered cells that do not express a receptor for a CAR. CAR-expressing cells (e.g., CAR-expressing cells). In some embodiments, the methods described herein involve administering a population of genetically engineered cells alone or in combination with a non-targeting cytokine or functional fragment or variant thereof that does not have a cell-binding domain. In contrast to the increase in Treg cells when the population of genetically engineered cells is administered in combination with a targeted cytokine construct, there is no increase in Treg cells. In some embodiments, the methods described herein provide a population of genetically engineered cells alone or in combination with a non-targeting cytokine or a functional fragment or variant thereof that does not have a cell binding domain. or to reduce the rate and / or extent of depletion of a population of genetically engineered cells administered to a subject relative to administration in combination with a cell binding domain without a cytokine.

[0081] In some embodiments, the methods described herein are directed to administering a population of genetically engineered cells without a targeted cytokine construct or with a cell binding domain without a cytokine. increases the in vivo persistence of a population of genetically engineered cells administered to a subject relative to when administered. In some embodiments, the methods described herein improve the in vitro persistence of a population of genetically engineered cells when not contacted with a targeted cytokine construct or when contacted with a cell binding domain that does not have a cytokine. versus increasing the in vitro persistence of a population of genetically engineered cells when contacted with the targeted cytokine constructs described herein. In some embodiments, the increase in in vivo persistence or in vitro persistence is at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times larger. In some examples, the in vivo persistence of the population of genetically engineered cells when administered to a subject in combination with a targeting cytokine construct is about 45 days, about 60 days, about 90 days, about 120 days, About 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 18 months or more, etc., at least about 30 days to about 1 year That's all.

[0082] The methods, compositions, combinations, kits, and therapies provided herein, in some embodiments, include preconditioning regimens (e.g., lymphodepletion therapy) necessary for effective engraftment of genetically engineered cells. ) can be made possible to reduce the severity of Thus, in one example, the subject is lymphodepleted prior to administering (eg, injecting) the population of genetically engineered cells. In other examples, lymphocyte depletion is not necessary and the population of genetically engineered cells is rapidly injected into the subject.

[0083] In some embodiments, the method includes administering a preconditioning agent, such as a lymphocyte depleting agent, or a chemotherapeutic agent, such as cyclophosphamide, fludarabine, or a combination thereof, or a depleting agent prior to initiation of genetically engineered cell therapy. This involves administering to the subject an antibody such as alemtuzumab, which is a sexual antibody. In some embodiments, the methods, compositions, combinations, kits, and regimens provided herein provide a preconditioning agent / regimen in combination with a targeting cytokine construct prior to administering the genetically engineered cell therapy. (e.g., lymphocyte depleting agents / regimen) to the subject.

[0084] In subjects who are administered a preconditioning agent, the subject may be administered the preconditioning agent at least 2 days, such as at least 3, 4, 5, 6, or 7 days before the initiation of cell therapy. In some embodiments, the subject is administered the preconditioning agent up to 7 days, such as up to 6, 5, 4, 3, or 2 days before the start of cell therapy.

[0085] In some embodiments, the subject receives a dose of cyclophosphamide of less than 100 mg / kg, such as less than about 20 mg / kg, less than about 40 mg / kg, or less than about 80 mg / kg per kg of the subject's body weight. preconditioned with. Accordingly, the preconditioning agent may be a genetically engineered cell therapy that can be used alone or with (a) a cell-binding domain that does not have a cytokine, or (b) a non-targeted cytokine or a functional fragment thereof or a functional fragment thereof that does not have a cell-binding domain. When administered in combination with at least one of the variants, the genetically engineered cell therapy is administered at a lower dosage than when administered in combination with the targeted cytokine construct. In some embodiments, the subject is preconditioned or administered a preconditioning agent, such as cyclophosphamide, in a single dose or in multiple doses, such as every day, every other day, or every three days. In some embodiments, the preconditioning agent, such as cyclophosphamide, is administered at about 400 mg / m2 per body surface area of ​​the subject. 2 Less than 300mg / m 2 less than about 250mg / m 2 less than about 200mg / m 2 Less than or about 100mg / m 2 administered to the subject at a dose of less than about 500 mg / m2, such as less than 500 mg / m2.

[0086] Exemplary lymphodepletion therapies are shown in the table below.

[0087] [Table 1]

[0088] [Table 2]

[0089] The methods, compositions, combinations, kits, and therapies provided herein can treat proliferative diseases such as cancer. In some embodiments, the combination therapies provided herein have beneficial therapeutic effects in the treatment of proliferative diseases such as cancer.

[0090] In some embodiments of the methods, compositions, combinations, kits, and uses provided herein, the combination therapy provided includes any of the therapies or parameters associated with the treatments, as described below. or one or more associated characteristics.

[0091] In some embodiments, parameters related to therapy or treatment outcome include parameters that can be evaluated for screening processes and / or assessment of treatment outcome and / or monitoring of treatment outcome, including tumor burden or disease burden. It will be done. Administration of a therapy comprising the genetically engineered cells and / or targeted cytokine constructs described herein, such as T cell therapy (e.g., CAR-expressing T cells), reduces the spread or burden of disease or disease in a subject. Or it can be prevented. For example, if the disease or disease is a tumor, the method generally reduces tumor size, bulk, metastasis, percentage of blasts in the bone marrow, or molecularly detectable cancer, and / or Prognosis or other symptoms related to survival or tumor burden can be improved.

[0092] In some embodiments, the provided combination therapy reduces tumor burden in the treated subject compared to an alternative method in which genetically engineered cell therapy (e.g., CAR-expressing T cells) is given without administration of targeted cytokine constructs. resulting in a decrease in Although it is not necessary that there is an actual reduction in tumor burden in all subjects receiving combination therapy, at least 50%, 60%, 70%, 80%, 90%, 95% of subjects treated with combination therapy Based on clinical data showing that the majority of subjects treated with such combination therapy showed a reduction in tumor burden, on average the tumor burden was reduced in treated subjects. .

[0093] Disease burden can include the total number of cells of the disease in the subject or in an organ, tissue, or body fluid of the subject, such as a tumor organ or tissue, or another location exhibiting metastasis, for example. For example, tumor cells in the blood, lymph, or bone marrow can be detected and / or quantified in connection with a particular hematological malignancy. Disease burden, in some embodiments, can include tumor mass, number or extent of metastases, and / or percentage of blast cells present in the bone marrow.

[0094] In some embodiments, the subject has myeloma, lymphoma, or leukemia. In some embodiments, the subject has non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), or myeloma. , for example, suffers from multiple myeloma (MM). In some embodiments, the subject suffers from MM or DBCBL. In some embodiments, the subject is suffering from a solid tumor.

[0095] In the case of multiple myeloma, exemplary parameters for assessing the extent of disease burden include the number of clonal plasma cells (e.g., >10% in bone marrow biopsies, or any Parameters such as amount, plasmacytoma), monoclonal protein (paraprotein) in either serum or urine ), evidence of end-organ damage felt to be related to plasma cell dysfunction (e.g., hypercalcemia (corrected calcium >2.75 mmol / 1), renal failure due to myeloma, anemia (hemoglobin <10 g / dL) , and / or bone lesions (osteoporosis with disseminated lesions or compression fractures)). For diffuse large B-cell lymphoma, exemplary parameters to assess the extent of disease burden include cell morphology (e.g., centroblasts, immunoblasts, and undifferentiated cells), gene expression, miRNA Parameters such as expression, and protein expression (eg, expression of BCL2, BCL6, MUM1, LM02, MYC, and p21) are included. In the case of leukemia, the extent of disease burden can be determined by assessment of residual leukemia in the blood or bone marrow. In some embodiments, the subject exhibits morphological disease if there are 5% or more blasts in the bone marrow, for example, as detected by light microscopy. In some embodiments, the subject exhibits complete remission or clinical remission if there are less than 5% blasts in the bone marrow. In some embodiments, in the case of leukemia, the subject may exhibit complete remission, but there are small amounts of residual leukemic cells that are morphologically undetectable (by light microscopy techniques). A subject is said to exhibit minimal residual disease (MRD) if the subject exhibits less than 5% blasts in the bone marrow and exhibits molecularly detectable cancer. In some embodiments, molecularly detectable cancer can be assessed using any of a variety of molecular techniques that allow sensitive detection of small numbers of cells. In some embodiments, such techniques include PCR assays that can determine unique Ig / T cell receptor gene rearrangements or fusion transcripts produced by chromosomal translocations. In some embodiments, flow cytometry can be used to identify cancer cells based on leukemia-specific immunophenotype. In some embodiments, molecular detection of cancer can detect as few as 1 leukemic cell in 100,000 normal cells. In some embodiments, the subject exhibits molecularly detectable MRD when at least 1 or more leukemic cells in 100,000 cells are detected, such as by PCR or flow cytometry. In some embodiments, the subject's disease burden is molecularly undetectable or the subject exhibits minimal residual disease (MRD) such that, in some cases, PCR or flow cytometry techniques are cannot detect leukemia cells in subjects using

[0096] In some embodiments, the combination therapy provided herein reduces disease burden as compared to the disease burden at the time immediately prior to administration of the combination therapy. In some embodiments, administration of the combination therapy prevents an increase in disease burden, as evidenced by no change in disease burden. In some embodiments, the method reduces the reduction observed in equivalent methods using alternative therapies, such as those receiving genetically engineered cell therapy alone, when the subject does not administer the targeted cytokine construct. In comparison, the disease or disease burden, eg, the number of tumor cells, tumor size, patient survival, or symptom-free survival, is reduced to a greater extent and / or for a longer period of time. In some embodiments, the disease burden is determined by administering each of the agents alone, e.g., by administering the targeted cytokine construct to a subject who has not received genetically engineered cell therapy or who has not received the targeted cytokine construct. Compared to the reduction produced by administering a genetically engineered cell therapy to a subject, a reduction occurs to a greater extent or for a longer period of time following combined therapy of a genetically engineered cell therapy and administration of a targeted cytokine construct.

[0097] In some embodiments, a disease or disease burden in a subject is detected, assessed, or measured. Disease burden, in some embodiments, is detected by detecting the total number of diseased or disease-associated cells, e.g., tumor cells, in an organ, tissue, or body fluid, such as blood or serum, within or of the subject. I can do that. In some embodiments, disease burden, eg, tumor burden, is assessed by measuring solid tumor mass and / or number or extent of metastases. In some embodiments, a subject's survival, survival within a specified time period, degree of survival, presence or duration of symptom-free survival or symptom-free survival, or relapse-free survival is assessed. In some embodiments, any symptom of a disease or disorder is assessed. In some embodiments, a disease burden or a measure of disease burden is identified. In some embodiments, exemplary parameters for determination include certain clinical outcomes indicative of improvement or improvement of a disease or disease, eg, a tumor. Such parameters include complete response (CR), partial response (PR), or stable disease (SD) (see e.g. Response Evaluation Criteria in Solid Tumors (RECIST) guidelines), objective response rate (ORR), ), progression-free survival (PFS), and duration of disease control, including overall survival (OS). Certain threshold values ​​of parameters can be established to determine the effectiveness of the combination therapy methods provided herein.

[0098] In some embodiments, the disease burden is determined before administration of the genetically engineered cell therapy, after administration of the genetically engineered cell therapy but before administration of the targeted cytokine construct, or after administration of the targeted cytokine construct but at the genetically engineered cell. Measured or detected prior to administration of the therapy and / or after administration of both the genetically engineered cell therapy and the targeted cytokine construct. In connection with multiple administrations of one or more steps of a combination therapy, the disease burden in some embodiments is determined before or after administration of any of these steps, before or after the dose and / or cycle of administration. It may be determined at a later time, or during the administration of any of these steps, at any time during the dose and / or cycle of administration.

[0099] In some embodiments, the load is at least about 10, 20, 30, 40, 50, 60, 70, 80, compared to immediately before administration of the targeted cytokine construct and genetically engineered cell therapy, by the provided methods. , 90, or 100 percent decrease. In some embodiments, the disease burden, tumor size, tumor volume, tumor mass, and / or tumor burden or size is lower than immediately after administration of the genetically engineered cell therapy and / or targeted cytokine construct. reduced by at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90% or more after administration of cell therapy and targeted cytokine constructs.

[0100] In some embodiments, the reduction in disease burden by the method occurs at 1 month, 2 months, 3 months, or more than 3 months after administration of the combination therapy, e.g., after initiation of the combination therapy. Includes induction of complete morphological remission as assessed. In some embodiments, the assay for minimal residual disease is negative, or the level of minimal residual disease is less than about 0.3%, less than about 0.2%, for example, as measured by multiparameter flow cytometry. , less than about 0.1%, or less than about 0.05%.

[0101] In some embodiments, the subject's symptom-free survival or overall survival rate is improved by the method as compared to other methods. For example, in some embodiments, six months after the methods of combination therapy provided herein, the rate or probability of symptom-free survival of a subject treated by the methods is greater than about 40%, about 50% greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95%. In some embodiments, the overall survival rate is greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95%. In some embodiments, a subject treated with the method has a symptom-free survival of at least 6 months, or up to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years. period, recurrence-free survival, or survival time. In some embodiments, the tumor growth arrest time is at least 6 months or about 6 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years. Time etc. will be improved.

[0102] In some embodiments, the probability of recurrence following treatment with the present method is reduced compared to other methods. For example, in some embodiments, the probability of recurrence six months after the method of combination therapy is less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%. %, less than about 20%, or less than about 10%.

[0103] In some embodiments, the genetically engineered cells described herein are modified (eg, contacted with targeted cytokine constructs of the present disclosure) at a point-of-care location. In some cases, the point-of-care location is at a hospital or facility (eg, a medical facility) near the subject in need of treatment. The subject undergoes apheresis and the resulting peripheral blood mononuclear cells (PBMCs) can be enriched, for example, by elutriation. In one example, the elutriation process is performed using a buffer containing human serum albumin. Genetically engineered cells, eg, CAR+ T cells, TCR-transduced T cells, can be isolated by the selection methods described herein. In one example, methods for selecting genetically engineered cells include beads specific for markers such as CD3 and CD8 on genetically engineered cells (eg, CAR+ T cells). In some cases, the beads can be paramagnetic beads. Harvested and modified cells can be cryopreserved in any suitable cryopreservation solution prior to modification. Cells can be thawed up to 24, 36, 48, 72, or 96 hours before injection. Thawed cells are placed in a cell culture buffer, e.g., cell culture buffer supplemented with fetal bovine serum (FBS) (e.g., RPMI), or in a buffer containing IL-2 and IL-21, prior to modification. You can put it in. In another embodiment, harvested cells can be modified immediately without the need for cryopreservation.

[0104] In some cases, the harvested cells are contacted with a chimeric receptor, genetically engineered / introduced with one or more cell tag(s), and / or targeted cytokine constructs of the present disclosure, and then rapidly administered to the subject. modified by injecting into In some cases, the source of cells can include both allogeneic and autologous sources. In some cases, the cells can be T cells or NK cells. In some cases, the chimeric receptor can be a CAR or a TCR. In some cases, the cell comprises a sequence selected from the group consisting of SEQ ID NOs: 11-90, or a sequence that is at least about 75% to about 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 11-90. , modified by contacting with a targeted cytokine construct comprising a functional fragment or variant of the cytokine protein.

