Methods and compositions for suppressing immune cell activation

Abstract

Disclosed herein, in some aspects, are compositions and methods for enhancing immunosuppressive functions of a cell, polynucleotides encoding polypeptides for effectuating the same, cells comprising the same, and compositions comprising the polypeptides, polynucleotides, and / or cells. In some aspects, the engineered cells show enhanced immunosuppression. Also disclosed are methods for disease treatment, such as graft-versushost disease, sepsis, transplant rejection, and / or autoimmune diseases, comprising administering engineered cells and / or compositions of the disclosure to a subject in need thereof.

Description

METHODS AND COMPOSITIONS FOR SUPPRESSING IMMUNE CELL ACTIVATIONCLAIM OF PRIORITY

[0001] This Application claims the benefit of U.S. Provisional Patent Application No. 63 / 635,106, filed on 17 April 2024, which is incorporated by reference herein in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in ST26 format and is hereby incorporated by reference in its entirety. Said ST26 copy, created on April 17, 2024, is named BAYM_P0422US_Pl_Sequence_Listing.xml and is 474,231 bytes in size.TECHNICAL FIELD AND BACKGROUNDI. Technical Field

[0003] Aspects of this disclosure relate to at least the fields of immunology, immunotherapy, autoimmune disease, transplantation, and medicine.IL Background

[0004] Activation of alloreactive T cells resulting in transplant rejection or graft-versus- host-disease (GVHD) is a leading cause of morbidity and mortality following allogeneic organ transplants, and avoidance of this morbidity / mortality often requires intensive immunosuppressive prophylaxis. In addition, more than 23 million Americans suffer from autoimmune diseases, and this number is steadily rising each year h Many of these conditions are caused by unwanted activation of allo- or auto-reactive T cells which proliferate and induce systemic organ damage.

[0005] In both allo- and auto-reactive settings, uncontrolled activated T cell activity results from evasion of suppression or absence of functional regulatory T cells (Treg). The current standard of care for these pathologies relies on immunosuppressive medication, such as corticosteroids, to dampen inflammation, but many patients are unresponsive to these treatments, which often produce significant side effects. Infusion of unmodified or engineered Treg is currently being investigated for the treatment of treatment-refractory allo- and autoimmune complications. Although several clinical trials have described the safety of polyclonal Treg in graft versus host disease, diabetes2, and solid organ transplantation3’4, their efficacyhas yet to be established. In addition, Treg as a cell platform has three important limitations. First, since Treg are a rare population of lymphocytes found in blood (e.g., 1-3% of peripheral blood mononuclear cells (PBMC)), and thus they generally require large amounts of starting materials, expensive purification steps, and / or lengthy expansion steps. Second, Tregs are inherently plastic and can lose their immunosuppressive function in an inflammatory environment5. Third, repeated stimulation is often used to enhance Treg expansion and yield sufficient counts for clinical translation6, yet this sometimes causes Treg to lose their suppressive potency and lineage identity7

[0006] Mesenchymal stem cells (MSC) are low-immunogenic, non-cytotoxic, mildly immunosuppressive cells that are easy to expand ex vivo. Infusion of MSC has demonstrated safety and some efficacy in patients with various inflammatory conditions, including GvHD8. However, the potency of MSC against pathogenic T-cells is still inferior to that of Tregs, engineered payload B-cells, and certain irradiated cell lines. In some cases, B-cells can be used for immune cell therapy, but suffer from limitations related to expansion, differentiation, and engraftment.

[0007] There is a need for methods or compositions for the treatment of immune diseases, such as but not limited to autoimmune disease, graft versus host disease, sepsis, or transplant rejection, that overcome the limitations of existing therapies.SUMMARY

[0008] This disclosure describes a solution to at least some of the problems associated with unwanted activation of immune cells, e.g., allo- or auto-reactive immune cells. In some aspects, the solution resides in engineered polypeptides and / or cells comprising one or more engineered interleukin receptors, one or more heterologous costimulatory antagonists, one or more heterologous immunosuppressive cytokines, and / or one or more co-inhibitor engagers. In some aspects, polypeptides of the disclosure can inhibit immune cell activation, alone or in combination, and in some cases synergistically. In some aspects, a cell, e.g., a mesenchymal stem cell, comprises one or more polypeptides of the disclosure.

[0009] In some aspects, this disclosure provides methods and compositions for engineering cells or preparing such cells, e.g., MSCs, to perform immunosuppressive functions, e.g., Treg- like functions, engineered cells, and methods of using engineered cells. Described herein are methods and compositions to engineer cells, e.g., MSCs, to enhance their immunosuppressive functions. Described herein are methods and compositions to engineer cells, e.g., MSCs, to deploy potent immunosuppressive programs and, optionally, to detect and engage pathogenicand / or activated immune cells, e.g., T cells. The detection and engaging of a pathogenic and / or activated immune cell can initiate immunosuppressive programs within the pathogenic and / or activated immune cell, such as through engagement of a coinhibitory receptor (e.g., PD1, TIGIT, etc.). Also, described herein in some aspects, are methods of treatment for patients suffering from graft-versus-host disease (GVHD), disorders driven by auto- or allo-reactive immune cells, transplant rejection, bacterial sepsis, viral sepsis, fungal sepsis, and / or autoimmune diseases. In some aspects, described herein are methods and compositions for decreasing chronic inflammation without eliminating beneficial circulating immune cells, e.g., T cells, in an individual. Also described herein in some aspects, are methods and compositions for developing cryopreserved cell therapies against disorders associated with auto- or allo- reactive immune cells. The methods and compositions described herein can provide solutions for the current limitations of adoptive Treg cell therapy as described above.

[0010] Aspects of the present disclosure address certain needs by providing at least nucleic acids, cells, proteins, methods, compositions, and / or kits useful for immunotherapy and for the treatment of graft-versus-host disease, transplant rejection, bacterial sepsis, viral sepsis, fungal sepsis, autoimmune diseases, and / or disorders associated with auto- or allo-reactive immune cells. Aspects of the present disclosure include nucleic acids encoding polypeptides for engineering cells to comprise enhanced immunosuppressive functions.

[0011] In certain aspects, compositions, polynucleotides, cells, and / or kits provided herein comprise an engineered interleukin receptor, wherein the engineered interleukin receptor comprises, a) a first interleukin receptor extracellular domain, b) a second interleukin receptor extracellular domain, and c) an interleukin receptor transmembrane and intracellular signaling domain, wherein the domains are operably linked to form a heterologous engineered interleukin receptor. In some aspects, a first interleukin receptor extracellular domain and a second interleukin receptor extracellular domain are from the same protein. In some aspects, a first interleukin receptor extracellular domain and a second interleukin receptor extracellular domain are each from different proteins. In some aspects, a first interleukin receptor extracellular domain and a second interleukin receptor extracellular domain are each from different proteins, and the interleukin receptor transmembrane and intracellular signaling domain is from another different protein. In some aspects, an engineered interleukin receptor domain comprises a sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) sequence identity to any one of SEQ ID NOs: 60-69.

[0012] In some aspects, a first interleukin receptor extracellular domain is or is not from CD25 (interleukin-2 receptor subunit alpha) or CD 122 (interleukin-2 receptor subunit beta). Insome aspects, a second interleukin receptor extracellular domain is or is not from CD25 or CD122. In some aspects, an interleukin receptor transmembrane and intracellular signaling domain is or is not from CD 132 (interleukin-2 receptor subunit gamma). In some aspects, a first interleukin receptor extracellular domain is from CD25, a second interleukin receptor extracellular domain is from CD122, and an interleukin receptor transmembrane and intracellular signaling domain is from CD 132. In some aspects, a first interleukin receptor extracellular domain is from CD122, a second interleukin receptor extracellular domain is from CD25, and an interleukin receptor transmembrane and intracellular signaling domain is from CD 132. In some aspects, the domains of an engineered interleukin receptor are operably linked by one or more linkers. In some aspects, a linker comprises a sequence of, or of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) sequence identity to any one of SEQ ID NOs: 88-97. In some aspects, an engineered interleukin receptor comprises a signal peptide. In some aspects, a signal peptide comprises a sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) sequence identity to any one of SEQ ID NOs: 98-103. In some aspects, an engineered interleukin receptor comprises an amino acid sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) identical to any one of SEQ ID NOs: 50-53. In some aspects, an engineered interleukin receptor is encoded by a polynucleotide sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) sequence identity to any one of SEQ ID NOs: 5-8, 20-23, or 37-40.

[0013] In some aspects, compositions, polynucleotides, cells, and / or kits comprising an immunosuppressive system are provided herein. In some aspects, an immunosuppressive system comprises, or does not comprises, one or more of an engineered interleukin receptor, one or more of a heterologous costimulatory antagonist, one or more of a heterologous immunosuppressive cytokine, and / or one or more of a co-inhibitor engager. In some aspects, an immunosuppressive system comprises an engineered interleukin receptor, a heterologous costimulatory antagonist, and a heterologous immunosuppressive cytokine. In some aspects, an immunosuppressive system comprises an engineered interleukin receptor, a heterologous costimulatory antagonist, a heterologous immunosuppressive cytokine, and a co-inhibitor engager. In some aspects, a co-inhibitor engager comprises a T-cell checkpoint activator and / or T-cell inhibitor. In some aspects, an immunosuppressive system comprises an engineered interleukin receptor comprising a first interleukin receptor extracellular domain from CD25, a second interleukin receptor extracellular domain from CD 122, and an interleukin receptortransmembrane and intracellular signaling domain from CD 132. In some aspects, an immunosuppressive system comprises an engineered interleukin receptor comprising a first interleukin receptor extracellular domain from CD 122, a second interleukin receptor extracellular domain from CD25, and an interleukin receptor transmembrane and intracellular signaling domain from CD 132. In some aspects, a heterologous costimulatory antagonist comprises CTLA-4. In some aspects, CTLA-4 comprises an amino acid sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) identity to any one of SEQ ID NOs: 47-48. In some aspects, CTLA-4 is encoded by a polynucleotide of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) sequence identity to any one of SEQ ID NOs: 1-2, 15-17, or 32-33. In some aspects, a heterologous immunosuppressive cytokine comprises, or does not comprise, IL-10, IL-35, and / or TGF-p. In some aspects, a heterologous immunosuppressive cytokine comprises an amino acid sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) identity to any one of SEQ ID NOs: 49, 55-59, 72-73, or 76-77. In some aspects, a heterologous immunosuppressive cytokine is encoded by a polynucleotide sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) sequence identity to any one of SEQ ID NOs: 3-4, 11-14, 18-19, 28-31, 35-36, 42-46, 70-71, or 74-75. In some aspects, a heterologous immunosuppressive cytokine comprises TGF-p. In some aspects, a TGF-P comprises an amino acid sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) identity to any one of SEQ ID NOs: 55-56, or 72-73. In some aspects, a TGF-P is encoded by a polynucleotide sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) sequence identity to any one of SEQ ID NOs: 11-12, 26-27, 42-43, or 70-71. In some aspects, a co-inhibitor engager comprises affinity to an inflammatory marker. In some aspects, a co-inhibitor engager comprises, or does not comprises, a PD-1, TIGIT, LAG3, BTLA, CEACAM-1, 2B4, CD200, CD 160, and / or TIM-3 engager. In some aspects, a co-inhibitor engager comprises a ligand for a T cell checkpoint receptor and / or T cell inhibitory receptor.

[0014] In some aspects, a co-inhibitor engager comprises a TIGIT co-inhibitor engager. In some aspects, a co-inhibitor engager comprises an amino acid sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) identity to any one of SEQ ID NOs: 140-148, 156-162, or 182-190. In some aspects, a co-inhibitor engager is encoded by a polynucleotide sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) sequence identity to any one ofSEQ ID NOs: 113-139, 149-155, or 173-181. In some aspects, a TIGIT co-inhibitor engager comprises an amino acid sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) identity to any one of SEQ ID NOs: 146, 159- 160, or 188. In some aspects, a TIGIT co-inhibitor engager is encoded by a polynucleotide sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) sequence identity to any one of SEQ ID NOs: 119, 128, 137, 152-153, or 179.

[0015] In some aspects, a co-inhibitor engager comprises a PD1 co-inhibitor engager. In some aspects, a PD1 co-inhibitor engager comprises an amino acid sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) identity to any one of SEQ ID NOs: 140-142, 145, 156-158, or 182-187. In some aspects, a PD1 co-inhibitor engager is encoded by a polynucleotide sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) sequence identity to any one of SEQ ID NOs: 131-133, 136, 149, 150-151, 173-175, and 178. In some aspects, a co-inhibitor engager comprises a TIM3 co-inhibitor engager. In some aspects, a TIM3 co-inhibitor engager comprises an amino acid sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) identity to any one of SEQ ID NOs: 147, 161-162, 189, or 195-196. In some aspects, a TIM3 co-inhibitor engager is encoded by a polynucleotide sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) sequence identity to any one of SEQ ID NOs: 138, 154-155, 180, or 191-194.

[0016] In some aspects, an immunosuppressive system is encoded, or is not encoded, by 1, 2, 3, 4, or more polynucleotides. In some aspects, an immunosuppressive system is encoded by 1 polynucleotide. In some aspects, an immunosuppressive system is encoded by one or more polycistronic polynucleotides. In some aspects, one or more polynucleotides encoding an immunosuppressive system are operatively under the control of one or more heterologous promoters. In some aspects, a heterologous promoter is constitutively active and / or inducible. In some aspects, one or more heterologous promoters are substantially the same promoter of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) sequence identity, or wherein the one or more heterologous promoters are different. In some aspects, one or more polycistronic polynucleotides comprise, or do not comprise, one or more of an internal ribosomal entry site (IRES) and / or a self-cleaving peptide. In some aspects, an IRES comprises a polynucleotide sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) sequence identity to SEQ ID NO:80. In some aspects, a self-cleaving sequence is a 2A sequence. In some aspects, a 2A sequence comprises an amino acid sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) identity to any one of SEQ ID NOs: 83-85. In some aspects, a 2A sequence is encoded by a polynucleotide sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) sequence identity to any one of SEQ ID NOs: 81-82. In some aspects, an encoding polynucleotide comprises, or does not comprise, one or more regulatory regions. In some aspects, a regulatory region comprises one or more of a long-terminal repeat, a promoter, and / or a 3' untranslated region (UTR). In some aspects, one or more polynucleotides are comprised in a vector. In some aspects, a vector is a viral vector. In some aspects, a viral vector is, or is not, a lentivirus. In some aspects, a cell comprises a polynucleotide, polypeptide, composition, or vector comprising an immunosuppressive system. In some aspects, a cell is, or is not, a fibroblast, adipocyte, myeloid cell, B-cell, T cell, immortalized cell line, division-incompetent cell line, epithelial cell, mesenchymal stem cell, or a combination thereof. In some aspects, a cell is a mesenchymal stem cell (MSC). In some aspects, a composition comprising a polynucleotide, polypeptide, vector, or cell comprising an immunosuppressive system is, or is not, frozen (e.g., cryopreserved). In some aspects, a composition comprising a polynucleotide, polypeptide, vector, or cell comprising an immunosuppressive system is, or is not, comprised in a delivery device.

[0017] In some aspects, a method of treating a disease in an individual is described herein, the method comprising the step of administering to the individual in need thereof a therapeutically effective amount of a composition, polynucleotide, polypeptide, or cell described herein. In some aspects, an individual has been diagnosed, is suspected of having, or has one or more symptoms of an autoimmune disease, transplant rejection, bacterial sepsis, viral sepsis, fungal sepsis, and / or graft-versus-host disease. In some aspects, an individual is administered an engineered cell described herein. In some aspects, an engineered cell described herein displays an increase in in vivo immunosuppressive functionality relative to a nonengineered cell. In some aspects, an increase in in vivo immunosuppressive functionality comprises a reduction in immune cell (e.g., T cell) proliferation, reduction in response to foreign antigens, reduction in response to self-antigens, reduction in inflammatory responses, reduction in organ damage, prolongation in the individual’ s survival, reduction in graft toxicity, reduction in weight loss, or a combination thereof.

[0018] In some aspects, a method of providing an individual with an immunosuppressive response is described herein, the method comprising administering to the individual in needthereof a therapeutically effective amount of a polynucleotide, polypeptide, cell, and / or composition described herein. In some aspects, the method may further comprise administering a second therapeutic agent to the individual.

[0019] In some aspects, a kit comprising polynucleotide(s), polypeptide(s), cell(s), composition(s), or any combination thereof as described in the disclosure are provided herein. In some aspects, a kit may be used for performing any of the methods described herein. In some aspects, described herein are one or more uses for a polynucleotide, polypeptide, cell, composition, kit, or a combination thereof described herein.

[0020] Certain aspects of the present disclosure are characterized through the following enumerated aspects.

[0021] Aspect 1 is a polynucleotide encoding an engineered interleukin receptor, wherein the encoded engineered interleukin receptor comprises, a) a first interleukin receptor extracellular domain, b) a second interleukin receptor extracellular domain, and c) an interleukin receptor transmembrane and intracellular signaling domain, wherein the domains are operably linked to form a heterologous engineered interleukin receptor.

[0022] Aspect 2 is the polynucleotide according to aspect 1, wherein the first interleukin receptor extracellular domain and the second interleukin receptor extracellular domain are from the same protein.

[0023] Aspect 3 is the polynucleotide according to aspect 1, wherein the first interleukin receptor extracellular domain and the second interleukin receptor extracellular domain are each from different proteins.

[0024] Aspect 4 is the polynucleotide according to aspect 1, wherein the first interleukin receptor extracellular domain and the second interleukin receptor extracellular domain are each from different proteins, and the interleukin receptor transmembrane and intracellular signaling domain is from a different protein.

[0025] Aspect 5 is the polynucleotide according to any one of aspects 1-4, wherein the first interleukin receptor extracellular domain is from CD122 (interleukin-2 receptor subunit beta) or CD25 (interleukin-2 receptor subunit alpha).

[0026] Aspect 6 is the polynucleotide according to any one of aspects 1-5, wherein the second interleukin receptor extracellular domain is from CD25 or CD122.

[0027] Aspect 7 is the polynucleotide according to any one of aspects 1-6, wherein the interleukin receptor transmembrane and intracellular signaling domain is from CD 132 (interleukin-2 receptor subunit gamma).

[0028] Aspect 8 is the polynucleotide according to any one of aspects 1-7, wherein a) the first interleukin receptor extracellular domain is from CD122, b) the second interleukin receptor extracellular domain is from CD25, and c) the interleukin receptor transmembrane and intracellular signaling domain is from CD 132.

[0029] Aspect 9 is the polynucleotide according to any one of aspects 1-7, wherein a) the first interleukin receptor extracellular domain is from CD25, b) the second interleukin receptor extracellular domain is from CD122, and c) the interleukin receptor transmembrane and intracellular signaling domain is from CD 132.

[0030] Aspect 10 is the polynucleotide according to any one of aspects 1-9, wherein the domains are operably linked by one or more linkers.

[0031] Aspect 11 is the polynucleotide according to aspect 10, wherein the linker comprises an amino acid sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) identity to any one of SEQ ID NOs: 89-97.

[0032] Aspect 12 is the polynucleotide according to aspect 10 or 11, wherein the linker is encoded by a polynucleotide sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) sequence identity to any one of SEQ ID NOs: 86-88.

[0033] Aspect 13 is the polynucleotide according to any one of aspects 1-12, further comprising a signal peptide.

[0034] Aspect 14 is the polynucleotide according to aspect 13, wherein the signal peptide comprises an amino acid sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) identity to any one of SEQ ID NOs: 101-103.

[0035] Aspect 15 is the polynucleotide according to aspect 13 or 14, wherein the signal peptide is encoded by a polynucleotide sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) sequence identity to any one of SEQ ID NOs: 98-100.

[0036] Aspect 16 is the polynucleotide according to any one of aspects 1-15, wherein the engineered interleukin receptor comprises an amino acid sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) identity to any one of SEQ ID NOs: 50-53.

[0037] Aspect 17 is the polynucleotide according to any one of aspects 1-16, wherein the engineered interleukin receptor is encoded by a polynucleotide sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) sequence identity to any one of SEQ ID NOs: 5-8, 20-23, or 37-40.

[0038] Aspect 18 is a polypeptide encoded by the polynucleotide according to any one of aspects 1-17.

[0039] Aspect 18.1 is a composition comprising the polynucleotide and / or polypeptide according to any one of aspects 1-18.

[0040] Aspect 19 is an immunosuppressive system comprising one or more polynucleotides and / or polypeptides according to any one of aspects 1-18.

[0041] Aspect 20 is one or more polynucleotides encoding an immunosuppressive system, wherein the encoded immunosuppressive system comprises one or more of an engineered interleukin receptor, one or more of a heterologous costimulatory antagonist, one or more of a heterologous immunosuppressive cytokine, and / or one or more of a co-inhibitor engager.

[0042] Aspect 21 is the one or more polynucleotides according to aspect 20, wherein the immunosuppressive system comprises an engineered interleukin receptor, a heterologous costimulatory antagonist, and a heterologous immunosuppressive cytokine.

[0043] Aspect 22 is the one or more polynucleotides according to aspect 20, wherein the immunosuppressive system comprises an engineered interleukin receptor, a heterologous costimulatory antagonist, a heterologous immunosuppressive cytokine, and a co-inhibitor engager.

[0044] Aspect 23 is the one or more polynucleotides according to any one of aspects 20-22, wherein the co-inhibitor engager comprises a T-cell checkpoint activator and / or a T-cell inhibitor.

[0045] Aspect 24 is the one or more polynucleotides according to any one of aspects 20-23, wherein the engineered interleukin receptor comprises, a) a first interleukin receptor extracellular domain from CD 122, b) a second interleukin receptor extracellular domain from CD25, and c) an interleukin receptor transmembrane and intracellular signaling domain from CD132.

[0046] Aspect 25 is the one or more polynucleotides according to any one of aspects 20- 23, wherein the engineered interleukin receptor comprises, a) a first interleukin receptor extracellular domain from CD25, b) a second interleukin receptor extracellular domain from CD 122, and c) an interleukin receptor transmembrane and intracellular signaling domain from CD132.

[0047] Aspect 26 is the one or more polynucleotides according to any one of aspects 20- 25, wherein the engineered interleukin receptor comprises an amino acid sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) identity to any one of SEQ ID NOs: 50-53.

[0048] Aspect 27 is the one or more polynucleotides according to any one of aspects 20-26, wherein the engineered interleukin receptor is encoded by a polynucleotide sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) sequence identity to any one of SEQ ID NOs: 5-8, 20-23, or 37-40.

[0049] Aspect 28 is the one or more polynucleotides according to any one of aspects 20-27, wherein the heterologous costimulatory antagonist comprises CTLA-4.

[0050] Aspect 29 is the one or more polynucleotides according to any one of aspects 20-28, wherein CTLA-4 comprises an amino acid sequence of, or at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) identity to any one of SEQ ID NOs: 47-48.

[0051] Aspect 30 is the one or more polynucleotides according to any one of aspects 20-29, wherein CTLA-4 is encoded by a polynucleotide sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) sequence identity to any one of SEQ ID NOs: 1-2, 15-17, or 32-33.

[0052] Aspect 31 is the one or more polynucleotides according to any one of aspects 20-30, wherein the heterologous immunosuppressive cytokine comprises TGF-P, IL-10, and / or IL-35.

[0053] Aspect 32 is the one or more polynucleotides according to any one of aspects 20-31, wherein the heterologous immunosuppressive cytokine comprises an amino acid sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) identity to any one of SEQ ID NOs: 49, 55-59, 72-73, or 76-77.

[0054] Aspect 33 is the one or more polynucleotides according to any one of aspects 20-32, wherein the heterologous immunosuppressive cytokine is encoded by a polynucleotide sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) sequence identity to any one of SEQ ID NOs: 3-4, 11-14, 18-19, 28- 31, 35-36, 42-46, 70-71, or 74-75.

[0055] Aspect 34 is the one or more polynucleotides according to any one of aspects 20-33, wherein the heterologous immunosuppressive cytokine comprises TGF-p.

[0056] Aspect 35 is the one or more polynucleotides according to any one of aspects 20-34, wherein the TGF-P comprises an amino acid sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) identity to any one of SEQ ID NOs: 55-56, or 72-73.

[0057] Aspect 36 is the one or more polynucleotides according to any one of aspects 20-35, wherein the TGF-P is encoded by a polynucleotide sequence of, of at least, or of at most80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) sequence identity to any one of SEQ ID NOs: 11-12, 26-27, 42-43, or 70-71.

[0058] Aspect 37 is the one or more polynucleotides according to any one of aspects 20-36, wherein the co-inhibitor engager comprises affinity to an inflammatory marker.

[0059] Aspect 38 is the one or more polynucleotides according to any one of aspects 20-37, wherein the co-inhibitor engager is a TIGIT, PD-1, CD5, CD3, LAG3, BTLA, CEACAM- 1, 2B4, CD200, CD 160, and / or TIM-3 co-inhibitor engager.

[0060] Aspect 39 is the one or more polynucleotides according to any one of aspects 20-38, wherein the co-inhibitor engager comprises a ligand for a T cell checkpoint receptor and / or T cell inhibitory receptor.

[0061] Aspect 40 is the one or more polynucleotides according to any one of aspects 20-39, wherein the co-inhibitor engager comprises an amino acid sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) identity to any one of SEQ ID NOs: 140-148, 156-162, 182-190, or 195-196.

[0062] Aspect 41 is the one or more polynucleotides according to any one of aspects 20-40, wherein the co-inhibitor engager is encoded by a polynucleotide sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) sequence identity to any one of SEQ ID NOs: 113-139, 149-155, 173-181, or 191-194.

[0063] Aspect 42 is the one or more polynucleotides according to any one of aspects 20-41, wherein the co-inhibitor engager comprises a TIGIT co-inhibitor engager.

[0064] Aspect 43 is the one or more polynucleotides according to any one of aspects 20-42, wherein the TIGIT engager comprises an amino acid sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) identity to any one of SEQ ID NOs: 146, 159-160, or 188.

[0065] Aspect 44 is the one or more polynucleotides according to any one of aspects 20-43, wherein the TIGIT co-inhibitor engager is encoded by a polynucleotide sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) sequence identity to any one of SEQ ID NOs: 119, 128, 137, 152-153, or 179.

[0066] Aspect 45 is the one or more polynucleotides according to any one of aspects 20-44, wherein the co-inhibitor engager comprises a PD1 co-inhibitor engager.

[0067] Aspect 46 is the one or more polynucleotides according to any one of aspects 20-45, wherein the PD1 co-inhibitor engager comprises an amino acid sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) identity to any one of SEQ ID NOs: 140-142, 145, 156-158, or 182-187.

[0068] Aspect 47 is the one or more polynucleotides according to any one of aspects 20-46, wherein the PD1 co-inhibitor engager is encoded by a polynucleotide sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) sequence identity to any one of SEQ ID NOs: 131-133, 136, 149, 150-151, 173-175, and 178.

[0069] Aspect 48 is the one or more polynucleotides according to any one of aspects 20-47, wherein the co-inhibitor engager comprises a TIM3 co-inhibitor engager.

[0070] Aspect 49 is the one or more polynucleotides according to any one of aspects 20-48, wherein the TIM3 co-inhibitor engager comprises an amino acid sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) identity to any one of SEQ ID NOs: 147, 161-162, 189, or 195-196.

[0071] Aspect 50 is the one or more polynucleotides according to any one of aspects 20-49, wherein the TIM3 co-inhibitor engager is encoded by a polynucleotide sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) sequence identity to any one of SEQ ID NOs: 138, 154-155, 180, or 191-194.

[0072] Aspect 51 is the one or more polynucleotides according to any one of aspects 20-50, wherein the immunosuppressive system is encoded by 1, 2, 3, 4, or more polynucleotides.

[0073] Aspect 52 is the one or more polynucleotides according to any one of aspects 20-51, wherein the immunosuppressive system is encoded by 1 polynucleotide.

[0074] Aspect 53 is the one or more polynucleotides according to any one of aspects 20-52, wherein the one or more polynucleotides are polycistronic.

[0075] Aspect 54 is the one or more polynucleotides according to any one of aspects 20-53, wherein the one or more polynucleotides are operatively under the control of one or more heterologous promoters.

[0076] Aspect 55 is the one or more polynucleotides according to aspect 54, wherein the one or more heterologous promoters are constitutively active and / or are inducible.

[0077] Aspect 56 is the one or more polynucleotides according to aspect 54 or 55, wherein the one or more heterologous promoters are substantially the same promoter of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) sequence identity, or wherein the one or more heterologous promoters are different.

[0078] Aspect 57 is the one or more polynucleotides according to any one of aspects 53- 56, wherein the one or more polycistronic polynucleotides comprise one or more of an internal ribosomal entry site (IRES) and / or a self-cleaving peptide.

[0079] Aspect 58 is the one or more polynucleotides according to aspect 57, wherein the IRES comprises a polynucleotide sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) sequence identity to any one of SEQ ID NO: 80.

[0080] Aspect 59 is the one or more polynucleotides according to aspect 57 or 58, wherein the self-cleaving sequence is a 2A sequence.

[0081] Aspect 60 is the one or more polynucleotides according to aspect 59, wherein the 2A sequence comprises an amino acid sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) identity to any one of SEQ ID NOs: 83-85.

[0082] Aspect 61 is the one or more polynucleotides according to aspect 59 or 60, wherein the 2A sequence is encoded by a polynucleotide sequence of, of at least, or of at most 80%, 85%, 90%, 95%, 99%, or 100% (or any range or value derivable therein) sequence identity to any one of SEQ ID NOs: 81-82.

[0083] Aspect 62 is the one or more polynucleotides according to any one of aspects 20- 61, wherein the encoding polynucleotide comprises one or more regulatory regions.

[0084] Aspect 63 is the one or more polynucleotides according to aspect 62, wherein the regulatory regions comprise one or more of a long-terminal repeat, a promoter, and / or a 3' untranslated region (UTR).

[0085] Aspect 64 is the one or more polynucleotides according to any one of aspects 20- 63, wherein the encoding polynucleotides are comprised in a vector.

[0086] Aspect 65 is the one or more polynucleotides according to aspect 64, wherein the vector is a viral vector.

[0087] Aspect 66 is the one or more polynucleotides according to aspect 64 or 65, wherein the vector is a lentivirus.

[0088] Aspect 67 is a cell comprising the one or more polynucleotides, polypeptides, compositions, and / or vectors according to any one of aspects 1-66.

[0089] Aspect 68 is the cell according to aspect 67, wherein the cell is a mesenchymal stem cell (MSC), fibroblast, adipocyte, myeloid cell, B-cell, T cell, immortalized cell line, divisionincompetent cell line, epithelial cell, or a combination thereof.

[0090] Aspect 69 is the cell according to aspect 67 or 68, wherein the cell is a MSC.

[0091] Aspect 69. l is a method of preparing the cell according to any one of aspects 67-69 comprising the step of introducing the polynucleotide, polypeptide, and / or immunosuppressive system according to any one of aspects 1-66.

[0092] Aspect 70 is a composition comprising the polynucleotide, polypeptide, cell, composition, or a combination thereof according to any one of aspects 1-69.

[0093] Aspect 71 is the composition according to aspect 70, wherein the composition is frozen.

[0094] Aspect 72 is the composition according to aspect 70 or 71, wherein the composition is comprised in a delivery device.

[0095] Aspect 73 is a method of treating a disease in an individual, the method comprising the step of administering to the individual in need thereof a therapeutically effective amount of the composition according to aspect 70 or 72.

[0096] Aspect 74 is the method according to aspect 73, wherein the individual has been diagnosed, is suspected of having, or has one or more symptoms of an autoimmune disease, transplant rejection, bacterial sepsis, viral sepsis, fungal sepsis, and / or graft-versus-host disease.

[0097] Aspect 75 is the method according to aspect 73 or 74, wherein the individual is administered an engineered cell.

[0098] Aspect 76 is the method according to aspect 75, wherein the engineered cell displays an increase in in vivo immunosuppressive functionality relative to a non-engineered cell.

[0099] Aspect 77 is the method according to aspect 76, wherein the increase in in vivo immunosuppressive functionality comprises a reduction in immune cell proliferation, reduction in response to foreign antigens, reduction in response to self-antigens, reduction in inflammatory responses, reduction in organ damage, prolongation in the individual’s survival, reduction in graft toxicity, reduction in weight loss, or a combination thereof.

[0100] Aspect 78 is a method of providing an individual with an immunosuppressive response, the method comprising administering to the individual in need thereof a therapeutically effective amount of the polynucleotide, polypeptide, cell, and / or composition according to any one of aspects 1-72.

[0101] Aspect 79 is the method according to any one of aspects 73-78, further comprising administering at least a second therapeutic agent to the individual.

[0102] Aspect 80 is a kit comprising the polynucleotide, polypeptide, cell, composition, or a combination thereof according to any one of aspects 1-72.

[0103] Aspect 81 is a kit for the means of performing the methods according to any one of aspects 73-78.

[0104] Aspect 82 is the use of the polynucleotide, polypeptide, cell, composition, kit, or a combination thereof according to any one of aspects 1-72 or 80-81 for production of a medicament for treatment of a disease and / or disorder.

[0105] Aspect 83 is a polynucleotide encoding an engineered interleukin receptor, wherein the encoded engineered interleukin receptor comprises, a) a first interleukin receptor extracellular domain from CD 122, b) a second interleukin receptor extracellular domain from CD25, and c) an interleukin receptor transmembrane and intracellular signaling domain from CD 132, wherein the domains are operably linked in a 5' to 3' order of (a), (b), and (c) to form a heterologous engineered interleukin receptor.

[0106] Aspect 84 is an engineered interleukin receptor comprising, a) a first interleukin receptor extracellular domain from CD 122, b) a second interleukin receptor extracellular domain from CD25, and c) an interleukin receptor transmembrane and intracellular signaling domain from CD 132, wherein the domains are operably linked in a 5' to 3' order of (a), (b), and (c) to form a heterologous engineered interleukin receptor.

[0107] Aspect 85 is one or more polynucleotides encoding an immunosuppressive system, wherein the encoded immunosuppressive system comprises: i) an engineered interleukin receptor comprising a) a first interleukin receptor extracellular domain from CD122, b) a second interleukin receptor extracellular domain from CD25, and c) an interleukin receptor transmembrane and intracellular signaling domain from CD 132; ii) a heterologous costimulatory antagonist comprising CTLA-4, iii) a heterologous immunosuppressive cytokine comprising TGF-P, and optionally iv) a co-inhibitor engager targeting TIGIT.

[0108] Aspect 86 is a mesenchymal stem cell comprising i) an engineered interleukin receptor comprising a) a first interleukin receptor extracellular domain from CD122, b) a second interleukin receptor extracellular domain from CD25, and c) an interleukin receptor transmembrane and intracellular signaling domain from CD 132; ii) a heterologous costimulatory antagonist comprising CTLA-4, iii) a heterologous immunosuppressive cytokine comprising TGF-P, and optionally iv) a co-inhibitor engager targeting TIGIT.

[0109] Aspect 87 is a method of treating graft-versus-host disease in an individual in need thereof comprising administering to the individual a mesenchymal stem cell comprising i) an engineered interleukin receptor comprising a) a first interleukin receptor extracellular domain from CD122, b) a second interleukin receptor extracellular domain from CD25, and c) an interleukin receptor transmembrane and intracellular signaling domain from CD 132; ii) a heterologous costimulatory antagonist comprising CTLA-4, iii) a heterologousimmunosuppressive cytokine comprising TGF-P, and optionally iv) a co-inhibitor engager targeting TIGIT.

[0110] It is specifically contemplated that any limitation discussed with respect to one aspect of the disclosure may apply to any other aspect of the disclosure. Furthermore, any composition of the disclosure may be used in any method of the disclosure, and any method of the disclosure may be used to produce or to utilize any composition of the disclosure. Any aspect discussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa. For example, any step in a method described herein can apply to any other method. Moreover, any method described herein may have an exclusion of any step or combination of steps. Aspects of an aspect set forth in the Examples are also aspects that may be implemented in the context of aspects discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary, Detailed Description, Claims, and Brief Description of the Drawings.

[0111] Any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “Use of’ any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect.

[0112] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific aspects of the inventions provided herein, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0113] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The inventions provided herein may be better understood by reference to one or more of these drawings in combination with the detailed description of specific aspects presented herein.

[0114] FIG. 1 shows a schematic of certain aspects provided herein. Merely as an example, mesenchymal stem cells (MSCs) may be engineered to comprise enhanced immunosuppressive functions, e.g., uptake of stimulatory cytokines by chimeric interleukin receptors, such as chimeric interleukin receptor 2 (cIL2R); heterologous expression of costimulatory antagonists, such as CTLA-4; heterologous expression of a co-inhibitor engager directed to an immune cell,and / or heterologous expression of immunosuppressive cytokines, such as TGF-P, which would act on antigen-presenting cells, dendritic cells, and / or effector T cells. Tetr: effector T cell, DC: dendritic cell, cIL2R: chimeric IL2 receptor.

[0115] FIGs. 2A-2B. Schematic representations. FIG. 2A shows mechanisms of immune cell suppression by regulatory T cells (Treg). FIG. 2B shows a schematic of a mesenchymal stem cell (MSC) engineered to comprise heterologous gene expression to mimic Treg function.

[0116] FIGs. 3A-3E. Activated T cells were immunosuppressed by engineered regulatory T (Treg) cells that were guided to the activated T cells using PD-1 or TIGIT co-inhibitor engagers. FIG. 3A shows upregulation of TIGIT and PD-1 in activated allo- reactive (5 days after allogeneic stimulation) and polyclonally activated T cells compared to resting T cells. FIG. 3B shows a schematic representation of an exemplary co-inhibitor engager. Merely as an example, an extracellular scFv that targeted co-inhibitory molecules, such as PD-1 or TIGIT, was anchored to the cell membrane and may be optionally linked to a signaling endodomain, e.g., a CD28 / CD3 chimeric signaling endodomain. FIG. 3C shows the frequency of Treg cells successfully engineered, e.g., transduced, to express a co-inhibitor engager. The results showed a high efficiency of Treg cells engineered to express a surface coinhibitor engager specific to PD-1 (sPD-1) or TIGIT (sTIGIT). FIG. 3D shows expression of PD-1 (x-axis) and TIGIT (y-axis) by engineered Treg cells. The results showed that, compared to non-transduced (NT) controls, Treg cells engineered with sPD-1 had reduced PD-1 expression, while those engineered with sTIGIT had reduced TIGIT expression. FIG. 3E shows immunosuppressive function of engineered Treg cells. Treg cells were co-cultured with resting T cells labeled with a proliferation dye and activating allogeneic dendritic cells. The results showed a robust suppression of activated T-cell proliferation (reflected by a higher level of the proliferation dye) by all Treg groups (NT, sPD-1, and sTIGIT) at a 1 : 1 ratio of engineered Treg cells to T-cells. However, at a lower ratio of engineered Treg cells to T-cells (1:4), sTIGIT Treg cells were significantly more potent suppressors than NT controls. Suppression of proliferation was also tested in a setting where NT Treg cells are not effective suppressors: against previously expanded allo-reactive T-cells or previously activated T-cells. The results showed that sTIGIT significantly suppressed proliferation of pre-activated allo-reactive T cells or polyclonal activated T cells compared to NT Treg controls.