[0105] In certain cases, genetically engineered cells are modified by expressing a cell tag on the cell surface that is distinct from the CAR or TCR molecules, such as truncated epidermal growth factor, EGFRt. In some examples, cell tags include cell tags such as the truncated epidermal growth factor receptor tag described herein, for conditional in vivo ablation of modified genetically engineered cells, e.g., cetuximab. activated through. In some embodiments, the cell is modified by expressing a chimeric antigen receptor or T cell receptor, or a portion thereof, that includes a polypeptide tag sequence, such as a myc tag, having the following sequence: EQKLISEEDL . The targeted cytokine constructs of the present disclosure are, in some examples, specific for a tag, such as those described above, expressed by a cell or expressed as part of a CAR or TCR molecule expressed by a cell. including a binding domain (such as an antibody or antigen-binding fragment thereof). Examples include anti-EGFR antibodies that can be used to bind to the EGFRt tag expressed on genetically engineered cells, or myc expressed as part of a CAR or TCR molecule on genetically engineered cells. Included are anti-myc antibodies that can be used to bind the tag.

[0106] In some embodiments, harvested cells are modified with targeted cytokine constructs through electroporation. In one example, electroporation is performed using an electroporator, such as Lonza's Nucleofector™ electroporator. In other embodiments, the vector containing the above construct is a non-viral vector or a viral vector. In some instances, the non-viral vector includes the Sleeping Beauty transposon-transposase system. In one example, cells are electroporated using a specific sequence. For example, cells can be electroporated with one transposon, followed by DNA encoding a transposase, followed by a second transposon. In another example, immune effector cells can be electroporated with all transposons and transposases simultaneously. In another example, cells can be electroporated with a transposase followed by both transposons or one transposon at once. During continuous electroporation, cells may be allowed to rest for a period of time before the next electroporation step.

[0107] In some cases, modified cells do not undergo proliferation and activation steps. In some cases, the modified cells do not undergo an incubation or culture step (eg, ex vivo expansion). In other examples, the modified immune effector cells are placed in a cell culture buffer, e.g., cell culture buffer (e.g., RPMI) supplemented with fetal bovine serum (FBS), or left undisturbed prior to injection. . Prior to injection, modified cells can be harvested, washed, and formulated in saline buffer for injection into a subject. Targeted cytokine constructs

[0108] The present disclosure provides, in some embodiments, a targeted cytokine construct comprising: (i) a chimeric antigen receptor (CAR) or a T cell receptor (TCR) exogenously introduced into a genetically engineered cell; domain, (ii) a tag molecule selectively expressed on the surface of genetically engineered cells, and (iii) exogenously introduced into genetically engineered cells. (iv) a polypeptide tag that is part of a TCR; or (vi) any combination of (i) to (v). Targeted cytokine constructs are provided that include a cell binding domain and a cytokine protein or functional fragment or variant thereof.

[0109] One embodiment provides a targeted cytokine construct for use in combination therapy with genetically engineered cells, wherein the fusion protein comprises (i) a cell-binding domain, and (ii) a cytokine protein or functional fragment or fragment thereof. including variants, the cell-binding domain (a) comprises an antibody or antigen-binding fragment thereof specific for a domain of an antigen receptor expressed on a genetically engineered cell (e.g., a CAR or TCR); and (c) tag The tag may be a surface molecule that is co-expressed (“separately expressed”) by the genetically engineered cell, or a tag may be a surface molecule that is specific for an antigen receptor expressed by the genetically engineered cell (e.g. (d) a domain derived from an antigen targeted by the genetically engineered cell, or (e )(a)~(d). In some embodiments, the receptor expressed by the genetically engineered cell is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0110] In some embodiments, the targeting cytokine construct comprises two parts, i) the first part comprises antibody heavy chain VH-CH1-hinge-CH2-CH3 monomers and antibody light chain VL-CL Polypes containing VH is a variable heavy chain, CH2-CH3 are Fc domains, VL is a variable light chain, and CL is a constant light chain. chain ii) the N-terminus of the mutant cytokine is fused to the C-terminus of the Fc domain via a linker, and ii) the second part is linked to the antibody heavy chain VH-CH1-hinge-CH2-CH3 monomer. , an antibody light chain VL-CL, wherein the first portion and the second portion are both selectively expressed on genetically engineered cells over non-genetically engineered cells. Bind to a domain.

[0111] In some embodiments, the targeting cytokine construct comprises two parts: i) the first part is a polypeptide comprising an antibody hinge-CH2-CH3 monomer and a variant cytokine polypeptide; CH2-CH3 is the Fc domain, and the N-terminus of the mutant cytokine polypeptide has a linker. ii) the second portion is a polypeptide comprising an antibody heavy chain VH-CH1-hinge-CH2-CH3 monomer and an antibody light chain VL-CL; and the second portion binds to a domain that is preferentially expressed on genetically engineered cells over non-genetically engineered cells.

[0112] In some embodiments, the targeting cytokine construct comprises two parts: i) the first part is a polypeptide comprising an antibody hinge-CH2-CH3 monomer and a variant cytokine polypeptide; CH2-CH3 is the Fc domain, and the C-terminus of the mutant cytokine polypeptide has a linker. ii) the second portion is a polypeptide comprising an antibody heavy chain VH-CH1-hinge-CH2-CH3 monomer and an antibody light chain VL-CL; and the second portion binds to a domain that is preferentially expressed on genetically engineered cells over non-genetically engineered cells.

[0113] The present disclosure provides, in some embodiments, targeted cytokine constructs comprising (i) a cell binding domain, and (ii) a cytokine protein or functional fragment or variant thereof, wherein the cell binding domain comprises (a) a gene (b) an antibody or antigen-binding fragment thereof specific for a receptor or domain expressed exogenously on the surface of the engineered cell; (c) a tag coexpressed by the genetically engineered cell, or a tag on the receptor (e.g., a chimeric antigen receptor-CAR, or a genetically engineered is specific for the T cell receptor (TCR) expressed by the cell, (d) is a domain derived from an antigen targeted by the genetically engineered cell, or (e) is a domain derived from an antigen targeted by the genetically engineered cell. ).

[0114] The cell-binding domain, in some examples, is an antibody or antigen-binding fragment thereof specific for a receptor or domain expressed exogenously on the surface of a genetically engineered cell. In some embodiments, the cell binding domain comprises an anti-idiotypic antibody specific for the genetically engineered cell. In some embodiments, the cell binding domain is a domain of an antigen binding protein that is expressed on a genetically engineered cell, e.g., the cell binding domain is specific for a VH-VL interface, a VH, a VL, or a linker of a scFv. is specific for the domain of the scFv expressed on the genetically engineered cells. In some embodiments, the cell binding domain is specific for a tag co-expressed by the genetically engineered cell, such as an EGFRt tag. In some embodiments, the cell binding domain is specific for a tag on a CAR construct expressed by the genetically engineered cell. An example is shown in Figure 5A. In some embodiments, such tags on the CAR construct include the following structure, scFv-tag-transmembrane domain-additional domain (CD3ζ signaling domain, costimulatory domain (e.g., CD28 domain, 41-BB domain) ) etc.).

[0115] In some embodiments, the cell binding domain includes a domain derived from an antigen targeted by the genetically engineered cell, non-limiting examples of which include neoepitopes from tumor-associated antigens, TSHR, CD19 , CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvlll, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, L ewisY, CD24, PDGFR-β, SSEA-4, CD20, folate receptor α, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, prostase, PAP, ELF2M, ephrinB2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor β, TEM1 / CD248, TEM7R, CL DN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51 E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 variant, prostein, survivin and telomerase, PCTA-1 / galectin 8, melan A / MART1, Ras variant, hTERT, Sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1 B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut Examples include hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, or IGLL1.

[0116] Non-limiting examples of cytokines include IL-2, IL-7, IL-10, IL-15, and IL-21, or a functional fragment or variant thereof, or any combination thereof. . In some embodiments, the cytokine is an IL-2 polypeptide, a fragment or variant thereof. In some embodiments, the cytokine is an IL-7 polypeptide, a fragment or variant thereof. In some embodiments, the cytokine is an IL-10 polypeptide, a fragment or variant thereof. In some embodiments, the cytokine is an IL-21 polypeptide, a fragment or variant thereof. Combining multiple modifications to the original cytokine peptide, e.g., wild-type IL-2, IL-7, IL-10, IL-15, or IL-21, such as reducing affinity or improving biophysical properties. , the desired activity modification may be achieved. As a non-limiting example, an amino acid sequence for consensus N-linked glycosylation may be incorporated into a polypeptide to enable glycosylation. Another non-limiting example is that lysine may be incorporated into a polypeptide to enable pegylation. In some embodiments, a mutation or mutations are introduced into a polypeptide to reduce its affinity for its receptor, thereby modifying its activity. Interleukin-2 (IL-2)

[0117] In some embodiments, the IL-2 polypeptide is a variant IL-2 polypeptide described herein. In some embodiments, provided herein is a mutant cytokine (e.g., an IL-2 polypeptide), which mutant cytokine has a receptor thereof, e.g., IL-2Rα (e.g., SEQ ID NO: 2 or 6B) with a binding affinity of less than 50%.

[0118] "Interleukin-2" or "IL-2", used interchangeably herein, can refer to any naturally occurring IL-2, unless otherwise indicated. "IL-2" can include unprocessed IL-2 (such as precursor IL-2), and "mature IL-2", which can be a form of IL-2 resulting from processing within a cell. The sequence of human "mature IL-2" is provided as SEQ ID NO:1. One exemplary form of intact human IL-2 may consist of an additional N-terminal amino acid signal peptide attached to mature IL-2. "IL-2" can also include naturally occurring variants of IL-2, such as, but not limited to, allelic or splice variants or variants. An exemplary human IL-2 amino acid sequence is described in UniProt P60568 (IL2_HUMAN). A "variant IL-2 polypeptide" can refer to an IL-2 polypeptide that may have an altered affinity for its receptor, such as a decreased affinity for its receptor; such decreased affinity , resulting in a decrease in the biological activity of the mutant. Alterations in affinity, such as reductions in affinity and thereby activity, can be obtained by introducing small amino acid mutations or substitutions. Variant IL-2 polypeptides can also have other modifications to the peptide backbone, such as amino acid deletions, rearrangements, cyclizations, disulfide bonds, or post-translational modifications (e.g., glycosylation or modification of the polypeptide). carbohydrates), chemical or enzymatic modifications to polypeptides to the protein or protein domain to produce a final construct with desired properties, such as attachment of PEG to the polypeptide backbone, addition of peptide tags or labels, or reduced affinity for IL-2Rβγ. Examples include, but are not limited to, fusion. Desired activities can also include improved biophysical properties compared to wild-type IL-2 polypeptide. Multiple modifications may be combined to achieve a desired activity modification, such as decreasing or increasing affinity or improving biophysical properties. As a non-limiting example, an amino acid sequence for consensus N-linked glycosylation may be incorporated into a polypeptide to enable glycosylation. Another non-limiting example is that lysine may be incorporated into a polypeptide to enable pegylation. In some cases, a mutation or mutations are introduced into a polypeptide to modify its activity.

[0119] In some embodiments, the wild-type IL-2 polypeptide comprises the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 1).

[0120] In some embodiments, the variant IL-2 polypeptide also exhibits less than 50% binding affinity for IL-2Rβ (eg, comprising the amino acid sequence of SEQ ID NO: 3). In some embodiments, the variant IL-2 polypeptide has less than 50% increased resistance to IL-2Rα compared to a wild-type IL-2 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1). (eg, comprising the amino acid sequence of SEQ ID NO: 3 or the amino acid sequence shown in FIG. 6C) exhibiting a binding affinity of less than 50% for IL-2Rβ. In some embodiments, the variant IL-2 polypeptide has less than 50% increased resistance to IL-2Rα compared to a wild-type IL-2 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 1). (eg, comprising the amino acid sequence of SEQ ID NO: 4 or the amino acid sequence shown in FIG. 6D) exhibiting a binding affinity of less than 50% for IL-2Rγ. In some embodiments, the variant IL-2 polypeptide exhibits less than 50% binding affinity for IL-2Rα and less than 50% binding affinity for IL-2Rβ compared to the wild-type IL-2 polypeptide. Shows less than 50% binding affinity and shows less than 50% binding affinity for IL-2Rγ. Differences in the binding affinities of the wild-type and disclosed variant polypeptides for IL-2Rα and IL-2Rβ can be determined, for example, by measuring the affinity of protein-protein interactions, which are well known to those skilled in the art. can be measured using a standard surface plasmon resonance (SPR) assay. The difference in binding affinity for IL-2Rγ between the wild-type polypeptide and the disclosed variant polypeptides is not reliable in SPR assays due to the very low affinity of the wild-type IL-2 polypeptide for IL-2Rγ. It cannot be measured in terms of gender. Instead, their reduced affinity for IL-2Rγ was determined by measuring pSTAT5, an IL-2 polypeptide with a substitution that reduces the affinity of IL-2Rγ, and an IL-2 polypeptide without that substitution. can be inferred by performing in vitro assays comparing the activity of IL-2R-expressing cells.

[0121] In some embodiments, the cytokine is (i) an IL-2Rβγ agonist polypeptide that binds to and / or activates an IL-2Rβ polypeptide comprising the amino acid sequence of SEQ ID NO: 3; At least one IL-2Rβγ polypeptide agonist polypeptide that binds to and / or activates an IL-2Rγ polypeptide comprising the amino acid sequence number 4.

[0122] In some embodiments, the IL-2Rβ polypeptide is MAAPALSWRLPLLILLLPLATSWASAAVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLVLCREGVRWRVMAI QDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRN TGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDA Contains the sequence YCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV (SEQ ID NO: 3).

[0123] In some embodiments, the IL-2Rα polypeptide is MDSYLLMWGLLTFIMVPGCQAELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPP Contains the sequence PWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQVAVAGCVFLLISVLLLSGLTWQRRQRKSRRTI (SEQ ID NO: 2)

[0124] In some embodiments, the cytokine is an IL-2 polypeptide, or a functional fragment thereof, or a variant thereof. In some embodiments, the cytokine comprises the amino acid sequence of SEQ ID NO: 2 compared to the binding affinity of a wild-type IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 to an IL-2Rα polypeptide. A mutant IL-2 polypeptide that exhibits a 50% or more reduced binding affinity for an IL-2Rα polypeptide. In some embodiments, the cytokine comprises the amino acid sequence of SEQ ID NO: 3 compared to the binding affinity of a wild-type IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 to an IL-2Rβ polypeptide. A mutant IL-2 polypeptide that exhibits a 50% or more reduced binding affinity for an IL-2Rβ polypeptide.

[0125] In some embodiments, the cytokine has a binding affinity of a wild-type IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 to an IL-2Rγ polypeptide comprising the amino acid sequence of SEQ ID NO: 4. A mutant IL-2 polypeptide exhibiting a 50% or more reduced binding affinity for an IL-2Rα polypeptide comprising the amino acid sequence of SEQ ID NO: 4 and a 50% or more reduced binding affinity for an IL-2Rγ polypeptide. It is a peptide.

[0126] In some embodiments, the IL-2Rγ polypeptide is MLKPSLPFTSLLFLQLPLLGVGLNTTILTPNGNEDTTADFFLTTMPTDSLSVSTLPLPEVQCFVFNVEYMNCTWNS SSEPQPTNLTLHYWYKNSDNDKVQKCSHYLFSEEITSGCQLQKKEIHLYQTFVVQLQDPREPRRQATQMLKLQNLVIPWAPENLTLHKLSESQLELNWNNRF Contains the sequence LNHCLEHLVQYRTDWDHSWTEQSVDYRHKFSLPSVDGQKRYTFRVRSRFNPLCGSAQHWSEWSHPIHWGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET (SEQ ID NO: 4).