[0117] FIGs. 4A-4E. Novel chimeric IL-2 receptors (cIL2R) effectively sequestered IL-2. FIG. 4A shows a schematic of a MSC engineered (e.g., transduced) to express a cIL2R. FIG. 4B shows schematics of cIL2R constructs comprised in certain aspects of this disclosure(CD25: IL2Ra; CD122: IL2RP; CD132: common y). FIG. 4C shows engineering (e.g., transduction) efficiency of MSCs comprising a cIL2R. The results showed that cIL2Rs (aP- gamma, or Pa-gamma) or control P-only were expressed on the cell surface of engineered MSC, with high expression of CD25 by cIL2R groups and high expression of CD 122 by all groups compared to non-transduced (NT) controls. FIG. 4D shows assessment of MSC phenotype, e.g., CD90 and / or CD73 expression, in engineered MSCs. The results showed that engineering of MSCs with cIL2Rs or P-only did not affect MSC phenotype, as seen by the expression of CD73 and CD90 compared to NT controls. FIG. 4E shows sequestration of IL-2 by Treg, NT MSC, or engineered MSCs. Cells were cultured in media supplemented with increasing concentrations of exogenous IL-2 (10, 50, and 100 U / mL), and sequestration was assessed at the 2- or 24-hour timepoints (top and bottom graphs, respectively). The results showed that at the 2-hour timepoint, MSC expressing the Pa-gamma cIL2R significantly decreased IL-2 concentration in the media when compared at least to NT MSC, for all tested concentrations. The results also showed that at the 24-hour timepoint, both aP-gamma and Pa-gamma significantly decreased IL-2 concentration in the media, decreasing IL-2 concentration to nearzero, when compared at least to NT controls, whereas P-only control did not. These results showed that both the aP-gamma and Pa-gamma construct were strikingly effective at sequestering IL-2 and that engineered MSCs sequestered higher levels of IL-2 relative to Tregs.

[0118] FIGs. 5A-5C. CTLA-4 enhanced immunosuppressive functions of MSCs and cIL2R-engineered MSCs. FIG. 5A shows a schematic of a MSC engineered (e.g., transduced) to comprise expression of a cIL2R and heterologous CTLA-4. FIG. 5B shows engineering (e.g., transduction) efficiency of constructs comprising the Pa-gamma construct (cIL2R), heterologous CTLA-4 (CTLA-4), or cIL2R and CTLA-4 (CTLA4+cIL2R), as measured by CTLA-4 or CD 122 expression. The results showed that CTLA-4 was highly expressed on the cell surface of engineered CTLA4-MSC and CTLA4+cIL2R-MSC, while cIL2R was detected on the surface of engineered cIL2R-MSC and CTLA4+cIL2R-MSC. FIG. 5C shows suppression of proliferation of allogeneic T cells by NT and engineered MSC, and Treg cells. The results showed that engineering of MSC to comprise cIL2R or CTLA4 alone enhanced the immunosuppressive functions of MSC in comparison to NT MSC, and resulted in similar potency to Treg cells. The results also showed that CTLA4+cIL2R-MSC were significantly more suppressive than NT, or cIL2R-MSC, and apparently more suppressive than CTLA4- MSC or Treg cells.

[0119] FIGs. 6A-6C. Immunosuppressive cytokines enhanced suppressive functions of MSCs. FIG. 6A shows a schematic of a MSC engineered (e.g., transduced) to compriseexpression of IL-35, IL-10, and / or TGF-p. FIG. 6B shows engineering (e.g., transduction) efficiency of constructs carrying IL-10, TGF-P, or IL-35, measured by NGFR (reporter for IL- 10) or CD34-Q8 (reporter for IL-25 and TGF-P) expression. The results showed that engineered MSCs showed increased surrogate marker expression for IL-10, IL-35, and TGF- p. FIG. 6C shows suppression of proliferation of allogeneic T cells by non-transduced (NT) MSCs, MSCs engineered to express IL-10, IL-35, or TGF-P, and Treg cells. The results showed that, compared to NT controls, MSC engineered to comprise and secrete heterologous IL-10 significantly suppressed proliferation of allogeneic T cells. Additionally, the results surprisingly showed that MSC engineered to secrete heterologous IL-10 were more suppressive than Tregs.

[0120] FIGs. 7A-7C. Heterologous expression of CTLA-4, cIL2R, and an immunosuppressive cytokine produced a synergistic effect in immunosuppressive functions of MSCs. FIG. 7A shows a schematic of a MSC engineered (e.g., transduced) to comprise expression of heterologous TGF-P, CTLA-4, and a cIL-2R. FIG. 7B shows engineering (e.g., transduction) efficiency of MSCs with CTLA-4, cIL2R, and / or an immunosuppressive cytokine, including IL-10, TGF-P, and / or IL-35. The results showed that engineering efficiency was high for all combinations as evidenced by flow cytometry analysis of NGFR (reporter for CTLA-4 and IL10), Q8 (reporter for TGF-P and IL-35), CTLA-4, or CD25 (IL2Ra) expression. FIG. 7C shows suppression of proliferation of allogeneic T cells by non-transduced (NT) MSC, engineered MSC, and Treg cells at a MSC or Treg:T cell ratio of 1 : 1 (top graph) or 1 :2 (bottom graph). The results showed that MSC engineered to comprise CTLA-4+cIL2R, CTLA-4+cIL2R+IL10, CTLA-4+cIL2R+TGF-P, or CTLA-4+cIL2R+IL35 showed significant suppression of T cell proliferation compared to NT controls at a 1 : 1 ratio. The results also showed that MSC engineered to comprise CTLA-4+cIL2R, CTLA- 4+cIL2R+IL10, CTLA-4+cIL2R+TGF-P, or CTLA-4+cIL2R+IL35 showed significant suppression of T cell proliferation compared to NT controls at a 1 :2 ratio, although it was apparent that MSC engineered to comprise CTLA-4+cIL2R+TGF-P showed the highest potency of suppression. Collectively, these results demonstrated that cells, e.g., MSC, engineered to comprise a cytokine uptake molecule, such as a cIL2R, a costimulatory antagonist, such as heterologous CTLA4, and an immunosuppressive cytokine, such as IL10, IL35 or TGF-P, drastically enhanced the immunosuppressive capabilities of said engineered cells, exceeding the immunosuppressive capabilities of Treg cells.DETAILED DESCRIPTION

[0121] It is to be understood that the present disclosure is not limited to particular aspects described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0122] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this technology belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present technology, the preferred methods, devices and materials are now described. All technical and patent publications cited herein are incorporated herein by reference in their entirety.

[0123] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Green and Sambrook eds. (2012) Molecular Cloning: A Laboratory Manual, 4th edition; the series Ausubel et al. eds. (2015) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (2015) PCR 1 : A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; McPherson et al. (2006) PCR: The Basics (Garland Science); Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Greenfield ed. (2014) Antibodies, A Laboratory Manual; Freshney (2010) Culture of Animal Cells: A Manual of Basic Technique, 6th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Herdewijn ed. (2005) Oligonucleotide Synthesis: Methods and Applications; Hames and Higgins eds. (1984) Transcription and Translation; Buzdin and Lukyanov ed. (2007) Nucleic Acids Hybridization: Modern Applications; Immobilized Cells and Enzymes (IRL Press (1986)); Grandi ed. (2007) In Vitro Transcription and Translation Protocols, 2nd edition; Guisan ed. (2006) Immobilization of Enzymes and Cells; Perbal (1988) A Practical Guide to Molecular Cloning, 2nd edition; Miller and Calos eds, (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Lundblad and Macdonald eds. (2010) Handbook of Biochemistry and Molecular Biology, 4th edition; and Herzenberg et al. eds(1996) Weir’s Handbook of Experimental Immunology, 5th edition; all of which are incorporated herein by reference.

[0124] It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art. It is to be inferred without explicit recitation and unless otherwise intended, that when the present technology relates to a polypeptide, protein, polynucleotide, cell, or antibody, an equivalent or a biologically equivalent of such is intended within the scope of the present technology.

[0125] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the measurement or quantitation method.

[0126] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0127] The phrase “and / or” means “and” or “or”. To illustrate, A, B, and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, “and / or” operates as an inclusive or.

[0128] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0129] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. Compositions and methods “consisting essentially of’ any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed invention.

[0130] The term “engineered” as used herein refers to an entity that is generated by the hand of man, including a cell, nucleic acid, polypeptide, vector, and so forth. In at least some cases, an engineered entity is synthetic and comprises elements that are not naturally present or configured in the manner in which it is utilized in the disclosure. In specific aspects, a vector is engineered through recombinant nucleic acid technologies, and a cell is engineered, e.g., through transfection or transduction of an engineered vector. Cells may be engineered to express heterologous proteins that are not naturally expressed by the cells, either because theheterologous proteins are recombinant or synthetic or because the cells do not naturally express the proteins.

[0131] The term “heterologous” when used in reference to a gene refers to a gene encoding a factor that is not in its natural environment (e.g., has been altered by the hand of man). For example, a heterologous gene includes a gene from one species introduced into another species. A heterologous gene also includes a gene native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, linked to a non-native promoter or enhancer sequence, etc.). Heterologous genes may comprise gene sequences that comprise cDNA forms of a gene; the cDNA sequences may be expressed in either a sense (to produce mRNA) or anti-sense orientation (to produce an anti-sense RNA transcript that is complementary to the mRNA transcript). Heterologous genes are distinguished from endogenous genes in that the heterologous gene sequences are typically joined to nucleotide sequences comprising regulatory elements such as promoters that are not found naturally associated with the gene for the protein encoded by the heterologous gene or with gene sequences in the chromosome, or are associated with portions of the chromosome not found in nature (e.g., genes expressed in loci where the gene is not normally expressed).

[0132] “Individual,” “subject,” and “patient” are used interchangeably and can refer to a human or non-human.

[0133] “Treating” or treatment of a disease or condition refers to executing a protocol, which may include administering one or more pharmaceutical compositions to a patient, in an effort to alleviate signs or symptoms of the disease or disorder. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. Alleviation can occur prior to signs or symptoms of the disease or condition appearing, as well as after their appearance. Thus, “treating” or “treatment” may include “preventing” or “prevention” of disease or undesirable condition. In addition, “treating” or “treatment” does not require complete alleviation of signs or symptoms, does not require a cure, and specifically includes protocols that have only a marginal effect on the patient.

[0134] The term “therapeutic benefit” or “therapeutically effective” as used throughout this application refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of this condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease. For example, treatment of cancer may involve, for example, a reduction in the size of a tumor, a reduction inthe invasiveness of a tumor, reduction in the growth rate of the cancer, or prevention of metastasis. Treatment of cancer may also refer to prolonging survival of a subject with cancer.

[0135] In certain aspects, there are disclosed compositions and methods for therapies intended to treat organ transplant rejection, graft-versus-host disease, autoimmune pathologies, bacterial sepsis, viral sepsis, fungal sepsis, and / or disorders of auto- or allo-reactive immune cells. In some aspects, there are disclosed compositions of engineered cells, which may or may not be for example, but not limited to, mesenchymal stem cells (MSC), fibroblasts, adipocytes, myeloid cells, B-cell, T cell, immortalized cell lines, division-incompetent cell lines, and / or epithelial cells, engineered to recognize and / or suppress activated immune cells, e.g., T cells, and methods of producing the same. In some aspects, cells may be engineered to comprise, consist essentially of, or consist of a set of heterologous, engineered, and / or artificial genes, polynucleotides, and / or polypeptides that produce potent immunosuppression, e.g., suppression of T cell activation and / or function. In some aspects, cells (e.g., MSCs, fibroblasts, adipocytes, etc.), may be engineered to mimic mechanisms of immunosuppression found in regulatory T cells (Treg). In some aspects, a cell is an MSC and / or derivative thereof.

[0136] In some aspects, methods and compositions to target engineered cells to an activated immune cell using a co-inhibitor engager are disclosed. In some aspects, a co-inhibitor engager may target a marker of cell inflammation and / or a co-inhibitory marker, and may or may not be for example, but not limited to PD-1, CD3, TIGIT, LAG3, BTLA, CEACAM-1, 2B4, CD200, CD 160, CD5, and / or TIM-3. In some aspects, a cell (e.g., MSC, Treg, fibroblast, etc.) may be engineered to comprise a co-inhibitor engager that is a ligand for and engages a T cell inhibitory receptor such as a checkpoint or another inhibitory receptor.

[0137] In some aspects, compositions comprising a chimeric interleukin receptor capable of sequestering soluble interleukin from the extracellular space are disclosed. In some aspects, a chimeric interleukin receptor may comprise, consist essentially of, or consist of, one or more extracellular domains, transmembrane domains, and intracellular domains of one or more interleukin receptors. In some aspects, a domain may be from the same protein or from different proteins as the other domains. In some aspects, a chimeric interleukin receptor may be a chimeric IL-2 receptor. In some aspects, a chimeric IL-2 receptor may comprise, consist essentially of, or consist of domains from CD25, CD122, and / or CD133. In some aspects, a cell (e.g., a MSC, fibroblast, etc.) may be engineered to comprise a chimeric interleukin receptor. In some aspects, a cell may be engineered to comprise a chimeric interleukin 2 receptor (cIL2R). In some aspects, a cell is an MSC.

[0138] In some aspects, compositions comprising, consisting essentially of, or consisting of a heterologous co-stimulatory antagonist are disclosed. In some aspects, a heterologous costimulatory antagonist may be a heterologous CTLA-4 gene or protein. In some aspects, a cell (e.g., a MSC, fibroblast, T cell, etc.) may be engineered to comprise, a heterologous costimulatory antagonist.

[0139] In some aspects, compositions comprising, consisting essentially of, or consisting of one or more heterologous immunosuppressive cytokines are disclosed. In some aspects, a heterologous immunosuppressive cytokine may be or may not be IL-10, IL-35, and / or TGF-p. In some aspects, a cell (e.g., a MSC, fibroblast, T cell, etc.) may be engineered to comprise a heterologous immunosuppressive cytokine. In some aspects, a heterologous immunosuppressive cytokine may or may not be secreted. In some aspects, a heterologous immunosuppressive cytokine may or may not be membrane bound.

[0140] In some aspects, methods and / or compositions for engineering a cell (e.g., a MSC, fibroblast, T cell, etc.) comprise, consist essentially of, or consist of engineering a cell to express one or more chimeric interleukin receptors, one or more co-inhibitor engagers, one or more heterologous immunosuppressive cytokines, one or more heterologous co-stimulatory antagonists, or a combination thereof. In some aspects, a cell may be engineered to include one or more polynucleotides and / or one or more polypeptides that comprise, consist essentially of, or consist of one or more chimeric interleukin receptors, one or more co-inhibitor engagers, one or more heterologous immunosuppressive cytokines, one or more heterologous co- stimulatory antagonists, or a combination thereof. In some aspects, a cell can be engineered to include one or more polynucleotides and / or one or more polypeptides that comprise, consist of, or consist essentially of an engineered interleukin receptor. In some aspects, a cell can be engineered to include one or more polynucleotides and / or one or more polypeptides that may comprise, consist of, or consist essentially of a heterologous costimulatory antagonist. In some aspects, a cell can be engineered to include one or more polynucleotides and / or one or more polypeptides that may comprise, consist of, or consist essentially of a heterologous immunosuppressive cytokine. In some aspects, a cell can be engineered to include one or more polynucleotides and / or one or more polypeptides that may comprise, consist of, or consist essentially of a co-inhibitor engager. In some aspects, a cell can be engineered to include one or more polynucleotides and / or one or more polypeptides that may comprise, consist of, or consist essentially of an engineered interleukin receptor and a heterologous costimulatory antagonist. In some aspects, a cell can be engineered to include one or more polynucleotides and / or one or more polypeptides that may comprise, consist of, or consist essentially of anengineered interleukin receptor, and a heterologous immunosuppressive cytokine. In some aspects, a cell can be engineered to include one or more polynucleotides and / or one or more polypeptides that may comprise, consist of, or consist essentially of an engineered interleukin receptor, and a co-inhibitor engager. In some aspects, a cell can be engineered to include one or more polynucleotides and / or one or more polypeptides that may comprise, consist of, or consist essentially of a heterologous costimulatory antagonist, and a heterologous immunosuppressive cytokine. In some aspects, a cell can be engineered to include one or more polynucleotides and / or one or more polypeptides that may comprise, consist of, or consist essentially of a heterologous costimulatory antagonist, and a co-inhibitor engager. In some aspects, a cell can be engineered to include one or more polynucleotides and / or one or more polypeptides that may comprise, consist of, or consist essentially of a heterologous immunosuppressive cytokine, and a co-inhibitor engager. In some aspects, a cell can be engineered to include one or more polynucleotides and / or one or more polypeptides that may comprise, consist of, or consist essentially of a chimeric interleukin receptor, a heterologous immunosuppressive cytokine, and a heterologous co-stimulatory antagonist. In some aspects, a cell, can be engineered to include one or more polynucleotides and / or one or more polypeptides that may comprise, consist of, or consist essentially of a chimeric interleukin receptor, a co-inhibitor engager, a heterologous immunosuppressive cytokine, and a heterologous co-stimulatory antagonist.

[0141] Accordingly, disclosed herein are polynucleotides, polypeptides, compositions, and / or cells comprising, consisting essentially of, consisting of, and / or encoding one or more of a chimeric interleukin receptor, one or more of a co-inhibitor engager, one or more of a heterologous immunosuppressive cytokine, one or more of a heterologous co-stimulatory antagonist, or a combination thereof, as well as methods for use of the same in treatment of diseases or disorders (e.g., graft-versus-host disease, transplant rejection, autoimmune diseases, etc.) or in the manufacture of a medicament to treat a disease or disorder. In various aspects, a chimeric interleukin receptor, a co-inhibitor engager, an immunosuppressive cytokine, and / or a co-stimulatory antagonist are heterologous to the cell in which they are comprised. In various aspects, a chimeric interleukin receptor, a co-inhibitor engager, an immunosuppressive cytokine, and / or a co-stimulatory antagonist are heterologous to the cell in which they are comprised and are not appreciably expressed (e.g., to the extent required for relative functionality as described herein) from an endogenous gene in the genome of the cell. In various aspects, the one or more chimeric interleukin receptor, co-inhibitor engager, heterologous immunosuppressive cytokine, heterologous co-stimulatory antagonist, or acombination thereof, may be expressed from a synthetic vector generated by the hand of man. In some aspects, the one or more chimeric interleukin receptors, one or more of co-inhibitor engagers, one or more of heterologous immunosuppressive cytokines, one or more of heterologous co-stimulatory antagonists, or a combination thereof, may be stably integrated into the genome of the host cell.

[0142] In some aspects, a protein, peptide, domain, domain sequence, amino acid sequence, polynucleotide, nucleic acid sequence, and / or gene product can be obtained or be substantially identical to all or part of a protein, peptide, domain, amino acid sequence, nucleic acid sequence, and / or gene product of a gene recited in the disclosure, including or excluding, but not limited to, CD122, CD25, CD132, CTLA-4, IL-10, IL-35, TGF-P, TIGIT, PD-1, CD28, or a combination thereof. Any gene referenced in the disclosure can refer to a mammalian gene, e.g., human or murine, or to a homolog or analog in any other species.

[0143] CD122: An example of an interleukin 2 receptor subunit beta (CD122; IMD63;IL15RB; P70-75; IL2RB) gene sequence can be found in the US NIH, National Library of Medicine, National Center for Biotechnology Information (NCBI) GENBANK® Database under Gene ID: 3560 (IL2RB) on chromosome 22ql2.3, at NC_000022. l l Reference GRCh38.pl4 Primary Assembly (range, 37125838 to 37175118, complement) for the genomic sequence which is incorporated herein by reference in its entirety. A CD122 mRNA transcript can be identified as NM_000878.5, NM_001346222.1, or NM_001346223.2, and the associated protein product can be identified as NP 000869.1, NP 001333151.1, or NP_001333152.1, respectively, each of which are incorporated by reference herein in their entirety. The protein encoded by this gene is involved in T cell-mediated immune responses.

[0144] CD25: An example of an interleukin 2 receptor subunit alpha (CD25; IL2RA; p55;CD25; IL2R; IMD41; TCGFR; IDDM10) gene sequence can be found in the US NIH, National Library of Medicine, National Center for Biotechnology Information (NCBI) GENBANK® Database under Gene ID: 3559 (IL2RA) on chromosome 1 Op 15.1, atNC OOOOlO.11 Reference GRCh38.pl4 Primary Assembly (range, 6010689 to 6062367, complement) for the genomic sequence which is incorporated herein by reference in its entirety. A CD25 mRNA transcript can be identified as NM_000417.3, NM_001308242.2, or NM_001308243.2, and the associated protein product can be identified as NP_000408.1, NP_001295171.1, or NP_001295172.1, respectively, each of which are incorporated by reference herein in their entirety. The protein encoded by this gene is involved in T cell-mediated immune responses.

[0145] CD132: An example of an interleukin 2 receptor subunit gamma (CD 132; IL2RG;P64; CIDX; IMD4; CD132; SCIDX; IL-2RG; SCIDX1) gene sequence can be found in the USNIH, National Library of Medicine, National Center for Biotechnology Information (NCBI) GENBANK® Database under Gene ID: 3561 (IL2RG) on chromosome Xql3.1, at NC_000023. l l Reference GRCh38.pl4 Primary Assembly (range, 71107404 to 71111577, complement) for the genomic sequence which is incorporated herein by reference in its entirety. A CD132 mRNA transcript can be identified as NM_000206.3, and the associated protein product can be identified as NP 000197.1, each of which are incorporated by reference herein in their entirety. The protein encoded by this gene is an important signaling component of many interleukin receptors.

[0146] CTLA-4 : An example of a cytotoxic T-lymphocyte associated protein 4 (CTLA-4; CD; GSE; GRD4; ALPS5; CD152; CTLA4; IDDM12; CELIAC3) gene sequence can be found in the US NIH, National Library of Medicine, National Center for Biotechnology Information (NCBI) GENBANK® Database under Gene ID: 1493 (CTLA4) on chromosome 2q33.2, at NC_000002.12 Reference GRCh38.pl4 Primary Assembly (range, 203867771 to 203873965) for the genomic sequence which is incorporated herein by reference in its entirety. A CTLA-4 mRNA transcript can be identified as NM_001037631.3 or NM_005214.5, and the associated protein product can be identified as NP_001032720.1 or NP_005205.2, respectively, each of which are incorporated by reference herein in their entirety. The protein encoded by this gene is a member of the immunoglobulin superfamily and encodes a protein which transmits an inhibitory signal to T cells.

[0147] IL-10: An example of an interleukin 10 (IL-10; CSIF; TGIF; GVHDS; IL-10;IL10A) gene sequence can be found in the US NIH, National Library of Medicine, National Center for Biotechnology Information (NCBI) GENBANK® Database under Gene ID: 3586 (IL-10) on chromosome lq32.1, at NC_000001. l l Reference GRCh38.pl4 Primary Assembly (range, 206767602 to 206772494, complement) for the genomic sequence which is incorporated herein by reference in its entirety. An IL- 10 mRNA transcript can be identified as NM_000572.3 or NM_001382624.1, and the associated protein product can be identified as NP_000563.1 or NP_001369553.1, respectively, each of which are incorporated by reference herein in their entirety. The protein encoded by this gene is a cytokine produced primarily by monocytes and to a lesser extent by lymphocytes.

[0148] IL-35: Interleukin 35 (IL-35) is a dimeric protein composed of IL-12a and IL-27P chains, which are encoded by two separate genes called IL12A and EBI3 (Epstein-Barr virus- induced gene 3), respectively. An example of an interleukin 12A (IL12A; P35; CLMF; NFSK; NKSF1; IL-12A) gene sequence can be found in the US NIH, National Library of Medicine, National Center for Biotechnology Information (NCBI) GENBANK® Database under GeneID: 3592 (IL-12A) on chromosome 3q25.33, at NC_000003.12 Reference GRCh38.pl4 Primary Assembly (range, 159988835 to 159996019) for the genomic sequence which is incorporated herein by reference in its entirety. An IL-12A mRNA transcript can be identified as NM_000882.4, NM_001354582.2, NM_001354583.2, or NM_001397992.1, and the associated protein product can be identified as NP_000873.2, NP_001341511.1, NP_001341512.1, or NP_001384921.1, respectively, each of which are incorporated by reference herein in their entirety. The protein encoded by this gene is a subunit of a cytokine that acts on T and natural killer cells, and has a broad array of biological activities. An example of an Epstein-Barr virus induced 3 (EBB; IL27B; IL35B; IL-27B) gene sequence can be found in the US NIH, National Library of Medicine, National Center for Biotechnology Information (NCBI) GENBANK® Database under Gene ID: 10148 (EBB) on chromosome 19pl3.3, at NC_000019.10 Reference GRCh38.pl4 Primary Assembly (range, 4229523 to 4237528) for the genomic sequence which is incorporated herein by reference in its entirety. An EBB mRNA transcript can be identified as NM 005755.3, and the associated protein product can be identified as NP 005746.2, each of which are incorporated by reference herein in their entirety. The protein encoded by this gene is a secreted glycoprotein belonging to the hematopoietin receptor family.

[0149] TGF-P: An example of a transforming growth factor beta 1 (TGF-P; TGFB 1 ; CED; LAP; DPD1; TGFB; IBDIMDE; TGFbeta; TGF-betal) gene sequence can be found in the US NIH, National Library of Medicine, National Center for Biotechnology Information (NCBI) GENBANK® Database under Gene ID: 7040 (TGFB1) on chromosome 19ql3.2, at NC_000019.10 Reference GRCh38.pl4 Primary Assembly (range, 41330323 to 41353922, complement) for the genomic sequence which is incorporated herein by reference in its entirety. A TGF-P mRNA transcript can be identified as NM_000660.7, and the associated protein product can be identified as NP 000651.3, each of which are incorporated by reference herein in their entirety. The protein encoded by this gene is a secreted ligand of the TGF-beta (transforming growth factor-beta) superfamily of proteins.

[0150] TIGIT : An example of a T cell immunoreceptor with Ig and ITIM domains (TIGIT ; VSIG9; VSTM3; WUCAM) gene sequence can be found in the US NIH, National Library of Medicine, National Center for Biotechnology Information (NCBI) GENBANK® Database under Gene ID: 201633 (TIGIT) on chromosome 3ql3.31, at NC 000003.12 Reference GRCh38.pl4 Primary Assembly (range, 114294028 to 114310288) for the genomic sequence which is incorporated herein by reference in its entirety. A TIGIT mRNA transcript can be identified as NM 173799.4, and the associated protein product can be identified asNP_776160.2, each of which are incorporated by reference herein in their entirety. The protein encoded by this gene is a member of the PVR (poliovirus receptor) family of immunoglobin proteins.

[0151] PD-1: An example of a programmed cell death 1 (PD-1; PDCD1; PD1; CD279;SLEB2; hPD-1 ; hPD-1; hSLEl ; AIMTBS) gene sequence can be found in the US NIH, National Library of Medicine, National Center for Biotechnology Information (NCBI) GENBANK® Database under Gene ID: 5133 (PDCD1) on chromosome 2q37.3, atNC_000002.12 Reference GRCh38.pl4 Primary Assembly (range, 241849884 to 241858894, complement) for the genomic sequence which is incorporated herein by reference in its entirety. A PD-1 mRNA transcript can be identified as NM 005018.3, and the associated protein product can be identified as NP 005009.2, each of which are incorporated by reference herein in their entirety. The protein encoded by this gene is an immune-inhibitory receptor expressed in activated T cells; it is involved in the regulation of T-cell functions, including those of effector CD8+ T cells.

[0152] CD28: An example of a CD28 molecule (CD28; Tp44; IMD123) gene sequence can be found in the US NIH, National Library of Medicine, National Center for Biotechnology Information (NCBI) GENBANK® Database under Gene ID: 940 (CD28) on chromosome 2q33.2, at NC_000002.12 Reference GRCh38.pl4 Primary Assembly (range, 203706482 to 203738912) for the genomic sequence which is incorporated herein by reference in its entirety. A CD28 mRNA transcript can be identified as NM_001243077.2, NM_001243078.2, NM_001410981.1, or NM_006139.4, and the associated protein product can be identified as NP_001230006.1, NP_001230007.1, NP_001397910.1 , or NP_006130.1, respectively, each of which are incorporated by reference herein in their entirety. The protein encoded by this gene is essential for T-cell proliferation and survival, cytokine production, and T-helper type-2 development.A. Nucleic Acids

[0153] Aspects of the disclosure include nucleic acids. In certain aspects, nucleic acid sequences can exist in a variety of instances such as: isolated segments and recombinant vectors of incorporated sequences or recombinant polynucleotides encoding one or more chimeric interleukin receptors, one or more of co-inhibitor engagers, one or more of heterologous immunosuppressive cytokines, one or more of heterologous co-stimulatory antagonists, one or both chains of an antibody, or a fragment, derivative, mutein, or variant thereof, chimeric antigen receptor, polynucleotides sufficient for use as hybridization probes, PCR primers orsequencing primers for identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide, anti-sense nucleic acids for inhibiting expression of a polynucleotide, and complementary sequences of the foregoing described herein. The nucleic acids can be single-stranded or double-stranded and can comprise RNA and / or DNA nucleotides and artificial variants thereof (e.g., peptide nucleic acids).

[0154] The term “polynucleotide” refers to a nucleic acid molecule that either is recombinant or has been isolated from total genomic nucleic acid. Included within the term “polynucleotide” are oligonucleotides (nucleic acids 100 residues or less in length), recombinant vectors, including, for example, plasmids, cosmids, phages, viruses, and the like. Polynucleotides include, in certain aspects, regulatory sequences, isolated substantially away from their naturally occurring genes or protein encoding sequences. Polynucleotides may be single- stranded (coding or antisense) or double- stranded, and may be RNA, DNA (genomic, cDNA or synthetic), analogs thereof, or a combination thereof. Additional coding or noncoding sequences may, but need not, be present within a polynucleotide.

[0155] As will be understood by those in the art, the term “nucleic acid” or “polynucleotide” encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants. A nucleic acid encoding all or part of a polypeptide may contain a contiguous nucleic acid sequence encoding all or a portion of such a polypeptide. It also is contemplated that a particular polypeptide may be encoded by nucleic acids containing variations having slightly different nucleic acid sequences but, nonetheless, encode the same or substantially similar protein.

[0156] In certain aspects, there are polynucleotide variants having substantial identity to the sequences disclosed herein; those of, of at least, or of at most 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity, including all values and ranges there between, compared to a polynucleotide sequence provided herein using the methods described herein (e.g., BLAST analysis using standard parameters). In certain aspects, the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide of, of at least, or of at most 90%, preferably 95% and above, identity to an amino acid sequence described herein, over the entire length of the sequence; or a nucleotide sequence complementary to said isolated polynucleotide.

[0157] The nucleic acid segments, regardless of the length of the coding sequence itself, may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, andthe like, such that their overall length may vary considerably. The nucleic acids can be any length. They can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000 or more nucleotides in length, and / or can comprise one or more additional sequences, for example, regulatory sequences, and / or be a part of a larger nucleic acid, for example, a vector. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, a nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example to allow for purification of the polypeptide, transport, secretion, post-translational modification, or for therapeutic benefits such as targeting or efficacy. A tag or other heterologous polypeptide may be added to the modified polypeptide-encoding sequence, wherein “heterologous” refers to a polypeptide that is not the same as the modified polypeptide.1. Polycistronic Expression Constructs

[0158] Polycistronic expression constructs allow for the expression of a plurality of gene products from a single nucleic acid and are useful in many basic research and therapeutic applications. One beneficial feature of polycistronic expression constructs is the possibility to express two or more gene products from the same nucleic acid construct, achieving simultaneous expression of the two or more gene products in a target cell. To co-express multiple gene products from the same polycistronic unit, a polycistronic unit may comprise an internal ribosome entry site (IRES) and / or a sequence coding for at least one protease cleavage site and / or a self-cleaving peptide. a. Internal Ribosomal Entry Site (IRES)

[0159] In certain aspects of the invention, the use of one or more internal ribosome entry sites (IRES) elements is disclosed to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988; incorporated herein by reference). IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, 1991; incorporated herein by reference). IRES elements may be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronicmessages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter / enhancer to transcribe a single polycistronic message (see U.S. Pat. Nos. 5,925,565 and 5,935,819, each incorporated herein by reference).

[0160] In certain aspects, an IRES sequence comprises, consists essentially of, or consists of a polynucleotide sequence of, of at least, or of at most 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range or value derivable therein, identity to SEQ ID NO: 80.

[0161] SEQ ID NO: 80 IRES DNA:CGGGATCAATTCCGCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTG TGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGA AACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATG CAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAAC GTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCC AAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGT TGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGA TGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACAT GTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTT GAAAAAC AC GAT AAT AC C b. Self-cleaving Peptides

[0162] Self-cleaving” peptides are about 18-22 amino acids long peptides that mediate ‘ribosomal skipping’ between proline and glycine residues, thereby inhibiting peptide bond formation without affecting downstream translation. These peptides allow multiple proteins to be encoded as polyproteins, which dissociate into component proteins upon translation. Use of the term “self-cleaving” is not intended to imply a proteolytic cleavage reaction. Self-cleaving peptides are found in members of the Picomaviridae virus family, including aphthoviruses such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV), Thosea asigna virus (TaV) and porcine teschovirus-1 (PTV-1) (Donnelly, M L, et al., J. Gen. Virol., 82, 1027-101 (2001); Ryan, M D, et al., J. Gen. Virol., 72, 2727-2732 (2001); incorporated herein by reference), and cardioviruses such as Theilovirus (e.g., Theiler’s murine encephalomyelitis) and encephalomyocarditis viruses.

[0163] The 2A peptides from FMDV, ERAV, PTV-1, and TaV are sometimes referred to herein as “F2A”, “E2A”, “P2A”, and “T2A”, respectively. Aphthovirus 2A polypeptides are typically about 18-22 amino acids long and contain a DxlEx2NPG, where xl is often valine or isoleucine. As noted above, the 2A sequence is believed to mediate ‘ribosomal skipping’between the proline and glycine, impairing normal peptide bond formation between the P and G without affecting downstream translation. An exemplary 2A sequence is VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 85) from FMDV. The C terminus of cardiovirus 2A peptides is conserved, shows a high degree of similarity with FMDV 2A peptide, and has been shown to also mediate self-cleavage (Donnelly, M L, et al., J. Gen. Virol., 78, 13-21 (1997); incorporated herein by reference). FDMV 2A peptide has been shown to mediate cleavage of an artificial polyprotein (Ryan, M D and Drew, J., EMBO J., 13, 928-933 (1994); incorporated herein by reference). The ability to express four proteins efficiently and stoichiometrically from one polycistron in vivo was demonstrated using self-processing 2A peptides to express the four CD3 proteins (Szymczak et al., Nature Biotech. 5, 589-594, 2004); incorporated herein by reference). Polycistronic transgenes in which the individual cDNAs are separated by 2A peptides have been shown to promote polycistronic gene expression in transfected cells including human embryonic stem cells (Hasegawa, K., et al., Stem Cells. 2007 July; 25(7): 1707-12, 2007); incorporated herein by reference).

[0164] In certain aspects, an self-cleaving peptide is encoded by a polynucleotide that comprises, consists essentially of, or consists of a sequence of, of at least, or of at most 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range or value derivable therein, identity to any one of SEQ ID NOs: 81 or 82.

[0165] SEQ ID NO: 81 T2A DNA:GAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCC

[0166] SEQ ID NO: 82 2A TGFB DNA:GCCACAAATTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAAGAGAATCCCGGCCCT

[0167] In certain aspects, a self-cleaving peptide comprises, consists essentially of, or consists of an amino acid sequence of, of at least, or of at most 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range or value derivable therein, identity to any one of SEQ ID NOs: 83 or 84.

[0168] SEQ ID NO: 83 T2A AA:EGRGSLLTCGDVEENPGP

[0169] SEQ ID NO: 84 2A TGFB AA:ATNFSLLKQAGDVEENPGPc. Protease Cleavage Site

[0170] In certain aspects of the invention, the use of one or more protease cleavage sites are used to create multiple proteins or protein fragments from one larger protein.

[0171] As used herein, the term “furin protease cleavage site” (also referred to as “furin cleavage site” or “furin cleavage sequence”) refers to the amino acid sequence of a peptide or protein that serves as a recognition sequence for enzymatic protease cleavage by furin or furinlike proteases. Typically, a furin protease cleavage site has a consensus sequence Arg-X-X- Arg, wherein X is any amino acid. The cleavage site is positioned after the carboxy-terminal arginine (Arg) residue in the sequence. A furin cleavage site may have a consensus sequence Lys / Arg-X-X-X-Lys / Arg-Arg, wherein X is any amino acid. The cleavage site is positioned after the carboxy-terminal arginine (Arg) residue in the sequence.

[0172] As used herein, the term “furin” refers to any protease that can recognize and cleave the furin protease cleavage site as defined herein, including furin or furin-like protease. Furin is also known as paired basic amino acid cleaving enzyme (PACE). Furin belongs to the subtili sin-like proprotein convertase family. The gene encoding furin was known as FUR (FES Upstream Region).

[0173] In certain aspects, a furin cleavage site is encoded by a polynucleotide that comprises, consists essentially of, or consists of a sequence of, of at least, or of at most 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range or value derivable therein, identity to SEQ ID NO: 78.

[0174] SEQ ID NO: 78 furin-cleavage-site DNA:AGAGCCAAAAGA

[0175] In certain aspects, a furin cleavage site comprises, consists essentially of, or consists of an amino acid sequence of, of at least, or of at most 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range or value derivable therein, identity to SEQ ID NO: 79.

[0176] SEQ ID NO: 79 furin-cleavage-site AA:RAKR2. Expression

[0177] The nucleic acid molecules may be used to express large quantities of recombinant (e.g., heterologous) polypeptides. Expression systems suitable for carrying out aspects of the present disclosure are presented herein, merely as example, and should not be understood to constitute the only possible or contemplated means of expression. One skilled in the art willrecognize that a variety of different expression systems may be employed to produce the same or substantively similar results as those described herein. a. Vectors

[0178] In some aspects, contemplated are expression vectors comprising a nucleic acid molecule encoding a polypeptide of the desired sequence or a portion thereof (e.g., a fragment containing one or more CDRs or one or more variable region domains). Expression vectors comprising the nucleic acid molecules may encode one or more portions of a polypeptide, e.g., a heavy chain, light chain, or an antigen-binding portion thereof. In some aspects, expression vectors comprising nucleic acid molecules may encode fusion proteins (also referred to as chimeric proteins), modified antibodies, antibody fragments, cytokines, heterologous proteins, or combinations thereof. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well.

[0179] To express the polypeptides, DNAs encoding partial or full polypeptides are inserted into expression vectors such that the gene area is operatively linked to transcriptional and translational control sequences. Typically, expression vectors used in any of the host cells contain sequences for plasmid or virus maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to as “flanking sequences” typically include one or more of the following operatively linked nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, long-terminal repeat sequences, or a selectable marker element (also referred to as reporter). Such sequences and methods of using the same are well known in the art.

[0180] It is contemplated that an engineered construct described herein, such as a construct comprising one or more components of an immunosuppressive system, can be introduced into a cell as naked DNA or RNA, a transposon, or in a suitable vector, such as but not limited to viral vectors.

[0181] In some aspects, a viral vector (for example but not limited to a retroviral vector, adenoviral vector, adeno-associated viral vector, gammaretroviral vector, or lentiviral vector) can be used to introduce an engineered construct into cells of any kind, including at leastfibroblasts, adipocytes, myeloid cells, B-cells, T cells, immortalized cell lines, divisionincompetent cell lines, epithelial cells, mesenchymal stem cells, or any combination thereof. Suitable vectors for use in accordance with the method of the present disclosure are nonreplicating in the cell. A large number of vectors are known that are based on viruses, where the copy number of the virus maintained in the cell is low enough to maintain the viability of the cell, such as, for example, vectors based on HIV, MoMLV, MSCV, SV40, EBV, HSV, and / or BPV.

[0182] In some aspects, engineered polynucleotides described herein may be packaged in viral vectors for engineering cells of interest. In some aspects, viral vectors comprising engineered polynucleotides described herein are used to transduce fibroblasts, adipocytes, myeloid cells, B-cells, T cells, immortalized cells, division-incompetent cells, epithelial cells, mesenchymal stem cells, or a combination thereof.

[0183] In certain aspects, a vector comprises, consists essentially of, or consists of a polynucleotide sequence of, of at least, or of at most 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range or value derivable therein, identity to any one of SEQ ID NOs: 1-14, 113-121, or 191. In some aspects, one or more non-coding regions such as but not limited to an LTR, a promoter, a spacer, and / or a UTR may be removed or substituted for another sequence. In some aspects, one or more coding regions such as a linker, hinge, transmembrane domain, intracellular domain, or reporter domain (e.g., a fluorescent protein and / or tag) may be removed or substituted for another sequence.(1) Promoter

[0184] The term “promoter,” as used herein, refers to a polynucleotide sequence capable of driving transcription of a DNA sequence in a cell. Thus, promoters may include cis- and trans-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and / or rate of transcription of a gene. For example, a promoter may be a cis-acting transcriptional control element, including an enhancer, a repressor binding sequence and the like. These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on / off, regulate, modulate, etc.) gene transcription. Most often the core promoter sequences lie within 1-2 kilo-basepairs (kbp) of the translation start site, more often within 1 kbp and often within 500 basepairs (bp) or 200 bp or fewer, of the translation start site. By convention, promoter sequences are usually provided asthe sequence on the coding strand of the gene it controls. In the context of this application, a promoter is typically referred to by the name of the gene for which it naturally regulates expression. Reference to a promoter by name includes a wild type, native promoter as well as variants of the promoter that retain the ability to induce expression. Reference to a promoter by name is not restricted to a particular species, but also encompasses a promoter from a corresponding gene in other species. A large number of promoters including constitutive, inducible and repressible promoters from a variety of different sources, are well known in the art (and identified in databases such as GenBank) and are available as or within cloned polynucleotides (from, e.g., repositories such as ATCC™, and / or ADDGENE™, as well as other commercial or individual sources).

[0185] As used herein, a “constitutive promoter” refers to an unregulated promoter that allows for continual transcription of an associated gene in any suitable host cell or organism. In some aspects, a promoter may comprise a polynucleotide sequence of, of at least, or of at most 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range or value derivable therein) identity to SEQ ID NO: 104. b. Expression Systems

[0186] Numerous expression systems exist that comprise at least a part or all of the expression vectors discussed above. Prokaryote- and / or eukaryote-based systems can be employed for use with an aspect to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Commercially and widely available systems include in but are not limited to bacterial, mammalian, yeast, and insect cell systems. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. Those skilled in the art are able to express a vector to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide using an appropriate expression system. c. Methods of Gene Transfer

[0187] Suitable methods for nucleic acid delivery to effect expression of compositions are anticipated to include virtually any method by which a nucleic acid (e.g., DNA, including viral and nonviral vectors) can be introduced into a cell, a tissue or an organism, as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are notlimited to, direct delivery of DNA such as by injection (U.S. Patents 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Patent 5,789,215, incorporated herein by reference); by electroporation (U.S. Patent No. 5,384,253, incorporated herein by reference); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE dextran followed by polyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimer et al., 1987); by liposome mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); by microprojectile bombardment (PCT Application Nos. WO 94 / 09699 and 95 / 06128; U.S. Patents 5,610,042; 5,322,783, 5,563,055, 5,550,318, 5,538,877 and 5,538,880, and each incorporated herein by reference); by agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Patents 5,302,523 and 5,464,765, each incorporated herein by reference); by Agrobacterium mediated transformation (U.S. Patents 5,591,616 and 5,563,055, each incorporated herein by reference); by PEG mediated transformation (Omirulleh et al., 1993; U.S. Patents 4,684,611 and 4,952,500, each incorporated herein by reference); or by desiccation / inhibition mediated DNA uptake (Potrykus et al., 1985). Other methods include viral transduction, such as gene transfer by lentiviral or retroviral transduction.

[0188] In some aspects, a viral vector (e.g., a retroviral vector, adenoviral vector, adeno- associated viral vector, gammaretroviral vector, lentiviral vector, etc.) can be used to introduce an engineered construct into cells of any kind, including at least fibroblasts, adipocytes, myeloid cells, B-cells, T cells, immortalized cell lines, division-incompetent cell lines, epithelial cells, mesenchymal stem cells, or any combination thereof. Suitable vectors for use in accordance with the method of the present disclosure are non-replicating in the cell. A large number of vectors are known that are based on viruses, where the copy number of the virus maintained in the cell is low enough to maintain the viability of the cell, such as, for example, vectors based on HIV, MoMLV, MSCV, SV40, EBV, HSV, or BPV. In some aspects, introduction of an engineered construct into a cell results in stable integration and / or expression of the construct. d. Host Cells

[0189] In another aspect, contemplated are the use of host cells into which a recombinant expression vector has been introduced. An expression construct encoding a polypeptide can be introduced into cells according to a variety of methods known in the art. Vector DNA can beintroduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Some vectors may employ control sequences that allow it to be replicated and / or expressed in both prokaryotic and eukaryotic cells. One of skill in the art would understand the conditions under which to incubate host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.

[0190] For stable transfection of mammalian cells, it is known, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die), among other methods known in the arts.3. Isolation

[0191] The nucleic acid molecule encoding any one of the polypeptide sequences described herein may be obtained from any source that produces such polypeptides. Methods of isolating nucleic acid encoding a polypeptide are well known in the art. See e.g., Sambrook et al., supra.4. Mutation

[0192] Changes can be introduced by mutation into a nucleic acid, thereby leading to changes in the amino acid sequence of a polypeptide (e.g., an antibody or antibody derivative, chimeric antigen receptor, etc.) that it encodes. Mutations can be introduced using any technique known in the art. One or more particular amino acid residues may be changed using, for example, a site-directed mutagenesis protocol. However it is made, a mutant polypeptide can be expressed and screened for a desired property.

[0193] Mutations can be introduced into a nucleic acid without significantly altering the biological activity of a polypeptide that it encodes. For example, one can make nucleotide substitutions leading to amino acid substitutions at non-essential amino acid residues. Alternatively, one or more mutations can be introduced into a nucleic acid that selectively changes the biological activity of a polypeptide that it encodes. See, e.g., Romain Studer et al., Biochem. J. 449:581-594 (2013), incorporated herein by reference. For example, the mutation can quantitatively or qualitatively change the biological activity. Examples of quantitativechanges include increasing, reducing or eliminating the activity. Examples of qualitative changes include altering the antigen specificity of an antibody.5. Probes

[0194] In another aspect, nucleic acid molecules are suitable for use as primers or hybridization probes for the detection of nucleic acid sequences. A nucleic acid molecule can comprise only a portion of a nucleic acid sequence encoding a full-length polypeptide, for example, a fragment that can be used as a probe or primer or a fragment encoding an active portion of a given polypeptide.

[0195] Probes based on the desired sequence of a nucleic acid can be used to detect the nucleic acid or similar nucleic acids, for example, transcripts encoding a polypeptide of interest. The probe can comprise a label group, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used to identify a cell that expresses the polypeptide.B. Proteins

[0196] As used herein, a “protein” or “polypeptide” refers to a molecule comprising at least five amino acid residues. As used herein, a “peptide” refers to a molecule comprising at least three amino acid residues. As used herein, the term “wild-type” refers to the endogenous version of a molecule that occurs naturally in an organism. In some aspects, wild-type versions of a protein or polypeptide are employed. A “modified protein” or “modified polypeptide” or a “variant” refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is altered with respect to the wild-type protein or polypeptide. In some aspects, a modified / variant protein or polypeptide has at least one modified activity or function (recognizing that proteins or polypeptides may have multiple activities or functions). It is specifically contemplated that a modified / variant protein or polypeptide may be altered with respect to one activity or function yet retain a wild-type activity or function in other respects. In some aspects, a modified / variant protein or polypeptide may be a chimeric polypeptide, e.g., a combination of two or more domains of wild-type proteins.

[0197] Where a protein is specifically mentioned herein, it is in general a reference to a native (wild-type) or recombinant (modified) protein or, optionally, a protein in which any signal sequence has been removed or inserted. The protein may be isolated directly from the organism of which it is native, produced by recombinant DNA / exogenous expression methods, or produced by solid-phase peptide synthesis (SPPS) or other in vitro methods. In particularaspects, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode a polypeptide (e.g., a chimeric interleukin receptor, a co-inhibitor engager, an antibody or fragment thereof, a costimulatory antagonist, an immunosuppressive cytokine, etc.). The term “recombinant” may be used in conjunction with a polypeptide or the name of a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule.

[0198] In certain aspects the size of a protein, domain, amino acid sequence, domain sequence, or polypeptide (wild-type or modified) may comprise, but is not limited to, 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, 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, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 amino acid residues or greater, and any range derivable therein, or derivative of a corresponding amino sequence described or referenced herein. It is contemplated that polypeptides may be mutated by truncation, rendering them shorter than their corresponding wild-type form, also, they might be altered by fusing or conjugating a heterologous protein or polypeptide sequence with a particular function (e.g., for targeting or localization (e.g., a marker, such as dsRed mono), for enhanced immunogenicity, for purification purposes, etc.). As used herein, the term “domain” refers to any distinct functional or structural unit of a protein or polypeptide, and generally refers to a sequence of amino acids with a structure or function recognizable by one skilled in the art.

[0199] The polypeptides, domain, amino acid sequence, domain sequence, or proteins of the disclosure may comprise, consist essentially of, or consist of 1, 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any derivable range therein) or more variant amino acids, or be at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) similar, identical, or homologous with at least, or at most 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, 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, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218,219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237,238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256,257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275,276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294,295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313,314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332,333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351,352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370,371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389,390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408,409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 425, 450, 475, 500, 525,550, 575, 600, 625, 650, 675, 700, 725, or 750, or more contiguous amino acids, or any range or value derivable therein, of SEQ ID NOs: 47-59, 65-69, 72-73, 76-77, 79, 83-85, 89-97, 101- 103, 109-112, 140-148, 156-162, 168-172, 182-190, or 195-196, and / or of a protein product associated with CD122, CD25, CD132, CTLA-4, IL-10, IL-35, TGF-P, TIGIT, PD-1, CD28, or a combination thereof.

[0200] In some aspects, a protein, domain, amino acid sequence, domain sequence, or polypeptide may comprise, consist essentially of, or consist of amino acids 1 to 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, 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, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201,202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239,240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277,278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296,297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315,316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334,335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 351, 352, 353, 354,355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373,374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392,393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411,412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 425, 450, 475, 500, 525, 550, 575, 600,625, 650, 675, 700, 725, or 750 (or any derivable range therein) of SEQ ID NOs: 47-59, OS- 69, 72-73, 76-77, 79, 83-85, 89-97, 101-103, 109-112, 140-148, 156-162, 168-172, 182-190, or 195-196, and / or of a protein product associated with CD122, CD25, CD132, CTLA-4, IL- 10, IL-35, TGF-P, TIGIT, PD-1, CD28, or a combination thereof

[0201] In some aspects, a protein, domain, amino acid sequence, domain sequence, or polypeptide may comprise, consist essentially of, or consist of 1, 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, 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, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241,242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260,261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279,280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298,299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317,318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336,337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355,356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374,375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393,394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412,413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 425, 450, 475, 500, 525, 550, 575, 600, 625,650, 675, 700, 725, or 750 (or any derivable range therein) contiguous amino acids of SEQ IDNOs: 47-59, 65-69, 72-73, 76-77, 79, 83-85, 89-97, 101-103, 109-112, 140-148, 156-162, 168- 172, 182-190, or 195-196, and / or of a protein product associated with CD122, CD25, CD132, CTLA-4, IL-10, IL-35, TGF-P, TIGIT, PD-1, CD28, or a combination thereof

[0202] In some aspects, the polypeptide, domain, amino acid sequence, domain sequence, or protein may comprise, consist essentially of, or consist of at least, at most, or exactly 1, 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, 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, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217,218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236,237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255,256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274,275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293,294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312,313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331,332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350,351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369,370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388,389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407,408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 425, 450, 475, 500,525, 550, 575, 600, 625, 650, 675, 700, 725, or 750 (or any derivable range therein) contiguousamino acids of SEQ ID NOs: 47-59, 65-69, 72-73, 76-77, 79, 83-85, 89-97, 101-103, 109-112, 140-148, 156-162, 168-172, 182-190, or 195-196, and / or of a protein product associated with CD122, CD25, CD132, CTLA-4, IL-10, IL-35, TGF-P, TIGIT, PD-1, CD28, or a combination thereof that are at least, at most, or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) similar, identical, or homologous with one of SEQ ID NOs: 47-59, 65-69, 72-73, 76-77, 79, 83-85, 89-97, 101-103, 109-112, 140-148, 156-162, 168-172, 182-190, or 195-196, and / or of a protein product associated with CD122, CD25, CD132, CTLA-4, IL-10, IL-35, TGF-P, TIGIT, PD-1, CD28, or a combination thereof

[0203] In some aspects there is a polypeptide, domain, amino acid sequence, domain sequence, starting at position 1, 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, 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, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134,135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172,173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191,192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248,249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267,268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286,287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305,306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324,325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343,344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362,363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381,382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400,401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419,420, 421, 422, 425, 450, 475, 500, 525, 550, 575 , 600, 625, 650 675, 700, 725 , or 750 of any of SEQ ID NOs: 47-59, 65-69, 72-73, 76-77, 7 / ), 83-85, 89-97. 101-103, 109- 112, 140-148,156-162, 168-172, 182-190, or 195-196, and / or of a protein product associated with CD122, CD25, CD132, CTLA-4, IL-10, IL-35, TGF-P, TIGIT, PD-1, CD28, or a combination thereof, and comprising at least, at most, or exactly 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, 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, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189,190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246,247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265,266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284,285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303,304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322,323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341,342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360,361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379,380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398,399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417,418, 419, 420, 421, 422, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, or 750 (or any derivable range therein) contiguous amino acids of any of SEQ ID NOs: 47-59, 65-69, 72-73, 76-77, 79, 83-85, 89-97, 101-103, 109-112, 140-148, 156-162, 168-172, 182- 190, or 195-196, and / or of a protein product associated with CD122, CD25, CD132, CTLA-4, IL-10, IL-35, TGF-P, TIGIT, PD-1, CD28, or a combination thereof

[0204] In some aspects, provided herein are nucleotide as well as the protein, polypeptide, and peptide sequences for various genes. In certain aspects, wild type copies of certain proteins, polypeptides, and / or nucleotides described herein have been previously disclosed, and may be found in the recognized computerized databases. Two commonly used databases are the National Center for Biotechnology Information’s Genbank and GenPept databases (on the World Wide Web at ncbi.nlm.nih.gov / ) and The Universal Protein Resource (UniProt; on theWorld Wide Web at uniprot.org). The coding regions for these genes may be amplified and / or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art.

[0205] It is contemplated that in compositions of the disclosure, there is between about 0.001 mg and about 10 mg of total polypeptide, peptide, and / or protein per mL. The concentration of protein in a composition can be about, at least about or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / mL or more (or any range or value derivable therein).1. Variant Polypeptides

[0206] The following is a discussion of changing the amino acid subunits of a protein to create an equivalent, or even improved, second-generation variant polypeptide or peptide. For example, certain amino acids may be substituted for other amino acids in a protein or polypeptide sequence with or without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein’s functional activity, certain amino acid substitutions can be made in a protein sequence and in its corresponding DNA coding sequence, and nevertheless produce a protein with similar or desirable properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes which encode proteins without appreciable loss of their biological utility or activity.

[0207] The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six different codons for arginine. Also considered are “neutral substitutions” or “neutral mutations” which refers to a change in the codon or codons that encode biologically equivalent amino acids.

[0208] Amino acid sequence variants of the disclosure can be substitutional, insertional, or deletion variants. A variation in a polypeptide of the disclosure may affect 1, 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more non-contiguous or contiguous amino acids of the protein or polypeptide, as compared to a wild-type protein or a protein sequence provided herein. A variant can comprise an amino acid sequence that is at least 50%, 60%, 70%, 80%, or 90%, including all values and ranges there between, identicalto any sequence provided or referenced herein. A variant can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more substitute amino acids.

[0209] It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and yet still be essentially identical as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region.

[0210] Deletion variants typically lack one or more residues of the native, wild type, or reference protein. Individual residues can be deleted or a number of contiguous amino acids can be deleted. A stop codon may be introduced (by substitution or insertion) into an encoding nucleic acid sequence to generate a truncated protein.

[0211] Insertional mutants typically involve the addition of amino acid residues at a nonterminal point in the polypeptide. This may include the insertion of one or more amino acid residues. Terminal additions may also be generated and can include fusion proteins which are multimers or concatemers of one or more peptides or polypeptides described or referenced herein.

[0212] Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein or polypeptide, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar chemical properties. “Conservative amino acid substitutions” may involve exchange of a member of one amino acid class with another member of the same class. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics or other reversed or inverted forms of amino acid moieties.

[0213] Alternatively, substitutions may be “non-conservative”, such that a function or activity of the polypeptide is affected. Non-conservative changes typically involve substituting an amino acid residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa. Non-conservative substitutions may involve the exchange of a member of one of the amino acid classes for a member from another class.2. Considerations for Substitutions

[0214] One skilled in the art can determine suitable variants of polypeptides as set forth herein using well-known techniques. One skilled in the art may identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not believed to be important for activity. The skilled artisan will also be able to identify amino acid residues and portions of the molecules that are conserved among similar proteins or polypeptides. In further aspects, areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without significantly altering the biological activity or without adversely affecting the protein or polypeptide structure.

[0215] In making such changes, the hydropathy index of amino acids may be considered. The hydropathy profile of a protein is calculated by assigning each amino acid a numerical value (“hydropathy index”) and then repetitively averaging these values along the peptide chain. Each amino acid has been assigned a value based on its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (—0.4); threonine (—0.7); serine (—0.8); tryptophan (-0.9); tyrosine (-1.3); proline (1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The importance of the hydropathy amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte et al., J. Mol. Biol. 157: 105-131 (1982)). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein or polypeptide, which in turn defines the interaction of the protein or polypeptide with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and others. It is also known that certain amino acids may be substituted for other amino acids having a similar hydropathy index or score, and still retain a similar biological activity. In making changes based upon the hydropathy index, in certain aspects, the substitution of amino acids whose hydropathy indices are within ±2 is included. Insome aspects of the present disclosure, those that are within ±1 are included, and in other aspects of the present disclosure, those within ±0.5 are included.

[0216] It also is understood in the art that the substitution of like amino acids can be effectively made based on hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. In certain aspects, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigen binding, that is, as a biological property of the protein. The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0+1); glutamate (+3.0+1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (—0.4); proline (-0.5+1); alanine (—0.5); histidine (—0.5); cysteine (—1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). In making changes based upon similar hydrophilicity values, in certain aspects, the substitution of amino acids whose hydrophilicity values are within ±2 are included, in other aspects, those which are within ±1 are included, and in still other aspects, those within ±0.5 are included. In some instances, one may also identify epitopes from primary amino acid sequences based on hydrophilicity. These regions are also referred to as “epitopic core regions.” It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein.

[0217] Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides or proteins that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues important for activity or structure in similar proteins. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues.

[0218] One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar proteins or polypeptides. In view of such information, one skilled in the art may predict the alignment of amino acid residues of an antibody with respect to its three-dimensional structure. One skilled in the art may choose not to make changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desiredamino acid residue. These variants can then be screened using standard assays for binding and / or activity, thus yielding information gathered from such routine experiments, which may allow one skilled in the art to determine the amino acid positions where further substitutions should be avoided either alone or in combination with other mutations. Various tools available to determine secondary structure can be found on the world wide web at expasy . org / proteomi cs / protein_structure .

[0219] In some aspects of the disclosure, amino acid substitutions are made that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter ligand or antigen binding affinities, and / or (5) confer or modify other physicochemical or functional properties on such polypeptides. For example, single or multiple amino acid substitutions (in certain aspects, conservative amino acid substitutions) may be made in the naturally occurring sequence. Substitutions can be made in that portion of the antibody that lies outside the domain(s) forming intermolecular contacts. In such aspects, conservative amino acid substitutions can be used that do not substantially change the structural characteristics of the protein or polypeptide (e.g., one or more replacement amino acids that do not disrupt the secondary structure that characterizes the native antibody).

[0220] In some aspects, one or more polypeptides of the present disclosure may be encoded by a polynucleotide that comprises a sequence of, of at least, or of at most 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range or value derivable therein) identity to any one of SEQ ID NOs: 32-46, 113-139, 149-155, 163-167, 173- 181, or 191-194.

[0221] SEQ ID NO: 32 CTLA4-P2A-dNGFR DNA:ATGGCTTGCCTTGGATTTCAGCGGCACAAGGCTCAGCTGAACCTGGCTACCAGGACCTGGCC CTGCACTCTCCTGTTTTTTCTTCTCTTCATCCCTGTCTTCTGCAAAGCAATGCACGTGGCCC AGCCTGCTGTGGTACTGGCCAGCAGCCGAGGCATCGCCAGCTTTGTGTGTGAGTATGCATCT CCAGGCAAAGCCACTGAGGTCCGGGTGACAGTGCTTCGGCAGGCTGACAGCCAGGTGACTGA AGTCTGTGCGGCAACCTACATGATGGGGAATGAGTTGACCTTCCTAGATGATTCCATCTGCA CGGGCACCTCCAGTGGAAATCAAGTGAACCTCACTATCCAAGGACTGAGGGCCATGGACACG GGACTCTACATCTGCAAGGTGGAGCTCATGTACCCACCGCCATACTACCTGGGCATAGGCAA CGGAACCCAGATTTATGTAATTGATCCAGAACCGTGCCCAGATTCTGACTTCCTCCTCTGGA TCCTTGCAGCAGTTAGTTCGGGGTTGTTTTTTTATAGCTTTCTCCTCACAGCTGTTTCTTTG AGCAAAATGCTAAAGAAAAGAAGCCCTCTTACAACAGGGGTCTATGTGAAAATGCCCCCAAC AGAGCCAGAATGTGAAAAGCAATTTCAGCCTTATTTTATTCCCATCAATctcgagGGTAGTG GGGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCATGGGG GCAGGTGCCACCGGCCGCGCCATGGACGGGCCGCGCCTGCTGCTGTTGCTGCTTCTGGGGGT GTCCCTTGGAGGTGCCAAGGAGGCATGCCCCACAGGCCTGTACACACACAGCGGTGAGTGCT GCAAAGCCTGCAACCTGGGCGAGGGTGTGGCCCAGCCTTGTGGAGCCAACCAGACCGTGTGT GAGCCCTGCCTGGACAGCGTGACGTTCTCCGACGTGGTGAGCGCGACCGAGCCGTGCAAGCCGTGCACCGAGTGCGTGGGGCTCCAGAGCATGTCGGCGCCGTGCGTGGAGGCCGACGACGCCG TGTGCCGCTGCGCCTACGGCTACTACCAGGATGAGACGACTGGGCGCTGCGAGGCGTGCCGC GTGTGCGAGGCGGGCTCGGGCCTCGTGTTCTCCTGCCAGGACAAGCAGAACACCGTGTGCGA GGAGTGCCCCGACGGCACGTATTCCGACGAGGCCAACCACGTGGACCCGTGCCTGCCCTGCA CCGTGTGCGAGGACACCGAGCGCCAGCTCCGCGAGTGCACACGCTGGGCCGACGCCGAGTGC GAGGAGATCCCTGGCCGTTGGATTACACGGTCCACACCCCCAGAGGGCTCGGACAGCACAGC CCCCAGCACCCAGGAGCCTGAGGCACCTCCAGAACAAGACCTCATAGCCAGCACGGTGGCAG GTGTGGTGACCACAGTGATGGGCAGCTCCCAGCCCGTGGTGACCCGAGGCACCACCGACAAC CTCATCCCTGTCTATTGCTCCATCCTGGCTGCTGTGGTTGTGGGCCTTGTGGCCTACATAGC C T T CAAGAGGT GGAACAGC

[0222] SEQ ID NO: 33 CTLA4- DNA:ATGGCTTGCCTTGGATTTCAGCGGCACAAGGCTCAGCTGAACCTGGCTACCAGGACCTGGCC CTGCACTCTCCTGTTTTTTCTTCTCTTCATCCCTGTCTTCTGCAAAGCAATGCACGTGGCCC AGCCTGCTGTGGTACTGGCCAGCAGCCGAGGCATCGCCAGCTTTGTGTGTGAGTATGCATCT CCAGGCAAAGCCACTGAGGTCCGGGTGACAGTGCTTCGGCAGGCTGACAGCCAGGTGACTGA AGTCTGTGCGGCAACCTACATGATGGGGAATGAGTTGACCTTCCTAGATGATTCCATCTGCA CGGGCACCTCCAGTGGAAATCAAGTGAACCTCACTATCCAAGGACTGAGGGCCATGGACACG GGACTCTACATCTGCAAGGTGGAGCTCATGTACCCACCGCCATACTACCTGGGCATAGGCAA CGGAACCCAGATTTATGTAATTGATCCAGAACCGTGCCCAGATTCTGACTTCCTCCTCTGGA TCCTTGCAGCAGTTAGTTCGGGGTTGTTTTTTTATAGCTTTCTCCTCACAGCTGTTTCTTTG AGCAAAATGCTAAAGAAAAGAAGCCCTCTTACAACAGGGGTCTATGTGAAAATGCCCCCAAC AGAG C C AGAAT G T GAAAAG C AAT T T GAG CCTTATTTTATTCCCAT C AAT

[0223] SEQ ID NO: 34 dNGFR-IRES-ILlO DNA:ATGGGGGCAGGTGCCACCGGCCGCGCCATGGACGGGCCGCGCCTGCTGCTGTTGCTGCTTCT GGGGGTGTCCCTTGGAGGTGCCAAGGAGGCATGCCCCACAGGCCTGTACACACACAGCGGTG AGTGCTGCAAAGCCTGCAACCTGGGCGAGGGTGTGGCCCAGCCTTGTGGAGCCAACCAGACC GTGTGTGAGCCCTGCCTGGACAGCGTGACGTTCTCCGACGTGGTGAGCGCGACCGAGCCGTG CAAGCCGTGCACCGAGTGCGTGGGGCTCCAGAGCATGTCGGCGCCGTGCGTGGAGGCCGACG ACGCCGTGTGCCGCTGCGCCTACGGCTACTACCAGGATGAGACGACTGGGCGCTGCGAGGCG TGCCGCGTGTGCGAGGCGGGCTCGGGCCTCGTGTTCTCCTGCCAGGACAAGCAGAACACCGT GTGCGAGGAGTGCCCCGACGGCACGTATTCCGACGAGGCCAACCACGTGGACCCGTGCCTGC CCTGCACCGTGTGCGAGGACACCGAGCGCCAGCTCCGCGAGTGCACACGCTGGGCCGACGCC GAGTGCGAGGAGATCCCTGGCCGTTGGATTACACGGTCCACACCCCCAGAGGGCTCGGACAG CACAGCCCCCAGCACCCAGGAGCCTGAGGCACCTCCAGAACAAGACCTCATAGCCAGCACGG TGGCAGGTGTGGTGACCACAGTGATGGGCAGCTCCCAGCCCGTGGTGACCCGAGGCACCACC GACAACCTCATCCCTGTCTATTGCTCCATCCTGGCTGCTGTGGTTGTGGGCCTTGTGGCCTA CATAGCCTTCAAGAGGTGGAACAGCCGGGATCAATTCCGCCCCCCCCCTAACGTTACTGGCC GAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCG TCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGG TCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTC TGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCA CCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGC ACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAA GCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTG GGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCG AACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATAATACCATGCACAGCTCAGCACTGC TCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCAGCCCAGGCCAGGGCACCCAGTCTGAG AACAGCTGCACCCACTTCCCAGGCAACCTGCCTAACATGCTTCGAGATCTCCGAGATGCCTTCAGCAGAGT GAAGAC T T T C T T T CAAAT GAAGGAT CAGC T GGACAAC T T GT T GT TAAAGGAGT CCTTGCTGGAGGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCTGAGATGATCCAGTTT TACCTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAGACATCAAGGCGCATGTGAA CTCCCTGGGGGAGAACCTGAAGACCCTCAGGCTGAGGCTACGGCGCTGTCATCGATTTCTTC CCTGTGAAAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAATGCCTTTAATAAGCTCCAAGAG AAAGGCAT C T ACAAAGC CAT GAG T GAG T T T GACAT C T T CAT CAAC T ACAT AGAAGC C T ACAT GAC AAT GAAGAT AC GAAAC

[0224] SEQ ID NO: 35 IRES-IL10 DNA:CGGGATCAATTCCGCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTG TGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGA AACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATG CAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAAC GTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCC AAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGT TGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGA TGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACAT GTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTT GAAAAACACGATAATACCATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGG GGTGAGGGCCAGCCCAGGCCAGGGCACCCAGTCTGAGAACAGCTGCACCCACTTCCCAGGCA ACCTGCCTAACATGCTTCGAGATCTCCGAGATGCCTTCAGCAGAGTGAAGACTTTCTTTCAA ATGAAGGATCAGCTGGACAACTTGTTGTTAAAGGAGTCCTTGCTGGAGGACTTTAAGGGTTA CCTGGGTTGCCAAGCCTTGTCTGAGATGATCCAGTTTTACCTGGAGGAGGTGATGCCCCAAG CTGAGAACCAAGACCCAGACATCAAGGCGCATGTGAACTCCCTGGGGGAGAACCTGAAGACC CTCAGGCTGAGGCTACGGCGCTGTCATCGATTTCTTCCCTGTGAAAACAAGAGCAAGGCCGT GGAGCAGGTGAAGAATGCCTTTAATAAGCTCCAAGAGAAAGGCATCTACAAAGCCATGAGTG AG T T T GACAT C T T C AT CAAC T AC AT AGAAG C C TAG AT GAC AAT GAAGAT AC GAAAC

[0225] SEQ ID NO: 36 IL10 DNA:ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCAGCCCAGG CCAGGGCACCCAGTCTGAGAACAGCTGCACCCACTTCCCAGGCAACCTGCCTAACATGCTTC GAGAT C T CCGAGAT GCC T T CAG C AG AG T GAAGAC T T T C T T T CAAAT GAAGGAT CAGC T GGAC AACTTGTTGTTAAAGGAGTCCTTGCTGGAGGACTTTAAGGGTTACCTGGGTTGCCAAGCCTT GTCTGAGATGATCCAGTTTTACCTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAG ACATCAAGGCGCATGTGAACTCCCTGGGGGAGAACCTGAAGACCCTCAGGCTGAGGCTACGG CGCTGTCATCGATTTCTTCCCTGTGAAAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAATGC C T T T AAT AAGC T C CAAGAGAAAGGCAT C T ACAAAGC CAT GAG T GAG T T T GACAT C T T CAT CA AC TAG AT AGAAG C C TAG AT GAC AAT GAAGAT AC GAAAC

[0226] SEQ ID NO: 37 signal-peptide ab-gamma DNA:ATGGATTCATACCTGCTGATGTGGGGACTGCTCACGTTCATCATGGTGCCTGGCTGCCAGGC AGAGCTCTGTGACGATGACCCGCCAGAGATCCCACACGCCACATTCAAAGCCATGGCCTACA AGGAAGGAACCATGTTGAACTGTGAATGCAAGAGAGGTTTCCGCAGAATAAAAAGCGGGTCA CTCTATATGCTCTGTACAGGAAACTCTAGCCACTCGTCCTGGGACAACCAATGTCAATGCAC AAG C T C T G C C AC T C G GAAGAC AAC GAAAC AAG T GAC AC C T CAAC C T GAAGAAC AGAAAGAAA GGAAAACCACAGAAATGCAAAGTCCAATGCAGCCAGTGGACCAAGCGAGCCTTCCAGGTCAC TGCAGGGAACCTCCACCATGGGAAAATGAAGCCACAGAGAGAATTTATCATTTCGTGGTGGG GCAGATGGTTTATTATCAGTGCGTCCAGGGATACAGGGCTCTACACAGAGGTCCTGCTGAGA GCGTCTGCAAAATGACCCACGGGAAGACAAGGTGGACCCAGCCCCAGCTCATATGCACAGGT GAAATGGAGACCAGTCAGTTTCCAGGTGAAGAGAAGCCTCAGGCAAGCCCCGAAGGCCGTCCT GAGAG T GAGAC TTCCTGCCTCGT C AC AAC AAC AGAT T T T C AAAT AC AGAC AGAAAT G G C T G CAACCATGGAGACGTCCATATTTACAACAGAGTACCAGGGTGGCGGTGGCTCGGGCGGTGGT GGGTCGGGTGGCGGCGGATCTGGTGGCGGCGGATCTGCGGTGAATGGCACTTCCCAGTTCAC ATGCTTCTACAACTCGAGAGCCAACATCTCCTGTGTCTGGAGCCAAGATGGGGCTCTGCAGG ACACTTCCTGCCAAGTCCATGCCTGGCCGGACAGACGGCGGTGGAACCAAACCTGTGAGCTG CTCCCCGTGAGTCAAGCATCCTGGGCCTGCAACCTGATCCTCGGAGCCCCAGATTCTCAGAA ACTGACCACAGTTGACATCGTCACCCTGAGGGTGCTGTGCCGTGAGGGGGTGCGATGGAGGG TGATGGCCATCCAGGACTTCAAGCCCTTTGAGAACCTTCGCCTGATGGCCCCCATCTCCCTC CAAGTTGTCCACGTGGAGACCCACAGATGCAACATAAGCTGGGAAATCTCCCAAGCCTCCCA CTACTTTGAAAGACACCTGGAGTTCGAGGCCCGGACGCTGTCCCCAGGCCACACCTGGGAGG AGGCCCCCCTGCTGACTCTCAAGCAGAAGCAGGAATGGATCTGCCTGGAGACGCTCACCCCA GACACCCAGTATGAGTTTCAGGTGCGGGTCAAGCCTCTGCAAGGCGAGTTCACGACCTGGAG CCCCTGGAGCCAGCCCCTGGCCTTCAGGACAAAGCCTGCAGCCCTTGGGAAGGACACCGGCG GCGGGGGCAGTGGAGGAGGCGGAAGCGGTGGGGGGGGATCGCTGAACACGACAATTCTGACG CCCAATGGGAATGAAGACACCACAGCTGATTTCTTCCTGACCACTATGCCCACTGACTCCCT CAGTGTTTCCACTCTGCCCCTCCCAGAGGTTCAGTGTTTTGTGTTCAATGTCGAGTACATGA ATTGCACTTGGAACAGCAGCTCTGAGCCCCAGCCTACCAACCTCACTCTGCATTATTGGTAC AAGAAC T C G GAT AAT GAT AAAG T C C AGAAG T G C AG C C AC TATCTATTCTCT GAAGAAAT C AC TTCTGGCTGTCAGTTGCAAAAAAAGGAGATCCACCTCTACCAAACATTTGTTGTTCAGCTCC AGGACCCACGGGAACCCAGGAGACAGGCCACACAGATGCTAAAACTGCAGAATCTGGTGATC CCCTGGGCTC C AGAGAAC C T AAC AC T T C AC AAAC T GAG T GAAT C C C AG C T AGAAC T GAAC T G GAACAACAGATTCTTGAACCACTGTTTGGAGCACTTGGTGCAGTACCGGACTGACTGGGACC ACAGCTGGACTGAACAATCAGTGGATTATAGACATAAGTTCTCCTTGCCTAGTGTGGATGGG CAGAAACGCTACACGTTTCGTGTTCGGAGCCGCTTTAACCCACTCTGTGGAAGTGCTCAGCA TTGGAGTGAATGGAGCCACCCAATCCACTGGGGGAGCAATACTTCAAAAGAGAATCCTTTCC TGTTTGCATTGGAAGCCGTGGTTATCTCTGTTGGCTCCATGGGATTGATTATCAGCCTTCTC TGTGTGTATTTCTGGCTGGAACGGACGATGCCCCGAATTCCCACCCTGAAGAACCTAGAGGA TCTTGTTACTGAATACCACGGGAACTTTTCGGCCTGGAGTGGTGTGTCTAAGGGACTGGCTG AGAGTCTGCAGCCAGACTACAGTGAACGACTCTGCCTCGTCAGTGAGATTCCCCCAAAAGGA GGGGCCCTTGGGGAGGGGCCTGGGGCCTCCCCATGCAACCAGCATAGCCCCTACTGGGCCCC C C C AT G T T AC AC C C T AAAG C C T GAAAC C