[0127] The variant IL-2 polypeptides of the present disclosure, in some examples, have one or more, two or more, or three or more Contains amino acid substitutions that reduce affinity, with one or more, two or more, or three or more Replacement residues are Q11, H16, L18, L19, D20, Q22, R38, F42, K43, Y45, E62, P65, E68, V69, L72, D84, S87, N88, V91, I92, T123, Q126, S127 , I129, and S130. Location of potential amino acid substitutions in the sequence of wild-type mature IL-2 polypeptide. Decreased affinity for IL-2Rα results from substitution of one or more of the following residues in the sequence of the wild-type mature IL-2 polypeptide: R38, F42, K43, Y45, E62, P65, E68, V69, and L72. It can be obtained by doing. Reduced affinity for IL-2Rβ may be obtained by substituting one or more of the following residues: E15, H16, L19, D20, D84, S87, N88, V91, and I92. Decreased affinity for IL-2Rγ can be achieved by substituting one or more of the following residues in the sequence of the wild-type mature IL-2 polypeptide: Q11, L18, Q22, T123, Q126, S127, I129, and S130. may be obtained by

[0128] In some embodiments, the variant IL-2 polypeptide comprises a F42A or F42K amino acid substitution relative to the wild-type mature IL-2 amino acid sequence, eg, SEQ ID NO: 1, as shown in FIG. 6A. In some embodiments, the variant IL-2 polypeptide has a F42A or F42K amino acid substitution and R38A, R38D, R38E, E62Q, E68A, E68Q, E68K, or Contains E68R amino acid substitution. For example, in some embodiments, the variant IL-2 polypeptide is F42A;R38A and F42A;R38D and F42A;R38E and F42A;F42A and E62Q;F42A and E68A; 2A and E68Q; F42A and E68K; F42A and E68R; or R38A and F42K amino acid substitution(s) relative to the wild type mature IL-2 amino acid sequence, eg, as shown in SEQ ID NO:1. In some embodiments, the variant IL-2 polypeptide comprises R38E and F42A amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the variant IL-2 polypeptide comprises R38D and F42A amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the variant IL-2 polypeptide comprises F42A and E62Q amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the variant IL-2 polypeptide comprises R38A and F42K amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the variant IL-2 polypeptide comprises R38D and F42A amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the variant IL-2 polypeptide comprises R38A and F42K amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the variant IL-2 polypeptide comprises F42A and E62Q amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the variant IL-2 polypeptide is H16E, H16D, D20N, M23A, M23R, M23K, D84L, D84N, D84V, D84H, D84Y, D84R relative to the wild-type IL-2 amino acid sequence. , D84K, S87K, S87A, N88A, N88D, N88G, N 88S, N88T, N88R, N88I, V91A, V91T, V91E, I92A, E95S, E95A, E95R, T123A, T123E, T1 Contains 23K, T123Q, Q126A, Q126S, Q126T, Q126E, S127A, S127E, S127K, or S127Q amino acid substitution. In some embodiments, the variant IL-2 polypeptide is F42A; R38A and F42A; R38D and F42A; R38E and F42A; F42A and E62Q; F42A and E68A; ;F42A and E68R; or R38A and F42K amino acid substitution(s) to the wild-type mature IL-2 amino acid sequence, e.g., as shown in SEQ ID NO:1; M23R, M23K, D 84L, D84N, D84V, D84H, D84Y, D84R, D84K, S87K, S87A, N88A, N88D, N88G, N88S, N88T, N88R, N88I, V91A, V91T, V91E, I92A, E95S, E95A , E95R, T123A, T123E, T123K, T123Q, Q126A, Q126S, Q126T, Q126E, S127A, S127E, S127K, or S127Q amino acid substitutions relative to the wild-type mature IL-2 amino acid sequence. For example, in some embodiments, a variant IL-2 polypeptide comprises R38E, F42A, and H16E amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the variant IL-2 polypeptide comprises R38E, F42A, and H16D amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the variant IL-2 polypeptide comprises R38E, F42A, and D84K amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the variant IL-2 polypeptide comprises R38E, F42A, and D84R amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the variant IL-2 polypeptide comprises R38E, F42A, and N88S amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the variant IL-2 polypeptide comprises R38E, F42A, and N88A amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the variant IL-2 polypeptide comprises R38E, F42A, and N88G amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the variant IL-2 polypeptide comprises R38E, F42A, and N88R amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the variant IL-2 polypeptide comprises R38E, F42A, and N88T amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the variant IL-2 polypeptide comprises R38E, F42A, and N88D amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the variant IL-2 polypeptide comprises R38E, F42A, and V91E amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the variant IL-2 polypeptide comprises R38E, F42A, and Q126S amino acid substitutions relative to the wild-type IL-2 amino acid sequence.In some embodiments, the variant IL-2 polypeptide comprises (relative to the sequence of SEQ ID NO: 1) R38E and F42A; R38D and F42A; F42A and E62Q; R38A and F42K; R38E, F42A, and N88S; R38E, F42A, and N88A; R38E, F42A, and N88G; R38E, F42A, and N88R; R38 E, F42A, and N88T; R38E, F42A, and N88D; R38E, F42A, and V91E; R38E, F42A, and D84H; R38E, F42A, and D 84K; R38E, F42A, and D84R; H16D, R38E, and F42A; H16E, R38E, and F42A; R38E, F42A, and Q126S; R38D, F42 A and N88S; R38D, F42A and N88A; R38D, F42A and N88G; R38D, F42A and N88R; R38D, F42A and N88T; R38D, F 42A and N88D; R38D, F42A and V91E; R38D, F42A, and D84H; R38D, F42A, and D84K; R38D, F42A, and D84R; H 16D, R38D and F42A; H16E, R38D and F42A; R38D, F42A and Q126S; R38A, F42K, and N88S; R38A, F42K, and N 88A; R38A, F42K, and N88G; R38A, F42K, and N88R; R38A, F42K, and N88T; R38A, F42K, and N88D; R38A, F4 2K, and V91E; R38A, F42K, and D84H; R38A, F42K, and D84K; R38A, F42K, and D84R; H16D, R38A, and F42K; H16E, R38A, and F42K; R38A, F42K, and Q126S; F42A, E62Q, and N88S; F42A, E62Q, and N88A; F42A, E62Q , and N88G; F42A, E62Q, and N88R; F42A, E62Q, and N88T; F42A, E62Q, and N88D; F42A, E62Q, and V91E; F4 2A, E62Q, and D84H; F42A, E62Q, and D84K; F42A, E62Q, and D84R; H16D, F42A, and E62Q; H16E, F42A, and E 62Q;F42A, E62Q, and Q126S;R38E, F42A, and C125A;R38D, F42A, and C125A;F42A, E62Q, and C125A;R3 8A, F42K, and C125A; R38E, R42A, N88S, and C125A; R38E, F42A, N88A, and C125A; R38E, F42A, N88G, and and C125A; R38E, F42A, N88R, and C125A; R38E, F42A, N88T, and C125A; R38E, F42A, N88D, and C125A; R3 8E, F42A, V91E, and C125A; R38E, F42A, D84H, and C125A; R38E, F42A, D84K, and C125A; R38E, F42A, D8 4R, and C125A; H16D, R38E, F42A, and C125A; H16E, R38E, F42A, and C125A; R38E, F42A, C125A, and Q126 S; R38D, F42A, N88S, and C125A; R38D, F42A, N88A, and C125A; R38D, F42A, N88G, and C125A; R38D, F4 2A, N88R, and C125A; R38D, F42A, N88T, and C125A; R38D, F42A, N88D, and C125A; R38D, F42A, V91E, and and C125A; R38D, F42A, D84H, and C125A; R38D, F42A, D84K, and C125A; R38D, F42A, D84R, and C125A; H1 6D, R38D, F42A, and C125A; H16E, R38D, F42A, and C125A; R38D, F42A, C125A, and Q126S; R38A, F42K, N88S, and C125A; R38A, F42K, N88A, and C125A; R38A, F42K, N88G, and C125A; R38A, F42K, N88R, and C1 25A; R38A, F42K, N88T, and C125A; R38A, F42K, N88D, and C125A; R38A, F42K, V91E, and C125A; R38A, F42K, D84H, and C125A; R38A, F42K, D84K, and C125A; R38A, F42K, D84R, and C125A; H16D, R38A, F42K, and C125A; H16E, R38A, F42K, and C125A; R38A, F42K, C125A and Q126S; F42A, E62Q, N88S, and C125A; F 42A, E62Q, N88A, and C125A; F42A, E62Q, N88G, and C125A; F42A, E62Q, N88R, and C125A; F42A, E62Q, N88T, and C125A; F42A, E62Q, N88D, and C125A; F42A, E62Q, V91E, and C125A; F42A, E62Q, and D84H, and C125A; F42A, E62Q, and D84K, and C125A; F42A, E62Q, and D84R, and C125A; H16D, F42A, and E62Q, and C125 A; H16E, F42A, and E62Q, and C125A; F42A, E62Q, C125A, and Q126S; F42A, N88S, and C125A; F42A, N88A, and C125A; F42A, N88G, and C125A; F42A, N88R, and C125A; F42A, N88T, and C125A; F42A, N88D, and C125 A;F42A, V91E, and C125A;F42A, D84H, and C125A;F42A, D84K, and C125A;F42A, D84R, and C125A;H16D , F42A, and C125A; H16E, F42A, and C125A; and F42A, C125A, and Q126S. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 1 with 1, 2, 3, 4, or 5 amino acid substitutions relative to SEQ ID NO: 1; 4 or 5 substitution(s) are Q11, H16, L18, L19 , D20, Q22, R38, F42, K43, Y45, E62, P65, E68, V69, L72, D84, S87, N88, V91, I92, T123, Q126, S127, I129, and S130 , sequence number. Contains substitution(s) in position 1. In some embodiments, the IL-2 polypeptide comprises (relative to the sequence of SEQ ID NO: 1) R38E and 42A; R38D and F42A; F42A and E62Q; R38A and F42K; , and N88A;R3 8E, F42A, and N88G; R38E, F42A, and N88R; R38E, F42A, and N88T; R38E, F42A, and N88D; R38E, F42A, and V91E; R38E, F42A, and D84H; R38E, F42A, and D84K; R38E, F42A, and D84R; H16D, R38E, and F42A; H16E, R38E, and F42A; R38E, F42A, and Q126S; R38D, F42A, and N88S; R38D, F42A, and N88A; D, F42A and N88R; R38D, F42A and N88T; R38D, F42A and N88D; R38D, F42A and V91E; R38D, F42A and D84H; R38D, F42A and D84K; 38D and F42A; H16E, R38D and F42A; R38D, F42A and Q126S; R38A, F42K, and N88S; R38A, F42K, and N88A; R38A, F42K, and N88G; 42K and N88T; R38A, F42K, and N88D; R38A, F42K, and V91E; R38A, F42K, and D84H; R38A, F42K, and D84K; R38A, F42K, and D84R; H16D, R38A, and F42K; H16E, R38A and F42K; R38A, F42K and Q126S; F42A, E62Q and N88S; F42A, E62Q and N88A; F42A, E62Q and N88G; F42A, E62Q and N88R; F42A, E62Q and N88T; F4 SEQ ID NO: 1 having one of the following amino acid substitutions: 2A, E62Q, and N88D; F42A, E62Q, and V91E; F42A, E62Q, and D84H; F42A, E62Q, and D84K; Contains an array of .

[0129] In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 1 with an additional amino acid substitution relative to SEQ ID NO: 1 at position C125. In some embodiments, the IL-2 polypeptide comprises (relative to the sequence of SEQ ID NO: 1) R38E, F42A, and C125A; R38D, F42A, and C125A; F42A, E62Q, and C125A; 42K, and C125A; R38E, R42A, N88S, and C125A; R38E, F42A, N88A, and C125A; R38E, F42A, N88G, and C125A; R38E, F42A, N88R, and C 125A; R38E, F42A, N88D, and C125A; R38E, F42A, N88T, and C125A; R38E, F42A, V91E, and C125A; R38E, F42A, D84H, and C125A; R3 8E, F42A, D84K, and C125A; R38E, F42A, D84R, and C125A; H16D, R38E, F42A, and C125A; H16E, R38E, F42A, and C125A; R38E, F42A , C125A and Q126S; R38D, F42A, N88S, and C125A; R38D, F42A, N88A, and C125A; R38D, F42A, N88G, and C125A; R38D, F42A, N88R, and C125A; R38D, F42A, N88T, and C125A; R38D, F42A, N88D, and C125A; R38D, F42A, V91E, and C125A; R38D, F42A, D84H, and C125A R38D, F42A, D84K, and C125A; R38D, F42A, D84R, and C125A; H16D, R38D, F42A, and C125A; H16E, R38D, F42A, and C125A; R38D, F 42A, C125A, and Q126S; R38A, F42K, N88S, and C125A; R38A, F42K, N88G, and C125A; R38A, F42K, N88R, and C125A; R38A, F42K, N8 8T, and C125A; R38A, F42K, N88D, and C125A; R38A, F42K, N88A, and C125A; R38A, F42K, V91E, and C125A; R38A, F42K, D84H, and C 125A; R38A, F42K, D84K, and C125A; R38A, F42K, D84R, and C125A; H16D, R38A, F42K, and C125A; H16E, R38A, F42K, and C125A; R3 8A, F42K, C125A and Q126S; F42A, E62Q, N88S, and C125A; F42A, E62Q, N88A, and C125A; F42A, E62Q, N88G, and C125A; F42A, E62Q , N88R, and C125A; F42A, E62Q, N88T, and C125A; F42A, E62Q, N88D, and C125A; F42A, E62Q, V91E, and C125A; F42A, E62Q, and D84H , and C125A; F42A, E62Q, and D84K, and C125A; F42A, E62Q, and D84R, and C125A; H16D, F42A, and E62Q, and C125A; H16E, F42A, and E6 2Q, and C125A; F42A, E62Q, C125A, and Q126S; F42A, N88S, and C125A; F42A, N88A, and C125A; F42A, N88D, and C125A; F42A, V91E, and C125A; F42A, D84H, and C125A; F42A, D84K, and C125A; F4 2A, D84R, and C125A; H16D, F42A, and C125A; H16E, F42A, and C125A; and F42A, C125A, and Q126S.

[0130] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 11).

[0131] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 12).

[0132] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 13).

[0133] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 14).

[0134] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 15).

[0135] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 16).

[0136] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 17).

[0137] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 18).

[0138] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 19).

[0139] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 20).

[0140] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCSSIISTLT (SEQ ID NO: 21).

[0141] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 22).

[0142] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 23).

[0143] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 24).

[0144] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 25).

[0145] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 26).

[0146] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 27).

[0147] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCSSIISTLT (SEQ ID NO: 28).

[0148] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 29).

[0149] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 30).

[0150] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 31).

[0151] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 32).

[0152] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 33).

[0153] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 34).

[0154] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCSSIISTLT (SEQ ID NO: 35).

[0155] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 36).

[0156] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 37).

[0157] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 38).

[0158] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPHRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 39).

[0159] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 40).

[0160] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 41).

[0161] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCSSIISTLT (SEQ ID NO: 42).

[0162] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 43).

[0163] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 44).

[0164] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 45).

[0165] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 46).

[0166] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 47).

[0167] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 48).

[0168] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 49).

[0169] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 50).

[0170] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 51).

[0171] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 52).

[0172] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFASSIISTLT (SEQ ID NO: 53).

[0173] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 54).

[0174] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 55).

[0175] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 56).

[0176] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 57).

[0177] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 58).

[0178] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 59).

[0179] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFASSIISTLT (SEQ ID NO: 60).

[0180] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 61).

[0181] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 62).

[0182] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 63).

[0183] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 64).

[0184] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 65).

[0185] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 66).

[0186] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFASSIISTLT (SEQ ID NO: 67).

[0187] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 68).

[0188] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 69).

[0189] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 70).

[0190] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPHRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 71).

[0191] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 72).

[0192] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 73).

[0193] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFASSIISTLT (SEQ ID NO: 74).

[0194] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 75).

[0195] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 76).

[0196] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 77).

[0197] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 78).

[0198] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 79).

[0199] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 80).

[0200] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFASSIISTLT (SEQ ID NO: 81).

[0201] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 82).

[0202] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 83).

[0203] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 84).

[0204] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 85)

[0205] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 86).

[0206] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 87).

[0207] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 88).

[0208] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 89).

[0209] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 90).

[0210] In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 137). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 138). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 139). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 140). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 141). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 142). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 143). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 144). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 145). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 146). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 147). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 148). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 149). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 150). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 151). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 152). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 153). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 154). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 156). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 157). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 158). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 159). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 160). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 161). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 162).In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 163). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 164). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 165). In some embodiments, the variant IL-2 polypeptide is APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATEL. Contains the amino acid sequence of KHLQCLEEELKPLEEVLNLAQSKNFHLRPRLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 166). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 167). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 168). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 169). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 170). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 174). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 175). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 176). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 177). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 178). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 179). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 180). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 181). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 182). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 183). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 184). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 185). In some embodiments, the variant IL-2 polypeptide comprises the amino acid sequence of an IL-2 polypeptide listed in Table 7.

[0211] [Table 3-1]

[0212] [Table 3-2]

[0213] In some embodiments, the variant IL-2 polypeptides of the present disclosure also include mutations and deletions that provide additional benefits such as improved biophysical properties, including, but not limited to, other mutations and deletions. Also includes modifications. Improved biophysical properties include, but are not limited to, improved thermal stability, tendency to agglomerate, acid reversibility, viscosity, and production in mammalian or bacterial or yeast cells. For example, residue C125 may be substituted with a neutral amino acid such as serine, alanine, threonine, or valine, and the N-terminal A1 residue may be deleted, both of which are described in U.S. Pat. listed in the number. Variant IL-2 polypeptides may also include mutations at residue M104, such as M104A, as described in US Pat. No. 5,206,344. Thus, in certain embodiments, variant IL-2 polypeptides of the present disclosure include the amino acid substitution C125A. In other embodiments, 1, 2, or 3 N-terminal residues are deleted. Interleukin-10 (IL-10)

[0214] Interleukin-10 (IL-10) is a cytokine that regulates many immune cell subsets, some of which include monocytes, macrophages, dendritic cells, B cells, T cells, and NK cells. It will be done. IL-10 is comprised of two subunits: IL-10RA, which is specific for IL-10 and expressed primarily on immune cells, and IL-10RB, which is shared with other cytokines and is more widely expressed. It binds to a heterodimeric receptor (IL-10 receptor, IL-10R) consisting of Binding of IL-10 to its receptor induces phosphorylation of the receptor-associated Janus kinase, JAK1, and tyrosine kinase, TYK2, which in turn induces phosphorylation of the STAT3 transcription factor (pSTAT3), which controls transcription of many genes in lymphocytes. Oxidation is promoted.

[0215] The terms "interleukin-10" or "IL-10," used interchangeably herein, refer to primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. ) may refer to any naturally occurring IL-10 from any vertebrate source, including mammals such as ). IL-10 can exist as a homodimer. "IL-10" can include unprocessed IL-10 and "mature IL-10," which is the form of IL-10 that results from intracellular processing. The sequence of "mature IL-10" is shown in Figure 9A. One exemplary form of intact human IL-10 is composed of an additional N-terminal amino acid signal peptide attached to mature IL-10. "IL-10" can also include naturally occurring variants of IL-10, such as allelic or splice variants or variants. An exemplary human IL-10 amino acid sequence is described in UniProt P22301 (IL10_HUMAN).

[0216] "IL-10 homodimer" or "IL-10 dimer", used interchangeably herein, refers to two molecules of the IL-10α chain (IL-10RA) and two molecules of the IL-10Rβ chain (IL-10RA) 10RB), which binds to the tetrameric IL-10 receptor (IL-10R) complex on cells. The α-helices of each IL-10 polypeptide chain are intertwined such that the first four helices of one chain (A-D) associate with the last two helices of the other chain (E and F), thereby , the structural integrity of each domain is maintained upon dimerization (Walter & Nagabhushan,Biochemistry.1995 Sep 26;34(38):12118-25). "IL-10 monomer" may refer to a monomeric form of IL-10 that can be produced by extending loops connecting exchanged secondary structure elements. Insertion of six amino acids into the loop prevents dimerization and reduces IL-10 monomerization, as described in Josephson et al, Biochemistry 1995 Sep 26;34(38):12118-25. was sufficient to induce body formation. The resulting IL-10 monomer is biologically active, binding to a single IL-10RA molecule and recruiting a single IL-10RB molecule into the signaling complex to induce IL-10 activity. can induce cellular responses. Thus, a short amino acid sequence or a short linker can be inserted into the IL-10 polypeptide (e.g., the wild-type IL-10 of the present disclosure or into the sequence of any mutant IL-10 polypeptide) to produce a "monomeric isomer" of that IL-10 polypeptide. This additional amino acid sequence or linker can be inserted immediately after C114, E115, N116, K117, S118, K119, or A120. As described herein, the amino acid numbering of the IL-10 monomeric polypeptide is set to SEQ ID NO: 95 (i.e., IL-10 dimeric polypeptide) so that the linker sequence / amino acid(s) are not counted. based on the number of body polypeptides).

[0217] A "variant IL-10 polypeptide" can refer to an IL-10 polypeptide that has an amino acid sequence that differs from wild-type IL-10. For example, variant IL-10 polypeptides may have amino acid substitutions, deletions, and insertions. In some embodiments, the variant IL-10 polypeptide has reduced affinity for its receptor, and such reduced affinity results in reduced biological activity of the variant. A reduction in affinity and therefore activity can be obtained by introducing a small number of amino acid mutations or substitutions. Variant IL-10 polypeptides can also have other modifications to the peptide backbone, such as amino acid deletions, rearrangements, cyclizations, disulfide bonds, or post-translational modifications (e.g., glycosylation or modification of the polypeptide). carbohydrates), chemical or enzymatic modifications to polypeptides. to proteins or protein domains to produce final constructs with desired properties, such as decoration (e.g., attachment of PEG to the polypeptide backbone), addition of peptide tags or labels, or reduced affinity for IL-10R. Examples include, but are not limited to, the fusion of Desired activities can also include improved biophysical properties compared to wild-type IL-10 polypeptide.

[0218] In some embodiments, the present disclosure relates to variant IL-10 polypeptides and targeted cytokines comprising variant IL-10 polypeptides. In some embodiments, the variant IL-10 polypeptide has increased binding affinity for an IL-10RB polypeptide (e.g., comprising the sequence SEQ ID NO: 97). Including the above mutations. In some embodiments, the variant IL-10 polypeptide has reduced binding affinity for an IL-10RA polypeptide (e.g., comprising the sequence SEQ ID NO: 96). Including the above mutations. In some embodiments, the variant IL-10 polypeptide has increased binding affinity for an IL-10RB polypeptide (e.g., comprising the sequence SEQ ID NO: 97). and one or more mutations (eg, to SEQ ID NO: 95) that reduce binding affinity for an IL-10RA polypeptide (eg, including the sequence of SEQ ID NO: 96). In some embodiments, the variant IL-10 polypeptide is wild-type mature IL-10 (e.g., SEQ ID NO: 95 shown in Figure 9A), or the mature monomeric IL-10 shown (e.g., in Figure 9D). at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least also 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 %, at least 98%, or at least 99% amino acid sequence identity.

[0219] In some embodiments, the variant IL-10 polypeptide is wild-type mature IL-10 (e.g., SEQ ID NO: 95 shown in FIG. 9A), or mature monomeric IL-10 (e.g., the sequence shown in FIG. 9D). No. 98), at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , comprising amino acid sequences having at least 98%, or at least 99% amino acid sequence identity. In some embodiments, the variant IL-10 polypeptide exhibits reduced binding affinity for i) an IL-10RA polypeptide having an amino acid sequence as shown by SEQ ID NO: 96 shown in FIG. 9B; ii) a wild-type IL-10 polypeptide as shown by SEQ ID NO: 95 shown in Figure 9A, or as shown by SEQ ID NO: 98 shown in Figure 9D and shown in Figures 10A-11B; As shown, P20, L23, R24, R27, D28, K34, T35, Q38, M39, D41, L43, D44, N45, L46, K49, I87, V91, L94, L98, K1 38, S141, E142, D144, N148, E151, and I158 to the amino acid sequence of mature monomeric IL-10. In some embodiments, the one or more amino acid substitutions are R24A, R27A, K34A, K34D, K34E, K34S, K34P, K34G, K34T, K34H, K 34L, K34N, K34F, K34R, K34Q, K34V, K34Y, Q38A, Q38D, Q38P, Q38G, Q38H, Q38I, Q38L, Q38R, Q38 K, Q38N, Q38F, Q38T, Q38E, Q38S, Q38V, Q38Y, D44A, D44E, D44S, D44V, D44G, D44H, D44I, D44K, D44P, D44L, D44N, D44F, D44T, D44R, D44Q, I87A, K138A, E142A, E142G, E142N, E142L, E142F, E 142I, E142V, E142K, E142R, E142P, E142Q, E142T, E142S, E142Y, D144A, D144E, D144G, D144H , D144R, D144I, D144K, D144N, D144Q, D144P, D144S, D144L, D144T, D144V, D144Y, N148G, N148 P, N148S, N148D, N148T, N148K, N148V, N148I, N148E, N148F, E151A, E151G, E151H, E151I, E15 1N, E151F, E151L, E151V, E151R, E151K, E151P, E151Q, E151S, E151T, and E151Y. In some embodiments, the one or more amino acid substitutions are R24A, R27A, K34A, K34D, K34E, K34S, K34P, K34G, K34T, K34H, K34L, K34N, K34F, K34V, K34Y, Q38A, Q38D, Q38P ,Q38G , Q38I, Q38L, Q38R, Q38K, Q38F, Q38T, Q38E, Q38S, Q38V, Q38Y, I87A, K13 8A, E142A, E142G, E142N, E142L, E142F, E142I, E142V, E142K, E142R, E14 2P, E142Q, E142T, E142S, E142Y, D144A, D144E, D144G, D144H, D144R, D1 44I, D144K, D144N, D144Q, D144P, D144S, D144L, D144T, D144V, D144Y, N1 48P, N148D, N148I, E151A, E151G, E151H, E151I, E151N, E151F, E151L, E151V, E151R, E151K, E151P, E151Q, E151S, E151T, and E151Y. In some embodiments, a variant IL-10 polypeptide of the present disclosure exhibits 50% or more reduced binding to an IL-10RA polypeptide having an amino acid sequence as set forth in SEQ ID NO: 96 shown in Figure 9B. Shows affinity. The difference in binding affinity for IL-10RA between wild-type and mutant IL-10 polypeptides is determined by standard methods for measuring the affinity of protein-protein interactions, which are well known to those skilled in the art. measured using a standard SPR assay. In some embodiments, the variant IL-10 polypeptide is wild-type mature IL-10 (e.g., SEQ ID NO: 95 shown in FIG. 9A), or mature monomeric IL-10 (e.g., the sequence shown in FIG. 9D). No. 98), at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , comprising amino acid sequences having at least 98%, or at least 99% amino acid sequence identity. In some embodiments, the variant IL-10 polypeptide exhibits increased binding affinity for i) an IL-10RB polypeptide having an amino acid sequence as shown by SEQ ID NO: 97 shown in FIG. 9C; ii) Denoted by SEQ ID NO: 95 shown in Figure 9A, N18, N21, M22, R24, D25, S31, R32, D55, M68, I69, L73, E 74, M77, P78, Q79, E81, N82, K88, A89, H90, N92, S93, G95, E96, N97, K99, T100, L101, L103, R104, R1 07, R110, and F111 (FIGS. 10A-11B) relative to the amino acid sequence of the wild-type mature IL-10 polypeptide. In some embodiments, the one or more amino acid substitutions are at position(s) selected from the group consisting of N18, D28, N92, K99, and L103, numbered according to SEQ ID NO:95. In some embodiments, the one or more amino acid substitutions are N18F, N18L, N18Y, D28Q, D28R, N92F, N, numbered according to SEQ ID NO:95. 92H, N92I, N92K, N92L, N92R, N92S, N92T, N92V, N92Y, K99N, L103N, and L103Q. In yet other embodiments, the variant IL-10 polypeptide exhibits 150% or more increased binding affinity for an IL-10RB polypeptide having an amino acid sequence as set forth in SEQ ID NO: 97 shown in Figure 9C. .

[0220] The positions of potential amino acid substitutions in the sequence of wild type mature IL-10 polypeptide are shown in Figures 10A-10B. In some embodiments, as shown in FIGS. 11A-11B, designated amino acids in the sequence of the wild type mature IL-10 polypeptide are substituted with alanine or another amino acid.

[0221] In some embodiments, variant IL-10 polypeptides also include other modifications, including but not limited to mutations and deletions, that provide additional benefits such as improved biophysical properties. include. Improved biophysical properties include, but are not limited to, improved thermal stability, tendency to agglomerate, acid reversibility, viscosity, and production in mammalian or bacterial or yeast cells.

[0222] In some embodiments, the variant IL-10 polypeptide further comprises an amino acid substitution to the amino acid sequence of SEQ ID NO: 1 at position R107. In some embodiments, the variant IL-10 polypeptide further comprises the R107A mutation numbered according to SEQ ID NO:95.

[0223] In some embodiments, the variant IL-10 polypeptide has a linkage between N116 and K117 (e.g., as shown in FIG. 9D), e.g., to enable folding and expression as a monomer. A monomer containing an amino acid insertion or a peptide insertion. In some embodiments, insertions are 1-15 amino acids in length. In some embodiments, the insertion is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids long. In certain embodiments, the insertion is 6 amino acids long. In some embodiments, the variant IL-10 monomeric polypeptide comprises an amino acid insertion or peptide insertion of 1 to 15 amino acids immediately following residues C114, E115, N116, K117, S118, K119, or A120. and the numbering is based on SEQ ID NO: 95. Examples of insertions are G, GG, GGG, GGGG, GGGSG, GGGGG , GGGGGG , and GGGSGG. In some embodiments, the variant IL-10 monomeric polypeptide comprises the amino acid sequence of SEQ ID NO:98. In some embodiments, a variant monomeric IL-10 polypeptide of the present disclosure has reduced binding affinity for an IL-10RA polypeptide having the amino acid sequence shown in Figure 1B. , P20, L23, R24, R27, D28, K34, T35, Q38, M39, D41, L43, D44, N45, L46, K49, I87, V91, L94, L98, K138 , S141, E142, D144, N148, E151, and I158 (or R24, R27, K34, Q38, D44, I87, K138, E142, D14 4, N148, and E151), the amino acid numbering refers to the corresponding amino acid in the wild-type IL-10 polypeptide without the 6 linker insertion. In some embodiments, the variant monomeric IL-10 polypeptides of the present disclosure also have increased binding affinity for an IL-10RB polypeptide having the amino acid sequence shown in FIG. 1C, with N18, N21, M22, R24, D25, D28, S31, R32, D55, M68, I69, L73, E74, M77, P 78, Q79, E81, N82, K88, A89, H90, N92, S93, G95, E96, N97, K99, T100, L101, L103, R104 , R107, R110 and F111 (or selected from the group N18, D28, N92, K99 and L103).