[0227] SEQ ID NO: 38 ab-gamma DNA:GAGCTCTGTGACGATGACCCGCCAGAGATCCCACACGCCACATTCAAAGCCATGGCCTACAA GGAAGGAACCATGTTGAACTGTGAATGCAAGAGAGGTTTCCGCAGAATAAAAAGCGGGTCAC TCTATATGCTCTGTACAGGAAACTCTAGCCACTCGTCCTGGGACAACCAATGTCAATGCACA AG C T C T G C C AC T C G GAAC AC AAC GAAAC AAG T GAC AC C T C AAC C T GAAGAAC AGAAAGAAAG GAAAACCACAGAAATGCAAAGTCCAATGCAGCCAGTGGACCAAGCGAGCCTTCCAGGTCACT GCAGGGAACCTCCACCATGGGAAAATGAAGCCACAGAGAGAATTTATCATTTCGTGGTGGGG CAGATGGTTTATTATCAGTGCGTCCAGGGATACAGGGCTCTACACAGAGGTCCTGCTGAGAG CGTCTGCAAAATGACCCACGGGAAGACAAGGTGGACCCAGCCCCAGCTCATATGCACAGGTG AAATGGAGACCAGTCAGTTTCCAGGTGAAGAGAAGCCTCAGGCAAGCCCCGAAGGCCGTCCT GAGAG T GAGAC TTCCTGCCTCGT C AC AAC AAC AGAT T T T C AAAT AC AGAC AGAAAT G G C T G C AACCATGGAGACGTCCATATTTACAACAGAGTACCAGGGTGGCGGTGGCTCGGGCGGTGGTG GGTCGGGTGGCGGCGGATCTGGTGGCGGCGGATCTGCGGTGAATGGCACTTCCCAGTTCACA TGCTTCTACAACTCGAGAGCCAACATCTCCTGTGTCTGGAGCCAAGATGGGGCTCTGCAGGA CACTTCCTGCCAAGTCCATGCCTGGCCGGACAGACGGCGGTGGAACCAAACCTGTGAGCTGC TCCCCGTGAGTCAAGCATCCTGGGCCTGCAACCTGATCCTCGGAGCCCCAGATTCTCAGAAA CTGACCACAGTTGACATCGTCACCCTGAGGGTGCTGTGCCGTGAGGGGGTGCGATGGAGGGT GATGGCCATCCAGGACTTCAAGCCCTTTGAGAACCTTCGCCTGATGGCCCCCATCTCCCTCCAAGTTGTCCACGTGGAGACCCACAGATGCAACATAAGCTGGGAAATCTCCCAAGCCTCCCAC TACTTTGAAAGACACCTGGAGTTCGAGGCCCGGACGCTGTCCCCAGGCCACACCTGGGAGGA GGCCCCCCTGCTGACTCTCAAGCAGAAGCAGGAATGGATCTGCCTGGAGACGCTCACCCCAG ACACCCAGTATGAGTTTCAGGTGCGGGTCAAGCCTCTGCAAGGCGAGTTCACGACCTGGAGC CCCTGGAGCCAGCCCCTGGCCTTCAGGACAAAGCCTGCAGCCCTTGGGAAGGACACCGGCGG CGGGGGCAGTGGAGGAGGCGGAAGCGGTGGGGGGGGATCGCTGAACACGACAATTCTGACGC CCAATGGGAATGAAGACACCACAGCTGATTTCTTCCTGACCACTATGCCCACTGACTCCCTC AGTGTTTCCACTCTGCCCCTCCCAGAGGTTCAGTGTTTTGTGTTCAATGTCGAGTACATGAA TTGCACTTGGAACAGCAGCTCTGAGCCCCAGCCTACCAACCTCACTCTGCATTATTGGTACA AGAAC T C G GAT AAT GAT AAAG T C C AGAAG T G GAG C GAG TATCTATTCTCT GAAGAAAT GAG T TCTGGCTGTCAGTTGCAAAAAAAGGAGATCCACCTCTACCAAACATTTGTTGTTCAGCTCCA GGACCCACGGGAACCCAGGAGACAGGCCACACAGATGCTAAAACTGCAGAATCTGGTGATCC CCTGGGCTCCAGAGAACCTAACACTTCACAAACTGAGTGAATCCCAGCTAGAACTGAACTGG AACAACAGATTCTTGAACCACTGTTTGGAGCACTTGGTGCAGTACCGGACTGACTGGGACCA CAGCTGGACTGAACAATCAGTGGATTATAGACATAAGTTCTCCTTGCCTAGTGTGGATGGGC AGAAACGCTACACGTTTCGTGTTCGGAGCCGCTTTAACCCACTCTGTGGAAGTGCTCAGCAT TGGAGTGAATGGAGCCACCCAATCCACTGGGGGAGCAATACTTCAAAAGAGAATCCTTTCCT GTTTGCATTGGAAGCCGTGGTTATCTCTGTTGGCTCCATGGGATTGATTATCAGCCTTCTCT GTGTGTATTTCTGGCTGGAACGGACGATGCCCCGAATTCCCACCCTGAAGAACCTAGAGGAT CTTGTTACTGAATACCACGGGAACTTTTCGGCCTGGAGTGGTGTGTCTAAGGGACTGGCTGA GAGTCTGCAGCCAGACTACAGTGAACGACTCTGCCTCGTCAGTGAGATTCCCCCAAAAGGAG GGGCCCTTGGGGAGGGGCCTGGGGCCTCCCCATGCAACCAGCATAGCCCCTACTGGGCCCCC C C AT G T T AC AC C C T AAAG C C T GAAAC C

[0228] SEQ ID NO: 39 signal-peptide ba-gamma DNA:ATGGCGGCCCCTGCTCTGTCCTGGCGTCTGCCCCTCCTCATCCTCCTCCTGCCCCTGGCTAC CTCTTGGGCATCTGCAGCGGTGAATGGCACTTCCCAGTTCACATGCTTCTACAACTCGAGAG CCAACATCTCCTGTGTCTGGAGCCAAGATGGGGCTCTGCAGGACACTTCCTGCCAAGTCCAT GCCTGGCCGGACAGACGGCGGTGGAACCAAACCTGTGAGCTGCTCCCCGTGAGTCAAGCATC CTGGGCCTGCAACCTGATCCTCGGAGCCCCAGATTCTCAGAAACTGACCACAGTTGACATCG TCACCCTGAGGGTGCTGTGCCGTGAGGGGGTGCGATGGAGGGTGATGGCCATCCAGGACTTC AAGCCCTTTGAGAACCTTCGCCTGATGGCCCCCATCTCCCTCCAAGTTGTCCACGTGGAGAC CCACAGATGCAACATAAGCTGGGAAATCTCCCAAGCCTCCCACTACTTTGAAAGACACCTGG AGTTCGAGGCCCGGACGCTGTCCCCAGGCCACACCTGGGAGGAGGCCCCCCTGCTGACTCTC AAGCAGAAGCAGGAATGGATCTGCCTGGAGACGCTCACCCCAGACACCCAGTATGAGTTTCA GGTGCGGGTCAAGCCTCTGCAAGGCGAGTTCACGACCTGGAGCCCCTGGAGCCAGCCCCTGG CCTTCAGGACAAAGCCTGCAGCCCTTGGGAAGGACACCGGTGGCGGTGGCTCGGGCGGTGGT GGGTCGGGTGGCGGCGGATCTGGTGGCGGCGGATCTGAGCTCTGTGACGATGACCCGCCAGA GATCCCACACGCCACATTCAAAGCCATGGCCTACAAGGAAGGAACCATGTTGAACTGTGAAT GCAAGAGAGGTTTCCGCAGAATAAAAAGCGGGTCACTCTATATGCTCTGTACAGGAAACTCT AGCCACTCGTCCTGGGACAACCAATGTCAATGCACAAGCTCTGCCACTCGGAACACAACGAA AC AAG T GAC AC C T C AAC C T GAAGAAC AGAAAGAAAG GAAAAC C AC AGAAAT G C AAAG T C C AA TGCAGCCAGTGGACCAAGCGAGCCTTCCAGGTCACTGCAGGGAACCTCCACCATGGGAAAAT GAAGCCACAGAGAGAATTTATCATTTCGTGGTGGGGCAGATGGTTTATTATCAGTGCGTCCA GGGATACAGGGCTCTACACAGAGGTCCTGCTGAGAGCGTCTGCAAAATGACCCACGGGAAGA CAAGGTGGACCCAGCCCCAGCTCATATGCACAGGTGAAATGGAGACCAGTCAGTTTCCAGGT GAAGAGAAGCCTCAGGCAAGCCCCGAAGGCCGTCCTGAGAGTGAGACTTCCTGCCTCGTCAC AACAACAGAT T T T CAAAT ACAGACAGAAAT GGC T GCAAC CAT GGAGAC G T C CAT AT T T ACAA CAGAGTACCAGGGCGGCGGGGGCAGTGGAGGAGGCGGAAGCGGTGGGGGGGGATCGCTGAAC AC GAC AAT T C T GAC G C C C AAT G G GAAT GAAGAC AC C AC AG CTGATTTCTTCCT GAC C AC TATGCCCACTGACTCCCTCAGTGTTTCCACTCTGCCCCTCCCAGAGGTTCAGTGTTTTGTGTTCA ATGTCGAGTACATGAATTGCACTTGGAACAGCAGCTCTGAGCCCCAGCCTACCAACCTCACT CTGCATTATTGGTACAAGAACTCGGATAATGATAAAGTCCAGAAGTGCAGCCACTATCTATT C T C T GAAGAAAT C AC T T C T G G C T G T C AG T T G C AAAAAAAG GAGAT C C AC C T C T AC C AAAC AT TTGTTGTTCAGCTCCAGGACCCACGGGAACCCAGGAGACAGGCCACACAGATGCTAAAACTG CAGAATCTGGTGATCCCCTGGGCTCCAGAGAACCTAACACTTCACAAACTGAGTGAATCCCA GCTAGAACTGAACTGGAACAACAGATTCTTGAACCACTGTTTGGAGCACTTGGTGCAGTACC GGAC T GAC T GGGACCACAGC T GGAC T GAACAAT CAGT GGAT TATAGACATAAGT T C T CC T T G CCTAGTGTGGATGGGCAGAAACGCTACACGTTTCGTGTTCGGAGCCGCTTTAACCCACTCTG TGGAAGTGCTCAGCATTGGAGTGAATGGAGCCACCCAATCCACTGGGGGAGCAATACTTCAA AAGAGAATCCTTTCCTGTTTGCATTGGAAGCCGTGGTTATCTCTGTTGGCTCCATGGGATTG ATTATCAGCCTTCTCTGTGTGTATTTCTGGCTGGAACGGACGATGCCCCGAATTCCCACCCT GAAGAACCTAGAGGATCTTGTTACTGAATACCACGGGAACTTTTCGGCCTGGAGTGGTGTGT CTAAGGGACTGGCTGAGAGTCTGCAGCCAGACTACAGTGAACGACTCTGCCTCGTCAGTGAG ATTCCCCCAAAAGGAGGGGCCCTTGGGGAGGGGCCTGGGGCCTCCCCATGCAACCAGCATAG CCCCTACTGGGCCCCCCCATGTTACACCCTAAAGCCTGAAACC

[0229] SEQ ID NO: 40 ba-gamma DNA:GCGGTGAATGGCACTTCCCAGTTCACATGCTTCTACAACTCGAGAGCCAACATCTCCTGTGT CTGGAGCCAAGATGGGGCTCTGCAGGACACTTCCTGCCAAGTCCATGCCTGGCCGGACAGAC GGCGGTGGAACCAAACCTGTGAGCTGCTCCCCGTGAGTCAAGCATCCTGGGCCTGCAACCTG ATCCTCGGAGCCCCAGATTCTCAGAAACTGACCACAGTTGACATCGTCACCCTGAGGGTGCT GTGCCGTGAGGGGGTGCGATGGAGGGTGATGGCCATCCAGGACTTCAAGCCCTTTGAGAACC TTCGCCTGATGGCCCCCATCTCCCTCCAAGTTGTCCACGTGGAGACCCACAGATGCAACATA AGCTGGGAAATCTCCCAAGCCTCCCACTACTTTGAAAGACACCTGGAGTTCGAGGCCCGGAC GCTGTCCCCAGGCCACACCTGGGAGGAGGCCCCCCTGCTGACTCTCAAGCAGAAGCAGGAAT GGATCTGCCTGGAGACGCTCACCCCAGACACCCAGTATGAGTTTCAGGTGCGGGTCAAGCCT CTGCAAGGCGAGTTCACGACCTGGAGCCCCTGGAGCCAGCCCCTGGCCTTCAGGACAAAGCC TGCAGCCCTTGGGAAGGACACCGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCG GATCTGGTGGCGGCGGATCTGAGCTCTGTGACGATGACCCGCCAGAGATCCCACACGCCACA TTCAAAGCCATGGCCTACAAGGAAGGAACCATGTTGAACTGTGAATGCAAGAGAGGTTTCCG CAGAATAAAAAGCGGGTCACTCTATATGCTCTGTACAGGAAACTCTAGCCACTCGTCCTGGG AC AAC C AAT G T C AAT G C AC AAG C T C T G C C AC T C G GAAC AC AAC GAAAC AAG T GAC AC C T C AA CCTGAAGAACAGAAAGAAAGGAAAACCACAGAAATGCAAAGTCCAATGCAGCCAGTGGACCA AGCGAGCCTTCCAGGTCACTGCAGGGAACCTCCACCATGGGAAAATGAAGCCACAGAGAGAA TTTATCATTTCGTGGTGGGGCAGATGGTTTATTATCAGTGCGTCCAGGGATACAGGGCTCTA CACAGAGGTCCTGCTGAGAGCGTCTGCAAAATGACCCACGGGAAGACAAGGTGGACCCAGCC CCAGCTCATATGCACAGGTGAAATGGAGACCAGTCAGTTTCCAGGTGAAGAGAAGCCTCAGG CAAGCCCCGAAGGCCGTCCTGAGAGTGAGACTTCCTGCCTCGTCACAACAACAGATTTTCAA ATACAGACAGAAATGGCTGCAACCATGGAGACGTCCATATTTACAACAGAGTACCAGGGCGG CGGGGGCAGTGGAGGAGGCGGAAGCGGTGGGGGGGGATCGCTGAACACGACAATTCTGACGC CCAATGGGAATGAAGACACCACAGCTGATTTCTTCCTGACCACTATGCCCACTGACTCCCTC AGTGTTTCCACTCTGCCCCTCCCAGAGGTTCAGTGTTTTGTGTTCAATGTCGAGTACATGAA TTGCACTTGGAACAGCAGCTCTGAGCCCCAGCCTACCAACCTCACTCTGCATTATTGGTACA AGAAC T C G GAT AAT GAT AAAG T C C AGAAG T G C AG C C AC TATCTATTCTCT GAAGAAAT C AC T TCTGGCTGTCAGTTGCAAAAAAAGGAGATCCACCTCTACCAAACATTTGTTGTTCAGCTCCA GGACCCACGGGAACCCAGGAGACAGGCCACACAGATGCTAAAACTGCAGAATCTGGTGATCC CCTGGGCTCCAGAGAACCTAACACTTCACAAACTGAGTGAATCCCAGCTAGAACTGAACTGG AACAACAGATTCTTGAACCACTGTTTGGAGCACTTGGTGCAGTACCGGACTGACTGGGACCA CAGCTGGACTGAACAATCAGTGGATTATAGACATAAGTTCTCCTTGCCTAGTGTGGATGGGCAGAAACGCTACACGTTTCGTGTTCGGAGCCGCTTTAACCCACTCTGTGGAAGTGCTCAGCAT TGGAGTGAATGGAGCCACCCAATCCACTGGGGGAGCAATACTTCAAAAGAGAATCCTTTCCT GTTTGCATTGGAAGCCGTGGTTATCTCTGTTGGCTCCATGGGATTGATTATCAGCCTTCTCT GTGTGTATTTCTGGCTGGAACGGACGATGCCCCGAATTCCCACCCTGAAGAACCTAGAGGAT CTTGTTACTGAATACCACGGGAACTTTTCGGCCTGGAGTGGTGTGTCTAAGGGACTGGCTGA GAGTCTGCAGCCAGACTACAGTGAACGACTCTGCCTCGTCAGTGAGATTCCCCCAAAAGGAG GGGCCCTTGGGGAGGGGCCTGGGGCCTCCCCATGCAACCAGCATAGCCCCTACTGGGCCCCC C C AT G T T AC AC C C T AAAG C C T GAAAC C

[0230] SEQ ID NO: 41 beta-CD19 DNA:ATGGCGGCCCCTGCTCTGTCCTGGCGTCTGCCCCTCCTCATCCTCCTCCTGCCCCTGGCTAC CTCTTGGGCATCTGCAGCGGTGAATGGCACTTCCCAGTTCACATGCTTCTACAACTCGAGAG CCAACATCTCCTGTGTCTGGAGCCAAGATGGGGCTCTGCAGGACACTTCCTGCCAAGTCCAT GCCTGGCCGGACAGACGGCGGTGGAACCAAACCTGTGAGCTGCTCCCCGTGAGTCAAGCATC CTGGGCCTGCAACCTGATCCTCGGAGCCCCAGATTCTCAGAAACTGACCACAGTTGACATCG TCACCCTGAGGGTGCTGTGCCGTGAGGGGGTGCGATGGAGGGTGATGGCCATCCAGGACTTC AAGCCCTTTGAGAACCTTCGCCTGATGGCCCCCATCTCCCTCCAAGTTGTCCACGTGGAGAC CCACAGATGCAACATAAGCTGGGAAATCTCCCAAGCCTCCCACTACTTTGAAAGACACCTGG AGTTCGAGGCCCGGACGCTGTCCCCAGGCCACACCTGGGAGGAGGCCCCCCTGCTGACTCTC AAGCAGAAGCAGGAATGGATCTGCCTGGAGACGCTCACCCCAGACACCCAGTATGAGTTTCA GGTGCGGGTCAAGCCTCTGCAAGGCGAGTTCACGACCTGGAGCCCCTGGAGCCAGCCCCTGG CCTTCAGGACAAAGCCTGCAGCCCTTGGGAAGGACACCATTCCGTGGCTCGGCCACCTCCTC GTGGGCCTCAGCGGGGCTTTTGGCTTCATCATCTTAGTGTACTTGCTGATCAACTGCAGGAA CACCGGGCCATGGCTGAAGAAGGTCCTGAAGTGTAACACCCCAGACCCCTCGAAGTTCTTTT CCCAGCTGAGCTCAGAGCATGGAGGAGACGTCCAGAAGTGGCTCTCTTCGCCCTTCCCCTCA TCGTCCTTCAGCCCTGGCGGCCTGGCACCTGAGATCTCGCCACTAGAAGTGCTGGAGAGGGA CAAGGTGACGCAGCTGCTCCTGCAGCAGGACAAGGTGCCTGAGCCCGCATCCTTAAGCAGCA ACCACTCGCTGACCAGCTGCTTCACCAACCAGGGTTACTTCTTCTTCCACCTCCCGGATGCC TTGGAGATAGAGGCCTGCCAGGTGTACTTTACTTACGACCCCTACTCAGAGGAAGACCCTGA TGAGGGTGTGGCCGGGGCACCCACAGGGTCTTCCCCCCAACCCCTGCAGCCTCTGTCAGGGG AGGACGACGCCTACTGCACCTTCCCCTCCAGGGATGACCTGCTGCTCTTCTCCCCCAGTCTC CTCGGTGGCCCCAGCCCCCCAAGCACTGCCCCTGGGGGCAGTGGGGCCGGTGAAGAGAGGAT GCCCCCTTCTTTGCAAGAAAGAGTCCCCAGAGACTGGGACCCCCAGCCCCTGGGGCCTCCCA CCCCAGGAGTCCCAGACCTGGTGGATTTTCAGCCACCCCCTGAGCTGGTGCTGCGAGAGGCT GGGGAGGAGGTCCCTGACGCTGGCCCCAGGGAGGGAGTCAGTTTCCCCTGGTCCAGGCCTCC TGGGCAGGGGGAGTTCAGGGCCCTTAATGCTCGCCTGCCCCTGAACACTGATGCCTACTTGT CCCTCCAAGAACTCCAGGGTCAGGACCCAACTCACTTGGTGctcgaGGAGGGCAGGGGAAGT CTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCATGCCACCTCCTCGCCTCCTCTT CTTCCTCCTCTTCCTCACCCCCATGGAAGTCAGGCCCGAGGAACCTCTAGTGGTGAAGGTGG AAGAGGGAGATAACGCTGTGCTGCAGTGCCTCAAGGGGACCTCAGATGGCCCCACTCAGCAG CTGACCTGGTCTCGGGAGTCCCCGCTTAAACCCTTCTTAAAACTCAGCCTGGGGCTGCCAGG CCTGGGAATCCACATGAGGCCCCTGGCCATCTGGCTTTTCATCTTCAACGTCTCTCAACAGA TGGGGGGCTTCTACCTGTGCCAGCCGGGGCCCCCCTCTGAGAAGGCCTGGCAGCCTGGCTGG ACAGTCAATGTGGAGGGCAGCGGGGAGCTGTTCCGGTGGAATGTTTCGGACCTAGGTGGCCT GGGCTGTGGCCTGAAGAACAGGTCCTCAGAGGGCCCCAGCTCCCCTTCCGGGAAGCTCATGA GCCCCAAGCTGTATGTGTGGGCCAAAGACCGCCCTGAGATCTGGGAGGGAGAGCCTCCGTGT CTCCCACCGAGGGACAGCCTGAACCAGAGCCTCAGCCAGGACCTCACCATGGCCCCTGGCTC CACACTCTGGCTGTCCTGTGGGGTACCCCCTGACTCTGTGTCCAGGGGCCCCCTCTCCTGGA CCCATGTGCACCCCAAGGGGCCTAAGTCATTGCTGAGCCTAGAGCTGAAGGACGATCGCCCG GCCAGAGATATGTGGGTAATGGAGACGGGTCTGTTGTTGCCCCGGGCCACAGCTCAAGACGCTGGAAAGTATTATTGTCACCGTGGCAACCTGACCATGTCATTCCACCTGGAGATCACTGCTC GGCCAGTACTATGGCACTGGCTGCTGAGGACTGGTGGCTGGAAGGTCTCAGCTGTGACTTTG GCTTATCTGATCTTCTGCCTGTGTTCCCTTGTGGGCATTCTTCATCTTCAAAGAGCCCTGGT C C T GAG GAG GAAAAGAAAG C GAAT GAG T GAG C C GAG GAG GAGAT T C

[0231] SEQ ID NO: 42 signal-peptide_TGFbl-Q8 DNA:ATGCCGCCCTCCGGGCTGCGGCTGCTGCCGCTGCTGCTACCGCTGCTGTGGCTACTGGTGCT GACGCCTGGCCGGCCGGCCGCGGGACTATCCACCTGCAAGACTATCGACATGGAGCTGGTGA AGCGGAAGCGCATCGAGGCCATCCGCGGCCAGATCCTGTCCAAGCTGCGGCTCGCCAGCCCC CCGAGCCAGGGGGAGGTGCCGCCCGGCCCGCTGCCCGAGGCCGTGCTCGCCCTGTACAACAG CACCCGCGACCGGGTGGCCGGGGAGAGTGCAGAACCGGAGCCCGAGCCTGAGGCCGACTACT ACGCCAAGGAGGTCACCCGCGTGCTAATGGTGGAAACCCACAACGAAATCTATGACAAGTTC AAGCAGAGTACACACAGCATATATATGTTCTTCAACACATCAGAGCTCCGAGAAGCGGTACC TGAACCCGTGTTGCTCTCCCGGGCAGAGCTGCGTCTGCTGAGGCTCAAGTTAAAAGTGGAGC AGCACGTGGAGCTGTACCAGAAATACAGCAACAATTCCTGGCGATACCTCAGCAACCGGCTG CTGGCACCCAGCGACTCGCCAGAGTGGTTATCTTTTGATGTCACCGGAGTTGTGCGGCAGTG GTTGAGCCGTGGAGGGGAAATTGAGGGCTTTCGCCTTAGCGCCCACTGCTCCTGTGACAGCA GGGATAACACACTGCAAGTGGACATCAACGGGTTCACTACCGGCCGCCGAGGTGACCTGGCC ACCATTCATGGCATGAACCGGCCTTTCCTGCTTCTCATGGCCACCCCGCTGGAGAGGGCCCA GCATCTGCAAAGCTCCCGGCACCGCCGAGCCCTGGACACCAACTATTGCTTCAGCTCCACGG AGAAGAACTGCTGCGTGCGGCAGCTGTACATTGACTTCCGCAAGGACCTCGGCTGGAAGTGG ATCCACGAGCCCAAGGGCTACCATGCCAACTTCTGCCTCGGGCCCTGCCCCTACATTTGGAG CCTGGACACGCAGTACAGCAAGGTCCTGGCCCTGTACAACCAGCATAACCCGGGCGCCTCGG CGGCGCCGTGCTGCGTGCCGCAGGCGCTGGAGCCGCTGCCCATCGTGTACTACGTGGGCCGC AAGCCCAAGGTGGAGCAGCTGTCCAACATGATCGTGCGCTCCTGCAAGTGCAGCAGAGCCAA AAGAGGAAGCGGCGCCACAAATTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAAGAGAATC CCGGCCCTATGGGACTCGTGCGCAGAGGCGCTAGAGCCGGCCCTAGAATGCCTAGAGGATGG ACCGCCCTGTGCCTGCTGTCTCTGCTGCCTAGCGGCTTCATGGCCGAGCTGCCTACTCAGGG CACCTTCAGCAACGTGTCCACCAATGTGTCCCCAGCCAAGCCCACCACAACCCCTGCTCCTA GACCTCCTACCCCAGCCCCTACCATTGCCTCCCAGCCACTGTCTCTGAGGCCCGAGGCTTGT AGACCTGCTGCAGGCGGAGCCGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTATAT CTGGGCCCCTCTGGCCGGCACCTGTGGCGTGCTGCTGCTGTCACTCGTGATCACCCTGTACT GCAACCACCGGAACCGGCGGAGAGTGTGCAAGTGCCCTAGACCCGTCGTG

[0232] SEQ ID NO: 43 TGFbl-Q8 DNA:CTATCCACCTGCAAGACTATCGACATGGAGCTGGTGAAGCGGAAGCGCATCGAGGCCATCCG CGGCCAGATCCTGTCCAAGCTGCGGCTCGCCAGCCCCCCGAGCCAGGGGGAGGTGCCGCCCG GCCCGCTGCCCGAGGCCGTGCTCGCCCTGTACAACAGCACCCGCGACCGGGTGGCCGGGGAG AGTGCAGAACCGGAGCCCGAGCCTGAGGCCGACTACTACGCCAAGGAGGTCACCCGCGTGCT AAT G G T G GAAAC C C AC AAC GAAAT C T AT GAG AAG T T C AAG C AGAG TAG AC AC AG CAT AT AT A TGTTCTTCAACACATCAGAGCTCCGAGAAGCGGTACCTGAACCCGTGTTGCTCTCCCGGGCA GAGCTGCGTCTGCTGAGGCTCAAGTTAAAAGTGGAGCAGCACGTGGAGCTGTACCAGAAATA CAGCAACAATTCCTGGCGATACCTCAGCAACCGGCTGCTGGCACCCAGCGACTCGCCAGAGT GGTTATCTTTTGATGTCACCGGAGTTGTGCGGCAGTGGTTGAGCCGTGGAGGGGAAATTGAG GGCTTTCGCCTTAGCGCCCACTGCTCCTGTGACAGCAGGGATAACACACTGCAAGTGGACAT CAACGGGTTCACTACCGGCCGCCGAGGTGACCTGGCCACCATTCATGGCATGAACCGGCCTT TCCTGCTTCTCATGGCCACCCCGCTGGAGAGGGCCCAGCATCTGCAAAGCTCCCGGCACCGC CGAGCCCTGGACACCAACTATTGCTTCAGCTCCACGGAGAAGAACTGCTGCGTGCGGCAGCT GTACATTGACTTCCGCAAGGACCTCGGCTGGAAGTGGATCCACGAGCCCAAGGGCTACCATG CCAACTTCTGCCTCGGGCCCTGCCCCTACATTTGGAGCCTGGACACGCAGTACAGCAAGGTCCTGGCCCTGTACAACCAGCATAACCCGGGCGCCTCGGCGGCGCCGTGCTGCGTGCCGCAGGC GCTGGAGCCGCTGCCCATCGTGTACTACGTGGGCCGCAAGCCCAAGGTGGAGCAGCTGTCCA ACATGATCGTGCGCTCCTGCAAGTGCAGCAGAGCCAAAAGAGGAAGCGGCGCCACAAATTTC AGCCTGCTGAAGCAGGCCGGCGACGTGGAAGAGAATCCCGGCCCTATGGGACTCGTGCGCAG AGGCGCTAGAGCCGGCCCTAGAATGCCTAGAGGATGGACCGCCCTGTGCCTGCTGTCTCTGC TGCCTAGCGGCTTCATGGCCGAGCTGCCTACTCAGGGCACCTTCAGCAACGTGTCCACCAAT GTGTCCCCAGCCAAGCCCACCACAACCCCTGCTCCTAGACCTCCTACCCCAGCCCCTACCAT TGCCTCCCAGCCACTGTCTCTGAGGCCCGAGGCTTGTAGACCTGCTGCAGGCGGAGCCGTGC ACACCAGAGGACTGGATTTCGCCTGCGACATCTATATCTGGGCCCCTCTGGCCGGCACCTGT GGCGTGCTGCTGCTGTCACTCGTGATCACCCTGTACTGCAACCACCGGAACCGGCGGAGAGT GTGCAAGTGCCCTAGACCCGTCGTG

[0233] SEQ ID NO: 44 IL12A-2A-EBI3-Q8 DNA:ATGTGGCCCCCTGGGTCAGCCTCCCAGCCACCGCCCTCACCTGCCGCGGCCACAGGTCTGCA TCCAGCGGCTCGCCCTGTGTCCCTGCAGTGCCGGCTCAGCATGTGTCCAGCGCGCAGCCTCC TCCTTGTGGCTACCCTGGTCCTCCTGGACCACCTCAGTTTGGCCAGAAACCTCCCCGTGGCC ACTCCAGACCCAGGAATGTTCCCATGCCTTCACCACTCCCAAAACCTGCTGAGGGCCGTCAG CAACAT GC T C CAGAAGGC CAGACAAAC T C T AGAAT T T TAG C C T T GCAC T T C T GAAGAGAT T G ATCATGAAGATATCACAAAAGATAAAACCAGCACAGTGGAGGCCTGTTTACCATTGGAATTA AC C AAGAAT GAGAG T T G C C T AAAT T C C AGAGAGAC C T C T T T C AT AAC T AAT G G GAG T T G C C T GGCCTCCAGAAAGACCTCTTTTATGATGGCCCTGTGCCTTAGTAGTATTTATGAAGACTTGA AGATGTACCAGGTGGAGTTCAAGACCATGAATGCAAAGCTTCTGATGGATCCTAAGAGGCAG ATCTTTCTAGATCAAAACATGCTGGCAGTTATTGATGAGCTGATGCAGGCCCTGAATTTCAA GAG T GAGAG T G T G C C AC AAAAAT CCTCCCTT GAAGAAC C G GAT T T T T AT AAAAC T AAAAT C A AGCTCTGCATACTTCTTCATGCTTTCAGAATTCGGGCAGTGACTATTGATAGAGTGATGAGC TATCTGAATGCTTCCAGAGCCAAAAGAGGTAGTGGGGAGGGCAGGGGAAGTCTTCTAACATG CGGGGACGTGGAGGAAAATCCCGGGCCCATGACCCCGCAGCTTCTCCTGGCCCTTGTCCTCT GGGCCAGCTGCCCGCCCTGCAGTGGAAGGAAAGGGCCCCCAGCAGCTCTGACACTGCCCCGG GTGCAATGCCGAGCCTCTCGGTACCCGATCGCCGTGGATTGCTCCTGGACCCTGCCGCCTGC TCCAAACTCCACCAGCCCCGTGTCCTTCATTGCCACGTACAGGCTCGGCATGGCTGCCCGGG GCCACAGCTGGCCCTGCCTGCAGCAGACGCCAACGTCCACCAGCTGCACCATCACGGATGTC CAGCTGTTCTCCATGGCTCCCTACGTGCTCAATGTCACCGCCGTCCACCCCTGGGGCTCCAG CAGCAGCTTCGTGCCTTTCATAACAGAGCACATCATCAAGCCCGACCCTCCAGAAGGCGTGC GCCTAAGCCCCCTCGCTGAGCGCCAGCTACAGGTGCAGTGGGAGCCTCCCGGGTCCTGGCCC TTCCCAGAGATCTTCTCACTGAAGTACTGGATCCGTTACAAGCGTCAGGGAGCTGCGCGCTT CCACCGGGTGGGGCCCATTGAAGCCACGTCCTTCATCCTCAGGGCTGTGCGGCCCCGAGCCA GGTACTACGTCCAAGTGGCGGCTCAGGACCTCACAGACTACGGGGAACTGAGTGACTGGAGT CTCCCCGCCACTGCCACAATGAGCCTGGGCAAGAGAGCCAAAAGAGGAAGCGGCGCCACAAA TTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAAGAGAATCCCGGCCCTATGGGACTCGTGC GCAGAGGCGCTAGAGCCGGCCCTAGAATGCCTAGAGGATGGACCGCCCTGTGCCTGCTGTCT CTGCTGCCTAGCGGCTTCATGGCCGAGCTGCCTACTCAGGGCACCTTCAGCAACGTGTCCAC CAATGTGTCCCCAGCCAAGCCCACCACAACCCCTGCTCCTAGACCTCCTACCCCAGCCCCTA CCATTGCCTCCCAGCCACTGTCTCTGAGGCCCGAGGCTTGTAGACCTGCTGCAGGCGGAGCC GTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTATATCTGGGCCCCTCTGGCCGGCAC CTGTGGCGTGCTGCTGCTGTCACTCGTGATCACCCTGTACTGCAACCACCGGAACCGGCGGA GAGTGTGCAAGTGCCCTAGACCCGTCGTG

[0234] SEQ ID NO: 45 IL12A-2A-EBI3 DNA:ATGTGGCCCCCTGGGTCAGCCTCCCAGCCACCGCCCTCACCTGCCGCGGCCACAGGTCTGCA TCCAGCGGCTCGCCCTGTGTCCCTGCAGTGCCGGCTCAGCATGTGTCCAGCGCGCAGCCTCCTCCTTGTGGCTACCCTGGTCCTCCTGGACCACCTCAGTTTGGCCAGAAACCTCCCCGTGGCC ACTCCAGACCCAGGAATGTTCCCATGCCTTCACCACTCCCAAAACCTGCTGAGGGCCGTCAG CAACAT GC T C CAGAAGGC CAGACAAAC T C T AGAAT T T TAG C C T T GCAC T T C T GAAGAGAT T G ATCATGAAGATATCACAAAAGATAAAACCAGCACAGTGGAGGCCTGTTTACCATTGGAATTA AC C AAGAAT GAGAG T T G C C T AAAT T C C AGAGAGAC C T C T T T C AT AAC T AAT G G GAG T T G C C T GGCCTCCAGAAAGACCTCTTTTATGATGGCCCTGTGCCTTAGTAGTATTTATGAAGACTTGA AGATGTACCAGGTGGAGTTCAAGACCATGAATGCAAAGCTTCTGATGGATCCTAAGAGGCAG ATCTTTCTAGATCAAAACATGCTGGCAGTTATTGATGAGCTGATGCAGGCCCTGAATTTCAA GAG T GAGAG T G T G C C AC AAAAAT CCTCCCTT GAAGAAC C G GAT T T T T AT AAAAC T AAAAT C A AGCTCTGCATACTTCTTCATGCTTTCAGAATTCGGGCAGTGACTATTGATAGAGTGATGAGC TATCTGAATGCTTCCAGAGCCAAAAGAGGTAGTGGGGAGGGCAGGGGAAGTCTTCTAACATG CGGGGACGTGGAGGAAAATCCCGGGCCCATGACCCCGCAGCTTCTCCTGGCCCTTGTCCTCT GGGCCAGCTGCCCGCCCTGCAGTGGAAGGAAAGGGCCCCCAGCAGCTCTGACACTGCCCCGG GTGCAATGCCGAGCCTCTCGGTACCCGATCGCCGTGGATTGCTCCTGGACCCTGCCGCCTGC TCCAAACTCCACCAGCCCCGTGTCCTTCATTGCCACGTACAGGCTCGGCATGGCTGCCCGGG GCCACAGCTGGCCCTGCCTGCAGCAGACGCCAACGTCCACCAGCTGCACCATCACGGATGTC CAGCTGTTCTCCATGGCTCCCTACGTGCTCAATGTCACCGCCGTCCACCCCTGGGGCTCCAG CAGCAGCTTCGTGCCTTTCATAACAGAGCACATCATCAAGCCCGACCCTCCAGAAGGCGTGC GCCTAAGCCCCCTCGCTGAGCGCCAGCTACAGGTGCAGTGGGAGCCTCCCGGGTCCTGGCCC TTCCCAGAGATCTTCTCACTGAAGTACTGGATCCGTTACAAGCGTCAGGGAGCTGCGCGCTT CCACCGGGTGGGGCCCATTGAAGCCACGTCCTTCATCCTCAGGGCTGTGCGGCCCCGAGCCA GGTACTACGTCCAAGTGGCGGCTCAGGACCTCACAGACTACGGGGAACTGAGTGACTGGAGT CTCCCCGCCACTGCCACAATGAGCCTGGGCAAGAGAGCCAAAAGA

[0235] SEQ ID NO: 46 IL12A furin-cleavage-site DNA:ATGTGGCCCCCTGGGTCAGCCTCCCAGCCACCGCCCTCACCTGCCGCGGCCACAGGTCTGCA TCCAGCGGCTCGCCCTGTGTCCCTGCAGTGCCGGCTCAGCATGTGTCCAGCGCGCAGCCTCC TCCTTGTGGCTACCCTGGTCCTCCTGGACCACCTCAGTTTGGCCAGAAACCTCCCCGTGGCC ACTCCAGACCCAGGAATGTTCCCATGCCTTCACCACTCCCAAAACCTGCTGAGGGCCGTCAG CAACAT GC T C CAGAAGGC CAGACAAAC T C T AGAAT T T TAG C C T T GCAC T T C T GAAGAGAT T G ATCATGAAGATATCACAAAAGATAAAACCAGCACAGTGGAGGCCTGTTTACCATTGGAATTA AC C AAGAAT GAGAG T T G C C T AAAT T C C AGAGAGAC C T C T T T CAT AAC T AAT G G GAG T T G C C T GGCCTCCAGAAAGACCTCTTTTATGATGGCCCTGTGCCTTAGTAGTATTTATGAAGACTTGA AGATGTACCAGGTGGAGTTCAAGACCATGAATGCAAAGCTTCTGATGGATCCTAAGAGGCAG ATCTTTCTAGATCAAAACATGCTGGCAGTTATTGATGAGCTGATGCAGGCCCTGAATTTCAA C AG T GAGAG T G T G C C AC AAAAAT CCTCCCTT GAAGAAC C G GAT T T T TAT AAAAC T AAAAT C A AGCTCTGCATACTTCTTCATGCTTTCAGAATTCGGGCAGTGACTATTGATAGAGTGATGAGC TAT C T GAAT GC T T C CAGAGC CAAAAGA

[0236] SEQ ID NO: 131 PDL1 380bBB coding DNA:ATGAGGATATTTGCTGTCTTTATATTCATGACCTACTGGCATTTGCTGAACGCATTTACTGT CACGGTTCCCAAGGACCTATATGTGGTAGAGTATGGTAGCAATATGACAATTGAATGCAAAT TCCCAGTAGAAAAACAATTAGACCTGGCTGCACTAATTGTCTATTGGGAAATGGAGGATAAG AACAT TAT T CAAT T T GT GCAT GGAGAGGAAGACC T GAAGGT T CAGCATAGTAGC TACAGACA GAGGGCCCGGCTGTTGAAGGACCAGCTCTCCCTGGGAAATGCTGCACTTCAGATCACAGATG TGAAATTGCAGGATGCAGGGGTGTACCGCTGCATGATCAGCTATGGTGGTGCCGACTACAAG C GAAT T AC T G T GAAAG T CAAT G C C C CAT AC AAC AAAAT C AAC C AAAGAAT TTTGGTTGTGGA TCCAGTCACCTCTGAACATGAACTGACATGTCAGGCTGAGGGCTACCCCAAGGCCGAAGTCA T C T G GAG AAG GAG T GAG CAT C AAG T C C T GAG T G G T AAGAC C AC C AC C AC CAAT T C C AAGAGA GAGGAGAAGC T T T T CAAT GT GAG GAG C AC AC T GAGAAT CAACACAACAAC TAAT GAGAT T T TCTACTGCACTTTTAGGAGATTAGATCCTGAGGAAAACCATACAGCTGAATTGGTCATCCCAG AACTACCTCTGGCACATCCTCCAAATGAAAGGGAGTCTAAATATGGCCCACCTTGCCCACCG TGCCCAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAC CAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGG AGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCC GACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAA CGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCT CCCTGTCTCCGGGTAAAAAAGATCCCAAATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTG GCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAGAGTGAA GTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGC TCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAG ATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGA TAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGC ACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATG CAGGCCCTGCCTCCTCGC

[0237] SEQ ID NO: 132 VHVL PD1 380b_coding DNA:ATGGAGGCTCCCGCTCAATTGCTTTTTCTTCTGCTTCTGTGGTTGCCTGACACAACTGGCCA GGTGCAGCTGCAGGAGTCGGGCCCAGGAGTGGTGAAGCCTTCGGGGACCCTGTCCCTCACCT GCGCTATTTCTGGTGGCTCCATCGGCTCTGGTGGCTCCATCAGAAGTACTAGGTGGTGGAGT TGGGTCCGCCAGTCCCCAGGGAAGGGGCTGGAGTGGATAGGCGAAATCTATCATAGTGGGAG GAG C AAC T AC AAC CCGTCCCT C AAGAG T C G C G T GAG CAT AT C AC T AGAC AAG T C T AG GAAT C ACTTCTCCCTGAGGCTGAACTCTGTGACCGCCGCGGACACGGCCGTTTATTACTGTGCGAGA CAGGACTACGGTGACTCCGGCGACTGGTACTTCGATCTGTGGGGCAAGGGGACAATGGTCAC CGTCTCCTCAGGCGGCGGGGGCAGTGGAGGAGGCGGAAGCGGTGGGGGGGGATCGAATTTTA TGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCATCTCCTGCACC CGCAGCAGTGGCAGCATTGCCAGCAACTCTGTGCAGTGGTACCAGCAGCGCCCGGGCAGTTC CCCCACCACTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTG GCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCGTCTCTGGACTGAAGACTGAGGAC GAGGCTGACTACTACTGTCAGTCTTCTGATAGCAGCGCTGTGGTATTCGGCAGTGGGACCAA GCTGACCGTCCTAGAGTCTAAATATGGCCCACCTTGCCCACCGTGCCCAGGGCAGCCCCGAG AACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTG ACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCA ACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCT ACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTG ATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAAA AGATCCCAAATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAG TAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAGAGTGAAGTTCAGCAGGAGCGCAGAC GCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGA GGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAA GGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTAC AGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGC

[0238] SEQ ID NO: 133 VLVH PD1 380b_coding DNA:ATGGAGGCTCCCGCTCAATTGCTTTTTCTTCTGCTTCTGTGGTTGCCTGACACAACTGGCAA TTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCATCTCCT GCACCCGCAGCAGTGGCAGCATTGCCAGCAACTCTGTGCAGTGGTACCAGCAGCGCCCGGGC AGTTCCCCCACCACTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGTT CTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCGTCTCTGGACTGAAGACTGAGGACGAGGCTGACTACTACTGTCAGTCTTCTGATAGCAGCGCTGTGGTATTCGGCAGTGGG ACCAAGCTGACCGTCCTAGGCGGCGGGGGCAGTGGAGGAGGCGGAAGCGGTGGGGGGGGATC GCAGGTGCAGCTGCAGGAGTCGGGCCCAGGAGTGGTGAAGCCTTCGGGGACCCTGTCCCTCA CCTGCGCTATTTCTGGTGGCTCCATCGGCTCTGGTGGCTCCATCAGAAGTACTAGGTGGTGG AGTTGGGTCCGCCAGTCCCCAGGGAAGGGGCTGGAGTGGATAGGCGAAATCTATCATAGTGG GAG GAG C AAC T AC AAC CCGTCCCT C AAGAG T C G C G T GAG CAT AT C AC T AGAC AAG T C T AG GA ATCACTTCTCCCTGAGGCTGAACTCTGTGACCGCCGCGGACACGGCCGTTTATTACTGTGCG AGACAGGACTACGGTGACTCCGGCGACTGGTACTTCGATCTGTGGGGCAAGGGGACAATGGT CACCGTCTCCTCAGAGTCTAAATATGGCCCACCTTGCCCACCGTGCCCAGGGCAGCCCCGAG AACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTG ACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCA ACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCT ACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTG ATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAAA AGATCCCAAATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAG TAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAGAGTGAAGTTCAGCAGGAGCGCAGAC GCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGA GGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAA GGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTAC AGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGG TCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGC

[0239] SEQ ID NO: 134 VHVL TIGIT 380b_coding DNA:ATGGAGGCTCCCGCTCAATTGCTTTTTCTTCTGCTTCTGTGGTTGCCTGACACAACTGGCGA GGTGCAGCTGCAGCAGAGCGGCCCCGAGCTGGTGAAGCCCGGCAGCAGCGTGAAGATGAGCT GCAAGGCTAGCGGCTACACCTTCAGCAGCTACGTGATGCACTGGGTGAAGCAGAAGCCCGGC CAAGGCCTGGAGTGGATCGGCTACATCGACCCCTACAACGACGGCGCCAAGTACAACGAGAA GTTCAAGGGCAAGGCCACCCTCACAAGCGACAAGAGCAGCAGCACCGCCTACATGGAGCTGA GCAGCCTGACAAGCGAGGACAGCGCCGTGTACTACTGCGCTAGAGGCGGCCCCTACGGCTGG TACTTCGACGTGTGGGGCGCCGGCACCACCGTGACCGTGAGCAGCGGCGGCGGGGGCAGTGG AGGAGGCGGAAGCGGTGGGGGGGGATCGGACATTCAGATGACACAGAGCCCCGCTAGCCTGA GCGCTAGCGTGGGCGAGACCGTGACCATCACCTGCAGAGCTAGCGAGCACATCTACAGCTAC CTGAGCTGGTATCAGCAGAAGCAAGGCAAGAGCCCTCAGCTGCTGGTGTACAACGCCAAGAC CCTGGCCGAGGGCGTGCCTAGCAGATTCAGCGGCAGCGGCAGCGGCACACAGTTCAGCCTGA AGATCAACAGCCTGCAGCCCGAGGACTTCGGCACCTACTACTGTCAGCACCACTTCGGCAGC CCCCTGACCTTCGGCGCCGGCACCACCCTGGAGCTGAAGGAGTCTAAATATGGCCCACCTTG CCCACCGTGCCCAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATG AGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATC GCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCT GGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGC AGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAG AGCCTCTCCCTGTCTCCGGGTAAAAAAGATCCCAAATTTTGGGTGCTGGTGGTGGTTGGTGG AGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTA GAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTAT AACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGA CCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGC AGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGC AAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCT TCACATGCAGGCCCTGCCTCCTCGC

[0240] SEQ ID NO: 135 VLVH TIGIT 380b_coding DNA:ATGGAGGCTCCCGCTCAATTGCTTTTTCTTCTGCTTCTGTGGTTGCCTGACACAACTGGCGA CATTCAGATGACACAGAGCCCCGCTAGCCTGAGCGCTAGCGTGGGCGAGACCGTGACCATCA CCTGCAGAGCTAGCGAGCACATCTACAGCTACCTGAGCTGGTATCAGCAGAAGCAAGGCAAG AGCCCTCAGCTGCTGGTGTACAACGCCAAGACCCTGGCCGAGGGCGTGCCTAGCAGATTCAG CGGCAGCGGCAGCGGCACACAGTTCAGCCTGAAGATCAACAGCCTGCAGCCCGAGGACTTCG GCACCTACTACTGTCAGCACCACTTCGGCAGCCCCCTGACCTTCGGCGCCGGCACCACCCTG GAGCTGAAGGGCGGCGGGGGCAGTGGAGGAGGCGGAAGCGGTGGGGGGGGATCGGAGGTGCA GCTGCAGCAGAGCGGCCCCGAGCTGGTGAAGCCCGGCAGCAGCGTGAAGATGAGCTGCAAGG CTAGCGGCTACACCTTCAGCAGCTACGTGATGCACTGGGTGAAGCAGAAGCCCGGCCAAGGC CTGGAGTGGATCGGCTACATCGACCCCTACAACGACGGCGCCAAGTACAACGAGAAGTTCAA GGGCAAGGCCACCCTCACAAGCGACAAGAGCAGCAGCACCGCCTACATGGAGCTGAGCAGCC TGACAAGCGAGGACAGCGCCGTGTACTACTGCGCTAGAGGCGGCCCCTACGGCTGGTACTTC GACGTGTGGGGCGCCGGCACCACCGTGACCGTGAGCAGCGAGTCTAAATATGGCCCACCTTG CCCACCGTGCCCAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATG AGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATC GCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCT GGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGC AGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAG AGCCTCTCCCTGTCTCCGGGTAAAAAAGATCCCAAATTTTGGGTGCTGGTGGTGGTTGGTGG AGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTA GAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTAT AACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGA CCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGC AGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGC AAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCT TCACATGCAGGCCCTGCCTCCTCGC

[0241] SEQ ID NO: 136 VLVH PD 1 148b CH3 28z t2A NGFR coding DNA:ATGGAGGCTCCCGCTCAATTGCTTTTTCTTCTGCTTCTGTGGTTGCCTGACACAACTGGCAA TTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCATCTCCT GCACCCGCAGCAGTGGCAGCATTGCCAGCAACTCTGTGCAGTGGTACCAGCAGCGCCCGGGC AGTTCCCCCACCACTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGTT CTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCGTCTCTGGACTGAAGACTG AGGACGAGGCTGACTACTACTGTCAGTCTTCTGATAGCAGCGCTGTGGTATTCGGCAGTGGG ACCAAGCTGACCGTCCTAGGCGGCGGGGGCAGTGGAGGAGGCGGAAGCGGTGGGGGGGGATC GCAGGTGCAGCTGCAGGAGTCGGGCCCAGGAGTGGTGAAGCCTTCGGGGACCCTGTCCCTCA CCTGCGCTATTTCTGGTGGCTCCATCGGCTCTGGTGGCTCCATCAGAAGTACTAGGTGGTGG AGTTGGGTCCGCCAGTCCCCAGGGAAGGGGCTGGAGTGGATAGGCGAAATCTATCATAGTGG GAG GAG C AAC T AC AAC CCGTCCCT C AAGAG T C G C G T GAG CAT AT C AC T AGAC AAG T C T AG GA ATCACTTCTCCCTGAGGCTGAACTCTGTGACCGCCGCGGACACGGCCGTTTATTACTGTGCG AGACAGGACTACGGTGACTCCGGCGACTGGTACTTCGATCTGTGGGGCAAGGGGACAATGGT CACCGTCTCCTCAGAGTCTAAATATGGCCCACCTTGCCCACCGTGCCCAGGGCAGCCCCGAG AACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTG ACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCA ACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCT ACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTG ATGCATGAGGCTCTGCACAACGCCTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAAA AGATCCCAAATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAG TAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGCCTCGAGAGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCATGGGGGCAGGTGCCACCGGCCGCGCCATGGACGGGCCGCGCCTGCTGCTGTTGCTGCTTCTGGGGGTGTCCCTTGGAGGTGCCAAGGAGGCATGCCCCACAGGCCTGTACACACACAGCGGTGAGTGCTGCAAAGCCTGCAACCTGGGCGAGGGTGTGGCCCAGCCTTGTGGAGCCAACCAGACCGTGTGTGAGCCCTGCCTGGACAGCGTGACGTTCTCCGACGTGGTGAGCGCGACCGAGCCGTGCAAGCCGTGCACCGAGTGCGTGGGGCTCCAGAGCATGTCGGCGCCGTGCGTGGAGGCCGACGACGCCGTGTGCCGCTGCGCCTACGGCTACTACCAGGATGAGACGACTGGGCGCTGCGAGGCGTGCCGCGTGTGCGAGGCGGGCTCGGGCCTCGTGTTCTCCTGCCAGGACAAGCAGAACACCGTGTGCGAGGAGTGCCCCGACGGCACGTATTCCGACGAGGCCAACCACGTGGACCCGTGCCTGCCCTGCACCGTGTGCGAGGACACCGAGCGCCAGCTCCGCGAGTGCACACGCTGGGCCGACGCCGAGTGCGAGGAGATCCCTGGCCGTTGGATTACACGGTCCACACCCCCAGAGGGCTCGGACAGCACAGCCCCCAGCACCCAGGAGCCTGAGGCACCTCCAGAACAAGACCTCATAGCCAGCACGGTGGCAGGTGTGGTGACCACAGTGATGGGCAGCTCCCAGCCCGTGGTGACCCGAGGCACCACCGACAACCTCATCCCTGTCTATTGCTCCATCCTGGCTGCTGTGGTTGTGGGCCTTGTGGCCTAC ATAGCCTTCAAGAGGTGGAACAGC

[0242] SEQ ID NO: 137 VLVH TIGIT 148b CH3 28z t2A NGFR codingDNA:ATGGAGGCTCCCGCTCAATTGCTTTTTCTTCTGCTTCTGTGGTTGCCTGACACAACTGGCGACATTCAGATGACACAGAGCCCCGCTAGCCTGAGCGCTAGCGTGGGCGAGACCGTGACCATCACCTGCAGAGCTAGCGAGCACATCTACAGCTACCTGAGCTGGTATCAGCAGAAGCAAGGCAAGAGCCCTCAGCTGCTGGTGTACAACGCCAAGACCCTGGCCGAGGGCGTGCCTAGCAGATTCAGCGGCAGCGGCAGCGGCACACAGTTCAGCCTGAAGATCAACAGCCTGCAGCCCGAGGACTTCGGCACCTACTACTGTCAGCACCACTTCGGCAGCCCCCTGACCTTCGGCGCCGGCACCACCCTGGAGCTGAAGGGCGGCGGGGGCAGTGGAGGAGGCGGAAGCGGTGGGGGGGGATCGGAGGTGCAGCTGCAGCAGAGCGGCCCCGAGCTGGTGAAGCCCGGCAGCAGCGTGAAGATGAGCTGCAAGGCTAGCGGCTACACCTTCAGCAGCTACGTGATGCACTGGGTGAAGCAGAAGCCCGGCCAAGGCCTGGAGTGGATCGGCTACATCGACCCCTACAACGACGGCGCCAAGTACAACGAGAAGTTCAAGGGCAAGGCCACCCTCACAAGCGACAAGAGCAGCAGCACCGCCTACATGGAGCTGAGCAGCCTGACAAGCGAGGACAGCGCCGTGTACTACTGCGCTAGAGGCGGCCCCTACGGCTGGTACTTCGACGTGTGGGGCGCCGGCACCACCGTGACCGTGAGCAGCGAGTCTAAATATGGCCCACCTTGCCCACCGTGCCCAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACgcCTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAAAAGATCCCAAATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGC ACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATG CAGGCCCTGCCtCCTCGCctcgagAGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGG GGACGTGGAGGAAAATCCCGGGCCCATGGGGGCAGGTGCCACCGGCCGCGCCATGGACGGGC CGCGCCTGCTGCTGTTGCTGCTTCTGGGGGTGTCCCTTGGAGGTGCCAAGGAGGCATGCCCC ACAGGCCTGTACACACACAGCGGTGAGTGCTGCAAAGCCTGCAACCTGGGCGAGGGTGTGGC CCAGCCTTGTGGAGCCAACCAGACCGTGTGTGAGCCCTGCCTGGACAGCGTGACGTTCTCCG ACGTGGTGAGCGCGACCGAGCCGTGCAAGCCGTGCACCGAGTGCGTGGGGCTCCAGAGCATG TCGGCGCCGTGCGTGGAGGCCGACGACGCCGTGTGCCGCTGCGCCTACGGCTACTACCAGGA TGAGACGACTGGGCGCTGCGAGGCGTGCCGCGTGTGCGAGGCGGGCTCGGGCCTCGTGTTCT CCTGCCAGGACAAGCAGAACACCGTGTGCGAGGAGTGCCCCGACGGCACGTATTCCGACGAG GCCAACCACGTGGACCCGTGCCTGCCCTGCACCGTGTGCGAGGACACCGAGCGCCAGCTCCG CGAGTGCACACGCTGGGCCGACGCCGAGTGCGAGGAGATCCCTGGCCGTTGGATTACACGGT CCACACCCCCAGAGGGCTCGGACAGCACAGCCCCCAGCACCCAGGAGCCTGAGGCACCTCCA GAACAAGACCTCATAGCCAGCACGGTGGCAGGTGTGGTGACCACAGTGATGGGCAGCTCCCA GCCCGTGGTGACCCGAGGCACCACCGACAACCTCATCCCTGTCTATTGCTCCATCCTGGCTG CTGTGGTTGTGGGCCTTGTGGCCTACATAGCCTTCAAGAGGTGGAACAGC

[0243] SEQ ID NO: 138 VLVH TIM3 148b CH3 28z t2A NGFR coding DNA:ATGGAGGCTCCCGCTCAATTGCTTTTTCTTCTGCTTCTGTGGTTGCCTGACACAACTGGCAT GGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTTCTGCTGCTCTGGCTCCCAGGTGCCAGAT GTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACC ATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGG GAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGT TCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGAT TTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCTCTCACTTTCGGCGGAGGGACCAA GGTGGAGATCAAACTGACGGGCGGCGGGGGCAGTGGAGGAGGCGGAAGCGGTGGGGGGGGAT CGATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGCTCTTTTAAGAGGTGTCCAGTGTCAG GTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTG TGCAGCGTCTGGATTCACCTCCAATAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCA AGGGGCTGGAGTGGGTGGCAGTTATATGGTATGATGGAAGTAATAAATACTATGGAGACTCC GTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAA CAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGATATGGTTCGGGGAGATGTTTT CCGAATACTTCCAGCACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCTAGCGAGTCT AAATATGGCCCACCTTGCCCACCGTGCCCAGGGCAGCCCCGAGAACCACAGGTGTACACCCT GCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCT TCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAG ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGA CAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACA ACGCCTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAAAAGATCCCAAATTTTGGGTG CTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTAT TTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCC GCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCC TATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAA CCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGAC GTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTAC AATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCG CCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCT ACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGCCTCGAGAGCAGAGCCGAGGGCAGGGGA AGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCATGGGGGCAGGTGCCACCGGCCGCGCCATGGACGGGCCGCGCCTGCTGCTGTTGCTGCTTCTGGGGGTGTCCCTTGGAGGTG CCAAGGAGGCATGCCCCACAGGCCTGTACACACACAGCGGTGAGTGCTGCAAAGCCTGCAAC CTGGGCGAGGGTGTGGCCCAGCCTTGTGGAGCCAACCAGACCGTGTGTGAGCCCTGCCTGGA CAGCGTGACGTTCTCCGACGTGGTGAGCGCGACCGAGCCGTGCAAGCCGTGCACCGAGTGCG TGGGGCTCCAGAGCATGTCGGCGCCGTGCGTGGAGGCCGACGACGCCGTGTGCCGCTGCGCC TACGGCTACTACCAGGATGAGACGACTGGGCGCTGCGAGGCGTGCCGCGTGTGCGAGGCGGG CTCGGGCCTCGTGTTCTCCTGCCAGGACAAGCAGAACACCGTGTGCGAGGAGTGCCCCGACG GCACGTATTCCGACGAGGCCAACCACGTGGACCCGTGCCTGCCCTGCACCGTGTGCGAGGAC ACCGAGCGCCAGCTCCGCGAGTGCACACGCTGGGCCGACGCCGAGTGCGAGGAGATCCCTGG CCGTTGGATTACACGGTCCACACCCCCAGAGGGCTCGGACAGCACAGCCCCCAGCACCCAGG AGCCTGAGGCACCTCCAGAACAAGACCTCATAGCCAGCACGGTGGCAGGTGTGGTGACCACA GTGATGGGCAGCTCCCAGCCCGTGGTGACCCGAGGCACCACCGACAACCTCATCCCTGTCTA TTGCTCCATCCTGGCTGCTGTGGTTGTGGGCCTTGTGGCCTACATAGCCTTCAAGAGGTGGA ACAGC

[0244] SEQ ID NO: 139 TC_TIGIT 148b CH3 28z t2A NGFR coding DNA:ATGGAGGCTCCCGCTCAATTGCTTTTTCTTCTGCTTCTGTGGTTGCCTGACACAACTGGCGA CATTCAGATGACACAGAGCCCCGCTAGCCTGAGCGCTAGCGTGGGCGAGACCGTGACCATCA CCTGCAGAGCTAGCGAGCACATCTACAGCTACCTGAGCTGGTATCAGCAGAAGCAAGGCAAG AGCCCTCAGCTGCTGGTGTACAACGCCAAGACCCTGGCCGAGGGCGTGCCTAGCAGATTCAG CGGCAGCGGCAGCGGCACACAGTTCAGCCTGAAGATCAACAGCCTGCAGCCCGAGGACTTCG GCACCTACTACTGTCAGCACCACTTCGGCAGCCCCCTGACCTTCGGCGCCGGCACCACCCTG GAGCTGAAGGGCGGCGGGGGCAGTGGAGGAGGCGGAAGCGGTGGGGGGGGATCGGAGGTGCA GCTGCAGCAGAGCGGCCCCGAGCTGGTGAAGCCCGGCAGCAGCGTGAAGATGAGCTGCAAGG CTAGCGGCTACACCTTCAGCAGCTACGTGATGCACTGGGTGAAGCAGAAGCCCGGCCAAGGC CTGGAGTGGATCGGCTACATCGACCCCTACAACGACGGCGCCAAGTACAACGAGAAGTTCAA GGGCAAGGCCACCCTCACAAGCGACAAGAGCAGCAGCACCGCCTACATGGAGCTGAGCAGCC TGACAAGCGAGGACAGCGCCGTGTACTACTGCGCTAGAGGCGGCCCCTACGGCTGGTACTTC GACGTGTGGGGCGCCGGCACCACCGTGACCGTGAGCAGCGAGTCTAAATATGGCCCACCTTG CCCACCGTGCCCAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATG AGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATC GCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCT GGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGC AGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACgcCTACACGCAGAAG AGCCTCTCCCTGTCTCCGGGTAAAAAAGATCCCAAATTTTGGGTGCTGGTGGTGGTTGGTGG AGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTA AGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACC CGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAA GTTCAGCAGGAGCctcgagAGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACG TGGAGGAAAATCCCGGGCCCATGGGGGCAGGTGCCACCGGCCGCGCCATGGACGGGCCGCGC CTGCTGCTGTTGCTGCTTCTGGGGGTGTCCCTTGGAGGTGCCAAGGAGGCATGCCCCACAGG CCTGTACACACACAGCGGTGAGTGCTGCAAAGCCTGCAACCTGGGCGAGGGTGTGGCCCAGC CTTGTGGAGCCAACCAGACCGTGTGTGAGCCCTGCCTGGACAGCGTGACGTTCTCCGACGTG GTGAGCGCGACCGAGCCGTGCAAGCCGTGCACCGAGTGCGTGGGGCTCCAGAGCATGTCGGC GCCGTGCGTGGAGGCCGACGACGCCGTGTGCCGCTGCGCCTACGGCTACTACCAGGATGAGA CGACTGGGCGCTGCGAGGCGTGCCGCGTGTGCGAGGCGGGCTCGGGCCTCGTGTTCTCCTGC CAGGACAAGCAGAACACCGTGTGCGAGGAGTGCCCCGACGGCACGTATTCCGACGAGGCCAA CCACGTGGACCCGTGCCTGCCCTGCACCGTGTGCGAGGACACCGAGCGCCAGCTCCGCGAGT GCACACGCTGGGCCGACGCCGAGTGCGAGGAGATCCCTGGCCGTTGGATTACACGGTCCACA CCCCCAGAGGGCTCGGACAGCACAGCCCCCAGCACCCAGGAGCCTGAGGCACCTCCAGAACAAGACCTCATAGCCAGCACGGTGGCAGGTGTGGTGACCACAGTGATGGGCAGCTCCCAGCCCGTGGTGACCCGAGGCACCACCGACAACCTCATCCCTGTCTATTGCTCCATCCTGGCTGCTGTGGTTGTGGGCCTTGTGGCCTACATAGCCTTCAAGAGGTGGAACAGC

[0245] In some aspects, one or more polypeptides of the present disclosure may comprise an amino acid sequence of, of at least, or of at most 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range or value derivable therein) identity to any one of SEQ ID NOs: 47-59, 140-148, 156-162, 168-172, 183-190, or 195-196.

[0246] SEQ ID NO: 47 CTLA4-P2A-dNGFR AA:MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAWLASSRGIASFVCEYAS PGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDS ICTGTSSGNQVNLTIQGLRAMDT GLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSL SKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPINLEGSGEGRGSLLTCGDVEENPGPMG AGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVC EPCLDSVTFSDWSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACR VCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAEC EEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGWTTVMGSSQPWTRGTTDN L I PVYCS I LAAVWGLVAY I AFKRWNS

[0247] SEQ ID NO: 48 CTLA4- AA:MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAWLASSRGIASFVCEYAS PGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDS ICTGTSSGNQVNLTIQGLRAMDT GLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSL SKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN

[0248] SEQ ID NO: 49 IL10 AA:MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLD NLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLR RCHRFLPCENKSKAVEQVKNAFNKLQEKGI YKAMSEFDI FINYIEAYMTMKIRN

[0249] SEQ ID NO: 50 signal-peptide ab-gamma AA:MDSYLLMWGLLTFIMVPGCQAELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGS LYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGH CREPPPWENEATERI YHFWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTG EMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETS I FTTEYQGGGGSGGG GSGGGGSGGGGSAVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCEL LPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISL QWHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTP DTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSGGGGSGGGGSLNTTILT PNGNEDTTADFFLTTMPTDSLSVSTLPLPEVQCFVFNVEYMNCTWNSSSEPQPTNLTLHYWY KNSDNDKVQKCSHYLFSEEITSGCQLQKKEIHLYQTFWQLQDPREPRRQATQMLKLQNLVI PWAPENLTLHKLSESQLELNWNNRFLNHCLEHLVQYRTDWDHSWTEQSVDYRHKFSLPSVDG QKRYTFRVRSRFNPLCGSAQHWSEWSHPIHWGSNTSKENPFLFALEAWISVGSMGLI ISLL CVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKG GALGEGPGASPCNQHSPYWAPPCYTLKPET

[0250] SEQ ID NO: 51 ab-gamma AA:ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCT SSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERI YHFWG QMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRP ESETSCLVTTTDFQIQTEMAATMETS I FTTEYQGGGGSGGGGSGGGGSGGGGSAVNGTSQFT CFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQK LTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQWHVETHRCNISWEISQASH YFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWS PWSQPLAFRTKPAALGKDTGGGGSGGGGSGGGGSLNTTILTPNGNEDTTADFFLTTMPTDSL SVSTLPLPEVQCFVFNVEYMNCTWNSSSEPQPTNLTLHYWYKNSDNDKVQKCSHYLFSEEIT SGCQLQKKEIHLYQTFWQLQDPREPRRQATQMLKLQNLVIPWAPENLTLHKLSESQLELNW NNRFLNHCLEHLVQYRTDWDHSWTEQSVDYRHKFSLPSVDGQKRYTFRVRSRFNPLCGSAQH WSEWSHPIHWGSNTSKENPFLFALEAWISVGSMGLI ISLLCVYFWLERTMPRIPTLKNLED LVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAP PCYTLKPET

[0251] SEQ ID NO: 52 signal-peptide ba-gamma AA:MAAPALSWRLPLLILLLPLATSWASAAVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVH AWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDF KPFENLRLMAPISLQWHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTL KQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSGGG GSGGGGSGGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNS SHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWEN EATERI YHFWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPG EEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETS I FTTEYQGGGGSGGGGSGGGGSLN TTILTPNGNEDTTADFFLTTMPTDSLSVSTLPLPEVQCFVFNVEYMNCTWNSSSEPQPTNLT LHYWYKNSDNDKVQKCSHYLFSEEITSGCQLQKKEIHLYQTFWQLQDPREPRRQATQMLKL QNLVIPWAPENLTLHKLSESQLELNWNNRFLNHCLEHLVQYRTDWDHSWTEQSVDYRHKFSL PSVDGQKRYTFRVRSRFNPLCGSAQHWSEWSHPIHWGSNTSKENPFLFALEAWISVGSMGL I ISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSE IPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET

[0252] SEQ ID NO: 53 ba-gamma AA:AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNL ILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQWHVETHRCNI SWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKP LQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSGGGGSGGGGSGGGGSELCDDDPPEIPHAT FKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQ PEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFWGQMVYYQCVQGYRAL HRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQ IQTEMAATMETS I FTTEYQGGGGSGGGGSGGGGSLNTTILTPNGNEDTTADFFLTTMPTDSL SVSTLPLPEVQCFVFNVEYMNCTWNSSSEPQPTNLTLHYWYKNSDNDKVQKCSHYLFSEEIT SGCQLQKKEIHLYQTFWQLQDPREPRRQATQMLKLQNLVIPWAPENLTLHKLSESQLELNW NNRFLNHCLEHLVQYRTDWDHSWTEQSVDYRHKFSLPSVDGQKRYTFRVRSRFNPLCGSAQH WSEWSHPIHWGSNTSKENPFLFALEAWISVGSMGLI ISLLCVYFWLERTMPRIPTLKNLED LVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAP PCYTLKPET

[0253] SEQ ID NO: 54 beta-CD19 AA:MAAPALSWRLPLLILLLPLATSWASAAVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVH AWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDF KPFENLRLMAPISLQWHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTL KQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTIPWLGHLL VGLSGAEGEI ILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPS SSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDA LEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSL LGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREA GEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLVLEEGRGS LLTCGDVEENPGPMPPPRLLFFLLFLTPMEVRPEEPLWKVEEGDNAVLQCLKGTSDGPTQQ LTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFI FNVSQQMGGFYLCQPGPPSEKAWQPGW TVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPC LPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRP ARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMS FHLE I TARPVLWHWLLRTGGWKVSAVTL AYLI FCLCSLVGILHLQRALVLRRKRKRMTDPTRRF

[0254] SEQ ID NO: 55 signal-peptide_TGFbl-Q8 AA:MPPSGLRLLPLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKLRLASP PSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKF KQSTHS IYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRL LAPSDSPEWLSFDVTGWRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLA TIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKW IHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGR KPKVEQLSNMIVRSCKCSRAKRGSGATNFSLLKQAGDVEENPGPMGLVRRGARAGPRMPRGW TALCLLSLLPSGFMAELPTQGTFSNVSTNVSPAKPTTTPAPRPPTPAPTIASQPLSLRPEAC RPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPW

[0255] SEQ ID NO: 56 TGFbl-Q8 AA:LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGE SAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHS IYMFFNTSELREAVPEPVLLSRA ELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGWRQWLSRGGEIE GFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHR RALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKV LALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCSRAKRGSGATNF SLLKQAGDVEENPGPMGLVRRGARAGPRMPRGWTALCLLSLLPSGFMAELPTQGTFSNVSTN VSPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTC GVLLLSLVITLYCNHRNRRRVCKCPRPW

[0256] SEQ ID NO: 57 IL12A-2A-EBI3-Q8 AA:MWPPGSASQPPPSPAAATGLHPAARPVSLQCRLSMCPARSLLLVATLVLLDHLSLARNLPVA TPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLEL TKNESCLNSRETSFITNGSCLASRKTSFMMALCLSS IYEDLKMYQVEFKTMNAKLLMDPKRQ I FLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMS YLNASRAKRGSGEGRGSLLTCGDVEENPGPMTPQLLLALVLWASCPPCSGRKGPPAALTLPR VQCRASRYPIAVDCSWTLPPAPNSTSPVSFIATYRLGMAARGHSWPCLQQTPTSTSCTITDV QLFSMAPYVLNVTAVHPWGSSSSFVPFITEHI IKPDPPEGVRLSPLAERQLQVQWEPPGSWP FPEI FSLKYWIRYKRQGAARFHRVGPIEATSFILRAVRPRARYYVQVAAQDLTDYGELSDWS LPATATMSLGKRAKRGSGATNFSLLKQAGDVEENPGPMGLVRRGARAGPRMPRGWTALCLLSLLPSGFMAELPTQGTFSNVSTNVSPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPW

[0257] SEQ ID NO: 58 IL12A-2A-EBI3 AA:MWPPGSASQPPPSPAAATGLHPAARPVSLQCRLSMCPARSLLLVATLVLLDHLSLARNLPVA TPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLEL TKNESCLNSRETSFITNGSCLASRKTSFMMALCLSS IYEDLKMYQVEFKTMNAKLLMDPKRQ I FLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMS YLNASRAKRGSGEGRGSLLTCGDVEENPGPMTPQLLLALVLWASCPPCSGRKGPPAALTLPR VQCRASRYPIAVDCSWTLPPAPNSTSPVSFIATYRLGMAARGHSWPCLQQTPTSTSCTITDV QLFSMAPYVLNVTAVHPWGSSSSFVPFITEHI IKPDPPEGVRLSPLAERQLQVQWEPPGSWP FPEI FSLKYWIRYKRQGAARFHRVGPIEATSFILRAVRPRARYYVQVAAQDLTDYGELSDWS LPATATMSLGKRAKR

[0258] SEQ ID NO: 59 IL12A furin-cleavage-site AA:MWPPGSASQPPPSPAAATGLHPAARPVSLQCRLSMCPARSLLLVATLVLLDHLSLARNLPVA TPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLEL TKNESCLNSRETSFITNGSCLASRKTSFMMALCLSS IYEDLKMYQVEFKTMNAKLLMDPKRQ I FLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMS YLNASRAKR

[0259] SEQ ID NO: 140 PDL1 380bBB coding AA:MRI FAVFI FMTYWHLLNAFTVTVPKDLYWEYGSNMTIECKFPVEKQLDLAALIVYWEMEDK NI IQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYK RITVKVNAPYNKINQRILWDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKR EEKLFNVTSTLRINTTTNEI FYCTFRRLDPEENHTAELVIPELPLAHPPNERESKYGPPCPP CPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWVLVWGGVL ACYSLLVTVAFI I FWVRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPR

[0260] SEQ ID NO: 141 VHVL PD1 380b_coding AA:MEAPAQLLFLLLLWLPDTTGQVQLQESGPGWKPSGTLSLTCAISGGS IGSGGS IRSTRWWS WVRQSPGKGLEWIGEIYHSGSTNYNPSLKSRVTISLDKSRNHFSLRLNSVTAADTAVYYCAR QDYGDSGDWYFDLWGKGTMVTVSSGGGGSGGGGSGGGGSNFMLTQPHSVSESPGKTVTISCT RSSGS IASNSVQWYQQRPGSSPTTVI YEDNQRPSGVPDRFSGS IDSSSNSASLTVSGLKTED EADYYCQSSDSSAWFGSGTKLTVLESKYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGKKDPKFWVLVWGGVLACYSLLVTVAFI I FWVRSRVKFSRSAD APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAY SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0261] SEQ ID NO: 142 VLVH PD1 380b_coding AA:MEAPAQLLFLLLLWLPDTTGNFMLTQPHSVSESPGKTVTISCTRSSGS IASNSVQWYQQRPG SSPTTVI YEDNQRPSGVPDRFSGS IDSSSNSASLTVSGLKTEDEADYYCQSSDSSAWFGSG TKLTVLGGGGSGGGGSGGGGSQVQLQESGPGWKPSGTLSLTCAISGGS IGSGGS IRSTRWW SWVRQSPGKGLEWIGEIYHSGSTNYNPSLKSRVTISLDKSRNHFSLRLNSVTAADTAVYYCA RQDYGDSGDWYFDLWGKGTMVTVSSESKYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGKKDPKFWVLVWGGVLACYSLLVTVAFI I FWVRSRVKFSRSAD APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAY SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0262] SEQ ID NO: 143 VHVL TIGIT 380b_coding AA:MEAPAQLLFLLLLWLPDTTGEVQLQQSGPELVKPGSSVKMSCKASGYTFSSYVMHWVKQKPG QGLEWIGYIDPYNDGAKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGGPYGW YFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTITCRASEHIYSY LSWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGTYYCQHHFGS PLTFGAGTTLELKESKYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGKKDPKFWVLVWGGVLACYSLLVTVAFI I FWVRSRVKFSRSADAPAYQQGQNQLY NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRG KGHDGLYQGLSTATKDTYDALHMQALPPR

[0263] SEQ ID NO: 144 VLVH TIGIT 380b_coding AA:MEAPAQLLFLLLLWLPDTTGDIQMTQSPASLSASVGETVTITCRASEHIYSYLSWYQQKQGK SPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGTYYCQHHFGSPLTFGAGTTL ELKGGGGSGGGGSGGGGSEVQLQQSGPELVKPGSSVKMSCKASGYTFSSYVMHWVKQKPGQG LEWIGYIDPYNDGAKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGGPYGWYF DVWGAGTTVTVSSESKYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGKKDPKFWVLVWGGVLACYSLLVTVAFI I FWVRSRVKFSRSADAPAYQQGQNQLY NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRG KGHDGLYQGLSTATKDTYDALHMQALPPR

[0264] SEQ ID NO: 145 VLVH PD 1 148b CH3 28z t2A NGFR coding AA:MEAPAQLLFLLLLWLPDTTGNFMLTQPHSVSESPGKTVTISCTRSSGS IASNSVQWYQQRPG SSPTTVI YEDNQRPSGVPDRFSGS IDSSSNSASLTVSGLKTEDEADYYCQSSDSSAWFGSG TKLTVLGGGGSGGGGSGGGGSQVQLQESGPGWKPSGTLSLTCAISGGS IGSGGS IRSTRWW SWVRQSPGKGLEWIGEIYHSGSTNYNPSLKSRVTISLDKSRNHFSLRLNSVTAADTAVYYCA RQDYGDSGDWYFDLWGKGTMVTVSSESKYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNAYTQKSLSLSPGKKDPKFWVLVWGGVLACYSLLVTVAFI I FWVRSKRSRLLHSD YMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEY DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS TATKDTYDALHMQALPPRLESRAEGRGSLLTCGDVEENPGPMGAGATGRAMDGPRLLLLLLL GVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDWSATEPC KPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTV CEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDS TAPSTQEPEAPPEQDLIASTVAGWTTVMGSSQPWTRGTTDNLIPVYCS ILAAVWGLVAY IAFKRWNS

[0265] SEQ ID NO: 146 VLVH TIGIT 148b CH3 28z t2A NGFR codingAA:MEAPAQLLFLLLLWLPDTTGDIQMTQSPASLSASVGETVTITCRASEHIYSYLSWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGTYYCQHHFGSPLTFGAGTTLELKGGGGSGGGGSGGGGSEVQLQQSGPELVKPGSSVKMSCKASGYTFSSYVMHWVKQKPGQGLEWIGYIDPYNDGAKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGGPYGWYFDVWGAGTTVTVSSESKYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKKDPKFWVLVWGGVLACYSLLVTVAFI I FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLESRAEGRGSLLTCGDVEENPGPMGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDWSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDL IAS TVAGWT TVMGS S QPWTRGT TDNL I PVYCS I LAAVWGLVAY I AFKRWNS

[0266] SEQ ID NO: 147 VLVH TIM3 148b CH3 28z t2A NGFR coding AA:MEAPAQLLFLLLLWLPDTTGMDMRVPAQLLGLLLLWLPGARCAIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKLTGGGGSGGGGSGGGGSMEFGLSWVFLVALLRGVQCQVQLVESGGGWQPGRSLRLSCAASGFTSNSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIWFGEMFSEYFQHWGQGTLVTVSSASESKYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKKDPKFWVLVWGGVLACYSLLVTVAFI I FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLESRAEGRGSLLTCGDVEENPGPMGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDWSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGWTTVMGSSQPWTRGTTDNLIPVYCS ILAAVWGLVAYIAFKRWNS

[0267] SEQ ID NO: 148 TC_TIGIT 148b CH3 28z t2A NGFR coding AA:MEAPAQLLFLLLLWLPDTTGDIQMTQSPASLSASVGETVTITCRASEHIYSYLSWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGTYYCQHHFGSPLTFGAGTTLELKGGGGSGGGGSGGGGSEVQLQQSGPELVKPGSSVKMSCKASGYTFSSYVMHWVKQKPGQGLEWIGYIDPYNDGAKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGGPYGWYFDVWGAGTTVTVSSESKYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKKDPKFWVLVWGGVLACYSLLVTVAFI I FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSLESRAEGRGSLLTCGDVEENPGPMGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGWTTVMGSSQPWTRGTTDNLIPVYCS ILAAVWGLVAY I AFKRWNS3. Co-inhibitor engager

[0268] As used herein “co-inhibitor engager” refers to a molecule, e.g., a protein or polypeptide, including but not limited to, antibodies, receptors, receptor ligands, and / or fragments thereof, that is capable of binding an antigen target. In some aspects, a co-inhibitorengager may be localized to the extracellular space of a cell while remaining anchored to the plasma membrane. In some aspects, a transmembrane domain may be operably linked to the co-inhibitor engager to anchor said co-inhibitor engager to the plasma membrane. In some aspects, a signal peptide may be operably linked to the co-inhibitor engager to target said coinhibitor engager to the extracellular space and / or plasma membrane. In some aspects, a coinhibitor engager can be directed to a T-cell checkpoint activator and / or T-cell inhibitor. In some aspects, a co-inhibitor engager may recognize and bind inflammatory markers, also referred to as activation markers. In some aspects, a co-inhibitor engager may engage and antagonize an activation receptor and / or maker. In some aspects, a co-inhibitor engager may engage a co-inhibitory receptor in an agonistic manner. In some aspects, inflammatory markers comprise, consist essentially of, consist of, or do not comprise LAG3, CD80, CD3, CD5, CD86, PD1, CD160, CD200, BTLA, PD1H, LAIR1, TIM1, TIM3, TIM4, 2B4, CEACAM-1, and / or TIGIT. In certain aspects, a co-inhibitor engager may be directed to an inhibitory receptor, and may comprise a ligand for an inhibitory receptor. In certain aspects, a co-inhibitor engager may be directed to an inhibitory receptor as described in Odorizzi & Wherry 2012, “Inhibitory Receptors on Lymphocytes: Insights from Infections” J. Immunol,' which is incorporated herein by reference in its entirety for the purposes described herein. In some aspects, a co-inhibitor engager guides a cell, which comprises said co-inhibitor engager, to a target cell expressing the molecule recognized by the co-inhibitor engager. In some aspects, a co-inhibitor engager is capable of activating its target, such as an inhibitory receptor, including but not limited to PD-1 and / or TIGIT. In some aspects, a co-inhibitor engager is capable of both guiding a cell and activating its target.