[0224] Table 3 shows exemplary amino acid insertions and insertion positions for IL-10 monomeric polypeptides of the present disclosure (inserts are underlined). In some embodiments, the variant IL-10 monomeric polypeptide comprises an amino acid sequence listed in Table 3. In some embodiments, the variant IL-10 monomeric polypeptide comprises an amino acid insertion as listed in Table 3 and / or at a position listed in Table 3. In some embodiments, the insertion is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids long. In some embodiments, the variant IL-10 monomeric polypeptide comprises an amino acid insertion or peptide insertion of 1 to 15 amino acids immediately following residues C114, E115, N116, K117, S118, K119, or A120. The amino acid sequence of a variant IL-10 monomeric polypeptide of the present disclosure having the numbering is based on SEQ ID NO: 95. Examples of insertions are G, GG, GGG, GGGG, GGGSG, GGGGG , GGGGGG , and GGGSGG.

[0225] [Table 4-1]

[0226] [Table 4-2]

[0227] [Table 4-3]

[0228] [Table 4-4]

[0229] In some embodiments, the variant IL-10 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 99-112 provided in Table 4.

[0230] [Table 5-1]

[0231] [Table 5-2]

[0232] [Table 5-3]

[0233] [Table 5-4]

[0234] [Table 5-5]

[0235] An exemplary amino acid sequence of an IL-7 polypeptide is provided below (SEQ ID NO: 91), with positions K10, Q11, S14, V15, V18, Q22, L35, N36, D74, L77, L80, At least one of K81, E84, I88, R133, Q136, E137, T140, N143, and K144 can be mutated (eg, K81A and T140A). DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (SEQ ID NO: 91).

[0236] For example, exemplary mutant IL-7 peptides include amino acid substitutions K81A and T140A. In some embodiments, the variant IL-7 peptide comprises the sequence of DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLAVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKACWNKILMGTKEH (SEQ ID NO: 113).

[0237] In some embodiments, the cytokine comprises the amino acid sequence of SEQ ID NO: 94 compared to the binding affinity of a wild-type IL-7 polypeptide comprising the amino acid sequence of SEQ ID NO: 91 to the IL-7Rα polypeptide. A mutant IL-7 polypeptide that exhibits a 50% or more reduced binding affinity for an IL-7Rα polypeptide. In some embodiments, the variant IL-7 polypeptide has a binding affinity for the IL-7Rγ polypeptide of a wild-type IL-7 polypeptide comprising the amino acid sequence of SEQ ID NO:91. 4 shows a 50% or more reduced binding affinity for the IL-7Rγ polypeptide containing the amino acid sequence of 4.

[0238] In some embodiments, the IL-7Rα polypeptide is MTILGTTFGMVFSLLQVVSGESGYAQNGDLEDAELDDYSFSCYSQLEVNGSQHSLTCAFEDPDVNITNLEFEICGALVEVKCLNFRKLQEIYFIETKKF LLIGKSNICVKVGEKSLTCKKIDLTTIVKPEAPFDLSVVYREGANDFVVTFNTSHLQKKYVKVLMHDVAYRQEKDENKWTHVNLSSTKLTLLQRKLQPAAMYEIKVRSIPDHYFKGFWSEWSPSY YFRTPEINNSSGEMDPILLTISILSFFSVALLVILACVLWKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNC PSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ (SEQ ID NO: 94). Interleukin-21 (IL-21)

[0239] In some embodiments, the cytokine has a binding affinity of SEQ ID NO: 93 as compared to the binding affinity of a wild-type IL-21 polypeptide comprising the amino acid sequence of SEQ ID NO: 92 or SEQ ID NO: 115 to an IL-21R polypeptide. A mutant IL-21 polypeptide that exhibits a 50% or more reduced binding affinity for an IL-21Rγ polypeptide comprising an amino acid sequence. In some embodiments, the binding affinity of a wild-type IL-21 polypeptide comprising the amino acid sequence of SEQ ID NO: 92 or SEQ ID NO: 115 to an IL-21Rγ polypeptide is compared to exhibits a 50% or more reduced binding affinity for the IL-21Rγ polypeptide comprising the amino acid sequence of SEQ ID NO:93.

[0240] "Interleukin-21" or "IL-21", used interchangeably herein, may refer to any naturally occurring IL-21, unless otherwise indicated. "IL-21" can include unprocessed IL-21 (such as precursor IL-21), as well as "mature IL-21", which can be the form of IL-21 resulting from intracellular processing. The sequence of human "mature IL-21" is provided as SEQ ID NO: 92 or SEQ ID NO: 115. "IL-21" can also include naturally occurring variants of IL-21, such as, but not limited to, allelic or splice variants or variants. An exemplary human IL-21 amino acid sequence is described in UniProt Q9HBE4 (IL21_HUMAN). A "mutant IL-2 polypeptide" can refer to an IL-2 polypeptide that may have an altered affinity for its receptor, such as a decreased affinity for its receptor; such decreased affinity , resulting in a decrease in the biological activity of the mutant. Alterations in affinity, such as reductions in affinity and thereby activity, can be obtained by introducing small amino acid mutations or substitutions. Variant IL-21 polypeptides can also have other modifications to the peptide backbone, such as amino acid deletions, rearrangements, cyclizations, disulfide bonds, or post-translational modifications (e.g., glycosylation or modification of the polypeptide). carbohydrates), chemical or enzymatic modifications to polypeptides. to proteins or protein domains to produce final constructs with desired properties, such as decoration (e.g., attachment of PEG to the polypeptide backbone), addition of peptide tags or labels, or reduced affinity for IL-21R. Examples include, but are not limited to, the fusion of Desired activities can also include improved biophysical properties compared to wild-type IL-21 polypeptide. Multiple modifications may be combined to achieve a desired activity modification, such as decreasing or increasing affinity or improving biophysical properties. As a non-limiting example, an amino acid sequence for consensus N-linked glycosylation may be incorporated into a polypeptide to enable glycosylation. Another non-limiting example is that lysine may be incorporated into a polypeptide to enable pegylation. In some cases, a mutation or mutations are introduced into a polypeptide to modify its activity.

[0241] IL-21 has a four-helix bundle structure and exists as a monomer. In humans, two isoforms of IL-21 are known, each derived from a precursor molecule. The first IL-21 isoform contains 162 amino acids (AA), of which the first 29 constitute the signal peptide, and the second IL-21 isoform contains 153 aa. the first 29 of which constitute the signal peptide, similar to the first isoform.

[0242] IL-21 binds to the heterodimeric IL-21 receptor complex, which is composed of the IL-21 receptor (IL-21R) and the common γ chain (γc). The IL-21 receptor complex is expressed on the surface of T cells, B cells, and NK cells. The IL-21 receptor complex is similar in structure to the IL-2 receptor complex in that each of these cytokine receptor complexes contains γc.

[0243] When IL-21 binds to the IL-21 receptor complex, the JAK / STAT signaling pathway is activated and target genes are activated. Although IL-21-induced signaling may be therapeutically desirable, given the broad expression profile of IL-21, IL-21 not only enhances CD8+ T-cell responses but also stimulates antigen presentation and T-cell priming. The fact that it also has the ability to suppress the effects of cancer requires careful consideration of the timing and location of signal transduction.

[0244] In some embodiments, the IL-21 comprises the sequence HKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 92).

[0245] In some embodiments, the IL-21 comprises the sequence QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 115).

[0246] In some embodiments, the IL-21R polypeptide is MPRGWAAPLLLLLLQGGWGCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQECGSFLLAESI KPAPPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKEGWNPHLLLLLLVIVFIPAFWSLKTHPLWRL WKKIWAVPSPERFFMPLYKGCSGDFKKWVGAPFTGSSLELGPWSPEVPSTLEVYSCHPPRSPAKRLQLTELQEPAELVESDGVPKPSFWPTAQNSGGSAYSEERDRPYGLVSIDTVTVLDAEGPCTWPCSCEDDGYPALDLDAGL EPSPGLEDPLLDAGTTVLSCGCVSAGSPGLGGPLGSLLDRLKPPLADGEDWAGGLPWGGRSPGVSESEAGSPLAGLDMDTFDSGFVGSDCSSPVECDFTSPGDEGPPRSYLRQWVVIPPPLSSPGPQAS (SEQ ID NO: 93).

[0247] The present disclosure provides, in some embodiments, for IL-21 polypeptides or Provide functional fragments or variants thereof. Such IL-21 polypeptides containing at least one amino acid substitution relative to SEQ ID NO: 92 or SEQ ID NO: 115 are also referred to herein as IL-21 muteins. In an exemplary embodiment, an IL-21 polypeptide or functional fragment or variant thereof described herein comprises at least one and no more than X amino acid substitutions, where X is 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 , 33, 34 or more. In some embodiments, an IL-21 polypeptide or functional fragment or variant thereof described herein comprises at least 35 amino acid substitutions compared to SEQ ID NO: 115. In an exemplary embodiment, an IL-21 polypeptide described herein or a functional fragment or variant thereof has the amino acid sequence of human IL-21 (e.g., SEQ ID NO: 115) and 10 amino acids, 15 amino acids, amino acids, 20 amino acids, or 25 amino acids. In an exemplary embodiment, an IL-21 polypeptide described herein or a functional fragment or variant thereof has the amino acid sequence of human IL-21 (e.g., SEQ ID NO: 115) and up to 7 amino acids or 5 Contains amino acid sequences that differ by no more than 1 amino acid. In an exemplary embodiment, an IL-21 polypeptide described herein or a functional fragment or variant thereof has the amino acid sequence of human IL-21 (e.g., SEQ ID NO: 115) and 3, 4, 5, or Contains amino acid sequences that differ by six amino acids. In an exemplary embodiment, an IL-21 polypeptide or functional fragment or variant thereof described herein has the amino acid sequence of human IL-21 (e.g., SEQ ID NO: 115) and 3 to 6 amino acids or Contains amino acid sequences that differ by 1 to 5 amino acids. In an exemplary embodiment, an IL-21 polypeptide described herein or a functional fragment or variant thereof comprises the amino acid sequence of human IL-21 (e.g., SEQ ID NO: 115) and one or two amino acids. Contains amino acid sequences that differ only by Reduced IL-21R binding

[0248] In some embodiments, the IL-21 polypeptide or functional fragment or variant thereof comprises at least one mutation that reduces its binding affinity for IL-21R. Such mutations include, in some embodiments, R5, I8, R9, R11, Q12, I14, D15, D18, Q19, Y23, R65, S70, K72, K73, K75, at one or more positions selected from the group consisting of R76, K77, S80, Q116, and K117, where the position numbering is according to the amino acid sequence of SEQ ID NO: 115.

[0249] In some embodiments, the mutation at position R5 comprises an amino acid substitution selected from the group consisting of A, D, E, S, T, N, Q, V, I, L, Y, or F. In some embodiments, the mutation at position I8 comprises an amino acid substitution selected from the group consisting of Q, H, E. In some embodiments, the mutation at position R9 comprises an amino acid substitution selected from the group consisting of A, D, E, S, T, N, Q, V, I, L, Y, or F. In some embodiments, the mutation at position R11 comprises an amino acid substitution selected from the group consisting of D or E. In some embodiments, the mutation at position Q12 comprises an amino acid substitution selected from the group consisting of L, I, or Y. In some embodiments, the mutation at position I14 comprises an amino acid substitution selected from the group consisting of D or E. In some embodiments, the mutation at position D15 comprises an amino acid substitution selected from the group consisting of R, K, H, L, Y, or F. In some embodiments, the mutation at position D18 comprises an amino acid substitution selected from the group consisting of A, K, or R. In some embodiments, the mutation at position Q19 comprises an amino acid substitution selected from the group consisting of L, or Y. In some embodiments, the mutation at position Y23 comprises an E amino acid substitution. In some embodiments, the mutation at position R65 comprises an amino acid substitution selected from the group consisting of G, S, E, D, or A. In some embodiments, the mutation at position S70 comprises an amino acid substitution selected from the group consisting of H, Y, L, V, or F. In some embodiments, the mutation at position K72 comprises an amino acid substitution selected from the group consisting of G, S, E, D or A. In some embodiments, the mutation at position K73 comprises an amino acid substitution selected from the group consisting of A, Y, L, F, G, S, T, E, or D. In some embodiments, the mutation at position K75 comprises an amino acid substitution selected from the group consisting of G, S, E, D or A. In some embodiments, the mutation at position R76 comprises an amino acid substitution selected from the group consisting of A, D, E, S, T, N, Q, V, I, L, Y, or F. In some embodiments, the mutation at position K77 comprises an amino acid substitution selected from the group consisting of G, S, E, D or A. In some embodiments, the mutation at position S80 comprises an amino acid substitution selected from the group consisting of H, A, G, E, or D. In some embodiments, the mutation at position Q116 comprises an amino acid substitution that is Y. In some embodiments, the mutation at position K117 comprises an amino acid substitution selected from the group consisting of A, D, or E.

[0250] Exemplary sequences of IL-21 polypeptides or functional fragments or variants thereof of the present disclosure include QGQDX 1 HMX 2 X 3 MX 4 X 5 LX 6 X 7 IVX 8 X 9 LKNX 10 VNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNEX 11 IINVX 12 IX 13 X 14 LX 15 X 16 X 17 PPX 18 TNAGRRQKHRLTTCPSCDSYEKPPPKEFLERFKSLLX 19 X 20 MIHQHLSSRTHGSEDS (SEQ ID NO: 136). In some embodiments, X 1 =R, A, D, E, S, T, N, Q, V, I, L, Y, or F. In some embodiments, X 2 =I, Q, H, E. In some embodiments, X 3 =R, A, D, E, S, T, N, Q, V, I, L, Y, or F. In some embodiments, X 4 =R, D or E. In some embodiments, X 5 =Q, L, I, or Y. In some embodiments, X 6 =I, D or E. In some embodiments, X 7 =D, R, K, H, L, Y, or F. In some embodiments, X 8 =D, A, K, or R. In some embodiments, X 9 =Q, L, or Y. In some embodiments, X 10 =Y or E. In some embodiments, X 11 =R, G, S, E, D, or A. In some embodiments, X 12 =S, H, Y, L, V, or F. In some embodiments, X 13 =K, G, S, E, D, or A. In some embodiments, X 14 =K, A, Y, L, F, G, S, T, E, A, or D. In some embodiments, X 15 =K, G, S, E, D, or A. In some embodiments, X 16 =R, A, D, E, S, T, N, Q, V, I, L, Y, or F. In some embodiments, X 17 =K, G, S, E, D, or A. In some embodiments, X 18 =S, H, A, G, E, or D. In some embodiments, X 19 =Q or Y. In some embodiments, X 20 =K, A, D, or E.