[0269] In some aspects, allo- or auto-reactive immune cells, e.g. T cells, are screened for upregulation of co-inhibitory molecules, e.g., co-inhibitory receptors. In some aspects, allo- or auto-reactive T cells are screened for upregulation of PD-1 and / or TIGIT.

[0270] In some aspects, engineered cells provided herein express a co-inhibitor engager. In some aspects, a co-inhibitor engager is specific to PD1 or TIGIT. In some aspects, engineered cells expressing a co-inhibitor engager described herein, show reduced expression of co-inhibitory receptors via masking of the target molecules. In some aspects, engineered cells expressing a co-inhibitor engager provided herein have improved immunosuppressive functions and / or engagement of allo- or auto-reactive immune cells. In some aspects, engineered cells provided herein inhibit proliferation of allo- or auto-reactive immune cells. In some aspects, an engineered cell is a Treg cell, mesenchymal stem cells (MSC), fibroblasts,adipocytes, myeloid cells, B-cell, T cell, immortalized cell lines, division-incompetent cell lines, and / or epithelial cells.

[0271] In some aspects, a co-inhibitor engager may comprise a sequence of, of at least, or of at most 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range or value derivable therein) identity to any one of SEQ ID NOs: 131- 196.

[0272] In some aspects, a polypeptide or peptide of the disclosure, e.g., a co-inhibitor engager, may comprise a combination of domains, e.g., comprising a sequence of, of at least, or of at most 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range or value derivable therein) identity to any one of SEQ ID NOs: 149- 162, may or may not be operably linked to each other by one or more linker regions. In some aspects, a polypeptide or peptide of the disclosure, e.g., a co-inhibitor engager, may be operably linked to a hinge domain, e.g., comprising a sequence of, of at least, or of at most 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range or value derivable therein) identity to any one of SEQ ID NOs: 163-164, or 168-169. In some aspects, a polypeptide or peptide of the disclosure, e.g., a co-inhibitor engager, may or may not be operably linked to a transmembrane domain directly or through one or more hinge domains and / or linker regions.

[0273] SEQ ID NO: 149 PDL1 DNA:ATGAGGATATTTGCTGTCTTTATATTCATGACCTACTGGCATTTGCTGAACGCATTTACTGT CACGGTTCCCAAGGACCTATATGTGGTAGAGTATGGTAGCAATATGACAATTGAATGCAAAT TCCCAGTAGAAAAACAATTAGACCTGGCTGCACTAATTGTCTATTGGGAAATGGAGGATAAG AACAT TAT T CAAT T T GT GCAT GGAGAGGAAGACC T GAAGGT T CAGCATAGTAGC TACAGACA GAGGGCCCGGCTGTTGAAGGACCAGCTCTCCCTGGGAAATGCTGCACTTCAGATCACAGATG TGAAATTGCAGGATGCAGGGGTGTACCGCTGCATGATCAGCTATGGTGGTGCCGACTACAAG C GAAT T AC T G T GAAAG T CAAT G C C C C AT AC AAC AAAAT C AAC C AAAGAAT TTTGGTTGTGGA TCCAGTCACCTCTGAACATGAACTGACATGTCAGGCTGAGGGCTACCCCAAGGCCGAAGTCA T C T G GAC AAG C AG T GAC CAT C AAG T C C T GAG T G G T AAGAC C AC C AC C AC CAAT T C C AAGAGA GAGGAGAAGC T T T T CAAT GT GAC C AG C AC AC T GAGAAT CAACACAACAAC TAAT GAGAT T T T CTACTGCACTTTTAGGAGATTAGATCCTGAGGAAAACCATACAGCTGAATTGGTCATCCCAG AAC T AC C T C T G G C AC AT C C T C C AAAT GAAAG G

[0274] SEQ ID NO: 156 PDL1 AA:MRI FAVFI FMTYWHLLNAFTVTVPKDLYWEYGSNMT IECKFPVEKQLDLAALIVYWEMEDK NT IQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQI TDVKLQDAGVYRCMI SYGGADYKRI TVKVNAPYNKINQRILWDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKR EEKLFNVTSTLRINTTTNE I FYCTFRRLDPEENHTAELVI PELPLAHPPNER

[0275] SEQ ID NO: 173 PDL1 380bBB ex DNA:ATGAGGATATTTGCTGTCTTTATATTCATGACCTACTGGCATTTGCTGAACGCATTTACTGT CACGGTTCCCAAGGACCTATATGTGGTAGAGTATGGTAGCAATATGACAATTGAATGCAAAT TCCCAGTAGAAAAACAATTAGACCTGGCTGCACTAATTGTCTATTGGGAAATGGAGGATAAG AACAT TAT T CAAT T T GT GCAT GGAGAGGAAGACC T GAAGGT T CAGCATAGTAGC TACAGACA GAGGGCCCGGCTGTTGAAGGACCAGCTCTCCCTGGGAAATGCTGCACTTCAGATCACAGATG TGAAATTGCAGGATGCAGGGGTGTACCGCTGCATGATCAGCTATGGTGGTGCCGACTACAAG C GAAT T AC T G T GAAAG T CAAT G C C C C AT AC AAC AAAAT C AAC C AAAGAAT TTTGGTTGTGGA TCCAGTCACCTCTGAACATGAACTGACATGTCAGGCTGAGGGCTACCCCAAGGCCGAAGTCA T C T G GAC AAG C AG T GAC CAT C AAG T C C T GAG T G G T AAGAC C AC C AC C AC CAAT T C C AAGAGA GAGGAGAAGC T T T T CAAT GT GAC C AG C AC AC T GAGAAT CAACACAACAAC TAAT GAGAT T T T CTACTGCACTTTTAGGAGATTAGATCCTGAGGAAAACCATACAGCTGAATTGGTCATCCCAG AACTACCTCTGGCACATCCTCCAAATGAAAGGGAGTCTAAATATGGCCCACCTTGCCCACCG TGCCCAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGAC CAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGG AGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCC GACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAA CGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCT CCCTGTCTCCGGGTAAA

[0276] SEQ ID NO: 174 VHVL PD1 380b_ex DNA:ATGGAGGCTCCCGCTCAATTGCTTTTTCTTCTGCTTCTGTGGTTGCCTGACACAACTGGCCA GGTGCAGCTGCAGGAGTCGGGCCCAGGAGTGGTGAAGCCTTCGGGGACCCTGTCCCTCACCT GCGCTATTTCTGGTGGCTCCATCGGCTCTGGTGGCTCCATCAGAAGTACTAGGTGGTGGAGT TGGGTCCGCCAGTCCCCAGGGAAGGGGCTGGAGTGGATAGGCGAAATCTATCATAGTGGGAG C AC C AAC TAG AAC CCGTCCCT C AAGAG T C G C G T C AC CAT AT C AC T AGAC AAG T C T AG GAAT C ACTTCTCCCTGAGGCTGAACTCTGTGACCGCCGCGGACACGGCCGTTTATTACTGTGCGAGA CAGGACTACGGTGACTCCGGCGACTGGTACTTCGATCTGTGGGGCAAGGGGACAATGGTCAC CGTCTCCTCAGGCGGCGGGGGCAGTGGAGGAGGCGGAAGCGGTGGGGGGGGATCGAATTTTA TGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCATCTCCTGCACC CGCAGCAGTGGCAGCATTGCCAGCAACTCTGTGCAGTGGTACCAGCAGCGCCCGGGCAGTTC CCCCACCACTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTG GCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCGTCTCTGGACTGAAGACTGAGGAC GAGGCTGACTACTACTGTCAGTCTTCTGATAGCAGCGCTGTGGTATTCGGCAGTGGGACCAA GCTGACCGTCCTAGAGTCTAAATATGGCCCACCTTGCCCACCGTGCCCAGGGCAGCCCCGAG AACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTG ACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCA ACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCT ACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTG ATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

[0277] SEQ ID NO: 175 VLVH PD1 380b_ex DNA:ATGGAGGCTCCCGCTCAATTGCTTTTTCTTCTGCTTCTGTGGTTGCCTGACACAACTGGCAA TTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCATCTCCT GCACCCGCAGCAGTGGCAGCATTGCCAGCAACTCTGTGCAGTGGTACCAGCAGCGCCCGGGC AGTTCCCCCACCACTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGTT CTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCGTCTCTGGACTGAAGACTGAGGACGAGGCTGACTACTACTGTCAGTCTTCTGATAGCAGCGCTGTGGTATTCGGCAGTGGG ACCAAGCTGACCGTCCTAGGCGGCGGGGGCAGTGGAGGAGGCGGAAGCGGTGGGGGGGGATC GCAGGTGCAGCTGCAGGAGTCGGGCCCAGGAGTGGTGAAGCCTTCGGGGACCCTGTCCCTCA CCTGCGCTATTTCTGGTGGCTCCATCGGCTCTGGTGGCTCCATCAGAAGTACTAGGTGGTGG AGTTGGGTCCGCCAGTCCCCAGGGAAGGGGCTGGAGTGGATAGGCGAAATCTATCATAGTGG GAG GAG C AAC T AC AAC CCGTCCCT C AAGAG T C G C G T GAG CAT AT C AC T AGAC AAG T C T AG GA ATCACTTCTCCCTGAGGCTGAACTCTGTGACCGCCGCGGACACGGCCGTTTATTACTGTGCG AGACAGGACTACGGTGACTCCGGCGACTGGTACTTCGATCTGTGGGGCAAGGGGACAATGGT CACCGTCTCCTCAGAGTCTAAATATGGCCCACCTTGCCCACCGTGCCCAGGGCAGCCCCGAG AACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTG ACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCA ACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCT ACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTG ATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

[0278] SEQ ID NO: 176 VHVL TIGIT 380b_ex DNA:ATGGAGGCTCCCGCTCAATTGCTTTTTCTTCTGCTTCTGTGGTTGCCTGACACAACTGGCGA GGTGCAGCTGCAGCAGAGCGGCCCCGAGCTGGTGAAGCCCGGCAGCAGCGTGAAGATGAGCT GCAAGGCTAGCGGCTACACCTTCAGCAGCTACGTGATGCACTGGGTGAAGCAGAAGCCCGGC CAAGGCCTGGAGTGGATCGGCTACATCGACCCCTACAACGACGGCGCCAAGTACAACGAGAA GTTCAAGGGCAAGGCCACCCTCACAAGCGACAAGAGCAGCAGCACCGCCTACATGGAGCTGA GCAGCCTGACAAGCGAGGACAGCGCCGTGTACTACTGCGCTAGAGGCGGCCCCTACGGCTGG TACTTCGACGTGTGGGGCGCCGGCACCACCGTGACCGTGAGCAGCGGCGGCGGGGGCAGTGG AGGAGGCGGAAGCGGTGGGGGGGGATCGGACATTCAGATGACACAGAGCCCCGCTAGCCTGA GCGCTAGCGTGGGCGAGACCGTGACCATCACCTGCAGAGCTAGCGAGCACATCTACAGCTAC CTGAGCTGGTATCAGCAGAAGCAAGGCAAGAGCCCTCAGCTGCTGGTGTACAACGCCAAGAC CCTGGCCGAGGGCGTGCCTAGCAGATTCAGCGGCAGCGGCAGCGGCACACAGTTCAGCCTGA AGATCAACAGCCTGCAGCCCGAGGACTTCGGCACCTACTACTGTCAGCACCACTTCGGCAGC CCCCTGACCTTCGGCGCCGGCACCACCCTGGAGCTGAAGGAGTCTAAATATGGCCCACCTTG CCCACCGTGCCCAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATG AGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATC GCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCT GGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGC AGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAG AGCCTCTCCCTGTCTCCGGGTAAA

[0279] SEQ ID NO: 177 VLVH TIGIT 380b_ex DNA:ATGGAGGCTCCCGCTCAATTGCTTTTTCTTCTGCTTCTGTGGTTGCCTGACACAACTGGCGA CATTCAGATGACACAGAGCCCCGCTAGCCTGAGCGCTAGCGTGGGCGAGACCGTGACCATCA CCTGCAGAGCTAGCGAGCACATCTACAGCTACCTGAGCTGGTATCAGCAGAAGCAAGGCAAG AGCCCTCAGCTGCTGGTGTACAACGCCAAGACCCTGGCCGAGGGCGTGCCTAGCAGATTCAG CGGCAGCGGCAGCGGCACACAGTTCAGCCTGAAGATCAACAGCCTGCAGCCCGAGGACTTCG GCACCTACTACTGTCAGCACCACTTCGGCAGCCCCCTGACCTTCGGCGCCGGCACCACCCTG GAGCTGAAGGGCGGCGGGGGCAGTGGAGGAGGCGGAAGCGGTGGGGGGGGATCGGAGGTGCA GCTGCAGCAGAGCGGCCCCGAGCTGGTGAAGCCCGGCAGCAGCGTGAAGATGAGCTGCAAGG CTAGCGGCTACACCTTCAGCAGCTACGTGATGCACTGGGTGAAGCAGAAGCCCGGCCAAGGC CTGGAGTGGATCGGCTACATCGACCCCTACAACGACGGCGCCAAGTACAACGAGAAGTTCAA GGGCAAGGCCACCCTCACAAGCGACAAGAGCAGCAGCACCGCCTACATGGAGCTGAGCAGCC TGACAAGCGAGGACAGCGCCGTGTACTACTGCGCTAGAGGCGGCCCCTACGGCTGGTACTTC GACGTGTGGGGCGCCGGCACCACCGTGACCGTGAGCAGCGAGTCTAAATATGGCCCACCTTGCCCACCGTGCCCAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATG AGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATC GCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCT GGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGC AGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAG AGCCTCTCCCTGTCTCCGGGTAAA

[0280] SEQ ID NO: 178 VLVH PD 1 148b CH3 28z t2A NGFR ex DNA:ATGGAGGCTCCCGCTCAATTGCTTTTTCTTCTGCTTCTGTGGTTGCCTGACACAACTGGCAA TTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCATCTCCT GCACCCGCAGCAGTGGCAGCATTGCCAGCAACTCTGTGCAGTGGTACCAGCAGCGCCCGGGC AGTTCCCCCACCACTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGTT CTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCGTCTCTGGACTGAAGACTG AGGACGAGGCTGACTACTACTGTCAGTCTTCTGATAGCAGCGCTGTGGTATTCGGCAGTGGG ACCAAGCTGACCGTCCTAGGCGGCGGGGGCAGTGGAGGAGGCGGAAGCGGTGGGGGGGGATC GCAGGTGCAGCTGCAGGAGTCGGGCCCAGGAGTGGTGAAGCCTTCGGGGACCCTGTCCCTCA CCTGCGCTATTTCTGGTGGCTCCATCGGCTCTGGTGGCTCCATCAGAAGTACTAGGTGGTGG AGTTGGGTCCGCCAGTCCCCAGGGAAGGGGCTGGAGTGGATAGGCGAAATCTATCATAGTGG GAG GAG C AAC T AC AAC CCGTCCCT C AAGAG T C G C G T GAG CAT AT C AC T AGAC AAG T C T AG GA ATCACTTCTCCCTGAGGCTGAACTCTGTGACCGCCGCGGACACGGCCGTTTATTACTGTGCG AGACAGGACTACGGTGACTCCGGCGACTGGTACTTCGATCTGTGGGGCAAGGGGACAATGGT CACCGTCTCCTCAGAGTCTAAATATGGCCCACCTTGCCCACCGTGCCCAGGGCAGCCCCGAG AACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTG ACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCA ACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCT ACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTG ATGCATGAGGCTCTGCACAACGCCTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAAA AGATC

[0281] SEQ ID NO: 179 VLVH TIGIT 148b CH3 28z t2A NGFR ex DNA:ATGGAGGCTCCCGCTCAATTGCTTTTTCTTCTGCTTCTGTGGTTGCCTGACACAACTGGCGA CATTCAGATGACACAGAGCCCCGCTAGCCTGAGCGCTAGCGTGGGCGAGACCGTGACCATCA CCTGCAGAGCTAGCGAGCACATCTACAGCTACCTGAGCTGGTATCAGCAGAAGCAAGGCAAG AGCCCTCAGCTGCTGGTGTACAACGCCAAGACCCTGGCCGAGGGCGTGCCTAGCAGATTCAG CGGCAGCGGCAGCGGCACACAGTTCAGCCTGAAGATCAACAGCCTGCAGCCCGAGGACTTCG GCACCTACTACTGTCAGCACCACTTCGGCAGCCCCCTGACCTTCGGCGCCGGCACCACCCTG GAGCTGAAGGGCGGCGGGGGCAGTGGAGGAGGCGGAAGCGGTGGGGGGGGATCGGAGGTGCA GCTGCAGCAGAGCGGCCCCGAGCTGGTGAAGCCCGGCAGCAGCGTGAAGATGAGCTGCAAGG CTAGCGGCTACACCTTCAGCAGCTACGTGATGCACTGGGTGAAGCAGAAGCCCGGCCAAGGC CTGGAGTGGATCGGCTACATCGACCCCTACAACGACGGCGCCAAGTACAACGAGAAGTTCAA GGGCAAGGCCACCCTCACAAGCGACAAGAGCAGCAGCACCGCCTACATGGAGCTGAGCAGCC TGACAAGCGAGGACAGCGCCGTGTACTACTGCGCTAGAGGCGGCCCCTACGGCTGGTACTTC GACGTGTGGGGCGCCGGCACCACCGTGACCGTGAGCAGCGAGTCTAAATATGGCCCACCTTG CCCACCGTGCCCAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATG AGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATC GCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCT GGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGC AGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACgcCTACACGCAGAAG AGCCTCTCCCTGTCTCCGGGTAAAAAAGATC

[0282] SEQ ID NO: 180 VLVH TIM3 148b CH3 28z t2A NGFR ex DNA:ATGGAGGCTCCCGCTCAATTGCTTTTTCTTCTGCTTCTGTGGTTGCCTGACACAACTGGCAT GGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTTCTGCTGCTCTGGCTCCCAGGTGCCAGAT GTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACC ATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGG GAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGT TCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGAT TTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCTCTCACTTTCGGCGGAGGGACCAA GGTGGAGATCAAACTGACGGGCGGCGGGGGCAGTGGAGGAGGCGGAAGCGGTGGGGGGGGAT CGATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGCTCTTTTAAGAGGTGTCCAGTGTCAG GTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTG TGCAGCGTCTGGATTCACCTCCAATAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCA AGGGGCTGGAGTGGGTGGCAGTTATATGGTATGATGGAAGTAATAAATACTATGGAGACTCC GTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAA CAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGATATGGTTCGGGGAGATGTTTT CCGAATACTTCCAGCACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCTAGCGAGTCT AAATATGGCCCACCTTGCCCACCGTGCCCAGGGCAGCCCCGAGAACCACAGGTGTACACCCT GCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCT TCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAG ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGA CAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACA ACGCCTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAAAAGATC

[0283] SEQ ID NO: 181 TC_TIGIT 148b CH3 28z t2A NGFR ex DNA:ATGGAGGCTCCCGCTCAATTGCTTTTTCTTCTGCTTCTGTGGTTGCCTGACACAACTGGCGA CATTCAGATGACACAGAGCCCCGCTAGCCTGAGCGCTAGCGTGGGCGAGACCGTGACCATCA CCTGCAGAGCTAGCGAGCACATCTACAGCTACCTGAGCTGGTATCAGCAGAAGCAAGGCAAG AGCCCTCAGCTGCTGGTGTACAACGCCAAGACCCTGGCCGAGGGCGTGCCTAGCAGATTCAG CGGCAGCGGCAGCGGCACACAGTTCAGCCTGAAGATCAACAGCCTGCAGCCCGAGGACTTCG GCACCTACTACTGTCAGCACCACTTCGGCAGCCCCCTGACCTTCGGCGCCGGCACCACCCTG GAGCTGAAGGGCGGCGGGGGCAGTGGAGGAGGCGGAAGCGGTGGGGGGGGATCGGAGGTGCA GCTGCAGCAGAGCGGCCCCGAGCTGGTGAAGCCCGGCAGCAGCGTGAAGATGAGCTGCAAGG CTAGCGGCTACACCTTCAGCAGCTACGTGATGCACTGGGTGAAGCAGAAGCCCGGCCAAGGC CTGGAGTGGATCGGCTACATCGACCCCTACAACGACGGCGCCAAGTACAACGAGAAGTTCAA GGGCAAGGCCACCCTCACAAGCGACAAGAGCAGCAGCACCGCCTACATGGAGCTGAGCAGCC TGACAAGCGAGGACAGCGCCGTGTACTACTGCGCTAGAGGCGGCCCCTACGGCTGGTACTTC GACGTGTGGGGCGCCGGCACCACCGTGACCGTGAGCAGCGAGTCTAAATATGGCCCACCTTG CCCACCGTGCCCAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATG AGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATC GCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCT GGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGC AGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACgcCTACACGCAGAAG AGCCTCTCCCTGTCTCCGGGTAAAAAAGATC

[0284] SEQ ID NO: 182 PDL1 380bBB ex AA:MRI FAVFI FMTYWHLLNAFTVTVPKDLYWEYGSNMT IECKFPVEKQLDLAALIVYWEMEDK NI IQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQI TDVKLQDAGVYRCMI SYGGADYK RI TVKVNAPYNKINQRILWDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKR EEKLFNVTSTLRINTTTNE I FYCTFRRLDPEENHTAELVI PELPLAHPPNERESKYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0285] SEQ ID NO: 183 VHVL PD1 380b_ex AA:MEAPAQLLFLLLLWLPDTTGQVQLQESGPGWKPSGTLSLTCAISGGS IGSGGS IRSTRWWS WVRQSPGKGLEWIGEIYHSGSTNYNPSLKSRVTISLDKSRNHFSLRLNSVTAADTAVYYCAR QDYGDSGDWYFDLWGKGTMVTVSSGGGGSGGGGSGGGGSNFMLTQPHSVSESPGKTVTISCT RSSGS IASNSVQWYQQRPGSSPTTVI YEDNQRPSGVPDRFSGS IDSSSNSASLTVSGLKTED EADYYCQSSDSSAWFGSGTKLTVLESKYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK

[0286] SEQ ID NO: 184 VLVH PD1 380b_ex AA:MEAPAQLLFLLLLWLPDTTGNFMLTQPHSVSESPGKTVTISCTRSSGS IASNSVQWYQQRPG SSPTTVI YEDNQRPSGVPDRFSGS IDSSSNSASLTVSGLKTEDEADYYCQSSDSSAWFGSG TKLTVLGGGGSGGGGSGGGGSQVQLQESGPGWKPSGTLSLTCAISGGS IGSGGS IRSTRWW SWVRQSPGKGLEWIGEIYHSGSTNYNPSLKSRVTISLDKSRNHFSLRLNSVTAADTAVYYCA RQDYGDSGDWYFDLWGKGTMVTVSSESKYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK

[0287] SEQ ID NO: 185 VHVL TIGIT 380b_ex AA:MEAPAQLLFLLLLWLPDTTGEVQLQQSGPELVKPGSSVKMSCKASGYTFSSYVMHWVKQKPG QGLEWIGYIDPYNDGAKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGGPYGW YFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTITCRASEHIYSY LSWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGTYYCQHHFGS PLTFGAGTTLELKESKYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK

[0288] SEQ ID NO: 186 VLVH TIGIT 380b_ex AA:MEAPAQLLFLLLLWLPDTTGDIQMTQSPASLSASVGETVTITCRASEHIYSYLSWYQQKQGK SPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGTYYCQHHFGSPLTFGAGTTL ELKGGGGSGGGGSGGGGSEVQLQQSGPELVKPGSSVKMSCKASGYTFSSYVMHWVKQKPGQG LEWIGYIDPYNDGAKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGGPYGWYF DVWGAGTTVTVSSESKYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK

[0289] SEQ ID NO: 187 VLVH PD 1 148b CH3 28z t2A NGFR ex AA:MEAPAQLLFLLLLWLPDTTGNFMLTQPHSVSESPGKTVTISCTRSSGS IASNSVQWYQQRPG SSPTTVI YEDNQRPSGVPDRFSGS IDSSSNSASLTVSGLKTEDEADYYCQSSDSSAWFGSG TKLTVLGGGGSGGGGSGGGGSQVQLQESGPGWKPSGTLSLTCAISGGS IGSGGS IRSTRWW SWVRQSPGKGLEWIGEIYHSGSTNYNPSLKSRVTISLDKSRNHFSLRLNSVTAADTAVYYCA RQDYGDSGDWYFDLWGKGTMVTVSSESKYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNAYTQKSLSLSPGKKD

[0290] SEQ ID NO: 188 VLVH TIGIT 148b CH3 28z t2A NGFR ex AA:MEAPAQLLFLLLLWLPDTTGDIQMTQSPASLSASVGETVTITCRASEHIYSYLSWYQQKQGK SPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGTYYCQHHFGSPLTFGAGTTL ELKGGGGSGGGGSGGGGSEVQLQQSGPELVKPGSSVKMSCKASGYTFSSYVMHWVKQKPGQG LEWIGYIDPYNDGAKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGGPYGWYF DVWGAGTTVTVSSESKYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQK SLSLSPGKKD

[0291] SEQ ID NO: 189 VLVH TIM3 148b CH3 28z t2A NGFR ex AA:MEAPAQLLFLLLLWLPDTTGMDMRVPAQLLGLLLLWLPGARCAIQLTQSPSSLSASVGDRVT ITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQFNSYPLTFGGGTKVEIKLTGGGGSGGGGSGGGGSMEFGLSWVFLVALLRGVQCQ VQLVESGGGWQPGRSLRLSCAASGFTSNSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYGDS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIWFGEMFSEYFQHWGQGTLVTVSSASES KYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKKD

[0292] SEQ ID NO: 190 TC_TIGIT 148b CH3 28z t2A NGFR ex AA:MEAPAQLLFLLLLWLPDTTGDIQMTQSPASLSASVGETVTITCRASEHIYSYLSWYQQKQGK SPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGTYYCQHHFGSPLTFGAGTTL ELKGGGGSGGGGSGGGGSEVQLQQSGPELVKPGSSVKMSCKASGYTFSSYVMHWVKQKPGQG LEWIGYIDPYNDGAKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGGPYGWYF DVWGAGTTVTVSSESKYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQK SLSLSPGKKD a. Antibodies

[0293] As used herein, the terms “antibody” or “antibodies” collectively refer to immunoglobulins or immunoglobulin-like molecules including by way of example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, for example, in mammals such as humans, goats, rabbits and mice, as well as non-mammalian species, such as shark immunoglobulins. Unless specifically noted otherwise, the term “antibody” includes intact immunoglobulins and “antibody fragments” or “antigen binding fragments” that specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules (for example, antibodies and antibody fragments that have a binding constant for the molecule of interest that is at least 103M'1greater, at least 104M'1greater or at least 105M'1greater than a binding constant for other molecules in a biological sample). The term “antibody” also includes genetically engineered forms such as chimeric antibodies (for example, murine or humanized non-primate antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce ChemicalCo., Rockford, Ill.); Owen et al., Kuby Immunology, 7thEd., W.H. Freeman & Co., 2013; Murphy, Janeway’s Immunobiology, 8thEd., Garland Science, 2014; Male et al., Immunology (Raitt), 8thEd., Saunders, 2012; Parham, The Immune System, 4thEd., Garland Science, 2014.

[0294] In some aspects, a co-inhibitor engager of the disclosure may comprise, consist essentially of, or consist of an antibody, or a fragment thereof. An antibody or fragment thereof, may bind and activate co-inhibitory ligands and / or receptors. In some aspects, an antibody or fragment thereof may be an agonist or an antagonist of a coinhibitory molecule selected from the group comprising, consisting essentially of, or consisting of LAG3, CD80, CD86, PD1, CD160, CD200, BTLA, PD1H, LAIR1, TIM1, TIM3, TIM4, 2B4, CEACAM-1, CD5, CD3, and / or TIGIT. In certain aspects, a co-inhibitory engager may be directed to a stimulatory molecule, co-stimulatory molecule, and / or immune cells specific maker, for example but no limited to, those as described in Deng et al., “ITPRIPL1 binds CD3s to impede T cell activation and enable tumor immune evasion” Cell (2024), 5:80092-8674(24)00310-6; which is incorporated herein by reference in its entirety for the purposes described herein.

[0295] In certain aspects, an antibody or fragment thereof, consists essentially of, or consists of a sequence of, of at least, or of at most 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range or value derivable therein, identity to SEQ ID NOs: 150-155, or 157-162.

[0296] SEQ ID NO: 150 VH PDl DNA:CAGGTGCAGCTGCAGGAGTCGGGCCCAGGAGTGGTGAAGCCTTCGGGGACCCTGTCCCTCAC CTGCGCTATTTCTGGTGGCTCCATCGGCTCTGGTGGCTCCATCAGAAGTACTAGGTGGTGGA GTTGGGTCCGCCAGTCCCCAGGGAAGGGGCTGGAGTGGATAGGCGAAATCTATCATAGTGGG AG GAG C AAC T AC AAC CCGTCCCT C AAGAG T C G C G T GAG CAT AT C AC T AGAC AAG T C T AG GAA TCACTTCTCCCTGAGGCTGAACTCTGTGACCGCCGCGGACACGGCCGTTTATTACTGTGCGA GACAGGACTACGGTGACTCCGGCGACTGGTACTTCGATCTGTGGGGCAAGGGGACAATGGTC ACCGTCTCCTCA

[0297] SEQ ID NO: 151 VL PD1 DNA:AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCATCTC CTGCACCCGCAGCAGTGGCAGCATTGCCAGCAACTCTGTGCAGTGGTACCAGCAGCGCCCGG GCAGTTCCCCCACCACTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGG TTCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCGTCTCTGGACTGAAGAC TGAGGACGAGGCTGACTACTACTGTCAGTCTTCTGATAGCAGCGCTGTGGTATTCGGCAGTG GGACCAAGCTGACCGTCCTA

[0298] SEQ ID NO: 152 VH TIGIT DNA:GAGGTGCAGCTGCAGCAGAGCGGCCCCGAGCTGGTGAAGCCCGGCAGCAGCGTGAAGATGAG CTGCAAGGCTAGCGGCTACACCTTCAGCAGCTACGTGATGCACTGGGTGAAGCAGAAGCCCG GCCAAGGCCTGGAGTGGATCGGCTACATCGACCCCTACAACGACGGCGCCAAGTACAACGAGAAGTTCAAGGGCAAGGCCACCCTCACAAGCGACAAGAGCAGCAGCACCGCCTACATGGAGCTGAGCAGCCTGACAAGCGAGGACAGCGCCGTGTACTACTGCGCTAGAGGCGGCCCCTACGGCTGGTACTTCGACGTGTGGGGCGCCGGCACCACCGTGACCGTGAGCAGC

[0299] SEQ ID NO: 153 VL TIGIT DNA:GACATTCAGATGACACAGAGCCCCGCTAGCCTGAGCGCTAGCGTGGGCGAGACCGTGACCATCACCTGCAGAGCTAGCGAGCACATCTACAGCTACCTGAGCTGGTATCAGCAGAAGCAAGGCAAGAGCCCTCAGCTGCTGGTGTACAACGCCAAGACCCTGGCCGAGGGCGTGCCTAGCAGATTCAGCGGCAGCGGCAGCGGCACACAGTTCAGCCTGAAGATCAACAGCCTGCAGCCCGAGGACTT CGGCACCTACTACTGTCAGCACCACTTCGGCAGCCCCCTGACCTTCGGCGCCGGCACCACCC TGGAGCTGAAG

[0300] SEQ ID NO: 154 VH TIM3 DNA:ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGCTCTTTTAAGAGGTGTCCAGTGTCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTCCAATAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGATGGAAGTAATAAATACTATGGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACA GCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGATATGGTTCGGGGAGATGTTTTCC GAATACTTCCAGCACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCTAGC

[0301] SEQ ID NO: 155 VL TIM3 DNA:ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTTCTGCTGCTCTGGCTCCCAGGTGCCAGATGTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAG ATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCTCTCACTTTCGGCGGAGGGACC AAGGTGGAGATCAAACTGACG

[0302] SEQ ID NO: 157 VH PDl AA:QVQLQESGPGWKPSGTLSLTCAISGGS IGSGGS IRSTRWWSWVRQSPGKGLEWIGEIYHSG STNYNPSLKSRVTISLDKSRNHFSLRLNSVTAADTAVYYCARQDYGDSGDWYFDLWGKGTMV TVSS

[0303] SEQ ID NO: 158 VL PDl AA:NFMLTQPHSVSESPGKTVTISCTRSSGS IASNSVQWYQQRPGSSPTTVIYEDNQRPSGVPDR ESGS IDSSSNSASLTVSGLKTEDEADYYCQSSDSSAWFGSGTKLTVL

[0304] SEQ ID NO: 159 VH TIGIT AA:EVQLQQSGPELVKPGSSVKMSCKASGYTFSSYVMHWVKQKPGQGLEWIGYIDPYNDGAKYNE KFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGGPYGWYFDVWGAGTTVTVSS

[0305] SEQ ID NO: 160 VL TIGIT AA:DIQMTQSPASLSASVGETVTITCRASEHIYSYLSWYQQKQGKSPQLLVYNAKTLAEGVPSRF SGSGSGTQFSLKINSLQPEDFGTYYCQHHFGSPLTFGAGTTLELK

[0306] SEQ ID NO: 161 VL TIM3 AA:MEFGLSWVFLVALLRGVQCQVQLVESGGGWQPGRSLRLSCAASGFTSNSYGMHWVRQAPGK GLEWVAVIWYDGSNKYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIWFGEMFS EYFQHWGQGTLVTVSSAS

[0307] SEQ ID NO: 162 VL TIM3 AA:MDMRVPAQLLGLLLLWLPGARCAIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKP GKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGT KVEIKLT b. Transmembrane (TM) Domain

[0308] Polypeptides of the present disclosure may comprise a transmembrane domain. In some aspects, a transmembrane domain is a hydrophobic alpha helix that spans the membrane. Different transmembrane domains may result in different protein, e.g., co-inhibitor engager or chimeric interleukin receptors, stability. In certain aspects, the transmembrane domain is interposed between an extracellular and an intracellular domain.

[0309] In certain aspects, engineered polypeptides, e.g., co-inhibitor engager or chimeric interleukin receptors, may or may not comprise a transmembrane domain sequence. In some aspects, a transmembrane domain sequence may have a functional impact on the expression, localization, and / or activity of a polypeptide comprising the same. In some aspects, a transmembrane domain sequence may or may not be all or part of a cluster of differentiation 28 (CD28) sequence.

[0310] Any transmembrane domain that provides for insertion of a polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell may be suitable for use. In some aspects, the transmembrane domain may or may not be all or part of cluster of differentiation 28 (CD28), cluster of differentiation 8 (CD8), cluster of differentiation (CD25), cluster of differentiation 4 (CD4), cluster of differentiation 3 zeta (CD3Q, cluster of differentiation 134 (CD 134), and / or cluster of differentiation 7 (CD7).

[0311] In certain aspects, a transmembrane domain consists essentially of, or consists of a sequence of, of at least, or of at most 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range or value derivable therein, identity to SEQ ID NOs: 163-165, or 168-170.4. Signal peptide

[0312] Polypeptides of the present disclosure may comprise a signal peptide. A “signal peptide” refers to a peptide sequence that directs the transport and localization of the proteinwithin a cell, e.g., to a certain cell organelle (such as the endoplasmic reticulum) and / or the cell surface. In some aspects, a signal peptide directs the nascent protein into the endoplasmic reticulum. This is essential if a receptor is to be glycosylated and anchored in the cell membrane. Generally, the signal peptide natively attached to the amino-terminal most component is used (e.g., in an scFv with orientation light chain - linker - heavy chain, the native signal of the light-chain is used).

[0313] In certain aspects, engineered polypeptides of the disclosure may comprise one or more signal peptides. In some aspects, a signal peptide may have a functional impact on the expression, localization, and / or activity of a polypeptide comprising the same. In some aspects, a signal peptide sequence may be or may not be part or all of CD34.

[0314] In some aspects, the signal peptide is cleaved after passage of the endoplasmic reticulum (ER), e.g., is a cleavable signal peptide. In some aspects, a restriction site is at the carboxy end of the signal peptide to facilitate cleavage.

[0315] In certain aspects, a signal peptide comprises, consists essentially of, or consists of a sequence of, of at least, or of at most 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range or value derivable therein, identity to any one of SEQ ID NOs: 98-103.