[0251] [Table 6]

[0252] Certain aspects of the present disclosure relate to methods of treating cancer or chronic infections. In some embodiments, the method includes administering to the patient an effective amount of a targeted cytokine construct, or a pharmaceutical composition comprising a targeted cytokine construct and a pharmaceutically acceptable carrier. In some embodiments, the patient in need of such treatment has been diagnosed with cancer.

[0253] In some embodiments, the targeted cytokine construct or composition is administered in combination with a genetically engineered cell therapy (e.g., T cell therapy), a cancer vaccine, a chemotherapeutic agent, or an immune checkpoint inhibitor (ICI). Ru. In some embodiments, the chemotherapeutic agent is a kinase inhibitor, an antimetabolite, a cytotoxic or cytostatic agent, an anti-hormonal agent, a platinum-based chemotherapeutic agent, a methyltransferase inhibitor, an antibody, or an anti-inflammatory agent. It is a peptide. In some embodiments, the immune checkpoint inhibitor is PD-L1, PD-1, CTLA-4, CEACAM, LAIR1, CD160, 2B4, CD80, CD86, CD276, VTCN1, HVEM, KIR, A2AR, MHC class I, MHC class II, GALS, adenosine, TGFR, OX40, CD137, CD40, IDO, CSF1R, TIM-3, BTLA, VISTA, LAG-3, TIGIT, IDO, MICA / B, LILRB4, SIGLEC-15, or arginase, and inhibitors of PD-1 (e.g., anti-PD-1 antibodies), PD- Examples include, but are not limited to, inhibitors of L1 (eg, anti-PD-L1 antibodies), or inhibitors of CTLA-4 (eg, anti-CTLA-4 antibodies). Examples of T-cell therapies include CD4+ T-cell-based therapy or CD8+ T-cell-based therapy, adoptive T-cell therapy, chimeric antigen receptor (CAR)-based T-cell therapy, and tumor-infiltrating lymphocyte (TIL)-based therapy. , autologous T cell therapy, and allogeneic T cell therapy. Exemplary cancer vaccines include dendritic cell vaccines, vaccines that include one or more polynucleotides encoding one or more cancer antigens, and vaccines that include one or more cancer antigen peptides. , but not limited to.

[0254] In some embodiments, a targeted cytokine construct of the present disclosure is part of a pharmaceutical composition that includes, for example, a targeted cytokine construct and one or more pharmaceutically acceptable carriers. The pharmaceutical compositions and formulations described herein contain an active ingredient (such as a targeted cytokine construct) of desired purity, in the form of a lyophilized formulation or an aqueous solution, in one or more optional pharmaceutically acceptable carriers. (Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids, ascorbic acid, and methionine. antioxidants, preservatives, low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids, monosaccharides, disaccharides, and other carbohydrates such as cose, mannose, or dextrin, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium, metal complexes ( Examples include, but are not limited to, Zn-protein complexes), and / or nonionic surfactants such as polyethylene glycol (PEG). In some embodiments, targeted cytokine constructs of the present disclosure are lyophilized. Genetically engineered cell therapy

[0255] In some embodiments, the present disclosure relates to administering genetically engineered cell therapy, eg, genetically engineered cell therapy, to a subject in need thereof. Genetically engineered cell therapy can be a type of immunotherapy in which cells (eg, T cells) are administered to a subject to help the body fight diseases such as cancer. In cancer treatment, T cells are typically taken from a patient's own blood or tumor tissue, grown in large numbers in a laboratory, and then returned to the patient to help the immune system fight cancer. In some cases, T cells are changed in the lab to generate genetically engineered cells that have an improved ability to target and kill cancer cells. Types of genetically engineered cell therapies include, but are not limited to, chimeric antigen receptor T cell (CAR T cell) therapy, T cell receptor (TCR) therapy, and tumor-infiltrating lymphocyte (TIL) therapy.

[0256] In some embodiments, the genetically engineered cell therapy for use in the combination therapy described herein recognizes and / or specifically binds to a disease or disease-associated molecule to administration of genetically engineered cells that express recombinant receptors designed to, upon binding to molecules, produce a response, such as an immune response, against such molecules. Receptors can include chimeric receptors, such as chimeric antigen receptors (CARs), and other transgenic antigen receptors, including transgenic T cell receptors (TCRs). In some embodiments, T cells having endogenous T cell receptors that recognize and / or specifically bind molecules associated with a disease or disease are isolated for T cell therapy. In some embodiments, the cell contains or has been genetically engineered to contain a receptor, e.g., a genetically engineered antigen receptor, such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR). Ru. Also, populations of such cells, compositions comprising such cells, and / or T cells or CD8 + cells or CD4 + Compositions enriched for particular types of cells, such as cells, are also provided, such as where such cells are enriched or selected. Also provided are therapeutic methods for administering cells and compositions to a subject, eg, a patient. In some embodiments, the cell comprises one or more nucleic acids introduced via genetic engineering, thereby expressing a recombinant or genetically engineered product of such nucleic acids. In some embodiments, gene transfer is performed, such as by combining the cells with a stimulus that induces a response such as proliferation, survival, and / or activation, as measured by the expression of cytokines or activation markers. , is achieved by first stimulating cells and subsequently transducing and expanding the activated cells in culture to numbers sufficient for clinical application.

[0257] In some embodiments, the cells used in the cell therapies described herein have functional non-TCR antigen receptors, such as antigen receptors, including chimeric antigen receptors (CARs). or receptors such as recombinant receptors, and other antigen-binding receptors such as transgenic T-cell receptors (TCRs). Other chimeric receptors may also be included among the receptors. In some embodiments, the cell can express receptors specific for the cytokines of the targeted cytokine constructs described herein. Chimeric antigen receptor (CAR)

[0258] Exemplary antigen receptors, including CARs, exemplary CAR-T cells, and methods of genetically engineering and introducing such receptors into cells include, for example, WO 2000 / 14257; WO 2000 / 14257; / 126726, 2012 / 129514 , 2014 / 031687, 2013 / 166321, 2013 / 071154, 2013 / 123061, US Patent Application Publication No. 2002131960, 2013287748, 20130149337 No. 6,451,995, No. 7,446,190, No. 8,252,592, No. 8,339,645, No. 8,398,282, No. 7,446,179, No. 6,410,319, No. 7,070,995 , 7,265,209, 7,354,762, 7,446,191, 8,324,353 and 8,479,118, and EP 2537416, and / or Sadelain et al,Cancer Discov.,3(4):388-398(2013);Davila et al,PLoS ONE 8(4):e61338(2013);Turtle et al,Curr.Opin.Immunol,24(5): 633-39(2012);Wu et al, Cancer, 18(2):160-75 (2012). In some embodiments, antigen receptors include the CARs described in US Pat. No. 7,446,190 and the CARs described in WO 2014055668 (A1). Examples of CARs include WO 2014031687, US Patent No. 8,339,645, US Patent No. 7,446,179, US Patent Application No. 2013 / 0149337, US Patent No. 7,446,190, US Patent No. 8,389,282, Kochenderfer et al, Nature Reviews Clinical Oncology, 10, 267-276 (2013); Wang et al, J. Immunother. 35 (9): 689-701 (2012); and Brentjens et al, Sci Transl Med. 5 (177) (2013). Examples include CARs disclosed in any of the above. See also WO 2014031687, US Pat. No. 8,339,645, US Pat. No. 7,446,179, US Pat. Chimeric receptors, such as CAR, contain a portion of an antibody molecule, typically an antibody's variable weight (V H ) chain region and / or variable light (V L) chain regions, eg, extracellular antigen binding domains such as scFv antibody fragments. Non-limiting examples of CAR or CAR-T cells that can be used in the methods or compositions provided herein include tisagene lecleucel, axicabtagene cilol yucel, lysocabtagene malal yucel. cells, and CD19, CD22, WT1, CD171(L1CAM), MUC16, ROR1, or Lweis These include CAR-T cells that are genetically engineered to target antigens such as the Y (LeY) blood group antigen.

[0259] The antigen binding domain of a chimeric transmembrane receptor polypeptide can include any protein or molecule capable of binding an antigen. Antigen binding domains of the chimeric transmembrane receptor polypeptides disclosed herein include Fab, Fab', F(ab') 2 , Fv, single-chain Fv (scFv), minibodies, diabodies, and single domains such as heavy chain variable domains (VH), light chain variable domains (VL), and variable domains of camelid nanobodies (VHH). The antibody may be a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, or a functional derivative, variant, or fragment thereof, including but not limited to. In some embodiments, the antigen binding domain is Fab, Fab', F(ab') 2 , Fv, and scFv. In some embodiments, the antigen binding domain comprises an antibody mimetic. An antibody mimetic refers to a molecule that can bind to a target molecule with an affinity comparable to that of antibodies, and includes single-chain binding molecules, cytochrome B562-based binding molecules, fibronectin or fibronectin-like protein scaffolds (e.g., Adnectin), lipocalin scaffolds, Including calixarene scaffolds, A domains, and other scaffolds. In some embodiments, the antigen binding domain comprises a transmembrane receptor, or any derivative, variant, or fragment thereof. For example, the antigen binding domain can include at least one ligand binding domain of a transmembrane receptor.

[0260] In some embodiments, the antigen binding domain comprises a humanized antibody. Humanized antibodies can be made using a variety of techniques including, but not limited to, CDR grafting, veneering or resurfacing, chain shuffling, and other techniques. Human variable domains, including light and heavy chains, can be selected to reduce the immunogenicity of humanized antibodies. In some embodiments, the antigen-binding domain of the chimeric transmembrane receptor polypeptide binds antigen with high affinity and exhibits other favorable biological properties, such as reduced and / or minimized immunogenicity. Contains fragments of humanized antibodies with A humanized antibody or antibody fragment can retain similar antigen specificity to the corresponding non-humanized antibody.

[0261] In some embodiments, the antigen binding domain comprises a single chain variable fragment (scFv). scFv molecules are assembled using flexible linkers, such as polypeptide linkers, to create immunoglobulin heavy chains (V H ) region and light chain (V L ) regions together. scFv can be prepared according to a variety of methods.

[0262] In some embodiments, the antigen binding domain is genetically engineered to bind a particular target antigen. For example, the antigen binding domain can be a genetically engineered scFv. Antigen binding domains containing scFvs can be found in phage display libraries, yeast display libraries, cell-based display libraries (e.g., mammalian cells), protein-nucleic acid fusions, ribosome display libraries, and / or E. coli periplasmic libraries. Display libraries, such as display libraries, can be genetically engineered using a variety of methods, including but not limited to display libraries. In some embodiments, a genetically engineered antigen binding domain can bind an antigen with higher affinity than a similar antibody or an antibody that has not been genetically engineered.

[0263] In some embodiments, the antigen binding domain binds multiple antigens, eg, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 antigens. The antigen binding domain is capable of binding two related antigens, such as the two subtypes of botulinum toxin (eg, botulinum neurotoxin subtype A1 and subtype A2). The antigen-binding domain is capable of binding two unrelated proteins, such as the receptor tyrosine kinase erbB-2 (also called Neu, ERBB2, HER2) and vascular endothelial growth factor (VEGF). Antigen binding domains capable of binding two antigens can include antibodies that are genetically engineered to bind two unrelated protein targets at different but overlapping sites on the antibody. In some embodiments, the antigen binding domain that binds multiple antigens comprises a bispecific antibody molecule. A bispecific antibody molecule has a first immunoglobulin variable domain sequence with binding specificity for a first epitope and a second immunoglobulin variable domain sequence with binding specificity for a second epitope. I can do that. In some embodiments, the first epitope and the second epitope are on the same antigen, eg, the same protein (or subunit of a multimeric protein). The first epitope and the second epitope may overlap. In some embodiments, the first epitope and the second epitope do not overlap. In some embodiments, the first epitope and the second epitope are on different antigens, eg, different proteins (or different subunits of a multimeric protein). In some embodiments, a bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence that have binding specificity for a first epitope and a heavy chain variable domain sequence that has binding specificity for a second epitope. a variable domain sequence and a light chain variable domain sequence. In some embodiments, a bispecific antibody molecule comprises a half antibody that has binding specificity for a first epitope and a half antibody that has binding specificity for a second epitope. In some embodiments, a bispecific antibody molecule comprises a half antibody or fragment thereof that has binding specificity for a first epitope and a half antibody or fragment thereof that has binding specificity for a second epitope. include.

[0264] In some embodiments, the extracellular region of the chimeric transmembrane receptor polypeptide comprises multiple antigen binding domains, e.g., at least two antigen binding domains (e.g., at least 3, 4, 5, 6, 7, 8, 9 or 10 antigen-binding domains). Multiple antigen binding domains can exhibit binding to the same antigen or different antigens. In some embodiments, the extracellular region comprises at least two antigen binding domains, eg, at least two scFvs linked in tandem. In some embodiments, two scFv fragments are joined by a peptide linker.

[0265] In some embodiments, the antigen that can be bound by a chimeric antigen receptor (CAR) is a polypeptide. In some embodiments, the antigen is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on diseased or diseased cells, such as tumor cells or pathogenic cells, as compared to normal cells or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or on genetically engineered cells.

[0266] In some embodiments, the antigen binding domain expressed on the genetically engineered cells comprises a neoepitope from a tumor-associated antigen, TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvllll, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, L ewisY, CD24, PDGFR-β, SSEA-4, CD20, folate receptor α, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, prostase, PAP, ELF2M, ephrinB2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor β, TEM1 / CD248, TEM7R, CL DN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51 E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 variant, prostein, survivin and telomerase, PCTA-1 / galectin 8, melan A / MART1, Ras variant, hTERT, Sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1 B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, 1-40-β-amyloid, 4-1B B, 5AC, 5T4, activin receptor-like kinase 1, ACVR2B, adenocarcinoma antigen, AGS-22M6, α-fetoprotein, angiopoietin 2, angiopoietin 3, anthrax toxin, AOC3 (VAP-1), B7-H3, Bacillus anthracis, BAFF, β-amyloid, B-lymphoma cells, C242 antigen, C5, CA-125, canine IL31, carbonic anhydrase 9 (CA-IX), cardiac myosin, CCL11 (eotaxin-1) , CCR4, CCR5, CD11, CD18, CD125, CD140a, CD147 (Basigin), CD15, CD152, CD154 (CD40L), CD19, CD2, CD20, CD200, CD22, CD 221, CD23 (IgE receptor), CD25 (α chain of IL-2 receptor), CD27, CD274, CD28, CD3, CD3ε, CD30, CD33, CD37, CD38, CD4, CD40, CD40 ligand, CD41, CD44v6, CD5, CD51, CD52, CD56, CD6, CD70, CD74, CD79B, CD80, CEA, CEA-related antigen, CFD, ch4D5, CLDN18.2, Clostridium difficile, clumping factor A, CSF1R, CSF2, CTLA-4, C-X-C chemokine receptor type 4, cytomegalovirus, cytomegalovirus glycoprotein B, dabigatran, DLL4, DPP4, DR5, Escherichia coli Shiga toxin type 1, large intestine Bacillus Shiga toxin type 2, EGFL7, EGFR, endotoxin, EpCAM, episialin, ERBB3, Escherichia coli, respiratory syncytial virus F protein, FAP, fibrin II β chain, fibronectin extra domain-B, folate hydrolase, folate receptor 1 , folate receptor alpha, Frizzled receptor, ganglioside GD2, GD2, GD3 ganglioside, glypican 3, GMCSF receptor alpha chain, GPNMB, growth and differentiation factor 8, GUCY2C, hemagglutinin, hepatitis B surface antigen, hepatitis B virus, HER1 , HER2 / neu, HER3, HGF, HHGFR, histone complex, HIV-1, HLA-DR, HNGF, Hsp90, human scatter factor receptor kinase, human TNF, human β-amyloid, ICAM-1 (CD54), IFN-α , IFN-γ, IgE, IgE Fc region, IGF-1 receptor, IGF-1, IGHE, IL 17A, IL 17F, IL 20, IL-12, IL-13, IL-17, IL-1β, IL-22, IL-23, IL-31RA, IL-4, IL-5, IL-6, IL-6 receptor, IL- 9, ILGF2, influenza A hemagglutinin, influenza A virus rus hemagglutinin, insulin-like growth factor I receptor, integrin α4β7, integrin α4, integrin α5β1, integrin α7β7, integrin αIIbβ3, integrin αvβ3, interferon α / β receptor, - Feron gamma induced protein, ITGA2, ITGB2 (CD18), KIR2D, Lewis-Y antigen, LFA-1 (CD11a), LINGO-1, lipoteichoic acid, LOXL2, L-selectin (CD62L), LTA, MCP-1, Mesothelin, MIF, MS4A1, MSLN, MUC1, mucin CanAg, myelin-associated glycoprotein, myostatin, NCA-90 (granulocyte antigen), neuroapoptosis-regulating proteinase 1, NGF, N-glycolylneuraminic acid, N OGO-A, Notch receptor, NRP1, rabbit, OX-40, oxLDL, PCSK9, PD-1, PDCD1, PDGF-Rα, sodium phosphate cotransporter, phosphatidylserine, platelet-derived growth factor receptor β, prostate Fine details cells, Pseudomonas aeruginosa, rabies virus glycoprotein, RANKL, respiratory syncytial virus, RHD, rhesus factor, RON, RTN4, sclerostin, SDC1, selectin P, SLAMF7, SOST, sphingosine-1-phosphate, Staphylococcus aureus , STEAP1, TAG-72, T-cell receptor, TEM1, tenascin-C, TFPI, TGF-β1, TGF-β2, TGF-β, TNF-α, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, Tumor specificity of MUC1 glycosylation, tumor-associated calcium signal transducer 2, TWEAK receptor, TYRP1 (glycoprotein 75), VEGFA, VEGFR1, VEGFR2, vimentin, and VWF.

[0267] In some embodiments, the antigen to which the chimeric antigen receptor (CAR) can bind is 707-AP, biotinylated molecule, alpha-actinin-4, abl-bcr alb-b3(b2a2), abl-bcr alb-b4(b3a2), adipophilin, AFP, AIM-2, annexin II, ART-4, BAGE, β-catenin, bcr-abl, bcr-abl p190(e1a2), bcr-abl p210(b2a2), bcr -abl p210(b3a2), BING-4, CAG-3, CAIX, CAMEL, caspase-8, CD171, CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44v7 / 8, CDC27, CDK-4, CEA, CLCA2, Cyp-B, DAM-10, DAM-6, DEK-CAN, EGFRvIII, EGP-2, EGP-40, ELF2, Ep-CAM, EphA2, EphA3 , erb-B2, erb-B3, erb-B4, ES-ESO-1a, ETV6 / AML, FBP, fetal acetylcholine receptor, FGF-5, FN, G250, GAGE-1, GAGE-2, GAGE-3 , G.A. GE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, GD2, GD3, GnT-V, Gp100, gp75, Her-2, HLA-A*0201-R170I, HMW-MAA, HSP70- 2 M, HST-2(FGF6), HST-2 / neu, hTERT, iCE, IL-11Rα, IL-13Rα2, KDR, KIAA0205, K-RAS, L1-cell adhesion molecule, LAGE-1, LDLR / FUT, Lewis Y, MAGE-1, MAGE-10, MAGE-12, MAGE-2, MAGE-3, MAGE-4, MAGE-6, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A6, MAGE-B1, MAGE-B2, malic enzyme, mammaglobin-A, MART-1 / Metal Ran-A, MART-2, MC1R, M-CSF, mesothelin, MUC1, MUC16, MUC2, MUM-1, MUM-2, MUM-3, myosin, NA88-A, Neo-PAP, NKG2D, NPM / ALK, N-RAS, NY-ESO-1, OA1, OGT, carcinoembryonic antigen (h5T4), OS-9, P polypeptide, P15, P53, PRAME, PSA, PSCA, PSMA, PTPRK, RAGE, ROR1, RU1, RU2, SART-1, SART-2, SART-3, SOX10, SSX- 2, Survivin, Survivin-2B, SYT / SS X, TAG-72, TEL / AML1, TGFaRII, TGFbRII, TP1, TRAG-3, TRG, TRP-1, TRP-2, TRP-2 / INT2, TRP-2-6b, tyrosinase, VEGF-R2, WT1, selected from the group consisting of α-folate receptor, and κ-light chain. In some embodiments, the antigen binding domain binds a tumor-associated antigen.

[0268] In some embodiments, the antigen binding domain binds an antibody, eg, an antibody that binds a cell surface protein or polypeptide. The cell surface proteins or polypeptides bound by antibodies may be associated with viral, bacterial, and / or parasitic infections, inflammatory and / or autoimmune diseases, or neoplasms such as cancers and / or tumors. It can include antigens associated with the disease. In some embodiments, the antibody binds a tumor-associated antigen (eg, protein or polypeptide). In some embodiments, the antigen binding domain of the chimeric transmembrane receptor polypeptides disclosed herein is Fab, Fab', F(ab') 2, Fc, Fv, scFv, minibody, diabody, and single domain antibodies such as heavy chain variable domain (VH), light chain variable domain (VL), and camelid nanobody variable domain (VHH). The antibody can bind to a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, or a functional derivative, variant, or fragment thereof, including, but not limited to. In some embodiments, the antigen binding domain is Fab, Fab', F(ab') 2 , Fc, Fv, and scFv. In some embodiments, the antigen binding domain binds to the Fc domain of an antibody.

[0269] In some embodiments, the antigen binding domain is 20-(74)-(74) (milatuzumab, veltuzumab), 20-2b-2b, 3F8, 74-(20)-(20) (milatuzumab, veltuzumab), 8H9, A33, AB-16B5, Avagovomab, Abciximab, Abituzumab, ABP494 (Cetuximab biosimilar), Abrilumab, ABT-700, ABT-806, Actimab-A (Actinium Ac-225 Lintuzumab), Actoxumab, Adalimumab, ADC-1013, ADCT-301, ADCT-402, Adecatumumab, Aducanumab, Afelimomab, AFM 13, aftuzumab, AGEN1884, AGS15E, AGS-16C3F, AGS67E, alacizumab pegol, ALD518, alemtuzumab, alirocumab, artumomab pentetate, amatuximab, AMG228, AMG820, anatumomab mafenatox, anetumab brutansine, Anifrolumab, Anlukinsumab, APN301, APN311, Apolizumab, APX003 / SIM-BD0801 (cevacizumab), APX005M, Arsitumomab, ARX788, Asculinbacumab, Acerizumab, ASG-15ME, Atezolizumab, Atinumab, ATL101, Trizumab (also known as tocilizumab), atrolimumab, avelumab, B-701, bapineuzumab, basiliximab, bavituximab, BAY1129980, BAY1187982, bectumomab, vegelomab, belimumab, benralizumab, vertilimumab, becilesomab, betaltin (177 Lu-tetraxetane-tetulomab), bevacizumab, BEVZ92 (bevacizumab biosimilar), bezlotoxumab, BGB-A317, BHQ880, BI836880, BI-505, bicilomab, bimagrumab, bimekizumab, bivacizumab mertansine, BIW-8962, blinatumoma brosozumab, BMS-936559, BMS-986012, BMS-986016, BMS-986148, BMS-986178, BNC101, bococizumab, brentuximab vedotin, brevalex, briakinumab, brodalumab, brolucizumab, bronticutuzumab, C2 -2 b-2b, canakinumab, cantuzumab mertansine, cantuzumab brutansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, CBR96-doxorubicin immunoconjugate, CBT124 (bevacizumab), CC-90002, CDX-014, CDX-1401, Sederisma Bu, certolizumab pegol, cetuximab, CGEN-15001T, CGEN-15022, CGEN-15029, CGEN-15049, CGEN-15052, CGEN-15092, Ch\.14\.18, sitatuzumab bogatox, cixutumumab , clazakizumab, crenoliximab, Clivatuzumab tetraxetane, CM-24, codrituzumab, cortuximab brutansine, conatumumab, concizumab, Kotara (iodine I-131 derlotuximab biotin), cR6261, crenezumab, DA-3111 (trastuzumab biosimilar), dacetuzumab, daclizumab, da Rotuzumab, dapilrolizumab pegol, daratumumab, daratumumab enhance (daratumumab), darleukin, decrekumab, demcizumab, denintuzumab mafodotin, denosumab, depatuxizumab, depatuxizumab mafodotin, derlotuximab Biotin, detumomab, DI-B4, dinutu ximab, diridabumab, DKN-01, DMOT4039A, dorlimomab alitox, drogitumab, DS-1123, DS-8895, durigotumab, dupilumab, durvalumab, dusigitumab, eclomeximab, eculizumab, edvacomab, edrecolomab, efalizumab, Efungumab, eldelumab, ergemtuzumab, elotuzumab, ercilimomab, emactuzumab, emibetuzumab, enabatuzumab, enfortumab vedotin, enrimomab pegol, enobrituzumab, enokizumab, enotikumab, encituximab, epitumomab cituxetan, epratuzumab Bu, erlizumab, ertumaxomab, etalacizumab, etrolizumab, evinacumab, evolocumab, exvivirumab, fanoresomab, faralimomab, farletuzumab, fasinumab, FBTA05, felbizumab, fezakinumab, FF-21101, FGFR2 antibody-drug conjugate, Fibromun, ficlatuzumab, figitumumab, filibumab, franbotumab, fletikumab, fontolizumab, foralumab, foravilumab, FPA144, fresolimumab, FS102, flulanumab, futuximab, galiximab, ganitumab, gantenerumab, gabilimomab, Gem Tuzumab ozogamicin, gerilimuzumab, gevokizumab, gilentuximab, glembatumumab vedotin, GNR-006, GNR-011, golimumab, gomiliximab, GSK2849330, GSK2857916, GSK3174998, GSK3359609, guselkumab, Hu14\.18K322A MAb, hu3S193, Hu8F4, HuL2G7, HuMab-5B1, ibalizumab, ibritumomab tiuxetan, icurucumab, idarucizumab, IGN002, IGN523, igobomab, IMAB362, IMAB362( claudiximab), imalumab, IMC-CS4, IMC-D11, imcilumab, imgatuzumab, IMGN529, IMMU-102 (yttrium Y-90 epratuzumab tetraxetane), IMMU-114, ImmuTune MP701 antagonist antibody, INCAGN1876, incracumab, INCSHR1210, indatuximab lavtansine, indusatumab vedotin, infliximab, inorimo Mab, inotuzumab ozogamicin, intetumumab, ipaflicept, IPH4102, ipilimumab, iratumumab, isatuximab, istiratumab, itilizumab, ixekizumab , JNJ-56022473, JNJ-61610588, keliximab, KTN3379, L19IL2 / L19TNF, labetuzumab, labetuzumab govitecan, LAG525, Lambro Rizumab, lampalizumab, L-DOS47, lebrikizumab, remaresomab, lentilumab, lerdelimumab, leukotuximab, lexatumumab, ribivirumab, rifastuzumab vedotin , ligelizumab, rilotomab satetraxetane, lintuzumab, rililumab, LKZ145, lodelcizumab, rokivetumab, lorbotuzumab mertansine, lucatumumab, lulizumab pegol , lumiliximab, rumletuzumab, LY3164530, mapatumumab, margetuximab, maslimomab, matuzumab, mavrilimumab, MB311, MCS-110, MEDI0562 , MEDI-0639, MEDI0680, MEDI-3617, MEDI-551 (inebilizumab), MEDI-565, MEDI6469, mepolizumab, metelimumab, MGB453 , MGD006 / S80880, MGD007, MGD009, MGD011, Milatuzumab, Milatuzumab-SN-38, Minretumomab, Mirvetuximab Soravtansine, Mitumomab, M K-4166, MM-111, MM-151, MM-302, Mogamulizumab, MOR202, MOR208, MORAb-066, Morolimumab, Motavizumab, Moxetumomab Passed Top muromonab-CD3, nacolomab tafenatox, namilumab, naptumomab estafenatox, narnatumab, natalizumab, nebacumab, necitumumab, nemolizumab, nele Rimomab, Nesbacumab, Nimotuzumab, Nivolumab, Nofetumomab Merpentane, NOV-10, Obiltokiaximab, Obinutuzumab, Ocaratuzumab, Ocrelizumab, Odulimo Mab, ofatumumab, olaratumab, orokizumab, omalizumab, OMP-131R10, OMP-305B83, onartuzumab, ontuxizumab, opicinumab, oportuzumab mona TOX, Oregovomab, Orticumab, Otelixizumab, Otreltuzumab, OX002 / MEN1309, Oxelumab, Ozanezumab, Ozolarizumab, Pagibaximab, Paliviz Mab, panitumumab, pancomab, pancomab-GEX, panobacumab, palsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, PAT-SC1, PAT-SM 6, Pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, PF-05082566 (Utomilumab), PF-06647263, PF-06671008, PF -06801591, pidilizumab, pinatuzumab vedotin, pintumomab, plaklumab, polatuzumab vedotin, ponezumab, priliximab, pritoxaximab, pritumumab, PRO 140, Proxinium, PSMA ADC, kilizumab, racotomumab, radrezumab, raffivirumab, ralpancizumab, ramucirumab, ranibizumab, raxibacumab, refanezumab, regavilumab, REGN1400, REGN2810 / SAR439684, reslizumab, RFM-203, RG7356, RG7386, RG7802, RG7813, RG 7841, RG7876, RG7888, RG7986, rilotumumab, rinucumab, rituximab, RM-1929, RO7009789, lobatumumab, lorezumab , romosozumab, rontalizumab, lobelizumab, luplizumab, sacituzumab govitecan, samalizumab, SAR408701, SAR566658, sarilumab, S AT012, Satumomab pendetide, SCT200, SCT400, SEA-CD40, Secukinumab, Seribantumab, Cetoxaximab, Sevilumab, SGN-CD19A, SGN-CD19B, SGN-CD33A, SGN-CD70A, SGN-LIV1A, Sibrotuzumab, Sifalimumab, Siltuximab, Simtuzumab , ciplizumab, sirukumab, sofituzumab vedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, slesomab, suvizumab, SYD 985, SYM004 (futuximab and modotuximab), Sym015, TAB08, tabalumab, takatuzumab tetraxetane, tadocizumab, talizumab, tane Zumab, Tanibilumab, Tapritumomab Paptox, Tarextumab, TB-403, Tefibazumab, Tereukin, Telimomab Alitox, Tenatumomab, Teneliximab, Teplizumab, Teprotumumab, Tesidolumab, Tetulomab, TG-1303, TGN1412, Thorium-227-Ep Ratuzumab conjugate, chi cilimumab, tigatuzumab, tiludrakizumab, tisotumab vedotin, TNX-650, tocilizumab, tralizumab, tosatoxumab, tositumomab, tovezumab, tralokinumab, trastuzumab, trastuzumab emtansine, TRBS07, TRC105, tregalizumab, tremelimumab, trevoglumab, TRPH011, T RX518, TSR-042, TTI-200\.7, tukotzumab sermolleukin, tubilumab, U3-1565, U3-1784, ublituximab, urokupulumab, urelumab, ultoxazumab, ustekinumab, vadastuximab tarilin, bundle tuzumabvedo Chin, banticutumab, vanucizumab, vapariximab, val From rilumab, batelizumab, VB6-845, vedolizumab, veltuzumab, bepalimomab, besenkumab, bicilizumab, volociximab, vorcetuzumab mafodotin, botumumab, YYB-101, zaltumumab, zanolimumab, zatuximab, diralimumab, and zolimomab alitox binds to an antibody selected from the group consisting of: In certain embodiments, the antigen binding domain binds to the Fc domain of the aforementioned antibody. \.