[0316] SEQ ID NO: 98 signal-peptide abgamma DNA:ATGGATTCATACCTGCTGATGTGGGGACTGCTCACGTTCATCATGGTGCCTGGCTGCCAGGC A

[0317] SEQ ID NO: 99 signal-peptide bagamma DNA:ATGGCGGCCCCTGCTCTGTCCTGGCGTCTGCCCCTCCTCATCCTCCTCCTGCCCCTGGCTAC CTCTTGGGCATCTGCA

[0318] SEQ ID NO: 100 CD34-signal-peptide_Q8 DNA:ATGGGACTCGTGCGCAGAGGCGCTAGAGCCGGCCCTAGAATGCCTAGAGGATGGACCGCCCT GTGCCTGCTGTCTCTGCTGCCTAGCGGCTTCATG

[0319] SEQ ID NO: 101 signal-peptide abgamma AA:MDSYLLMWGLLTFIMVPGCQA

[0320] SEQ ID NO: 102 signal-peptide bagamma AA:MAAPALSWRLPLLILLLPLATSWASA

[0321] SEQ ID NO: 103 CD34-signal-peptide_Q8 AA:MGLVRRGARAGPRMPRGWTALCLLSLLPSGFM5. Cytokine

[0322] The term “cytokine” is a generic term for proteins released by one cell population, which act on another cell population as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included or excluded among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha and -beta; mullerian- inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF- alpha; platelet-growth factor; transforming growth factors (TGFs) such as TGF-alpha and TGF- beta; insulin-like growth factor-1 and -11; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-alpha, -beta and -gamma colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte macrophage-CSF (GM-CSF); and granulocyte- CSF (G-CSF); interleukins (ILs) such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-18, IL-23, IL-35; a tumor necrosis factor such as TNF- alpha or TNF-beta; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.

[0323] The term “immunosuppressive cytokine” refers to any cytokine capable of suppressing immune cell activation, e.g., T cell activation, directly or indirectly. Suppression of immune cell activation by an immunosuppressive cytokine may occur before or after immune cell activation occurs, e.g., immune cell activation may be inhibited or reversed. Immunosuppressive cytokines, include or exclude, but are not limited to, IL-IRa, IL-4, IL- 10, IL-11, IL-13, IL-33, IL-35, IL-37, and / or TGF-p. In some aspects, immunosuppressive cytokines may directly bind to receptors on T cells and suppress T cell activation, e.g., but not limited to, IL- 10, IL-35, and / or TGF-p. In some aspects, immunosuppressive cytokines are or are not IL- 10, IL-35, and / or TGF-B. In some aspects, an immunosuppressive cytokine is IL- 10. In some aspects, an immunosuppressive cytokine is IL-35. In some aspects, an immunosuppressive cytokine is TGF-B.

[0324] In some aspects, engineered cells described herein comprise constitutive or inducible expression of a heterologous immunosuppressive cytokine, such as IL-10, IL-35, or TGF-p. In some aspects, a heterologous immunosuppressive cytokine is or is not secreted. Insome aspects, cells engineered to comprise heterologous expression of IL-10 have enhanced immunosuppressive functions relative to non-engineered cells. In some aspects, cells engineered to comprise heterologous expression of IL-35 have enhanced immunosuppressive functions relative to non-engineered cells. In some aspects, cells engineered to comprise heterologous expression of TGF-P have enhanced immunosuppressive functions relative to non-engineered cells. In some aspects, expression of heterologous immunosuppressive cytokines functions synergistically with other components of an immunosuppressive system as described herein.

[0325] In certain aspects, a cytokine comprises, consists essentially of, or consists of a sequence of, of at least, or of at most 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range or value derivable therein, identity to any one of SEQ ID NOs: 36, 49, or 70-77.

[0326] SEQ ID NO: 36 IL10 DNA:ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCAGCCCAGG CCAGGGCACCCAGTCTGAGAACAGCTGCACCCACTTCCCAGGCAACCTGCCTAACATGCTTC GAGAT C T CCGAGAT GCC T T CAGCAGAGT GAAGAC T T T C T T T CAAAT GAAGGAT CAGC T GGAC AACTTGTTGTTAAAGGAGTCCTTGCTGGAGGACTTTAAGGGTTACCTGGGTTGCCAAGCCTT GTCTGAGATGATCCAGTTTTACCTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAG ACATCAAGGCGCATGTGAACTCCCTGGGGGAGAACCTGAAGACCCTCAGGCTGAGGCTACGG CGCTGTCATCGATTTCTTCCCTGTGAAAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAATGC C T T T AAT AAGC T C CAAGAGAAAGGCAT C T ACAAAGC CAT GAG T GAG T T T GAGAT C T T CAT CA AC T AC AT AGAAG C C TAG AT GAC AAT GAAGAT AC GAAAC

[0327] SEQ ID NO: 49 IL10 AA:MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLD NLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLR RCHRFLPCENKSKAVEQVKNAFNKLQEKGI YKAMSEFDI FINYIEAYMTMKIRN

[0328] SEQ ID NO: 70 TGFbl-LAP DNA:CTATCCACCTGCAAGACTATCGACATGGAGCTGGTGAAGCGGAAGCGCATCGAGGCCATCCG CGGCCAGATCCTGTCCAAGCTGCGGCTCGCCAGCCCCCCGAGCCAGGGGGAGGTGCCGCCCG GCCCGCTGCCCGAGGCCGTGCTCGCCCTGTACAACAGCACCCGCGACCGGGTGGCCGGGGAG AGTGCAGAACCGGAGCCCGAGCCTGAGGCCGACTACTACGCCAAGGAGGTCACCCGCGTGCT AAT G G T G GAAAC C C AC AAC GAAAT C T AT GAC AAG T T C AAG C AGAG TAG AC AC AG CAT AT AT A TGTTCTTCAACACATCAGAGCTCCGAGAAGCGGTACCTGAACCCGTGTTGCTCTCCCGGGCA GAGCTGCGTCTGCTGAGGCTCAAGTTAAAAGTGGAGCAGCACGTGGAGCTGTACCAGAAATA CAGCAACAATTCCTGGCGATACCTCAGCAACCGGCTGCTGGCACCCAGCGACTCGCCAGAGT GGTTATCTTTTGATGTCACCGGAGTTGTGCGGCAGTGGTTGAGCCGTGGAGGGGAAATTGAG GGCTTTCGCCTTAGCGCCCACTGCTCCTGTGACAGCAGGGATAACACACTGCAAGTGGACAT CAACGGGTTCACTACCGGCCGCCGAGGTGACCTGGCCACCATTCATGGCATGAACCGGCCTT TCCTGCTTCTCATGGCCACCCCGCTGGAGAGGGCCCAGCATCTGCAAAGCTCCCGGCACCGC CGA

[0329] SEQ ID NO: 71 TGFbl-TGFB DNA:GCCCTGGACACCAACTATTGCTTCAGCTCCACGGAGAAGAACTGCTGCGTGCGGCAGCTGTA CATTGACTTCCGCAAGGACCTCGGCTGGAAGTGGATCCACGAGCCCAAGGGCTACCATGCCA ACTTCTGCCTCGGGCCCTGCCCCTACATTTGGAGCCTGGACACGCAGTACAGCAAGGTCCTG GCCCTGTACAACCAGCATAACCCGGGCGCCTCGGCGGCGCCGTGCTGCGTGCCGCAGGCGCT GGAGCCGCTGCCCATCGTGTACTACGTGGGCCGCAAGCCCAAGGTGGAGCAGCTGTCCAACA TGATCGTGCGCTCCTGCAAGTGCAGC

[0330] SEQ ID NO: 72 TGFbl-LAP AA:LSTCKT IDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGE SAEPEPEPEADYYAKEVTRVLMVETHNE IYDKFKQSTHS IYMFFNTSELREAVPEPVLLSRA ELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLS FDVTGWRQWLSRGGE IE GFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLAT IHGMNRPFLLLMATPLERAQHLQSSRHR R

[0331] SEQ ID NO: 73 TGFbl-TGFB AA:ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVL ALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS

[0332] SEQ ID NO: 74 IL12A DNA:ATGTGGCCCCCTGGGTCAGCCTCCCAGCCACCGCCCTCACCTGCCGCGGCCACAGGTCTGCA TCCAGCGGCTCGCCCTGTGTCCCTGCAGTGCCGGCTCAGCATGTGTCCAGCGCGCAGCCTCC TCCTTGTGGCTACCCTGGTCCTCCTGGACCACCTCAGTTTGGCCAGAAACCTCCCCGTGGCC ACTCCAGACCCAGGAATGTTCCCATGCCTTCACCACTCCCAAAACCTGCTGAGGGCCGTCAG CAACAT GC T C CAGAAGGC CAGACAAAC T C T AGAAT T T TAG C C T T GCAC T T C T GAAGAGAT T G ATCATGAAGATATCACAAAAGATAAAACCAGCACAGTGGAGGCCTGTTTACCATTGGAATTA AC C AAGAAT GAGAG T T G C C T AAAT T C C AGAGAGAC C T C T T T C AT AAC T AAT G G GAG T T G C C T GGCCTCCAGAAAGACCTCTTTTATGATGGCCCTGTGCCTTAGTAGTATTTATGAAGACTTGA AGATGTACCAGGTGGAGTTCAAGACCATGAATGCAAAGCTTCTGATGGATCCTAAGAGGCAG ATCTTTCTAGATCAAAACATGCTGGCAGTTATTGATGAGCTGATGCAGGCCCTGAATTTCAA GAG T GAGAG T G T G C C AC AAAAAT CCTCCCTT GAAGAAC C G GAT T T T T AT AAAAC T AAAAT C A AGCTCTGCATACTTCTTCATGCTTTCAGAATTCGGGCAGTGACTATTGATAGAGTGATGAGC TATCTGAATGCTTCC

[0333] SEQ ID NO: 75 EBI3 DNA:ATGACCCCGCAGCTTCTCCTGGCCCTTGTCCTCTGGGCCAGCTGCCCGCCCTGCAGTGGAAG GAAAGGGCCCCCAGCAGCTCTGACACTGCCCCGGGTGCAATGCCGAGCCTCTCGGTACCCGA TCGCCGTGGATTGCTCCTGGACCCTGCCGCCTGCTCCAAACTCCACCAGCCCCGTGTCCTTC ATTGCCACGTACAGGCTCGGCATGGCTGCCCGGGGCCACAGCTGGCCCTGCCTGCAGCAGAC GCCAACGTCCACCAGCTGCACCATCACGGATGTCCAGCTGTTCTCCATGGCTCCCTACGTGC TCAATGTCACCGCCGTCCACCCCTGGGGCTCCAGCAGCAGCTTCGTGCCTTTCATAACAGAG CACATCATCAAGCCCGACCCTCCAGAAGGCGTGCGCCTAAGCCCCCTCGCTGAGCGCCAGCT ACAGGTGCAGTGGGAGCCTCCCGGGTCCTGGCCCTTCCCAGAGATCTTCTCACTGAAGTACT GGATCCGTTACAAGCGTCAGGGAGCTGCGCGCTTCCACCGGGTGGGGCCCATTGAAGCCACG TCCTTCATCCTCAGGGCTGTGCGGCCCCGAGCCAGGTACTACGTCCAAGTGGCGGCTCAGGA CCTCACAGACTACGGGGAACTGAGTGACTGGAGTCTCCCCGCCACTGCCACAATGAGCCTGG GCAAG

[0334] SEQ ID NO: 76 IL12A AA:MWPPGSASQPPPSPAAATGLHPAARPVSLQCRLSMCPARSLLLVATLVLLDHLSLARNLPVA TPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLEL TKNESCLNSRETSFITNGSCLASRKTSFMMALCLSS IYEDLKMYQVEFKTMNAKLLMDPKRQ I FLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMS YLNAS

[0335] SEQ ID NO: 77 EBI3 AA:MTPQLLLALVLWASCPPCSGRKGPPAALTLPRVQCRASRYPIAVDCSWTLPPAPNSTSPVSF IATYRLGMAARGHSWPCLQQTPTSTSCTITDVQLFSMAPYVLNVTAVHPWGSSSSFVPFITE HI IKPDPPEGVRLSPLAERQLQVQWEPPGSWPFPEI FSLKYWIRYKRQGAARFHRVGPIEAT SFILRAVRPRARYYVQVAAQDLTDYGELSDWSLPATATMSLGK6. Interleukin Receptor

[0336] As used herein, an “interleukin (IL) receptor” refers to a cytokine receptor for an interleukin. There are two main families of IL receptors, type 1 and type 2 cytokine receptors. Type 1 interleukin receptors include IL-2 receptor, IL-3 receptor, IL-4 receptor, IL-5 receptor, IL-6 receptor, IL-7 receptor, IL-9 receptor, IL- 11 receptor, IL- 12 receptor, IL- 13 receptor, IL- 15 receptor, IL-21 receptor, IL-23 receptor and IL-27 receptor. Type 2 IL receptors include IL- 10 receptor, IL-20 receptor, IL-22 receptor and IL-28 receptor. An IL receptor may comprise multiple polypeptide chains. In some aspects, for example, the IL-2 receptor chains comprise CD25 (a-chain), CD122 (P-chain), and CD132 (y-chain), sometimes abbreviated as IL2Ra, IL- 2Rb, and IL-2Rg, respectively.

[0337] As used herein, “a chimeric interleukin receptor” refers a molecule which comprises a cytokine receptor endodomain and one or more of a cytokine receptor exodomain, operably linked to each other. The exo- and endo-domains may be from the same or from different proteins. The chimeric interleukin receptor may bind an interleukin with more or less affinity than a natural interleukin receptor.

[0338] In some aspects, a chimeric interleukin receptor is described herein. In some aspects, a chimeric interleukin receptor comprises linked heterologous domains of interleukin receptors to induce uptake of extracellular interleukins. In some aspects, a chimeric interleukin receptor is a chimeric interleukin 2 receptor (cIL2R) which comprises domains of CD25 (a- subunit), CD 122 (P-subunit), and / or CD 132 (common y-subunit). In some aspects, a cIL2R comprises domains in the order of aP-gamma or Pa-gamma.

[0339] In some aspects, cells are engineered to comprise a chimeric interleukin receptor, e.g., cIL2R. In some aspects, engineered cells comprising a cIL2R have an unchanged cell phenotype. In some aspects, engineered cells comprising a cIL2R have improved uptake ofextracellular IL-2 relative to non-engineered cells. In some aspects, engineered cells comprising a cIL2R reduce extracellular concentrations of IL-2 no near zero. In some aspects, a Pa-gamma ordered cIL2R has improved IL-2 uptake than an aP-gamma ordered cIL2R.

[0340] In certain aspects, a chimeric interleukin receptor comprises, consists essentially of, or consists of a sequence of, of at least, or of at most 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range or value derivable therein, identity to any one of SEQ ID NOs: 37-41, 50-54, or 60-69.

[0341] SEQ ID NO: 60 alpha-CD25 DNA:GAGCTCTGTGACGATGACCCGCCAGAGATCCCACACGCCACATTCAAAGCCATGGCCTACAA GGAAGGAACCATGTTGAACTGTGAATGCAAGAGAGGTTTCCGCAGAATAAAAAGCGGGTCAC TCTATATGCTCTGTACAGGAAACTCTAGCCACTCGTCCTGGGACAACCAATGTCAATGCACA AG C T C T G C GAG T C G GAAC AC AAC GAAAC AAG T GAG AC C T C AAC C T GAAGAAC AGAAAGAAAG GAAAACCACAGAAATGCAAAGTCCAATGCAGCCAGTGGACCAAGCGAGCCTTCCAGGTCACT GCAGGGAACCTCCACCATGGGAAAATGAAGCCACAGAGAGAATTTATCATTTCGTGGTGGGG CAGATGGTTTATTATCAGTGCGTCCAGGGATACAGGGCTCTACACAGAGGTCCTGCTGAGAG CGTCTGCAAAATGACCCACGGGAAGACAAGGTGGACCCAGCCCCAGCTCATATGCACAGGTG AAATGGAGACCAGTCAGTTTCCAGGTGAAGAGAAGCCTCAGGCAAGCCCCGAAGGCCGTCCT GAGAG T GAGAC TTCCTGCCTCGT C AC AAC AAC AGAT T T T C AAAT AC AGAC AGAAAT G G C T G CAAC C AT G GAGAC GTCCATATT T AC AAC AGAG TAG C AG

[0342] SEQ ID NO: 61 beta-CD122 DNA:GCGGTGAATGGCACTTCCCAGTTCACATGCTTCTACAACTCGAGAGCCAACATCTCCTGTGT CTGGAGCCAAGATGGGGCTCTGCAGGACACTTCCTGCCAAGTCCATGCCTGGCCGGACAGAC GGCGGTGGAACCAAACCTGTGAGCTGCTCCCCGTGAGTCAAGCATCCTGGGCCTGCAACCTG ATCCTCGGAGCCCCAGATTCTCAGAAACTGACCACAGTTGACATCGTCACCCTGAGGGTGCT GTGCCGTGAGGGGGTGCGATGGAGGGTGATGGCCATCCAGGACTTCAAGCCCTTTGAGAACC TTCGCCTGATGGCCCCCATCTCCCTCCAAGTTGTCCACGTGGAGACCCACAGATGCAACATA AGCTGGGAAATCTCCCAAGCCTCCCACTACTTTGAAAGACACCTGGAGTTCGAGGCCCGGAC GCTGTCCCCAGGCCACACCTGGGAGGAGGCCCCCCTGCTGACTCTCAAGCAGAAGCAGGAAT GGATCTGCCTGGAGACGCTCACCCCAGACACCCAGTATGAGTTTCAGGTGCGGGTCAAGCCT CTGCAAGGCGAGTTCACGACCTGGAGCCCCTGGAGCCAGCCCCTGGCCTTCAGGACAAAGCCTGCAGCCCTTGGGAAGGACACC

[0343] SEQ ID NO: 62 gamma-IL2g DNA:C T GAAC AC GAC AAT T C T GAC G C C C AAT G G GAAT GAAGAC AC C AC AG CTGATTTCTTCCT GAC CACTATGCCCACTGACTCCCTCAGTGTTTCCACTCTGCCCCTCCCAGAGGTTCAGTGTTTTG TGTTCAATGTCGAGTACATGAATTGCACTTGGAACAGCAGCTCTGAGCCCCAGCCTACCAAC CTCACTCTGCATTATTGGTACAAGAACTCGGATAATGATAAAGTCCAGAAGTGCAGCCACTA TCTATTCTCTGAAGAAATCACTTCTGGCTGTCAGTTGCAAAAAAAGGAGATCCACCTCTACC AAACATTTGTTGTTCAGCTCCAGGACCCACGGGAACCCAGGAGACAGGCCACACAGATGCTA AAACTGCAGAATCTGGTGATCCCCTGGGCTCCAGAGAACCTAACACTTCACAAACTGAGTGA ATCCCAGCTAGAACTGAACTGGAACAACAGATTCTTGAACCACTGTTTGGAGCACTTGGTGC AGTACCGGACTGACTGGGACCACAGCTGGACTGAACAATCAGTGGATTATAGACATAAGTTC TCCTTGCCTAGTGTGGATGGGCAGAAACGCTACACGTTTCGTGTTCGGAGCCGCTTTAACCCACTCTGTGGAAGTGCTCAGCATTGGAGTGAATGGAGCCACCCAATCCACTGGGGGAGCAATA CTTCAAAAGAGAATCCTTTCCTGTTTGCATTGGAAGCCGTGGTTATCTCTGTTGGCTCCATG GGATTGATTATCAGCCTTCTCTGTGTGTATTTCTGGCTGGAACGGACGATGCCCCGAATTCC CACCCTGAAGAACCTAGAGGATCTTGTTACTGAATACCACGGGAACTTTTCGGCCTGGAGTG GTGTGTCTAAGGGACTGGCTGAGAGTCTGCAGCCAGACTACAGTGAACGACTCTGCCTCGTC AGTGAGATTCCCCCAAAAGGAGGGGCCCTTGGGGAGGGGCCTGGGGCCTCCCCATGCAACCA GCATAGCCCCTACTGGGCCCCCCCATGTTACACCCTAAAGCCTGAAACC

[0344] SEQ ID NO: 63 IL2Rb DNA:ATGGCGGCCCCTGCTCTGTCCTGGCGTCTGCCCCTCCTCATCCTCCTCCTGCCCCTGGCTAC CTCTTGGGCATCTGCAGCGGTGAATGGCACTTCCCAGTTCACATGCTTCTACAACTCGAGAG CCAACATCTCCTGTGTCTGGAGCCAAGATGGGGCTCTGCAGGACACTTCCTGCCAAGTCCAT GCCTGGCCGGACAGACGGCGGTGGAACCAAACCTGTGAGCTGCTCCCCGTGAGTCAAGCATC CTGGGCCTGCAACCTGATCCTCGGAGCCCCAGATTCTCAGAAACTGACCACAGTTGACATCG TCACCCTGAGGGTGCTGTGCCGTGAGGGGGTGCGATGGAGGGTGATGGCCATCCAGGACTTC AAGCCCTTTGAGAACCTTCGCCTGATGGCCCCCATCTCCCTCCAAGTTGTCCACGTGGAGAC CCACAGATGCAACATAAGCTGGGAAATCTCCCAAGCCTCCCACTACTTTGAAAGACACCTGG AGTTCGAGGCCCGGACGCTGTCCCCAGGCCACACCTGGGAGGAGGCCCCCCTGCTGACTCTC AAGCAGAAGCAGGAATGGATCTGCCTGGAGACGCTCACCCCAGACACCCAGTATGAGTTTCA GGTGCGGGTCAAGCCTCTGCAAGGCGAGTTCACGACCTGGAGCCCCTGGAGCCAGCCCCTGG CCTTCAGGACAAAGCCTGCAGCCCTTGGGAAGGACACCATTCCGTGGCTCGGCCACCTCCTC GTGGGCCTCAGCGGGGCTTTTGGCTTCATCATCTTAGTGTACTTGCTGATCAACTGCAGGAA CACCGGGCCATGGCTGAAGAAGGTCCTGAAGTGTAACACCCCAGACCCCTCGAAGTTCTTTT CCCAGCTGAGCTCAGAGCATGGAGGAGACGTCCAGAAGTGGCTCTCTTCGCCCTTCCCCTCA TCGTCCTTCAGCCCTGGCGGCCTGGCACCTGAGATCTCGCCACTAGAAGTGCTGGAGAGGGA CAAGGTGACGCAGCTGCTCCTGCAGCAGGACAAGGTGCCTGAGCCCGCATCCTTAAGCAGCA ACCACTCGCTGACCAGCTGCTTCACCAACCAGGGTTACTTCTTCTTCCACCTCCCGGATGCC TTGGAGATAGAGGCCTGCCAGGTGTACTTTACTTACGACCCCTACTCAGAGGAAGACCCTGA TGAGGGTGTGGCCGGGGCACCCACAGGGTCTTCCCCCCAACCCCTGCAGCCTCTGTCAGGGG AGGACGACGCCTACTGCACCTTCCCCTCCAGGGATGACCTGCTGCTCTTCTCCCCCAGTCTC CTCGGTGGCCCCAGCCCCCCAAGCACTGCCCCTGGGGGCAGTGGGGCCGGTGAAGAGAGGAT GCCCCCTTCTTTGCAAGAAAGAGTCCCCAGAGACTGGGACCCCCAGCCCCTGGGGCCTCCCA CCCCAGGAGTCCCAGACCTGGTGGATTTTCAGCCACCCCCTGAGCTGGTGCTGCGAGAGGCT GGGGAGGAGGTCCCTGACGCTGGCCCCAGGGAGGGAGTCAGTTTCCCCTGGTCCAGGCCTCC TGGGCAGGGGGAGTTCAGGGCCCTTAATGCTCGCCTGCCCCTGAACACTGATGCCTACTTGT CCCTCCAAGAACTCCAGGGTCAGGACCCAACTCACTTGGTG

[0345] SEQ ID NO: 64 CD19 DNA:ATGCCACCTCCTCGCCTCCTCTTCTTCCTCCTCTTCCTCACCCCCATGGAAGTCAGGCCCGA GGAACCTCTAGTGGTGAAGGTGGAAGAGGGAGATAACGCTGTGCTGCAGTGCCTCAAGGGGA CCTCAGATGGCCCCACTCAGCAGCTGACCTGGTCTCGGGAGTCCCCGCTTAAACCCTTCTTA AAACTCAGCCTGGGGCTGCCAGGCCTGGGAATCCACATGAGGCCCCTGGCCATCTGGCTTTT CATCTTCAACGTCTCTCAACAGATGGGGGGCTTCTACCTGTGCCAGCCGGGGCCCCCCTCTG AGAAGGCCTGGCAGCCTGGCTGGACAGTCAATGTGGAGGGCAGCGGGGAGCTGTTCCGGTGG AATGTTTCGGACCTAGGTGGCCTGGGCTGTGGCCTGAAGAACAGGTCCTCAGAGGGCCCCAG CTCCCCTTCCGGGAAGCTCATGAGCCCCAAGCTGTATGTGTGGGCCAAAGACCGCCCTGAGA TCTGGGAGGGAGAGCCTCCGTGTCTCCCACCGAGGGACAGCCTGAACCAGAGCCTCAGCCAG GACCTCACCATGGCCCCTGGCTCCACACTCTGGCTGTCCTGTGGGGTACCCCCTGACTCTGT GTCCAGGGGCCCCCTCTCCTGGACCCATGTGCACCCCAAGGGGCCTAAGTCATTGCTGAGCC TAGAGCTGAAGGACGATCGCCCGGCCAGAGATATGTGGGTAATGGAGACGGGTCTGTTGTTGCCCCGGGCCACAGCTCAAGACGCTGGAAAGTATTATTGTCACCGTGGCAACCTGACCATGTCATTCCACCTGGAGATCACTGCTCGGCCAGTACTATGGCACTGGCTGCTGAGGACTGGTGGCT GGAAGGTCTCAGCTGTGACTTTGGCTTATCTGATCTTCTGCCTGTGTTCCCTTGTGGGCATT CTTCATCTTCAAAGAGCCCTGGTCCTGAGGAGGAAAAGAAAGCGAATGACTGACCCCACCAG GAGATTC

[0346] SEQ ID NO: 65 alpha-CD25 AA:ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCT SSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERI YHFWG QMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRP ESETSCLVTTTDFQIQTEMAATMETS I FTTEYQ

[0347] SEQ ID NO: 66 beta-CD122 AA:AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNL ILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQWHVETHRCNI SWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKP LQGEFTTWSPWSQPLAFRTKPAALGKDT

[0348] SEQ ID NO: 67 gamma-IL2g AA:LNTTILTPNGNEDTTADFFLTTMPTDSLSVSTLPLPEVQCFVFNVEYMNCTWNSSSEPQPTN LTLHYWYKNSDNDKVQKCSHYLFSEEITSGCQLQKKEIHLYQTFWQLQDPREPRRQATQML KLQNLVIPWAPENLTLHKLSESQLELNWNNRFLNHCLEHLVQYRTDWDHSWTEQSVDYRHKF SLPSVDGQKRYTFRVRSRFNPLCGSAQHWSEWSHPIHWGSNTSKENPFLFALEAWISVGSMGLI ISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLV SEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET

[0349] SEQ ID NO: 68 IL2RbAA:MAAPALSWRLPLLILLLPLATSWASAAVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVH AWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDF KPFENLRLMAPISLQWHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTL KQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTIPWLGHLLVGLSGAFGFI ILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPS SSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDA LEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSL LGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV

[0350] SEQ ID NO: 69 CD19 AAMPPPRLLFFLLFLTPMEVRPEEPLWKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFL KLSLGLPGLGIHMRPLAIWLFI FNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRW NVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQ DLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYLI FCLCSLVGI LHLQRALVLRRKRKRMTDPTRRF7. Co-signaling Receptors and Ligands

[0351] Immune cells have receptors that transmit immunomodulatory (e.g., costimulatory and coinhibitory) signals. For example, T cells have T cell receptors and the CD3 complex, Bcells have B cell receptors, and myeloid cells have Fc receptors. In addition, immune cells bear receptors that transmit signals that provide costimulatory signals, or receptors that transmit signals that inhibit receptor-mediated signaling. For example, CD28 transmits a costimulatory signal to T cells. After ligation of the T cell receptor, ligation of CD28 results in a costimulatory signal characterized by, e.g., upregulation of IL-2ra, IL-2rP, and IL-2ry receptor, increased transcription of IL-2 messenger RNA, and increased expression of cytokine genes (including IL-2, IFN-y, GM-CSF, and TNF-a). Transmission of a costimulatory signal allows the cell to progress through the cell cycle and, thus, increases T cell proliferation (Greenfield et al. (1998) Crit. Rev. Immunol. 18:389; incorporated herein by reference). For example, binding of a receptor on a T cell which transmits a costimulatory signal to the cell (e.g., ligation of a costimulatory receptor that leads to cytokine secretion and / or proliferation of the T cell) by a B7 family molecule, such as B7-1 or B7-2, results in costimulation. Thus, inhibition of an interaction between a B7 family molecule, such as B7-1 or B7-2, and a receptor that transmits a costimulatory signal on an immune cell results in a downmodulation of the immune response, specific unresponsiveness, termed immune cell anergy, clonal deletion, and / or exhaustion. Inhibition of this interaction can be accomplished using, e.g., anti-CD28 Fab fragments, antibodies to B7-1 or B7-2, or by using a soluble form of a receptor to which a B7 family member molecule can bind as a competitive inhibitor (e.g., CTLA4-Ig).

[0352] Inhibitory receptors that bind to costimulatory molecules have also been identified on immune cells. Activation of CTLA4, for example, transmits a negative signal to a T cell. Engagement of CTLA4 inhibits IL-2 production and can induce cell cycle arrest (Krummel and Allison (1996) J. Exp. Med. 183:2533). In addition, mice that lack CTLA4 develop lymphoproliferative disease (Tivol, et al. (1995) Immunity 3:541; Waterhouse et al. (1995) Science 270:985). The blockade of CTLA4 with antibodies can block an inhibitory signal, whereas aggregation of CTLA4 with antibody transmits an inhibitory signal. Therefore, depending upon the receptor to which a costimulatory molecule binds (e.g., a costimulatory receptor such as CD28 or an inhibitory receptor such as CTLA4), certain B7 molecules including B7-4 can promote T cell costimulation or inhibition.

[0353] As used herein, the term “costimulatory antagonist” refers to a molecule that stops the action or effect of a costimulatory receptor or costimulatory ligand whether directly or indirectly. For example, CTLA-4 may bind CD80, CD86, or both (CD80 / 86) on an antigen presenting cell, internalize CD80 / 86, and thereby prevent activation of immune cells, such as T cells, by antigen presenting cells. In such a case, for example, CTLA-4 is considered anantagonist of the costimulatory ligand(s) CD80 / 86 and / or the costimulatory receptors for CD80 / 86.

[0354] In some aspects, engineered cells described herein comprise heterologous expression of CTLA-4. In some aspects, cells engineered to comprise heterologous expression of CTLA-4 boost immunosuppressive functions of engineered cells relative to non-engineered cells. In some aspects, cells engineered to comprise expression of cIL2R are further engineered to comprise heterologous CTLA-4. In some aspects, concurrent expression of cIL2R and heterologous CTLA-4 synergistically improves immunosuppressive functions of an engineered cell relative to a non-engineered cell or a cell engineered to express only a cIL2R or heterologous CTLA-4.

[0355] In certain aspects, a costimulatory antagonist comprises, consists essentially of, or consists of a sequence of, of at least, or of at most 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range or value derivable therein, identity to any one of SEQ ID NOs: 32-33, or 47-48.8. Peptide Linker

[0356] In some aspects, the polypeptides of the disclosure include peptide linkers (sometimes referred to as a linker). A peptide linker may be used to separate any of the peptide domain / regions described herein. As an example, a linker may be between a signal peptide and a co-inhibitor engager, between a VH and a VL domain of a co-inhibitor engager, and / or between the extracellular, transmembrane, or intracellular domains of a polypeptide. The peptide linker may have any of a variety of amino acid sequences. Domains and regions can be joined by a peptide linker that is generally of a flexible nature, although other chemical linkages are not excluded. A linker can be a peptide of between about 6 and about 40 amino acids in length, or between about 6 and about 25 amino acids in length. These linkers can be produced by using synthetic, linker-encoding oligonucleotides to couple the proteins.

[0357] Peptide linkers with a degree of flexibility can be used. The peptide linkers may have virtually any amino acid sequence, bearing in mind that suitable peptide linkers will have a sequence that results in a generally flexible peptide. The use of small amino acids, such as glycine and alanine, are of use in creating a flexible peptide. The creation of such sequences is routine to those of skill in the art.

[0358] Suitable linkers can be readily selected and can be of any suitable length, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0359] Example flexible linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n, (G4S)n, and (GGGS)n, where n is an integer of at least one). In some aspects, n is at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any derivable range therein). Glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between components. Glycine polymers can be used; glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains. Exemplary spacers can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 92), GGSGG (SEQ ID NO: 93), GSGSG (SEQ ID NO: 94), GSGGG (SEQ ID NO: 95), GGGSG (SEQ ID NO: 96), or GSSSG (SEQ ID NO: 97).

[0360] In certain aspects, a peptide linker comprises, consists essentially of, or consists of a sequence of, of at least, or of at most 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range or value derivable therein, identity to any one of SEQ ID NOs: 86-90.

[0361] SEQ ID NO: 86 41inker ab DNA:GGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGGTGGCGGCGGATCT

[0362] SEQ ID NO: 87 31inker bgamma DNA:GGCGGCGGGGGCAGTGGAGGAGGCGGAAGCGGTGGGGGGGGATCG

[0363] SEQ ID NO: 88 GSG DNA:GGAAGCGGC

[0364] SEQ ID NO: 89 41inker bgamma AA:GGGGSGGGGSGGGGSGGGGS

[0365] SEQ ID NO: 90 31inker bgamma AA:GGGGSGGGGSGGGGS

[0366] SEQ ID NO: 91 GSG AA:GSG9. Reporters

[0367] As used herein, a “reporter”, or “reporter gene” refers to a polynucleotide sequence encoding a protein product, or a protein that can generate, under appropriate conditions, a detectable signal that allows detection for indicating the presence and / or quantity of the reporter gene protein product. In certain aspects, any sequence described herein that includes a reporter gene may optionally not comprise said reporter gene.

[0368] In certain aspects, a reporter comprises, consists essentially of, or consists of a sequence of, of at least, or of at most 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range or value derivable therein, identity to any one of SEQ ID NOs: 105-112.

[0369] SEQ ID NO: 105 dNGFR DNA:ATGGGGGCAGGTGCCACCGGCCGCGCCATGGACGGGCCGCGCCTGCTGCTGTTGCTGCTTCT GGGGGTGTCCCTTGGAGGTGCCAAGGAGGCATGCCCCACAGGCCTGTACACACACAGCGGTG AGTGCTGCAAAGCCTGCAACCTGGGCGAGGGTGTGGCCCAGCCTTGTGGAGCCAACCAGACC GTGTGTGAGCCCTGCCTGGACAGCGTGACGTTCTCCGACGTGGTGAGCGCGACCGAGCCGTG CAAGCCGTGCACCGAGTGCGTGGGGCTCCAGAGCATGTCGGCGCCGTGCGTGGAGGCCGACG ACGCCGTGTGCCGCTGCGCCTACGGCTACTACCAGGATGAGACGACTGGGCGCTGCGAGGCG TGCCGCGTGTGCGAGGCGGGCTCGGGCCTCGTGTTCTCCTGCCAGGACAAGCAGAACACCGT GTGCGAGGAGTGCCCCGACGGCACGTATTCCGACGAGGCCAACCACGTGGACCCGTGCCTGC CCTGCACCGTGTGCGAGGACACCGAGCGCCAGCTCCGCGAGTGCACACGCTGGGCCGACGCC GAGTGCGAGGAGATCCCTGGCCGTTGGATTACACGGTCCACACCCCCAGAGGGCTCGGACAG CACAGCCCCCAGCACCCAGGAGCCTGAGGCACCTCCAGAACAAGACCTCATAGCCAGCACGG TGGCAGGTGTGGTGACCACAGTGATGGGCAGCTCCCAGCCCGTGGTGACCCGAGGCACCACC GACAACCTCATCCCTGTCTATTGCTCCATCCTGGCTGCTGTGGTTGTGGGCCTTGTGGCCTA CATAGCCTTCAAGAGGTGGAACAGC

[0370] SEQ ID NO: 106 Q8 DNA: atgggactcgtgcgcagaggcgctagagccggccctagaatgcctagaggatggaccgccct gtgcctgctgtctctgctgcctagcggcttcatggccgagctgcctactcagggcaccttca gcaacgtgtccaccaatgtgtccccagccaagcccaccacaacccctgctcctagacctcct accccagcccctaccattgcctcccagccactgtctctgaggcccgaggcttgtagacctgc tgcaggcggagccgtgcacaccagaggactggatttcgcctgcgacatctatatctgggccc ctctggccggcacctgtggcgtgctgctgctgtcactcgtgatcaccctgtactgcaaccac cggaaccggcggagagtgtgcaagtgccctagacccgtcgtg

[0371] SEQ ID NO: 107 CD34-epitope_Q8 DNA:GAGCTGCCTACTCAGGGCACCTTCAGCAACGTGTCCACCAATGTGTCC

[0372] SEQ ID NO: 108 CD34a_Q8 DNA:CCAGCCAAGCCCACCACAACCCCTGCTCCTAGACCTCCTACCCCAGCCCCTACCATTGCCTC CCAGCCACTGTCTCTGAGGCCCGAGGCTTGTAGACCTGCTGCAGGCGGAGCCGTGCACACCA GAGGACTGGATTTCGCCTGCGACATCTATATCTGGGCCCCTCTGGCCGGCACCTGTGGCGTGCTGCTGCTGTCACTCGTGATCACCCTGTACTGCAACCACCGGAACCGGCGGAGAGTGTGCAAGTGCCCTAGACCCGTCGTG

[0373] SEQ ID NO: 109 dNGFR AA:MGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQT VCEPCLDSVTFSDWSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEA CRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADA ECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGWTTVMGSSQPWTRGTT DNL I PVYCS I LAAVWGLVAY I AFKRWNS

[0374] SEQ ID NO: 110 Q8 AA:MGLVRRGARAGPRMPRGWTALCLLSLLPSGFMAELPTQGTFSNVSTNVSPAKPTTTPAPRPP TPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNH RNRRRVCKCPRPW

[0375] SEQ ID NO: 111 CD34-epitope_Q8 AA:ELPTQGTFSNVSTNVS

[0376] SEQ ID NO: 112 CD34a_Q8 AA:PAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGV LLLSLVITLYCNHRNRRRVCKCPRPW10. Hinge

[0377] As used herein, the term “hinge” refers to a flexible polypeptide connector region (also referred to herein as “hinge region”) providing structural flexibility and spacing to flanking polypeptide regions and can consist of natural or synthetic polypeptides. A “hinge” from an immunoglobulin (e.g., IgGl, or IgG4) is generally defined as stretching from Glu216 to Pro230 of human IgGl (Burton (1985) Molec. Immunol., 22: 161- 206). Hinge regions of other IgG isotypes may be aligned with the IgGl sequence by placing the first and last cysteine residues forming inter-heavy chain disulfide (S-S) bonds in the same positions. The hinge region may be of natural occurrence or non-natural occurrence, including but not limited to an altered hinge region as described in U.S. Pat. No. 5,677,425, incorporated by reference herein. The hinge region can include a complete or partial hinge region from an antibody of a different class or subclass from that of the CHI domain. The term “hinge” can also include regions from CD8 and other receptors that provide a similar function in providing flexibility and spacing to flanking regions.