[0270] In some embodiments, the antigen binding domain binds to an antibody mimetic. Antibody mimetics can bind target molecules with affinity comparable to antibodies, as described elsewhere herein. In some embodiments, the antigen binding domain is capable of binding humanized antibodies described elsewhere herein. In some embodiments, the antigen binding domain of the chimeric transmembrane receptor polypeptide is capable of binding a fragment of a humanized antibody. In some embodiments, the antigen binding domain is capable of binding a single chain variable fragment (scFv).

[0271] In some embodiments, the antigen binding domain also comprises the Fc of a suitable mammalian (e.g., human, mouse, rat, goat, sheep, or monkey) immunoglobulin (e.g., IgG, IgA, IgM, or IgE). Also joins parts. Suitable Fc binding domains may be derived from naturally occurring proteins such as mammalian Fc receptors or certain bacterial proteins (eg, protein A and protein G). Additionally, the Fc binding domain may be a synthetic polypeptide specifically genetically engineered to bind to the Fc portion of any of the Ig molecules described herein with the desired affinity and specificity. For example, such an Fc-binding domain can be an antibody or antigen-binding fragment thereof that specifically binds to the Fc portion of an immunoglobulin. Examples include, but are not limited to, single chain variable fragments (scFv), domain antibodies, and Nanobodies. Alternatively, the Fc-binding domain may be a synthetic peptide that specifically binds to the Fc portion, such as a Kunitz domain, small modular immunotherapy (SMIP), Adnectin, Avimer, Affibody, DARPin, or Anticalin, which can be identified by screening peptide libraries for binding activity to Fc.

[0272] In some embodiments, the antigen binding domain comprises an Fc binding domain that includes an extracellular ligand binding domain of a mammalian Fc receptor. Fc receptors are cell surface receptors commonly expressed on the surface of many immune cells, including B cells, dendritic cells, natural killer (NK) cells, macrophages, neutrophils, mast cells, and eosinophils. It exhibits binding specificity for the Fc domain of antibodies. In some cases, binding of Fc receptors to the Fc portion of antibodies can cause antibody-dependent cellular cytotoxicity (ADCC) effects. The Fc receptors used to construct the chimeric transmembrane receptor polypeptides described herein have an altered (e.g., increased or decreased) affinity for the Fc domain compared to their wild-type counterparts. It may also be a naturally occurring polymorphic variant, such as a variant that may have Alternatively, the Fc receptor may be a functional variant of the wild-type counterpart with one or more mutations (e.g., up to 10 amino acid residue substitutions) that alter the binding affinity for the Fc portion of the Ig molecule. good. In some embodiments, mutations may alter the glycosylation pattern of the Fc receptor and, therefore, the binding affinity for the Fc domain.

[0273] Fc receptors can be classified based on the isotype of antibodies to which they can bind. For example, Fc-γ receptors (FcγR) commonly bind IgG antibodies (eg, IgG1, IgG2, IgG3, and IgG4). Fc-α receptors (FcαR) generally bind IgA antibodies, and Fc-ε receptors (FcεR) generally bind IgE antibodies. In some embodiments, the antigen binding domain comprises an Fcγ receptor, or any derivative, variant, or fragment thereof. In some embodiments, the antigen binding domain is FcγRI(CD64), FcγRIa, FcγRIb, FcγRIc, FcγRIIA(CD32) including allotypes H131 and R131, FcγRIIB(CD32) including FcγRIIB-1 and FcγRIIB-2, allotype V158. Reach and FcγRIIIB (CD16b), including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2, any derivative thereof, any variant thereof, and any fragment thereof. Contains a binding domain. FcγRs can be derived from any organism including, but not limited to, human, mouse, rat, rabbit, and monkey. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcδRIII (CD16), and FcγRIII-2 (CD16-2). In some embodiments, the antigen binding domain comprises an Fcε receptor, or any derivative, variant, or fragment thereof. In some embodiments, the antigen binding domain comprises an FcR selected from FcεRI, FcεRII (CD23), any derivative thereof, any variant thereof, and any fragment thereof. In some embodiments, the antigen binding domain comprises an Fcα receptor, or any derivative, variant, or fragment thereof. In some embodiments, the antigen binding domain comprises an FcR selected from FcαRI (CD89), Fcα / μR, any derivative thereof, any variant thereof, and any fragment thereof. In some embodiments, the antigen binding domain comprises an FcR selected from FcRn, any derivative thereof, any variant thereof, and any fragment thereof. The selection of a ligand-binding domain of an Fc receptor for use in a chimeric transmembrane receptor polypeptide depends on a variety of factors, including the isotype of the antibody to which binding of the Fc-binding domain is desired and the desired affinity of the binding interaction. can depend on it.

[0274] The immune cell signaling domain of the intracellular region of the chimeric transmembrane receptor polypeptide of the subject system can include a primary signaling domain. The primary signaling domain can be any signaling domain involved in immune cell signaling, or a derivative, variant, or fragment thereof. For example, signaling domains are involved in controlling the primary activation of TCR complexes, either stimulatory or inhibitory. The primary signaling domains are Fcγ receptor (FcγR), Fcε receptor (FcεR), Fcα receptor (FcαR), neonatal Fc receptor (FcRn), CD3, CD 3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L(CD154), CD45, CD66d, CD 79a, CD79b, CD80, CD86, CD278 (also known as ICOS), CD247ζ, CD247η, DAP10, DAP12, FYN, LAT, Lck, MAPK, MHC complex, NFAT, NF-κB, PLC-γ, iC3b , C3dg, C3d, and Zap70 signaling domains. In some embodiments, the primary signaling domain comprises an immunoreceptor tyrosine-based activation motif or ITAM. The ITAM-containing primary signaling domain can contain two repeats of the amino acid sequence YxxL / I separated by 6-8 amino acids, where each x is independently any amino acid, and the conserved motif YxxL / Ix (6-8) Generate YxxL / I. Primary signaling domains, including ITAMs, can be modified, for example, by phosphorylation when the antigen-binding domain is bound to an antigen. Phosphorylated ITAM can serve as a docking site for other proteins, such as proteins involved in various signal transduction pathways. In some embodiments, the primary signaling domain is a modified ITAM domain, e.g., mutated, truncated, and / or optimized, with altered (e.g., increased or decreased) activity compared to the native ITAM domain. Contains the ITAM domain that was created.

[0275] In some embodiments, the primary signaling domain comprises an FcγR signaling domain (eg, ITAM). The FcγR signaling domain can be selected from FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b). In some embodiments, the primary signaling domain comprises an FcεR signaling domain (eg, ITAM). The FcεR signaling domain can be selected from FcεRI and FcεRII (CD23). In some embodiments, the primary signaling domain comprises an FcαR signaling domain (eg, ITAM). The FcαR signaling domain can be selected from FcαRI (CD89) and Fcα / μR. In some embodiments, the primary signaling domain comprises a CD3ζ signaling domain. In some embodiments, the primary signaling domain comprises the ITAM of CD3ζ.

[0276] In some embodiments, the primary signaling domain comprises an immunoreceptor tyrosine-based inhibition motif or ITIM. The primary signaling domain, including ITIM, can include a conserved sequence of amino acids (S / I / V / LxYxxI / V / L) found in the cytoplasmic tail of several inhibitory receptors of the immune system. Primary signaling domains, including ITIMs, can be modified, eg, phosphorylated, by enzymes such as members of the Src kinase family (eg, Lck). After phosphorylation, other proteins including enzymes can be recruited to ITIM. These other proteins include, but are not limited to, enzymes such as the phosphotyrosine phosphatases SHP-1 and SHP-2, inositol phosphatases designated SHIP, and proteins with one or more SH2 domains (e.g., ZAP70). Not done. The primary signaling domains are BTLA, CD5, CD31, CD66a, CD72, CMRF35H, DCIR, EPO-R, FcγRIIB (CD32), Fc receptor-like protein 2 (FCRL2), and Fc receptor receptor-like protein 3 (FCRL3). , Fc receptor-like protein 4 (FCRL4 ), Fc receptor-like protein 5 (FCRL5), Fc receptor-like protein 6 (FCRL6), protein G6b (G6B), interleukin 4 ...

Claims

1. 1. A composition comprising a targeting cytokine construct for targeting a genetically engineered cell, the targeting cytokine construct comprising: (a) a cell binding domain comprising an antibody or antigen-binding fragment thereof specific for a receptor or domain exogenously expressed on the surface of the engineered cell, the cell binding domain targeting at least one of: (i) a domain of a chimeric antigen receptor (CAR) or a T cell receptor (TCR) exogenously introduced into the engineered cell; (ii) a tag molecule selectively expressed on the surface of the engineered cell, the tag molecule being a cell tag separate from a CAR or an exogenous TCR; (iii) a polypeptide tag that is a part of a CAR that is exogenously introduced into the engineered cell; (iv) a polypeptide tag that is a part of a TCR that is exogenously introduced into the engineered cell; or (vi) any combination of (i)-(v); (b) a cytokine protein, wherein the cytokine is selected from the group consisting of IL-2, IL-7, IL-10, IL-15, IL-21, and any combination thereof.

2. 2. The composition of claim 1, wherein the targeted cytokine construct selectively activates the engineered cells with 10-fold or greater potency compared to activation of non-engineered cells, the potency being measured by a pSTAT5 activation assay or a pSTAT3 activation assay, and the non-engineered cells do not express the CAR, the TCR, or the tag molecule on their surface.

3. The composition described in claim 1 or 2, wherein the domain of the CAR is an scFv.

4. The composition of any one of claims 1 to 3, wherein the cytokine is an IL-2 polypeptide, (1) the IL-2 polypeptide exhibits a binding affinity for the IL-2Rα polypeptide having the amino acid sequence of SEQ ID NO:2 that is reduced by at least about 50% compared to the binding affinity of a wild-type IL-2 polypeptide having the amino acid sequence of SEQ ID NO:1 to the IL-2Rα polypeptide, (2) the IL-2 polypeptide exhibits a binding affinity for the IL-2Rβ polypeptide having the amino acid sequence of SEQ ID NO:3 that is reduced by at least about 50% compared to the binding affinity of a wild-type IL-2 polypeptide having the amino acid sequence of SEQ ID NO:1 to the IL-2Rβ polypeptide, and / or (3) the composition of any one of claims 1 to 3 exhibits a binding affinity for the IL-2Rγ polypeptide having the amino acid sequence of SEQ ID NO:4 that is reduced by at least about 50% compared to the binding affinity of a wild-type IL-2 polypeptide having the amino acid sequence of SEQ ID NO:1 to the IL-2Rγ polypeptide.

5. The composition of any one of claims 1 to 4, wherein the IL-2 polypeptide comprises an amino acid sequence that is at least about 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 11-90, and the amino acid sequence comprises one or more substitutions at positions in SEQ ID NO: 1 selected from the group consisting of Q11, H16, L18, L19, D20, Q22, R38, F42, K43, Y45, E62, P65, E68, V69, L72, D84, S87, N88, V91, I92, T123, Q126, S127, I129, and S130.

6. The composition described in claim 5, wherein the one or more substitutions further include an amino acid mutation C125A compared to sequence number 1.

7. The composition of any one of claims 1 to 3, wherein the cytokine is an IL-10 polypeptide, and the IL-10 polypeptide comprises an amino acid sequence having at least about 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO:

95.

8. The composition of claim 7, wherein the IL-10 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 99-112.

9. The composition of claim 1, wherein the cytokine is an IL-21 polypeptide, the IL-21 polypeptide comprising the sequence of SEQ ID NO:115 or a sequence comprising amino acid substitutions at one or more positions selected from positions R5, I8, R9, R11, Q12, I14, D15, D18, Q19, Y23, R65, S70, K72, K73, K75, R76, K77, S80, Q116, and K117, and the numbering of the positions is numbered according to the amino acid sequence of SEQ ID NO:

115.

10. The composition of any one of claims 1 to 9, wherein the tag molecule, the polypeptide tag that is part of a CAR exogenously introduced into the genetically engineered cell, or the polypeptide tag that is part of a TCR exogenously introduced into the genetically engineered cell, is an EGFRt tag or a myc tag.

11. A composition according to any one of claims 1 to 10 for use as a pharmaceutical in the treatment of a disease or disorder in a subject.

12. The composition of claim 11, wherein the targeted cytokine construct results in increased activation of a population of engineered cells compared to activation of a population of non-engineered cells, the activation being measured by a pSTAT5 activation assay or a pSTAT3 activation assay, and the non-engineered cells do not express the CAR exogenously introduced into the engineered cells, the TCR exogenously introduced into the engineered cells, or the tag molecule.

13. The composition of claim 11 or 12, wherein the targeted cytokine construct results in increased expansion and / or proliferation of a population of the engineered cells compared to expansion and / or proliferation of a population of non-engineered cells, and the non-engineered cells do not express the CAR exogenously introduced to the engineered cells, the TCR exogenously introduced to the engineered cells, or the tag molecule.

14. The composition of any one of claims 11 to 13, wherein the targeted cytokine construct results in increased in vivo persistence of a population of the engineered cells compared to the in vivo persistence of a population of the non-engineered cells, and the non-engineered cells do not express the CAR exogenously introduced into the engineered cells, the TCR exogenously introduced into the engineered cells, or the tag molecule.

15. The composition of any one of claims 11 to 14, wherein the targeted cytokine construct reduces the rate and / or extent of depletion of the genetically engineered cell population compared to the rate and / or extent of depletion of the genetically engineered cell population without the targeted cytokine construct.

16. The composition of any one of claims 11 to 15, wherein the targeted cytokine construct does not increase the number of Treg cells in a biological sample isolated from the subject compared to the number of Treg cells in a biological sample isolated from a subject administered a non-targeted cytokine, or a functional fragment or variant thereof.

17. The composition of any one of claims 11 to 16, wherein the subject has previously been administered a preconditioning regimen, and the targeted cytokine construct enables a reduction in at least one of the severity or duration of the preconditioning regimen, and the preconditioning regimen is used to reduce an endogenous lymphocyte population so that the population of the genetically engineered cells can be expanded.

18. The composition described in any one of claims 11 to 17, wherein the subject is experiencing loss of B cell aplasia.

19. The composition of any one of claims 11 to 18, wherein the targeted cytokine construct reduces the effective dose of the genetically engineered cells in the subject relative to the effective dose of the genetically engineered cells in a subject lacking the targeted cytokine construct.

20. The composition described in any one of claims 11 to 19, wherein the disease or illness is cancer.