[0378] A hinge, such as an extracellular spacer may link a targeting moiety (e.g., an antibody, a fragment thereof, a ligand, etc.) component of a co-inhibitor engager to a transmembrane domain. In some aspects, a hinge is flexible enough to allow the targetingmoiety of a co-inhibitor engager to orient in different directions to facilitate target binding. In some aspects, the spacer comprises the hinge region from IgGl, and / or IgG4.

[0379] In certain aspects, an engineered polypeptides may comprise one or more hinges and / or linker sequences. In some aspects, a hinge and / or linker sequence may have a functional impact on the expression, localization, and / or activity of a polypeptide comprising the same.

[0380] A hinge can have a length of at least, at most, or exactly 4, 5, 6, 7, 8, 9, 10, 12, 15, 16, 17, 18, 19, 20, 20, 25, 30, 35, 40, 45, 50, 75, 100, 110, 119, 120, 130, 140, 150, 160, 170,180, 190, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,217, 218, 219, 220, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239,240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 260, 270, 280, 290, 300, 325, 350, or400 amino acids (or any derivable range therein). In some aspects, a shorter spacer such as less than 50, 45, 40, 30, 35, 30, 25, 20, 15, 14, 13, 12, 11, or 10 amino acids may be used. In some aspects, a longer spacer, such as one that is at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217,218, 219, 220, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 260, 270, 280, or 290 amino acids may be used.

[0381] When the hinge comprises multiple parts, there may be anywhere from 0-50 amino acids in between the various parts. For example, there may be at least, at most, or exactly 0, 1, 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, 35, 40, 45, or 50 amino acids (or any derivable range therein) between the hinge and the CH2 or CH3 region or between the CH2 and CH3 region when both are present. In some aspects, the extracellular spacer consists essentially of a hinge, CH2, and / or CH3 region, meaning that the hinge, CH2, and / or CH3 region is the only identifiable region present and all other domains or regions are excluded, but further amino acids not part of an identifiable region may be present.C. Cellular Therapies

[0382] Aspects of the present disclosure include cellular therapies, including engineering and formulating cellular therapies as well as using such therapies for treatment of various conditions such as but not limited to, cancer, autoimmune disorders / diseases, transplant rejection, cytokine storm, sepsis, GVHD, etc.1. Cell Culture

[0383] In some aspects, cells may be cultured for at least between about 0 and 9 days, for at least between about 10 days and about 40 days, for at least between about 15 days and about 35 days, for at least between about 15 days and 21 days, such as for at least about 15, 16, 17, 18, 19 or 21 days. In some aspects, the cells of the disclosure may be cultured for no longer than 60 days, or no longer than 50 days, or no longer than 45 days. The cells may be cultured for 1, 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, 35, 36, 37, 38, 39, or 40 days. The cells may be cultured in the presence of a liquid culture medium. Typically, the medium may comprise a basal medium formulation as known in the art. Many basal media formulations can be used to culture cells herein, including but not limited to Eagle’s Minimum Essential Medium (MEM), Dulbecco’s Modified Eagle’s Medium (DMEM), alpha modified Minimum Essential Medium (alpha- MEM), Basal Medium Essential (BME), Iscove’s Modified Dulbecco’s Medium (IMDM), BGJb medium, F-12 Nutrient Mixture (Ham), Liebovitz L-15, DMEM / F-12, Essential Modified Eagle’s Medium (EMEM), RPMI-1640, and modifications and / or combinations thereof. Compositions of the above basal media are generally known in the art, and it is within the skill of one in the art to modify or modulate concentrations of media and / or media supplements as necessary for the cells cultured. In some aspects, a culture medium formulation may be explants medium (CEM) which is composed of IMDM supplemented with 10% fetal bovine serum (FBS), 100 U / ml penicillin G, 100 pg / ml streptomycin and 2 mmol / L L- glutamine. Other aspects may employ further basal media formulations, such as chosen from the ones above.

[0384] Any medium capable of supporting cells in vitro may be used to culture the cells. Media formulations that can support the growth of cells include, but are not limited to, Dulbecco’s Modified Eagle’s Medium (DMEM), alpha modified Minimal Essential Medium (aMEM), and Roswell Park Memorial Institute Media 1640 (RPMI Media 1640) and the like. Typically, up to 20% fetal bovine serum (FBS) or 1-20% horse serum is added to the above medium in order to support the growth of cells. A defined medium, however, also can be used if the growth factors, cytokines, and hormones necessary for culturing cells are provided at appropriate concentrations in the medium. Media useful in the methods of the disclosure may comprise one or more compounds of interest, including, but not limited to, antibiotics, mitogenic compounds, or differentiation compounds useful for the culturing of cells. The cells may be grown at temperatures between 27° C to 40° C, such as 31° C to 37° C, and may be in a humidified incubator. The carbon dioxide content may be maintained between 2% to 10%and the oxygen content may be maintained between 1% and 22%. The disclosure, however, should in no way be construed to be limited to any one method of isolating and culturing cells. Rather, any method of isolating and culturing cells should be construed to be included in the present disclosure.

[0385] For use in the cell culture, media can be supplied with one or more further components. For example, additional supplements can be used to supply the cells with the necessary trace elements and substances for optimal growth and expansion. Such supplements include insulin, transferrin, selenium salts, and combinations thereof. These components can be included in a salt solution such as, but not limited to, Hanks’ Balanced Salt Solution (HBSS), Earle’s Salt Solution. Further antioxidant supplements may be added, e.g., P-mercaptoethanol. While many media already contain amino acids, some amino acids may be supplemented later, e.g., L-glutamine, which is known to be less stable when in solution. A medium may be further supplied with antibiotic and / or antimycotic compounds, such as, typically, mixtures of penicillin and streptomycin, and / or other compounds, exemplified but not limited to, amphotericin, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tylosin, and zeocin. Also contemplated is supplementation of cell culture medium with mammalian plasma or sera. Plasma or sera often contain cellular factors and components that are necessary for viability and expansion. The use of suitable serum replacements is also contemplated.

[0386] Reference to particular buffers, media, reagents, cells, culture conditions and the like, or to some subclass of same, is not intended to be limiting, but should be read to include all such related materials that one of ordinary skill in the art would recognize as being of interest or value in the particular context in which that discussion is presented. For example, it is often possible to substitute one buffer system or culture medium for another, such that a different but known way is used to achieve the same goals as those to which the use of a suggested method, material or composition is directed. In particular aspects, cells are cultured in a cell culture system comprising a cell culture medium, preferably in a culture vessel, in particular a cell culture medium supplemented with a substance suitable and determined for protecting the cells from in vitro aging and / or inducing in an unspecific or specific reprogramming.2. Cell Generation

[0387] Certain methods of the disclosure concern culturing the cells obtained from human tissue samples. In certain aspects, cells are fibroblasts, adipocytes, myeloid cells, B-cells, Tcells, immortalized cell lines, division-incompetent cell lines, epithelial cells, mesenchymal stem cells, or any combination thereof. In particular aspects, cells are mesenchymal stem cells. In particular aspects of the present disclosure, cells are plated onto a substrate that allows for adherence of cells thereto. This may be carried out, for example, by plating the cells in a culture plate that displays one or more substrate surfaces compatible with cell adhesion. When the one or more substrate surfaces contact the suspension of cells (e.g., suspension in a medium) introduced into the culture system, cell adhesion between the cells and the substrate surfaces may ensue. Accordingly, in certain aspects cells are introduced into a culture system that features at least one substrate surface that is generally compatible with adherence of cells thereto, such that the plated cells can contact the said substrate surface, such aspects encompass plating onto a substrate, which allows adherence of cells thereto.

[0388] Cells of the present disclosure may be identified and characterized by their expression of specific marker proteins, such as cell-surface markers. Detection and isolation of these cells can be achieved, for example, through flow cytometry, ELISA, and / or magnetic beads. Reverse-transcription polymerase chain reaction (RT-PCR) may be used to quantify cell-specific genes and / or to monitor changes in gene expression in response to differentiation.3. Cells

[0389] Aspects of the disclosure include the use of immunoregulatory cells to prevent or dampen unwanted auto- and alloimmune responses and / or overt inflammation, such as, but not limited to, graft versus host disease, immune rejection, autoimmune diabetes, multiple sclerosis, inflammatory bowel disease, and / or sepsis. In some aspects, the engineered cells may be localized to specific organs, tissues, or cells via the use of an artificial targeting construct (e.g., co-inhibitor engager). The targeting construct may be non-signaling or signaling, and may or may not produce additional effects in the parental and / or target cell.

[0390] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), human embryonic kidney (HEK) 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3),HLHepG2 cells, Hut-78, Jurkat, HL-60, NK cell lines (e.g., NKL, NK92, and YTS), and the like.

[0391] In some instances, the cell is not an immortalized cell line, but is instead a cell obtained from an individual (e.g., a primary cell). For example, in some aspects, the cells comprise, consist of, or consist essentially of mesenchymal stem cells (MSCs), fibroblasts, adipocytes, myeloid cells, B-cells, T-cells, epithelial cells or combinations thereof. In some cases, the cell is an immune cell obtained from an individual. As an example, the cell is a stem cell (e.g., peripheral blood stem cell, mesenchymal stem cell, etc.) or progenitor cell obtained from an individual. In some aspects, the cell is a mesenchymal stem cell. In some aspects, the cell is an immortalized cell line and / or division-incompetent cell line. In some aspects, a cell (such as but not limited to a B-cell, an MSC, a fibroblast, etc.) can be engineered in a manner similar to that as described in Evan Kleinboehl et al., 2022, “Primary B cell Engineering for Therapeutic Research” Trends Mol Med. 2022 Jun; 28(6) 528-529; which is incorporated herein by reference in its entirety for the purposes described herein. In some aspects, a cell is engineered to express polypeptides or peptides of the disclosure, antibodies and / or cytokines to treat a disorder.

[0392] The term “allogeneic” as used herein, refers to HLA or MHC loci that are antigenically distinct. a. Mesenchymal Stem Cells (MSCs)

[0393] As used herein, a “mesenchymal stem cell” is a mesenchymal cell having the ability to proliferate and to differentiate into one or more mesenchymal cells. Like a mesodermal cell, a mesenchymal stem cell is pluripotent, capable of differentiating into osteoblasts, cartilage cells, myoblasts, fat cells, stroma cells, tendon cells, and the like. Mesenchymal stem cells are known to persist for a long time in the adult body after it has passed through development.

[0394] As used herein, “mesenchymal cells” may be osteoblasts, cartilage cells, myoblasts, fat cells, stroma cells, tendon cells and other cells that form mesenchymal tissue, and mesenchymal stem cells which may differentiate into these. Mesenchymal cells occurring during embryogenesis, mesenchymal cells in individual animals, and mesenchymal cells generated by differentiation from pluripotent stem cells in vitro or in vivo are all included in the term “mesenchymal cells.”

[0395] Mesenchymal stem cells may be obtained from any source available to one skilled in the art, and by a number of methods well known in the art, see for example, U.S. Pat. Nos. 5,486,358; 6,387,367; and 7,592,174, and U.S. Pat App. Pub. No. 2003 / 0211602, the contentsof which are all incorporated herein by reference. Mesenchymal cells can include autologous mesenchymal stem cells, e.g., a cell or cells taken from an individual who is in need of treatment (e.g., the donor and recipient are the same individual). Autologous mesenchymal stem cells have the advantage of avoiding any immunologically-based rejection of the cells. Alternatively, the cells can be heterologous or allogeneic, e.g., taken from a donor. The second subject can be of the same or different species. Typically, when the cells come from a donor, they will be from a donor who is sufficiently immunologically compatible with the recipient, e.g., will not be subject to transplant rejection, to lessen or remove the need for immunosuppression. In some aspects, the cells are taken from a xenogeneic source, e.g., a nonhuman mammal that has been genetically engineered to be sufficiently immunologically compatible with the recipient, or the recipient’s species. Methods for determining immunological compatibility are known in the art, and include tissue typing to assess donorrecipient compatibility for HLA and ABO determinants. See, e.g., Transplantation Immunology, Bach and Auchincloss, Eds. (Wiley, John & Sons, Incorporated 1994).

[0396] In some aspects, the mesenchymal stem cell is derived from a de-differentiated somatic cell (a reprogrammed cell). For example, a somatic cell de-differentiated to a pluripotent stem cell, for example by direct reprogramming of a cell of endodermal origin. Without wishing to be bound by theory, a de-differentiated cell has a morphology that resembles a more primitive cell type from which it was derived, e.g., mesenchymal morphology.

[0397] In some aspects, the mesenchymal stem cell is a re-differentiated mesenchymal stem cell. As used herein, the term “re-differentiated mesenchymal stem cell” refers to a mesenchymal stem cell that is differentiated from a de-differentiated cell.

[0398] In some aspects, the mesenchymal stem cells are in a stabilized state, e.g., the cells were taken from a subject and treated in such a manner as to allow them to be stored for some period of time. For example, the cells can be frozen, e.g., using methods known in the art for freezing primary cells, such that the cells are viable when thawed. For example, methods known in the art to freeze and thaw embryos to generate live mammals can be adapted for use in the present methods. Such methods may include the use of liquid nitrogen, e.g., with one or more cryoprotectants, e.g., agents that prevent freeze-thaw damage to the cell.

[0399] The population of mesenchymal stem cells obtained from a subject or donor can be substantially pure. The purity of the population can be determined, and manipulated, using methods known in the art. For example, methods using fluorescence activated cell sorting can be used.

[0400] Without wishing to be bound by a theory, any suitable cell culture media can be used for in vitro or ex vivo methods of the disclosure. For example, MSCs can be maintained in a-MEM medium containing 10% fetal bovine serum.

[0401] In some aspects of this and other aspects of the disclosure, mesenchymal stem cells may be bone marrow-derived mesenchymal stem cells (BMSC).

[0402] In some aspects of this and other aspects of the disclosure, mesenchymal stem cells may be murine marrow derived mesenchymal stem cells.

[0403] In some aspects of this and other aspects of the disclosure, mesenchymal stem cells may be human mesenchymal stem cells (hMSC). b. T Cells

[0404] In some aspects, a cell may be a T cell. T cells (also referred to as T lymphocytes) belong to a group of white blood cells referred to as lymphocytes. Lymphocytes generally are involved in cell-mediated immunity. The “T” in “T cells” refers to cells from or whose maturation is influence by the thymus. T cells can be distinguished from other lymphocytes types such as B cells and Natural Killer (NK) cells by the presence of cell surface proteins known as T cell receptors. “T cell” includes all types of immune cells expressing CD3 including, but not limited to, T-helper cells, invariant natural killer T (iNKT) cells, cytotoxic T cells, T-regulatory cells (Tregs), alpha-beta (aP) T cells, and gamma-delta (y5) T cells. The T cell may refer to a CD4+or CD8+T cell.

[0405] The term “activated T cells” as used herein, refers to T cells that have been stimulated to produce an immune response (e.g., clonal expansion of activated T cells) by recognition of an antigenic determinant presented in the context of a Class II major histocompatibility (MHC) marker. T-cells are activated by the presence of an antigenic determinant, cytokines and / or lymphokines and cluster of differentiation cell surface proteins (e.g., CD3, CD4, CD8, the like and combinations thereof). Cells that express a cluster of differential protein often are said to be “positive” for expression of that protein on the surface of T-cells (e.g., cells positive for CD3 or CD4 expression are referred to as CD3+or CD4+). CD3 and CD4 proteins are cell surface receptors or co-receptors that may be directly and / or indirectly involved in signal transduction in T cells. c. NK Cells

[0406] In some aspects, a cell may be a natural killer (NK) cell. “NK cell” includes any cell along the NK-cell hematopoietic lineage, including, for example, CD56bright, CD56dim(CD16+KIRs+), CD57+, CD56brightCD16’, and / or CD56dimCD16+NK cells. NK cells may be identified morphologically, e.g., by the presence of cytoplasmic lytic granules, or by the use of markers, such as, but not limited to, T-BET, EOMES, perforin, granzyme, CD56, CD57, CD16, CD94, KIR, and / or NKG2C. d. Mononuclear Phagocytes

[0407] In some aspects, a cell may be a mononuclear phagocyte. “Mononuclear phagocytes” includes any type of monocytes, macrophages, or dendritic cells (DC). Mononuclear phagocyte subtypes may be identified morphologically or by the use of markers. For example, macrophages may be defined as cells expressing CSF1, CSF2, and / or IL-34; monocytes may be defined as cells expressing CSF1, CSF2, CD14, CD16, NR4A1, CEBPP, KLF2, CD209a, and / or Ly6C; dendritic cells may be defined as cells expressing CD8, CD103, CDl lb, CD172a, CD141, BDCA1, BATF3, IRF4, and / or E2-2. Monocytes may be CD 14+CD 16- (“classical”), CD14+CD16+(“intermediate”), or CD14lowCD16+(“non- classical”). DC may be conventional / classical (eDC) or plamacytoid DC (pDC). eDC may be further defined as CD141+(“cDCl”), or BDAC1+(“cDC2”).4. Cell Engineering

[0408] Certain aspects relate to cells comprising polypeptides or nucleic acids of the disclosure.

[0409] The term “engineered” as used herein refers to an entity that is generated by the hand of man, including a cell, nucleic acid, polypeptide, vector, and so forth. In at least some cases, an engineered entity is synthetic and comprises elements that are not naturally present or configured in the manner in which it is utilized in the disclosure. In specific aspects, a cell is engineered through transfection or transduction of an engineered vector. Cells may be engineered to express heterologous proteins that are not naturally expressed by the cells, either because the heterologous proteins are recombinant or synthetic or because the cells do not naturally express the proteins.

[0410] Methods for producing cells comprising vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York; incorporated herein by reference.

[0411] In some aspects, engineered polynucleotides described herein may be packaged in viral vectors for engineering cells of interest. In some aspects, viral vectors comprisingengineered polynucleotides described herein are used to transduce fibroblasts, adipocytes, myeloid cells, B-cells, T cells, immortalized cells, division-incompetent cells, epithelial cells, mesenchymal stem cells, or a combination thereof.

[0412] In some aspects, engineered cells comprise expression of a heterologous CTLA-4, a heterologous cytokine, and an engineered IL receptor (e.g., cIL2R) (e.g., triple-engineered cells). In some aspects, triple-engineered cells show enhanced immunosuppressive functions compared to non-engineered cells, e.g., Treg cells, or cells engineered to comprise one or two of a heterologous CTLA-4, a heterologous cytokine, or an engineered IL receptor (e.g., cIL2R). In some aspects, an engineered cell is engineered to comprise a heterologous co-inhibitor engager. In some aspects, engineered cells comprise expression of a heterologous CTLA-4, a heterologous cytokine, a heterologous IL receptor (e.g., cIL2R), and a heterologous coinhibitor engager (e.g., quadruple-engineered cells). In some aspects, quadruple-engineered cells show enhanced immunosuppressive functions compared to non-engineered cells, e.g., Treg cells, or cells engineered to comprise one, two or three, immunosuppressive system components.

[0413] In certain aspects, a cell is engineered to comprise a vector that comprises, consists essentially of, or consists of a sequence of, of at least, or of at most 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range or value derivable therein, identity to any one of SEQ ID NOs: 1-46, 60-64, 70-71, or 74-75. a. Transduction

[0414] As used herein the term “transduce” or “transduction” refers to the process whereby a foreign nucleotide sequence is introduced into a cell by means of a virus or viral vector. In some aspects, this transduction is done via a viral vector.

[0415] In certain aspects, the term “viral vector” intends a recombinant vector that retains the ability to infect and transduce nondividing and / or slowly-dividing cells and may integrate into the target cell’s genome. In some aspects, the vector may be derived from or based on a wild-type virus. In some aspects, the vector may be derived from or based on a wild-type lentivirus. Examples of such include without limitation, human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), simian immunodeficiency virus (SIV) and feline immunodeficiency virus (Hy). Alternatively, it is contemplated that other retrovirus can be used as a basis for a vector backbone such as murine stem cell virus (MSCV) or Moloney Murine Leukemia Virus (MoMLV). It will be evident that a viral vector according to thedisclosure need not be confined to the components of a particular virus. The viral vector may comprise components from two or more different viruses, and may also comprise synthetic components. Viral vector components may be manipulated to obtain desired characteristics, such as target cell specificity.

[0416] The recombinant viral vectors of this disclosure may be from primates and nonprimates. Examples of primate lentiviruses include the human immunodeficiency virus (HIV), the causative agent of human acquired immunodeficiency syndrome (AIDS), and the simian immunodeficiency virus (SIV). The non-primate lentiviral group includes the proto-type “slow virus” visna / maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV) and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV). Prior art recombinant lenti-viral vectors are known in the art, e.g., see U.S. Pat. Nos. 6,924,123; 7,056,699; 7,419,829 and 7,442,551, incorporated herein by reference. b. Transfection

[0417] As used herein, the term “transfection” is defined as the introduction of an extracellular nucleic acid into a host cell by any means known in the art, including calcium phosphate co-precipitation, viral transduction, liposome fusion, microinjection, microparticle bombardment, electroporation, etc. The terms “uptake of nucleic acid by a host cell”, “taking up of nucleic acid by a host cell”, “uptake of particles comprising nucleic acid by a host cell”, and “taking up of particles comprising nucleic acid by a host cell” denote any process wherein an extracellular nucleic acid, with or without accompanying material, enters a host cell.

[0418] A variety of methods are known in the art and suitable for transfection of nucleic acid into a cell. The polynucleotides of the present disclosure may be formulated, using the methods described herein. The formulations may contain polynucleotides which may be modified and / or unmodified. The formulations may further include, but are not limited to, cell penetration agents, a pharmaceutically acceptable carrier, a delivery agent, a bioerodible or biocompatible polymer, a solvent, and / or a sustained-release delivery depot.

[0419] The formulated polynucleotides may be delivered to the cell using routes of administration known in the art and described herein. Examples of typical methods include, but are not limited to, naked delivery, lipidoid mediate transfer, liposome-, lipoplexes, and / or lipid nanoparticle-mediated transfer, electroporation, calcium phosphate mediated transfer, nucleofection, sonoporation, heat shock, magnetofection, microinjection, microprojectilemediated transfer (nanoparticles), cationic polymer mediated transfer (DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like) or cell fusion. c. Cryopreserved Cells

[0420] While autologous cells can be used for some aspects of the disclosure, their availability can be limiting and obtaining cells from a subject can be onerous and / or expensive. In some aspects, a frozen therapeutic cell preparation may be prepared by placing cultured cells in an appropriate cryopreservative for cryopreservation. Cells to be used for individual doses may be frozen, e.g., cryopreserved for later use. Individual doses may comprise, consist essentially of, or consist of about 1 million to about 100 million cells per mL. Individual doses may comprise, consist of, or consist essentially of between about 106and about 109cells in total.

[0421] Cells described herein may be cryopreserved, e.g., in cryopreservation medium in small containers, e.g., ampoules. Suitable cry opreservation medium includes, but is not limited to, culture medium including, e.g., growth medium, or cell freezing medium, for example commercially available cell freezing medium, e.g., C2695, C2639 or C6039 (SIGMA™). Cryopreservation medium may comprises DMSO (dimethyl sulfoxide), at a concentration of, e.g., about 10-20% (v / v). Cryopreservation medium may comprise additional agents, for example, Plasmalyte, methylcellulose with or without glycerol. Cells are preferably cooled at about 1° C. / min during cry opreservation. A preferred cry opreservation temperature is about -80° C. to about -180° C., preferably about -125° C. to about -140° C. Cryopreserved cells may be transferred to liquid nitrogen prior to thawing for use. For example, once the ampoules have reached about -90° C., they are transferred to a liquid nitrogen storage area. Cryopreserved cells preferably are thawed at a temperature of about 25° C. to about 40° C., preferably to a temperature of about 37° C.

[0422] Other preservation methods are described in U.S. Pat. Nos. 5,656,498, 5,004,681, 5,192,553, 5,955,257, and 6,461,645, all of which are incorporated herein by reference.D. Immune Disorder

[0423] The immune system comprises innate and adaptive immunity. Innate immunity refers to an early system of defense that depends on invariant receptors recognizing common features of pathogens. The innate immune system provides barriers and mechanisms to inhibit foreign substances, in particular through the action of macrophages and neutrophils. The inflammatory response is considered part of innate immunity. The innate immune system isinvolved in initiating adaptive immune responses and removing pathogens that have been targeted by an adaptive immune response. However, innate immunity can be evaded or overcome by many pathogens, and does not lead to immunological memory. Adaptive immunity refers to the ability to recognize pathogens specifically and to provide enhanced protection against reinfection due to immunological memory based on clonal selection of lymphocytes bearing antigen-specific receptors. A process of random recombination of variable receptor gene segments and the pairing of different variable chains generates a population of lymphocytes, each bearing a distinct receptor, forming a repertoire of receptors that can recognize virtually any antigen. If the receptor on a lymphocyte is specific for a ubiquitous self-antigen, the cell is normally eliminated by encountering the antigen early in its development. Adaptive immunity is normally initiated when an innate immune response fails to eliminate a new infection, and antigen and activated antigen-presenting cells are delivered to draining lymphoid tissues. When a recirculating lymphocyte encounters its specific foreign antigen in peripheral lymphoid tissues, it is induced to proliferate and its progeny then differentiate into e...

Claims

WHAT IS CLAIMED IS:

1. A polynucleotide encoding an engineered interleukin receptor, wherein the encoded engineered interleukin receptor comprises, a) a first interleukin receptor extracellular domain, b) a second interleukin receptor extracellular domain, and c) an interleukin receptor transmembrane and intracellular signaling domain, wherein the domains operably linked by one or more linkers form a heterologous engineered interleukin receptor.

2. The polynucleotide of claim 1, wherein the first interleukin receptor extracellular domain and the second interleukin receptor extracellular domain are from different proteins.

3. The polynucleotide of claim 1, wherein the first interleukin receptor extracellular domain and the second interleukin receptor extracellular domain are each from the same proteins.

4. The polynucleotide of claim 1, wherein the first interleukin receptor extracellular domain and the second interleukin receptor extracellular domain are each from different proteins, and the interleukin receptor transmembrane and intracellular signaling domain is from a different protein.

5. The polynucleotide of claim 1, wherein the first interleukin receptor extracellular domain is from CD122 (interleukin-2 receptor subunit beta) or CD25 (interleukin-2 receptor subunit alpha).

6. The polynucleotide of claim 1, wherein the second interleukin receptor extracellular domain is from CD25 or CD122.

7. The polynucleotide of claim 1, wherein the interleukin receptor transmembrane and intracellular signaling domain is from CD 132 (interleukin-2 receptor subunit gamma).

8. The polynucleotide of claim 1, wherein a) the first interleukin receptor extracellular domain is from CD122, b) the second interleukin receptor extracellular domain is from CD25, and c) the interleukin receptor transmembrane and intracellular signaling domain is from CD132.

9. The polynucleotide of claim 1, wherein a) the first interleukin receptor extracellular domain is from CD25, b) the second interleukin receptor extracellular domain is from CD122, and c) the interleukin receptor transmembrane and intracellular signaling domain is from CD132.

10. The polynucleotide of claim 1, wherein the domains are operably linked by one or more linkers, wherein the linker comprises a glycine polymer, glycine-alanine polymer, alanineserine polymer, or glycine-serine polymer.

11. The polynucleotide of claim 1, wherein the linker comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 89-97.

12. The polynucleotide of claim 1, wherein the liker is encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 86-88.

13. The polynucleotide of claim 1, further comprising a signal peptide.

14. The polynucleotide of claim 13, wherein the signal peptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 101-103.

15. The polynucleotide of claim 13, wherein the signal peptide is encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 98-100.

16. The polynucleotide of claim 1, wherein the engineered interleukin receptor comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 50-53.

17. The polynucleotide of claim 1, wherein the engineered interleukin receptor is encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 5-8, 20-23, or 37-40.

18. A polypeptide encoded by the polynucleotide of claim 1.

19. A composition comprising the polynucleotide or polypeptide of any one of claims 1-18.

20. An immunosuppressive system comprising one or more polynucleotides, and / or polypeptides of any one of claims 1-18.

21. One or more polynucleotides encoding an immunosuppressive system, wherein the encoded immunosuppressive system comprises one or more of an engineered interleukin receptor, one or more of a heterologous costimulatory antagonist, one or more of a heterologous immunosuppressive cytokine, and / or one or more of a co-inhibitor engager.

22. The one or more polynucleotides of claim 21, wherein the immunosuppressive system comprises an engineered interleukin receptor, a heterologous costimulatory antagonist, and a heterologous immunosuppressive cytokine.

23. The one or more polynucleotides of claim 21, wherein the immunosuppressive system comprises an engineered interleukin receptor, a heterologous costimulatory antagonist, a heterologous immunosuppressive cytokine, and a co-inhibitor engager.

24. The one or more polynucleotides of claim 21, wherein the co-inhibitor engager comprises a T-cell checkpoint activator and / or a T-cell inhibitor.

25. The one or more polynucleotides claim 21, wherein the engineered interleukin receptor comprises, a) a first interleukin receptor extracellular domain from CD 122, b) a second interleukin receptor extracellular domain from CD25, and c) an interleukin receptor transmembrane and intracellular signaling domain from CD132.

26. The one or more polynucleotides of claim 21, wherein the engineered interleukin receptor comprises, a) a first interleukin receptor extracellular domain from CD25, b) a second interleukin receptor extracellular domain from CD 122, and c) an interleukin receptor transmembrane and intracellular signaling domain from CD132.

27. The one or more polynucleotides of claim 21, wherein the engineered interleukin receptor comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 50-53.

28. The one or more polynucleotides of claim 21, wherein the engineered interleukin receptor is encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 5-8, 20-23, or 37-40.

29. The one or more polynucleotides of claim 21, wherein the heterologous costimulatory antagonist comprises CTLA-4.

30. The one or more polynucleotides of claim 29, wherein CTLA-4 comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 47-48.

31. The one or more polynucleotides of claim 29, wherein CTLA-4 is encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-2, 15-17, or 32-33.

32. The one or more polynucleotides of claim 21, wherein the heterologous immunosuppressive cytokine comprises TGF-P, IL-10, and / or IL-35.

33. The one or more polynucleotides of claim 21, wherein the heterologous immunosuppressive cytokine comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 49, 55-59, 72-73, or 76-77.

34. The one or more polynucleotides of claim 21, wherein the heterologous immunosuppressive cytokine is encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 3-4, 11- 14, 18-19, 28-31, 35-36, 42-46, 70-71, or 74-75.

35. The one or more polynucleotides of claim 21, wherein the heterologous immunosuppressive cytokine comprises TGF-p.

36. The one or more polynucleotides of claim 35, wherein the TGF-P comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 55-56, or 72-73.

37. The one or more polynucleotides of claim 35, wherein the TGF-P is encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 11-12, 26-27, 42-43, or 70-71.

38. The one or more polynucleotides of claim 21, wherein the co-inhibitor engager comprises affinity to an inflammatory marker.

39. The one or more polynucleotides of claim 21, wherein the co-inhibitor engager is a TIGIT, PD-1, CD5, CD3, LAG3, BTLA, CEACAM-1, 2B4, CD200, CD160, and / or TIM-3 co-inhibitor engager.

40. The one or more polynucleotides of claim 21, wherein the co-inhibitor engager comprises a ligand for a T cell checkpoint receptor and / or T cell inhibitory receptor.

41. The one or more polynucleotides of claim 21, wherein the co-inhibitor engager comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 140-148, 156-162, 182-190, or 195-196.

42. The one or more polynucleotides of claim 21, wherein the co-inhibitor engager is encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 113-139, 149-155, 173-181, or 191-194.

43. The one or more polynucleotides of claim 21, wherein the co-inhibitor engager comprises a TIGIT co-inhibitor engager.

44. The one or more polynucleotides of claim 43, wherein the TIGIT co-inhibitor engager comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 146, 159-160, or 188.

45. The one or more polynucleotides of claim 43, wherein the TIGIT co-inhibitor engager is encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 119, 128, 137, 152-153, or 179.

46. The one or more polynucleotides of claim 21, wherein the co-inhibitor engager comprises a PD1 co-inhibitor engager.

47. The one or more polynucleotides of claim 46, wherein the PD1 co-inhibitor engager comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 140-142, 145, 156-158, or 182-187.

48. The one or more polynucleotides of claim 46, wherein the PD1 co-inhibitor engager is encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 131-133, 136, 149, 150-151, 173-175, and 178.

49. The one or more polynucleotides of claim 21, wherein the co-inhibitor engager comprises a TIM3 co-inhibitor engager.

50. The one or more polynucleotides of claim 49, wherein the TIM3 co-inhibitor engager comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 147, 161-162, 189, or 195-196.

51. The one or more polynucleotides of claim 49, wherein the TIM3 co-inhibitor engager is encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 138, 154-155, 180, or 191-194.

52. The one or more polynucleotides of claim 21, wherein the immunosuppressive system is encoded by 1, 2, 3, 4, or more polynucleotides.

53. The one or more polynucleotides of claim 21, wherein the immunosuppressive system is encoded by 1 polynucleotide.

54. The one or more polynucleotides of claim 21, wherein the one or more polynucleotides are polycistronic.

55. The one or more polynucleotides of claim 21, wherein the one or more polynucleotides are operatively under the control of one or more heterologous promoters.

56. The one or more polynucleotides of claim 55, wherein the one or more heterologous promoters are constitutively active and / or are inducible.

57. The one or more polynucleotides of claim 54, wherein the one or more heterologous promoters are substantially the same promoter comprising greater than or equal to 80%, 85%,90%, 95%, 99%, or 100% sequence identity, or wherein the one or more heterologous promoters are different.

58. The one or more polynucleotides of claim 54, wherein the one or more polycistronic polynucleotides comprise one or more of an internal ribosomal entry site (IRES) and / or a selfcleaving peptide.

59. The one or more polynucleotides of claim 58, wherein the IRES comprises a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 80.

60. The one or more polynucleotides of claim 58, wherein the self-cleaving sequence is a 2A sequence.

61. The one or more polynucleotides of claim 60, wherein the 2A sequence comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 83-85.

62. The one or more polynucleotides of claim 60, wherein the 2A sequence is encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 81-82.

63. The one or more polynucleotides of claim 21, wherein the one or more polynucleotides comprises one or more regulatory regions.

64. The one or more polynucleotides of claim 63, wherein the regulatory regions comprise one or more of a long-terminal repeat, a promoter, and / or a 3' untranslated region (UTR).

65. The one or more polynucleotides of claim 21, wherein the one or more encoding polynucleotides are comprised in a vector.

66. The one or more polynucleotides of claim 65, wherein the vector is a viral vector.

67. The one or more polynucleotides of claim 65, wherein the vector is a lentivirus.

68. A cell comprising the one or more polynucleotides, polypeptides, compositions and / or vectors of any one of claims 1-67.

69. The cell of claim 68, wherein the cell is a mesenchymal stem cell (MSC), fibroblast, adipocyte, myeloid cell, B-cell, T cell, immortalized cell line, division-incompetent cell line, epithelial cell, or a combination thereof.

70. The cell of claim 68, wherein the cell is a MSC.

71. A method of preparing a cell comprising the step of introducing the polynucleotide, polypeptide, and / or immunosuppressive system according to any one of aspects 1-66.

72. A composition comprising, the polynucleotide, polypeptide, cell, composition, or a combination thereof of any one of claims 1-70.

73. The composition of claim 72, wherein the composition is frozen.

74. The composition of claim 72, wherein the composition is comprised in a delivery device.

75. A method of treating a disease in an individual, the method comprising the step of administering to the individual in need thereof a therapeutically effective amount of the composition of claim 72.

76. The method of claim 75, wherein the individual has been diagnosed, is suspected of having, or has one or more symptoms of an autoimmune disease, transplant rejection, bacterial sepsis, viral sepsis, fungal sepsis, and / or graft-versus-host disease.

77. The method of claim 75, wherein the individual is administered an engineered cell.

78. The method of claim 77, wherein the engineered cell displays an increase in in vivo immunosuppressive functionality relative to a non-engineered cell.

79. The method of claim 78, wherein the increase in in vivo immunosuppressive functionality comprises a reduction in immune cell proliferation, reduction in response to foreign antigens, reduction in response to self-antigens, reduction in inflammatory responses, reduction in organ damage, prolongation in the individual’ s survival, reduction in graft toxicity, reduction in weight loss, or a combination thereof.

80. A method of providing an individual with an immunosuppressive response, the method comprising administering to the individual in need thereof a therapeutically effective amount of the polynucleotide, polypeptide, cell, and / or composition, of any one of claims 1-74.

81. The method of claim 75 or 80, further comprising administering at least a second therapeutic agent to the individual.

82. A kit comprising the polynucleotide, polypeptide, cell, composition, or a combination thereof of any one of claims 1-74.

83. A kit for the means of performing the methods of claim 75 or 80.

84. Use of the polynucleotide, polypeptide, cell, composition, kit, or a combination thereof of any one of claims 1-74 or 82-83 for production of a medicament for treatment of a disease and / or disorder.

85. A polynucleotide encoding an engineered interleukin receptor, wherein the encoded engineered interleukin receptor comprises, a) a first interleukin receptor extracellular domain from CD 122, b) a second interleukin receptor extracellular domain from CD25, and c) an interleukin receptor transmembrane and intracellular signaling domain from CD 132, wherein the domains are operably linked in a 5' to 3' order of (a), (b), and (c) to form a heterologous engineered interleukin receptor.

86. An engineered interleukin receptor comprising, a) a first interleukin receptor extracellular domain from CD 122, b) a second interleukin receptor extracellular domain from CD25, and c) an interleukin receptor transmembrane and intracellular signaling domain from CD 132, wherein the domains are operably linked in a 5' to 3' order of (a), (b), and (c) to form a heterologous engineered interleukin receptor.

87. One or more polynucleotides encoding an immunosuppressive system, wherein the encoded immunosuppressive system comprises i) an engineered interleukin receptor comprisinga) a first interleukin receptor extracellular domain from CD 122, b) a second interleukin receptor extracellular domain from CD25, and c) an interleukin receptor transmembrane and intracellular signaling domain from CD 132; ii) a heterologous costimulatory antagonist comprising CTLA-4, iii) a heterologous immunosuppressive cytokine comprising TGF-P, and optionally iv) a co-inhibitor engager targeting TIGIT.

88. A mesenchymal stem cell comprising i) an engineered interleukin receptor comprising a) a first interleukin receptor extracellular domain from CD 122, b) a second interleukin receptor extracellular domain from CD25, and c) an interleukin receptor transmembrane and intracellular signaling domain from CD 132; ii) a heterologous costimulatory antagonist comprising CTLA-4, iii) a heterologous immunosuppressive cytokine comprising TGF-P, and optionally iv) a co-inhibitor engager targeting TIGIT.

89. A method of treating graft-versus-host disease in an individual in need thereof comprising administering to the individual a mesenchymal stem cell comprising i) an engineered interleukin receptor comprising a) a first interleukin receptor extracellular domain from CD 122, b) a second interleukin receptor extracellular domain from CD25, and c) an interleukin receptor transmembrane and intracellular signaling domain from CD 132; ii) a heterologous costimulatory antagonist comprising CTLA-4, iii) a heterologous immunosuppressive cytokine comprising TGF-P, and optionally iv) a co-inhibitor engager targeting TIGIT.

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